Patent Application
20210189418
The nucleic and amino acid sequences listed in the accompanying Sequence Listing are presented in accordance with 37 C.F.R. 1.822. The Sequence Listing is submitted as an ASCII computer readable text file, created on May 4, 2017, as 65 KB, which is incorporated by reference herein in its entirety.
The present invention relates to chimeric transgene constructs and methods for generating multi-genic resistances to cereal viruses using the same.
The limitation of any host resistance mechanism (genetic or transgenic) based on a single gene or single target (monogenic or vertical resistance) is the development of pathogen resistance (a genetic arms race). There are several published reports of the high capacity of RNA virus populations to evolve relatively rapidly (e.g., the TuMV mutation rate=˜6×10{circumflex over ( )}(−5)) mutations per replication event in Arabidopsis, especially when selective pressures (and genetic bottlenecks) are imposed on the population to favor/accumulate variants that persist under these pressures. More specifically related to our approach, there is also one report of the build-up of resistance in an RNA virus population (Turnip mosaic virus, TuMV) that was serially and mechanically inoculated on transgenic Arabidopsis plants that expressed ONE antiviral amiRNA 21-nt-sequence to target the HC-Pro gene of TuMV for degradation. In this report, ultra-deep sequencing of the virus population revealed that i) variants (haplotype sequences) already existed in the population prior to challenge with the plant-expressed amiRNA that differed enough in the viral target sequence (HC-Pro) to ‘break-resistance’, and ii) after up to 20 passages through the transgenic plant, single nucleotide mutations in the viral HC-Pro sequence accumulated at every site of the amiRNA sequence, rendering the amiRNA sequence ineffective after the multiple passages of the virus population and accumulation of the ‘escape’ mutants in the transgenic plants.
There remains a need for techniques that induce broad spectrum, durable resistance in plants to viral infection.
Our research has shown that plants in the field are often infected by multiple damaging viruses. Thus, effective control measures that control more than one virus that limits wheat yields are needed to maximize crop yield. The challenge lies in the fact that viruses have highly variable genomic sequences, and the existence of highly conserved sequences of suitable length for targeting resistance traits has not been well documented until the present invention. In the past, most studies of virus diseases in cereal crops have focused on one disease and one viral species at a time, neglecting the fact that plants in the field are frequently infected not only by multiple forms of the same virus, but by multiple virus species. The variability of a single virus targeted by a single RNAi construct is likely to defeat the resistance in a single geographic location over time, and it is likely to limit the geographic range in which the resistance can be useful.
Similarly, a lack of awareness of the multiplicity and variability of virus infections in the field, coupled with visual diagnostic techniques, has led to a substantial underestimation of the prevalence of virus infections and the losses they truly cause year after year. In wheat, for example, virus diseases such as Barley Yellow Dwarf, are often described as sporadic and unlikely to cause significant losses, except in epidemic years. Field studies leading to the present invention have demonstrated the presence of a sizable class of plants that appear to be healthy, but nevertheless carry sizable loads of multiple virus species. Monitoring grain production from such plants has shown that they yield substantially less than apparently healthy plants. As shown herein, accounting for all the classes of infected plants has produced a new and very different understanding of yield losses due to virus infection. The losses are not sporadic and generally minor. Rather, they are chronic, significant and previously unrecognized. Restoring these yield losses requires simultaneous control of all the damaging viruses that attack the crop.
In one or more embodiments, highly conserved cDNA from viral sequences for RNA-mediated resistance in plants are described herein. The conserved sequences can be expressed as double-stranded RNA (dsRNA) hairpins and/or a “string” of multiple artificially-synthesized, short lengths of viral dsRNA sequences (e.g., artificial microRNAs, amiRNAs), and/or co-expressed cDNAs. The approach aims to target multiple virus species and species variants simultaneously using multi-genic (chimeric) transgene constructs.
In one or more embodiments, the invention is concerned with cDNA sequences encoding for highly conserved domains of plant viral genomes. The cDNA sequences are selected from the group consisting of those listed in Table I (i.e., any one of SEQ ID NOs: 1 to 68).
In one or more embodiments, the invention is concerned with cDNA fusion constructs or chimeric transgene constructs comprising at least two different cDNA sequences selected from the list in Table I, and more specifically cDNA corresponding to at least two different viruses. In one or more embodiments, chimeric constructs are selected from: Wheat-A (SEQ ID NO:69), Wheat-B (SEQ ID NO:70), Wheat-C(SEQ ID NO:71), Wheat-D (SEQ ID NO:72), Wheat-E (SEQ ID NO:73), Wheat-F (SEQ ID NO:74), Wheat-G (SEQ ID NO:75), Wheat-H (SEQ ID NO:76), Wheat-I (SEQ ID NO:77), Wheat-J (SEQ ID NO:78), and Wheat-K (SEQ ID NO:79). Plant expression vectors or transformation vectors comprising multiple expression cassettes are also contemplated herein. In one or more embodiments, the construct or vectors comprise at least two different cDNA sequences operably linked to one or more regulatory sequences for expression in a plant cell.
Transgenic plants with broad spectrum and durable resistance to multiple pathogenic plant viruses of agronomic importance are also described herein. In one or more embodiments, the transgenic plant has been transformed with the cDNA fusion construct. In one or more embodiments, the transgenic plant has the cDNA fusion construct stably incorporated in its genome. In one or more embodiments, the transgenic plant is resistant to at least two viruses, selected from the group consisting of Barley yellow dwarf virus (BYDV) (PAV and PAS), Wheat streak mosaic virus (WSMV), Cereal yellow dwarf virus-RPV (CYDV-RPV), Soil-borne wheat mosaic virus (SBWMV), and Wheat spindle streak mosaic virus (WSSMV).
