Patent Grant
11041166
The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 070294-0127SEQLST.TXT, created on Dec. 11, 2017, and having a size of 440 kilobytes, and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
The present invention relates to the fields of gene isolation and plant improvement, particularly to enhancing the resistance of plants to plant disease through the use of disease resistance genes.
Late blight, caused by oomycete pathogen Phytophthora infestans, is a devastating disease of cultivated potato (Solanum tuberosum) and tomato (Solanum lycopersicum), causing several billion dollars annual losses (Jones (2014) Philos. Trans. R. Soc. Lond. B Biol. Sci. 369:20130087-20130087). It was estimated that only in Europe late blight cost in potato production is over 1 billion euros including costs of control and damage caused by the pathogen (Haverkort (2008) Potato Res. 51:47-57).
Plant breeders have typically introduced one Rpi (i.e. Resistance to Phytophthora infestans) gene at a time from wild relatives into cultivated potato. However, this process is laborious and slow, and so far has resulted in an Rpi gene that is overcome by new P. infestans races in less time than it took to breed the new potato variety that contains it (Jones et al. 2014). A transgenic approach allows introduction of several genes at the same time (‘gene stacking’), providing more durable resistance. Several major genes conferring resistance against late blight has been reported, however due to quick P. infestans evolution, there is still need to clone additional Rpi genes.
Cloned Rpi genes and their functional alleles include, for example: Rpi-blb1/RB from Solanum demissum (van der Vossen et al. (2003) Plant J. 36:867-882; Song et al. (2003) PNAS 100:9128-9133) and its homologues Rpi-sto1 and Rpi-pta1 from S. stoloniferum and S. papita, respectively (Vleeshouwers et al. (2008) PLOS ONE 3:e2875); Rpi-blb2 from S. demissum (van der Vossen E A et al. (2005) Plant J. 44:208-222); Rpi-blb3 and its homologues Rpi-abpt and R2-like from S. bulbocastanum and R2 from S. demissum (Lokossou et al. (2009) MPMI 22:630-641) and additional homologues Rpi-edn1.1. Rpi-edn1.2. Rpi-snk1.1. Rpi-snk1.2 and Rpi-hjt1.1-Rpi-hjt1.3 from S. edinense, S. schenckii and S. hjertingii, respectively, described by Champouret ((2010) “Functional genomics of Phytophthora infestans effectors and Solanum resistance genes,” Ph.D. Thesis, Wageningen Univ., Wageningen); Rpi-bt1 from S. demissum (Oosumi et al. (2009) Amer. J. Potato Res. 86:456-465); R1 from S. demissum (Ballvora et al. (2002) Plant J. 30:361-71); R3a and R3b from S. demissum (Huang et al. (2005) Plant J. 42:261-271; Li et al. (2011) MPMI 24:1132-1142; respectively); Rpi-vnt1.1, Rpi-vnt1.2. Rpi-vnt1.3 from S. venturii (Foster et al. (2009) MPMI22:589-600; Pel et al. (2009) MPMI 22:601-615; WO2009013468); Rpi-mcq1 from S. mochiquense (WO2009013468); Rpi-chc from S. chacoense (WO 2011/034433) and Ph-3 from S. pimpinellifolium (Zhang et al. (2014) Theor. Appl. Genet. 127:1353-1364).
Solanum nigrum and closely related species are generally regarded as non-hosts for infection by P. infestans. They are not infected under laboratory conditions, and infections are very rarely observed in the field (Lebecka (2009) Eur. J. Plant Pathol. 124:345-348). However, there is one report of S. nigrum susceptibility to P. infestans infection, and of Mendelian segregation for resistance when a susceptible line is crossed to a resistant line, and the F1 selfed to produce F2 progeny (Lebecka (2008) Eur. J. Plant Pathol. 120:233-240; Lebecka (2009) Eur. J. Plant Pathol. 124:345-348). This resistance under strong pathogen pressure suggests that resistance genes present in S. nigrum might have unique efficacy and recognition specificities, making them valuable to clone and characterize. S. nigrum is a hexaploid plant of complex polyploid origin, making classical map-based cloning laborious and time consuming.
Recently, the cloning of a new Rpi gene, Rpi-amr3i, from a Mexican accession of Solanum americanum was reported (Witek et al. (2016) Nat. Biotechnol. 34: 656). S. americanum is an herbaceous flowering plant growing worldwide that has been reported to be a putative diploid ancestor of S. nigrum (Poczai and Hyvonen (2010) Mol. Biol. Rep. 38:1171-1185). Due to the rapid evolution of P. infestans races that can overcome the existing Rpi genes, additional new Rpi genes will be needed soon to combat late blight disease in potatoes and tomatoes. Because the cloning of new Rpi genes from diploid Solanaceous species like S. americanum is expected to be less time consuming than cloning Rpi genes from a Solanaceous species with a complex polyploid genome like S. nigrum, the use of diploid Solanaceous species as a source of Rpi genes may allow researchers to clone new Rpi genes more quickly to provide plant breeders with new sources of resistance against late blight caused by P. infestans.
The present invention provides nucleic acid molecules for resistance (R) genes that are capable of conferring to a plant, particularly a solanaceous plant, resistance to at least one race of a Phytophthora species (sp.) that is known to cause a plant disease in the plant. In one embodiment, the present invention provides nucleic acid molecules comprising an R gene, which is referred to herein as Rpi-amr1e, and its variants including, for example, alleles of Rpi-amr1e, homologs of Rpi-amr1e, and other naturally and non-naturally occurring variants of Rpi-amr1e. In another embodiment, the present invention provides nucleic acid molecules comprising an R gene, which is referred to herein as Rpi-amr6b, and its variants including, for example, alleles of Rpi-amr6b, homologs of Rpt-amr6b, and other naturally and non-naturally occurring variants of Rpi-amr6b. In yet another embodiment, the present invention provides nucleic acid molecules comprising an R gene, which is referred to herein as Rpi-amr7d, and its variants including, for example, alleles of Rpi-amr7d, and homologs of Rpi-amr7d, and other naturally and non-naturally occurring variants of Rpi-amr7d. In a further embodiment, the present invention provides nucleic acid molecules comprising an R gene, which is referred to herein as Rpi-amr8c, and its variants including, for example, alleles of Rpi-amr8c, homologs of Rpi-amr8c, and other naturally and non-naturally occurring variants of Rpi-amr8c.
