RNAS FROM PATHOGENS INHIBIT PLANT IMMUNITY

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing named “081906-1099170-214920US_SEQ.txt”, created on Sep. 24, 2018, 224,616 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Botrytis cinerea is a fungal pathogen that infects almost all vegetable and fruit crops and annually causes $10-100 billion losses worldwide. With its broad host range, B. cinerea is a useful model for studying the pathogenicity of aggressive fungal pathogens. Many pathogens of plants and animals deliver effectors into host cells to suppress host immunity (H. Ashida et al., Curr. Opin. Microbiol. 14, 16 (2011); M. Rafiqi et al., Curr. Opin. Plant Biol. 15, 477 (2012); T. O. Bozkurt et al., Curr. Opin. Plant Biol. 15, 483 (2012); H. Hilbi, et al., Traffic 13, 1187 (2012)).

sRNAs induce gene silencing by binding to Argonaute (AGO) proteins and directing the RNA-induced silencing complex (RISC) to genes with complementary sequences. sRNAs from both plant and animal hosts have been recognized as regulators in host-microbial interaction (5-8). Although sRNAs are also present in various fungi and oomycetes, including many pathogens (9-14), it has not been clear whether they regulate host-pathogen interaction.

BRIEF SUMMARY OF THE INVENTION

The present application provides for plants (or a plant cell, seed, flower, leaf, fruit, or other plant part from such plants or processed food or food ingredient from such plants) comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide that is complementary to, or mediates destruction, of a plant immunity suppressing sRNA of a pathogen, wherein the plant is less susceptible to the pathogen compared to a control plant lacking the expression cassette.

In some embodiments, the polynucleotide encodes a short tandem target mimic (STTM) of the sRNA. In some embodiments, the STTM is engineered from primers (a forward primer and a reverse primer) listed in Table 2. In some embodiments, the polynucleotide encodes an antisense nucleic acid that is complementary to the sRNA.

The present application also provides for plants (or a plant cell, seed, flower, leaf, fruit, or other plant part from such plants or processed food or food ingredient from such plants) comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide that is an sRNA-resistant target that encodes a protein that functions in plant immunity, wherein the promoter is heterologous to the polynucleotide. In some embodiments, a plant into which the expression cassette has been introduced has enhanced pathogen resistance compared to a control plant lacking the expression cassette.

In some embodiments, the polynucleotide is substantially (e.g., at least 60, 70, 75, 80, 85, 90, or 95%) identical to any of SEQ ID NOS:4-13. In some embodiments, the polynucleotide is an sRNA-resistant target encoding mitogen activated protein kinase 1 (MPK1), mitogen activated protein kinase 2 (MPK2), peroxiredoxin (PRXIIF), cell-wall associated kinase (WAK), or tomato mitogen activated protein kinase kinase kinase 4 (MAPKKK4). In some embodiments, the polynucleotide is an sRNA-resistant target of a gene listed in FIG. 1, Table 1, or Table 3. In some embodiments, the polynucleotide is resistant to gene silencing by an sRNA listed in Table 1. In some embodiments, the polynucleotide is resistant to gene silencing by Bc-siR3.1, Bc-siR3.2, or Bc-siR5.

In some embodiments, the sRNA comprises a sequence listed in Table 1. In some embodiments, the sRNA comprises the sequence of Bc-siR3.1, Bc-siR3.2, or Bc-siR5.

In some embodiments, the pathogen is Botrytis. In some embodiments, the pathogen is Botrytis cinera.

In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is pathogen inducible. In some embodiments, the promoter is induced upon infection by Botrytis. In some embodiments, the promoter is substantially (e.g., at least 60, 70, 75, 80, 85, 90, or 95%) identical to Arabidopsis BIK1 (SEQ ID NO:1), Arabidopsis PDF1.2 (SEQ ID NO:2), or tomato TPK1b (SEQ ID NO:3). In some embodiments, the promoter is stress-inducible. In some embodiments, the promoter is tissue-specific. In some embodiments, the promoter is specifically expressed in the epidermis. In some embodiments, the promoter is substantially (e.g., at least 60, 70, 75, 80, 85, 90, or 95%) identical to Arabidopsis ML1 (SEQ ID NO:14) or tomato ML1 (SEQ ID NO:15).

In another aspect, the present invention provides for expression cassettes comprising: a promoter operably linked to a polynucleotide that is complementary to, or mediates destruction, of a plant immunity suppressing sRNA of a pathogen, wherein the plant is less susceptible to the pathogen compared to a control plant lacking the expression cassette; or comprising a promoter operably linked a polynucleotide that is an sRNA-resistant target that encodes a protein that functions in plant immunity, wherein the promoter is heterologous to the polynucleotide. Isolated nucleic acids comprising said expression cassettes are also provided.

In still another aspect, the present invention provides for expression vectors comprising an expression cassette as described herein.

In another aspect, methods of making a pathogen-resistant plant are provided. In some embodiments, the method comprises:

- introducing the nucleic acid comprising an expression cassette as described herein into a plurality of plants; and
- selecting a plant comprising the expression cassette

In yet another aspect, methods of cultivating a plurality of pathogen-resistant plants are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G. Bc-sRNAs silence host target genes in both Arabidopsis and S. lycopersicum during B. cinerea infection. (A) Bc-siR3.1, Bc-siR3.2, and Bc-siR5 were expressed during infection of Arabidopsis as detected at 18, 24, 48, and 72 hpi and, (B) S. lycopersicum leaves at 18, 24, 32, 48 hpi by RT-PCR. Actin genes of B. cinerea, and Arabidopsis and S. lycopersicum were used as internal controls. Similar results were obtained from three biological replicates. (C) The Arabidopsis targets of Bc-siRNAs were suppressed at 24, 32, and 48 hpi of B. cinerea infection. PDF1.2, BIK1 and β-tubulin were used as controls. (D) The S. lycopersicum target gene MAPKKK4 was suppressed upon B. cinerea infection. Expression (C and D) was measured by quantitative RT (qRT)-PCR using actin as an internal control. Error bars indicate standard deviation of three technical replicates. Similar results were seen in three biological replicates. (E) Co-expression of Bc-siR3.2 or Bc-siR5 with their host targets (HA-tagged) in N. benthamiana revealed target silencing by Western blot analysis. Co-expression of AtmiR395 or target site-mutated versions of target genes was used as controls. (F) Expression of YFP-MPK2 or its synonymously mutated version (YFP-MPK2-m) after infection of B. cinerea was observed by confocal microscopy. Co-expression of YFP-MPK2 and Bc-siR3.2 was used as a control. (G) Expression of the YFP sensors carrying a Bc-siR3.2 target site of MPK2 or a Bc-siR3.2 target site-m was analyzed after infection of B. cinerea. Samples were examined at 24 hpi. Upper panel: YFP; bottom panel: YFP/bright field overlay; scale bars (F, G), 37.5 μm. Error bars indicate standard deviation of 20 images (F, G). The asterisk indicates significant difference (two-tail t-test; p<0.01). Similar results were obtained in three biological replicates in E-G.

FIGS. 2A-2F. Bc-sRNAs trigger silencing of host targets that are involved in host immunity. (A) Expression of Bc-siR3.1, BcsiR3.2, or Bc-siR5 in transgenic Arabidopsis ectopically expressing Bc-siRNAs under the Cauliflower Mosaic Virus promoter 35S (Bc-sRNAox) was examined by Northern blot analysis. Highly expressed lines were selected for the following experiments. (B) Bc-sRNAox lines showed constitutive silencing of respective Bc-siRNA target genes measured by qRT-PCR. Two independent lines for each Bc-sRNAs were examined. Similar results were observed in two generations of the selected transgenic lines. (C) Bc-sRNAox plants exhibited enhanced disease susceptibility to B. cinerea compared to the wild type. (D) Loss-of-function mutants of Bc-siR3.2 and Bc-siR5 targets mpk1 mpk2 and wak displayed enhanced disease susceptibility. In all pathogen assays (C and D), lesion sizes were measured at 96 hpi. Error bars indicate the standard deviation of 20 leaves. (E) Biomass of B. cinerea was measured by qPCR at 96 hpi. Error bars indicate standard deviation of three technical replicates. For C, D and E, similar results were obtained from three biological repeats. (F) Virus-induced gene silencing (VIGS) of MAPKKK4 exhibited enhanced disease susceptibility to B. cinerea in S. lycopersicum (examined at 72 hpi) compared to control plants (TRV-RB). RB is a late-blight resistance gene that is not present in tomato. We chose to use a TRV vector with a fragment from a foreign gene as a control to eliminate the potential side effect of viral disease symptoms caused by TRV empty vector. Spray inoculation was used because silencing sectors are not uniform within the VIGS plants. Three sets of experiments with each of 6-10 plants for each construct were performed, and similar results were obtained. The asterisk indicates significant difference (two-tail t-test, p<0.01) in C-F.

FIGS. 3A-3D. Bc-sRNAs hijack Arabidopsis AGO1 to suppress host immunity genes. (A) Loading of Bc-siR3.1, Bc-siR3.2 and Bc-siR5 into Arabidopsis AGO1 during infection was detected by AGO1-IP followed by RT-PCR. AGO1 from B. cinerea-infected leaves harvested at 24, 32 and 48 hpi was pulled down by AGO1 peptide antibody, and RNA was extracted from the AGO1-IP fraction. As a control, non-infected leaves mixed with B. cinerea mycelium (at least twice as much as that in B. cinerea-infected leaves at 48 hpi) were used to rule out any binding between AGO1 and Bc-sRNAs during the experimental procedures. Similar results were obtained from at least three biological repeats. (B) Arabidopsis ago1-27 exhibited reduced disease susceptibility to B. cinerea compared to the wild type. Lesion size of at least 20 leaves and fungal biomass were measured at 96 hpi. (C) Silencing of MPK2, MPK1, PRXIIF, and WAK during B. cinerea infection was abolished in ago1-27. (D) Arabidopsis dcl1-7 exhibited enhanced disease susceptibility to B. cinerea compared to the wild type. Similar results were obtained from three biological repeats (B-D). The asterisk indicates significant difference (two-tail t-test, p<0.01) in B, D.

FIGS. 4A-4G. B. cinerea dcl1 dcl2 double mutant is compromised in virulence. (A) B. cinerea dcl1 dcl2 double mutant, but not dcl1 or dcl2 single mutants were impaired in generating Bc-siR3.1, Bc-siR3.2, and Bc-siR5 as revealed by RT-PCR. B. cinerea dcl1 dcl2 double mutant, but not dcl1 or dcl2 single mutants, produced much weaker disease symptoms than the wild type in Arabidopsis (B) and S. lycopersicum (C), as demonstrated by the lesion size measured of 20 leaves at 96 hpi and 48 hpi, respectively. Similar results were obtained from three biological repeats. (D) Expression of the sensor YFP-Bc-siR3.2 target site was silenced by wild type B. cinerea upon infection, but not by the dcl1 dcl2 mutant at 24 hpi (scale bar: 75 μm). Error bars indicate standard deviation of 20 images. Experiments were repeated two times with similar results. (E) B. cinerea dcl1 dcl2 mutant was compromised in suppression of MPK2, MPK1, PRXIIF in Arabidopsis, and MAPKKK4 in S. lycopersicum. Similar results were seen in two biological repeats. (F) Arabidopsis Bc-siR3.1ox and Bc-siR3.2ox lines were more susceptible to B. cinerea dcl1 dcl2 strain than Col-0 wild type. (G) Enhanced disease phenotype of dcl1 dcl2 infection was also observed on three TRV-MAPKKK4 silenced S. lycopersicum plants. Experiments in F and G were repeated three times with similar results. B. cinerea biomass was quantified at 96 hpi. The asterisk (in B, C, D, F, G) indicates significant difference (two-tail t-test; p<0.01).

FIG. 5. Genomic map and read distribution of Bc-SIR3 and Bc-SIR5 loci. The genomic regions of 60 nt up- and downstream of the Bc-sRNA of interest were included. Sequence reads of Bc-siR3 and Bc-siR5 in B. cinerea-infected Arabidopsis (0, 24, 48, 72 hpi), B. cinerea-infected S. lycopersicum (leaf/fruit 0, 24, 72 hpi), or in vitro culture B. cinerea sRNA libraries (conidiospores, mycelia, total biomass) (see, FIG. 15) are shown in three individual panels. Bc-siR3 and Bc-siR5 reads are in red. In vitro culture B. cinerea sRNA libraries did not show a clear peak for Bc-siR3.1 or Bc-siR3.2 compared to B. cinerea-infected Arabidopsis and S. lycopersicum libraries, indicating that those Bc-siRNAs were induced during infection. Similarly, Bc-SIR5 showed induction upon infection.

FIGS. 6A-6C. (A) Target site and target site mutated versions of Bc-siRNA Arabidopsis target genes that were used in this study (SEQ ID NOS:16, 17-19, 17 and 20-23, respectively). (B) B. cinerea mycelium coincided with target gene suppression of YFP-MPK2 (center), but not YFP-MPK2-m (right) in N. benthamiana at 24 hpi; YFP-MPK2 without fungal infection was used as a control (left). Upper panel: YFP; bottom panel: YFP/bright field overlay; scale bar: 50 μm. (C) A schematic diagram of the YFP sensor carrying a Bc-siR3.2 target site.

FIGS. 7A-7B. Isolation and characterization of Bc-siRNA target mutants and Bc-siRNAox lines. (A) Isolation of a loss-of function mutant line for WAK gene (At5g50290). Expression of WAK was completely knocked out in the T-DNA insertion line shown by RT-PCR. (B) Induction of BIK1 expression in response to B. cinerea infection was reduced in Bc-siR3.1ox and Bc-iR3.2ox lines, mpk1 mpk2, and wak mutant lines. Relative transcript levels of BIK1 were measured by real time RT-PCR. Error bars indicate standard deviation (SD) of three technical replicates. Similar results were obtained from two biological repeats.

FIGS. 8A-8B. S. lycopersicum MAPKKK4 gene knockdown by TRV-induced gene silencing. (A) Expression of MAPKKK in S. lycopersicum TRV-MAPKKK4 silenced plants was measured by qRT-PCR using actin as an internal control. Error bars indicate SD of three technical replicates. Similar results were obtained from three biological repeats. (B) TRV-MAPKKK4 silenced plants exhibited a dwarf phenotype as compared with control plants (TRV-RB).

FIG. 9. Bc-siR3.1 and Bc-siR5 were specifically loaded into Arabidopsis AGO1 during infection, but not into AGO2 or AGO4, as revealed by AGO-IP followed by RT-PCR. Endogenous plant sRNAs were used as internal controls for IP: At-miR398a for AGO1, At-miR393b* for AGO2, and At-siR1003 for AGO4.

FIG. 10. The sRNAs that have no predicted plant targets (Bc-siR394, Bc-siR233, Bc-siR269) or have predicted targets that were not down-regulated (Bc-siR9, Bc-siR24, Bc-siR67) by B. cinerea infection are not present in the AGO-associated fractions.

FIGS. 11A-11B. Arabidopsis ago1-27 is more resistant to B. cinerea infection than wild-type. (A) ago1-27 displayed reduced disease phenotype upon B. cinerea infection. (B) Induction of BIK1 in response to B. cinerea infection was increased in ago1-27.

FIG. 12. The phylogenetic tree of DCL proteins in pathogenic fungi. Schizosaccharomyces pombe and Neurospora crassa were used as references. An oomycete pathogen Phytophthora infestans was also included.

FIGS. 13A-13D. Generation of B. cinerea dcl1, dcl2 single mutants and the dcl1 dcl2 double mutant by homologous recombination. (A) Schematic diagram of Bc-DCL1 and Bc-DCL2 knockout strategy by homologous recombination. Black arrows indicate primers used for genotyping. (B) The dcl1, dcl2, and dcl1 dcl2 knockout strains were confirmed by RT-PCR. (C) B. cinerea dcl1, dcl2, and dcl1 dcl2 mutant strains showed gradual growth retardation and delayed development of conidiospores: upper panel shows radial growth after 3 days, bottom panel shows condition at 21 days. (D) Two Bc-sRNAs, Bc-microRNA-like RNA2 (Bc-milR2) and Bc-siR1498, were identified as Dicer-independent and were expressed in dcl1 dcl2.

FIGS. 14A-14B. The biomass of the B. cinerea dcl1 dcl2 mutant strain was strongly reduced as compared with the wild-type strain during infection of both Arabidopsis (A) and S. lycopersicum (B), as quantified by qPCR at 72 hpi and 48 hpi, respectively.

FIG. 15. Statistical analysis of the sRNA libraries from cultured B. cinerea, B. cinerea-infected Arabidopsis, and B. cinerea-infected S. lycopersicum.

FIG. 16. The predicted host targets of sRNAs Bc-siR3.1, Bc-siR3.2, and Bc-siR5 (SEQ ID NOS:24, 25, 24, 26, 24, 27-31, 30, 32, 30, 33, 30, 34, 30, 35-37, 36, 38, 36, 39, 36, 40, 36, 41, 36 and 42, respectively). Normalized read counts are given in reads per million B. cinerea sRNAs. Reads were summed from individual sRNA libraries for each category: cultured B. cinerea, B. cinerea-infected Arabidopsis, B. cinerea-infected S. lycopersicum. Target gene alignment was scored as described in Materials and Methods.

DEFINITIONS

The term “pathogen-resistant” or “pathogen resistance” refers to an increase in the ability of a plant to prevent or resist pathogen infection or pathogen-induced symptoms. Pathogen resistance can be increased resistance relative to a particular pathogen species or genus (e.g., Botrytis), increased resistance to multiple pathogens, or increased resistance to all pathogens (e.g., systemic acquired resistance).

“Pathogens” include, but are not limited to, viruses, bacteria, nematodes, fungi or insects (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, Calif. (1988)). In some embodiments, the pathogen is a fungal pathogen. In some embodiments, the pathogen is Botrytis.

The term “plant immunity suppressing sRNA” refers to an sRNA that induces gene silencing in a plant of one or more genes that function or are predicted to function in plant immunity. For example, in some embodiments a plant immunity suppressing sRNA is an sRNA that induces gene silencing of a mitogen-activated protein kinase (e.g., MPK1, MPK2, or MAPKKK4), an oxidative stress-related gene (e.g., periredoxin (PRXIIF), or a cell wall-associated kinase (WAK). Exemplary plant immunity suppressing sRNAs are listed, for example, in FIG. 16 and Table 1.

The term “sRNA” refers to “small RNA,” a short non-coding RNA sequence. In some embodiments, an sRNA sequence comprises less than about 250 nucleotides (e.g., less than 250 nucleotides, less than 200 nucleotides, less than 150 nucleotides, less than 100 nucleotides, or less than 50 nucleotides). In some embodiments, an sRNA sequence comprises about 50-250 nucleotides, about 15-250 nucleotides, about 20-200 nucleotides, about 50-200 nucleotides, about 20-100 nucleotides, about 20-50 nucleotides, or about 20-30 nucleotides. In some embodiments, a sRNA sequence induces gene silencing, e.g., in a host plant. For example, in some embodiments a sRNA sequence induces gene silencing by directing a host's (e.g., host plant's) RNA-induced silencing complex (RISC) to genes with complementary sequences (“target genes”).

The term “sRNA-resistant target,” as used with reference to a polynucleotide sequence, refers to a polynucleotide sequence having a synonymous mutation relative to a sRNA target gene, wherein the polynucleotide sequence of the sRNA-resistant target comprises one or more nucleotide mutations relative to the polynucleotide sequence of the sRNA target gene that decreases the ability of the sRNA (e.g., a pathogen sRNA) to induce gene silencing of the sRNA-resistant target gene and wherein the amino acid sequence (e.g., protein sequence) that is encoded by the polynucleotide sequence of the sRNA-resistant target is identical to the amino acid sequence that is encoded by the polynucleotide sequence of the sRNA target gene. In some embodiments, the polynucleotide sequence of the sRNA-resistant target comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide mutations relative to the polynucleotide sequence of the sRNA target gene.

The term “nucleic acid” or “polynucleotide” refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. Nucleic acids may also include modified nucleotides that permit correct read through by a polymerase and do not significantly alter expression of a polypeptide encoded by that nucleic acid.

The phrase “nucleic acid encoding” or “polynucleotide encoding” refers to a nucleic acid which directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein. The nucleic acid sequences include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length sequences. It should be further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.

Two nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

The term “substantial identity” or “substantially identical,” as used in the context of polynucleotide or polypeptide sequences, refers to a sequence that has at least 60% sequence identity to a reference sequence. Alternatively, percent identity can be any integer from 60% to 100%. Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection.

Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10⁻⁵, and most preferably less than about 10⁻²⁰.

The term “complementary to” is used herein to mean that a polynucleotide sequence is complementary to all or a portion of a reference polynucleotide sequence. In some embodiments, a polynucleotide sequence is complementary to at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, or more contiguous nucleotides of a reference polynucleotide sequence. In some embodiments, a polynucleotide sequence is “substantially complementary” to a reference polynucleotide sequence if at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the polynucleotide sequence is complementary to the reference polynucleotide sequence.

A polynucleotide sequence is “heterologous” to an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety).

An “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of a RNA or polypeptide, respectively. Antisense constructs or sense constructs that are not or cannot be translated are expressly included by this definition. One of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only substantially similar to a sequence of the gene from which it was derived.

The term “promoter,” as used herein, refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell. Thus, promoters used in the polynucleotide constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5′ and 3′ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. A “constitutive promoter” is one that is capable of initiating transcription in nearly all tissue types, whereas a “tissue-specific promoter” initiates transcription only in one or a few particular tissue types. An “inducible promoter” is one that initiates transcription only under particular environmental conditions or developmental conditions.

The term “plant” includes whole plants, shoot vegetative organs and/or structures (e.g., leaves, stems and tubers), roots, flowers and floral organs (e.g., bracts, sepals, petals, stamens, carpels, anthers), ovules (including egg and central cells), seed (including zygote, embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), seedlings, plant tissue (e.g., vascular tissue, ground tissue, and the like), cells (e.g., guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid, and hemizygous.

DETAILED DESCRIPTION OF THE INVENTION
I. Introduction

As described in the Examples section below, it has been surprisingly discovered that small RNAs (sRNAs) from a plant pathogen can suppress genes involved in plant immunity. Without being bound to a particular theory, it is believed that the pathogen sRNAs suppress immunity in a host plant by using the host plant's own gene silencing mechanisms to suppress genes that function in plant immunity.

Thus, one aspect of the present invention relates to enhancing a plant's pathogen resistance by blocking, attenuating, or targeting for destruction the pathogen sRNAs. In some embodiments, a pathogen sRNA is blocked, attenuated, or targeted for destruction using a complementary polynucleotide sequence (e.g., an antisense nucleic acid sequence that is complementary or substantially complementary to the sRNA) or using a short tandem target mimic (STTM) targeting the sRNA. In some embodiments, the complementary polynucleotide sequence or STTM that targets the pathogen sRNA is expressed in a plant (e.g., in an expression cassette operably linked to a promoter), wherein the plant is less susceptible to the pathogen as compared to a control plant in which complementary polynucleotide sequence or STTM is not expressed.

In another aspect, the present invention relates to enhancing a plant's pathogen resistance by expressing sRNA-resistant target genes involved in plant immunity in plants to overcome the effect of the pathogen sRNAs. In some embodiments, the sRNA-resistant target genes are expressed under the control of a promoter (e.g., a pathogen-inducible promoter, a stress-inducible promoter, or a tissue-specific promoter).

II. Pathogen sRNAs and Attenuation of Pathogen sRNAs

In one aspect, methods of blocking or attenuating plant immunity-suppressing sRNAs of pathogens are provided. In some embodiments, the method comprises expressing in a plant a polynucleotide that is complementary or substantially complementary to the pathogen sRNA or that mediates destruction of the pathogen sRNA. In some embodiments, the polynucleotide encodes a short tandem target mimic (STTM) targeting the sRNA. In some embodiments, the polynucleotide encodes an antisense nucleic acid that is complementary or substantially complementary to the sRNA. In some embodiments, the method comprises expressing in the plant the polynucleotide that is complementary or substantially complementary to the pathogen sRNA or that mediates destruction of the pathogen sRNA under the control of a promoter, e.g., a constitutively active promoter, an inducible promoter, or tissue-specific promoter (e.g., a stress inducible promoter, a pathogen inducible promoter, or an epidermis-specific promoter).

In another aspect, plants having blocked or attenuated function of pathogen sRNAs are provided. In some embodiments, the plant comprises a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide that is complementary or substantially complementary to the pathogen sRNA or that mediates destruction of the pathogen sRNA, wherein the plant is less susceptible to the pathogen relative to a control plant lacking the expression cassette. In some embodiments, the expression cassette comprises a polynucleotide that encodes a short tandem target mimic (STTM) targeting the sRNA. In some embodiments, the expression cassette comprises a polynucleotide that encodes an antisense nucleic acid that is complementary or substantially complementary to the sRNA. In some embodiments, the expression cassette comprises a promoter that is an inducible promoter (e.g., stress inducible or pathogen inducible). In some embodiments, the expression cassette comprises a promoter that is a constitutively active promoter. In some embodiments, the promoter is tissue-specific (e.g., epidermis-specific).

In yet another aspect, expression cassettes comprising a promoter operably linked to a polynucleotide that is complementary to, or mediates destruction, of a plant immunity suppressing sRNA of a pathogen, wherein the promoter is heterologous to the polynucleotide, or isolated nucleic acids comprising said expression cassettes, are provided. In some embodiments, the expression cassette comprises a polynucleotide that encodes a short tandem target mimic (STTM) targeting the sRNA. In some embodiments, the expression cassette comprises a polynucleotide that encodes an antisense nucleic acid that is complementary or substantially complementary to the sRNA. In some embodiments, the expression cassette comprises a promoter that is an inducible promoter (e.g., stress inducible or pathogen inducible). In some embodiments, the expression cassette comprises a promoter that is a constitutively active promoter. In some embodiments, the promoter is tissue-specific (e.g., epidermis-specific). In some embodiments, a plant in which the expression cassette is introduced is less susceptible to the pathogen compared to a control plant lacking the expression cassette.

Pathogen sRNAs

In some embodiments, the plant immunity suppressing sRNA is from a viral, bacterial, fungal, nematode, or insect pathogen. In some embodiments, the sRNA is from a fungal pathogen. Examples of plant fungal pathogens include, but are not limited to, Botyritis, Magnaporthe, Sclerotinia, Puccinia, Fusarium, Mycosphaerella, Blumeria, Colletotrichum, Ustilago, and Melampsora. See, e.g., Dean et al., Mol Plant Pathol 13:804 (2012). In some embodiments, the pathogen is Botyritis. In some embodiments, the pathogen is Botyritis cinera.

In some embodiments, the pathogen sRNA comprises a sequence of about 15-250 nucleotides, about 15-150 nucleotides, about 15-100 nucleotides, about 15-50 nucleotides, about 20-50 nucleotides, about 15-30, or about 20-30 nucleotides. In some embodiments, the pathogen sRNA comprises a sequence of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

In some embodiments, the pathogen sRNA comprises a sequence of about 15-250 nucleotides that specifically targets (e.g., induces gene silencing of) a gene encoding a protein that functions or is predicted to function in plant immunity. In some embodiments, the pathogen sRNA comprises a sequence of about 15-250 nucleotides that specifically targets a gene that encodes mitogen activated protein kinase 1 (MPK1), mitogen activated protein kinase 2 (MPK2), peroxiredoxin (PRXIIF), cell-wall associated kinase (WAK), or mitogen activated protein kinase kinase kinase 4 (MAPKKK4). In some embodiments, the pathogen sRNA comprises a sequence of about 15-250 nucleotides that specifically targets any of SEQ ID NOs:4-13 or a portion thereof.

In some embodiments, the pathogen sRNA comprises a sequence listed in FIG. 16 (e.g., Bc-siR3.2, Bc-siR3.1, or Bc-siR5) or Table 1 (e.g., Bc-siR1, Bc-siR1010, Bc-siR3.1, Bc-siR3.2, Bc-siR1008, Bc-siR5, Bc-siR9, Bc-siR10, Bc-siR18, Bc-siR15, Bc-siR17, Bc-siR22, Bc-siR24, Bc-siR25, Bc-siR1015, Bc-siR20, Bc-siR1021, Bc-siR1002, Bc-siR28, Bc-siR31, Bc-siR29, Bc-siR41, Bc-siR35, Bc-siR57, Bc-siR43, Bc-siR40, Bc-siR38, Bc-siR46, Bc-siR48, Bc-siR1007, Bc-siR56, Bc-siR49, Bc-siR58, Bc-siR63, Bc-siR1005, Bc-siR60, Bc-siR61, Bc-siR62, Bc-siR65, Bc-siR67, Bc-siR68, Bc-siR73, Bc-siR81, Bc-siR82, Bc-siR86, Bc-siR91, Bc-siR92, Bc-siR95, Bc-siR1017, Bc-siR97, Bc-siR99, Bc-siR1013, Bc-siR102, Bc-siR1011, Bc-siR109, Bc-siR1018, Bc-siR114, Bc-siR1020, Bc-siR1016, Bc-siR1003, Bc-siR124, Bc-siR127, Bc-siR128, Bc-siR130, Bc-siR1004, Bc-siR144, Bc-siR137, Bc-siR140, Bc-siR141, Bc-siR156, Bc-siR161, Bc-siR163, or Bc-siR1001). In some embodiments, the pathogen sRNA comprises the sequence of Bc-siR3.1 (TTGTGGATCTTGTAGGTGGGC; SEQ ID NO:43), Bc-siR3.2 (TACATTGTGGATCTTGTAGGT; SEQ ID NO:44), or Bc-siR5 (TTTGACTCGGAATGTATACTT; SEQ ID NO:45).

Polynucleotides Targeting Pathogen sRNAs

In some embodiments, the function of a pathogen sRNA as described herein in a plant is blocked, attenuated, or reduced by expressing in the plant a polynucleotide that is complementary or substantially complementary to the sRNA or that mediates the destruction of the sRNA. As used herein, the term “mediates destruction of an sRNA” refers to inducing or promoting the degradation of a small RNA (e.g., by a small RNA degrading nuclease). In some embodiments, the polynucleotide encodes a short tandem target mimic (STTM) that targets the sRNA. In some embodiments, the polynucleotide encodes an antisense nucleic acid that is complementary or substantially complementary to the sRNA.

Short Tandem Target Mimics

In some embodiments, a short tandem target mimic (STTM) construct is used to block or attenuate function or activity of the pathogen sRNA. STTMs are composed of two short polynucleotide sequences mimicking small RNA target sites (e.g., one or more pathogen sRNA sites as described herein), separated by a linker of an empirically determined optimal size. STTMs trigger efficient degradation of targeted sRNAs by small RNA degrading nucleases. See Yan et al., Plant Cell 24:415-427 (2012).

Typically, the STTM is designed to have two noncleavable sRNA binding sites separated by a spacer. The two noncleavable sRNA binding sites can be either identical (to target one specific sRNA) or slightly different to target two slightly different sRNAs. The optimal length of the spacer is typically from about 48 to 88 nucleotides, although shorter or longer spacer sequences can be used. The sequences of the spacer should be relatively AT rich and able to form a stable stem. Methods of designing and testing STTM constructs are described, e.g., in Yan et al., Plant Cell 24:415-427 (2012), and in Tang et al., Methods 58:118-125 (2012), incorporated by reference herein.

In some embodiments, the polynucleotide comprises an STTM construct that targets an sRNA sequence listed in FIG. 16 (e.g., Bc-siR3.2, Bc-siR3.1, or Bc-siR5) or Table 1 (e.g., Bc-siR1, Bc-siR1010, Bc-siR3.1, Bc-siR3.2, Bc-siR1008, Bc-siR5, Bc-siR9, Bc-siR10, Bc-siR18, Bc-siR15, Bc-siR17, Bc-siR22, Bc-siR24, Bc-siR25, Bc-siR1015, Bc-siR20, Bc-siR1021, Bc-siR1002, Bc-siR28, Bc-siR31, Bc-siR29, Bc-siR41, Bc-siR35, Bc-siR57, Bc-siR43, Bc-siR40, Bc-siR38, Bc-siR46, Bc-siR48, Bc-siR1007, Bc-siR56, Bc-siR49, Bc-siR58, Bc-siR63, Bc-siR1005, Bc-siR60, Bc-siR61, Bc-siR62, Bc-siR65, Bc-siR67, Bc-siR68, Bc-siR73, Bc-siR81, Bc-siR82, Bc-siR86, Bc-siR91, Bc-siR92, Bc-siR95, Bc-siR1017, Bc-siR97, Bc-siR99, Bc-siR1013, Bc-siR102, Bc-siR1011, Bc-siR109, Bc-siR1018, Bc-siR114, Bc-siR1020, Bc-siR1016, Bc-siR1003, Bc-siR124, Bc-siR127, Bc-siR128, Bc-siR130, Bc-siR1004, Bc-siR144, Bc-siR137, Bc-siR140, Bc-siR141, Bc-siR156, Bc-siR161, Bc-siR163, or Bc-siR1001).

In some embodiments, the polynucleotide comprises an STTM construct that is generated using a pair of primers (a forward primer and a reverse primer) listed in Table 2. The STTM primers (e.g., the primers listed in Table 2) are used to amplify and clone into an expression vector a STTM construct having a sequence that targets an sRNA of interest (e.g., an sRNA listed in FIG. 16 or Table 1, e.g., any of Bc-siR1, Bc-siR1010, Bc-siR3.1, Bc-siR3.2, Bc-siR1008, Bc-siR5, Bc-siR9, Bc-siR10, Bc-siR18, Bc-siR15, Bc-siR17, Bc-siR22, Bc-siR24, Bc-siR25, Bc-siR1015, Bc-siR20, Bc-siR1021, Bc-siR1002, Bc-siR28, Bc-siR31, Bc-siR29, Bc-siR41, Bc-siR35, Bc-siR57, Bc-siR43, Bc-siR40, Bc-siR38, Bc-siR46, Bc-siR48, Bc-siR1007, Bc-siR56, Bc-siR49, Bc-siR58, Bc-siR63, Bc-siR1005, Bc-siR60, Bc-siR61, Bc-siR62, Bc-siR65, Bc-siR67, Bc-siR68, Bc-siR73, Bc-siR81, Bc-siR82, Bc-siR86, Bc-siR91, Bc-siR92, Bc-siR95, Bc-siR1017, Bc-siR97, Bc-siR99, Bc-siR1013, Bc-siR102, Bc-siR1011, Bc-siR109, Bc-siR1018, Bc-siR114, Bc-siR1020, Bc-siR1016, Bc-siR1003, Bc-siR124, Bc-siR127, Bc-siR128, Bc-siR130, Bc-siR1004, Bc-siR144, Bc-siR137, Bc-siR140, Bc-siR141, Bc-siR156, Bc-siR161, Bc-siR163, or Bc-siR1001). In some embodiments, the STTM construct is expressed under the control of a promoter as described in Section IV below, e.g., a constitutively active promoter, an inducible promoter, or a tissue-specific promoter.