A method of producing a plant with broad spectrum, durable resistance to multiple pathogenic plant viruses is also described herein. The method generally comprises transforming a plant cell with a multi-genic construct or vector as described herein.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Highly conserved domains of viral sequences for RNA-mediated resistance to viral pathogens in plants are described herein. As used herein the phrase “highly conserved domain” means a domain, or a stretch of (RNA) sequence that is conserved (i.e., relatively unchanged) among the different species and/or strains of a target virus. In other words, such domains are substantially invariant across different strains of a given virus, such that any substantive change in the sequence of the domain results in loss of viral function. It will be appreciated that in some cases even a single base pair change can cause a frame shift resulting in abnormal protein products or loss of expression altogether. Such highly conserved sequences are thus substantially invariant across geographic regions and over time, providing the key to stable, broad spectrum and durable resistance. The term “broad spectrum” is used herein to denote that the invention is effective against a wide variety of viral species and species isolates across geographic regions. The term “stable” is used herein to denote that the transgene is passed to progeny plants without change in transgene sequence or loss of activity, such that resistance is passed to progeny plants and remains effective over time from generation to generation. The term “durable” is used herein to denote that the resistance remains effective against viral isolates in a population from season to season (preferably over a period of at least 10 years) despite viral mutations, due to the highly conserved nature of the target domains.
Thus, the invention permits simultaneous control of multiple viruses, restoring plant yields to a higher level than could be achieved if a single virus were being targeted with the construct. Preferably, a highly conserved domain is one in which sequence identity is at least about 80% across strains of a given species, preferably at least about 85%, more preferably at least about 89%, and more preferably from about 89% to about 100% across strains of a given species. The conserved sequences are introduced into the plant, where they can be expressed in the plant as double-stranded RNA (dsRNA) hairpins and/or a “string” of multiple artificially-synthesized, short lengths of viral dsRNA sequences (e.g., artificial microRNAs, amiRNAs, etc.) or single cDNAs co-expressed.
The approach aims to target multiple RNA virus species and species variants simultaneously using multi-genic (chimeric) transgene constructs to generate plants having increased resistance to viral pathogens. Unless otherwise indicated by the context, references herein to a “plant” or “plants” includes tissues, organs, or parts thereof (e.g., leaves, stems, tubers), fruit, seeds, or cells thereof.
Exemplary virus targets for use in the invention include the Luteoviridae family, Potyviridae family, and/or Furovirus family of pathogens.
Family Luteoviridae (B/CYDV)—BYDV-PAV and -PAS
The luteovirus genome comprises a single positive strand RNA of approximately 5.6 kb. At the 5′ proximal ORF, the 39K ORF encodes a subunit of the RNA dependent RNA polymerase (RDRP) and this ORF is sometimes extended due to RdRp frameshifting to produce a protein with a C-terminal extension and this is the RdRp. The “luteovirid gene block” is located at 3′ terminal half of the virus genome. The capsid protein (CP) transcribed from a subgenomic promoter. The minor component of the virion is the CP-readthrough protein (CP-RTP), generated by translational readthrough of the CP stop codon resulting in a C-terminal extension of the CP. Another subgenomic promoter drives transcription of the movement protein. There are two other predicted ORFs at the extreme 3′ end of the viral genome and are predicted to encode very small proteins 7 kDa and 6 kDa.
Family Potyviridae, Genus Tritimovirus
Wheat streak mosaic virus (WSMV) is a positive-strand RNA virus with one large ORF that extends the length of the genome. The 5′ terminus has a genome-linked protein (VPg), and the 3′ terminus has a poly(A) tract. The long ORF is translated a single polyprotein that is cleaved by proteases to generate the individual proteins. From the 5′ end to the 3′ end the genes and their functions are: P1 protein (putative protease), HC-Pro (helper component protease), P3 protein (unknown function), 6K1 (unknown function), CI (Cylindrical inclusion putative helicase), 6K2 (unknown function), VPg (Genome-linked protein), NIa (Nuclear Inclusion putative protease), NIb (Nuclear Inclusion putative polymerase), CP (coat protein). Another ORF has been identified and is generated by frameshifting in the third (P3) cistron for translation of the protein named PIPO. This ORF overlaps with the large ORF.
Family Potyviridae, Genus Bymovirus
Wheat spindle streak virus is in the family Potyviridae but unlike WSMV, the genome is divided into two genome segments, a characteristic of all members of the genus Bymovirus. The total genome is 10000-12000 nucleotides long. Like other potyviruses, 3′ terminus has a poly(A) tract. 5′ terminus has a genome-linked protein (VPg). RNA1 is believed to produce 8 peptides: P3 protein (unknown function), 6K1 (unknown function), CI (cylindrical inclusion protein with helicase activity and possibly involved in cell to cell movement), 6K2 (unknown function), VPg (putative genome-linked protein), NIa-Pro (serine-like protease), NIb (homologous to potyvirus Nuclear Inclusion polymerase), CP (coat protein). RNA2 produces 2 peptides: P1 (protease domain similar to that of the potyvirus HC-Pro) and P2 protein (involved in fungus transmission). An additional small ORF (PIPO) is generated by frameshifting within the first cistron of RNA1 (P3).