The present invention additionally provides plants, plant cells, and seeds comprising in their genomes one or more heterologous polynucleotides of the invention. The heterologous polynucleotides comprise a nucleotide sequence encoding a resistance (R) protein of the present invention. Such R proteins are encoded by the R genes of the present invention, particularly Rpi-amr1e, Rpi-amr6b, Rpi-amr7d, and Rpi-amr8c, and alleles, homologs, and other naturally and non-naturally occurring variants of such R genes. In a preferred embodiment, the plants and seeds are transgenic solanaceous plants and seeds that have been transformed with one or more heterologous polynucleotides of the invention. Preferably, such solanaceous plants comprise enhanced resistance to at least one race of a Phytophthora sp. that is known to cause a plant disease in a solanaceous plant, when compared to the resistance of a control plant that does not comprise the heterologous polynucleotide. Solanaceous plants of the invention include, but are not limited to, domesticated solanaceous plants including, for example, domesticated varieties of potato and tomato.
The present invention provides methods for enhancing the resistance of a plant, particularly a solanaceous plant, to a plant disease caused by at least one race of at least one Phytophthora sp. Such methods comprise introducing into at least one plant cell a heterologous polynucleotide comprising a nucleotide sequence of an R gene of the present invention. Preferably, the heterologous polynucleotide or part thereof is stably incorporated into the genome of the plant cell. The methods can optionally further comprise regenerating the plant cell into a plant that comprises in its genome the heterologous polynucleotide. Preferably, such a plant comprises enhanced resistance to a plant disease caused by at least one race of a Phytophthora sp., relative to a control plant not comprising the heterologous polynucleotide. More preferably, such a plant comprises enhanced resistance to plant disease(s) caused by at least two, three, four, five, or more races of a Phytophthora sp., relative to a control plant not comprising the heterologous polynucleotide.
The present invention additionally provides methods for identifying a solanaceous plant that displays newly conferred or enhanced resistance to a plant disease caused by at least one race of a Phytophthora sp. The methods comprise detecting in the solanaceous plant the presence of Rpi-amr1e, Rpi-amr6b, Rpi-amr7d, and/or Rpi-amr8c, and/or alleles, homologs, and other naturally and non-naturally occurring variants of such R genes.
Methods of using the plants of the present invention in agricultural crop production to limit plant disease caused by at least one race of a Phytophthora sp. are also provided. The methods comprise planting a plant (e.g. a seedling), a tuber, or a seed of the present invention, wherein the plant, tuber, or seed comprises at least one R gene nucleotide sequence of the present invention. The methods further comprise growing a plant under conditions favorable for the growth and development of the plant, and optionally harvesting at least one fruit, tuber, leaf, or seed from the plant.
Additionally provided are plants, plant parts, seeds, plant cells, other host cells, expression cassettes, and vectors comprising one or more of the nucleic acid molecules of the present invention.
The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5′ end of the sequence and proceeding forward (i.e. from left to right in each line) to the 3′ end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e. from left to right in each line) to the carboxy terminus.
SEQ ID NO: 1 sets forth a nucleotide sequence of the R gene, Rpi-amr1e, Solanum americanum.
SEQ ID NO: 2 sets forth the amino acid sequence of Rpi-amr1e, the R protein encoded by Rpi-amr1e.
SEQ ID NO: 3 sets forth the nucleotide sequence of the coding region of the Rpi-amr1e cDNA. If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 3. The native stop codon of this cDNA is TGA.
SEQ ID NO: 4 sets forth a nucleotide sequence of the Rpi-amr1e allele from S. americanum accession A14750130.
SEQ ID NO: 5 sets forth the amino acid sequence of the R protein encoded by the Rpi-amr1e allele from S. americanum accession A14750130.
SEQ ID NO: 6 sets forth the nucleotide sequence of the coding region of the cDNA of the Rpi-amr1e allele from S. americanum accession A14750130. If desired, a stop codon (e.g. TAA, TAG. TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 6. The native stop codon of this cDNA is TGA.
SEQ ID NO: 7 sets forth a nucleotide sequence of the Rpi-amr1e allele from S. americanum accession Veg422.
SEQ ID NO: 8 sets forth the amino acid sequence of the R protein encoded by the Rpi-amr1e allele from S. americanum accession Veg422.
SEQ ID NO: 9 sets forth the nucleotide sequence of the coding region of the cDNA of the Rpi-amr1e allele from S. americanum accession Veg422. If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 9. The native stop codon of this cDNA is TGA.
SEQ ID NO: 10 sets forth a nucleotide sequence of the Rpi-amr1e allele from S. americanum accession Wang2058.
SEQ ID NO: 11 sets forth the amino acid sequence of the R protein encoded by the Rpi-amr1e allele from S. americanum accession Wang2058.
SEQ ID NO: 12 sets forth the nucleotide sequence of the coding region of the cDNA of the Rpi-amr1e allele from S. americanum accession Wang2058. If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 12. The native stop codon of this cDNA is TGA.
SEQ ID NO: 13 sets forth a nucleotide sequence of the Rpi-amr1e allele from S. americanum accession sn27.
SEQ ID NO: 14 sets forth the amino acid sequence of the R protein encoded by the Rpi-amr1e allele from S. americanum accession sn27.
SEQ ID NO: 15 sets forth the nucleotide sequence of the coding region of the cDNA of the Rpi-amr1e allele from S. americanum accession sn27. If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 15. The native stop codon of this cDNA is TGA.
SEQ ID NO: 16 sets forth a nucleotide sequence of the Rpi-amr1e allele from S. americanum accession SOLA425.