Antisense Technology

In some embodiments, antisense technology is used to block or attenuate function or activity of the pathogen sRNA. The antisense nucleic acid sequence that is transformed into plants is substantially identical to the pathogen sRNA sequence to be blocked. In some embodiments, the antisense polynucleotide sequence is complementary to the pathogen sRNA sequence to be blocked. However, the sequence does not have to be perfectly identical to inhibit expression. Thus, in some embodiments, an antisense polynucleotide sequence that is substantially complementary (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% complementary) to the pathogen sRNA sequence to be blocked can be used (e.g., in an expression cassette under the control of a heterologous promoter, which is then transformed into plants such that the antisense nucleic acid is produced). In some embodiments, the antisense polynucleotide is expressed under the control of a promoter as described in Section IV below, e.g., a constitutively active promoter, an inducible promoter, or a tissue-specific promoter.

In some embodiments, the polynucleotide encodes an antisense nucleic acid sequence that is complementary or substantially (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) complementary to an sRNA sequence listed in FIG. 16 (e.g., an antisense nucleic acid sequence that is complementary or substantially complementary to Bc-siR3.2, Bc-siR3.1, or Bc-siR5) or Table 1 (e.g., an antisense nucleic acid sequence that is complementary or substantially complementary to Bc-siR1, Bc-siR1010, Bc-siR3.1, Bc-siR3.2, Bc-siR1008, Bc-siR5, Bc-siR9, Bc-siR10, Bc-siR18, Bc-siR15, Bc-siR17, Bc-siR22, Bc-siR24, Bc-siR25, Bc-siR1015, Bc-siR20, Bc-siR1021, Bc-siR1002, Bc-siR28, Bc-siR31, Bc-siR29, Bc-siR41, Bc-siR35, Bc-siR57, Bc-siR43, Bc-siR40, Bc-siR38, Bc-siR46, Bc-siR48, Bc-siR1007, Bc-siR56, Bc-siR49, Bc-siR58, Bc-siR63, Bc-siR1005, Bc-siR60, Bc-siR61, Bc-siR62, Bc-siR65, Bc-siR67, Bc-siR68, Bc-siR73, Bc-siR81, Bc-siR82, Bc-siR86, Bc-siR91, Bc-siR92, Bc-siR95, Bc-siR1017, Bc-siR97, Bc-siR99, Bc-siR1013, Bc-siR102, Bc-siR1011, Bc-siR109, Bc-siR1018, Bc-siR114, Bc-siR1020, Bc-siR1016, Bc-siR1003, Bc-siR124, Bc-siR127, Bc-siR128, Bc-siR130, Bc-siR1004, Bc-siR144, Bc-siR137, Bc-siR140, Bc-siR141, Bc-siR156, Bc-siR161, Bc-siR163, or Bc-siR1001).

Other methods of using oligonucleotide or polynucleotide constructs for blocking the function of small RNAs as described herein can also be used, such as target mimicry (see, e.g., Franco-Zorrilla et al., Nat Genet. 39:1033-1037 (2007)) and “sponges” (see, e.g., Ebert et al., Nat. Methods 4:721-726 (2007)).

III. Expression of sRNA-Resistant Targets

In another aspect, methods of making plants that are resistant to one or more pathogen sRNAs are provided. In some embodiments, the method comprises:

- introducing into a plant a heterologous expression cassette comprising a promoter operably linked to a polynucleotide that is an sRNA-resistant target that encodes a protein that functions in plant immunity, wherein the promoter is heterologous to the polynucleotide; and
- selecting a plant comprising the expression cassette.

In another aspect, expression cassettes comprising a promoter operably linked to a polynucleotide encoding a sRNA-resistant target, isolated nucleic acids comprising said expression cassettes, or plants comprising said expression cassettes, are provided. In some embodiments, a plant into which the expression cassette has been introduced has enhanced pathogen resistance relative to a control plant lacking the expression cassette. In some embodiments, a plant into which the expression cassette has been introduced has enhanced resistance to a fungal pathogen (e.g., Botrytis, e.g., B. cinera) relative to a control plant lacking the expression cassette.

In some embodiments, the promoter is heterologous to the polynucleotide. In some embodiments, the polynucleotide encoding the sRNA-resistant target is operably linked to an inducible promoter. In some embodiments, the promoter is pathogen inducible (e.g., a Botrytis inducible promoter). In some embodiments, the promoter is stress inducible (e.g., an abiotic stress inducible promoter). In some embodiments, the promoter is tissue-specific (e.g., epidermis-specific).

sRNA-Resistant Targets

In some embodiments, the polynucleotide is an sRNA-resistant target that encodes a protein that functions or is predicted to function in plant immunity. As used herein, an sRNA-resistant target is a polynucleotide sequence having a synonymous mutation of a sequence that is targeted by a pathogen sRNA. As used herein, the term “synonymous mutation” refers to a change, relative to a reference sequence, in a DNA sequence that encodes for a protein or peptide (e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides relative to the reference sequence), wherein the change does not alter the amino acid that is encoded. For example, in some embodiments, pathogen sRNAs target plant immunity genes such as mitogen-activated protein kinases (including but not limited to, mitogen-activated protein kinase 1 (MPK1) or mitogen-activated protein kinase 2 (MPK2)); accordingly, in some embodiments an sRNA-resistant target comprises a synonymous mutation of a plant gene that encodes a mitogen-activated protein kinase (e.g., a synonymous mutation of MPK1 or MPK2).

In some embodiments, a polynucleotide sequence is an sRNA-resistant target if the polynucleotide sequence if the amino acid encoded by the polynucleotide sequence is produced at a detectable level. In some embodiments, a polynucleotide sequence is an sRNA-resistant target if the polynucleotide sequence if the amount of amino acid produced by a plant expressing the polynucleotide sequence in the presence of a pathogen sRNA is decreased by no more than 50%, 40%, 30%, 20%, 10%, 5%, or less relative to the amount of amino acid produced by a control plant expressing the polynucleotide sequence in the absence of the pathogen sRNA. Whether a polynucleotide is an sRNA-resistant target can be tested, for example, using a coexpression assay in Nicotiana benthamiana in which the sRNA is coexpressed with a polynucleotide sequence (e.g., a target gene or a synonymous mutation of the target gene) and the level of gene silencing induced by sRNA is measured. See, e.g., Example 1.

In some embodiments, the polynucleotide encodes a protein that functions or is predicted to function in plant immunity. In some embodiments, the polynucleotide comprises an sRNA-resistant target gene or predicted target gene listed in FIG. 16, Table 1, or Table 3. In some embodiments, the polynucleotide comprises a synonymous mutation of an sRNA target gene that encodes mitogen activated protein kinase 1 (MPK1), mitogen activated protein kinase 2 (MPK2), peroxiredoxin (PRXIIF), cell-wall associated kinase (WAK), or mitogen activated protein kinase kinase kinase 4 (MAPKKK4). In some embodiments, the polynucleotide comprises a synonymous mutation of an sRNA target gene in tomato selected from Solyc08g081210.2.1, Solyc03g061650.1.1, Solyc01g108160.2.1, Solyc09g014790.2.1, Solyc03g112190.2.1, or Solyc07g066530.2.1. In some embodiments, the polynucleotide comprises a synonymous mutation of an sRNA target gene in Vitis selected from VIT_10s0092g00240, VIT_12s0028g01140, VIT_06s0009g01890, VIT_10s0116g00190, VIT_05s0020g01790, VIT_01s0011g01000, VIT_05s0077g01510.

In some embodiments, the polynucleotide is substantially identical (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to any of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13. In some embodiments, the polynucleotide is a homolog of any of SEQ ID NOS:4-13 (e.g., a homolog found in a species of Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malta, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, or Zea).

In some embodiments, the polynucleotide is substantially identical (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to any of SEQ ID NOS:4-13, comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide mutations relative to SEQ ID NOS:4-13, and encodes an identical protein as SEQ ID NOS:4-13. Non-limiting examples of nucleotide mutations (synonymous mutations) that can be made in the sequences of SEQ ID NOS:4-13 are described below in Example 3, as shown in the alignments of sRNA sequences to wild-type target gene sequences and mutated target gene sequences.

In some embodiments, the sRNA-resistant target gene comprises a polynucleotide sequence that is resistant to gene silencing by an sRNA listed in FIG. 16 or Table 1. In some embodiments, the sRNA-resistant target comprises a polynucleotide sequence that is resistant to gene silencing by Bc-siR3.1 (TTGTGGATCTTGTAGGTGGGC; SEQ ID NO:43), Bc-siR3.2 (TACATTGTGGATCTTGTAGGT; SEQ ID NO:44), or Bc-siR5 (TTTGACTCGGAATGTATACTT; SEQ ID NO:45).

IV. Polynucleotides and Recombinant Expression Vectors

The isolation of polynucleotides of the invention may be accomplished by a number of techniques. For instance, oligonucleotide probes based on the sequences disclosed here can be used to identify the desired polynucleotide in a cDNA or genomic DNA library from a desired plant species. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. Alternatively, cDNA libraries from plants or plant parts (e.g., flowers) may be constructed.

The cDNA or genomic library can then be screened using a probe based upon a sequence disclosed here. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Alternatively, antibodies raised against a polypeptide can be used to screen an mRNA expression library.

Alternatively, the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology to amplify the sequences of the genes directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. For a general overview of PCR see PCR Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990).

Polynucleotides can also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al., J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

Once a polynucleotide sequence that is complementary to the pathogen sRNA or that mediates destruction of the pathogen sRNA, or a polynucleotide that is a sRNA-resistant target, is obtained, it can be used to prepare an expression cassette for expression in a plant. In some embodiments, expression of the polynucleotide is directed by a heterologous promoter.

Any of a number of means well known in the art can be used to drive expression of the polynucleotide sequence of interest in plants. Any organ can be targeted, such as shoot vegetative organs/structures (e.g. leaves, stems and tubers), epidermis, roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit. Alternatively, expression can be conditioned to only occur under certain conditions (e.g., using an inducible promoter).

For example, a plant promoter fragment may be employed to direct expression of the polynucleotide sequence of interest in all tissues of a regenerated plant. Such promoters are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens, and other transcription initiation regions from various plant genes known to those of skill.

Alternatively, the plant promoter may direct expression of the polynucleotide sequence of interest in a specific tissue (tissue-specific promoters) or may be otherwise under more precise environmental control (inducible promoters). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues, such as the epidermis, leaves, or guard cells (including but not limited to those described in WO/2005/085449; U.S. Pat. Nos. 6,653,535; 7,834,243; EP Patent No. 1 888 754; Li et al., Sci China C Life Sci. 2005 April; 48(2):181-6; Husebye, et al., Plant Physiol, April 2002, Vol. 128, pp. 1180-1188; Plesch, et al., Gene, Volume 249, Number 1, 16 May 2000, pp. 83-89(7), and Sessions et al., Plant J, October 1999, Vol. 20, pp. 259-263, each of which is incorporated by reference). Examples of environmental conditions that may affect transcription by inducible promoters include the presence of a pathogen, anaerobic conditions, elevated temperature, or the presence of light.

In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is stress inducible (e.g., inducible by abiotic stress). In some embodiments, the promoter is pathogen inducible. In some embodiments, the promoter is induced upon infection by Botrytis. Non-limiting examples of pathogen inducible promoters include Botrytis-Induced Kinase 1 (BIK1) and the plant defensing gene PDF1.2. See, e.g., Penninckx et al., Plant Cell 10:2103-2113 (1998); see also Veronese et al., Plant Cell 18:257-273 (2006). In some embodiments, the promoter is A. thaliana BIK1 (SEQ ID NO:1) or is substantially identical to A. thaliana BIK1 (SEQ ID NO:1). In some embodiments, the promoter is A. thaliana PDF1.2 (SEQ ID NO:2) or is substantially identical to A. thaliana PDF1.2 (SEQ ID NO:2). In some embodiments, the promoter is TPK1b (SEQ ID NO:3) or is substantially identical to TPK1b (SEQ ID NO:3).

In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is specifically expressed in the epidermis. Non-limiting examples of epidermis-specific promoters include Meristem Layer 1 (ML1). See, e.g., Takada et al., Development 140:1919-1923 (2013). In some embodiments, the promoter is substantially (e.g., at least 60, 70, 75, 80, 85, 90, or 95%) identical to Arabidopsis ML1 (SEQ ID NO:14) or tomato ML1 (SEQ ID NO:15).

In some embodiments, a polyadenylation region at the 3′-end of the coding region can be included. The polyadenylation region can be derived from a NH3 gene, from a variety of other plant genes, or from T-DNA.

The vector comprising the sequences will typically comprise a marker gene that confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosluforon or Basta.

V. Production of Transgenic Plants

As detailed herein, embodiments of the present invention provide for transgenic plants comprising recombinant expression cassettes for expressing a polynucleotide sequence as described herein (e.g., a polynucleotide sequence that is complementary to the pathogen sRNA or that mediates destruction of the pathogen sRNA, or a polynucleotide encoding a sRNA-resistant target). In some embodiments, a transgenic plant is generated that contains a complete or partial sequence of a polynucleotide that is derived from a species other than the species of the transgenic plant. It should be recognized that transgenic plants encompass the plant or plant cell in which the expression cassette is introduced as well as progeny of such plants or plant cells that contain the expression cassette, including the progeny that have the expression cassette stably integrated in a chromosome.

In some embodiments, the transgenic plants comprising recombinant expression cassettes for expressing a polynucleotide sequence as described herein have increased or enhanced pathogen resistance compared to a plant lacking the recombinant expression cassette, wherein the transgenic plants comprising recombinant expression cassettes for expressing the polynucleotide sequence have about the same growth as a plant lacking the recombinant expression cassette. Methods for determining increased pathogen resistance are described, e.g., in Section VI below.

A recombinant expression vector as described herein may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA construct can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. Alternatively, the DNA construct may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. While transient expression of the polynucleotide sequence of interest is encompassed by the invention, generally expression of construction of the invention will be from insertion of expression cassettes into the plant genome, e.g., such that at least some plant offspring also contain the integrated expression cassette.

Microinjection techniques are also useful for this purpose. These techniques are well known in the art and thoroughly described in the literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. EMBO J. 3:2717-2722 (1984). Electroporation techniques are described in Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein et al. Nature 327:70-73 (1987).

Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example, Horsch et al. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983).

Transformed plant cells derived by any of the above transformation techniques can be cultured to regenerate a whole plant that possesses the transformed genotype and thus the desired phenotype such as enhanced pathogen resistance. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486 (1987).

One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

The expression cassettes of the invention can be used to confer enhanced pathogen resistance on essentially any plant. Thus, the invention has use over a broad range of plants, including species from the genera Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and Zea. In some embodiments, the plant is a tomato plant. In some embodiments, the plant is a vining plant, e.g., a species from the genus Vitis. In some embodiments, the plant is an ornamental plant. In some embodiments, the plant is a vegetable- or fruit-producing plant. In some embodiments, the plant is a monocot. In some embodiments, the plant is a dicot.

VI. Selecting for Plants with Enhanced Pathogen Resistance

Plants with enhanced pathogen resistance can be selected in many ways. One of ordinary skill in the art will recognize that the following methods are but a few of the possibilities. One method of selecting plants with enhanced pathogen resistance is to determine resistance of a plant to a specific plant pathogen. Possible pathogens include, but are not limited to, viruses, bacteria, nematodes, fungi or insects (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, Calif.) (1988)). One of skill in the art will recognize that resistance responses of plants vary depending on many factors, including what pathogen, compound, or plant is used. Generally, enhanced resistance is measured by the reduction or elimination of disease symptoms (e.g., reduction in the number or size of lesions or reduction in the amount of fungal biomass on the plant or a part of the plant) when compared to a control plant. In some cases, however, enhanced resistance can also be measured by the production of the hypersensitive response (HR) of the plant (see, e.g., Staskawicz et al. (1995) Science 268(5211): 661-7). Plants with enhanced pathogen resistance can produce an enhanced hypersensitive response relative to control plants.

Enhanced pathogen resistance can also be determined by measuring the increased expression of a gene operably linked a defense related promoter. Measurement of such expression can be measured by quantifying the accumulation of RNA or subsequent protein product (e.g., using northern or western blot techniques, respectively (see, e.g., Sambrook et al. and Ausubel et al.).

VII. Examples

The following examples are offered to illustrate, but not limit the claimed invention.

Example 1: Fungal Small RNAs Suppress Plant Immunity by Hijacking Host RNA Interference Pathways

To explore the role of B. cinerea sRNAs in pathogenicity, we profiled sRNA libraries prepared from B. cinerea (strain B05.10)-infected Arabidopsis thaliana Col-0 leaves collected at 0, 24, 48, and 72 h post inoculation (hpi) and from B. cinerea-infected Solanum lycopersicum (tomato) leaves and fruits at 0, 24, and 72 hpi. sRNA libraries prepared from B. cinerea mycelia, conidiospores and total biomass after 10 days of culture were used as controls. By using 100 normalized reads per million B. cinerea sRNA reads as a cutoff, we identified a total of 832 sRNAs that were present in both B. cinerea-infected Arabidopsis and S. lycopersicum libraries and had more reads in these two libraries than in the cultured B. cinerea libraries, with sequences exactly matching the B. cinerea B05.10 genome (15) but not Arabidopsis or S. lycopersicum genomes or cDNA (see, FIGS. 15 and 16 and Table 1). The closest sequence matches in Arabidopsis or S. lycopersicum contained a minimum of 2 mismatches. Among them, 27 had predicted microRNA-like precursor structures. A similar number of microRNA-like sRNAs was found in Sclerotinia sclerotiorum (9). We found that 73 Bc-sRNAs could target host genes in both Arabidopsis and S. lycopersicum under stringent target prediction criteria (FIG. 15). Among them, 52 were derived from 6 retrotransposon long terminal repeats (LTR) loci in the B. cinerea genome, 13 were from intergenic regions of 10 loci, and 8 were mapped to 5 protein coding genes.

Some of the predicted plant targets, such as MAPKs, are likely to function in plant immunity. To test whether Bc-sRNAs could indeed suppress host genes during infection, three Bc-sRNAs (Bc-siR3.1, Bc-siR3.2, and Bc-siR5) were selected for further characterization (FIG. 16). These Bc-sRNAs were among the most abundant sRNAs that were 21 nt in length and had potential targets likely to be involved in plant immunity in both Arabidopsis and S. lycopersicum. These sRNAs were also enriched after infection (FIGS. 1A-1B, FIG. 5, and FIG. 16), and were the major sRNA products from their encoding loci, LTR retrotransposons (FIG. 5). Bc-siR3.1 and Bc-siR3.2 were derived from the same locus with a four-nucleotide shift in sequence.

To determine whether Bc-sRNAs could trigger silencing of host genes, we examined the transcript levels of the predicted target genes after B. cinerea infection. The following Arabidopsis genes were targeted in the coding regions and were suppressed after B. cinerea infection: mitogen activated protein kinase 2 (MPK2) and MPK1, which are targeted by Bc-siR3.2; an oxidative stress-related gene peroxiredoxin (PRXIIF), which is targeted by Bc-siR3.1; and a putative cell wall-associated kinase gene (WAK), which is targeted by Bc-siR5 (FIG. 1C). In contrast, the plant defense marker genes PDF1.2 and BIK1 (P. Veronese et al., Plant Cell 18, 257 (2006)), which do not contain the Bc-sRNA target sites, were highly induced upon B. cinerea infection (FIG. 1C). We conclude that suppression of some but not all genes is a result of sequence-specific sRNA interaction and not due to cell death within infected lesions. Bc-siR3.2, which silences Arabidopsis MPK1 and MPK2, was enriched also in S. lycopersicum leaves upon B. cinerea infection and was predicted to target another member of the MAPK signaling cascade in S. lycopersicum, MAPKKK4 (FIG. 1B, FIG. 16). Expression of MAPKKK4 was indeed suppressed upon B. cinerea infection (FIG. 1D).

To confirm that the suppression of the targets was indeed triggered by Bc-sRNAs, we performed co-expression assays in Nicotiana benthamiana. Expression of HA-epitope tagged MPK2, MPK1, and WAK was reduced when they were co-expressed with the corresponding Bc-sRNAs but not when co-expressed with Arabidopsis miR395 that shared no sequence similarity (FIG. 1E). The silencing was abolished, however, when the target genes carried a synonymously mutated version of the relevant Bc-sRNA target sites (FIG. 6A, FIG. 1E). We also observed suppression of YFP-tagged target MPK2 by B. cinerea infection at 24 hpi (FIG. 1F and FIG. 6B); when the Bc-siR3.2 target site of MPK2 was mutated, infection by B. cinerea failed to suppress its expression (FIG. 1F). Thus, Bc-siR3.2 delivered from B. cinerea is sufficient for inducing silencing of wild type MPK2 but cannot silence target site-mutated MPK2. Similarly, of the YFP-sensors with wild type or mutated Bc-siR3.2 target sites (FIG. 6C), only the wild type sensor was suppressed after B. cinerea infection (FIG. 1G).

To test the effect of Bc-sRNAs on host plant immunity, we generated transgenic Arabidopsis plants that ectopically expressed Bc-siR3.1, Bc-siR3.2, or Bc-siR5 using a plant artificial miRNA vector (FIG. 2A) (17). These Bc-sRNA expression (Bc-sRNAox) lines showed normal morphology and development without pathogen challenge when compared to the wild type plants, and expression of the target genes was suppressed (FIG. 2B). With pathogen challenge, all of the Bc-sRNAox lines displayed enhanced susceptibility to B. cinerea (FIG. 2C, 2E). The results indicate that these Bc-sRNAs play a positive role in B. cinerea pathogenicity.

Enhanced disease susceptibility of the Bc-sRNAox lines suggests that the target genes of these Bc-sRNAs are likely to be involved in host immunity against B. cinerea. Plants with mutated target genes showed normal morphology and development without pathogen challenge. The Arabidopsis targets of Bc-siR3.2, MPK1 and MPK2, are homologs that share 87% amino acid identity. These genes are functionally redundant and are co-activated in response to various stress factors (18). The mpk1 mpk2 double mutant exhibited enhanced susceptibility to B. cinerea (FIG. 2D, 2E). A T-DNA knockout mutant of the Bc-siR5 target WAK (SALK_089827) (FIG. 7A) also displayed enhanced susceptibility to B. cinerea (FIG. 2D, 2E). Consistent with this, Bc-siRNAox lines as well as mpk1 mpk2 and wak showed lower induction of the defense marker gene BIK1 (FIG. 7B). These results suggest that the MPK1, MPK2, and WAK genes, all of which are targeted by Bc-sRNAs, participate in the plant's immune response to B. cinerea. To determine whether MAPKKK4 is involved in S. lycopersicum defense response against B. cinerea, we applied the virus-induced gene silencing (VIGS) approach to knock down MAPKKK4 in S. lycopersicum using tobacco rattle virus (TRV) (FIG. 8A) (19). VIGS of TRV-MAPKKK4 caused a dwarf phenotype (FIG. 8B). The MAPKKK4-silenced plants showed enhanced disease susceptibility in response to B. cinerea and contained >15 times more fungal biomass than the control plants (FIG. 2F). We conclude that Bc-sRNAs silence plant genes to suppress host immunity during early infection.

These fungal sRNAs hijack the plant's own gene silencing mechanism. 63 of the 73 Bc-sRNAs that had predicted Arabidopsis and S. lycopersicum targets were 20-22 nucleotides in length with a 5′ terminal U (see Table 1). This sRNA structure is favored for binding to AGO1 in Arabidopsis (S. J. Mi et al., Cell 133, 116 (2008); T. A. Montgomery et al., Cell 133, 128 (2008)). In order to determine whether Bc-sRNAs act through Arabidopsis AGO1, we immunoprecipitated AGO1 from B. cinerea-infected Arabidopsis collected at 24, 32 and 48 hpi and analyzed the AGO1-associated sRNAs. Bc-siR3.1, Bc-siR3.2 and Bc-siR5 were clearly detected in the AGO1-associated fraction pulled down from the infected plant samples but hardly in the control (FIG. 3A) or in the AGO2- and AGO4-associated sRNA fractions (FIG. 9). The sRNAs that had no predicted plant targets or had predicted targets that were not down-regulated by B. cinerea infection were not found in the AGO1-associated fractions (FIG. 10).

If AGO1 plays an essential role in Bc-sRNA-mediated host gene silencing, we would expect to see reduced disease susceptibility in the ago1 mutant since these Bc-sRNAs could no longer suppress host immunity genes. For plants carrying the ago1-27 mutant allele (J. B. Morel et al., Plant Cell 14, 629 (2002)) and were inoculated with B. cinerea, the disease level was significantly less than on the wild type (FIG. 3B and FIG. 11A). Consistent with this, BIK1 induction was increased compared to wild type (FIG. 11B). Furthermore, the expression of Bc-siR3.2 targets MPK2 and MPK1, Bc-siR3.1 target PRXIIF, and Bc-siR5 target WAK in ago1-27 was not suppressed compared to wild type infected plants after B. cinerea infection (FIG. 3C). On the contrary, Arabidopsis miRNA biogenesis mutant dicer-like (dcl) 1-7 that shows similar morphological defects to ago1-27 exhibited an enhanced disease level to B. cinerea (FIG. 3D). These results suggest that the increased resistance phenotype we observed in ago1-27 is not caused by any reduced vigor or pleiotropic phenotype, but due to the function of the Bc-siRNAs, and that Arabidopsis DCL1 is not required for the function of Bc-siRNAs. Thus, B. cinerea Bc-sRNAs evidently hijacked host RNAi machinery by loading into AGO1; the complex in turn suppressed host immunity genes.

To delete the siR3 and siR5 loci from the B. cinerea genome by homologous recombination would be an ideal way to confirm their function; however, it is not feasible because siR3 is from a LTR with 3 copies and siR5 is from a LTR with 13 copies. To better understand the function and biogenesis of the Bc-sRNAs, we chose to knock out the B. cinerea DCL genes, which encode the core sRNA processing enzymes. B. cinerea strain B05.10 possesses two Dicer-like genes (Bc-DCL1 and Bc-DCL2) (FIG. 12). We generated dcl1 and dcl2 single and dcl1 dcl2 double knockout mutant strains through homologous recombination (FIG. 13A-13B). We found that dcl1 and dcl2 single mutants showed reduced growth and delayed sporulation (FIG. 13C). The dcl1 dcl2 double mutant displayed a more obvious phenotype than each of the single mutants, suggesting partial functional redundancy between DCL1 and DCL2 in B. cinerea. Bc-siR3.1, Bc-siR3.2, and Bc-siR5 could not be detected in the dcl1 dcl2 double mutant (FIG. 4A), indicating that they were DCL-dependent, while two other Bc-siRNAs, Bc-milR2 and Bc-siR1498, could still be detected in dcl1 dcl2 double mutant (FIG. 13D). Fungi have diverse sRNA biogenesis pathways, and not all sRNAs are DCL-dependent (H. C. Lee et al., Mol. Cell 38, 803 (2010)). The dcl1 dcl2 double mutant caused significantly smaller lesions than the wild type or dcl1 and dcl2 single mutants on both Arabidopsis and S. lycopersicum leaves (FIG. 4B-4C), in consistence with the significantly reduced fungal biomass at 72 hpi in Arabidopsis and 48 hpi in S. lycopersicum (FIG. 14), which indicates that the virulence of the dcl1 dcl2 mutant was greatly reduced. These results further support the conclusion that Bc-siRNAs, particularly Bc-siR3.1, Bc-siR3.2 and Bc-siR5 that depend on DCL function, contribute to the pathogenicity of B. cinerea. Mutation of dcl1 or dcl2 in B. cinerea caused delayed growth and sporulation (FIG. 13C) but had no effect on pathogenicity (FIG. 4B-4C). Furthermore, expression of the YFP sensor carrying the Bc-siR3.2 target site in N. benthamiana was silenced when infected with wild type B. cinerea. The suppression was abolished when inoculated with the dcl1 dcl2 strain (FIG. 4D), indicating that the dcl1 dcl2 double mutant was unable to generate Bc-siR3.2 to suppress the target. We also confirmed the inability of dcl1 dcl2 to suppress Bc-siR3.1 and Bc-siR3.2 target genes MPK2, MPK1, and PRXIIF in Arabidopsis and MAPKKK4 in tomato upon infection (FIG. 4E). Consistent with this, the dcl1 dcl2 virulence was partially restored when infected on Arabidopsis Bc-siR3.1ox and Bc-siR3.2ox plants as well as in tomato TRV-MAPKKK4 silenced plants (FIG. 4F-4G).

Animal and plant pathogens have evolved virulence or effector proteins to counteract host immune responses. Various protein effectors have been predicted or discovered in fungal or oomycete pathogens from whole-genome sequencing and secretome analysis (M. Rafiqi et al., Curr. Opin. Plant Biol. 15, 477 (2012); T. O. Bozkurt et al., Curr. Opin. Plant Biol. 15, 483 (2012)), although delivery mechanisms are still under active investigation (D. Kale et al., Cell 142, 284 (2010); S. Wawra et al., Curr. Opin. Microbiol. 15, 685 (2012); M. Rafiqi et al., Plant Cell 22, 2017 (2010); S. Schornack et al., Proc. Natl. Acad. Sci. USA 107, 17421 (2010); S. Wawra et al., Proc. Natl. Acad. Sci. USA 109, 2096 (2012)). Here, we show that sRNAs as well can act as effectors through a mechanism that silences host genes in order to debilitate plant immunity and achieve infection. The sRNAs from B. cinerea hijack the plant RNAi machinery by binding to AGO proteins which in turn direct host gene silencing. Another fungal plant pathogen, Verticllium (V.) dahliae, also depends on AGO1 function for its pathogenicity (U. Ellendorff, et al., J. Exp. Bot. 60, 591 (2009)). The implications of these findings suggest an extra mechanism underlying pathogenesis promoted by sophisticated pathogens with the capability to generate and deliver small regulatory RNAs into hosts to suppress host immunity.

Material and Methods

Generation of dcl1, dcl2 Single and Double Mutants of B. cinerea

By using homologous recombination and the Agrobacterium tumefaciens-mediated transformation system adapted from Utermark and Karlovsky (U. Utermark, P. Karlovsky, Protocol Exchange, published online 20 Mar. 2008 (10.1038/nprot.2008.83)), we generated dcl1, dcl2 and dcl1 dcl2 deletion mutants in B. cinerea strain B05.10. Transformants were selected with 70 ppm hygromycin or 100 ppm NH⁴-glufosinate.

Plant Materials and Protocols

Plant materials used in this study are: Arabidopsis thaliana ecotype Col-0, Solanum lycopersicum (tomato) cultivar Moneymaker, and Nicotiana benthamiana, Arabidopsis knockout mutants mpk1 mpk2 (SALK_063847×SALK_019507) (D. Ortiz-Masia et al., FEBS Lett. 581, 1834-1840 (2007)) and wak (SALK_089827).

The Gateway pEarley vectors (with YFP & HA tags) were used for expression of Bc-sRNA target genes (K. W. Earley et al., Plant J. 45, 616-629 (2006)). Bc-sRNAs were cloned into the miRNA319a backbone vector (R. Schwab et al., Plant Cell 18, 1121-1133 (2006)) and transferred into the Gateway vector pEarley100 (without tag) for expression.

Transient co-expression assays in N. benthamiana were performed as described in (X. Zhang et al., Mol. Cell 42, 356-366 (2011)).

Virus-induced gene silencing (VIGS) was performed by cloning a 294-bp MPKKK4 gene fragment into the TRV2 vector (Y. L. Liu et al., Plant J. 31, 777-786 (2002)).

Pathogen Assay

Four-week-old plants were inoculated by applying a single 20 μl droplet per leaf or by spray-inoculating the entire plant, using 2×10⁵spores/ml for Arabidopsis and 1×10⁴spores/ml for S. lycopersicum and N. benthamiana. Disease was assessed by measuring lesion size (ImageJ software) and/or by quantifying B. cinerea biomass using quantitative PCR with B. cinerea-specific ITS primers (FIG. 8).

Confocal Microscopy

YFP-tagged protein expression in N. benthamiana was quantified using the confocal microscopy system Leica SP2. Z-series images (10 images in a distance of 0.7 μM) were merged to gain average signal intensity. Merged images were exported as TIFF files and YFP quantity was measured using the ImageJ software.

AGO Immunoprecipitation (IP)

Arabidopsis AGO IP (X. Zhang et al., Mol. Cell 42, 356-366 (2011)) was conducted with 5 g fresh leaves collected at 24, 32 and 48 h after spray inoculation with B. cinerea. Uninfected leaves mixed with at least double amount of B. cinerea biomass as in 48 hpi samples were used as a control. AGO1 was purified with a peptide-specific antibody. AGO2 and AGO4 IPs were conducted using native promoter-driven transgenic epitope HA-tagged and c-MYC-tagged lines, respectively and commercial HA and c-MYC antibodies.

sRNA RT-PCR

RNA was extracted from B. cinerea-infected plant tissue or the AGO pull-down fraction using the Trizol method. Purified RNA was treated with DNase I and then used in RT-PCR (E. Varkonyi-Gasic et al., Plant Methods 3, 12 (2007)) to detect Bc-sRNAs. 35-40 cycles were used for detecting Bc-sRNAs, 22-28 cycles were used for detecting actin genes from Arabidopsis, S. lycopersicum and B. cinerea. Primers used for reverse transcription and amplification of Bc-siRNAs are listed in Table 2.

sRNA Cloning and Illumina HiSeq Data Analysis

sRNAs (18-28 nucleotides) were isolated by 15% PAGE and libraries were constructed using the miRCat cloning system and deep sequencing was performed on an Illumina HiSeq 2000. The sequence datasets of sRNA libraries from B. cinerea (GSE45320), B. cinerea-infected Arabidopsis (GSE45323) and B. cinerea-infected S. lycopersicum (GSE45321) are available at the NCBI database. The sRNA sequencing reads were preprocessed with the procedure of quality control and adapter trimming by using fastx-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html). Following adapter trimming, sequences were mapped to B. cinerea B05.10, Arabidopsis (TAIR10), or S. lycopersicum (ITAG_SL2.40) genomes and only the reads that matched perfectly to each genome were used for further analysis. The read number for each distinct sRNA was normalized to the total B. cinerea mapped reads in B. cinerea-infected A. thaliana and S. lycopersicum libraries. The ratio of total B. cinerea mapped reads of A. thaliana and S. lycopersicum libraries is 2.5:1, so we divide the normalized siRNA read number of S. lycopersicum by 2.5.

The sRNAs we selected have satisfied the following conditions: 1) it must be present in both B. cinerea-infected A. thaliana and S. lycopersicum libraries; 2) its normalized read number was larger than 100 in A. thaliana or S. lycopersicum libraries; 3) its normalized reads must be higher than that in cultured B. cinerea libraries and 4) it has predicted targets in both A. thaliana and S. lycopersicum.