Genus Furovirus
Soilborne wheat mosaic virus (SBWMV) is a bipartite, positive-strand RNA virus and is the type member of the genus Furovirus. RNA1 encodes the viral replicase and putative viral movement protein (MP). The viral replicase is encoded by a single large open reading frame (ORF). The 3′ proximal ORF of RNA1 encodes the 37K MP. RNA2 has four ORFs that encode proteins. The 5′ proximal ORF of RNA2 encodes a 25K protein from a nonAUG start codon and its role in virus infection is unknown. The capsid protein (CP) ORF has an opal translational termination codon and readthrough of this codon produces a large 84K protein. The CP readthrough domain (RT) is essential for plasmodiophorid transmission of the virus. The 3′ proximal ORF of RNA2 encodes a 19K small cysteine-rich protein (CRP) that functions as a pathogenicity determinant and a suppressor of RNA silencing.
a=derived from KS direct amplicon sequences, c=derived from KS clone sequences, i=derived from Illumina deep sequencing reads of national isolates, BY=Barley yellow dwarf virus (BYDV-PAV and -PAS), WS=Wheat streak mosaic virus (WSMV), SB=Soil-borne wheat mosaic virus (SBWMV), SS=Wheat spindle streak mosaic virus (WSSMV), SB and SS sequences derived from Illumina deep sequencing reads of national samples, *KS sequence information obtained for this genome region, **longer contiguous sequences of viral polymerase gene from Illumina (U.S. isolate) data; bviral genome open reading frames or genes or domains targeted for degradation ( indicates “elite” sequences selected for chimeric transgene design in Examples.
In one or more embodiments, the invention is concerned with cDNA sequences encoding for highly conserved domains of RNA genomes of plant viruses. The cDNA sequences comprise, consist, or consist essentially of the sequences listed in Table I, or those having at least about 95%, and preferably at least about 99% sequence identity to those listed. It will be appreciated that viral cDNAs are artificially synthesized sequences that do not exist in nature for RNA viruses. Significant work was carried out herein to identify and isolate highly conserved RNA domains from virus-infected plant tissue. The highly conserved RNA sequences were reverse transcribed into cDNA. The cDNA is used to construct multi-genic, stable anti-virus plant expression vectors and chimeric transgene constructs. For example, the reverse transcribed cDNA can be amplified and then either sequenced directly or cloned into a plasmid and sequenced. These cDNA sequences can be used in various orders and orientations (sense or anti-sense, inverted, etc.) to construct different transgenes comprising concatenated cDNA sequences separated with or without linkers.
When expressed in plants these chimeric transgenes result in long dsRNA hairpins containing the multiple target (conserved) viral RNA sequences that launch the innate RNAi cellular process of the host plant to dice (via Dicer protein) this hairpin into small dsRNA duplexes that will seek and destroy (with the help of the RNA-induced silencing complex (RISC) holo-enzyme) the complementary sequence of viral RNA during an infection. That is, Dicer recognizes these dsRNAs and cleaves them into duplex small-interfering RNA (siRNA) comprising a guide strand (i.e., strand complementary to target mRNA) and a passenger strand. The guide strand is incorporated into the RISC complex, which is then programmed to degrade the target viral RNA and/or inhibit translation of target mRNA.
The chimeric transgenes can also be expressed as a “string” of multiple artificially-synthesized, short lengths of viral dsRNA sequences (e.g., artificial microRNAs, amiRNAs, etc.), which will likewise launch the RNAi process of the host plant as described. RNAi relies on sequence-specific, post-transcriptional gene silencing, and is broadly defined herein to include all post-transcriptional and transcriptional mechanisms of RNA-mediated inhibition of gene expression. Generally, in RNAi, all or a portion of a viral domain cDNA is duplicated in an expression vector in a sense/antisense or an antisense/sense orientation so that the resulting expressed RNA can be processed by the cell into the siRNAs. RNAi can be used to either partially or completely inhibit expression of the target gene. RNAi may also be considered to completely or partially inhibit the function of a target RNA. RNAi may also cleave viral genomic RNA. Thus, in one aspect, the nucleic acid construct preferably comprises a sense and/or an antisense sequence for the target viral domain and encodes double stranded RNA that inhibits the expression, activity, or function of the viral gene. In a further aspect, the nucleic acid construct will preferably comprise a sense sequence operably linked to its complementary antisense sequence and encoding double stranded RNA that inhibits expression, activity, or function of the target viral gene.
In one or more embodiments, the invention is concerned with cDNA fusion constructs or chimeric transgene constructs comprising at least two different cDNA sequences selected from the list in Table I above (SEQ ID NOs:1-68). More specifically, the constructs comprise at least a first cDNA sequence encoding a first highly conserved viral domain, and at least a second cDNA sequence encoding a second highly conserved viral domain, where the first and second highly conserved viral domains each correspond to a different target virus species or viral isolate. As used herein, a viral RNA domain or cDNA “corresponds” to a target virus when it is based upon an RNA sequence isolated from a same or similar virus species and/or when its expressed RNA will otherwise recognize (through perfect or imperfect complements) a viral RNA domain in the target virus to initiate RNAi in a host plant.
Plant expression vectors or transformation vectors comprising multiple expression cassettes are also contemplated herein. In one or more embodiments, the construct or vectors comprise at least two different cDNA sequences (one each corresponding to a different target virus) operably linked to one or more regulatory sequences for expression in a plant cell. The recombinant expression vectors of the invention comprise a cDNA of the invention, or complement thereof in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the cDNA sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the cDNA sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc.