SEQ ID NO: 17 sets forth the amino acid sequence of the R protein encoded by the Rpi-amr1e allele from S. americanum accession SOLA425.
SEQ ID NO: 18 sets forth the nucleotide sequence of the coding region of the cDNA of the Rpi-amr1e allele from S. americanum accession SOLA425. If desired, a stop codon (e.g. TAA, TAG. TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 18. The native stop codon of this cDNA is TAA.
SEQ ID NO: 19 sets forth a nucleotide sequence of the Rpi-amr1e allele from S. americanum accession A14750006.
SEQ ID NO: 20 sets forth the amino acid sequence of the R protein encoded by the Rpi-amr1e allele from S. americanum accession A14750006.
SEQ ID NO: 21 sets forth the nucleotide sequence of the coding region of the cDNA of the Rpi-amr1e allele from S. americanum accession A14750006. If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 21. The native stop codon of this cDNA is TGA.
SEQ ID NO: 22 sets forth a nucleotide sequence of the R gene, Rpi-amr1e. The promoter regions spans nucleotides 1-1633 and the terminator region spans nucleotides 6443-7349.
SEQ ID NO: 23 sets forth the nucleotide sequence of the coding region of the cDNA of splice variant 1 of Rpi-amr1e (SEQ ID NO: 22). If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 23. The native stop codon of this cDNA is TAA.
SEQ ID NO: 24 sets forth the amino acid sequence of the R protein encoded by the splice variant 1 cDNA set forth in SEQ ID NO: 23.
SEQ ID NO: 25 sets forth the nucleotide sequence of the coding region of the cDNA of splice variant 2 of Rpi-amr1e (SEQ ID NO: 22). If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 25. The native stop codon of this cDNA is TAA.
SEQ ID NO: 26 sets forth the nucleotide sequence of the coding region of the cDNA of splice variant 3 of Rpi-amr1e (SEQ ID NO: 22). If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 26. The native stop codon of this cDNA is TAA.
SEQ ID NO: 27 sets forth the nucleotide sequence of the coding region of the cDNA of splice variant 4 of Rpi-amr1e (SEQ ID NO: 22). If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 27. The native stop codon of this cDNA is TGA.
SEQ ID NO: 28 sets forth the amino acid sequence of the R protein encoded by the splice variant 2 cDNA set forth in SEQ ID NO: 25.
SEQ ID NO: 29 sets forth the amino acid sequence of the R protein encoded by the splice variant 3 cDNA set forth in SEQ ID NO: 26.
SEQ ID NO: 30 sets forth the amino acid sequence of the R protein encoded by the splice variant 4 cDNA set forth in SEQ ID NO: 27.
SEQ ID NO: 31 sets forth a nucleotide sequence of SP1032 allele of the R gene, Rpi-amr1e. The promoter regions spans nucleotides 1-1823 and the terminator region spans nucleotides 6944-7913.
SEQ ID NO: 32 sets forth a nucleotide sequence of SP1123 allele of the R gene. Rpi-amr1e. The promoter regions spans nucleotides 49-1577 and the terminator region spans nucleotides 6705-7662.
SEQ ID NO: 33 sets forth a nucleotide sequence of SP2272 allele of the R gene, Rpi-amr1e. The promoter regions spans nucleotides 641-1745 and the terminator region spans nucleotides 6802-7770.
SEQ ID NO: 34 sets forth a nucleotide sequence of SP2307 allele of the R gene, Rpi-amr1e. The promoter regions spans nucleotides 1-1991 and the terminator region spans nucleotides 9253-9596.
SEQ ID NO: 35 sets forth a nucleotide sequence of SP3408 allele of the R gene, Rpi-amr1e. The promoter regions spans nucleotides 1-1405 and the terminator region spans nucleotides 7567-8398.
SEQ ID NO: 36 sets forth the nucleotide sequence of the coding region of a cDNA of the SP1032 allele of Rpi-amr1e. If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 36. The native stop codon of this cDNA is TAA.
SEQ ID NO: 37 sets forth the nucleotide sequence of the coding region of a cDNA of the SP1123 allele of Rpi-amr1e. If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 37. The native stop codon of this cDNA is TAA.
SEQ ID NO: 38 sets forth the nucleotide sequence of the coding region of a cDNA of the SP2272 allele of Rpi-amr1e. If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 38. The native stop codon of this cDNA is TAA.
SEQ ID NO: 39 sets forth the nucleotide sequence of the coding region of a cDNA of the SP2307 allele of Rpi-amr1e. If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 39. The native stop codon of this cDNA is TAA.
SEQ ID NO: 40 sets forth the nucleotide sequence of the coding region of a cDNA of the SP3408 allele of Rpi-amr1e. If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 40. The native stop codon of this cDNA is TAA.
SEQ ID NO: 41 sets forth the amino acid sequence of the R protein encoded by the SP1032 cDNA sequence set forth in SEQ ID NO: 36.
SEQ ID NO: 42 sets forth the amino acid sequence of the R protein encoded by the SP1123 cDNA sequence set forth in SEQ ID NO: 37.
SEQ ID NO: 43 sets forth the amino acid sequence of the R protein encoded by the SP2272 cDNA sequence set forth in SEQ ID NO: 38.
SEQ ID NO: 44 sets forth the amino acid sequence of the R protein encoded by the SP2307 cDNA sequence set forth in SEQ ID NO: 39.
SEQ ID NO: 45 sets forth the amino acid sequence of the R protein encoded by the SP3408 cDNA sequence set forth in SEQ ID NO: 40.
SEQ ID NO: 46 sets forth a nucleotide sequence of the R gene, Rpi-amr6b, from Solanum nigrescens accession A14750423. The promoter regions spans nucleotides 1-2030 and the terminator region spans nucleotides 7162-8005.
SEQ ID NO: 47 sets forth the amino acid sequence of the R protein encoded by the splice variant 1 of Rpi-amr6b (SEQ ID NO: 46). A cDNA of splice variant 1 is set forth in SEQ ID NO: 49.