Target gene prediction for Bc-sRNA was performed using TAPIR1.1 (E. Bonnet et al., Bioinformatics 26, 1566-1568 (2010)) with more stringent requirement than described in (E. Bonnet et al., Bioinformatics 26, 1566-1568 (2010)). No gap or bulge within the alignment between the sRNA and the target was allowed, and the 10th nucleotide of the sRNA must perfectly match its target. At most one mismatch or two wobbles was allowed from position 2 to 12. A maximum of two continuous mismatches was allowed and a score of 4.5 was used as a cutoff. If a sRNA has predicted targets in both A. thaliana and S. lycopersicum, it was selected. The sRNAs were grouped if their 5′ end position and 3′ end position were within 3 nucleotides on the genomic loci. We presented the selected sRNAs with targets in both A. thaliana and S. lycopersicum in Table 1.

TABLE 1

Bc-sRNAs that have predicted targets in both Arabidopsis and S. lycopersicum.

Bc-siRNA

ID, locus,

and siRNA
Normalized read

SEQ

Putative

sequence
counts
Target gene alignment
ID

Target gene
function of

(5′-3′)
A
S
B
and aligned score
NO:
AS*
ID/target site
target gene

siR1
147.4
3015.92
36.4
Bc-siRNA
3′GTCTTAAGATGAGAACGAAGCT 5′
46
4.5
AT5G06290.1
2-cysteine

SIR1 LTR

| ||||| | ||||||:||||:

686~708(CDS)
peroxiredoxin B

transposon

Target
5′CTGAATTATCCTCTTGTTTCGG 3′
47

TCGAAG

CAAGAG

TAGAATT

CTG (SEQ

ID NO: 46)

Bc-siRNA
3′GTCTTAAGATGAGAACGAAGCT 5′
46
4.25
Solyc01g068070.2.1
Wd-repeat

|:|||||| :|||||||||:|:

1754~1776(cDNA)
protein

Target
5′CGGAATTCCGCTCTTGCTTTGG 3′
48

(AHRD V1 *-*-

C1FDE0_9CHLO);

contains

Interpro

domain(s)

IPR017986

WD40 repeat,

region

siR1010
2484.9
1644.16
2403.2
Bc-siRNA
3′TCGTTAGTTTTTAAGGGGGCT 5′
49
4.5
AT1G69330.1
RING/U-box

SIR1010

:|:|||| |||:|||||:||:

566~587(CDS)
superfamily

Intergenic

Target
5′GGTAATCTAAAGTTCCCTCGG 3′
50

protein

region

TCGGGG

GAATTTT

TGATTGC

T (SEQ ID

NO: 49)

Bc-siRNA
3′TCGTTAGTTTTTAAGGGGGCT 5′
49
4.5
Solyc07g018350.2.1
DNA

|| |:| |||||||:||:|||

581~602(cDNA)
mismatch

Target
5′AGAAGTGAAAAATTTCCTCGA 3′
51

repair protein

muts (AHRD

V1 *-*-

Q16P35_AEDAE);

contains

Interpro

domain(s)

IPR015536

DNA

mismatch

repair protein

MutS-

homolog

MSH6

siR3.1
812.1
1231.08
49.9
Bc-siRNA
3′CGGGTGGATGTTCTAGGTGTT 5′
52
3.25
AT1G50760.1
Aminotransferase-

SIR2 LTR

:|||| ||||||||||||||

86~107(CDS)
like, plant

transposon

Target
5′ATCCACATACAAGATCCACAA 3′
53

mobile

TTGTGGA

domain family

TCTTGTA

protein

GGTGGG

C (SEQ ID

NO: 52)

Bc-siRNA
3′CGGGTGGATGTTCTAGGTGTT 5′
52
4.5
AT3G06050.1
peroxiredoxin

|||:| |||||||| |||||

333~354(CDS)
IIF

Target
5′GCCTAGCTACAAGAGCCACAT 3′
54

Bc-siRNA
3′CGGGTGGATGTTCTAGGTGTT 5′
52
4
AT5G46795.1
microspore-

|:|| |:||||| ||||||||

401~422(CDS)
specific

Target
5′GTCCCCTTACAACATCCACAA 3′
55

promoter 2

Bc-siRNA
3′CGGGTGGATGTTCTAGGTGTT 5′
52
4.25
Solyc01g108160.2.1
Autophagy-

:||||:| |||||||||||:

3210~3231(cDNA)
related protein

Target
5′ATCCACTTTCAAGATCCACAG 3′
56

2 (AHRD V1

*-*-

C1GCV2_PARBD);

contains

Interpro

domain(s)

IPR015412

ATG2, C-

terminal

Bc-siRNA
3′CGGGTGGATGTTCTAGGTGTT 5′
52
4.5
Solyc09g014790.2.1
Class E

|||||||:||| ||||||:|

1194~1215(cDNA)
vacuolar

Target
5′ACCCACCTGCAACATCCACGA 3′
57

protein-sorting

machinery

protein hse1

(AHRD V1 *---

HSE1_EMENI);

contains

Interpro

domain(s)

IPR018205

VHS subgroup

siR3.2
202.1
996.52
33.1
Bc-siRNA
3′TGGATGTTCTAGGTGTTACAT 5′
58
4.5
AT1G10210.1
mitogen-

SIR2 LTR

|:| | |||||:||||||||

291~312(CDS)
activated

transposon

Target
5′ATCAAGAAGATTCACAATGTT 3′
59

protein kinase 1

TACATTG

TGGATCT

TGTAGGT

(SEQ ID

NO: 58)

Bc-siRNA
3′TGGATGTTCTAGGTGTTACAT 5′
58
3
AT1G59580.1
mitogen-

|:| | ||||||||||||||:

353~374(CDS)
activated

Target
5′ATCAAGAAGATCCACAATGTG 3′
60

protein kinase

homolog 2

Bc-siRNA
3′TGGATGTTCTAGGTGTTACAT 5′
58
4
AT3G16830.1
TOPLESS-

|:||:||||:||:|:||||||

585~606(CDS)
related 2

Target
5′ATCTGCAAGGTCTATAATGTA 3′
61

Bc-siRNA
3′TGGATGTTCTAGGTGTTACAT 5′
58
4.5
AT4G28300.1
Protein of

||:|:||||:||||||| ||:

1444~1465(CDS)
unknown

Target
5′ACTTGCAAGGTCCACAAGGTG 3′
62

function

(DUF1421)

Bc-siRNA
3′TGGATGTTCTAGGTGTTACAT 5′
58
3.5
Solyc03g061650.1.1
F-box/LRR-

|:||| |||||||| ||||||

907~928(cDNA)
repeat protein

Target
5′ATCTAGAAGATCCAAAATGTA 3′
63

At3g26922

(AHRD V1 *-*-

FBL47_ARATH);

contains

Interpro

domain(s)

IPR006566

FBD-like

Bc-siRNA
3′TGGATGTTCTAGGTGTTACAT 5′
58
4.5
Solyc09g091030.2.1
Beta-amylase

| | ||||||| ||||||||:

1510~1531(cDNA)
(AHRD V1

Target
5′AGCCACAAGATGCACAATGTG 3′
64

****

E0AE02_SOLLC);

contains

Interpro

domain(s)

IPR013781

Glycoside

hydrolase,

subgroup,

catalytic core

Bc-siRNA
3′-GUGGAUGUUCUAGGUGUUACA 5′
65
4.5
Solyc08g081210.2.1
MPKKK4

||::|| ||||||||| ||||

1936~1956(cDNA)

Target
5′CAUUUAAAAGAUCCACCAUGU 3′
66

siR1008
4255.7
635.28
299.8
Bc-siRNA
3′CGTATTTGACTAGTAGTAGTGT 5′
67
4
AT1G04650.1
unknown

SIR6 CDS

|| ||||| ||||||||||:|

2418~2440(CDS)
protein,

(spurious

Target
5′TCAGAAACTAATCATCATCATA 3′
68

hypothetical

gene)

protein

TGTGATG

ATGATCA

GTTTATG

C (SEQ ID

NO: 67)

Bc-siRNA
3′CGTATTTGACTAGTAGTAGTGT 5′
67
4
AT4G39180.2
Sec14p-like

|||||||| |||||:||||:|

1911~1933(3′UTR)
phosphatidylinositol

Target
5′TCATAAACTAATCATTATCATA 3′
69

transfer

family protein

Bc-siRNA
3′CGTATTTGACTAGTAGTAGTGT 5′
67
3.5
AT5G36940.1
cationic amino

||| |:||| |||||||||||

221~243(CDS)
acid

Target
5′GCAGAGACTCATCATCATCACC 3′
70

transporter 3

Bc-siRNA
3′CGTATTTGACTAGTAGTAGTGT 5′
67
4.25
Solyc05g012030.1.1
At1g69160/F4N2_9

||||| :||||||||||| |||

603~625(cDNA)
(AHRD

Target
5′GCATATGCTGATCATCATAACA 3′
71

V1 ***-

Q93Z37_ARATH)

Bc-siRNA
3′CGTATTTGACTAGTAGTAGTGT 5′
67
4.5
Solyc06g076130.2.1
Unknown

||| ||:| ||||||||| |||

1605~1627(cDNA)
Protein

Target
5′GCAAAAGCAGATCATCATGACA 3′
72

(AHRD V1)

siR5
1710
1380
302.6
Bc-siRNA
3′TTCATATGTAAGGCTCAGTTT 5′
73
4.5
AT3G05860.1
MADS-box

SIR3 LTR

:| | |||| |||||||||||

655~676(CDS)
transcription

transposon

Target
5′GAATTTACAATCCGAGTCAAA 3′
74

factor family

TTTGACT

protein

CGGAAT

GTATACT

T (SEQ ID

NO: 73)

Bc-siRNA
3′TTCATATGTAAGGCTCAGTTT 5′
73
4
AT3G07730.1
unknown

|| | || ||||||||||||

491~512(CDS)
protein,

Target
5′TAGGAAACTTTCCGAGTCAAA 3′
75

hypothetical

protein,

uncharacterized

protein

Bc-siRNA
3′TTCATATGTAAGGCTCAGTTT 5′
73
4
AT3G08530.1
Clathrin,

:||| |:|||||||:|||:||

3491~3512(CDS)
heavy chain

Target
5′GAGTTTGCATTCCGGGTCGAA 3′
76

Bc-siRNA
3′TTCATATGTAAGGCTCAGTTT 5′
73
4.5
Solyc03g112190.2.1
Pentatricopeptide

|:||| ||||||:||| ||||

1764~1785(cDNA)
repeat-

Target
5′AGGTAGACATTCTGAGGCAAA 3′
77

containing

protein

(AHRD V1

***-

D7LRK9_ARALY);

contains

Interpro

domain(s)

IPR002885

Pentatricopeptide

repeat

Bc-siRNA
3′TTCATATGTAAGGCTCAGTTT 5′
73
4
Solyc07g066530.2.1
Mitochondrial

|||||| |||||| ||||||

910~931(cDNA)
import

Target
5′CAGTATAGATTCCGTGTCAAA 3′
78

receptor

subunit

TOM34

(AHRD V1 *---

B5X380_SALSA);

contains

Interpro

domain(s)

IPR011990

Tetratricopeptide-

like helical

Bc-siRNA
3′-UUCAUAUGUAAGGCUCAGUUU 5′
79
4.25
AT5G50290
wall

::||||||||||||:||||::

495~515(CDS)
associated

Target
5′GGGUAUACAUUCCGGGUCAGG 3′
80

kinase

siR9
3847.8
120.16
231.7
Bc-siRNA
3′AGATTTTTACGAGTAGTATTTT 5′
81
4.5
AT1G73880.1
UDP-glucosyl

SIR6 CDS

|||:||| ||||||:||:|||

146~168(CDS)
transferase

(spurious

Target
5′ACTAGAAAAGCTCATTATGAAA 3′
82

89B1

gene)

TTTTATG

ATGAGC

ATTTTTA

GA (SEQ

ID NO: 81)

Bc-siRNA
3′AGATTTTTACGAGTAGTATTTT 5′
81
4
Solyc04g005540.2.1
Cc-nbs-lrr,

|:||:|||| |||| |||||||

1920~1942(cDNA)
resistance

Target
5′TTTAGAAATTCTCAGCATAAAA 3′
83

protein

Bc-siRNA
3 ′AGATTTTTACGAGTAGTATTTT 5′
81
4.25
Solyc05g007170.2.1
Cc-nbs-lrr,

||| :||| |:|||||||||||

7265~7287(cDNA)
resistance

Target
5′TCTTGAAACGTTCATCATAAAA 3′
84

protein with

an R1 specific

domain

Bc-siRNA
3′AGATTTTTACGAGTAGTATTTT 5′
81
4
Solyc07g017880.2.1
Peroxidase

|:|:| ||||||:||:||||||

780~802(cDNA)
(AHRD V1

Target
5′TTTGATAATGCTTATTATAAAA 3′
85

****

D4NYQ9_9ROSI);

contains

Interpro

domain(s)

IPR002016

Haem

peroxidase,

plant/fungal/bacterial

Bc-siRNA
3′AGATTTTTACGAGTAGTATTTT 5′
81
3.5
Solyc10g050580.1.1
Protein

||:||||||:|||||||:|||

306~328(cDNA)
binding

Target
5′GCTGAAAATGTTCATCATGAAA 3′
86

protein

(AHRD V1

***-

D7M3B0_ARALY)

Bc-siRNA
3′AGATTTTTACGAGTAGTATTTT 5′
81
4.5
Solyc11g013490.1.1
Beta-1,3-

|||:||:| ||||| ||||||:

561~583(cDNA)
galactosyltransferase 6

Target
5′TCTGAAGAAGCTCAACATAAAG 3′
87

(AHRD V1

***-

B6UBH3_MAIZE);

contains

Interpro

domain(s)

IPR002659

Glycosyl

transferase,

family 31

siR10
2234.2
689.6
56.5
Bc-siRNA
3′TCGTGGGATGTTGGATCTTTT 5′
88
4.25
AT1G63860.1
Disease

SIR2 LTR

||:| :||:||:|||||||||

1124~1145(CDS)
resistance

transposon

Target
5′AGTAATCTGCAGCCTAGAAAA 3′
89

protein (TIR-

TTTTCTA

NBS-LRR

GGTTGTA

class) family

GGGTGCT

(SEQ ID

NO: 88)

Bc-siRNA
3′TCGTGGGATGTTGGATCTTTT 5′
88
4
AT5G09260.1
vacuolar

|| |::| ||||||||||||:

511~532(CDS)
protein

Target
5′AGAATTCGACAACCTAGAAAG 3′
90

sorting-

associated

protein 20.2

Bc-siRNA
3′TCGTGGGATGTTGGATCTTTT 5′
88
4.5
Solyc04g050970.2.1
Receptor

||| |:| ||||||:|||||

19~40(cDNA)
protein kinase-

Target
5′TGCAACTTTCAACCTGGAAAA 3′
91

like protein

(AHRD V1

****

Q9LRYl_ARATH);

contains

Interpro

domain(s)

IPR002290

Serine/threonine

protein

kinase

Bc-siRNA
3′TCGTGGGATGTTGGATCTTTT 5′
88
4.25
Solyc05g014650.2.1
Iojap-like

||||: |||||||:||||:||

541~562(cDNA)
protein

Target
5′AGCATACTACAACTTAGAGAA 3′
92

(AHRD V1 *-*-

B5ZUF1_RHILW);

contains

Interpro

domain(s)

IPR004394

Iojap-related

protein

siR18
155.7
1260.68
16.2
Bc-siRNA
3′ACTAGCTGAGACAAAACCGAT 5′
93
4.5
AT2G01110.1
Sec-

SIR1 LTR

| ||| |||||||||||||:

511~532(CDS)
independent

transposon

Target
5′TATTCGTCTCTGTTTTGGCTG 3′
94

periplasmic

TAGCCA

protein

AAACAG

translocase

AGTCGAT

CA (SEQ

ID NO: 93)

Bc-siRNA
3′ACTAGCTGAGACAAAACCGAT 5′
93
4.5
AT2G31980.1
PHYTOCYSTATIN 2

|| |:||||||||| |||:||

490~511(CDS)

Target
5′TGTTTGACTCTGTTGTGGTTA 3′
95

Bc-siRNA
3′ACTAGCTGAGACAAAACCGAT 5′
93
4.5
AT3G26300.1
cytochrome

||:|||| |:|| ||||||||

1345~1366(CDS)
P450, family

Target
5′TGGTCGAGTTTGGTTTGGCTA 3′
96

71, subfamily

B, polypeptide

34

Bc-siRNA
3′ACTAGCTGAGACAAAACCGAT 5′
93
4.5
AT3G47440.1
tonoplast

||||:| ||||||| |||||

366~387(CDS)
intrinsic

Target
5′TGATTGCCTCTGTTATGGCTT 3′
97

protein 5; 1

Bc-siRNA
3′ACTAGCTGAGACAAAACCGAT 5′
93
4.5
AT4G37160.1
SKU5 similar

| | |:|||||||||||:||

52~73(CDS)
15

Target
5′GGTTAGGCTCTGTTTTGGTTA 3′
98

Bc-siRNA
3′ACTAGCTGAGACAAAACCGAT 5′
93
4
Solyc02g071770.2.1
DUF1264

||| | ||||||||||| |||

1000~1021(cDNA)
domain

Target
5′TGAGCAACTCTGTTTTGTCTA 3′
99

protein

(AHRD V1

**--

A1CBM4_ASPCL);

contains

Interpro

domain(s)

IPR010686

Protein of

unknown

function

DUF1264

Bc-siRNA
3′ACTAGCTGAGACAAAACCGAT 5′
93
4
Solyc03g059420.2.1
Sister

||||:||:||||||||| ||

2896~2917(cDNA)
chromatid

Target
5′TGATTGATTCTGTTTTGCCTT 3′
100

cohesion 2

(AHRD V1

**--

D7M7D7_ARALY);

contains

Interpro

domain(s)

IPR016024

Armadillo-

type fold

Bc-siRNA
3′ACTAGCTGAGACAAAACCGAT 5′
93
3.5
Solyc07g017240.1.1
Unknown

|||| | |||||||||||:|:

1~22(cDNA)
Protein

Target
5′TGATAGTCTCTGTTTTGGTTG 3′
101

(AHRD V1)

siR15
936.7
926.6
155
Bc-siRNA
3′AGTTTGTTGTTCCAAGTTGTGT 5′
102
4.5
AT2G23080.1
Protein kinase

SIR3 LTR

|:||| || ||||||| |||||

1250~1272(3′UTR)
superfamily

transposon

Target
5′TTAAAAAAAAAGGTTCCACACA 3′
103

protein

TGTGTTG

AACCTTG

TTGTTTG

A (SEQ ID

NO: 102)

Bc-siRNA
3′AGTTTGTTGTTCCAAGTTGTGT 5′
102
4
AT3G46920.1
Protein kinase

|||| ||||||| ||||||||

3478~3500(CDS)
superfamily

Target
5′CCAAAGAACAAGGCTCAACACA 3′
104

protein with

octicosapeptide/

Phox/Bem1p

domain

Bc-siRNA
3′AGTTTGTTGTTCCAAGTTGTGT 5′
102
3.5
AT5G48860.1
unknown

||:|| |||||||| |||||||

291~313(CDS)
protein,

Target
5′TCGAAAAACAAGGTGCAACACA 3′
105

hypothetical

protein,

uncharacterized

protein

Bc-siRNA
3′AGTTTGTTGTTCCAAGTTGTGT 5′
102
4.25
Solyc01g088020.2.1
Protein

| :||||||||||||||:||:|

786~808(cDNA)
transport

Target
5′TGGAACAACAAGGTTCAGCATA 3′
106

protein sec31

(AHRD V1

**--

C8V1I6_EMENI);

contains

Interpro

domain(s)

IPR017986

WD40 repeat,

region

siR17
1682.7
589.2
245.8
Bc-siRNA
3′GGTCACGGTAAGTAGTAAAAT 5′
107
4.5
AT1G56190.1
Phosphoglycerate

SIR6 CDS

||||| |||||:|:|||||:

1738~1759(3′UTR)
kinase

(spurious

Target
5′ACAGTGACATTCGTTATTTTG 3′
108

family protein

gene)

TAAAAT

GATGAA

TGGCACT

GG (SEQ

ID

NO: 107)

Bc-siRNA
3′GGTCACGGTAAGTAGTAAAAT 5′
107
4.5
AT1G72740.1
Homeodomain-

:|||| |||||:||||||| |

661~682(CDS)
like/winged-

Target
5′TCAGTTCCATTTATCATTTCA 3′
109

helix DNA-

binding family

protein

Bc-siRNA
3′GGTCACGGTAAGTAGTAAAAT 5′
107
4.5
Solyc05g005950.2.1
Solute carrier

| ||||||||||||||||:

262~283(cDNA)
family 15

Target
5′ACCATGCCATTCATCATTTTG 3′
110

member 4

(AHRD V1

**--

S15A4_XENLA);

contains

Interpro

domain(s)

IPR000109

TGF-beta

receptor, type

I/II

extracellular

region

Bc-siRNA
3′GGTCACGGTAAGTAGTAAAAT 5′
107
4.5
Solyc05g005960.2.1
Peptide

| ||||||||||||||||:

69~90(cDNA)
transporter 1

Target
5′ACCATGCCATTCATCATTTTG 3′
111

(AHRD V1

**-*

Q7XAC3_VICFA);

contains

Interpro

domain(s)

IPR000109

TGF-beta

receptor, type

I/II

extracellular

region

Bc-siRNA
3′GGTCACGGTAAGTAGTAAAAT 5′
107
4
Solyc08g075450.2.1
Nodulin-like

|:| ||:||||| ||||||||

222~243(cDNA)
protein

Target
5′CTACTGTCATTCTTCATTTTA 3′
112

(AHRD V1

***-

Q9FHJ9_ARATH);

contains

Interpro

domain(s)

IPR000620

Protein of

unknown

function

DUF6,

transmembrane

Bc-siRNA
3′GGTCACGGTAAGTAGTAAAAT 5′
107
4
Solyc08g075460.2.1
Nodulin-like

|:| ||:||||| ||||||||

424~445(cDNA)
protein

Target
5′CTACTGTCATTCTTCATTTTA 3′
113

(AHRD V1

***-

Q9FHJ9_ARATH);

contains

Interpro

domain(s)

IPR000620

Protein of

unknown

function

DUF6,

transmembrane

siR22
370
995.72
63.3
Bc-siRNA
3′TGATGTGGGAACTGGTGCAAT 5′
114
4.25
AT3G17360.1
phragmoplast

SIR3 LTR

: | ||||:|||||||||||:

625~646(CDS)
orienting

transposon

Target
5′GATTCACCTTTGACCACGTTG 3′
115

kinesin 1

TAACGTG

GTCAAG

GGTGTA

GT (SEQ

ID

NO: 114)

Bc-siRNA
3′TGATGTGGGAACTGGTGCAAT 5′
114
4.5
AT5G66510.1
gamma

| ||:|:|||| |||||||:

438~459(CDS)
carbonic

Target
5′ACAACGCTCTTGTCCACGTTG 3′
116

anhydrase 3

Bc-siRNA
3′TGATGTGGGAACTGGTGCAAT 5′
114
3.5
Solyc01g005240.2.1
Aspartokinase

||||| |||||| |||||||:

1912~1933(cDNA)
(AHRD V1

Target
5′ACTACTCCCTTGCCCACGTTG 3′
117

***-

B9RGY9_RICCO);

contains

Interpro

domain(s)

IPR001341

Aspartate

kinase region

siR24
1210.2
651.72
429.9
Bc-siRNA
3′CAGTTTGTCTCTCCTGGTTAGT 5′
118
3.5
AT5G04990.1
SAD1/UNC-

SIR3 LTR

|||::| ||||||||||||||

1226~1248(CDS)
84 domain

transposon

Target
5′ATCAGGCTGAGAGGACCAATCA 3′
119

protein 1

TGATTGG

TCCTCTC

TGTTTGA

C (SEQ ID

NO: 118)

Bc-siRNA
3′CAGTTTGTCTCTCCTGGTTAGT 5′
118
4
Solyc02g069090.2.1
Cathepsin B

|||||||| |||||:||||| |

2009~2031(cDNA)
(AHRD V1

Target
5′GTCAAACAAAGAGGGCCAATAA 3′
120

***-

Q1HER6_NICBE);

contains

Interpro

domain(s)

IPR015643

Peptidase

C1A,

cathepsin B

Bc-siRNA
3′CAGTTTGTCTCTCCTGGTTAGT 5′
118
4.5
Solyc03g007390.2.1
Pentatricopeptide

|||:| | ||||||:||||||

2085~2107(cDNA)
repeat-

Target
5′TTCAGAAATAGAGGATCAATCA 3′
121

containing

protein

(AHRD V1

***-

D7ML46_ARALY);

contains

Interpro

domain(s)

IPR002885

Pentatricopeptide

repeat

Bc-siRNA
3′CAGTTTGTCTCTCCTGGTTAGT 5′
118
4.5
Solyc03g097450.2.1
SWI/SNF

|| |:||||||||||| |:|||

1351~1373(cDNA)
complex

Target
5′GTGAGACAGAGAGGACAAGTCA 3′
122

subunit

SMARCC1

(AHRD V1 *---

SMRC1_HUMAN);

contains

Interpro

domain(s)

IPR007526

SWIRM

Bc-siRNA
3′CAGTTTGTCTCTCCTGGTTAGT 5′
118
4.5
Solyc09g089970.1.1
Unknown

|| | |:|||||||:||||||

287~309(cDNA)
Protein

Target
5′ATCTACCGGAGAGGATCAATCA 3′
123

(AHRD V1)

siR25
2747.8
15.64
20.8
Bc-siRNA
3′TTTTGGTTTTAAACTAAGTGAT 5′
124
3.75
AT5G41250.1
Exostosin

SIR2 LTR

:|:|:||: |||||||||||||

1349~1371(CDS)
family protein

transposon

Target
5′GAGATCAGTATTTGATTCACTA 3′
125

TAGTGA

ATCAAAT

TTTGGTT

TT (SEQ

ID

NO: 124)

Bc-siRNA
3′TTTTGGTTTTAAACTAAGTGAT 5′
124
4
AT5G44030.1
cellulose

|| || ||| |||||||||||

3330~3352(3′UTR)
synthase A4

Target
5′AATACAAAACTTTGATTCACTT 3′
126

Bc-siRNA
3′TTTTGGTTTTAAACTAAGTGAT 5′
124
4.5
Solyc01g044240.2.1
Unknown

|||::|||||||||||:|:||

1312~1334(cDNA)
Protein

Target
5′TAAATTAAAATTTGATTTATTA 3′
127

(AHRD V1)

Bc-siRNA
3′TTTTGGTTTTAAACTAAGTGAT 5′
124
3.5
Solyc12g005790.1.1
Peroxidase 27

|||| |||:|||||:||||:||

512~534(cDNA)
(AHRD V1

Target
5′AAAAACAAGATTTGGTTCATTA 3′
128

***-

D7LAI1_ARALY);

contains

Interpro

domain(s)

IPR002016

Haem

peroxidase,

plant/fungal/bacterial

siR1015
1200.3
574.4
2304
Bc-siRNA
3′TGGCTAGTCTGTTGGTAGTT 5′
129
4
AT2G45030.1
Translation

SIR1015

|| | ||| |||||||||||

2328~2348(3′UTR)
elongation

Intergenic

Target
5′ACGGTTCACACAACCATCAA 3′
130

factor

region

EFG/EF2

TTGATGG

protein

TTGTCTG

ATCGGT

(SEQ ID

NO: 129)

Bc-siRNA
3′TGGCTAGTCTGTTGGTAGTT 5′
129
4.5
AT5G02500.1
heat shock

||:| |||||| ||||||:|

954~974(CDS)
cognate

Target
5′ACTGCTCAGACCACCATCGA 3′
131

protein 70-1

Bc-siRNA
3′TGGCTAGTCTGTTGGTAGTT 5′
129
4.5
Solyc05g005180.2.1
Naphthoate

|::| | |||:|||||||||

437~457(cDNA)
synthase

Target
5′ATTGCTAAGATAACCATCAA 3′
132

(AHRD V1

***-

A8I2W2_CHLRE);

contains

Interpro

domain(s)

IPR010198

Naphthoate

synthase

Bc-siRNA
3′TGGCTAGTCTGTTGGTAGTT 5′
129
4.5
Solyc06g036150.1.1
Unknown

||:| || ||||:||||:||

564~584(cDNA)
Protein

Target
5′ACTGTTCTGACAGCCATTAA 3′
133

(AHRD V1)

Bc-siRNA
3′TGGCTAGTCTGTTGGTAGTT 5′
129
4.5
Solyc07g043250.1.1
Unknown

:|| |||||||:|| |||||

116~136(cDNA)
Protein

Target
5′GCCCATCAGACGACGATCAA 3′
134

(AHRD V1);

contains

Interpro

domain(s)

IPR008889

VQ

Bc-siRNA
3′TGGCTAGTCTGTTGGTAGTT 5′
129
3.5
Solyc08g063100.1.1
Ulp1 protease

||:| || |||||||||:||

438~458(cDNA)
family C-

Target
5′ACTGTTCTGACAACCATTAA 3′
135

terminal

catalytic

domain

containing

protein

(AHRD V1 *-*-

Q60D46_SOLDE)

Bc-siRNA
3′TGGCTAGTCTGTTGGTAGTT 5′
129
4
Solyc10g006090.2.1
Genomic

||:|||::|||||||||| |

2583~2603(cDNA)
DNA

Target
5′ACTGATTGGACAACCATCCA 3′
136

chromosome 5

P1 clone

MTE17

(AHRD V1

**--

Q9FJ71_ARATH);

contains

Interpro

domain(s)

IPR011011

Zinc finger,

FYVE/PHD-

type

Bc-siRNA
3′TGGCTAGTCTGTTGGTAGTT 5′
129
3.5
Solyc12g044780.1.1
F-box family

||:|:|::|||||||||||

816~836(cDNA)
protein

Target
5′ACTGGTTGGACAACCATCAC 3′
137

(AHRD V1 *-*-

D7LXD8_ARALY);

contains

Interpro

domain(s)

IPR001810

Cyclin-like F-

box

Bc-siRNA
3′TGGCTAGTCTGTTGGTAGTT 5′
129
3.5
Solyc12g044790.1.1
F-box family

||:|:|::|||||||||||

816~836(cDNA)
protein

Target
5′ACTGGTTGGACAACCATCAC 3′
138

(AHRD V1 *-*-

D7LXD8_ARALY);

contains

Interpro

domain(s)

IPR001810

Cyclin-like F-

box

siR20
1402.4
467.4
83.2
Bc-siRNA
3′TTAGTCTTTTTGTTCTTGTGAT 5′
139
4
AT3G18010.1
WUSCHEL

SIR2 LTR

::| |||||:|||||||||:||

1076~1098(CDS)
related

transposon

Target
5′GGTGAGAAAGACAAGAACATTA 3′
140

homeobox 1

TAGTGTT

CTTGTTT

TTCTGAT

T (SEQ ID

NO: 139)

Bc-siRNA
3′TTAGTCTTTTTGTTCTTGTGAT 5′
139
4.5
AT3G20660.1
organic

|| || |||||||| ||||||:

43~65(5′UTR)
cation/carnitine

Target
5′AAACACAAAAACAAAAACACTG 3′
141

transporter4

Bc-siRNA
3′TTAGTCTTTTTGTTCTTGTGAT 5′
139
4.5
AT4G23882.1
Heavy metal

||| |||:||:||||||||| |

549~571(CDS)
transport/detoxification

Target
5′AATAAGAGAAGCAAGAACACAA 3′
142

superfamily

protein

Bc-siRNA
3′TTAGTCTTTTTGTTCTTGTGAT 5′
139
4.5
A15G17680.1
disease

|:|||| ||||| ||||||||

3220~3242(CDS)
resistance

Target
5′AGTCAGCAAAACCAGAACACTC 3′
143

protein (TIR-

NBS-LRR

class),

putative

Bc-siRNA
3′TTAGTCTTTTTGTTCTTGTGAT 5′
139
4.5
Solyc02g076690.2.1
Cathepsin B-

|| ||| |:||||||| |||||

598~620(cDNA)
like cysteine

Target
5′AAACAGCAGAACAAGACCACTA 3′
144

proteinase

(AHRD V1

**-*

CYSP SCHMA);

contains

Interpro

domain(s)

IPR013128

Peptidase

C1A, papain

Bc-siRNA
3′TTAGTCTTTTTGTTCTTGTGAT 5′
139
4.5
Solyc03g117110.2.1
DCN1-like

|:|| ||||||||||:|:|||

462~484(cDNA)
protein 4

Target
5′AGTCTGAAAAACAAGGATACTT 3′
145

(AHRD V1

***-

B6TI85_MAIZE);

contains

Interpro

domain(s)

IPR014764

Defective in

cullin

neddylation

Bc-siRNA
3′TTAGTCTTTTTGTTCTTGTGAT 5′
139
4.5
Solyc03g120530.2.1
BHLH

|:| |||||||||| ||:||||

163~185(cDNA)
transcription

Target
5′AGTAAGAAAAACAATAATACTA 3′
146

factor-like

protein

(AHRD V1 *-**

Q5ZAK6_ORYSJ);

contains

Interpro

domain(s)

IPR011598

Helix-loop-

helix DNA-

binding

Bc-siRNA
3′TTAGTCTTTTTGTTCTTGTGAT 5′
139
4.5
Solyc11g039880.1.1
Nucleoporin

|||:| ||||||||| |||||

1821~1843(cDNA)
NUP188

Target
5′AATTATAAAAACAAGCACACTC 3′
147

homolog

(AHRD V1 *-*-

NU188_HUMAN)

siR1021
2041.3
137.44
94.1
Bc-siRNA
3′TGTACAAAACAAGTAGTGACAT 5′
148
3
AT2G40520.1
Nucleotidyltransferase

SIR1021

|||||| || |||||||||||

815~837(CDS)
family protein

CDS

Target
5′ACATGTCTTATTCATCACTGTC 3′
149

TACAGTG

ATGAAC

AAAACA

TGT (SEQ

ID

NO: 148)

Bc-siRNA
3′TGTACAAAACAAGTAGTGACAT 5′
148
3.5
AT3G11530.1
Vacuolar

| | ||||| |||||||||||:

682~704(3′UTR)
protein sorting

Target
5′AAAAGTTTTATTCATCACTGTG 3′
150

55 (VPS55)

family protein

Bc-siRNA
3′TGTACAAAACAAGTAGTGACAT 5′
148
4.5
Solyc05g009280.2.1
Fatty acid

||| || || |||||||:|||:

1339~1361(cDNA)
elongase 3-

Target
5′ACACGTCTTCTTCATCATTGTG 3′
151

ketoacyl-CoA

synthase

(AHRD VI

****

Q6DUV5_BRANA);

contains

Interpro

domain(s)

IPR012392

Very-long-

chain 3-

ketoacyl-CoA

synthase

siR1002
1408.4
360.44
239.1
Bc-siRNA
3′ACACAATGTTTCTAAACTTCTTA 5′
152
4.5
AT1G62940.1
acyl-CoA

SIR1002

|||| | |||:|| |||||||||

111~134(CDS)
synthetase 5

Intergenicregion

Target
5′TGTGCTCCAAGGAGTTGAAGAAT 3′
153

ATTCTTC

AAATCTT

TGTAACA

CA (SEQ

ID

NO: 152)