In order to improve host plant resistance to viral infection, this technology permits the construction of transgenes that combine multiple conserved sequences that specifically target multiple genes, for example, 4 genes and one intergenic region of BYDV genome (5-6 genes in genome), and 2 genes in WSMV genome (10 genes in genome) (
Thus, it will be appreciated that the invention facilitates multi-genic resistance in the transformed plant against a plurality of target viruses, and the likelihood that the target viruses will accumulate mutations in all of these gene targets is greatly reduced. The invention also addresses the problem of mutation because it targets highly conserved domains of diverse viruses found in samples from around the world. Genetic analysis of haplotypes in the virus population includes pre-existing variants in the generation of the most conserved sequences across the genomes of each virus species. Thus, the development of virus strains that are capable of infecting plants and overcoming the RNAi resistance is a negligible risk. Advantageously, many of the cDNA sequences listed herein target overlapping open reading frames (which may be involved in expressing multiple viral proteins from a single sequence by various mechanisms). These are advantageous targets for RNAi because they target one nucleotide sequence, yet disrupt protein expression of more than one viral gene, and they are under greater selection pressure and less likely to change.
The technology can involve artificial method to inoculate plants, and preferably leaf-rub inoculation. In contrast, and in the field, BYDV and WSMV are transmitted by arthropod vectors. It is well documented that arthropod vector transmission imposes a severe bottleneck on virus populations and will also contribute to the durability of resistance using the inventive multi-virus, multi-locus RNAi approach. However, it will be appreciated that any other suitable plant transformation techniques can be used, including, without limitation, a ballistic particle delivery system, microprojectile bombardment, viral infection, Agrobacterium-mediated transformation (Agrobacterium tumefaciens), electroporation, and liposomal delivery, to produce transformed cells. The term “bombardment” with respect to transformation refers to the process of accelerating particles towards a target biological sample (e.g., cell, tissue, etc.) to effect wounding of the cell membrane of a cell in the target biological sample and/or entry of the particles into the target biological sample.
Transgenic plants with broad spectrum resistance to multiple pathogenic plant viruses of agronomic importance are also described herein. Transgenic plants include wheat, barley, oat, rice, and/or sorghum. In one or more embodiments, the transgenic plant has been transformed with a cDNA fusion construct according to embodiments of the invention. In one or more embodiments, the transgenic plant has the cDNA fusion construct stably incorporated in its genome. The invention also provides a plant wherein expression of a single heterologous expression vector of the invention results in resistance to two or more viruses selected from the group consisting of: Barley yellow dwarf virus (BYDV) (PAV and PAS), Wheat streak mosaic virus (WSMV), Cereal yellow dwarf virus-RPV (CYDV-RPV), Soil-borne wheat mosaic virus (SBWMV), and Wheat spindle streak mosaic virus (WSSMV). That is, the broad spectrum resistance is achieved by utilizing silencing RNA expressed from a single transformed construct in the plant, wherein that single construct comprises more than one cDNA or expression cassette that produces silencing RNA that targets one or more functions necessary for viral infection, multiplication, transmission, and/or protein translation in the plant. Thus, resistance to multiple plant viruses may be achieved in a single transgenic “event,” which enables the use of simple genetic crossing for easy incorporation of broad spectrum, durable virus resistance into any cultivar of interest.
The transformed plant may also further comprise a non-transgenic plant virus resistance trait. That is, the transgene construct can be stacked with other genetic traits (e.g., from classical breeding or transgenic introduction) or genetic backgrounds to maximize yield. The underlying basis for the invention, as applied to wheat, can be applied to other cereal crops (grasses in monocot family Poaceae) susceptible to these particular virus species, such as oat, barley, rice, and sorghum. The present invention provides, as an example, a transformed host plant of a pathogenic target organism, transformed plant cells, and transformed plants and their progeny. The transformed plant cells and transformed plants may be engineered to express one or more of silencing RNA sequences, under the control of a regulatory sequence, described herein to provide resistance to multiple pathogenic plant viruses.
A method of producing a plant with broad spectrum resistance to multiple pathogenic plant viruses is also described herein. The method generally comprises introducing a cDNA fusion construct according to the invention into the plant. Methods include transforming a plant cell with a multi-genic construct or vector as described herein. A recombinant plant cell comprising the cDNA fusion construct, preferably stably incorporated into its genome, is also provided herein. In certain embodiments, increased resistance in the plant to at least one plant viral pathogen is provided by expression of a nucleic acid construct that produces a silencing RNA (e.g., dsRNA). The silencing RNAs may be either “sense” (identical) or “antisense” (complementary) to the viral genomic RNA. It is readily appreciated that the antisense silencing RNAs are capable of hybridizing directly to the genomic RNA ((+) RNA strand) of the target virus by base pairing, and so of inhibiting the genomic RNA either prior to or during replication, whereas the sense silencing RNAs are capable of hybridizing to the (−) replicative strand of the target virus which is produced during replication of the target virus, and thereby are capable of inhibiting replication of RNA viruses during viral replication, or subgenomic RNAs, thereby capable of degrading viral messenger RNA, i.e., post-transcriptional degradation.
The invention also provides resistant and/or transgenic cells, tissue, and seeds of plants produced by the methods described herein, and the progeny thereof. Methods of the invention include, culturing plant tissue (e.g., leaf, cotyledon, or hypocotyl explants) on a suitable media (e.g., Murashige and Skoog (MS), or Chu (N6)), followed by introduction of the cDNA construct into the tissue using suitable techniques, such as those described above and in the working examples. Expression of the construct results in transformed or modified tissue. As noted herein, reporter genes can be used to verify transformation. The transformed tissue can then be used to regenerate transgenic whole plants having increased resistance to multiple viral pathogens. Transgenic plants can be regenerated using various techniques depending upon the plant species involved. In one or more embodiments, regeneration comprises inducing callus formation from the transformed tissue, and regeneration of shoots, followed by rooting of the shoots in soil or other appropriate rooting media to generate the whole plant.