SEQ ID NO: 48 sets forth the amino acid sequence of the R protein encoded by the splice variant 2 of Rpi-amr6b (SEQ ID NO: 46). A cDNA of splice variant 2 is set forth in SEQ ID NO: 50.
SEQ ID NO: 49 sets forth the nucleotide sequence of the coding region of the cDNA of splice variant 1 of Rpi-amr6b (SEQ ID NO: 46). If desired, a stop codon (e.g. TAA. TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 49. The native stop codon of this cDNA is TAA.
SEQ ID NO: 50 sets forth the nucleotide sequence of the coding region of the cDNA of splice variant 1 of Rpi-amr6b (SEQ ID NO: 46). If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 50. The native stop codon of this cDNA is TAA.
SEQ ID NO: 51 sets forth a nucleotide sequence of the R gene, Rpi-amr7d, from S. americanum accession A54750014. The promoter regions spans nucleotides 1-1960 and the terminator region spans nucleotides 7032-7842.
SEQ ID NO: 52 sets forth the amino acid sequence of the R protein encoded by the splice variant 1 of Rpi-amr7d (SEQ ID NO: 51). A cDNA of splice variant 1 is set forth in SEQ ID NO: 54.
SEQ ID NO: 53 sets forth the amino acid sequence of the R protein encoded by the splice variant 2 of Rpi-amr7d (SEQ ID NO: 51). A cDNA of splice variant 2 is set forth in SEQ ID NO: 55.
SEQ ID NO: 54 sets forth the nucleotide sequence of the coding region of the cDNA of splice variant 1 of Rpi-amr7d (SEQ ID NO: 51). If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 54. The native stop codon of this cDNA is TAA.
SEQ ID NO: 55 sets forth the nucleotide sequence of the coding region of the cDNA of splice variant 2 of Rpi-amr7d (SEQ ID NO: 51). If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 55. The native stop codon of this cDNA is TAA.
SEQ ID NO: 56 sets forth a nucleotide sequence of the R gene, Rpi-amr8c, from S. americanum accession SOLA 226. The promoter regions spans nucleotides 1-1953 and the terminator region spans nucleotides 7078-7456.
SEQ ID NO: 57 sets forth the amino acid sequence of the R protein encoded by the splice variant 1 of Rpi-amr8c (SEQ ID NO: 56). A cDNA of splice variant 1 is set forth in SEQ ID NO: 60.
SEQ ID NO: 58 sets forth the amino acid sequence of the R protein encoded by the splice variant 2 of Rpi-amr8c (SEQ ID NO: 56). A cDNA of splice variant 2 is set forth in SEQ ID NO: 59.
SEQ ID NO: 59 sets forth the nucleotide sequence of the coding region of the cDNA of splice variant 2 of Rpi-amr8c (SEQ ID NO: 56). If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 59. The native stop codon of this cDNA is TAA.
SEQ ID NO: 60 sets forth the nucleotide sequence of the coding region of the cDNA of splice variant 1 of Rpi-amr8c (SEQ ID NO: 56). If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 60. The native stop codon of this cDNA is TAA.
SEQ ID NO: 61 sets forth a nucleotide sequence of the R gene, Rpi-amr9d, from S. americanum accession SOLA425. The promoter regions spans nucleotides 1-1991 and the terminator region spans nucleotides 9269-9596.
SEQ ID NO: 62 sets forth the amino acid sequence of the R protein encoded by the splice variant 1 of Rpi-amr9d (SEQ ID NO: 61). A cDNA of splice variant 1 is set forth in SEQ ID NO: 60.
SEQ ID NO: 63 sets forth the amino acid sequence of the R protein encoded by the splice variant 2 of Rpi-amr9d (SEQ ID NO: 61). A cDNA of splice variant 2 is set forth in SEQ ID NO: 60.
SEQ ID NO: 64 sets forth the nucleotide sequence of the coding region of the cDNA of splice variant 2 of Rpi-amr9d (SEQ ID NO: 61). If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 64. The native stop codon of this cDNA is TAA.
SEQ ID NO: 65 sets forth the nucleotide sequence of the coding region of the cDNA of splice variant 1 of Rpi-amr9d (SEQ ID NO: 61). If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3′ end of nucleic acid molecule comprising SEQ ID NO: 65. The native stop codon of this cDNA is TAA.
The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
The present invention relates to the isolation of plant resistance (R) genes, particularly R genes that confer upon a solanaceous plant resistance to late blight disease caused by one or more multiple races of Phytophthora infestans. As disclosed hereinbelow, an R gene, referred to herein as Rpi-amr1e, was isolated from Solanum americanum accession 954750184, a diploid, non-tuber-bearing relative of potato, using a map-based cloning approach with fine mapping on 1793 F2 plants and sequencing of co-segregating BAC clones. Additional Rpi-amr1e alleles from Veg422, A14750130, Wang 2058, sn27, A14750006 and SOLA425 S. americanum accessions were isolated using a method involving R gene sequence capture (RenSeq) with long-read sequencing that has been previously described (Eid et al. (2008) Science 323:133-138; Sharon et al. (2013) Nat. Biotechnol. 31:1009-14; both of which are herein incorporated by reference). The isolation of additional Rpi-amr1e alleles from S. americanum accessions 954750174, A14750130, and 954750172 is disclosed hereinbelow in Example 8. Also disclosed hereinbelow in Examples 9-16 is the isolation of three additional R genes that are homologs of Rpi-amr1e: Rpi-amr6b from Solanum nigrescens accession A14750423; Rpi-amr7d from S. americanum accession A54750014; and Rpi-amr8c from S. americanum accession SOLA 226.