Bc-siRNA
3′ACACAATGTTTCTAAACTTCTTA 5′
152
4.5
AT4G30420.1
nodulin

| ||| |:|||||| ||||||||

1039~1062(CDS)
MtN21/

Target
5′TCTGTAATAAAGATCTGAAGAAT 3′
154

EamA-like

transporter

family protein

Bc-siRNA
3′ACACAATGTTTCTAAACTTCTTA 5′
152
3.5
AT4G34380.1
Transducin/WD40

| ||| |:||||||||||||||

285~308(5′UTR)
repeat-

Target
5′TTTGTGATAAAGATTTGAAGAAA 3′
155

like

superfamily

protein

Bc-siRNA
3′ACACAATGTTTCTAAACTTCTTA 5′
152
4
Solyc08g060920.2.1
Xenotropic

|| ||||||||||| |||||||

98~121(cDNA)
and polytropic

Target
5′TGAGTTACAAAGATCTGAAGAAA 3′
156

retrovirus

receptor

(AHRD V1

**--

B2GU54_XENTR);

contains

Interpro

domain(s)

IPR004331

SPX, N-

terminal

Bc-siRNA
3′ACACAATGTTTCTAAACTTCTTA 5′
152
4
Solyc08g081380.2.1
At5g63850-

||| ||:|||:|||||||:|||

989~1012(cDNA)
like protein

Target
5′TGTATTGCAAGGATTTGAGGAAA 3′
157

(Fragment)

(AHRD V1 *-*-

Q3YI76_ARALY);

contains

Interpro

domain(s)

IPR000210

BTB/POZ-like

Bc-siRNA
3′ACACAATGTTTCTAAACTTCTTA 5′
152
4.5
Solyc12g009480.1.1
Xenotropic

||| |||||:|||||||||||

67~90(cDNA)
and polytropic

Target
5′TGTCATACAAGGATTTGAAGAAA 3′
158

retrovirus

receptor

(AHRD V1

**--

B2GU54_XENTR);

contains

Interpro

domain(s)

IPR004331

SPX, N-

terminal

siR28
415.5
727.44
29.8
Bc-siRNA
3′TTCTTCTAGTGTCAAAGTTTTT 5′
159
2.5
AT1G16760.1
Protein kinase

SIR1 LTR

|:|||||||||||||||| |||

1454~1476(CDS)
protein with

transposon

Target
5′AGGAAGATCACAGTTTCACAAA 3′
160

adenine

TTTTTGA

nucleotide

AACTGTG

alpha

ATCTTCT

hydrolases-

T (SEQ ID

like domain

NO: 159)

Bc-siRNA
3′TTCTTCTAGTGTCAAAGTTTTT 5′
159
2
AT1G78940.1
Protein kinase

|:|:||||||||||||||:|||

1425~1447(CDS)
protein with

Target
5′AGGGAGATCACAGTTTCAGAAA 3′
161

adenine

nucleotide

alpha

hydrolases-like domain

Bc-siRNA
3′TTCTTCTAGTGTCAAAGTTTTT 5′
159
4
Al2G28830.1
PLANT U-

||||||| || |||||||:|||

2571~2593(CDS)
BOX 12

Target
5′AAGAAGAACAAAGTTTCAGAAA 3′
162

Bc-siRNA
3′TTCTTCTAGTGTCAAAGTTTTT 5′
159
4.5
AT2G40720.1
Tetratricopeptide

|||||| |:|||||||:| |||

2191~2213(CDS)
repeat

Target
5′AAGAAGCTTACAGTTTTATAAA 3′
163

(TPR)-like

superfamily

protein

Bc-siRNA
3′TTCTTCTAGTGTCAAAGTTTTT 5′
159
4.5
AT3G20200.1
Protein kinase

|:||||||| || |||||||:|

1777~1799(CDS)
protein with

Target
5′AGGAAGATCTCAATTTCAAAGA 3′
164

adenine

nucleotide

alpha

hydrolases-

like domain

Bc-siRNA
3′TTCTTCTAGTGTCAAAGTTTTT 5′
159
3
AT4G31230.1
Protein kinase

|:| ||:|||||||||||:|||

1505~1527(CDS)
protein with

Target
5′AGGCAGGTCACAGTTTCAGAAA 3′
165

adenine

nucleotide

alpha

hydrolases-

like domain

Bc-siRNA
3′TTCTTCTAGTGTCAAAGTTTTT 5′
159
4.5
Solyc01g080610.2.1
Unknown

||||:| || |||||||||| |

852~874(cDNA)
protein

Target
5′AAGAGGTTCTCAGTTTCAAATA 3′
166

(AHRD V1);

contains

Interpro

domain(s)

IPR005508

Protein of

unknown

function

DUF313

Bc-siRNA
3′TTCTTCTAGTGTCAAAGTTTTT 5′
159
4.5
Solyc01g080720.2.1
Pentatricopeptide

||||:| || |||||||||| |

319~341(cDNA)
repeat-

Target
5′AAGAGGTTCTCAGTTTCAAATA 3′
167

containing

protein

(AHRD V1

***-

D7L610_ARALY);

contains

Interpro

domain(s)

IPR002885

Pentatricopeptide

repeat

Bc-siRNA
3′TTCTTCTAGTGTCAAAGTTTTT 5′
159
4.5
Solyc03g115850.2.1
NAC domain

| |:| |||| |||||||||||

934~956 (cDNA)
protein

Target
5′ACGGACATCAGAGTTTCAAAAA 3′
168

IPR003441

(AHRD V1

***-

B9I557_POPTR);

contains

Interpro

domain(s)

IPR003441

No apical

meristem

(NAM)

protein

Bc-siRNA
3′TTCTTCTAGTGTCAAAGTTTTT 5′
159
3
Solyc05g024450.1.1
Unknown

|||||| |||:||||||||:||

196~218(cDNA)
Protein

Target
5′AAGAAGTTCATAGTTTCAAGAA 3′
169

(AHRD V1)

Bc-siRNA
3′TTCTTCTAGTGTCAAAGTTTTT 5′
159
3.75
Solyc06g009200.2.1
Polygalacturonase

|| :| ||:|||||||||||||

664~686(cDNA)
(AHRD

Target
5′AATGACATTACAGTTTCAAAAA 3′
170

V1 ***-

Q2M4X6_LILLO);

contains

Interpro

domain(s)

IPR000743

Glycoside

hydrolase,

family 28

Bc-siRNA
3′TTCTTCTAGTGTCAAAGTTTTT 5′
159
4
Solyc06g031690.2.1
Ankyrin

|||:| ||:|||||||||:|:|

345~367(cDNA)
repeat family

Target
5′AAGGATATTACAGTTTCAGAGA 3′
171

protein

(AHRD V1

***-

D7LCV0_ARALY);

contains

Interpro

domain(s)

IPR002110

Ankyrin

Bc-siRNA
3′TTCTTCTAGTGTCAAAGTTTTT 5′
159
4
Solyc07g041780.2.1
OBP3-

||||||||| ||||| |||||

450~472(cDNA)
responsive

Target
5′AAGAAGATCCCAGTTACAAAAT 3′
172

gene 4

(AHRD V1

**--

D7L9C5_ARALY)

siR31
117
803.16
4.7
Bc-siRNA
3′GTAAGTGCTGGTGTTCTGAGT 5′
173
4.5
AT1G65550.1
Xanthine/uracil

SIR1 LTR

::||:|:||||||||| ||||

761~782(CDS)
permease

transposon

Target
5′TGTTTATGACCACAAGTCTCA 3′
174

family protein

TGAGTCT

TGTGGTC

GTGAAT

G (SEQ ID

NO: 173)

Bc-siRNA
3′GTAAGTGCTGGTGTTCTGAGT 5′
173
4
AT2G05970.1
F-box family

::||:|||| |||||||||||

569~590(CDS)
protein with a

Target
5′TGTTTACGAACACAAGACTCA 3′
175

domain of

unknown

function

(DUF295)

Bc-siRNA
3′GTAAGTGCTGGTGTTCTGAGT 5′
173
4.5
AT5G25420.1
Xanthine/uracil/

::||:|:||||||||| ||||

716~737(CDS)
vitamin C

Target
5′TGTTTATGACCACAAGCCTCA 3′
176

permease

Bc-siRNA
3′GTAAGTGCTGGTGTTCTGAGT 5′
173
3.5
Solyc01g011090.2.1
Phospholipid-

|||:| ||||||||||:|||

3435~3456(cDNA)
transporting

Target
5′AATTTAAGACCACAAGATTCA 3′
177

ATPase

(AHRD V1

***-

C5G6U4_AJEDR);

contains

Interpro

domain(s)

IPR001757

ATPase, P-

type,

K/Mg/Cd/Cu/

Zn/Na/Ca/Na/

H-transporter

Bc-siRNA
3′GTAAGTGCTGGTGTTCTGAGT 5′
173
4.5
Solyc01g110700.2.1
Unknown

|||:| ||:|||||||:|||

36445~36466(cDNA)
Protein

Target
5′AATTTAAGATCACAAGATTCA 3′
178

(AHRD V1)

Bc-siRNA
3′GTAAGTGCTGGTGTTCTGAGT 5′
173
4.5
Solyc01g111180.2.1
Unknown

|||:| ||:|||||||:|||

6734~6755(cDNA)
Protein

Target
5′AATTTAAGATCACAAGATTCA 3′
179

(AHRD V1)

siR29
1843
87.24
28.9
Bc-siRNA
3′GGGTTTTTCCTGATAGGTTGT 5′
180
4.5
AT2G45110.1
expansin B4

SIR2 LTR

||| ||:||||| ||:|||||

729~750(CDS)

transposon

Target
5′CCCTAAGAGGACCATTCAACA 3′
181

TGTTGGA

TAGTCCT

TTTTGGG

(SEQ ID

NO: 180)

Bc-siRNA
3′GGGTTTTTCCTGATAGGTTGT 5′
180
3.75
AT5G38990.1
Malectin/receptor-

: ||||:|||||||||||||

1156~1177(CDS)
like

Target
5′TACAAAGAGGACTATCCAACC 3′
182

protein kinase

family protein

Bc-siRNA
3′GGGTTTTTCCTGATAGGTTGT 5′
180
4
Solyc00g025660.1.1
Unknown

:||||:|||||| |||||:||

576~597(cDNA)
Protein

Target
5′TCCAAGAAGGACAATCCAGCA 3′
183

(AHRD V1)

Bc-siRNA
3′GGGTTTTTCCTGATAGGTTGT 5′
180
4
Solyc03g117510.2.1
Formamidopyrimidine-

:|||||:||||||:| |||||

745~766(cDNA)
DNA

Target
5′TCCAAAGAGGACTGTGCAACA 3′
184

glycosylase

(AHRD V1

****

C5JTH8_AJEDS);

contains

Interpro

domain(s)

IPR000191

DNA

glycosylase/AP

lyase

siR41
371.5
652.56
54.5
Bc-siRNA
3′AAGATGAGGGCTTTTGATAGT 5′
185
4.5
AT3G09530.1
exocyst

SIR3 LTR

|| |:|||||:|||||||||

826~847(CDS)
subunit exo70

transposon

Target
5′TTGGATTCCCGGAAACTATCA 3′
186

family protein

TGATAGT

H3

TTTCGGG

AGTAGA

A (SEQ ID

NO: 185)

Bc-siRNA
3′AAGATGAGGGCTTTTGATAGT 5′
185
4.5
AT3G19780.1

| | |||:|||||||||:||

1248~1269(CDS)

Target
5′TGCCACTTCCGAAAACTGTCC 3′
187

Bc-siRNA
3′AAGATGAGGGCTTTTGATAGT 5′
185
4
Solyc05g014050.2.1
Inner

||| |||:|:|||||:||||:

1422~1443(cDNA)
membrane

Target
5′TTCCACTTCTGAAAATTATCG 3′
188

protein oxaA

(AHRD V1 *-*-

B9L0L4_THERP);

contains

Interpro

domain(s)

IPR001708

Membrane

insertion

protein,

OxaA/YidC

siR35
149.7
727.44
21.2
Bc-siRNA
3′TTGCGCTGTACCGTGTCATGT 5′
189
4
AT3G52810.1
purple acid

SIR2 LTR

|| || |||||:||||||||

978~999(CDS)
phosphatase

transposon

Target
5′CACACGCCATGGTACAGTACA 3′
190

21

TGTACTG

TGCCATG

TCGCGTT

(SEQ ID

NO: 189)

Bc-siRNA
3′TTGCGCTGTACCGTGTCATGT 5′
189
3.5
Solyc11g017230.1.1
DNA

||| | |::||||||||||||

721~742(cDNA)
polymerase I

Target
5′AACACTATGTGGCACAGTACA 3′
191

(AHRD V1

***-

B6U7X8_MAIZE);

contains

Interpro

domain(s)

IPR002421

5′-3′

exonuclease,

N-terminal

siR57
114
728.28
13.8
Bc-siRNA
3′GGTTGCTTGGTCTCTAATAGAT 5′
192
4.5
AT3G28390.1
P-glycoprotein

SIR1 LTR

:|:|||||:|:||||||||| |

3253~3275(CDS)
18

transposon

Target
5′TCGACGAATCGGAGATTATCGA 3′
193

TAGATA

ATCTCTG

GTTCGTT

GG (SEQ

ID

NO: 192)

Bc-siRNA
3′GGTTGCTTGGTCTCTAATAGAT 5′
192
4.5
AT3G29575.1
ABI five

:|:| ||| ||||||||:|||:

350~372(CDS)
binding

Target
5′TCGAAGAAACAGAGATTGTCTG 3′
194

protein 3

Bc-siRNA
3′GGTTGCTTGGTCTCTAATAGAT 5′
192
2.5
Solyc03g007790.2.1
Receptor-like

||:| |||||||||:|||||||

2084~2106(cDNA)
protein kinase

Target
5′CCGAGGAACCAGAGGTTATCTA 3′
195

(AHRD V1

****

Q9FLV4_ARATH);

contains

Interpro

domain(s)

IPR002290

Serine/threonine

protein

kinase

siR43
645
501.16
122
Bc-siRNA
3′GGGTTGTTCTCTTTCGAGGGT 5′
196
4
AT1G19050.1
response

SIR1 LTR

:|:||||||||||||||:| |

592~613(CDS)
regulator 7

transposon

Target
5′TCTAACAAGAGAAAGCTTCAA 3′
197

TGGGAG

CTTTCTC

TTGTTGG

G (SEQ ID

NO: 196)

Bc-siRNA
3′GGGTTGTTCTCTTTCGAGGGT 5′
196
4.5
AT1G26450.1
Carbohydrate-

|:| ||| ||||||||:|||

401~422(CDS)
binding X8

Target
5′ACTATCAAAAGAAAGCTTCCA 3′
198

domain

superfamily

protein

Bc-siRNA
3′GGGTTGTTCTCTTTCGAGGGT 5′
196
4.5
AT1G51600.1
ZIM-LIKE 2

| |:|||||||||| |||||

1398~1419(3′UTR)

Target
5′ACAAGCAAGAGAAAGATCCCA 3′
199

Bc-siRNA
3′GGGTTGTTCTCTTTCGAGGGT 5′
196
4.25
AT1G70190.1
Ribosomal

::|:|: ||||||||||||||

202~223(CDS)
protein

Target
5′TTCGATCAGAGAAAGCTCCCA 3′
200

L7/L12,

oligomerisation;

Ribosomal

protein

L7/L12, C-

terminal/adaptor

protein

ClpS-like

Bc-siRNA
3′GGGTTGTTCTCTTTCGAGGGT 5′
196
4.25
AT3G19860.1
basic helix-

|:|||:|: |||||||||:||

979~1000(CDS)
loop-helix

Target
5′CTCAATAGAAGAAAGCTCTCA 3′
201

(bHLH) DNA-

binding

superfamily

protein

Bc-siRNA
3′GGGTTGTTCTCTTTCGAGGGT 5′
196
4.5
AT5G45030.1
Trypsin family

| |:||| ||||||| |||||

65~86(5′UTR)
protein

Target
5′CACGACATGAGAAAGATCCCA 3′
202

Bc-siRNA
3′GGGTTGTTCTCTTTCGAGGGT 5′
196
4.5
Solyc01g093970.2.1
Glycosyltransferase

||::| || ||||||:|||||

809~830(cDNA)
(AHRD

Target
5′CCTGAAAAAAGAAAGTTCCCA 3′
203

V1 **--

B9IC41_POPTR);

contains

Interpro

domain(s)

IPR002495

Glycosyl

transferase,

family 8

Bc-siRNA
3′GGGTTGTTCTCTTTCGAGGGT 5′
196
3.5
Solyc04g039950.2.1
Mediator of

||| |||:|:||:||||||||

2037~2058(cDNA)
RNA

Target
5′CCCTACAGGGGAGAGCTCCCA 3′
204

polymerase II

transcription

subunit 13

(AHRD V1 *-*-

MED13_DICDI);

contains

Interpro

domain(s)

IPR009401

Mediator

complex,

subunit

Med13

siR40
693.6
473.16
43
Bc-siRNA
3′TTGGTTATGTTCGGGTAAGGT 5′
205
4.5
AT1G06910.1
TRF-like 7

SIR2 LTR

|::|||| ||| |||||||||

756~777(CDS)

transposon

Target
5′AGTCAATTCAATCCCATTCCA 3′
206

TGGAAT

GGGCTTG

TATTGGT

T (SEQ ID

NO: 205)

Bc-siRNA
3′TTGGTTATGTTCGGGTAAGGT 5′
205
4.5
AT1G09350.1
galactinol

:|| |||||||||:||||||

723~744(CDS)
synthase 3

Target
5′GACATATACAAGCCTATTCCA 3′
207

Bc-siRNA
3′TTGGTTATGTTCGGGTAAGGT 5′
205
3.5
AT4G38550.1

Arabidopsis

|| ||||:|:||||||||:||

604~625(CDS)
phospholipase-like

Target
5′AAGCAATGCGAGCCCATTTCA 3′
208

protein

(PEARLI 4)

family

Bc-siRNA
3′TTGGTTATGTTCGGGTAAGGT 5′
205
4
Solyc02g037560.1.1
Ulp1 protease

|:|:||||||||| ||||:||

542~563(cDNA)
family C-

Target
5′AGCTAATACAAGCACATTTCA 3′
209

terminal

catalytic

domain

containing

protein

(AHRD V1

***-

Q60D46_SOLDE)

Bc-siRNA
3′TTGGTTATGTTCGGGTAAGGT 5′
205
4
Solyc08g074820.1.1
Unknown

:||:||||||||| ||||:||

86~107(cDNA)
Protein

Target
5′GACTAATACAAGCACATTTCA 3′
210

(AHRD V1)

siR38
1765.5
35.4
23.3
Bc-siRNA
3′TGCTATAGCAGAGGACTTAAT 5′
211
4.5
AT3G23130.1
C2H2 and

SIR2 LTR

|| || |:||||||| |||||

1039~1060(3′UTR)
C2HC zinc

transposon

Target
5′ACAATTTTGTCTCCTTAATTA 3′
212

fingers

TAATTCA

superfamily

GGAGAC

protein

GATATCG

T (SEQ ID

NO: 211)

Bc-siRNA
3′TGCTATAGCAGAGGACTTAAT 5′
211
4.25
Solyc04g081500.2.1
BRCA1-A

:|:|||| |||||||||||:

836~857(cDNA)
complex

Target
5′TTGGTATCTTCTCCTGAATTG 3′
213

subunit BRE

(AHRD V1

***-

BRE_XENTR);

contains

Interpro

domain(s)

IPR010358

Brain and

reproductive

organ-

expressed

siR46
1811.1
5.76
166.5
Bc-siRNA
3′CAACCACCGGAAGTTAGCAATC 5′
214
4
AT5G21430.1
Chaperone

SIR9

| ||| || ||||||||||||

703~725(CDS)
DnaJ-domain

Intergenic

Target
5′TTCGGTTGCGTTCAATCGTTAG 3′
215

superfamily

region

protein

CTAACG

ATTGAA

GGCCAC

CAAC

(SEQ ID

NO: 214)

Bc-siRNA
3′CAACCACCGGAAGTTAGCAATC 5′
214
3
Solyc09g007340.2.1
PWWP

|||||||||||||||||| |:|

938~960(cDNA)
domain-

Target
5′GTTGGTGGCCTTCAATCGCTGG 3′
216

containing

protein

(AHRD V1 *-*-

D7L8B3_ARALY);

contains

Interpro

domain(s)

IPR000313

PWWP

siR48
66.9
678.08
7.7
Bc-siRNA
3′AACTAGCTATGACAGTGAAGT 5′
217
4
AT2G03040.1
emp24/gp25L/

SIR1 LTR

||||| ||||||||:|:|||

444~465(CDS)
p24

transposon

Target
5′TTGATGGATACTGTTATTTCC 3′
218

family/GOLD

TGAAGT

family protein

GACAGT

ATCGATC

AA (SEQ

ID

NO: 217)

Bc-siRNA
3′AACTAGCTATGACAGTGAAGT 5′
217
4
AT2G03290.1
emp24/gp25L/

||||| ||||||||:|:|||

444~465(CDS)
p24

Target
5′TTGATGGATACTGTTATTTCC 3′
219

family/GOLD

family protein

Bc-siRNA
3′AACTAGCTATGACAGTGAAGT 5′
217
4
AT2G44430.1
DNA-binding

||| |||||||| ||:|||||

511~532(CDS)
bromodomain-

Target
5′TTGTTCGATACTATCGCTTCA 3′
220

containing

protein

Bc-siRNA
3′AACTAGCTATGACAGTGAAGT 5′
217
4.5
AT5G58160.1
actin binding

|| ||:| ||||||||| |||

1894~1915(CDS)

Target
5′TTCATTGTTACTGTCACCTCA 3′
221

Bc-siRNA
3′AACTAGCTATGACAGTGAAGT 5′
217
3
Solyc06g068240.2.1
Pyrophosphate-

||| |:|:|:|||||||||||

441~462(cDNA)
energized

Target
5′TTGCTTGGTGCTGTCACTTCA 3′
222

proton pump

(AHRD V1

***-

B0SRX3_LEPBP);

contains

Interpro

domain(s)

IPR004131

Inorganic H+

pyrophosphatase

Bc-siRNA
3′AACTAGCTATGACAGTGAAGT 5′
217
4.5
Solyc12g099250.1.1
Kinase family

|| || |:|:||||:||||||

1641~1662(cDNA)
protein

Target
5′TTCATGGGTGCTGTTACTTCA 3′
223

(AHRD V1

***-

D7KVQ9_ARALY);

contains

Interpro

domain(s)

IPR002290

Serine/threonine

protein

kinase

siR1007
1641.7
14
0
Bc-siRNA
3′TAGGAAGGCGTCCTAGTGGATG 5′
224
4.5
AT3G09370.1
myb domain

SIR1007

:|||| || ||| |||||||||

334~356(CDS)
protein 3r-3

LTR

Target
5′GTCCTGCCACAGTATCACCTAC 3′
225

transposon

GTAGGT

GATCCTG

CGGAAG

GAT (SEQ

ID

NO: 224)

Bc-siRNA
3′TAGGAAGGCGTCCTAGTGGATG 5′
224
3
Solyc12g099450.1.1
Genomic

||:||||| ||||||||:|||:

514~536(cDNA)
DNA

Target
5′ATTCTTCCACAGGATCATCTAT 3′
226

chromosome 5

TAC clone

K20J1

(AHRD V1 *-*-

Q9FH24_ARATH)

siR56
38.7
655.04
19.1
Bc-siRNA
3′TGCGTTGATGTCCTACTTGCT 5′
227
4
AT5G37010.1
unknown

SIR1 LTR

|:||||||:|||||| ||||

1380~1401(CDS)
protein,

transposon

Target
5′ATGCAACTGCAGGATCAACGT 3′
228

hypothetical

TCGTTCA

protein,

TCCTGTA

uncharacterized

GTTGCGT

protein

(SEQ ID

NO: 227)

Bc-siRNA
3′TGCGTTGATGTCCTACTTGCT 5′
227
4
Solyc03g019870.2.1
Cytochrome

| |||||||||||||||:| |

915~936(cDNA)
P450

Target
5′AAGCAACTACAGGATGAGCAA 3′
229

siR49
1079.5
228.76
50.6
Bc-siRNA
3′ATAGTTTTCTGTATTCGGTGT 5′
230
4.5
AT3G45700.1
Major

SIR2 LTR

|| |||| ||||||| ||||:

1535~1556(CDS)
facilitator

transposon

Target
5′TACCAAATGACATAAACCACG 3′
231

superfamily

TGTGGCT

protein

TATGTCT

TTTGATA

(SEQ ID

NO: 230)

Bc-siRNA
3′ATAGTTTTCTGTATTCGGTGT 5′
230
4.5
AT4G01410.1
Late

||:|||||:||||||||| |

940~961(3′UTR)
embryogenesis

Target
5′AATTAAAAGGCATAAGCCAAA 3′
232

abundant

(LEA)

hydroxyproline-

rich

glycoprotein

family

Bc-siRNA
3′ATAGTTTTCTGTATTCGGTGT 5′
230
4.5
Solyc01g107100.2.1
Beta-1,4-

||| ||:| ||| ||||||||

82~103(cDNA)
xylosidase

Target
5′TATGAAGAAACACAAGCCACA 3′
233

(AHRD V1

***-

D7LA14_ARALY)

Bc-siRNA
3′ATAGTTTTCTGTATTCGGTGT 5′
230
4.25
Solyc07g042160.2.1
Polygalacturonase

|| :|:|:|||||||||:|||

1440~1461(cDNA)
(AHRD

Target
5′TACTAGAGGACATAAGCTACA 3′
234

V1 **--

B6SZN5_MAIZE);

contains

Interpro

domain(s)

IPR012334

Pectin lyase

fold

siR58
39.5
636.12
7
Bc-siRNA
3′GTCTGTTACTTAGGGTTAAAT 5′
235
4.5
AT4G36080.1
phosphotransferases,

SIR1 LTR

||||||| |||||:|||| |:

4572~4593(CDS)
alcohol

transposon

Target
5′CAGACAAAGAATCTCAATATG 3′
236

group as

TAAATTG

acceptor; binding;

GGATTCA

inositol or

TTGTCTG

phosphatidylinositol

(SEQ ID

kinases

NO: 235)

Bc-siRNA
3′GTCTGTTACTTAGGGTTAAAT 5′
235
4.5
Solyc01g058540.2.1
WRKY

| ||:||||||||::||||||

1023~1044(cDNA)
transcription

Target
5′CTGATAATGAATCTTAATTTA 3′
237

factor 31

(AHRD V1 *-*-

C9DI20_9ROSI);

contains

Interpro

domain(s)

IPR003657

DNA-binding

WRKY

Bc-siRNA
3′GTCTGTTACTTAGGGTTAAAT 5′
235
4
Solyc01g109980.2.1
BEL1-like

:| |:|:| |||||||||||:

2186~2207(cDNA)
homeodomain

Target
5′TATATAGTCAATCCCAATTTG 3′
238

protein 6

(AHRD V1 *---

BLH6_ARATH);

contains

Interpro

domain(s)

IPR006563

POX

siR63
132.9
578.48
7.8
Bc-siRNA
3′TGTAAGAGAGTAGTTGATAAT 5′
239
4.5
AT5G04430.1
binding to

SIR1 LTR

| ||||:|:||||||||||

1461~1482(3′UTR)
TOMV RNA

transposon

Target
5′TCTTTCTTTTATCAACTATTT 3′
240

1L (long

TAATAGT

form)

TGATGA

GAGAAT

GT (SEQ

ID

NO: 239)

Bc-siRNA
3′TGTAAGAGAGTAGTTGATAAT 5′
239
4.5
AT5G48385.1
FRIGIDA-like

| |||:|||:||:||:|||||

2124~2145(3′UTR)
protein

Target
5′AGATTTTCTTATTAATTATTA 3′
241

Bc-siRNA
3′TGTAAGAGAGTAGTTGATAAT 5′
239
4.5
Solyc01g096910.2.1
Vacuolar

:|||| | |||||||| ||||

975~996(cDNA)
protein sorting

Target
5′GCATTGTATCATCAACAATTA 3′
242

36 family

protein

(AHRD V1

***-

D7LY74_ARALY);

contains

Interpro

domain(s)

IPR007286

EAP30

siR1005
441.4
452.6
277.5
Bc-siRNA
3′AGGATAACTTCTTTGAGAAAT 5′
243
4
AT1G20200.1
PAM domain

SIR1005

||||| | ||||| |||||||

1224~1245(CDS)
(PCI/PINT

Target
5′TCCTACTCAAGAATCTCTTTA 3′
244

associated

LTR

module)

transposon

protein

TAAAGA

GTTTCTT

CAATAG

GA (SEQ

ID

NO: 243)

Bc-siRNA
3′AGGATAACTTCTTTGAGAAAT 5′
243
4.5
AT1G20650.1
Protein kinase

||:|| |||||||:||| |||

1502~1523(CDS)
superfamily

Target
5′TCTTAATGAAGAAGCTCATTA 3′
245

protein

Bc-siRNA
3′AGGATAACTTCTTTGAGAAAT 5′
243
4.5
AT1G67540.1
unknown

|||||||:||||||||||:

352~373(CDS)
protein,

Target
5′GGCTATTGAGGAAACTCTTTG 3′
246

hypothetical

protein,

uncharacterized

protein

Bc-siRNA
3′AGGATAACTTCTTTGAGAAAT 5′
243
4.5
AT2G23790.1
Protein of

|::||| |||||||||||| |

82~103(CDS)
unknown

Target
5′TTTTATCGAAGAAACTCTTCA 3′
247

function

(DUF607)

Bc-siRNA
3′AGGATAACTTCTTTGAGAAAT 5′
243
4.5
AT3G50950.1
HOPZ-

||:|:|| ||||||||| ||:

2116~2137(CDS)
ACTIVATED

Target
5′TCTTGTTCAAGAAACTCCTTG 3′
248

RESISTANCE 1

Bc-siRNA
3′AGGATAACTTCTTTGAGAAAT 5′
243
4.5
AT4G14510.1
CRM family

| || |||:|||||||||| |

1862~1883(CDS)
member 3B

Target
5′TACTCTTGGAGAAACTCTTGA 3′
249

Bc-siRNA
3′AGGATAACTTCTTTGAGAAAT 5′
243
4.5
AT5G61290.1
Flavin-binding

||:| || ||||||||||| |

1366~1387(CDS)
monooxygenase

Target
5′TCTTCTTCAAGAAACTCTTCA 3′
250

family

protein

Bc-siRNA
3′AGGATAACTTCTTTGAGAAAT 5′
243
3.5
Solyc01g091200.2.1
NAD

||| |||||||||||:|||:

824~845(cDNA)
dependent

Target
5′CCCTCTTGAAGAAACTTTTTG 3′
251

epimerase/dehydratase

family protein

expressed

(AHRD V1

***-

Q2MJA7_ORYSJ);

contains

Interpro

domain(s)

IPR016040

NAD(P)-

binding

domain

Bc-siRNA
3′AGGATAACTTCTTTGAGAAAT 5′
243
4.5
Solyc04g028560.2.1
Zinc finger

|:| |||||||||||||| |

2604~2625(cDNA)
transcription

Target
5′TTCAATTGAAGAAACTCTGTT 3′
252

factor (AHRD

V1 *-**

Q7K9G4_DROME);

contains

Interpro

domain(s)

IPR013087

Zinc finger,

C2H2-

type/integrase,

DNA-binding

Bc-siRNA
3′AGGATAACTTCTTTGAGAAAT 5′
243
3.5
Solyc05g050990.1.1
UDP-D-

||| |||||||||||:|||:

478~499(cDNA)
glucuronate 4-

epimerase 2

Target
5′CCCTCTTGAAGAAACTTTTTG 3′
253

(AHRD V1

****

D7M5S7_ARALY);

contains

Interpro

domain(s)

IPR016040

NAD(P)-

binding

domain

Bc-siRNA
3′AGGATAACTTCTTTGAGAAAT 5′
243
4.5
Solyc10g005940.1.1
CT099

|| ||| |:||| ||||||||

191~212(cDNA)
(Fragment)

(AHRD V1 *---

Target
5′TCATATCGGAGATACTCTTTA 3′
254

Q4KR02_SOLCI);

contains

Interpro

domain(s)

IPR003245

Plastocyanin-

like

siR60
33.4
599.88
34.1
Bc-siRNA
3′GGCAGAAGCTTAAGGTAACGT 5′
255
4.5
AT1G55610.1
BRI1 like

SIR1 LTR

::||||||||||||:||| ||

817~838(CDS)

transposon

Target
5′TTGTCTTCGAATTCTATTTCA 3′
256

TGCAATG

GAATTCG

AAGACG

G (SEQ ID

NO: 255)

Bc-siRNA
3′GGCAGAAGCTTAAGGTAACGT 5′
255
4.25
Solyc08g067800.1.1
Acetyltransferase

|| |||| |||||||||||::

261~282(cDNA)
(AHRD

Target
5′CCATCTTGGAATTCCATTGTG 3′
257

V1 **-*

B4RG69_PHEZH);

contains

Interpro

domain(s)

IPR016181

Acyl-CoA N-

acyltransferase

siR61
230.9
515.12
10.3
Bc-siRNA
3′GTGCATATGCTAAGATAAGAT 5′
258
4.25
AT2G17510.2
ribonuclease II

SIR3 LTR

|: ||| |||||||||||||

1543~1564(CDS)
family protein

transposon

Target
5′AATTTATTCGATTCTATTCTA 3′
259

TAGAAT

AGAATC

GTATACG

TG (SEQ

ID

NO: 258)

Bc-siRNA
3′GTGCATATGCTAAGATAAGAT 5′
258
4
Solyc03g078160.2.1
POT family

|| ||:||:||||:|:|||||

896~917(cDNA)
domain

Target
5′CAAGTGTATGATTTTGTTCTA 3′
260

containing

protein

expressed

(AHRD V1

***-

D8L9H8_WHEAT);

contains

Interpro

domain(s)

IPR007493

Protein of

unknown

function

DUF538

Bc-siRNA
3′GTGCATATGCTAAGATAAGAT 5′
258
3.5
Solyc03g121810.2.1
Phospholipid-

:|:|||||:|||||||||| |

2888~2909(cDNA)
transporting

Target
5′TATGTATATGATTCTATTCAA 3′
261

ATPase 1

(AHRD V1

****

C5FPS3_NANOT);

contains

Interpro

domain(s)

IPR006539

ATPase, P-

type,

phospholipid-

translocating,

flippase

Bc-siRNA
3′GTGCATATGCTAAGATAAGAT 5′
258
4.5
Solyc04g082430.2.1
B-like cyclin

||:|| |||||||| |||:||

8~29(cDNA)
(AHRD V1

Target
5′CATGTTTACGATTCAATTTTA 3′
262

****

Q40337_MEDSA);

contains

Interpro

domain(s)