The resulting transgenic plants can be crossed to prepare progeny that are homozygous for the resistance trait. Further, resistant plants can be produced indirectly by breeding parent plants, one or both of which have broad spectrum resistance to multiple pathogenic plant viruses with other resistant plants, or with other cultivars having additional desired characteristics (e.g., drought tolerance, geographic adaptation, stalk strength, etc.). The resulting progeny can then be screened to identify resistant progeny with inhibited expression, activity, or function of the corresponding target susceptibility gene or gene products. In one or more embodiments, the invention is also concerned with a process of producing transgenic seed. In some embodiments, the method comprises self-pollination of a transgenic plant as described herein. In some embodiments, the method comprises crossing a first plant with a second plant, wherein at least one of the first or second plants is a transgenic plant having increased resistance to multiple viral pathogens as described herein. In some embodiments, the first and second plants are both transgenic plants as described herein. In one or more embodiments, the first and second plants can be crossed via cross-pollination using insects (e.g., in cloth cages), manual (hand) pollination, and the like.
Regardless of the embodiment, transgenic plants according to the invention preferably exhibit increased broad spectrum resistance to multiple pathogenic plant viruses as compared to a corresponding non-transformed or wild-type plant. However, unlike many other transgenic plants with similar improvements in pathogen resistance, plants according to the invention have a phenotype/morphology that is otherwise substantially similar to, and in some cases, nearly identical to wild-type plants of the same species. In other words, the transgenic techniques of the invention do not adversely affect the wild-type morphology or phenotype of the plant, such that the shape, size, and/or abundance of foliage and/or fruit/vegetable is substantially similar between the transgenic plants and wild-type plants. Plants are considered to be “substantially similar” herein if those skilled in the art have difficulty visually distinguishing between the genetically-modified plant and the control plant when grown under identical normal growing conditions. In contrast, when exposed to viral pathogens, transgenic plants according to the various embodiments of the invention, have significantly improved characteristics as compared to control plants grown under the same conditions. For example, the transgenic plant may have one or more of the following improved characteristics: vigorous growth, abundant foliage, verdant foliage color, longer primary roots, yield, height, and/or shoot water potential, when grown in the presence of one or more viral pathogens.
Additional advantages of the various embodiments of the invention will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and/or integrations of the specific embodiments described herein.
As used herein, the phrase “and/or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and/or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.
A “control” plant, as used in the present invention, refers to a plant used to compare against transgenic or genetically modified plants according to the invention for the purpose of identifying changes in the transgenic or genetically modified plant. The control plant is of the same species as the non-naturally occurring plant. In some cases, the control plant may be a wild-type (native) plant, although cultivars and genetically altered plants that otherwise have not be altered for viral resistance can also be used a references for comparison. A “wild-type” plant is a plant that has not been genetically modified or treated in an experimental sense. A “wild-type” gene is one that has the characteristics of a gene isolated from a naturally occurring source. A “wild-type” gene product is one that has the characteristics of a gene product isolated from a naturally occurring source, whereas “modified” genes or gene products are those having modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. Likewise, “genetically-modified” cells, tissues, seeds, plants etc. are those that have been altered to include a transgene and/or to change the expression, activity, or function of the target genes or gene products, as opposed to non-modified cells, tissues, etc. The term is synonymous with “genetically-engineered.”
The term “vector” refers to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The term includes recombinant DNA molecules containing a desired coding sequence(s) and appropriate nucleic acid sequences (e.g., promoters) necessary for the expression of the operably linked coding sequence in a particular host organism.
The term “operably linked” refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced
The term “transform” is used herein to refer to the introduction of foreign DNA into cells. Transformation may be accomplished by a variety of means known to the art and described herein.
The term “isolated” when used in relation to a nucleic acid, refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural environment. That is, an isolated nucleic acid is one that is present in a form or setting that is different from that in which it is found in nature.
The term “sequence identity” is used herein to describe the sequence relationships between two or more nucleic acid sequences when aligned for maximum correspondence over a specified comparison window. The percentage of “identity” is determined by comparing two optimally aligned sequences over the comparison window. For “optimal alignment” of the two sequences, it will be appreciated that the portion of the sequence in the comparison window may include gaps (e.g., deletions or additions) as compared to the reference sequence, which does not contain additions or deletions. After alignment, the number of matched positions (i.e., positions where the identical nucleic acid base or amino acid residue occurs in both sequences) is determined and then divided by the total number of positions in the comparison window. This result is then multiplied by 100 to calculate the percentage of sequence or amino acid identity. It will be appreciated that a sequence having a certain % of sequence identity to a reference sequence does not necessarily have to have the same total number of nucleotides or amino acids. Thus, a sequence having a certain level of “identity” includes sequences that correspond to only a portion (i.e., 5′ non-coding regions, 3′ non-coding regions, coding regions, etc.) of the reference sequence.
The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting “greater than about 10” (with no upper bounds) and a claim reciting “less than about 100” (with no lower bounds).
The following examples set forth methods in accordance with the invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.
Wheat leaf tissue was collected from all nine wheat crop reporting districts of Kansas. These regions have been delimited by the United States Department of Agriculture. The sample locations included multiple commercial field sites and 15 Kansas Agricultural Experiment Station wheat variety performance trial locations, each located in a different county. In total there were 50 and 42 counties surveyed during the first and second growing seasons, respectively. Symptomatic and asymptomatic tissue were subsampled from individual wheat plants at each location.