The present invention provides nucleic acid molecules comprising the nucleotide sequences of R genes, particularly the nucleotide sequences of Rpi-amr1e, Rpi-amr6b, Rpi-amr7d, and Rpi-amr8c and alleles, homologs, orthologs, and other naturally occurring variants of such R genes and synthetic or artificial (i.e. non-naturally occurring) variants thereof. As used herein, such nucleic acid molecules are referred to herein as “Rpi-amr nucleic acid molecules” or “Rpi-amr genes”, unless stated otherwise or apparent from the context of use. Likewise, the nucleotide sequences of Rpi-amr1e, Rpi-amr6b. Rpi-amr7d, and Rpi-amr8c and alleles, homologs, orthologs, and other naturally occurring variants of such R genes and synthetic or artificial (i.e. non-naturally occurring) variants thereof are referred to herein as “Rpi-amr nucleotide sequences” unless stated otherwise or apparent from the context of use.
The Rpi-amr nucleotide sequences of the present invention are nucleotide sequences of R genes, which are also referred to herein as R gene nucleotide sequences. Preferably, such nucleotide sequences of R genes encode R proteins. Rpi-amr nucleotide sequences of the invention include, but not limited to, the nucleotide sequences of wild-type Rpi-amr1e, Rpi-amr6b. Rpi-amr7d, and Rpi-amr8c genes comprising a native promoter and the 3′ adjacent region comprising the coding region, cDNA sequences, and nucleotide sequences comprising only the coding region. Examples of such Rpi-amr nucleotide sequences include the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 49, 50, 51, 54, 55, 56, 59, 60, 61, 64, and 65 and variants thereof. In embodiments in which the native Rpi-amr gene promoter is not used to drive the expression of the nucleotide sequence encoding the R protein, a heterologous promoter can be operably linked a nucleotide sequence encoding an R protein of the invention to drive the expression of nucleotide sequence encoding an R protein in a plant.
Preferably, the R proteins encoded by the Rpi-amr nucleotide sequences of the invention are functional R proteins, or part(s), or domain(s) thereof, which are capable of conferring on a plant, particularly a solanaceous plant, comprising the R protein enhanced resistance to a plant disease caused by at least one race of at least one Phytophthora sp. In certain preferred embodiments, the R proteins of the present invention are capable of conferring on a plant broad-spectrum resistance to at least one race, but preferably multiple races, of P. infestans and include, for example, Rpi-amr1e (SEQ ID NO: 2), the R protein encoded by Rpi-amr1e (SEQ ID NO: 1) and the R proteins (SEQ ID NOS: 5, 8, 11, 14, 17, 20, 41, 42, 43, 44, and 45) encoded by the alleles of Rpi-amr1e (SEQ ID NOS: 4, 7, 10, 13, 16, 19, 31, 32, 33, 34, and 35, respectively). Such R proteins of the present invention include, but are not limited to, the R proteins comprising the amino acid sequences set forth in SEQ ID NOS: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, and 63 and/or are encoded by the Rpi-amr nucleotide sequences set forth in SEQ ID NOS: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 49, 50, 51, 54, 55, 56, 59, 60, 61, 64, and 65.
Likewise, preferred Rpi-amr genes, Rpi-amr nucleic acid molecules, and Rpi-amr1e alleles of the present invention are capable of conferring on a plant, particularly a solanaceous plant, comprising the Rpi-amr gene, the Rpi-amr nucleic acid molecule, or Rpi-amr1e allele, enhanced resistance to a plant disease caused by at least one race of at least one Phytophthora sp. In certain preferred embodiments, the Rpi-amr genes. Rpi-amr nucleic acid molecules and Rpi-amr1e alleles of the present invention are capable of conferring on a plant broad-spectrum resistance to at least one race, but preferably multiple races, of P. infestans. Such Rpi-amr genes. Rpi-amr nucleic acid molecules and Rpi-amr1e alleles include, but are not limited to, Rpi-amr genes, Rpi-amr nucleic acid molecules, and Rpi-amr1e alleles comprising a nucleotide sequence selected from the group consisting of: a nucleotide sequences set forth in SEQ ID NO: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 49, 50, 51, 54, 55, 56, 59, 60, 61, 64, or 65; and a nucleotide sequence encoding an amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, or 63.
The present invention further provides plants comprising a heterologous polynucleotide which comprises an R gene nucleotide sequence of the present invention. Preferably, such an R gene nucleotide sequence encodes a full-length R protein of the present invention, or at least a functional part(s) or domain(s) thereof. In some embodiments, such a heterologous polynucleotide of the present invention is stably incorporated into the genome of the plant, and in other embodiments, the plant is transformed by a transient transformation method and the heterologous polynucleotide is not stably incorporated into the genome of the plant.
In other embodiments, a plant comprising a heterologous polynucleotide which comprises an R gene nucleotide sequence of the present invention is produced using a method of the present invention that involves genome editing to modify the nucleotide sequence of a native or non-native gene in the genome of the plant. The native or non-native gene comprises a nucleotide sequence that is different from (i.e. not identical to) an R gene nucleotide sequence of the present invention, and after modification by methods disclosed in further detail hereinbelow, the modified native or non-native gene comprises an R gene nucleotide sequence of the present invention. Generally, such methods comprise the use of a plant comprising in its genome a native or non-native gene wherein the native or non-native gene comprises a nucleotide sequence that is homologous to an R gene nucleotide sequence of the present invention and further comprises introducing into the plant a nucleic acid molecule comprising at least part of an R gene nucleotide sequence of the present invention. Preferably, a nucleotide sequence of native or non-native gene comprises about 70%, 75% 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater nucleotide sequence identity to at least one R gene nucleotide sequence of the present invention. Such a native or non-native gene can be, for example an R gene, particularly an Rpi-amr gene, or a non-functional homolog of such an R gene that is not, or is not known to be, capable of conferring to a plant, resistance to a plant disease. It is recognized that a plant produced by genome engineering as disclosed herein is a stably transformed plant when the native or non-native gene that is modified is stably incorporated in the genome of the plant.