IPR014400

Cyclin,

A/B/D/E

siR62
149.7
547.24
8.6
Bc-siRNA
3′AAATGAACGCTTAGGCAGCAT 5′
263
4.25
AT1G11620.1
F-box and

SIR2 LTR

|||: ||||||||||||:||

353~374(CDS)
associated

transposon

Target
5′TTTGGTTGCGAATCCGTTGTT 3′
264

interaction

TACGAC

domains-

GGATTCG

containing

CAAGTA

protein

AA (SEQ

ID

NO: 263)

Bc-siRNA
3′AAATGAACGCTTAGGCAGCAT 5′
263
4
AT4G10030.1
alpha/beta-

| || ||||||||:||||||

100~121(5′UTR)
Hydrolases

Target
5′TGTAATTGCGAATTCGTCGTT 3′
265

superfamily

protein

Bc-siRNA
3′AAATGAACGCTTAGGCAGCAT 5′
263
4
Solyc01g009570.2.1
Unknown

|||||||| |||||||| ||

236~257(cDNA)
Protein

Target
5′TTTACTTGGGAATCCGTAGTC 3′
266

(AHRD V1)

siR65
14.4
583.44
22.2
Bc-siRNA
3′TGATGTCTTAGGGAGAACGAT 5′
267
4
AT1G75950.1
S phase

SIR1 LTR

|| ||:||:|||||||| |||

282~303(CDS)
kinase-

transposon

Target
5 ′ACAACGGAGTCCCTCTTCCTA 3′
268

associated

TAGCAA

protein 1

GAGGGA

TTCTGTA

GT (SEQ

ID

NO: 267)

Bc-siRNA
3′TGATGTCTTAGGGAGAACGAT 5′
267
4
AT2G21330.1
fructose-

:| |||||||||||| ||||:

974~995(CDS)
bisphosphate

Target
5′GCAACAGAATCCCTCCTGCTG 3′
269

aldolase 1

Bc-siRNA
3′TGATGTCTTAGGGAGAACGAT 5′
267
4.5
AT3G23670.1
phragmoplast-

::| || |:|||:||||||||:

3292~3313(CDS)
associated

Target
5′GCAACTGGATCTCTCTTGCTG 3′
270

kinesin-related

protein,

putative

Bc-siRNA
3′TGATGTCTTAGGGAGAACGAT 5′
267
4.5
AT4G25980.1
Peroxidase

|| |:| |||:|||||||||

187~208(CDS)
superfamily

Target
5′TCTTCGGCATCTCTCTTGCTA 3′
271

protein

Bc-siRNA
3′TGATGTCTTAGGGAGAACGAT 5′
267
4.5
A14G27680.1
P-loop

|:||| |||||:||||||:|

1507~1528(3′UTR)
containing

Target
5′ATTACTGAATCTCTCTTGTTC 3′
272

nucleoside

triphosphate

hydrolases

superfamily

protein

Bc-siRNA
3′TGATGTCTTAGGGAGAACGAT 5′
267
4
Solyc07g007790.2.1
Sucrose

::||:||||||:||:||||||

3439~3460(cDNA)
phosphate

Target
5′GTTATAGAATCTCTTTTGCTA 3′
273

synthase

(AHRD V1

****

Q2HYI0_CUCME);

contains

Interpro

domain(s)

IPR012819

Sucrose

phosphate

synthase, plant

Bc-siRNA
3′TGATGTCTTAGGGAGAACGAT 5′
267
4.5
Solyc12g008370.1.1
Pre-mRNA-

::|||| ||||||||||| ||

496~517(cDNA)
processing

Target
5′GTTACACAATCCCTCTTGATA 3′
274

protein 45

(AHRD V1

**--

D6RKF6_COPC7);

contains

Interpro

domain(s)

IPR017862

SKI-

interacting

protein, SKIP

siR67
687.5
297.88
25.7
Bc-siRNA
3′TTTTTTAAGAGGCTAGCTAAAT 5′
275
4
AT1G27880.1
DEAD/DEAH

SIR2 LTR

| ||||||||||||| |||||

3~25(CDS)
(SEQ ID

transposon

Target
5′ATAAAATTCTCCGATGGATTTC 3′
276

NOS: 277 and

TAAATCG

278) box RNA

ATCGGA

helicase

GAATTTT

family protein

TT (SEQ

ID

NO: 275)

Bc-siRNA
3′TTTTTTAAGAGGCTAGCTAAAT 5′
275
3
Solyc05g055050.1.1
Calcium-

||:||||:||||||||||||

568~590(cDNA)
dependent

Target
5′CAAGAATTTTCCGATCGATTTC 3′
279

protein kinase

2 (AHRD V1

****

B4FZS4_MAIZE);

contains

Interpro

domain(s)

IPR002290

Serine/threonine

protein

kinase

Bc-siRNA
3′TTTTTTAAGAGGCTAGCTAAAT 5′
275
4
Solyc07g053900.2.1
Plant-specific

|:|:|| ||||||||||| |||

421~443(cDNA)
domain

Target
5′AGAGAAATCTCCGATCGACTTA 3′
280

TIGR01615

family protein

(AHRD V1 *-*-

B6UDN7_MAIZE);

contains

Interpro

domain(s)

IPR006502

Protein of

unknown

function

DUF506,

plant

siR68
20.5
534.88
6.4
Bc-siRNA
3′GTTAAGGCTAGTGACGTAGGT 5′
281
4.25
AT4G21700.1
Protein of

SIR1 LTR

: ||||||||||||||| |||

167~188(CDS)
unknown

transposon

Target
5′TTATTCCGATCACTGCAACCA 3′
282

function

TGGATGC

(DUF2921)

AGTGATC

GGAATT

G (SEQ ID

NO: 281)

Bc-siRNA
3′GTTAAGGCTAGTGACGTAGGT 5′
281
4
Solyc04g009560.2.1
TBC1 domain

|||| |:|:||||||:|||:|

2811~2832(cDNA)
family

Target
5′CAATACTGGTCACTGTATCTA 3′
283

member 8B

(AHRD V1 *---

B9A6K5_HUMAN);

contains

Interpro

domain(s)

IPR000195

RabGAP/TBC

Bc-siRNA
3′GTTAAGGCTAGTGACGTAGGT 5′
281
4.5
Solyc10g007340.2.1
Unknown

|:| ||||:|||||| ||||:

453~474(cDNA)
Protein

Target
5′CGAATCCGGTCACTGAATCCG 3′
284

(AHRD V1)

siR73
478.6
305.28
141.6
Bc-siRNA
3′AGGCTTTTATCTAACCCGTGT 5′
285
4
AT1G17020.1
senescence-

SIR3 LTR

|| |||| ||||||||||| |

459~480(CDS)
related gene 1

transposon

Target
5′TCAGAAACTAGATTGGGCAGA 3′
286

TGTGCCC

AATCTAT

TTTCGGA

(SEQ ID

NO: 285)

Bc-siRNA
3′AGGCTTTTATCTAACCCGTGT 5′
285
4.5
Solyc01g111250.2.1
Phosphatidylinositol-

|||||| | || ||||||||:

533~554(cDNA)
specific

Target
5′TCCGAACAGAGTTTGGGCACG 3′
287

phospholipase

c (AHRD V1

*-*-

B9UXN2_LISMO);

contains

Interpro

domain(s)

IPR017946

PLC-like

phosphodiesterase,

TIM

beta/alpha-

barrel domain

Bc-siRNA
3′AGGCTTTTATCTAACCCGTGT 5′
285
4.5
Solyc01g111260.2.1
Phosphatidylinositol-

|||||| | || ||||||||:

543~564(cDNA)
specific

Target
5′TCCGAACAGAGTTTGGGCACG 3′
288

phospholipase

c (AHRD V1

*-*-

B9UY71_LISMO);

contains

Interpro

domain(s)

IPR017946

PLC-like

phosphodiesterase,

TIM

beta/alpha-

barrel domain

Bc-siRNA
3′AGGCTTTTATCTAACCCGTGT 5′
285
4.5
Solyc06g069280.2.1
Protein

|| ||:|| |||| |||||||

1359~1380(cDNA)
LSM14

Target
5′TCAGAGAAGAGATGGGGCACA 3′
289

homolog A

(AHRD V1 *---

LS14A_PONAB);

contains

Interpro

domain(s)

IPR019053

FFD and TFG

box motifs

siR81
28.1
438.6
3.6
Bc-siRNA
3′GGGTTGCGAACTAATCTCTGT 5′
290
4.5
AT5G48670.1
AGAMOUS-

SIR1 LTR

||||:|:||||||:|| |||

403~424(CDS)
like 80

transposon

Target
5′TCCAATGTTTGATTGGAAACA 3′
291

TGTCTCT

AATCAA

GCGTTGG

G (SEQ ID

NO: 290)

Bc-siRNA
3′GGGTTGCGAACTAATCTCTGT 5′
290
4.5
Solyc03g082940.2.1
Importin

::||| || |||||:||||||

1376~1397(cDNA)
subunit beta

Target
5′TTCAAAGCCTGATTGGAGACA 3′
292

(AHRD V1

***-

BOWBR4_CULQU);

contains

Interpro

domain(s)

IPR011989

Armadillo-like

helical

Bc-siRNA
3′GGGTTGCGAACTAATCTCTGT 5′
290
4.5
Solyc08g062940.2.1
Calmodulin

:|||| ||||| ||||||||

810~831(cDNA)
binding

Target
5′TCCAAAGCTTGCTTAGAGACT 3′
293

protein

(AHRD V1

**-*

B6T951_MAIZE);

contains

Interpro

domain(s)

IPR000048

IQ

calmodulin-

binding region

siR82
275
335.76
26.9
Bc-siRNA
3′TAGTCAATTCTTTAGGCATAGT 5′
294
4.5
AT2G45540.1
WD-40 repeat

SIR1 LTR

||::||||| |||||:|||||

4598~4620(CDS)
family protein/

transposon

Target
5′ATTGGTTAAAAAATCTGTATCC 3′
295

beige-related

TGATACG

GATTTCT

TAACTGA

T (SEQ ID

NO: 294)

Bc-siRNA
3′TAGTCAATTCTTTAGGCATAGT 5′
294
4
Solyc11g006560.1.1
Glycosyl

||| ||||| :|||||||||||

922~944(cDNA)
transferase

Target
5′ATCTGTTAACGAATCCGTATCA 3′
296

group 1

(AHRD V1

***-

B6T775_MAIZE);

contains

Interpro

domain(s)

IPR001296

Glycosyl

transferase,

group 1

siR86
695.9
147.28
89.9
Bc-siRNA
3′TGGTAGTTTAGTCGATAGTTGT 5′
297
3.25
AT1G10180.1
uncharacterized

SIR2 LTR

:||: |||:|||||||||||||

2187~2209(CDS)
protein.

transposon

Target
5′GCCGCCAAGTCAGCTATCAACA 3′
298

hypothetical

TGTTGAT

protein

AGCTGAT

TTGATGG

T (SEQ ID

NO: 297)

Bc-siRNA
3′TGGTAGTTTAGTCGATAGTTGT 5′
297
4.5
AT5G66650.1
Protein of

|:||| ||||||| ||||:|||

734~756(CDS)
unknown

Target
5′ATCATAAAATCAGATATCGACA 3′
299

function

(DUF607)

Bc-siRNA
3′TGGTAGTTTAGTCGATAGTTGT 5′
297
4.5
Solyc01g058190.2.1
30S ribosomal

||:||:|:|||| ||||||||

1101~1123(cDNA)
protein S6

Target
5′ACTATTAGATCATCTATCAACC 3′
300

(AHRD V1 *-*-

B4WMV0_9

GAMM);

contains

Interpro

domain(s)

IPR000529

Ribosomal

protein S6

Bc-siRNA
3′TGGTAGTTTAGTCGATAGTTGT 5′
297
4.5
Solyc05g052280.2.1
Peroxidase

|| :|: |||||||||||||||

211~233(cDNA)
(AHRD V1

Target
5′ACAGTTCAATCAGCTATCAACA 3′
307

***-

B9VRK9_CAPAN);

contains

Interpro

domain(s)

IPR002016

Haem

peroxidase,

plant/fungal/bacterial

siR91
533.3
187.64
32.5
Bc-siRNA
3′TTTAGTCGATAGTTGTCGTGGT 5′
302
4
AT1G70620.1
cyclin-related

SIR2 LTR

:|:| ||||||||:|||||:||

654~676(CDS)

transposon

Target
5′GAGTAAGCTATCAGCAGCATCA 3′
303

TGGTGCT

GTTGATA

GCTGATT

T (SEQ ID

NO: 302)

Bc-siRNA
3′TTTAGTCGATAGTTGTCGTGGT 5′
302
4.5
Solyc01g006030.2.1
E3 ubiquitin-

:|| ||| |||||||||||| |

449~471(cDNA)
protein ligase

Target
5′GAAGCAGGTATCAACAGCACAA 3′
304

bre1 (AHRD

V1 *-*-

B6K254_SCHJY);

contains

Interpro

domain(s)

IPR018957

Zinc finger,

C3HC4

RING-type

Bc-siRNA
3′TTTAGTCGATAGTTGTCGTGGT 5′
302
4.5
Solyc01g060270.1.1
Os06g0207500

:| || |||||||||||||||

975~997(cDNA)
protein

Target
5′GATACAACTATCAACAGCACCA 3′
305

(Fragment)

(AHRD V1

***-

Q0DDQ9_ORYSJ);

contains

Interpro

domain(s)

IPR004253

Protein of

unknown

function

DUF231,

plant

Bc-siRNA
3′TTTAGTCGATAGTTGTCGTGGT 5′
302
4
Solyc05g026330.1.1
Caffeoyl-CoA

||:|:||||||||| ||:||||

322~344(cDNA)
O-

Target
5′AAGTTAGCTATCAAAAGTACCA 3′
306

methyltransferase

(AHRD

V1 ****

A2PZD5_IPONI);

contains

Interpro

domain(s)

IPR002935

O-

methyltransferase,

family 3

Bc-siRNA
3′TTTAGTCGATAGTTGTCGTGGT 5′
302
4
Solyc05g026350.1.1
Caffeoyl-CoA

||:|:||||||||| ||:||||

444~466(cDNA)
O-

Target
5′AAGTTAGCTATCAAAAGTACCA 3′
307

methyltransferase

(AHRD

V1 ****

A2PZD5_IPONI);

contains

Interpro

domain(s)

IPR002935

O-

methyltransferase,

family 3

Bc-siRNA
3′TTTAGTCGATAGTTGTCGTGGT 5′
302
4
Solyc05g041300.1.1
Caffeoyl-CoA

||:|:||||||||| ||:||||

183~205(cDNA)
O-

Target
5′AAGTTAGCTATCAAAAGTACCA 3′
308

methyltransferase

(AHRD

V1 ***-

A2PZD5_IPONI);

contains

Interpro

domain(s)

IPR002935

O-

methyltransferase,

family 3

Bc-siRNA
3′TTTAGTCGATAGTTGTCGTGGT 5′
302
4.5
Solyc05g041320.1.1
Caffeoyl-CoA

:|:|:||||||||| ||:||||

322~344(cDNA)
O-

Target
5′GAGTTAGCTATCAAAAGTACCA 3′
309

methyltransferase

(AHRD

V1 ****

A2PZD5_IPONI);

contains

Interpro

domain(s)

IPR02935

O-

methyltransferase,

family 3

Bc-siRNA
3′TTTAGTCGATAGTTGTCGTGGT 5′
302
4.5
Solyc05g041610.1.1
Caffeoyl-CoA

:|:|:||||||||| ||:||||

415~437(cDNA)
O-

Target
5′GAGTTAGCTATCAAAAGTACCA 3′
310

methyltransferase

(AHRD

V1 ****

A2PZD5_IPONI);

contains

Interpro

domain(s)

IPR002935

O-

methyltransferase,

family 3

Bc-siRNA
3′TTTAGTCGATAGTTGTCGTGGT 5′
302
4.5
Solyc05g041620.1.1
Caffeoyl-CoA

:|:|:||||||||| ||:||||

322~344(cDNA)
O-

Target
5′GAGTTAGCTATCAAAAGTACCA 3′
311

methyltransferase

(AHRD

V1 ****

A2PZD5_IPONI);

contains

Interpro

domain(s)

IPR002935

O-

methyltransferase,

family 3

Bc-siRNA
3′TTTAGTCGATAGTTGTCGTGGT 5′
302
4
Solyc05g041690.1.1
Caffeoyl-CoA

||:|:||||||||| ||:||||

475~497(cDNA)
O-

Target
5′AAGTTAGCTATCAAAAGTACCA 3′
312

methyltransferase

(AHRD

V1 ****

A2PZD5_IPONI);

contains

Interpro

domain(s)

IPR002935

O-

methyltransferase,

family 3

siR92
29.6
374.44
22.5
Bc-siRNA
3′GGATGCTATGGTCTTGTCATGT 5′
313
3.5
AT3G45620.1
Transducin/WD40

SIR3 LTR

:|||||||||:|||| ||||||

701~723(CDS)
repeat-

transposon

Target
5′TCTACGATACTAGAAGAGTACA 3′
314

like

TGTACTG

superfamily

TTCTGGT

protein

ATCGTAG

G (SEQ ID

NO: 313)

Bc-siRNA
3′GGATGCTATGGTCTTGTCATGT 5′
313
4
Solyc02g085760.2.1
Rhomboid

||| || ||:|||||||||||

491~513(cDNA)
family protein

Target
5′GCTAAGAAACTAGAACAGTACA 3′
315

(AHRD V1

***-

D7MJX8_ARALY);

contains

Interpro

domain(s)

IPR002610

Peptidase S54,

rhomboid

siR95
20.5
373.6
3.2
Bc-siRNA
3′AGATGATATGTATTGAAGCGT 5′
316
4.5
AT2G03060.1
AGAMOUS-

SIR1 LTR

|:| ||||||||||:||| ||

1405~1426(3′UTR)
like 30

transposon

Target
5′TTTTCTATACATAATTTCTCA 3′
317

TGCGAA

GTTATGT

ATAGTA

GA (SEQ

ID

NO: 316)

Bc-siRNA
3′AGATGATATGTATTGAAGCGT 5′
316
4
Solyc08g016050.2.1
Dedicator of

||||| ||:||||||||||

1697~1718(cDNA)
cytokinesis

Target
5′ACTACTTTATATAACTTCGCT 3′
318

family protein

(AHRD V1

***-

A8P5S7_BRUMA);

contains

Interpro

domain(s)

IPR010703

Dedicator of

cytokinesis

siR1017
711.8
95.44
113.1
Bc-siRNA
3′CTTACAGGCTTGAGAGAGGTGGGA 5′
319
4.5
AT3G11910.1
ubiquitin-

SIR1017

||:|||||| | ||||| ||||||

1418~1442(CDS)
specific

Intergenic

Target
5′GAGTGTCCGCAATCTCTACACCCT 3′
320

protease 13

region

AGGGTG

GAGAGA

GTTCGGA

CATTC

(SEQ ID

NO: 319)

Bc-siRNA
3′CTTACAGGCTTGAGAGAGGTGGGA 5′
319
4.5
Solyc03g007760.2.1
Cell division

||||||||||:||||:|:||| ||

1996~2020(cDNA)
protease ftsH

Target
5′GAATGTCCGAGCTCTTTTCACACT 3′
321

(AHRD V1 *---

FTSH_SHIFL);

contains

Interpro

domain(s)

IPR003959

ATPase,

AAA-type,

core

siR97
114
331.64
40.2
Bc-siRNA
3′GGGTTCTTCCTACCTGGGCTAT 5′
322
4.5
AT4G17505.1
Protein of

SIR3 LTR

:||:|||:|||||| ||||||

185~207(CDS)
Unknown

transposon

Target
5′TCCGAGAGGGATGGTCCCGATC 3′
323

Function

TATCGGG

(DUF239)

TCCATCC

TTCTTGG

G (SEQ ID

NO: 322)

Bc-siRNA
3′GGGTTCTTCCTACCTGGGCTAT 5′
322
4.5
Solyc01g091370.2.1
AT-hook

||:|:|||| ||||:||:|||:

1179~1201(cDNA)
motif nuclear

Target
5′CCTAGGAAGTATGGGCCTGATG 3′
324

localized

protein 1

(AHRD V1

***-

Q8VYJ2_ARATH);

contains

Interpro

domain(s)

IPR005175

Protein of

unknown

function

DUF296

Bc-siRNA
3′GGGTTCTTCCTACCTGGGCTAT 5′
322
3
Solyc01g094640.2.1
uncharacterized

:|||| |:|||||||||:||||

2690~2712(cDNA)
protein

Target
5′TCCAAAAGGGATGGACCTGATA 3′
325

LOC101249582

(related)

(AHRD V1

***-

Q2HTJ8_MEDTR)

siR99
366.9
216.44
13.1
Bc-siRNA
3′GAGGACTTAATCGACTGTGAT 5′
326
4.5
AT2G07360.1
SH3 domain-

SIR2 LTR

:|| ||||||||||| ||||

412~433(CDS)
containing

transposon

Target
5′TTCATGAATTAGCTGCCACTT 3′
327

protein

TAGTGTC

AGCTAAT

TCAGGA

G (SEQ ID

NO: 326)

Bc-siRNA
3′GAGGACTTAATCGACTGTGAT 5′
326
4.5
AT2G39100.1
RING/U-box

|:|| || |||||||||||

1127~1148(3′UTR)
superfamily

Target
5′ATTCTCAAATAGCTGACACTT 3′
328

protein

Bc-siRNA
3′GAGGACTTAATCGACTGTGAT 5′
326
3
AT5G13320.1
Auxin-

|||||||||||| |||||||

889~910(CDS)
responsive

Target
5′GTCCTGAATTAGCAGACACTA 3′
329

GH3 family

protein

Bc-siRNA
3′GAGGACTTAATCGACTGTGAT 5′
326
4.5
Solyc02g067320.1.1
Zinc finger-

| || ||||||||||| |||:

52~73(cDNA)
homeodomain

Target
5′CACCAGAATTAGCTGAAACTG 3′
330

protein 1

(Fragment)

(AHRD V1

**--

B0LK19_CUCSA);

contains

Interpro

domain(s)

IPR006456

ZF-HD

homeobox

protein

Cys/His-rich

dimerisation

region

Bc-siRNA
3′GAGGACTTAATCGACTGTGAT 5′
326
4.5
Solyc08g066940.2.1
Peptide

:||:||:| ||||||||:||

1557~1578(cDNA)
transporter 1

Target
5′TTCTTGGACTAGCTGACGCTT 3′
331

(AHRD V1

**-*

Q7XAC3_VICFA);

contains

Interpro

domain(s)

IPR000109

TGF-beta

receptor, type

I/II

extracellular

region

siR1013
521.1
149.76
24.4
Bc-siRNA
3′AAATTTCAAACAAGTAGTATATT 5′
332
4.5
AT1G79840.2
HD-ZIP IV

SIR1013

|||:|| |||| ||||||||||

77~100(5′UTR)
family of

CDS

Target
5′TTTGAATTTTGCTCATCATATAT 3′
333

homeobox-

TTATATG

leucine zipper

ATGAAC

protein with

AAACTTT

lipid-binding

AAA (SEQ

START

ID

domain

NO: 332)

Bc-siRNA
3′AAATTTCAAACAAGTAGTATATT 5′
332
4
Solyc03g098070.2.1
C2H2L

||| | ||||||||||:||||:|

1258~1281(cDNA)
domain class

Target
5′TTTTATGTTTGTTCATTATATGA 3′
334

transcription

factor (AHRD

V1 *--*

D9ZIU3_MALDO);

contains

Interpro

domain(s)

IPR007087

Zinc finger,

C2H2-type

siR102
827.3
20.56
101.5
Bc-siRNA
3′GTTACATAGTTAGAGGGGAGGT 5′
335
3.5
AT3G13750.1
beta

SIR13

||||||:| |||| ||||||||

3258~3280(3′UTR)
galactosidase 1

Intergenic

Target
5′CAATGTGTGAATCACCCCTCCA 3′
336

region

TGGAGG

GGAGAT

TGATACA

TTG (SEQ

ID

NO: 335)

Bc-siRNA
3′GTTACATAGTTAGAGGGGAGGT 5′
335
4.5
AT5G43100.1
Eukaryotic

| ||| |||:||||:||||||

139~161(CDS)
aspartyl

Target
5′CCATGGATCGATCTTCCCTCCT 3′
337

protease

family protein

Bc-siRNA
3′GTTACATAGTTAGAGGGGAGGT 5′
335
4
Solyc11g067000.1.1
ATP-binding

|||| ||| ||||||:||||:|

2884~2906(cDNA)
cassette

Target
5′CAATCTATGAATCTCTCCTCTA 3′
338

transporter

(AHRD V1

***-

D8T797_SELML);

contains

Interpro

domain(s)

IPR013525

ABC-2 type

transporter

siR1011
413.3
172.8
117.2
Bc-siRNA
3′TGGTTAGAACGAGTAGTATAAT 5′
339
4.5
AT4G21215.1

SIR1011

|:||||||||:| ||||||||

724~746(CDS)

CDS

Target
5′ATCAATCTTGTTAATCATATTC 3′
340

TAATATG

ATGAGC

AAGATT

GGT (SEQ

ID

NO: 339)

Bc-siRNA
3′TGGTTAGAACGAGTAGTATAAT 5′
339
3
AT5G51530.1
Ubiquitin

|| ||| |||:|||||||||||

3078~3100(CDS)
carboxyl-

Target
5′ACAAATATTGTTCATCATATTA 3′
341

terminal

hydrolase-

related protein

Bc-siRNA
3′TGGTTAGAACGAGTAGTATAAT 5′
339
4
AT5G67140.1
F-box/RNI-

|:|:| ||||||||||||||

772~794(CDS)
like

Target
5′TCTAGTGTTGCTCATCATATTT 3′
342

superfamily

protein

Bc-siRNA
3′TGGTTAGAACGAGTAGTATAAT 5′
339
4.5
Solyc02g093150.2.1
AP2-like

| ||||:| |||||||| ||||

1404~1426(cDNA)
ethylene-

Target
5′AGCAATTTGGCTCATCAAATTA 3′
343

responsive

transcription

factor

At1g16060

(AHRD V1 *-*-

AP2L1_ARATH);

contains

Interpro

domain(s)

IPR001471

Pathogenesis-

related

transcriptional

factor and

ERF, DNA-

binding

siR109
437.6
160.48
150
Bc-siRNA
3′TGGTGCTTTTAGTGTGGTCGT 5′
344
4.5
AT5G64390.1
RNA-binding

SIR3 LTR

|:||||| ||||:||| ||||

377~398(CDS)
KH domain-

transposon

Target
5′ATCACGATAATCGCACAAGCA 3′
345

containing

TGCTGGT

protein

GTGATTT

TCGTGGT

(SEQ ID

NO: 344)

Bc-siRNA
3′TGGTGCTTTTAGTGTGGTCGT 5′
344
4.5
Solyc01g103350.2.1
Cell division

|:|||||||||:|| |||||

2540~2561(cDNA)
protein kinase

Target
5′TCTACGAAAATCGCAGCAGCA 3′
346

13 (AHRD V1

*-*-

CDK13_MOUSE);

contains

Interpro

domain(s)

IPR002290

Serine/threonine

protein

kinase

Bc-siRNA
3′TGGTGCTTTTAGTGTGGTCGT 5′
344
4.25
Solyc02g069630.2.1
Subtilisin-like

|:|:|||::|||||||||||

2706~2727(cDNA)
serine protease

Target
5′TCTATGAAGGTCACACCAGCA 3′
347

(AHRD V1

**-*

Q948Q4_ARATH);

contains

Interpro

domain(s)

IPR015500

Peptidase S8,

subtilisin-

related

Bc-siRNA
3′TGGTGCTTTTAGTGTGGTCGT 5′
344
3.5
Solyc05g015510.2.1
Squamosa

||||:| |||||| |||||||

3013~3034(cDNA)
promoter-

Target
5′ACCATGCAAATCAGACCAGCA 3′
348

binding-like

protein 11

(AHRD_V1

***-

B6TF72_MAIZE);

contains

Interpro

domain(s)

IPR004333

Transcription

factor, SBP-

box

Bc-siRNA
3′TGGTGCTTTTAGTGTGGTCGT 5′
344
4.5
Solyc09g007710.2.1
Tir-nbs-lrr,

|||:| |||||||:|||||

3351~3372(cDNA)
resistance

Target
5′TCCATGTAAATCACGCCAGCT 3′
349

protein

Bc-siRNA
3′TGGTGCTTTTAGTGTGGTCGT 5′
344
4
Solyc10g081020.1.1
Transcription

||||:|||:|||:|||:|||

3688~3709(cDNA)
elongation

Target
5′ACCATGAAGATCGCACTAGCT 3′
350

factor SPT6

(AHRD V1

***-

A8NF94_COPC7);

contains

Interpro

domain(s)

IPR017072

Transcription

elongation

factor Spt6

siR1018
618.4
51
288.2
Bc-siRNA
3′GGCTCGGCCCATACGTTGTAGT 5′
351
4.5
AT1G62970.1
Chaperone

SIR8

|:||||||| || ||||:||||

1017~1039(CDS)
DnaJ-domain

Intergenic

Target
5′CTGAGCCGGCTAGGCAATATCA 3′
352

superfamily

region

protein

TGATGTT

GCATACC

CGGCTCG

G (SEQ ID

NO: 351)

Bc-siRNA
3′GGCTCGGCCCATACGTTGTAGT 5′
351
4.5
Solyc04g007510.2.1
ATP-

:||||| || ||||||||| ||

3230~3252(cDNA)
dependent

Target
5′TCGAGCAGGATATGCAACACCA 3′
353

RNA helicase

A-like protein

(AHRD V1

***-

Q9FF84_ARATH);

contains

Interpro

domain(s)

IPR007502

Helicase-

associated

region

siR114
395.8
138.24
14.3
Bc-siRNA
3′GGATAAGGTTTTCCTGGGACCT 5′
354
4.5
AT1G78960.1
lupeol

SIR2 LTR

| ||||:| |||||| ||||||

1445~1467(CDS)
synthase 2

transposon

Target
5′CATATTTCCAAAGGAGCCTGGA 3′
355

TCCAGG

GTCCTTT

TGGAAT

AGG (SEQ

ID

NO: 354)

Bc-siRNA
3′GGATAAGGTTTTCCTGGGACCT 5′
354
4.5
Solyc12g006510.1.1
Cycloartenol

| ||||:||||||||:| ||||

1377~1399(cDNA)
Synthase

Target
5′CATATTTCAAAAGGATCGTGGA 3′
356

(AHRD V1

***-

O82139_PANGI);

contains

Interpro

domain(s)

IPR018333

Squalene

cyclase

siR1020
138.3
209
10.1
Bc-siRNA
3′ACAGGACCAAGCAGCACCGTT 5′
357
4
AT2G22810.1
1-

SIR1020

| ||||||||||||||| ||

1176~1197(CDS)
aminocyclopropane-

Intergenic

Target
5′TCTCCTGGTTCGTCGTGCCAT 3′
358

1-

region

carboxylate

TTGCCAC

synthase 4

GACGAA

CCAGGA

CA (SEQ

ID

NO: 357)

Bc-siRNA
3′ACAGGACCAAGCAGCACCGTT 5′
357
4
Solyc04g005650.1.1
Mitochondrial

| ||:||| ||||:||||||:

337~358(cDNA)
carrier family

Target
5′TTTCTTGGCTCGTTGTGGCAG 3′
359

(AHRD V1

***-

C1MWU5_MICPS);

contains

Interpro

domain(s)

IPR001993

Mitochondrial

substrate

carrier

Bc-siRNA
3′ACAGGACCAAGCAGCACCGTT 5′
357
4.5
Solyc09g091210.2.1
Disease

||||:|| ||| |:|||||||

861~882(cDNA)
resistance

Target
5′TGTCTTGTTTCATTGTGGCAA 3′
360

response/

dirigent-like

protein

(AHRD V1

***-

Q0WPQ6_ARATH);

contains

Interpro

domain(s)

IPR004265

Plant disease

resistance

response

protein

siR1016
22.8
255.08
5
Bc-siRNA
3′AGGCAAACTGAATCGAGAGTT 5′
361
3.5
AT1G23190.1
Phosphoglucomutase/

SIR1 LTR

||:| ||||||||||||| ||

1753~1774(CDS)
phosphomannomutase

transposon

Target
5′TCTGGTTGACTTAGCTCTAAA 3′
362

family

TTGAGA

protein

GCTAAGT

CAAACG

GA (SEQ

ID

NO: 361)

Bc-siRNA
3′AGGCAAACTGAATCGAGAGTT 5′
361
4.25
AT5G19260.1
Protein of

|| ||||::|||||||||||

184~205(CDS)
unknown

Target
5′ACCATTTGGTTTAGCTCTCAA 3′
363

function

(DUF3049)

Bc-siRNA
3′AGGCAAACTGAATCGAGAGTT 5′
361
3.5
Solyc01g101090.2.1
TBC1 domain

|||||| ||||:|||||||:

1040~1061(cDNA)
family

Target
5′CCCGTTTCACTTGGCTCTCAG 3′
364

member

CG11727

(AHRD V1

**-*

Y1727_DROME);

contains

Interpro

domain(s)

IPR000195

RabGAP/TBC

Bc-siRNA
3′AGGCAAACTGAATCGAGAGTT 5′
361
4
Solyc02g082060.1.1
PPPDE

|||| ||||:|| ||||||||

497~518(cDNA)
peptidase

Target
5′TCCGGTTGATTTTGCTCTCAA 3′
365

domain-

containing

protein 1

(AHRD V1 *---

PPDE1_XENLA);

contains

Interpro

domain(s)

IPR008580

Protein of

unknown

function

DUF862,

eukaryotic

Bc-siRNA
3′AGGCAAACTGAATCGAGAGTT 5′
361
3.5
Solyc04g076690.2.1
Unknown

|::||||| |||||||:||||

623~644(cDNA)
Protein

Target
5′TTTGTTTGTCTTAGCTTTCAA 3′
366

(AHRD V1)

siR1003
615.3
2.48
0.5
Bc-siRNA
3′CGTAGCGGTCAAGACCAATGG 5′
367
4
AT2G31220.1
basic helix-

SIR1003

|| ||| ||||||||||:||

223~244(CDS)
loop-helix

LTR

Target
5′CCACCGCAAGTTCTGGTTGCC 3′
368

(bHLH) DNA-

transposon

binding

GGTAAC

superfamily

CAGAAC

protein

TGGCGAT

GC (SEQ

ID

NO: 367)

Bc-siRNA
3′CGTAGCGGTCAAGACCAATGG 5′
367
4
Solyc06g050170.2.1
Potassium

||||:|:| ||| ||||||||

1771~1792(cDNA)
transporter

Target
5′GCATTGTCCGTTATGGTTACC 3′
369

(AHRD V1

****

Q1T761_PHRAU);

contains

Interpro

domain(s)