The virus content in each plant sample was determined by ELISA to detect coat proteins of six virus species most prevalent in Kansas (Table II). For each sample, 500 mg of leaf tissue was subsampled with scissors dipped in 10% household bleach for 10 s followed by 20 s in distilled water to decontaminate between samples. Triple antibody sandwich (TAS) enzyme-linked immunosorbant assay (ELISA) was performed to detect BYDV-PAV, CYDV-RPV, and SBWMV and double antibody sandwich (DAS)-ELISA was performed to detect WSMV, HPV, and WSSMV using Agdia® Pathoscreen Kits in a 96-well microtiter plate format following the manufacturer's protocol, with all incubation steps performed overnight at 6° C. The subsampled tissue was ground in 1 ml of general extract buffer (GEB) using a tabletop rolling tissue grinder. A Titertek Multiskan Plus plate reader set at λ=405 nm was used to determine absorbance at 30-min readings for HPV, WSMV, WSSMV, and WSBMV and 3-hr readings for BYDV-PAV and CYDV-RPV. Samples were determined to be positive for virus if the absorbance reading was at least 3 times the average absorbance of the negative controls included on each plate.
aLeaf tissue samples collected from symptomatic and asymptomatic winter wheat plants growing in statewide KAES variety performance trials, commercial grower fields, and KSU Rocky Ford Agriculture Station Barley yellow dwarf and soil-borne virus nurseries;
bVirus-infection status and virus species determined by performing Enzyme-Linked Immunosorbant Assays (ELISAs);
cHigh plains virus (HPV) and Cereal yellow dwarf virus-RPV (CYDV-RPV), detected but not included herein; BYDV = Barley yellow dwarf virus-PAV and Barley yellow dwarf virus-PAS; WMSV = Wheat streak mosaic virus; SBWMV = Soil-borne wheat mosaic virus; WSSMV = Wheat spindle streak mosaic virus, ND = not determined
3. Total RNA Extractions and cDNA Synthesis
Each of the subsamples kept at −80° C. consisted of 0.1 to 0.15 g of leaf tissue stored in a 2 mL tube resistant to high centrifugation forces. Using a ‘FastPrep-24’ instrument (MB Biomedicals, Inc., Santa Ana, Calif.), each tube containing the frozen tissue and one 6-mm ceramic bead was loaded into the instrument tube holder filled with dry ice to keep tissue frozen. The tissue was pulverized to a fine powder at rate of 4.0 m s-1 and immediately resuspended in 500 μL of the lysis solution RLT supplied in the RNeasy Plant Mini Kit (Qiagen, Valencia, Calif.) and supplemented with 1.0% 2-Mercaptoethanol. Total plant RNA was extracted using this kit by following the options: Microfuge centrifugation through spin columns to retain RNA on filters, two consecutive RPE-washes of the RNA-retention filter, and elution of RNA with 50 μL of RNase-free water according to the manufacturer's recommendations. This protocol usually yielded 200-500 ng μL-1 of total RNA per sample. 1.0 μg of the extracted total RNA was added as template in a 20 μL reaction for the synthesis of the cDNA. This was polymerized by MMLV reverse transcriptase and primed by random hexamers following the iScript cDNA synthesis (Bio-Rad, Hercules, Calif.) protocol.
Primers were designed with the aid of the Primer-BLAST (ncbi.nlm.nih.gov/tools/primer-blast/). Only those primers that did not match any of the sequences deposited in a wheat gene database (taxid: 4565) were selected by this program. Using SeaView v4.0, one consensus sequence per species was derived from multiple alignments of all publically-available GenBank full-length genome sequences for BYDV-PAV and -PAS and WSMV, and the RNA1 genome segment for SBWMV, and each consensus sequence served as the template for primer design. Primers used for generating high-fidelity amplicons for direct sequencing and for cloning and subsequent sequencing of virus haplotypes are listed in Table III. To cross-check primer alignment to each consensus in silico, alignments were performed with the ‘Muscle’ algorithm in MEGA 5. Primers that aligned in genomic regions with the lowest number of ambiguous symbols in any of the consensus sequences were selected for HF-PCR to maximize the spectrum of amplified genetic variants.
Several genomic regions of BYDV, WSMV, and SBWMV were DNA-amplified by HF-PCR using the following general conditions: 5.0 μL of the cDNA reaction was mixed with 500 nM of each primer, 200 μM of dNTP, and 0.4 U of ‘Phusion’ proofreading polymerase (New England BioLabs, Inc.) in a total volume of 50 μL including the standard Phusion buffer. DNA amplification took place during 30 cycles of denaturing at 95° C. for 30 s, annealing at the temperature defined for each template-primer combination for 30 s, and extension at 72° C. for 45 s for amplification of gene-specific templates or 75 s in the case of the ˜2.3 kb fragments amplified for cloning using the BY(L)1890/BY(R)4161 primer pair and the WS(L)1649/WS(R)3990 primer pair for BYDV-PAV/PAS and WSMV, respectively.
The concentration of HF-amplicons prepared for direct sequencing was normalized as follows: Three independent 50 μL HF-PCR reactions were performed for each field sample and then consolidated in a single suspension that was subjected to DNA clean up using the QIAquick PCR purification kit (Qiagen, Inc.) following the manufacturer's recommendations, including the DNA elution with 50 μL of 10 mM Tris-Cl pH 8.0. Then, the concentration of each DNA preparation was adjusted to 20 ng μL-1 with DEPC-treated H2O. HF-amplicons were direct-sequenced (consensus of haplotypes) for each KS isolate selected from different crop-reporting regions of the state (Table II)
5. cDNA Cloning and Sequencing (BYDV-PAV, BYDV-PAS and WSMV)
HF-PCR amplicons (˜2.3 Kb, BYDV and WSMV) were generated and cloned into the pCR-Blunt II-TOPO plasmid vector (Invitrogen, Inc. Carlsbad, Calif.). A 1:1 ratio of insert-vector was ligated in 300 mM NaCl and 15 mM MgCl2 solution incubated at room temperature for 5 min. Then, the recombinant plasmid was introduced by electroporation (1 pulse at 3.0 kV) into One Shot TOP10 Electrocomp E. coli cells (Invitrogen, Inc. Carlsbad, Calif.). Kanamycin-resistant colonies were picked for insert detection by PCR and those bacterial colonies with the expected insert size were amplified in selective media and frozen for direct cell sequencing according to the specifications of the sequencing company (Beckman Coulter Genomics, Inc., Danvers, Mass.). For the sequencing reactions, universal vector primers, M13L and M13R, were used to generate 5′ and 3′ reads of the insert and internal primers BY(L)2925/BY(R)3013 and WS(L)2791/WS(R)2948 were used to sequence the middle region of the 2.3 Kb inserts. These primers generated an overlapping sequence of around 88 bases long.