Methods for both the stable and transient transformation of plants and genome editing are disclosed elsewhere herein or otherwise known in the art. In a preferred embodiment of the invention, the plants are stably transformed potato or tomato plants comprising a heterologous polynucleotide of the present invention stably incorporated into their respective genomes and further comprising enhanced resistance to late blight disease caused by at least one race of P. infestans. In a more preferred embodiment of the invention, the plants are stably transformed potato or tomato plants comprising a heterologous polynucleotide of the present invention stably incorporated into their respective genomes and further comprising enhanced resistance to late blight disease caused by at least two, three, four, five, six or more races of P. infestans.
In certain embodiments, a plant of the invention comprises a heterologous polynucleotide which comprises a nucleotide sequence encoding an R protein of the present invention and a heterologous promoter that is operably linked for expression of the nucleotide sequence encoding an R protein. The choice of heterologous promoter can depend on a number of factors such as, for example, the desired timing, localization, and pattern of expression as well as responsiveness to particular biotic or abiotic stimulus. Promoters of interest include, but are not limited to, pathogen-inducible, constitutive, tissue-preferred, wound-inducible, and chemical-regulated promoters.
In certain embodiments of the invention, the plant of the invention, particularly a solanaceous plant, can comprise one, two, three, four, five, six, or more nucleotide sequences encoding an R protein. Typically, but not necessarily, the two or more R proteins will be different from each other. For the present invention, an R protein is different from another R protein when the two R proteins have non-identical amino acid sequences. In certain embodiments of the invention, each of the different R proteins for resistance to a plant disease caused by a Phytophthora sp. has one or more differences in resistance characteristics such as, for example, resistance against a different race and/or group of races of the same Phytophthora sp. or even a different Phytophthora sp. It is recognized that by combining two, three, four, five, six, or more nucleotide sequences with each nucleotide sequence encoding a different R protein for resistance to a different race of a Phytophthora sp. or Phytophthora species (spp.), a solanaceous plant can be produced that comprises broad spectrum resistance against multiple races of a single Phytophthora sp. or even multiple Phytophthora spp. Such a solanaceous plant, particularly a potato or tomato plant, finds use in agriculture in regions where multiple races of a Phytophthora sp., such as, for example, multiple races of P. infestans, are prevalent.
Examples of R genes that can be combined in single potato plant with one or more Rpi-amr nucleotide sequences of the present invention include, but are not limited to, the following cloned Rpi genes: Rpi-amr3i (Accession No. KT373889; SEQ ID NO: 1 of WO 2016/182881) Rpi-blb1 (also known as “RB”; Accession Nos. FB764493.1 and AY336128.1). Rpi-sto1 (Accession No. EU884421), Rpi-pta1 (Accession No. EU884422). Rpi-blb2 (Accession No. DQ122125), Rpi-blb3 (Accession No. FJ536326), Rpi-abpt (Accession No. FJ536324), R2-like (Accession No. FJ536323). R2 (Accession No. FJ536325), Rpi-edn1.1 (Accession No. GU563963). Rpi-edn1.2. Rpi-snk1.1, Rpi-snk1.2, Rpi-hjt1.1-Rpi-hjt1.3 (Accession No. GU563971-3), Rpi-bt1 (Accession No. FJ188415), R1 (Accession No. AF447489). R3a (Accession No. AY849382), R3b (Accession No. JF900492), Rpi-vnt1.1 (Accession No. FJ423044), Rpi-vnt1.2 (Accession No. FJ423045), Rpi-vnt1.3 (Accession No. FJ423046), Rpi-mcq1 (Accession No. GN043561), Rpi-chc, Ph-3 (Accession No. KJ563933), and R8 (Accession No. KU530153). The nucleotide sequences corresponding to the accession numbers of the genes listed above or of any genes or proteins disclosed elsewhere herein can be obtained from publicly accessible, online nucleotide and amino acid sequence databases such as, for example, the GenBank and EMBL databases (available on the World Wide Web at ncbi.nlm.nih.gov/genbank and ebi.ac.uk, respectively).
A plant of the invention comprising multiple R genes can be produced, for example, by transforming a plant that already comprises one or more other R gene nucleotide sequences with a heterologous polynucleotide comprising at least one Rpi-amr nucleotide sequence of the present invention including, for example, one or more of an Rpi-amr1e nucleotide sequence, an Rpi-amr6b nucleotide sequence, an Rpi-amr7d nucleotide sequence, and an Rpi-amr8c nucleotide sequence. Such a plant that already comprises one or more other R gene nucleotide sequences can comprise R genes that are native to the genome or the plant, that were introduced into the plant via sexual reproduction, or that were introduced by transforming the plant or a progenitor thereof with an R gene nucleotide sequence. Alternatively, the one or more other R gene nucleotide sequences can be introduced into a plant of the invention, which already comprises a heterologous polynucleotide of the invention, by, for example, transformation or sexual reproduction.
In other embodiments, two or more different R gene sequences can be introduced into a plant by stably transforming the plant with a heterologous polynucleotide or vector comprising two or more R gene nucleotide sequences. It is recognized that such an approach can be preferred for plant breeding as it is expected that the two or more R gene nucleotide sequences will be tightly linked and thus, segregate a single locus. Alternatively, a heterologous polynucleotide of the present invention can be incorporated into the genome of a plant in the immediate vicinity of another R gene nucleotide sequence using homologous recombination-based genome modification methods that are described elsewhere herein or otherwise known in the art.
The present invention further provides methods for enhancing the resistance of a plant to a plant disease caused by at least one race of at least one Phytophthora sp. The methods comprise modifying at least one plant cell to comprise a heterologous polynucleotide, and optionally regenerating a plant from the modified plant comprising the heterologous polynucleotide. In a first aspect, the methods for enhancing the resistance of a plant to a plant disease caused by at least one race of at least one Phytophthora sp. comprise introducing a heterologous polynucleotide of the invention into at least one plant cell, particular a plant cell from a solanaceous plant. In certain embodiments, the heterologous polynucleotide is stably incorporated into the genome of the plant cell.