IPR018519

Potassium

uptake

protein, kup

IPR003855

K+ potassium

transporter

siR124
17.5
232.88
3.6
Bc-siRNA
3′TGGAGGGGCCTCGAGACCAGT 5′
370
4
AT1G13270.1
methionine

SIR1 LTR

:|:|:|:|||||||| |||||

140~161(CDS)
aminopeptidase

transposon

Target
5′GCTTTCTCGGAGCTCCGGTCA 3′
371

1B

TGACCA

GAGCTCC

GGGGAG

GT (SEQ

ID

NO: 370)

Bc-siRNA
3′TGGAGGGGCCTCGAGACCAGT 5′
370
4.5
AT3G59040.1
Tetratricopeptide

|::|:|||||| ||||||||:

1252~1273(CDS)
repeat

Target
5′ATTTTCCCGGACCTCTGGTCG 3′
372

(TPR)-like

superfamily

protein

Bc-siRNA
3′TGGAGGGGCCTCGAGACCAGT 5′
370
4.5
Solyc02g065550.2.1
Coiled-coil

|||||:||||| ||| |||||

280~301(cDNA)
domain-

Target
5′ACCTCTCCGGATCTCCGGTCA 3′
373

containing

protein 109A

(AHRD V1 *---

C109A_MOUSE);

contains

Interpro

domain(s)

IPR006769

Protein of

unknown

function

DUF607

Bc-siRNA
3′TGGAGGGGCCTCGAGACCAGT 5′
370
4
Solyc04g045540.1.1
Ycf1

|||:|:| ||||||||||:|

127~148(cDNA)
(Fragment)

Target
5′TCCTTCTCCGAGCTCTGGTTA 3′
374

(AHRD V1

***-

A6YA36_9MAGN);

contains

Interpro

domain(s)

IPR008896

Ycf1

Bc-siRNA
3′TGGAGGGGCCTCGAGACCAGT 5′
370
4
Solyc05g047440.1.1
Ycf1

|||:|:| ||||||||||:|

127~148(cDNA)
(Fragment)

Target
5′TCCTTCTCCGAGCTCTGGTTA 3′
375

(AHRD V1

***-

A6Y9X6_HAMJA);

contains

Interpro

domain(s)

IPR008896

Ycf1

Bc-siRNA
3′TGGAGGGGCCTCGAGACCAGT 5′
370
4.25
Solyc05g055360.2.1
Unknown

||||||: |||||:||||||

1577~1598(cDNA)
Protein

Target
5′TCCTCCCTCGAGCTTTGGTCA 3′
376

(AHRD V1)

Bc-siRNA
3′TGGAGGGGCCTCGAGACCAGT 5′
370
4
Solyc10g062330.1.1
Hypothetical

|||:|:| ||||||||||:|

82~103(cDNA)
chloroplast

Target
5′TCCTTCTCCGAGCTCTGGTTA 3′
377

RF1 (AHRD

V1 **--

C3UP30_9MAGN);

contains

Interpro

domain(s)

IPR008896

Ycf1

Bc-siRNA
3′TGGAGGGGCCTCGAGACCAGT 5′
370
4
Solyc11g021310.1.1
Hypothetical

|||:|:| ||||||||||:|

127~148(cDNA)
chloroplast

Target
5′TCCTTCTCCGAGCTCTGGTTA 3′
378

RF1 (AHRD

V1 ***-

C3UP30_9MAGN);

contains

Interpro

domain(s)

IPR008896

Ycf1

siR127
451.3
54.32
19.2
Bc-siRNA
3′GCAGTTTGTTGTACAGTTTTGT 5′
379
4
AT5G10450.3
G-box

SIR2 LTR

| ||||| |||||||:|||||

932~954(3′UTR)
regulating

transposon

Target
5′CATCAAAGAACATGTTAAAACT 3′
380

factor 6

TGTTTTG

ACATGTT

GTTTGAC

G (SEQ ID

NO: 379)

Bc-siRNA
3′GCAGTTTGTTGTACAGTTTTGT 5′
379
4.5
Solyc01g068430.1.1
Os06g0207500

||:| | |||||||||||:||

871~893(cDNA)
protein

Target
5′AGTTACAAAACATGTCAAAGCA 3′
381

(Fragment)

(AHRD V1

**--

Q0DDQ9_ORYSJ);

contains

Interpro

domain(s)

IPR004253

Protein of

unknown

function

DUF231,

plant

siR128
574.3
3.28
7.7
Bc-siRNA
3′TAGAACTAAGACATAAGACAT 5′
382
3
AT1G48210.1
Protein kinase

SIR15

||:|||:||||||||| ||||

1343~1364(3′UTR)
superfamily

Intergenic

Target
5′ATTTTGGTTCTGTATTGTGTA 3′
383

protein

region

TACAGA

ATACAG

AATCAA

GAT (SEQ

ID

NO: 382)

Bc-siRNA
3′TAGAACTAAGACATAAGACAT 5′
382
4
AT2G23348.1
unknown

|||| | |||| |||||||||

402~423(3′UTR)
protein,

Target
5′ATCTAGTTTCTTTATTCTGTA 3′
384

hypothetical

protein,

uncharacterized

protein

Bc-siRNA
3′TAGAACTAAGACATAAGACAT 5′
382
3
AT4G08990.1
DNA

:|||||:||||| ||||||||

2536~2557(CDS)
(cytosine-5-)-

Target
5′GTCTTGGTTCTGGATTCTGTA 3′
385

methyltransferase

family

protein

Bc-siRNA
3′TAGAACTAAGACATAAGACAT 5′
382
4
AT4G14140.1
DNA

:||| |:||||| ||||||||

2560~2581(CDS)
methyltransferase 2

Target
5′GTCTAGGTTCTGGATTCTGTA 3′
386

Bc-siRNA
3′TAGAACTAAGACATAAGACAT 5′
382
4.5
Solyc04g005530.2.1
Unknown

:|||| | |:||||||:||||

1196~1217(cDNA)
Protein

Target
5′GTCTTTACTTTGTATTTTGTA 3′
387

(AHRD V1)

Bc-siRNA
3′TAGAACTAAGACATAAGACAT 5′
382
4.5
Solyc11g012550.1.1
F-box family

|| ||||| |||||||| ||:

49~70(cDNA)
protein

Target
5′ATATTGATCCTGTATTCCGTG 3′
388

(AHRD V1

***-

D7L4T6_ARALY);

contains

Interpro

domain(s)

IPR001810

Cyclin-like F-

box

siR130
400.4
65
6.5
Bc-siRNA
3′TGGTTATATCTGAACAACTTGT 5′
389
4
AT2G42340.1
unknown

SIR2 LTR

||:| |||:||||||||||| |

486~508(CDS)
protein,

transposon

Target
5′ACTACTATGGACTTGTTGAAAA 3′
390

hypothetical

TGTTCAA

protein,

CAAGTCT

uncharacterized

ATATTGG

protein

T (SEQ ID

NO: 389)

Bc-siRNA
3′TGGTTATATCTGAACAACTTGT 5′
389
4
Solyc01g008080.2.1
Ribosomal

| |:||:| ||||||||||||

2214~2236(cDNA)
protein S27

Target
5′AACGATGTCGACTTGTTGAACC 3′
391

(AHRD V1

***-

Q3HVK9_SOLTU);

contains

Interpro

domain(s)

IPR000592

Ribosomal

protein S27e

Bc-siRNA
3′TGGTTATATCTGAACAACTTGT 5′
389
3.5
Solyc01g095740.2.1
ATP-

|| |:|| |||||||||||||

2485~2507(cDNA)
dependent

Target
5′ACAAGTACAGACTTGTTGAACT 3′
392

RNA helicase

DBP4 (AHRD

V1 *-**

C1GZM0_PARBA);

contains

Interpro

domain(s)

IPR011545

DNA/RNA

helicase,

DEAD/DEAH

box type, N-

terminal

siR1004
485.4
14
32.8
Bc-siRNA
3′CTTGAGGAAGGAAGGTTAGTAA 5′
393
4.5
AT3G07990.1
serine

SIR15

||||:|||||||:|||||||

72~94(CDS)
carboxypeptidase-

Intergenic

Target
5′TTACTCTTTCCTTCTAATCATT 3′
394

like 27

region

AATGATT

GGAAGG

AAGGAG

TTC (SEQ

ID

NO: 393)

Bc-siRNA
3′CTTGAGGAAGGAAGGTTAGTAA 5′
393
4.5
AT4G21740.1
unknown

|||:| | |||||||:|||||

99~121(CDS)
protein,

Target
5′GAATTACGTCCTTCCGATCATG 3′
395

hypothetical

protein,

uncharacterized

protein

Bc-siRNA
3′CTTGAGGAAGGAAGGTTAGTAA 5′
393
4.25
Solyc07g042910.2.1
Genomic

||||| :|| |||:||||||||

1930~1952(cDNA)
DNA

Target
5′GAACTATTTGCTTTCAATCATT 3′
396

chromosome 5

TAC clone

K21L19

(AHRD V1

**--

Q9FGT4_ARATH)

siR144
471
9.88
46.1
Bc-siRNA
3′CTATTTAATTAGTAGTACAAT 5′
397
4
AT2G46330.1
arabinogalactan

SIR6 CDS

| ||| || |||||||||||

471~492(3′UTR)
protein 16

(spurious

Target
5′GTTAATTTCATCATCATGTTC 3′
398

gene)

TAACATG

ATGATTA

ATTTATC

(SEQ ID

NO: 397)

Bc-siRNA
3′CTATTTAATTAGTAGTACAAT 5′
397
4
AT4G12040.2
A20/AN1-like

|:| | ||:||:|||||||||

513~534(5′UTR)
zinc finger

Target
5′GGTTATTTGATTATCATGTTA 3′
399

family protein

Bc-siRNA
3′CTATTTAATTAGTAGTACAAT 5′
397
4.25
Solyc01g080260.2.1
At4g14280-

|| :||| ||||||||||||

2174~2195(cDNA)
like protein

Target
5′GAAGAATCAATCATCATGTTC 3′
400

(Fragment)

(AHRD V1 *-*-

C7FD87_ARALP);

contains

Interpro

domain(s)

IPR011989

Armadillo-like

helical

Bc-siRNA
3′CTATTTAATTAGTAGTACAAT 5′
397
4.5
Solyc01g098240.1.1
RNA

| |||||:||| ||||||||

3823~3844(cDNA)
polymerase

Target
5′CAGAAATTGATCTTCATGTTA 3′
401

Rpb1 C-

terminal

repeat

domain-

containing

protein

(AHRD V1 *---

C5GU31_AJEDR);

contains

Interpro

domain(s)

IPR012474

Frigida-like

Bc-siRNA
3′CTATTTAATTAGTAGTACAAT 5′
397
4.5
Solyc10g005650.2.1
Peroxisomal

| |||||:|| |||||||||

814~835(cDNA)
targeting

Target
5′TAGAAATTGATAATCATGTTA 3′
402

signal 1

receptor

(AHRD V1

****

Q9ZTK6_TOBAC);

contains

Interpro

domain(s)

IPR011990

Tetratricopeptide-

like helical

Bc-siRNA
3′CTATTTAATTAGTAGTACAAT 5′
397
4.5
Solyc12g007150.1.1
Pollen-

|| | | | |||||||||||:

73~94(cDNA)
specific kinase

Target
5′GACATACTCATCATCATGTTG 3′
403

partner

protein-like

protein

(Fragment)

(AHRD V1 *---

Q5DK68_SOLLC);

contains

Interpro

domain(s)

IPR005512

Rop

nucleotide

exchanger,

PRONE

siR137
376.8
46.08
3
Bc-siRNA
3′ATGATGATCTTATCTTAGCAT 5′
404
4
AT1G22110.1
structural

SIR2 LTR

||||| || ||| ||||||||

1283~1304(3′UTR)
constituent of

transposon

Target
5′TACTAATAAAATCGAATCGTA 3′
405

ribosome

TACGATT

CTATTCT

AGTAGT

A (SEQ ID

NO: 404)

Bc-siRNA
3′ATGATGATCTTATCTTAGCAT 5′
404
4.5
AT3G25510.1
disease

|:|||||||||:|:|||||

5473~5494(CDS)
resistance

Target
5′GATTACTAGAATGGGATCGTT 3′
406

protein (TIR-

NBS-LRR

class),

putative

Bc-siRNA
3′ATGATGATCTTATCTTAGCAT 5′
404
4.5
Solyc04g063230.2.1
Dehydration-

| || ||||||| |||||||:

1354~1375(cDNA)
responsive

Target
5′TTCTCCTAGAATTGAATCGTG 3′
407

family protein

(AHRD V1

**--

D7LF23_ARALY);

contains

Interpro

domain(s)

IPR004159

Protein of

unknown

function

DUF248,

methyltransferase

putative

siR140
417.1
27.16
49.1
Bc-siRNA
3′TGTATGCTTTGCCGTTTTAGTT 5′
408
4.5
AT2G07360.1
SH3 domain-

SIR8

|| | | |||||||||||||:

3291~3313(CDS)
containing

Intergenic

Target
5′TCACAAGCAACGGCAAAATCAG 3′
409

protein

region

TTGATTT

TGCCGTT

TCGTATG

T (SEQ ID

NO: 408)

Bc-siRNA
3′TGTATGCTTTGCCGTTTTAGTT 5′
408
4.25
Solyc04g080720.2.1
Transferase

||: |||| ||||:||||||||

1084~1106(cDNA)
family protein

Target
5′ACGAACGATACGGTAAAATCAA 3′
410

(AHRD V1

**-*

D7KBT0_ARALY);

contains

Interpro

domain(s)

IPR003480

Transferase

Bc-siRNA
3′TGTATGCTTTGCCGTTTTAGTT 5′
408
4
Solyc07g017860.2.1
Acetyl-

|||| || ||:||||||||:||

436~458(cDNA)
coenzyme A

Target
5′ACATTCGCAATGGCAAAATTAA 3′
411

synthetase

(AHRD V1

***-

Q2J3D0_RHOP2);

contains

Interpro

domain(s)

IPR011904

Acetate--CoA

ligase

Bc-siRNA
3′TGTATGCTTTGCCGTTTTAGTT 5′
408
3.5
Solyc12g098610.1.1
Xyloglucan

|| ||:| |||:|||||||||||

641~663 (cDNA)
endotransglucosylase/

Target
5′AGATGCAAAATGGCAAAATCAA 3′
412

hydrolase

8 (AHRD

V1 ***-

C0IRG7_ACTDE);

contains

Interpro

domain(s)

IPR016455

Xyloglucan

endotransglucosylase/

hydrolase

siR141
11.4
187.64
3
Bc-siRNA
3′TGTCTTCAGGCTTACAAAGAT 5′
413
4
AT3G01350.1
Major

SIR1 LTR

|::|:|||| |||||||||||

1191~1212(CDS)
facilitator

transposon

Target
5′ATGGGAGTCGGAATGTTTCTA 3′
414

superfamily

TAGAAA

protein

CATTCGG

ACTTCTG

T (SEQ ID

NO: 413)

Bc-siRNA
3′TGTCTTCAGGCTTACAAAGAT 5′
413
4.5
Solyc03g113070.2.1
ATP-binding

||| || ||||||| |||||:

1358~1379(cDNA)
cassette

Target
5′ACATAATTCCGAATATTTCTG 3′
415

(ABC)

transporter 17

(AHRD V1 *-*-

Q4H493_RAT)

siR156
335
9.88
251.1
Bc-siRNA
3′AGGGTTAGGGTAGGGTAGGGT 5′
416
4.5
AT5G45973.1
unknown

SIR18

|||| |||:|||||:||| ||

62~83(CDS)
protein,

Intergenic

Target
5′TCCCTATCTCATCCTATCGCA 3′
417

hypothetical

region

protein,

TGGGAT

uncharacterized

GGGATG

protein

GGATTG

GGA (SEQ

ID

NO: 416)

Bc-siRNA
3′AGGGTTAGGGTAGGGTAGGGT 5′
416
2
Solyc01g112220.2.1
Serine/threonine

||:|||||:|||||||||||

163~184(cDNA)
protein

Target
5′TCTCAATCTCATCCCATCCCT 3′
418

kinase-like

(AHRD V1

****

Q5XWQ1_SOLTU);

contains

Interpro

domain(s)

IPR002290

Serine/threonine

protein

kinase

Bc-siRNA
3′AGGGTTAGGGTAGGGTAGGGT 5′
416
4.5
Solyc12g019040.1.1
Exostosin

|| || |::||||:|||||||

100~121(cDNA)
family protein

Target
5′TCACATTTTCATCTCATCCCA 3′
419

(AHRD V1 *-*-

D7LPB7_ARALY)

Bc-siRNA
3′AGGGTTAGGGTAGGGTAGGGT 5′
416
4.5
Solyc12g096410.1.1
Unknown

||:| ||| ||||||||:||

54~75(cDNA)
Protein

Target
5′TCTCCATCACATCCCATTCCT 3′
420

(AHRD V1)

siR161
9.9
120.16
5.7
Bc-siRNA
3′GGTTCCTTCTCTTACTACGGAT 5′
421
4.5
AT2G16270.1

SIR1 LTR

||||||||||||:|| ||:||:

295~317(CDS)

transposon

Target
5′CCAAGGAAGAGAGTGTTGTCTG 3′
422

TAGGCAT

CATTCTC

TTCCTTG

G (SEQ ID

NO: 421)

Bc-siRNA
3′GGTTCCTTCTCTTACTACGGAT 5′
421
4.5
AT3G18660.1
plant

|:|||| |||||| |||||:||

1168~1190(CDS)
glycogenin-

Target
5′CTAAGGCAGAGAAAGATGCTTA 3′
423

like starch

initiation

protein 1

Bc-siRNA
3′GGTTCCTTCTCTTACTACGGAT 5′
421
4
AT3G63380.1
ATPase E1-E2

:| | |||||||||||||:||:

1416~1438(CDS)
type family

Target
5′TCCATGAAGAGAATGATGTCTG 3′
424

protein/

haloacid

dehalogenase-

like hydrolase

family protein

Bc-siRNA
3′GGTTCCTTCTCTTACTACGGAT 5′
421
3.75
AT5G17400.1
endoplasmic

|: ||||:|||||||||||:||

863~885(CDS)
reticulum-

Target
5′CTTAGGAGGAGAATGATGCTTA 3′
425

adenine

nucleotide

transporter 1

Bc-siRNA
3′GGTTCCTTCTCTTACTACGGAT 5′
421
2.5
Solyc03g083340.1.1
Response

:|||| |:|:||||||||||||

1152~1174(cDNA)
regulator 8

Target
5′TCAAGTAGGGGAATGATGCCTA 3′
426

(AHRD V1 *-*-

Q9AV93_MAIZE);

contains

Interpro

domain(s)

IPR001789

Signal

transduction

response

regulator,

receiver

region

Bc-siRNA
3′GGTTCCTTCTCTTACTACGGAT 5′
421
4.5
Solyc04g005430.2.1
Dehydration-

||| ||||:|||| |||||||

1312~1334(cDNA)
responsive

Target
5′GCAAAGAAGGGAATCATGCCTA 3′
427

protein-like

(AHRD V1

**--

Q653G1_ORYSJ);

contains

Interpro

domain(s)

IPR004159

Protein of

unknown

function

DUF248,

methyltransferase

putative

Bc-siRNA
3′GGTTCCTTCTCTTACTACGGAT 5′
421
4.5
Solyc11g005760.1.1
Glycogenin-

|:|| | |||||| ||||||||

892~914(cDNA)
like protein

Target
5′CTAAAGCAGAGAAAGATGCCTA 3′
428

(AHRD V1

***-

Q5NA53_ORYSJ);

contains

Interpro

domain(s)

IPR002495

Glycosyl

transferase,

family 8

siR163
275
8.24
74.2
Bc-siRNA
3′ATGTGTAACATGAAACCTAGT 5′
429
2.5
AT3G07140.1
GPI

SIR8

| |:|||||||:|||||||||

1754~1775(CDS)
transamidase

Intergenic

Target
5′TTCGCATTGTATTTTGGATCA 3′
430

component

region

Gpi16 subunit

TGATCCA

family protein

AAGTAC

AATGTGT

A (SEQ ID

NO: 429)

Bc-siRNA
3′ATGTGTAACATGAAACCTAGT 5′
429
4.5
AT5G46640.1
AT hook

|||::||| |||||||||||

1159~1180(CDS)
motif DNA-

Target
5′AACATGTTGAACTTTGGATCA 3′
431

binding family

protein

Bc-siRNA
3′ATGTGTAACATGAAACCTAGT 5′
429
4.5
AT5G59810.1
Subtilase

||||:| ||||||| |||||

293~314(CDS)
family protein

Target
5′TACATAGTGTACTTGGGATCT 3′
432

Bc-siRNA
3′ATGTGTAACATGAAACCTAGT 5′
429
4.5
Solyc06g084310.2.1
Small nuclear

|:|||| | ||:||||||||

598~619(cDNA)
ribonucleoprotein

Target
5′TGCACAATTTATTTTGGATCT 3′
433

Sm D1

(AHRD V1

***-

B6TXH2_MAIZE);

contains

Interpro

domain(s)

IPR006649

Like-Sm

ribonucleoprotein,

eukaryotic

and archaea-

type, core

Bc-siRNA
3′ATGTGTAACATGAAACCTAGT 5′
429
4.25
Solyc08g079630.2.1
AT-hook

||||: |||||||||||| ||

1618~1639(cDNA)
motif nuclear

Target
5′TACATTTTGTACTTTGGACCA 3′
434

localized

protein 1

(AHRD V1

***-

Q8VYJ2_ARATH);

contains

Interpro

domain(s)

IPR005175

Protein of

unknown

function

DUF296

siR1001
218
7.4
8.4
Bc-siRNA
3′TAATACAAAATTATTAGTACACT 5′
435
3.5
AT1G77470.1
replication

SIR1001

|||||||||||||:||| ||||:

1437~1460(3′UTR)
factor C

CDS

Target
5′ATTATGTTTTAATGATCTTGTGG 3′
436

subunit 3

TCACATG

ATTATTA

AAACAT

AAT (SEQ

ID

NO: 435)

Bc-siRNA
3′TAATACAAAATTATTAGTACACT 5′
435
4.5
Solyc04g055110.2.1
Mitochondrial

|||:||| || |||||| |||||

1474~1497(cDNA)
import

Target
5′ATTGTGTCTTCATAATCCTGTGA 3′
437

receptor

subunit

TOM34

(AHRD V1 *---

TOM34_RAT);

contains

Interpro

domain(s)

IPR011990

Tetratricopeptide-

like helical

Normalized read counts are given in reads per million B. cinerea sRNAs. Reads were summed from individual sRNA libraries for each category: B. cinerea-infected Arabidopsis (“A”), B. cinerea-infected S. lycopersicum (“S”), and cultured B. cinerea (“B”).

*AS (aligned score): Target gene alignment was scored as described in Materials and Methods.

TABLE 2

Primers for constructing short tandem target mimic (STTM) against selected B.

cinerea sRNAs listed in Table 1

sRNA
Primer*
Primer sequence

Bc-
3.2-
GccATTTAAATatggtctaaagaagaagaatACCTACAAGATctaCCACAATGTAgaattcggtacgctgaaatcaccag

siR3.2
STTMSwa48
(SEQ ID NO: 438)

ntlink-PF

3.2-
GccATTTAAATtagaccataacaacaacaacTACATTGTGGtagATCTTGTAGGTaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 439)

ntlink-PR

Bc-
3.1-
GccATTTAAATatggtctaaagaagaagaatGCCCACCTACActaAGATCCACAAgaattcggtacgctgaaatcaccag

siR3.1
STTMSwa48
(SEQ ID NO: 440)

ntlink-PF

3.1-
GccATTTAAATtagaccataacaacaacaacTTGTGGATCTtagTGTAGGTGGGCaagcttgggctgtcctctccaaatg

STTMSwa49
(SEQ ID NO: 441)

ntlink-PR

Bc-siR5
5-
GccATTTAAATatggtctaaagaagaagaatAAGTATACATTctaCCGAGTCAAAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 442)

ntlink-PF

5-
GccATTTAAATtagaccataacaacaacaacTTTGACTCGGtagAATGTATACTTaagcttgggctgtcctctccaaatg

STTMSwa49
(SEQ ID NO: 443)

ntlink-PR

3.1-3.2-STTMSwa48ntlink-PF (=3.2-STTMSwa48ntlink-PF)

3.1-3.2-STTMSwa48ntlink-PR (=3.1-STTMSwa48ntlink-PR)

5-3.2-STTMSwa48ntlink-PF (=3.2-STTMSwa48ntlink-PF)

5-3.2-STTMSwa48ntlink-PR (=5-STTMSwa48ntlink-PR)

5-3.1-STTMSwa48ntlink-PF (=3.1-STTMSwa48ntlink-PF)

5-3.1-STTMSwa48ntlink-PR (=5-STTMSwa48ntlink-PR)

SiR1
SiR1-
GccATTTAAATatggtctaaagaagaagaatCAGAATTCTACTctaCTTGCTTCGAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 444)

ntlink-PF

SiR1-
GccATTTAAATtagaccataacaacaacaacTCGAAGCAAGtagAGTAGAATTCTGaagcttgggctgtcctctccaaatg

STTMSwa49
(SEQ ID NO: 445)

ntlink-PR

siR1010
1010-
GccATTTAAATatggtctaaagaagaagaatAGCAATCAAAActaATTCCCCCGAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 446)

ntlink-PF

1010-
GccATTTAAATtagaccataacaacaacaacTCGGGGGAATTAGTTTTGATTGCTaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 447)

ntlink-PR

siR1008
1008-
GccATTTAAATatggtctaaagaagaagaatGCATAAACTGATctaCATCATCACAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 448)

ntlink-PF

1008-
GccATTTAAATtagaccataacaacaacaacTGTGATGATGTAGATCAGTTTATGCaagcttgggctgtcctctccaaatg

STTMSwa48

ntlink-PR
(SEQ ID NO: 449)

siR9
9-
GccATTTAAATatggtctaaagaagaagaatTCTAAAAATGCTctaCATCATAAAAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 450)

ntlink-PF

9-
GccATTTAAATtagaccataacaacaacaacTTTTATGATGTAGAGCATTTTTAGAaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 451)

ntlink-PR

siR10
10-
GccATTTAAATatggtctaaagaagaagaatAGCACCCTACActaACCTAGAAAAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 452)

ntlink-PF

10-
GccATTTAAATtagaccataacaacaacaacTTTTCTAGGTTAGTGTAGGGTGCTaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 453)

ntlink-PR

siR18
18-
GccATTTAAATatggtctaaagaagaagaatTGATCGACTCTctaGTTTTGGCTAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 454)

ntlink-PF

18-
GccATTTAAATtagaccataacaacaacaacTAGCCAAAACTAGAGAGTCGATCAaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 455)

ntlink-PR

siR15
15-
GccATTTAAATatggtctaaagaagaagaatTCAAACAACAAGctaGTTCAACACAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 456)

ntlink-PF

15-
GccATTTAAATtagaccataacaacaacaacTGTGTTGAACTAGCTTGTTGTTTGAaagcttgggctgtcctaccaaatg

STTMSwa48
(SEQ ID NO: 457)

ntlink-PR

siR17
17-
GccATTTAAATatggtctaaagaagaagaatCCAGTGCCATTctaCATCATTTTAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 458)

ntlink-PF

17-
GccATTTAAATtagaccataacaacaacaacTAAAATGATGTAGAATGGCACTGGaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 459)

ntlink-PR

siR22
22-
GccATTTAAATatggtctaaagaagaagaatACTACACCCTTctaGACCACGTTAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 460)

ntlink-PF

22-
GccATTTAAATtagaccataacaacaacaacTAACGTGGTCTAGAAGGGTGTAGTaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 461)

ntlink-PR

siR24
24-
GccATTTAAATatggtctaaagaagaagaatGTCAAACAGAGActaGGACCAATCAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 462)

ntlink-PF

24-
GccATTTAAATtagaccataacaacaacaacTGATTGGTCCTAGTCTCTGTTTGACaagcttgggctgtcctaccaaatg

STTMSwa48
(SEQ ID NO: 463)

ntlink-PR

siR25
25-
GccATTTAAATatggtctaaagaagaagaatAAAACCAAAATTctaTGATTCACTAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 464)

ntlink-PF

25-
GccATTTAAATtagaccataacaacaacaacTAGTGAATCATAGAATTTTGGTTTTaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 465)

ntlink-PR

siR1015
1015-
GccATTTAAATatggtctaaagaagaagaatACCGATCAGActaCAACCATCAAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 466)

ntlink-PF

1015-
GccATTTAAATtagaccataacaacaacaacTTGATGGTTGTAGTCTGATCGGTaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 467)

ntlink-PR

siR20
20-
GccATTTAAATatggtctaaagaagaagaatAATCAGAAAAACctaAAGAACACTAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 468)

ntlink-PF

20-
GccATTTAAATtagaccataacaacaacaacTAGTGTTCTTTAGGTTTTTCTGATTaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 469)

ntlink-PR

siR1021
1021-
ccATTTAAATatggtctaaagaagaagaatACATGTTTTGTTctaCATCACTGTAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 470)

ntlink-PF

1021-
GccATTTAAATtagaccataacaacaacaacTACAGTGATGTAGAACAAAACATGTaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 471)

ntlink-PR

siR1002
1002-
GccATTTAAATatggtctaaagaagaagaatTGTGTTACAAAGActaTTTGAAGAATgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 472)

ntlink-PF

1002-
GccATTTAAATtagaccataacaacaacaacATTCTTCAAATAGTCTTTGTAACACAaagcttgggctgtcctctccaaat

STTMSwa48
g (SEQ ID NO: 473)

ntlink-PR

siR28
28-
GccATTTAAATatggtctaaagaagaagaatAAGAAGATCACActaGTTTCAAAAAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 474)

ntlink-PF

28-
GccATTTAAATtagaccataacaacaacaacTTTTTGAAACTAGTGTGATCTTCTTaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 475)

ntlink-PR

siR31
31-
GccATTTAAATatggtctaaagaagaagaatCATTCACGACCctaACAAGACTCAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 476)

ntlink-PF

31-
GccATTTAAATtagaccataacaacaacaacTGAGTCTTGTTAGGGTCGTGAATGaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 477)

ntlink-PR

siR29
29-
GccATTTAAATatggtctaaagaagaagaatCCCAAAAAGGActaCTATCCAACAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 478)

ntlink-PF

29-
GccATTTAAATtagaccataacaacaacaacTGTTGGATAGTAGTCCTTTTTGGGaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 479)

ntlink-PR

siR41
41-
GccATTTAAATatggtctaaagaagaagaatTTCTACTCCCGctaAAAACTATCAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 480)

ntlink-PF

41-
GccATTTAAATtagaccataacaacaacaacTGATAGTTTTTAGCGGGAGTAGAAaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 481)

ntlink-PR

siR35
35-
GccATTTAAATatggtctaaagaagaagaatAACGCGACATGctaGCACAGTACAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 482)

ntlink-PF

35-
GccATTTAAATtagaccataacaacaacaacTGTACTGTGCTAGCATGTCGCGTTaagcttgggctgtcctaccaaatg

STTMSwa48
(SEQ ID NO: 483)

ntlink-PR

siR57
57-
GccATTTAAATatggtctaaagaagaagaatCCAACGAACCAGctaAGATTATCTAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 484)

ntlink-PF

57-
GccATTTAAATtagaccataacaacaacaacCCAACGAACCAGctaAGATTATCTAaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 485)

ntlink-PR

siR43
43-
GccATTTAAATatggtctaaagaagaagaatCCCAACAAGAGctaAAAGCTCCCAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 486)

ntlink-PF

43-
GccATTTAAATtagaccataacaacaacaacTGGGAGCTTTTAGCTCTTGTTGGGaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 487)

ntlink-PR

siR40
40-
GccATTTAAATatggtctaaagaagaagaatAACCAATACAActaGCCCATTCCAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 488)

ntlink-PF

40-
GccATTTAAATtagaccataacaacaacaacTGGAATGGGCTAGTTGTATTGGTTaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 489)

ntlink-PR

siR48
48-
GccATTTAAATatggtctaaagaagaagaatTTGATCGATACctaTGTCACTTCAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 490)

ntlink-PF

48-
GccATTTAAATtagaccataacaacaacaacTGAAGTGACATAGGTATCGATCAAaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 491)

ntlink-PR

siR49
49-
GccATTTAAATatggtctaaagaagaagaatTATCAAAAGACctaATAAGCCACAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 492)

ntlink-PF

49-
GccATTTAAATtagaccataacaacaacaacTGTGGCTTATTAGGTCTTTTGATAaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 493)

ntlink-PR

siR58
58-
GccATTTAAATatggtctaaagaagaagaatCAGACAATGAActaTCCCAATTTAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 494)

ntlink-PF

58-
GccATTTAAATtagaccataacaacaacaacTAAATTGGGATAGTTCATTGTCTGaagcttgggctgtcctctccaaatg

STTMSwa48
(SEQ ID NO: 495)

ntlink-PR

siR1005
1005-
GccATTTAAATatggtotaaagaagaagaatTCCTATTGAAGctaAAACTCTTTAgaattcggtacgctgaaatcaccag

STTMSwa48
(SEQ ID NO: 496)

ntlink-PF

1005-
GccATTTAAATtagaccataacaacaacaacTAAAGAGTTTTAGCTTCAATAGGAaagcttgggctgtcctaccaaatg

STTMSwa48
(SEQ ID NO: 497)

ntlink-PR

*Forward primers are denoted as “PF.” Reverse primers are denoted as “PR.”