The initial sequence exploration of BYDV was realized on full-length genomic sequences publically available in the GenBank database. In total, they were 53, 10, 2, and 2 isolates classified as PAV, GAP, MAV, and PAS species, respectively. These sequences were aligned together or in sub groups using ClustalW as implemented in MEGA5 (Tamura et al., 2011), SeaView (Gouy et al., 2010), or BioEdit (Hall, 1999). For comparative purposes, sequence alignments were also created using the Muscle program included in MEGA5. Same alignment procedures were applied to the sequences produced in this work. Then, the FASTA files of aligned or unaligned sequences were imported into different applications for different purposes.
The sequences derived from BYDV isolates processed in this work were assembled and curated as follows: Chromatogram files derived from the same DNA clone were aligned together against a reference by ChromasPro v1.6 (Technelysium Pty Ltd, South Brisbane, Queensland, Australia) to create contigs of the cloned BYDV genomic region. Except for some indel mutants, the contig was 2187 bp long after removal of the primer sequences. NCBI GenBank reference sequences for BYDV-PAV, BYDV-PAS, and WSMV were used to guide contig assembly. Ambiguous base calls were manually resolved after visual inspection of the intensity and quality of the signal. All sites in the chromatograms with more than one base call in the same site were recorded and counted to estimate the genetic heterogeneity of the viral isolate.
The identification of highly conserved stretches of genomic sequences was performed using DNaSP. The sliding window was set at a window length of 10 sites with a sliding step of 5 sites. At each window, the number of polymorphic sites (S), nucleotide diversity (it), and mutation rate (0) was estimated along a sequence alignment. The parameters used to identify conserved sequences were a minimum size of 30 bases and a conservation threshold of 99 percent.
Deep Sequencing of U.S. Wheat Samples for RNA Viruses and Comparison to KS Conserved cDNA Sequences
To expand the virus sequence resources for identification of highly conserved sequence candidates, single-plant wheat samples were collected from 12 U.S. states (WA, OK, CA, TN, NC, ID, TX, AR, NY, MO, NE and SD) for deep sequencing. A total of 50 plant samples that tested positive by ELISA for at least one of the four virus species targets (single or joint infection: 28 BYDV-PAV/PAS isolates, 18 WSMV isolates, 11 SBWMV isolates, and 2 WSSMV isolates) were selected for RNA isolation and RNAseq library construction, and each of the 50 tagged libraries was prepared for Illumina paired-end RNA-sequencing. Raw sequence reads obtained for each library were trimmed and aligned to NCBI reference sequences (Refseq) of three full-length viral genomes (BYDV: NC_004750.1; WSMV: NC_001886.1; SBWMV: NC_002041.1) and the partial genome (3′-terminal half of RNA 1) of WSSMV (gb|X73883.1) using Bowtie2, mapped reads were BAM-indexed, and GATK was used to re-align BAM-indexed reads to the viral genome reference sequences for SNP analysis (variant call format).
For each virus species, conserved strings of nucleotides sequences (cDNA) were identified across the 50 U.S. wheat samples. These regions of conservation were aligned with the each of the conserved cDNA sequences identified from the KS wheat survey using MEGA and % conservation was determined for the KS and U.S. isolate collection (Table I, above). For BYDV, the regions of high conservation (agreement) were aligned to 55 NCBI GenBank BYDV-PAV and 2 BYDV-PAS genome sequences from isolates collected in the U.S., China, Japan, Sweden, Germany, and Pakistan to determine % conservation (Table I, 79% to 100%). For WSMV, the regions of high conservation were aligned to 15 NCBI GenBank WSMV genome sequences from isolates collected in the U.S., Mexico, Australia, Hungary, Austria, Turkey, Czech Republic, and Iran and % conservation was determined (Table I, 88% to 100%). Illumina deep sequencing of full-length viral genomes revealed other genomic regions of high conservation and these sequences were also aligned and compared to the GenBank viral genome sequences to determine % conservation (Table I, 76% to 100%). Together, the KS and U.S. sequencing effort produced 68 highly conserved cDNA sequences, and when utilized in multiple confirmations to produce multi-genic (chimeric) transgene constructs, have the potential to target concomitantly multiple BYDV, WSMV, SBWMV, and WSSMV virus populations described worldwide.
MVR Transgenic wheat expressing dsRNA sequences that have the potential of targeting multiple genes of four wheat virus species (BYDV-PAV/PAS, WSMV, SBWMV, and WSSMV) by RNA-interference were created.
1. Transgene Design.
A collection of 68 small, conserved cDNA sequences from research, which includes 17 for BYDV-PAV/PAS, 15 for WSMV, 33 for SBWMV, and 3 for WSSMV (Table I) were analyzed in silico for stringent filtering-out of sequences with 1) the potential off-target effects in wheat, honey bees, or humans, 2) prediction of producing inefficient siRNAs (i.e., poor silencing potential, based on antisense siRNA binding affinity to RISC complex), and 3) potential generation of siRNAs with ‘toxic motifs’ (G and U rich short sequences shown to mount immune-stimulatory responses and to be toxic to human cell lines).