In a second aspect, the methods for enhancing the resistance of a plant to a plant disease caused by at least one race of at least one Phytophthora sp. involve the use of a genome-editing method to modify the nucleotide sequences of a native or non-native gene in the genome of the plant cell to comprise a heterologous polynucleotide of the present invention. The methods comprise introducing a nucleic acid molecule into the plant cell, wherein the nucleic acid molecule comprises a nucleotide sequence comprising at least a part of the Rpi-amr nucleotide sequence of the present invention and wherein at least a part of the nucleotide sequence of the native or non-native gene is replaced with at least a part of the nucleotide sequence of the nucleic acid molecule. Thus, the methods of the invention involve gene replacement to produce a heterologous polynucleotide of the present invention in the genome of a plant cell.
If desired, the methods of the first and/or second aspect can further comprise regenerating the plant cell into a plant comprising in its genome the heterologous polynucleotide. Preferably, such a regenerated plant comprises enhanced resistance to a plant disease caused by at least one race of at least one Phytophthora sp., relative to the resistance of a control plant to the plant disease.
The methods of the present invention for enhancing the resistance of a plant to a plant disease caused by at least one race of at least one Phytophthora sp. can further comprise producing a plant comprising two, three, four, five, six, or more nucleotide sequences encoding an R protein, preferably each nucleotide sequence encoding a different R protein. Such a plant comprising multiple R gene nucleotide sequences comprises one or more additional R gene nucleotide sequences of the present invention and/or any other nucleotide sequence encoding an R protein known in the art. It is recognized that the methods of the first and/or second aspect can be used to produce such a plant comprising multiple nucleotide sequences encoding an R protein. Moreover, it is recognized that a heterologous polynucleotide of the present invention can comprise, for example, one or more Rpi-amr nucleotide sequences of the present invention or at least one Rpi-amr nucleotide sequences of the present invention and one or more nucleotide sequences encoding an R protein that is known in the art.
The plants disclosed herein find use in methods for limiting plant disease caused by at least one race of at least one Phytophthora sp. in agricultural crop production, particularly in regions where such a plant disease is prevalent and is known to negatively impact, or at least has the potential to negatively impact, agricultural yield. The methods of the invention comprise planting a plant (e.g. a seedling), tuber, or seed of the present invention, wherein the plant, tuber, or seed comprises at least one R gene nucleotide sequence of the present invention. The methods further comprise growing the plant that is derived from the seedling, tuber, or seed under conditions favorable for the growth and development of the plant, and optionally harvesting at least one fruit, tuber, leaf, or seed from the plant.
The present invention additionally provides methods for identifying a solanaceous plant that displays newly conferred or enhanced resistance to a plant disease caused by at least one race of a Phytophthora sp. The methods find use in breeding solanaceous plants for resistance to plant diseases caused by Phytophthora spp. such as, for example, late blight disease. Such resistant plants find use in the agricultural production of fruits, tubers, leaves, and/or seeds for human or livestock consumption or other use. The methods comprise detecting in a solanaceous plant, or in at least one part or cell thereof, the presence of an Rpi-amr nucleotide sequence of the present invention. In some embodiments of the invention, detecting the presence of the Rpi-amr nucleotide sequence comprises detecting the entire Rpi-amr nucleotide sequence in genomic DNA isolated from a solanaceous plant. In preferred embodiments, however, detecting the presence of an Rpi-amr nucleotide sequence comprises detecting the presence of at least one marker within the Rpi-amr nucleotide sequence. In other embodiments of the invention, detecting the presence of an Rpi-amr nucleotide sequence comprises detecting the presence of the R protein encoded by the Rpi-amr nucleotide sequence using, for example, immunological detection methods involving antibodies specific to the R protein.
In the methods for identifying a solanaceous plant that displays newly conferred or enhanced resistance to a plant disease caused by at least one race of a Phytophthora sp., detecting the presence of the Rpi-amr nucleotide sequence in the solanaceous plant can involve one or more of the following molecular biology techniques that are disclosed elsewhere herein or otherwise known in the art including, but not limited to, isolating genomic DNA and/or RNA from the plant, amplifying nucleic acid molecules comprising the Rpi-amr nucleotide sequence and/or marker therein by PCR amplification, sequencing nucleic acid molecules comprising the Rpi-amr nucleotide sequence and/or marker, identifying the Rpi-amr nucleotide sequence, the marker, or a transcript of the Rpi-amr nucleotide sequence by nucleic acid hybridization, and conducting an immunological assay for the detection of the R protein encoded by the Rpi-amr nucleotide sequence. It is recognized that oligonucleotide probes and PCR primers can be designed to identity the Rpi-amr nucleotide sequences of the present invention and that such probes and PCR primers can be utilized in methods disclosed elsewhere herein or otherwise known in the art to rapidly identify in a population of plants one or more plants comprising the presence of an Rpi-amr nucleotide sequence of the present invention.
Depending on the desired outcome, the heterologous polynucleotides of the invention can be stably incorporated into the genome of the plant cell or not stably incorporated into genome of the plant cell. If, for example, the desired outcome is to produce a stably transformed plant with enhanced resistance to a plant disease caused by at least one race of a Phytophthora sp., then the heterologous polynucleotide can be, for example, fused into a plant transformation vector suitable for the stable incorporation of the heterologous polynucleotide into the genome of the plant cell. Typically, the stably transformed plant cell will be regenerated into a transformed plant that comprises in its genome the heterologous polynucleotide. Such a stably transformed plant is capable of transmitting the heterologous polynucleotide to progeny plants in subsequent generations via sexual and/or asexual reproduction. Plant transformation vectors, methods for stably transforming plants with an introduced heterologous polynucleotide and methods for plant regeneration from transformed plant cells and tissues are generally known in the art for both monocotyledonous and dicotyledonous plants or described elsewhere herein.