TABLE 3

Predicted B. cinerea sRNA targets in V. vinifera

SEQ

sRNA and target in

ID

Target site

V. vinifera

Alignment
NO:
Molecular function
position

Bc-siR3.2

VIT_10s0092g00240
Target
5′ CCCUACAAGAUUAACAAUGUA
498
carbohydrate binding,
CDS + UTR

||||||||||: ||||||||

hydrolase activity

Bc-siR3.2
3′ UGGAUGUUCUAGGUGUUACAU
24
carbohydrate metabolic

process

Bc-siR3.1

VIT_12s0028g01140
Target
5′ ACCCAAUUACAAGAUCCACGA
499
Pentatricopeptide repeat
INTRON

|||| : ||||||||||||: |

Bc-siR3.1
3′ CGGGUGGAUGUUCUAGGUGUU
30

VIT_06s0009g01890
Target
5′ ACCAAUCUACAAAAUCCACAA
500
exonuclease
intron

|| |: |||||| ||||||||

Bc-siR3.1
3′ CGGGUGGAUGUUCUAGGUGUU
30

VIT_10s0116g00190
Target
5′ CCCCAAGUACAAGAACCACAA
501
KNOX1,2 domain
Intron

|||| ||||||| ||||||

containing protein

Bc-siR3.1
3′ CGGGUGGAUGUUCUAGGUGUU
30

Bc-siR5

VIT_05s0020g01790
target
5′ UAUAUUACAUUCCGAGUCAUG
502
Lipase
CDS

| |||||||||||||| :

Bc-siR5
3′ UUCAUAUGUAAGGCUCAGUUU
36

VIT_01s0011g01000
target
5′ AAGCAUACAUACCGAGUCAAU
503
NB-ARC and LRR
intron

||| |||||| |||||||||

domain

Bc-siR5
3′ UUCAUAUGUAAGGCUCAGUUU
36

VIT_05s0077g01510
target
5′ AAGTAATCATTCCAAGTCAAA
504
DUF7 domain
intron

||||| |||||| |||||||

Bc-siR5
3′ UUCAUAUGUAAGGCUCAGUUU
36

Example 2: Sequences of Promoters and sRNA-Resistant Targets

A. thaliana (At) BIK1 Promoter:

SEQ ID NO: 1

attttattatattatatagcgatgagagagacagagcttgaaggttcttt

ttagcgaaagagaaaaatccaggaagataggcgaaaaggaagatgaagcg

aagatgaggttaatataatactcatgttaaatgacaaaaatgcccttata

tgattaatgatattaccatttgagcttgctgtggaagctgtaacgaaccg

aaaattaaaaacagaataacgaacatagacggagaatatgatattattcg

ttttaccaaagaaactaacaaatagttttaactttatctaacaaaggggt

aaaacgggtaatttgtttgggatgaggtggagcgtagcggacaatcgaga

aattaaaagtttggcttggggacgaagttaaaggtgggctttaacgtttt

aaattggctgactcggacgatatttcttgtatttaataccaaaaatgaat

gactttataattcatttgtagattgaaagttacgtattgattcgaaaatc

aacacattgtgttttcaagtgggcataaactataacaccttgttgattga

ttaatagattacctaaagacattatggtttattactggtctttcaatata

tttttatcgcattgtcaatgatattgtttttgtatcccaagtccactgtt

ttggtctctacattcattttgattgggatttatctttttaaaatttcttc

taatgttttttcgatatggttattacttgctttgattttcttttcagtat

gtgtattgctttgcaaattgtttttttcttaagatgaaaaacaactcatt

aaattgtttgagaaatactactaaaacaaataaacaatgaggagaattat

ggaaaacaaagtgtaataggctttaattcattgctagtgggctttttggg

cctatgggcatattacttaccactatccaacccaaaatgccaaataaccg

acatgtctcaccaatccaattttgggccatacggccgaaattatttaaac

ctgtgctcataatttactttacaaattattacttttccataaattgtgga

aaagttatctgtaacatccgattcaactggagtctagactactatagaca

ttgatacgttttgagtttttagatacttggaagatatatgcatttatgaa

tacagattacagacacatactagtactactgtatgtctgtatatggatac

aaaaaaaatcatgtatgaatactaaaattttattagaaatctatttttca

attgttgcaacaatcaagttgtcaaatttatttttgtaaccgttaaacaa

acaaatatcgatttaggtttctaatctgaattgacatctcaaacaaaaaa

ggctgaatactttctgaaaatagtgtatggaatgaaggtggcttttagag

ccattataaccggaagaaaattcaggtgacttttagaaccattataaccg

gaagaaaaggtgaattttaatttttagctgtgtggaagacacggcaagtc

caagtagtaccttcgtacgtcaatattgtccaaccggccgtgtcgaaaat

cttcttgagaaaaattggattttcatctataaaaaaaaaaagtccaagta

ataccaaacaaagacagcgacgtgtaaaacaatacaagactcataatcac

aaacctaccacccaagtcaaacctatattccatttagtgaattcttgatt

atgacttcttgaaatcatttgtattcatatgtataattatttaagtcatt

tttctgtaagtaaaatttttatatatctagaataacgagttccctacgac

aagatacagttgaacgtaaatgtgacatctcaattttcattggtgtctag

tactctagtgattaggttttcgacatttattgtactgattaagtaaaaat

tcatggtacaaacatcgaatatatatttttctgcttacacacaccaatta

acgtggatagaccaattgaaatattttgttacgacaaagcaaaacaaaac

aaacgtcatgtttcgctgtttgtttgtcgtcccgttaatggtaatctttc

agacacatacagtacccaaacaagtaatttgactaaaattttctctctgt

ctaaatttcagaagaaaaaaaaactttaggatatattgccaaaagatctt

aaaaatgggtcatatcattttgatcatatagaatccaacgacctttatct

tttcgccgaactatacttttttgtgtccatttgtttgactttctttcaca

cacacatccacaaagaaaaaaggaccattcttctccttcttctagtcacc

cctcgtgcctctctttaacaccaaacccaaaactcccttctctttcttcc

ttcctctccgatctccgttcacatctctctctcatctttatcttcttctt

tttttgccttgtgggttgaaagtttctatattttctctttctcttctgtt

tacataatccattttcagctcaagcagctgaagaataacgatcaagaacc

aaaaaagaagaaaacgaatctgttcttagctttg

At PDF1.2 Promoter

SEQ ID NO: 2

ACGACGTTGGACTGTTTCATCATATCCCATAAAAATACATGATTGGGGTG

AAAATCTTGAACATATTAAAAAAATATTAAATCAAAATGATAAAGATAGG

GATTTATAAATGTAAAACGGGCGTGTCGAGAATTTTATGGACATTGGGAC

AAGCTTTATATGCAGCATGCATCGCCGCATCGATATCCCGAGGTGCATCG

TTTCTACTTTCATGTCCAAATTTGGGGTTAACTCACAATATATATCATGT

TGCCTATGTAAATTTATAATCATAAATCTAAACCCAAATTTTAATCCTCA

TTCCAAAGCAAAAGTTCTAAGCCCTACAAAAATATGTATTTCCCAAGTTT

AAAAAGAATTAATCTATACTTTTACAAATTTAAATTCTGATCTCTTATAA

TGTTCGGTTTTTCCTTTTTTATTTATTAAGTTAGTTAAAATTTGCAGTTA

TTTTGTTGAATGTCGTTGTTTACGAATTTACGAATAATACCTTTATAGCT

AATCTACAAAATTTTGATGACTGACAACACCGTTAATGTTTTTTTTTAAA

TTACCCTGAGCCTCTCACTTGCGGTCAGACCATGCATGTCGATAGTCCAT

TACGTTTAAGGCCACAATCAACTATAGTTTGTTTATCAATAGCCAACTAA

GCTAACTTTTAGGTTCCTGCCCTCTCCGTTCCTCCGGTACCAATCGTTTC

TTTGTCCCTTCGATAGTTTGAAAACCTACCGACGGTGAGAGCAAAATATT

GATGAATCATCCAATTTTCAGTAATAGGTGTGTCCCAGGGATATATAAAT

GGCGAAACTACGCGAGAACGGTTCCTTGTTCTGCAAACTTGGCGGAACAA

TGCTGCTCTTGAGATCAACCAAACCATATGTTTAGTCCACAACGATCTAT

ATGTCTAGGGGTGATCCTCTAATCGAAAAATGTTGTATTTGTTCGACGAT

GACGAAGGTCAGACTATGAACTGCACAGTCTGCACTTGTCCTAACCGCGA

GAATCTCTGACATCAATATACTTGTGTAACTATGGCTTGGTTAAGATATT

ATTTTCTTGAGTCTTAATCCATTCAGATTAACCAGCCGCCCATGTGAACG

ATGTAGCATTAGCTAAAAGCCGAAGCAGCCGCTTAGGTTACTTTAGATAT

CGACAGAGAAATATATGTGGTGGAGAAACCAGCCATCAACAAACAAAAAG

CAAGATCTTATCTTTTGATATTGGCTACGGGAAGATGATGTCTGTTTAAT

GTGTGGGGTTACCACGTTATTGTACGATGCACAAGTAGAAGATTAACCCA

CTACCATTTCATTATAAATAGACGTTGATCTTTGGCTTATTTCTTCACAC

AACACATACATCTATACATTGAAAACAAAATAGTAATAATCATC

At BIK1 homologous gene in tomato (TPK1b) Promoter

SEQ ID NO: 3

TTGCGTTTAATTTGTATGAATGTCATTTAATTTTTAGGATCGGCTTAAAT

TTGAAATTAAAAAAGCAAAATAATAATACTAGTATTTTCTAACTTTGTAT

TTTAATGCATGACATTATTTTTAGAAAAAATTGTAACGAAGAGAATCATA

TTTATGATAGAATTATTTGTAATTACTATTTGACTGATATTACTAGTTTA

ATTATTTCGCACACAAAGTATATTTTTTTAAAAAAAAATATTTTACATTG

ATTATTTTCTCTCTATCCCAACACCCCATCCCGTCTTTATTTTTATAGTA

TTTATTATACAAATATTTTAAAAGTATCTTATTGAACATCAAAATAATCT

TTTTTAAAAATTATTTATATCCCCAAAAAAATTATATGCACGTGTGAAAA

TGAGAAAATGTTGGTTGGGTGTGAATAATTTGTTGGTTCCCAAATATGAT

TATAATCCAAGAAAATTGGAAATTTGATTATTGCTTCCTTTTGACTTAAA

ACTCTTTGCTAAATTGCTAAGCATTCTTTTTAATTTTGTTTTTCCATTAA

TAACAATTTGGGTAATTCATATCCACTAGTCGGTGGATTTAATAGAAGTG

ATACATATTTTTTTGATGTTATTGTTAATTAATAGTGAAAGGTCCTTTTT

TCTCTCTCCTAATTTATATATAATTCATTTTTTAAAATCAATTTTGAAAG

AATGATATAGTTTCTATATTTAAGTAATGATTTATTTTATTGATAATAAA

ATAAGTTATAATCATATATATATTTTTAATATATTTAAAATTATAATTTA

AATTATTTATATCACATCAATTGAAACGGATGAAATTATTTATTTTAAAA

AAAATGATGAATGGGTGGCATCCATAAAAATGTGACATTTCTCCATGTGT

TTTGCTTAAATGAGATTTTTGACTATTTTTCTTGTGTTCATATTTATGAA

GAAGATCAACAATAAATTTTTATCAATAAAGAGGAAATTAAAAGTTGATT

AATATTAAAAATCACAAATATTTATTGAAAGTGAATAAATTTATAGTTAT

TACACATATATGGAGAGAGATCAAAATCAATATGCTAATTTTTTGTAATG

GAAGGGCACAATGAAAATAAAGTTAATTTTCATGACTAATTTAATCCATA

TAGTTAAATTCTAATCATATAAATTTCAGTGAATAAGTTCATTTGATTTT

TTTTAGATCTAATATTAATTATTAAGATGTAAATGTTAACTATGTTTTTA

TTAATGTTTCAATCACTGTGTCTATATTTGAATGATTACTACTTGTAATT

AAGTGAAAAAATTCAGTATTTTGTGTATTAAAATTTTTTATTATTGAAAG

AGATATAGATTTAAGTGGAAAGTTAATAAAGAAAATTGCAGTTCGCCCTC

AAATGAATTATCTTTAAAATTTGTTTAATAATATTTGGATCAATAAGTTA

ACGGAGTGGAGATTTTTAAAAGATGATAGTTAAAATTTGCACATAACCGA

ACAAATTGTCTATTTAGGTATGTAATTTAGAGAGTGTCTCTTTTGAGGTT

TGATGTTTAGGGTTCAAAAATTGTCCGTTTTGGTGCCAGAAACGTGCCTA

CAACCACCATCCAATCCATTCTCAATCACAATCACCATCACTGACACCCA

ATCACTACAATAAGTCGTCATTGCCGCCATCCTTATAACAAAAGTAATTT

YTTTGCAGTCATAACTATATACTTTAATAAAAAAATGTAAATTTTTATCG

ACATTACTTAAGTATCATTAAATTTACTATCGCTAAAATCTTTAGGGAAA

TTTATAAAGAGTGTTAATTGTTATTAAAAAAATTATATTTATCGATAATT

AAATTATTGTTGCTAATTACTTACCATTGACGACCATTTTCAATGTAGTA

CATCCAATATTATCGCAATAAATCATTATCACCCGTCATTACTATTAATT

ACTACTTATATATCGTCAATCACCATCATCATTAACCATTGCTCTTCATC

CACCATAGTTATTGTCTTTCAAGCATTACCATCATTCATCATCATTATTA

ACTACTTATATATCATCAATAACGATTTATCATTCATCACAATTATTATT

TATCAACATCACCTATCGCTCTTGATCATTACTATTAATCATCATTAACC

TTTAACTGCAACTTACACTATTGTTCTTAATCGATATTCACAATCACCAT

AGTTAGTCATCACCATGAGTCCTAGCCACAAATTCAAAGCAAAACACCCT

TAAAGCCTGGTAGTGTGTGTGAATTAAAGACCAGCAGTCCAAAGAGAGAG

AGAGAGAGAGAAAATGTAGACTTTAAAGATATGTAGTAGGACCAGTCTGC

CATTAATATCTCCTTCTACCAACCTTCCTCTCCTCTTTCACTACCCTACA

TTTAACATTTTCCTATAACCACTGCTTTAGATAAGTCAAATTTAGCTCTT

TGTTTTGATCTCTGTTTCAAAAGAAAACACCTATTAAGCAGCCATCATCT

TTCTTATCTTTTCCAAAACCAAAACTACTGACTTTTCTTGAAAAAAGAAG

AGGTGGGGTGCTTTCTTTTCTTCAAAAACCTTCTCTTTGTTCTTGAAAAA

ACAGGACTCATTCATTTTTTTTTGTGTGTTTCTTTCAGAAGAAATAACAA

AGACCCTTTCTCTGTTTTCTTCATATTTCAGCTTTGAGCTACTTGGATCT

GTTTTTTTTTTTTGAATATACAAGTAGTTTGTGTGTTCTGGGGTCTACAG

AAGAAGGAGAAGCTAAAAGGGGTGATTTTGTTTTTTGTTTGTTGTTGTTC

TA

AtML1 promoter (AT4G21750.1 promoter sequence)

SEQ ID NO: 14

aagcttatcaaagaaaaaacaagaacaaaacgatgcatagtttctaaaat

gtgctaaaattcagaaactgaaacatgattcattgtctgaaactttgttt

caaattactgaaaataatcattcactggaccaaaacaaataaataaaata

aaatcgaatttctgaatttggaaattggtttttggtttttaattttaaac

aaaacaaaaacgaaatttgaaggcaataaatgagttagttggtaggcaga

agtcactcgttcccactagctattattattagaagaaacgtccccacaac

tccaaggcgtttcagttcctttaatttactgaattaccctcctcatatct

ataaaaaatcacctcttgtaccaatgccccatttacacatcctgtcgttt

atttctagactaagtggactacatgtcggttatttgattcgcaccatgcg

tatttggattatcgctaacacaccccttcaaacaatacgcttaactcgta

ttacaaaatttcaagtgatgaattatctatgtataagatatagataggaa

caactaagcatcgagaaatttgtatataaatcaactagacttatatatat

ttcgatacagaatttatacgtattatatcaaattaattagtaattgtttc

ctctacgtgagtttaattaacaatgataagctacattgagtgtatcagtt

ctaaaactttatagtatgctacaatcaatttttctaagtaacaacttcaa

gcaaggaatcacacacacacagtggtacataataaacttgattttaatat

catatgatcagcatcattaacggaataagttaagtaattcgtcatccata

ctactaagtcatattaaaatcataatcaaacttaaaagccgattagaaag

agagcaaatatatctaaaaattcacgaggaagacgacaaatgcaaggaaa

cacagctagtattattaaacttaatagatattggatgaatgactgcataa

tatatatcacattaaaagtggacataaatttgcatatgtgtaatgtacct

ctccacaattaatcgcggaccatttattttactattacaagtcaagtaac

tttatattgttgatccataattcttttcgaacataaaatcatatacttag

gccattttcaactgtcaaaactcgaatccgagaaccaaatttcaccattt

tccaaaaatgatgagtgtcgaccaaatggggtactactgtctaatcagga

acttgtgaacaaattttcaaccttttccaaataagacgagtgtcaaccaa

ctttttccaaccaagagatattgggttgctacacaaatacttaatagcca

ttgcatatttatgcatatgcaaatgcagggtcgtggcgtcagaaagaaac

ataggaccctcaacatatttaatattttgggagctatatttgactatttc

atattagaaaataataataaaaaagtgttggttttatatcaaattgtaat

ttacgaaaaacttatgcttttgcgcaatgatttttgtaaagtatctacta

tgtttagtgtttacattgattagtaggctgccgttttttttcttgtgtat

tatgtactatatatgaatatgaacatttgtaaaagtgaatcttgtcattt

tcttgttgaaaacatatatagtatgtgcaaacaaagcataggttaatcca

ataccacacaaataacacgtcaggtaaatccaataataaatcgtatgtgc

atgtatgtgtattcatgtatgttacatgaatgtctgaatcagtcagtgta

cgtatatgatgtaggtgatgtaaatcttaatgtatgagctgtttcttgga

ccatggtccacaatggatattgctccccaactacattagtcaatcgactg

gccaatttttaattaagataattaatccaaactaccattaaatataactt

tgaccttttttctattcatttttagatattattggaacttacgtagttta

catgcatctcatccctttcttttgctccttgaaagtgggtccaatcacaa

aaaatgatcttatattttgtattttgtattttaaaaactcataattatat

aggttcaaaaatttaattaacatcagtgtatactataattactactctag

ccaacaagataaattcattttgacatcagccaaaagataaaaatttggtt

aaaaactattggattagcttttagtatttaatattttatgtactgattaa

atacgaatttagaaatctaggatataagtgagggtgtataataagggagg

ggtggaccattaatagcgatgtgcaattaaaaattatgattaagaatcta

ggaaatttgtagattgcttagttatttttatggcgatcgtcgtgtcaatg

tcatggattttgaaactttaaattaatctcttaaattagcacctaccttt

gaattttatagaatctttttattttatatgtttaattttatagaatctaa

ctagcttattttgagattaaattgtttagttacttttataacagtataaa

tgtataatgaggacctaagaatgtagtcctgtaatgttcttgctattcta

cttaatctcatcaccaatcaaccatcaaaagaagctagtactaataaaac

ctgcaggtattcgaataataattaagctcaaacactatactaatttatgg

aggattatatattcaatgaattaggaacctcatgatggacattattgact

gatataatgtgtatactaattgtgagtatttaaaaaccatacaaagcatt

tatatgtccacatatattggacacacatgcaatcaatgttcaatatgctc

cacacacagaaataaaaatactctttctgatcatatgatacatcatacat

atactaaaaaaatctaaaatgaactataaccacaagcatatataataaca

atgaaatggtaatgtttcttcatttttatttgttcaaattcttattcggt

tgttttttcttaccctacgagaatccgtgaggtcaaagggaaacagtgat

tttttttttgtattttgttttttaaattgatgaactgtaaaactctctct

ctagaaaaatatataagtagtagtatgaattttctctcactaaaagcatt

aatggacctttcgataatcataaatgcaatgcaccctctctatgcatttc

gcaataactccttttccttctgccacatcctcttcctcacctctttctct

tcttccctttctcctaagttcctcctccaccaaattctccatttatttcg

ttaactatcctccatttgttttcttctgaagagtgatatattctaccttt

ctctggttaaagaaactccctgaatccaccggttatgtcttgaccggcta

taagcctataaactgatgccctaagacacctttttaggtttctcaataat

tctccgcatctatcttttcttctccacaagtaagagaaccagaaaaccag

agaagaagccgagctagctagggtttcattgtgtgcacaaaagtaagatc

tctctctctaaccaatacttgtgtaatttgtctttgtttctttgagcaaa

tattgcatgtttgttcatattagccggatccgttttatattttttcatga

tctacattttatctttattttgtttgtaaattaatgagtttttttttttt

ttttctgtttttgtcacgatctaaaaaacaagcgttacaaagaagaagaa

aaacctttttggagttagaagtgtaaaaggggtttcagtttgacgaattt

tccttagtagttgtgtaaaaaaaggccattgacttaatgtcaactctata

tatctacacatttttttattaattagtttttgtttttttcccacttcatt

tacctttagtcaatgaatttttactgaaaacgttttttcaaggtcaattt

cactgagttaaaaaaaaaagttttatttttaaccaaaaattacgtttttt

cctaggcttcggtaacctgtgaattcctctatctcactagcttttatgta

gaagagagagaaggcaacattaaattcgatctaaaacttcaagaaaccaa

aacaacacttcaaaaaaaaaaagagatctgttctatagagttttaatctt

ttctttcgactcgagtttggctcaacaaagtttatatcgatttggcactc

taaaatgtaagtagaaccaaatgaatcttgtattttatgtacgttaataa

aaaattagggtttcctagacgacaatctcgtcatccgtttcttctttgtc

tacctctgcgttttcttgtagatccgatgatgtgctcagtcttgtgactt

tcaagattgattttatcgttattgtttgaagatatgtggtttgattattt

tctcaacacattgtgtccttttagcgctttacttcagtttctctctaatt

ttcataatattattattgaacattatgcttaattattcatccgaatattc

gtgtcccattttttaaattgaatttcaggataacttgtattttatatgca

acgaggttatgtcacgtagtgggtgcatttatattcataccctttttgat

aagatgaatgcatatgcttatataagcgtataggtataaataaccatcaa

aaatagagaaaaagaccaatattttgcttttcggttacttatgaaatgtg

aaaaagaccatataaatatatctattaaagggaagtatagtttcataaaa

tcttgaggattacattccataaaccaagattaccttccgtttttgctttg

atcctcttcttatcaaatatataaacatgaccatttgatctttcattttg

gatagtgggatatacaggcagaagaaaatcgagataaatcaactaaatga

tttggataatcatcttgaagatttgaaggaaaatccaagagcttcaaaaa

ctccaaaaattgataggcatccatcatcatc

Tomato ML1 Solyc10g005330.2.1 promoter sequence

SEQ ID NO: 15

ATTTTGACACACGAAAAAGTAGTACGAATATTGAACTCATGATAACTTTA

TCAGTTACTTCAAGACTCTCATTTTAACACAAGAAATATATTTTACAAAG

AAAAAGGGAACATATTTTACAAAGCTTTATTTTGTATTTTCATTAATAAT

TATTTTCAAGGCTTGAACTCATAATAATTTTATCAGTTTTTTCAAGATTT

TCATTTTAACACACGAAAAAGTAATATGAATATTGAACTCATGATAATTT

TATCAGTTACTTTAAGACACTTATTTTGACACACGAAAAAAGTAATACGA

ATATCAAACACCGAATACGAAAGAAAAAAAGAAATGAAAGCATTATAGTA

GTTGCCAACCGCCCCTTCCTCCTCCTCTCTCTCTTCAACAACAACATTAA

CACCTCTATAGCAAGTCATAAATGCTATTTCATCCTCTCTATACCCTTTG

CATTAACTCCTTTGCTTCCACAATCTCTTCTCCCACCTCTTCACCTTCCC

CTTTTCACACTTTCTTTCTCTTTCTTTTTTTCTTTCATCCTTAGCCTCAA

AACTATTCTTCTTAAATTCTAGTCACAAGAAAAGTGTTCAATTTCAACCT

AGCTTCACTAAAATATATACATGTTCATTCTCCAAAAAGTACTTCTTGTC

AAAACTTAGATTTAACCATTTTCTCAAAAACCCTAATAACATCAACAACA

AAAAAGAAGAAGAAGGTGTGTTCTTGCTTTTGTCACAAGGCTTCTCTACA

ACTCATGTAAGTCAAACATATACTATCATCTTCTTGAATTTGTTGAATTC

TTTTTTACTAGCTTATAAGTGTACTATATTGTTCGAATTTTCTAAAAATA

TTATCCGATCTTTTAGGAACAATATATATTTTTAAAGATCCAATACAAAT

ATAACATTAGTTTCACAGAGTCCGAGCAAAATAGATAAATAGTTGTAAAT

TCACTTGTATTTGACTTACCTTTTCATTTTTCCGTTATATTTTGCAGAAA

TAGAAATGCCAGTGAAGTTGGACTCTGCCTAGATACTCGTGGACGTTATA

TCATATACAAGTACCTAAGTTTTGAAAAAAAAATTAACAGTGAAAAAATA

TTAGTTTTTGAGTTCACACTATGTCAACTCTATCTTTGTTTTTTGCTAAA

TTTTTCTAGTTTCAAGTCTTTTTTTTTGTTTGACTTGTAAAACTTTTTTC

TTTTACATTATTTTTATCCCCTTAGAGATTCTATAAAAACTCTATGCCCT

AACAAAATTTCTTACTAAACAAACAGATATATCAACATATATAGAAACAA

AGGAGAGAGAAATTGTTTCTATGGCTTGAAGGGCTTATGTCATATATGTT

ATATATGGTGTAAACTCCATCACTATGAAGTTTCTGGCAAGCGGTGAATT

TCATCGTAGGTAATAGGAGGTAACAGGTATTCAGTAAGTCGTAATTTTAA

CATCGAATGTTTATACGAATCATTTTTATACAATAGATGTGAGTTCAATT

CTCTCTGTTATTCTTTGTCTAGAGAGTAGTAAAAAAAAAGATAAAAAGAT

CCGTTCGTTCTCATCTCTCTCCAATTGTTGAGATCTGTTTGGATCTTGAG

TTATTAGGTACTAATAAAGACCTTTCAAGTTGAATTATTCAATTTTATTA

TTATTTTTGCACTTTTGGACATCATTTTATGTTTTTAATCATGTCATAAT

TATATATGCATGTAGATGAAATAAATCAAAAAGTAGATTTTTATTCAAGA

ATCAAATAATTTCTTTATGTTTTTTTTCTTAAATTTATCTTCTTTTGCTT

TTTTTAGGGGCAGATTAAAA

Example 3: Sequences of sRNA Targets and Mutations for Making sRNA-Resistant Targets

Polynucleotide sequences for sRNA targets (MPK1, MPK2, WAK, PRXIIF, MAPKKK4, Sl F-box (Solyc03g061650.1.1), Autophagy-related protein 2 (Solyc01g108160.2.1), Sl Vacuolar protein-sorting (Solyc09g014790.2.1), Sl Pentatricopeptide (Solyc03g112190.2.1), and TOM34 (Solyc07g066530.2.1)) are provided. Underlined sequences represent target sequences for sRNAs. Alignments of sRNAs to wild-type target sequences and mutated target sequences (target site synonymous mutations) are also provided.

SEQ ID NO: 4—Bc-siR3.2 Target At-MPK1

GTCAACTGTCCGAGCGTTGGCCAAATCTCTCTCACTTCCACAGGTTTCTCTCTCCGGCCAAAT

embedded image

GGTTGATCCTCCTAATGGGATAAGGAATGAAGGGAAGCATTACTTCTCAATGTGGCAAACTC

TGTTCGAGATCGACACTAAGTACATGCCTATCAAGCCTATTGGTCGTGGAGCTTACGGTGTT

embedded image

GACATGAGAATGTCATTGCTTTGAAAGATGTCATGATGCCAATTCATAAGATGAGCTTCAAG

GATGTTTATCTTGTTTATGAGCTCATGGACACTGATCTCCACCAGATTATCAAGTCTTCTCAA

GTTCTTAGTAACGATCATTGCCAATACTTCTTGTTCCAGTTGCTTCGAGGGCTCAAGTATATT

embedded image

CGATTTAAAGATATGCGATTTTGGACTAGCGCGTGCGAGCAACACCAAGGGTCAGTTCATGA

CTGAATATGTTGTGACTCGTTGGTACCGAGCCCCAGAGCTTCTCCTCTGTTGTGACAACTATG

GAACATCCATTGATGTTTGGTCTGTTGGTTGCATTTTCGCCGAGCTTCTTGGTAGGAAACCGA

embedded image

AGAGAAGAAGATCTTGAGTTCATAGATAACCCGAAAGCTAAAAGATACATTAGATCACTTC

CGTACTCACCTGGGATGTCTTTATCCAGACTTTACCCGGGCGCTCATGTTTTGGCCATCGACC

TTCTGCAGAAAATGCTTGTITTTGATCCGTCAAAGAGGATTAGTGTCTCTGAAGCACTCCAG

CATCCATACATGGCGCCTCTATATGACCCGAATGCAAACCCTCCTGCTCAAGTTCCTATCGAT

CTCGATGTAGATGAGGATTTGAGAGAGGAGATGATAAGAGAAATGATGTGGAATGAGATGC

embedded image

GGGTAATTTACAGAAAACTTCTTCTTCTTATGTCTGATTGTCATCATAGACTCATAGTGTATA

TAGTCTTGAAAAATAAGATGAAGACTAACTTATAGTTTAAGCGAATAGTGATGCCATGGAA

GCTCTGTTTTATTTAATTACAAGCTTGATGTGTGTCTGTAACATATGTACATAGAGAGAGCTG

TTTTTTTTTTTTAATTACAAGTTTGATGTGTGTCTGTAACATATGTACATAGAAAGAGCTGTG

TTTTTTTTTTAATTACAAGCTTGATGTGTGTCTGTAACATATGTTCATAGAGAGAGCTGTGTT

TCTGTTTCTCTGTTTGTTTGTTGCGTTCTTGCAGAACTTTTAACCCTCTCATGCAATCCAAGCC

TTTTGATG

Alignments of sRNA sequence Bc-siR3.2 to wild-type (WT) At-MPK1 target and mutated

(MU) At-MPK1 target

miRNA:
3′UGGAUGUUCUAGGUGUUACAU5′ (SEQ ID NO: 24)

alignment:
|:| | |||||:||||||||

WT Target:
5′ATCAAGAAGATTCACAATGTT3′ SEQ ID NO: 59)

miRNA:
3′UGGAUGUUCUAGGUGUUACAU5′ (SEQ ID NO: 24)

alignment:
|: | || || ||:|| ||

MU Target:
5′ATAAAGAAAATACATAACGTT3′ (SEQ ID NO: 505)

SEQ ID NO: 5—Bc-siR3.2 Target At-MPK2

ATGGCGACTCCTGTTGATCCACCTAATGGAATTAGGAATCAAGGGAAGCATTACTTCTCAAT

GTGGCAAACACTTTTCGAGATCGATACCAAATACGTGCCTATCAAACCGATAGGCCGAGGC

embedded image

TACGTCATCTTCGACATGAGAATGTGGTTGCTCTTAAAGATGTAATGATGGCTAATCATAAGA

GAAGCTTTAAGGATGTTTATCTTGTTTATGAGCTTATGGATACTGATCTTCATCAGATTATTA

AGTCTTCTCAAGTTCTAAGTAATGACCATTGCCAATACTTCTTGTTCCAGTTGCTTCGAGGGC

TCAAGTATATTCATTCAGCAAACATTCTCCATCGGGATCTGAAACCCGGTAACCTCCTTGTG

AATGCAAACTGCGACTTAAAGATATGTGACTTTGGGCTAGCGAGGACGAGCAACACCAAAG

GTCAGTTCATGACTGAATATGTTGTGACTAGATGGTACCGAGCACCAGAGCTACTCCTCTGT

TGTGACAACTATGGAACCTCCATTGATGTCTGGTCAGTCGGTTGCATATTCGCCGAGCTTCTT

GGAAGAAAACCAGTATTCCCGGGAACAGAATGTCTAAACCAGATTAAACTCATCATTAACA

TTTTGGGTAGCCAGAGAGAGGAAGATCTCGAGTTTATAGATAACCCAAAAGCCAAAAGATA

CATAGAATCACTCCCTTACTCACCAGGGATATCATTCTCTCGTCTTTACCCGGGTGCAAATGT

TTTAGCCATTGATCTGCTTCAGAAAATGCTCGTTCTTGACCCTTCGAAAAGGATTAGTGTCAC

GGAAGCGCTTCAACATCCTTACATGGCGCCTTTATATGACCCGAGTGCAAATCCTCCTGCTC

AAGTTCCTATTGATCTCGATGTAGATGAAGACGAGGATTTGGGAGCAGAGATGATAAGAGA

ATTAATGTGGAAGGAAATGATTCATTATCATCCAGAAGCTGCTACCATAAACAACAATGAG

GTCTCTGAGTTTTGA

Alignments of sRNA sequence Bc-siR3.2 to wild-type (WT) At-MPK2 target and mutated

(MU) At-MPK2 target

miRNA:
3′UGGAUGUUCUAGGUGUUACAU5′ (SEQ ID NO: 24)

alignment:
|:| | ||||||||||||||:

Target:
5′ATCAAGAAGATCCACAATGTG3′ (SEQ ID NO: 60)

MUTANT

miRNA:
3′UGGAUGUUCUAGGUGUUACAU5′ (SEQ ID NO: 24)

alignment:
|: | || || ||:|| ||:

MU Target:
5′ATAAAGAAAATACATAACGTT3′ (SEQ ID NO: 505)

SEQ ID NO: 6—Bc-siR5 Target-WAK

ATGAAAATCTTGATCTTGATTCTATCCTTTGTGACACTCTTTGAGATTTGCGTTGTGGACGC

ATGTCGATCATACTGTGGAAACATAACCGTTGATTATCCGTTTGGGATCCGAAACGGATGT

GGGCATCCAGGGTATAGAGATCTATTGTTTTGTATGAACGATGTGTTGATGTTTCACATAAG

TTCAGGTTCTTATAGAGTTTTGGACATCGATTACGCATATCAGTCCATAACACTGCATGATC

CTCACATGTCGAACTGCGAAACCATCGTGCTCGGTGGCAAAGGCAATGGCTTTGAAGCTGA

GGATTGGAGAACTCCATATTTCAATCCTACCTCAGATAATGTCTTTATGTTGATCGGATGTT

CTCCTAAATCTCCTATATTTCAAGGCTTCCCGGAAAAGAAAGTGCCGTGCCGCAACATCTCT

GGAATGAGCTGCGAAGAATACATGTCATGTCCAGCTTGGGACATGGTCGGATACAGACAAC

embedded image

AGCGATTAATCTAAGTAAGTTGGAGTGTGAAGGATACAGTAGTGCGTATAATCTAGCACCC

TTGAAACTTAGAGGACCCTCTGATTGGGCTTATGGGATACGTGTTAAGTATGAACTCCAAG

GAAGTGATGCGTTTTGTCGTGCGTGTGTTGCAACTTCTGGGACTTGTGGCTATGAACCTGCT

GATGGTGGAGGGCTTAGACATGTTTGCATGTGTGACAACCATAATTCCACTACAAACTGTG

ATTCAGTTATATCACCAACCGGTGCATCATCAAGTGTTCGACCAAAAGCTATCGGATCACT

GATCATCTACTTCATAGCTATGAACATAGGCTTTCAGAGAAGACAGCGATGA

Alignments of sRNA sequence Bc-siR5 to wild-type WAK target (WAK) and mutated

WAK (WAK-m) target

WAK
5′GGGUAUACAUUCCGGGUCAGG3′ (SEQ ID NO: 21)

::||||||||||||:||||::

Bc-siR5
3′UUCAUAUGUAAGGCUCAGUUU5′ (SEQ ID NO: 22)

:| || || || |: || ::

WAK-m
5′UGGAAUUCACUCGGGCUCUGG3′ (SEQ ID NO: 23)