Publically-available sequence analysis tools used for this step were RNAiScan, pssRNAit, MEGA6, and NCBI GenBank. Those sequences predicted in any of the three filtering criteria were removed from further consideration. There were 31 small, conserved cDNA sequences selected as strong candidates (elite) for transgene construct design (See sequences marked ‡ in Table I.). For each virus species, multiple concatenated strings (˜100-150 nucleotides) of target sequence were identified by the maximum number of contiguous elite small, conserved cDNA sequences (Table IV).
SB
—
6
13
From these concatenated sequences, chimeric sequences were created to form the transgene hairpin arms (sense (left arm) and antisense (right arm) complementation) of 11 different transgene constructs, with chimeras designed to target two to four virus species (Table V). Single arm lengths of each hairpin construct ranged from 418 nucleotides to 1100 nucleotides to accommodate the insert size limit (including linker) of the plant transformation vector plasmid.
♦SEQ
♦= Entire construct (left arm, right arm) with GUS linker (SEQ ID NO: 109);
2. Construct Synthesis, Cloning, and Transformation
Five of the 11 transgene constructs (Wheat-A, -D, -G, —H, and -K) were synthesized (with BAMHI restriction sites added to 5′ and 3′ end of the construct) and sub-cloned by GenScript (Piscataway, N.J., USA) into the monocot transformation vector pAHC17. The five constructs and their virus targets are: Wheat-A (BYDV & WSMV, SEQ ID NO:69), Wheat-D (SBWMV, BYDV, WSMV, SEQ ID NO:72), Wheat-G (SEQ ID NO:75), Wheat-H (SEQ ID NO:76), and Wheat-K (WSSMV, SBWMV, BYDV, WSMV, SEQ ID NO:79). Constructs were synthesized as a hairpin and included a GUS-linker region (929 nucleotides from pANDA35HK plasmid sequence as a spacer between hairpin arms (SEQ ID NO:109).
The five resulting transformation plasmids (pAHC17::Wheat-transgene) were sequenced to confirm the correct transgene sequence and orientation (all 5 hairpin sequences were confirmed), and plasmid DNA was used to transform TOP10 Cells (Invitrogen). Plasmid DNA was purified using the PureLink® HiPure Plasmid Filter Maxiprep Kit (Invitrogen) at maximum yield for each of the 5 plasmids.
Particle bombardment transformation of the spring wheat cultivar ‘Bobwhite’ was carried out by the Kansas State University Plant Transformation Facility Service. For each construct, multiple co-bombardments of pAHC-17::Wheat-transgene & pAHC-20 (BAR gene, herbicide selection) were performed in ‘Bobwhite’ tissue culture.
3. Confirmation of Transgene Presence and Expression in T0 Transformants
Resulting transformants (T0 generation) were transplanted to soil, screened for resistance to Liberty herbicide (BAR gene-expressing), and leaf tissue sampled from 2 or 3 tillers per T0 herbicide-resistant plant. The presence of the transgene construct was verified by PCR, while RT-PCR was used to verify expression of the transgene (hairpin). Leaves were collected from young wheat plants and DNA and RNA were extracted from leaf samples using the DNeasy® Plant Mini Kit (Qiagen, Hilden, Germany) and Trizol Reagent method (Life Technologies/ThermoFisher Scientific), respectively. Construct specific primers (Table VI) were used to detect the transgene via polymerase chain reaction (PCR) with PCR being carried out using GoTaq® DNA Polymerase (Promega, Madison, Wis., USA) according to the following program: a 2-min heating step at 95° C. followed by 30 cycles of 30 sec melting at 95° C., 30 sec annealing at 51° C., and 1 min elongation at 72° C. with a final extension of 2 min at 72° C. cDNA was prepared from extracted total RNA using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, Mass., USA) using the RT-enhancer to remove possible contaminating DNA.
cDNA was then tested using the same primers according to the protocol described above for PCR. PCR and RT-PCR results were visualized on 1.0% agarose gels after staining with GelRed™ (Biotium, Fremont, Calif.). T0 plants that tested positive for the transgene and expression of the hairpin were grown to maturity, and seeds were collected (T1, progeny of T0). The number of T0 plants testing positive for the transgene presence and expression of the hairpin (dsRNA trigger of RNAi) are reported in Table VII.
1plants analyzed
2tillers analyzed
1plants have multiple tillers per plant; those selected for molecular analysis had tested positive for the BAR gene (selection marker for transformation) by exhibiting resistance to topically-applied Liberty herbicide (performed by the plant transformation facility service).
2collection of tillers over all plants analyzed.
4. Presence and Expression of Transgene in T1 Plants (Progeny of T0 Transgenics).
Multiple seeds from 4 lines of the Wheat-H transgenics (4 T0 parent plants) were sown in soil mix and grown under greenhouse conditions to produce T1 plants, providing a total of 33 lines. Non-transformed ‘Bobwhite’ plants were grown as negative controls. DNA and RNA were isolated from leaf tissue as described above. Using H arm-specific primers (Table VI, expected amplicon size=560 bp), 12 lines tested positive for the Wheat-H transgene (
The T1 plants expressing the H transgene (hairpin) appeared healthy, producing multiple tillers and heads comparable to the wild-type ‘Bobwhite’ controls (
The present application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 62/332,055, filed May 5, 2016, entitled MULTIGENIC TRANSGENIC RESISTANCE TO CEREAL VIRUSES BY RNA-INTERFERENCE, incorporated by reference in its entirety herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/031068 | 5/4/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62332055 | May 2016 | US |