SEQ ID NO: 7—Bc-siR3.1 Target AtPRXIIF

ATGGCGATGTCAATTCTAAAGCTAAGAAATTTATCGGCACTAAGATCGGCGGCAAATAGTG

CCCGGATCGGAGTTTCATCGAGGGGTTTCTCAAAGCTCGCGGAAGGCACTGACATAACCTC

GGCGGCGCCTGGCGTTTCTCTCCAGAAAGCTCGCAGCTGGGACGAAGGTGTTTCCTCCAAA

TTCTCCACCACGCCATTGTCAGATATCTTCAAGGGGAAGAAAGTCGTCATCTTTGGTCTTCC

embedded image

AAGTTTAAAGCCAAAGGCATTGATTCTGTCATCTGTGTCTCTGTTAATGATCCCTTTGCTAT

CAATGGTTGGGCAGAGAAGCTTGGTGCCAAAGATGCAATTGAGTTTTATGGAGATTTTGAT

GGGAAATTTCACAAAAGCTTGGGGCTAGACAAGGATCTCTCTGCTGCATTGCTCGGGCCAC

GGTCTGAGAGATGGTCGGCTTATGTAGAAGACGGGAAGGTTAAGGCGGTGAATGTGGAAG

AAGCACCGTCTGACTTCAAGGTTACAGGGGCAGAAGTCATCTTAGGACAGATCTAA

Alignments of sRNA sequence Bc-siR3.1 to wild-type AtPRXIIF target and mutated

AtPRXIIF (MU) target

embedded image

SEQ ID NO: 8—B-siR3.2 target in tomato: MAPKKK4 Solyc08g081210.2.1

ATGCGTTCATGGTGGGGGAAGTCTTCATCTAAGGATGTAAGGAGGAAATCCACTAAGGAGA

GTTTCATTGACATAATAAATCGGAAACTGAAGATTTTCACCACGGAAAAATCAAGTGGTAAA

TCTGGATCATCTCGAAGACGACGTAAAGATACAAATTCAGTGAAGGGTTCTCAATCAAGGGT

TTCAAGGTCACCATCACCATCTACTGGATCCATAATATTAGTGACCGGTGAAGTCTCCGAGC

CATCATTGACTTTGCCTCTTCCCATGCCCAGGCATCTTCCACATGGACCAACTGCTGCAGGAG

TTGACAGGGACTTACCAACTGCTTCTGTTTCTTGTGACAGCTCCAGTGACAGTGATGATCTTA

CTGACTCACGATTTCTAAGTCCCCAAACATCTGATTATGAAAACGGGAGCAGAACTGCCTTG

AATAGTCCTTCCAGTTTGAAGCAGAAGGTTCAGTCCCCTATTGCATCCAATGCAAGCTCAGG

AGAGATGCTGAAGTCAGCTACTCTTTTGTCAGACAATCAGGCGATCCCTACATCTCCTAGAC

AGAGGCTTTTAAGATCTCATGTACCACCAGGCTTACAGATTCCTCATCATGGCGCTTCCTACA

GTGCTCCTGACAGCTCGATGTCAAGTCCTTCAAGAAGTCCCATGAGGGTATTTGGGCATGAA

ACGGTCATGAACCCTGGTTTCTGGCTAGGGAAGCCACATGGAGAGATAACCTTCTTAGGATC

AGGGCACTGCTCCAGTCCAGGTTCTGGCCAAAACTCTGGGCACAATTCAATTGGAGGTGATA

TGTTAGCGCAGCCCTTTTGGCCACACAGCAGGTGTAGTCCTGAGTGTTCACCTGTACCTAGC

CCTAGAATGACTAGTCCTGGTCCTGGCTCTAGGATACATAGTGGTGCTGTAACTCCCTTGCAT

CCTCGAGCTGGAGGAACGTTGGCTGAGTCTTCCACAGCTTCACTTGATAATGGAAAACAACA

AAGTCATCGTCTGCCTCTTCCTCCCATATCAATCCCTCATTCTTCTACTTTTTCTTTGTCATGT

TCAATGACTCCTGCAATTCCACGAAGTCCTGGTAGAACAGGTAATCCTCCAAGCCCTGGGCC

ACGTTGGAAGAAAGGACGTCTGATTGGTAGTGGCACATTTGGACATGTGTACCTTGGTTTTA

ACAGTGAAAGCGGTGAAATGTGTGCAATGAAGGAAGTAACACTTTTTTCAGACGACCCAAAG

TCAAGAGAAAGTGCACAGCAGCTTGGACAAGAAATATCTCTGCTAAGTCGGTTACGCCATCCA

AATATTGTGCAATATTATGGCTCTGAAACGGTAGATGACAAGCTATACATATACCTTGAGTA

TGTTTCAGGTGGTTCGATCTATAAAATTCTTCAAGAATACGGTCAGTTGGGTGAGCTAGCAA

TTCAAAGTTACACTCAACAAATTCTGTCTGGACTTGCATATTTGCATGCTAAAAACACAGTG

CACAGAGATATTAAAGGAGCAAATATACTGGTTGACCCAAATGGCCGCGTTAAATTGGCAG

ACTTTGGGATGGCAAAACATATAACTGGTCACTACTGTCCTTTGTCTTTCAAGGGAAGTCCTT

ACTGGATGGCACCTGAGGTTATTAAAAATTCAAATGGTTGCAATCTTGCGGTAGATATATGG

AGCCTTGGATGCACGGTTTTGGAGATGGCAACAACAAAACCACCTTGGAGTCAGTATGAAG

GGGTCGCTGCTATTTTTAAGATTGGAAACAGCAAGGAAGTTCCAGCAATTCCCTATCACCTG

TCAGATAAGGGCAAGGATTTTGTGCGGCAATGTCTACAACGCAATCCACTCCACCGTCCAAC

AGCTTCTCAGCTCTTGAAACATCCCTTTGTCAAAAGTACTGCTCCAATGGAAAGATTCATTG

embedded image

CCTAGAAGTTCAATTTTTTTTCCTGGATTTAGCGACGTACCTGTTCCAAGATCTTGCCCAGTT

TCTCCAGTTGGGATAGAGAGCCCTGTTTACCATTCACAATCACCTAAACATATGAGTGGAAG

ATTGTCCCCCTCTACCATATCAAGCCCCCGTGCTGTATCTGGTTCATCAACACCTCTTAGCGG

TGGTGGTGGTGCTGTTCCACTATCTAACCCAATTATGCCTACAACTTCTTCATCAGAAGACAT

GGGAACATCACCAAAGGCCCAAAGTTGTTTTTACCCTGATGCTTACACTAGTCACGGTCTGA

AGTCTGACATGTCTCGAGAAGCACCTCCATATGGCAATGGTTTTTTTGGAGAAAATTTTGGG

GGCCATGCTCAAAGTGGTGTTAATGGACAACCATATCAGGGACAGTCAGTATTAGCTAATAG

GGTTGCTCAGCAGCTTTTAAGGGACCAAGTAAAATTGAGCCCATCGTTTGACCTGAACCCAG

GCTCTCCAGTTTTTAGTTGGGATAATGGGGTCTAA

Alignments of sRNA sequence Bc-siR3.2 to wild-type MAPKKK4 target and mutated

MAPKKK4 (MU) target

embedded image

SEQ ID NO: 9—Bc-siR3.2 Target in tomato: Sl F-box (Solyc03g061650.1.1)

embedded image

AAATGATATAGACAAAATCTCTAATTTGCCCATGGATATCCTTGATAAAATATTCAAGGACA

TGTCATTTCTAGAATTGGTAAAAACGTGCGTCTTGTCGAAGAAATGGGTACATTTCTGGGCT

ATGCATCCAATTCTTGTTCTAGATGGAGATTTTTTTAGAAAGATAAGTGGTAATATAAAATT

GATTGAAGATGGTTTTAGTGGCCTAATTGACAAAATTCTCTTTCAACATGTTGGATCAATAGT

CAAGTTTTCCCTTGATTTGTCAACTATCTATTATAATAATAATAGGGACCTTGGTCATTGGTT

GATTTGCGTAACAAGTAAGTGTGTCAAAGAACTTACCCTAAAAAATCACAAACACAAACAC

TATAATTTACCTTTTTGCGTATTTGATTGCCCAACTCTCACATATTTAGACGTAACCAATTTC

ATAGTTAAATTACCATCTTCCAAAACATTATTCCCAAATCTCCTTGAATTAACCTTGAAGTCC

ATCAAATTTCGCCCAACCAATGCAAATTATGTCTTGAATGCCCCTTTTCTTACCTCCTTAACA

TTAATTTCTTGCAATGGTGTTCATTGGCTCACCATATTTGCTCCCAGGATTAAGTTCTTGACA

ATTAATGATAGCCATGACATTTGCGCAAATTTTTTTGTAAATTTCTCAAATGTTAGGGAGTTG

TTATTCCGTGAAGAATCTTATTATGAAGAAGGGAGGTTCATCACATGGTCACATCTTCTTTCT

TTGTGCCCTAACCTAACAAGGCTTGTTTTGAATAATTCTTGCATTCAGGTTTTCAATACCTTG

embedded image

AATTTGAGAAGCTTGAATTTGTGGAACTAAGAAAGTTTGAGGGGACACACTTTGAGCTCATT

TTCTTAAAGAAAATATTGGGATATTCTCCTTCGCTTTCAAGGATTATTGTTGAACCTTCTGAT

GATATTGATGTTGCAGAGATATTGGATTTGTATGAAGAACTAATGATGTTTTTAAAAGCATC

embedded image

Alignments of sRNA sequence Bc-siR3.2 to wild-type S1F-box target and mutated S1F-box

(MU) target

embedded image

SEQ ID NO: 10—Bc-sRNA 3.1 target Autophagy-related protein 2 (Solyc01g108160.2.1)

ATGTGGAACTTCGCGAGGTCTGCGGAGAAGTTGTTCTCGCGCTGGGCAATCAAGAGGTTT

TGCAAGTTCTGGTTGAAGAAGAAATTGGGGAAATTTATACTTGGTGATATTGATCTCGAT

CAACTCGATGTGCAAGCCAGGGCCGGTATCATTCAGCTCTCTGATCTTGCCCTCAATGTT

GATTATCTCAATCAAAAGTTTGGTTCCGCAGCAGCCGTATATGTTCAAGAAGGATCAATC

GGCTCTCTGCTTATGAAAATGCCTTGGCAAGGGGATGGCTTTCGGATAGAGGTGGATGAA

CTTGAGCTTGTGCTTGCTCCTGAGGCAACCTTTTCTCCTAGCACATTTGGAAATTGTCTT

TCAACTCAAGATGGTGCTGCTTCGGTGAACCAAGAATCAGGAAACCGCAAGGATGTTGCT

GTCGATGATTGTGGGGCTAAAACAACTGCTTTTGATGTTCATGAAGGGGTCAAGACCATT

GCTAAAATGGTTAAATGGTTTCTTACTAGGTTGAATGTAGAAGTTAGAAAATTGATCATA

GTATTTGATCCCTGTTTAGGTGAGGAAAAACAGAGAGGGCTTTGCAGAACCTTAGTATTA

AGAGTAAGTGAAGTAGCCTGTGGGACATGCATCTCGGAAGGGGATTCTCTGGATACTGAA

GCAGCGGATGCTAACCTTTTGGGGTTGACTCAAATGACAAATTTTATCAAATTTAGTGGA

GCAGTTCTTGAATTCCTTCAAATTGATGAGGTTGTTGATAAGACACCAAATCCATGTGCT

TCAGGAACAGCTACAGGTGAGTGGTCAAGAAACTATTCACCAAATGTCACAACTCCTATA

ATAACCGGGGAAAGAGGCGGACTTTCTGGGAACCTAAAATTGACTATACCTTGGAGAAAT

GGTTCCTTAGATATCCGCGAAGTGGAGGTAGATGCTTCTATTGATCCTCTGGTAATCAAA

CTTCAACCTAGTAGCATCAGATGCCTAATACATTTGTGGGGAATTTTGAAAGATACGGGT

CAGAAGAAGGATACAGAATTTCCATTCTGTAATTCAGTAATGACTTGTGATTCAACAAAG

GCAGATACTTCTCTGCTCAGTATGGATGAGGTGCTTCCAGATTCTAAAGCAAATTCTGCT

GAATGTGCATTTGAGAGTGAACCTGTGAGGGAAGCTTTGCTGTCTGAGTCCCGTCTTATA

TCGAACTGGGTGAGTAGAAGCCGGAAAGTCAATGACGAAGAGGAACCAGACTTTGGGGAA

AGCGTGCACCAGTTTTTTGAGTGCTTTGATGGTCTGAGAAACTCGCAGTCAGCTCTAGGA

AACAGTGGGATGTGGAATTGGACTTGTTCTGTTTTTAGTGCGATAACTGCTGCTTCTAAT

CTTGCTTCTGGGTCGTTGCTTGTTCCTTCTGATCAGCAGCATCTTGAAACCAATATTAGG

GCTACAGTTGCCAAAGTATCTCTTCTTTTTTCTTTTATTGACGAGGAAGAGAGACATTGC

TGCACTGTGGATGCTGATAAAGGGAATGCTGGTTTTTATGTTCATTATATAAGTGCAAGT

TTTCAAGATTTGCTTCTGGTATTGCAGGTACAGCGCCAGGAAGTGAATTTTGAAGCAACA

GTTCAACATGTGGCACTTACTGATCACTTCTCAAGAGAAGATGACACTGTTGATTTCAAA

TGGTGTACATATAATAACATCAAAAAAATTCAAGACGCAATTCAAACTGCCATCCCACCT

CTTGATTGGTCCACCAAGAATGTTGATCTGGATAATCAGAGTGCATCTGCTGCTCCTTAT

CCATTAAGGATGAATTTTACTGATGGGTTCCCTCATCCAAGGAAGAAAATAAGTCTTTTT

GCTGACGATGGAGTGCAGGTAGAATTGCTTAAGACTTTTGGTGCTAGCCTCTGTCAAGCA

ACCATAAGTTCTTCAGGAAACTCATTTGTTGGGCCAACATCTTTTTCATTGAAGTTTCCA

CCATTTGTTTTCTGGGTGAACTTTAATTTGTTAACTAAAATCTCAGAATTTTTCAAGAAA

ATTGAGGATCCTATTGGAACATCTAGCACTCTGGCTCATGAGGATAAGTGTGTAGCTTCA

TCCAAAGGGAATGGAAGGACTAGCCCTTGCTCTGATACTAGAAGAAGTTCAGAACAAGAA

AGTTTCAGGGGCACTGTATCTCTTCCAACTGCCAGGATTATATTGGCTTTTCCTTGTGGA

AAAGGTGAAGATTTTAGGAGCTATTACTGTTGGCAACAGTTTATTTCTCTTGATGTTTCT

TCACCATCAGCTCCTGTGGACAAAGCAAGTCATGCAACTAAAAAATGTTCTGCTACTAGT

TCTAAAAGTTGGAATTCCGTGGCTAAATTGTGCTCTTTGTCCTTGAATTTTGGGAAGCTT

GATGTCAACTTAATCACACCATTGTCTGGAGAGAATGTTGAAATTACCTATGATAGTGTT

CTAAAGTATAGACTTTCAGCTCAGAAATTAATGACCACATCAAATGGAAGAGGGCCTTCT

GTTGTTACCTTTTCTTGGCAGGACTGTGCCAGTACTGGTCCTTGGATAATGAAGAGAGCC

AGACAGCTTGCTTGTTCAGAGAATGCAAGGTGCTTAGAGAAGTTCAGAGGAAAAGGATAT

GACTTTTCGTCTGTAACCACTGTCAAGGATTCTGGGGACATTGATAACATTCGACAAGAA

ATGATTATAAGCTCTGAGTTCTGCATTCATGCACATTTATCTCCCGTTATAATTTCTTTA

AGCAAATCAGAATTTCTTAAATTAAATGATATTGTGAGTCAGGTGATTGATAGGTTATCA

GGACTGGACTTAAATCTTGTTGATACTGAAAAAGTGACTGCTGCCTCTCAGTCATCAGTT

CTTGTTGAATGTGATTCTGTAACCATATCAATTAATGAGGAAGCCATGGAGAAGAATAAT

AAGGGTTCACTACAGAATGAAATTACTGGTTCTTGGCATAGCTTTACTCTGGAACTTCAG

AACTTTGGCCTATTATCTGTTTCAGATCTTGGAGGAACAAATGGTTCTAGCTTTCTCTGG

GTAACCCATGGTGAGGGCAACTTGTGGGGTTCAGTTACAGGGGTCCCGAGTGAAAAGTTT

CTCCTCATCTCCATCAATGACTCTTCCAGTAGCCGTGGTGACGGAGAAGGTTCAAATGTA

embedded image

GTGTCCATCACTGTCCGGTGCGGCACTGTTGTTGCAGTTGGTGGACGCTTGGATTGGTTT

GACACAATTTTCTCATTTTTCGCTTCACCCTCCCCTGAAGCTACACAAGAATGTGATAGT

AATGTGCAGAAAGAGGGTGAAACTAGTGTTCCTTTTGAATCTTCTTTTATCCTTAGCTTG

ATAGACATTGCCTTGAGTTACGAGCCATACTTAAATAAATTGACGATGCATGGATGCGCT

GATTCTCAGTCAAGTTCTCCCAATTGTGAGGAAGCAATAGATGAGCAACATGTAGCATGT

CTGTTGGCTGCATCTTCCTTGAGGTTTTCCAGTACAACCTTTGCTGATTCTGTTATCAAG

GATTACAAAATTACTGCGCAGGATCTGGGTCTGCTTCTTTCTGCAGTGCGTGCACCGAAC

TGTGCTGGCAGTGTCTACAGTGTGGAGCATCTTCGCAAGACGGGATATGTTAAAGTTGCT

CAAGGGTCAGATGTTGAAGCTCTTTTAAGAATCAGTTCTGGAAGTGGTGCTCTTTGGGAA

ATTGATTGTTCAGAGTCACAGATTGTTCTGAACACTTGCCATGATACAGCTAGTGGATTG

ACACGTTTAGCTGCTCAAATGCAACAGCTTTTTGCCCCTGACCTGGAAGAATCTGTGGTT

CACTTGCAGACAAGGTGGAATAATGTTCAGCATGCACGTGAGGGCAAAGAATTCTGCACT

TTTGACGTGGCTGTAGCATCAACTTCAGATATGCAGCCTATGACTGGTGATGTAAGTAGC

AAATGCGGTAATATCAACTTGATGGATGAAATCTGTGAAGATGCATTTCAATTGAACCAC

GAGGAGGATGACCAAGCTGATCATCTTGAATCACCCATTTACCTGTCACCTAATAATAGT

TTCATTGGCGAGACATTTTACTACAGTAATGAAGACTCTCCAAGGTTTTTGAATAGCTCG

CCTCTCACTTGCTCAGTCCCAGTAGGTGGACAAGAAACTAGTGAGACTCCATTATCACCT

GAACAGCCACCTCAGTTTATCGAAGAATATTTCTTGTCTGACCTATGTCCTCTGTCTGAA

CTAGCATTGACAGATCAGTCATCGAAGGATATTATTAGATACGCGCCCAGTCCTCTAAGG

AGTGGTGATGATTTTAGGGGAAGTACTGGATGGTATGGGGGCAACTGTTTAAGAATTTTA

GAGAATCATGTTTCAGAAGTCGACAGAAAAGCTGGTTCGGAGGAGTTGACAGAGTCTGAG

GCTTCTAGCATTCTCAGTGAACCTGATGAAAATAAAAATGTTAAGGGTCGCATAGTTCTT

AATAACATGAATATCATCTGGAGATTGTATGCGGGATCTGATTGGCAAAATGTTGAGAGT

AATACCCAGCAATCTACAGGAACTTGTGGGCGGGATACAACTGTTTGTTTAGAACTGACA

CTGTCTGGAATGCGATTTCTGTATGACATCTTTCCTGATGGTGGAACTCGGGTATCTAGG

CAGTCCATAACAGTTCATGATTTCTTTGTTAAAGACAACAGTAATGCTGCCCCTTGGAAA

CTGGTGCTGGGGTACTATCAATCAAAAGGCTGTTTAAGGAAGTCTTCTTCCAAAGCATTT

AAGCTGGATCTGGAAGCAGTAAGACCTGATCCTGCTATCCCTCTTGAGGAGTACCGGTTA

CGAATTGCATTCCTCCCGATGCGCTTACATCTTCATCAAAACCAGTTAGATTTTCTCATC

AGCTTTTTTGGAGGAACAAAGTCAGCAGTTACCCCCTCCCAAAGTTCTTCACAAAATTTG

AGTAAATCGGAAATAGTAGCAAAGAGAACTAAATTTGGGGGTAAAGCAGTCATTGAAGAG

GCACTGCTTCCTTATTTTCAGAAATTTGATATCTGGCCTGTTCATCTTCGGGTTGACTAT

AGCCCTTGCCGTGTTGATTTAGCTGCATTAAGGGGTGGCAAGTATGTTGAGCTTGTTAAC

CTTGTGCCTTGGAAGGGGGTTGACCTGCATCTCAAACATGTTCAAGCTCTAGGTGTCTAT

GGCTGGAGTGGCATAGGTGAAATAATAGTAGGTGAATGGTTGGAAGATATATCCCAAAAT

CAGATTCATAAACTATTGAAAGGCCTTCCTCCTATTCGGTCATTGGTAGCTGTTGGTTCT

AGTGCAGCAAAGTTGGTTTCTCTGCCTGTGAAGAGTTACAAGAAGGATCAAAAGTTGCTA

AAAGGAATGCAAAGAGGTACAATAGCGTTCCTTAGAAGTATTTCGCTTGAAGCAATTGGG

CTTGGAGTGCACTTGGCTGCTGGCGCTCATGAAATCCTTCTGCAAGCAGAATATATCCTT

ACAAGTGTTCCACCATCAGTAACATGGCCTGTGCAAAGTGGAGGAAACACTAGTGTGAGA

TTTAATCAACCTAGAGATTCCCGACAAGGGATCCAACAGGCTTATGAAAGTATGAGTGAT

GGCTTCAGTAAATCTGCTTCTGCTCTAATACGCACTCCCATCAAACGGTATCAGCGTGGT

GCTGGAATGGGATCTGCTTTTGCAACTGCTGTCCAAGCAGCTCCAGCAGCAGCCATTGCC

CCAGCTTCTGCCACAGCACGAGCTGTTCATTGTGCTCTTCTAGGTGTAAGGAACAGCCTC

AATCCGGAGCGTAAGAAAGAGTCTTTGGAGAAATATTTGGGGACAAATCCATCTCAGCAG

TACATGTATTTCTCCATGAAGAGCTCCAACAAAATTTGCAAGCCAGCATTAGTTTTGTAT

AGGTGTACAGATCGTAGGACAATTAGACAAATTCTTTTATCTGAGGAGACAGGTAATCAT

GTAAATTATGTAATATCAGAGTGGTAAACTTATTTTTATGTAATATCAGAGTGGTAAACT

TATTTTTTTTGCTCGTATGGCCGGGCCTGCCACTAGTTTCAATTTTTCGGTTATGTCAGC

TGTGTTATGTGCAAATTGTGAATATATTGATTCCCTTGGTTTTGCTGGCAGAATTGTCAT

CTGTACAACATTGTTTCTTGTAATTATCTTCTGTTTGAACTT

Alignments of sRNA sequence Bc-siR3.1 to wild-type Autophagy-related protein 2 target

and mutated Autophagy-related protein 2 (MU) target

embedded image

SEQ ID NO: 11—Bc-siR3.1 Target Sl Vacuolar protein-sorting (Solyc09g014790.2.1)

ATGATTTCATCATTGGGTGCAACTTCTTCTTCGTCTTCATCATCATCATCATCAGCTGCTGTTC

GTGTTGAGAAGGCAACGAGCGAGTTCTTGATAGGTCCTGATTGGACGATGAATATTGATATT

TGTGATACAATCAATTCTAACCAATGGTTGGCAAAAGATGTCGTCAAAGCTGTGAAAAAGA

GGTTGCAGCACAAGAACCCCAAAGTTCAGCTACTCGCTTTAACACTTATGGAGACAATGGTG

AAGAACTGTGGTGATAATGTGCATTTTCAAATTACTGAAAGAACTATACTGCAAGACATGGT

CAAAATTGTAAAGAAGAAGACTGATATGCATGTGAGAGATAAAGTGCTAGTACTACTGGAC

TCTTGGCAAGAAGCATTTGGTGGCCCTGGAGGAAAGTATCCCCAGTATTATTGGGCTTATGA

AGAATTGAGGCGCGCTGGTGTTGAATTTCCCAAGCGTTCATTTGATACAGCTCCTATCTTTAC

TCCTCCTGTTACTCATCCTGCACCAAGACAAGCGCAACCTGGTTATGGAATGCCAAACAATT

CCTCAACAAGACTTGACGAGGCAATGGCAGCAGACGTGGGAAACTTAAGCTTGTCCAGCAT

AAATTCTATGAGGGATGTTGCTGATCTGTTGGCTGATATGCTACAAGCTGTGACCCCAGGCG

ATCGTTTGGCTGTAAAGGATGAAGTTATAGCCGATCTTGTTGATCGGTGTCGCTCTAACCAG

AAGAAGTTGATGCAAATGTTAACAACAACAGGGGATGAAGAACTTCTTGCCCAGGGTCTTG

AATTGAATGACAACCTCCAAACTGTACTGGCTAAACATGATGCAATAGCTTCTGGTTCTCCA

CTCCCAACTCAAGTCCCAAATGACAACTTCTCTGCAAGAGAAATGCATGATCCAAGCCTCAA

ACCTGTTGAAGTTAAGCCACCCAGTCCAATAGCAGATGTCAAACCTTCTGCGCCAGTTCTTG

TAGCAACCGCAGGTCAAATTGATGAAGAGGAAGATGAGGAAGATGACTTTGCTCAACTAGC

TCGAAGACATTCAAAAACAAGTCCAGCAGCACAAACAAGTGAAGGAATGGTCTCTGCCAAT

embedded image

CAAGTGTTTATCCCGAAACATCACAAAATTCTGCTTCAGCTACTCAAAACACGCATCAGGAG

CCTCTTGCCTCAACCACACATGGAAATCCATATGCATCTCAAGCTTATATTGGGTATCAGGA

TCAGAGCTTTAACAGTTATGTAGCTCCTTGGGCTCAGCCCCAACCCCAGCATCAGTCACCAC

CCCAGTTTCATCCTCAATATCAACACCAAGGCCAACCTCAGTTTCATCCTCAATTTCAACACC

CAACCCAAGCCCAGGTCCAGTCCCAACCTCAACCTCATCCACAGCAACAACCTCAATCACAA

CTTCATCATCAATCCCGACCCCAACCATCCACTCAGCCTCAACGGCAGCAACCCCAAGAATC

TTCATTACAGTCTCAGCATACATCACAACAGCTTCCACAATCTCCTGTGCAACCTGAACTGA

ACCAACCTAGAACTCAGCAAGAACTTCATCCTCAGTCTCAACCGTTATCACCACGTACTCAG

ACTCAGTTCCCACAGTACTCTGCTTATCCACCTCCACCTTGGGCAGCAACTCCCGGATATCTG

AGCAATACAACATCTAGACCAACCTACATGTACCCAACTCCACAAGCAGCCACAAATACAC

CCATGTCTTTGCAAGCCACTAGACCCATACAGAATGTTAACTCGTTCCCTAATATGGGAAGC

AATGGTATAGCTATTAATGGTGACACTCAAGTTCATCCCCACCCCAAGACAACTCCTGCTTC

TGGTCAAAAAACCTTCATTCCATCTTATAGGCTGTTTGAAGATCTTAATGTTTTTGGCAACAG

CGATCAAAGACACAACTCATCTTCTGGTTTATCAGGAACTAACAGCCAAAGTATGGTTGGTG

GACGAAAATGA

Alignments of sRNA sequence Bc-siR3.1 to wild-type Sl Vacuolar protein-sorting target

and mutated Sl Vacuolar protein-sorting (MU) target

embedded image

SEQ ID NO: 12—Bc-siR5 target: Sl Pentatricopeptide (Solyc03g112190.2.1)

ATGAATCACGGCAAGAGAATACTGAGTTCGCTTCGATTGAGGAATTCTCTTTTTTTCACTCAG

CTTTCACGAGCCACTTCTTCCAATCATCAGGTGACTCAACACTTATATCTTTCTCCTTCACTTC

TCACGCAAATTTACACTTCTACTAGTATTCTCGGTTCAAGTCAAAATGTCTTCTTTTCATCAA

AAACTGAATCTTTTGTTGACATTATACTATCCAACGACTGGTCGAAACAATTAGAAAAGGAT

TTAGGAAAAAATGACTTTCCTGTGACCCATGAAGCTGTTATGTATTTGTTGAAGAAACTTGA

TAAAGAACCGCGAAAGGCAGGGGATTTCTTGAAATGGGTTGTTAAGCAAAAGGGGTTTAAA

CCTAGTTCTTCTATGTACAGTCTGATGCTTAGAATTTATGCTAACAGGGATTCAATGAAGGA

CTTTTGGACTACTATTAAGGAAATGAAAGAGAACGGGTTTTATATTGATGAGGAAACGTATA

AATCAATTTATTCTATTTTTCGGAATTTGAAAATGGAAACTGATGCCACTGCTTTGAAGCATT

TTTATGGGAGGATGATTAAAGATAATGCTATGGGTGATGTGGCGAAAGATGTGTCTGAATTG

ATTACAAAACAAGAATGGGGAGTTGAGGTGGAGAGACAATTAGGGGAGATGAAACTCTCGG

TGTCGGATAATTTTGTGCTTAGGGTGTTGAAGGAACTTAGAGAAGTAGGAAATCCACTGAAA

GCTTTCAGCTTTTTCAAATGGGTTGCGAGGAATTTAGATTTTCAGCACAGCACTGTTACTTAT

AATGGGATTCTTAGGGTTCTTTGCCGAGAAGAGTCGATTGAGGAGTTCTGGGGTGTAGTAAA

AGAGATGATGAGCCTTGGGTTTGAAATAGATCTTGATACATATATAAAGATCTCGAGGCATT

TTCAGAAGATTAAGATGTTGAAAGATGCAGTAGAACTATATGAACTGATGATGGATGGTCA

GTTTAAACCATCACTTGGGCATTCACGCTCAAAGATTATTTATGATGTCATTCATAGGTGTTT

GACTAACTTGGGGCGATTTGAGGAAGCAGAGAAGATAACAGAAGCTATGAGAGATGCAGG

ATTTGAACCTGACAATATTACCTATAGCCAATTAATATATGGACTTTGCAAAGTGAGGAGGC

TGGAGGAGGCATCAAAGGTGATAGATGTGATGGAAGAATGTGGATGCATTCCGGATATCAA

GACTTGGACTGTTCTAATACAAGGGCATTGTTTTGCTGGTGAAGTTGATAAGGCGCTGTTTTG

TTTTGCTAAGATGATGGAGAAAAATGTTGATACAGATGCTGATCTGTTGGATGTACTACTTA

ATGGTTTTTTGAGTCAAAGAAGAGTTTTTGGTGCATATCAGTTATTGACCGAGTTGGTGAAT

AAGTTTCAAATGCGCCCATGGCAAGCAACATACAAACTTGTCATCCAAAAGCTCTTGGGGGA

AAGGAAATTCGAAGAAGCGCTTGATCTACTCCGTCGGATGAAGAAACACAATTATCCACCTT

TTCCAGAACCCTTTCTTCAATATATTTCAAAGTCAGGAACAGTGGAAGATGCAGTGGAGTTT

TTAAAGGCGTTGAGCGTCAAGGACTATCCATCTGTTTCAGCCTATCAACATGTTTTCCAGTCC

embedded image

TCGGCAACACCCAGCAATTTGTGGCCTCTTTGGTTCGTCAAATTCTAACAGTGGAAAAATGA

AGAAAAAGCAGGAGCCTCATCAAGATGAAGAACATGATGTTGAAATCCTCAAGGCTGTGGC

ACAAGCCTGGCATGGACACTCGAGCAGCCGTGGAACTACTGCTGAATTCGACGCCCACCGC

CACAATTTCAAGAATAAGCCATCAAGATTCAAGCTTGAAGCTATGAACAAGGCAACCTCCA

GAGAATATGATGGAACAATTAGTAGATGGGATTTCAGCCAGTCTCTTTGGGATTCTTATGAG

ATACTCAATGTGTCCAAAAAGTTAGAAACTGGGCTAATGCTGGACCATCCATTGGATGGGTC

TATCCGAATTGGACAGAAGAGGAAAGAGAGTAAGAATAGCTTAAGAAATTTGCTCAATAGG

GTGTCTTCAAGAAGATATAATGATGCTGATTCAACACTAGACAAGGATGGTTAA

Alignments of sRNA sequence Bc-siR5 to wild-type Sl Pentatricopeptide target and

mutated Sl Pentatricopeptide (MU) target

embedded image

SEQ ID NO: 13—siR5 Target TOM34 (Solyc07g066530.2.1)

embedded image

Alignments of sRNA sequence siR5 to wild-type TOM34 target and mutated TOM34 (MU)

target

TargetMU
5′CAAUACAGGUUUCGUGUGAAG3′ (SEQ ID NO: 512)

| || | :||:|| || ||:

Bc-siRNA
3′UUCAUAUGUAAGGCUCAGUUU5′ (SEQ ID NO: 22)

|||||| |||||| ||||||

Target
5′CAGUAUAGAUUCCGUGUCAAA3′ (SEQ ID NO: 41)

Example 4: STTM Primers for Blocking the Function of Pathogen sRNAs

STTM primer sequences were designed against 30 Botyritis sRNAs (“Bc-sRNAs”) from Table 1 that were identified as having targets in both Arabidopsis and tomato. The designed STTM sequences can be used in other species which are also targeted by the Bc-sRNAs. The STTM primer sequences (forward primers and reverse primers) for generating STTM constructs, and the Bc-sRNAs targeted by each set of primers, are shown in Table 2.

STTM sequences can be expressed in plants according to the methods described in Yan et al., Plant Cell 24:415-427 (2012). Briefly, the STTM modules are inserted in a vector (e.g., the pOT2 vector) between the promoter and terminator. Insertion of the STTM modules is accomplished by PCR amplification of the vector with a proofreading Taq polymerase and a pair of long primers covering the entire STTM sequences (to minimize errors in STTM regions during the PCR reaction). The PCR product is and transformed into cells, e.g., XL1-blue. Single colonies are propagated for plasmid isolation and the recombinant constructs are verified, e.g., by linearization of the plasmids by a restriction enzyme. The recombinant plasmids are further amplified, and the PCR products containing the STTM and a selection marker (e.g., chloramphenicol) are introduced into a binary vector. Recombinant binary plasmids are selected on Luria-Bertani plates containing the appropriate selection antibiotics (e.g., chloramphenicol and kanamycin). The final constructs are verified by DNA sequencing before being used for plant transformation.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

	Number	Date	Country
Parent	14505378	Oct 2014	US
Child	16140179		US

RNAS FROM PATHOGENS INHIBIT PLANT IMMUNITY

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