In other embodiments of the invention in which it is not desired to stably incorporate the heterologous polynucleotide in the genome of the plant, transient transformation methods can be utilized to introduce the heterologous polynucleotide into one or more plant cells of a plant. Such transient transformation methods include, for example, viral-based methods which involve the use of viral particles or at least viral nucleic acids. Generally, such viral-based methods involve constructing a modified viral nucleic acid comprising a heterologous polynucleotide of the invention operably linked to the viral nucleic acid and then contacting the plant either with a modified virus comprising the modified viral nucleic acid or with the viral nucleic acid or with the modified viral nucleic acid itself. The modified virus and/or modified viral nucleic acids can be applied to the plant or part thereof, for example, in accordance with conventional methods used in agriculture, for example, by spraying, irrigation, dusting, or the like. The modified virus and/or modified viral nucleic acids can be applied in the form of directly sprayable solutions, powders, suspensions or dispersions, emulsions, oil dispersions, pastes, dustable products, materials for spreading, or granules, by means of spraying, atomizing, dusting, spreading or pouring. It is recognized that it may be desirable to prepare formulations comprising the modified virus and/or modified viral nucleic acids before applying to the plant or part or parts thereof. Methods for making pesticidal formulations are generally known in the art or described elsewhere herein.
The present invention provides nucleic acid molecules comprising Rpi-amr nucleotide sequences. Preferably, such nucleic acid molecules are capable of conferring upon a host plant, particularly a solanaceous host plant enhanced resistance to a plant disease caused by at least one race of a Phytophthora sp. Thus, such nucleic acid molecules find use in limiting a plant disease caused by at least one race of a Phytophthora sp. in agricultural production. The nucleic acid molecules of the present invention include, but are not limited to, nucleic acid molecules comprising at least one Rpi-amr nucleotide sequence disclosed herein but also additional orthologs and other variants of the Rpi-amr nucleotide sequences that are capable of conferring to a plant resistance to a plant disease caused by at least one race of a Phytophthora sp. Methods are known in the art or otherwise disclosed herein for determining resistance of a plant a plant disease caused by at least one race of a Phytophthora sp., including, for example, the detached leaf assay (DLA) utilizing detached Nicotiana benthamiana leaves that is described elsewhere herein.
The present invention further provides plants and cells thereof, particularly solanaceous plants and cells thereof, comprising Rpi-amr1e, Rpi-amr6b, Rpi-amr7d, and/or Rpi-amr8c, and/or alleles, homologs, and other naturally and non-naturally occurring variants of such R genes, and that are produced by methods that do not involve the introduction of recombinant DNA into the plant or a cell thereof. Such methods can comprise, for example, interspecific hybridizations involving two or more different plant species. In preferred embodiments, the plants are solanaceous plants.
In certain embodiments, the solanaceous plant is any solanaceous plant except a Solanum americanum plant or a Solanum nigrescens plant. In certain other embodiments, the solanaceous plant is any solanaceous plant neither a S. americanum plant nor a S. nigrescens plant. In other embodiments, the solanaceous plant is any solanaceous plant except a S. americanum plant comprising Rpi-amr1e having the nucleotide sequence set forth in SEQ ID NO: 1 and/or 22, and/or one or more of alleles of Rpi-amr1e having the nucleotide sequences set forth in SEQ ID NOS: 4, 7, 10, 13, 16, 19, 31, 32, 33, 34, and 35 wherein Rpi-amr1e and/or one or more of alleles of Rpi-amr1e are the endogenous or native genes in their natural location(s) in the genome.
While it is believed that Rpi-amr nucleotide sequences set forth in SEQ ID NOS: 4, 7, 10, 13, 16, 19, 31, 32, 33, 34, and 35 are the nucleotide sequences of alleles of Rpi-amr1e (SEQ ID NO: 1) of S. americanum, it is recognized that the present invention does not depend on such Rpi-amr nucleotide sequences corresponding to alleles that are present at the Rpi-amr1e locus of S. americanum and/or other solanaceous plant(s). Such Rpi-amr nucleotide sequences, and Rpi-amr nucleic acid molecules and Rpi-amr genes comprising such Rpi-amr nucleotide sequences, find use in the methods and compositions of the present invention as disclosed herein irrespective of whether any such Rpi-amr nucleotide sequence corresponds to an allele of Rpi-amr1e of S. americanum and/or other solanaceous plant.
In yet other embodiments, the solanaceous plant is any solanaceous plant except a S. americanum plant comprising Rpi-amr7d having the nucleotide sequence set forth in SEQ ID NO: 51, wherein Rpi-amr7d is the endogenous or native gene in its natural location(s) in the genome. In still other embodiments, the solanaceous plant is any solanaceous plant except a S. americanum plant comprising Rpi-amr8c having the nucleotide sequence set forth in SEQ ID NO: 56, wherein the Rpi-amr8c is the endogenous or native genes in their natural location(s) in the genome. In further embodiments, the solanaceous plant is any solanaceous plant except a S. nigrescens plant comprising Rpi-amr6b having the nucleotide sequence set forth in SEQ ID NO: 46.
Additionally provided are methods for introducing at least one Rpi-amr gene of present invention into a plant, particularly a solanaceous plant, lacking in its genome the at least one Rpi-amr gene. The Rpi-amr genes of the present invention include, for example, Rpi-amr1e, Rpi-amr6b, Rpi-amr7d, and Rpi-amr8c, and alleles, homologs, and other naturally and non-naturally occurring variants of such R genes, and/or R genes comprising a nucleotide sequence set forth in SEQ ID NO: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 49, 50, 51, 54, 55, 56, 59, 60, 61, 64, or 65 and/or encoding R protein comprising an amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, or 63. The methods comprise crossing (i.e. cross-pollinating) a first plant comprising in its genome at least one copy of an Rpi-amr gene of present invention with a second solanaceous plant lacking in its genome the Rpi-amr gene. The first and second plants can be the same species or can be different solanaceous species, although in preferred embodiments the first and second plants are solanaceous plants. For example, the first plant can be Solanum americanum and the solanaceous plant can be Solanum tuberosum or Solanum lycopersicum. Such a crossing of a first species of a plant to a second species of a plant is known as an interspecific hybridization and can be used to introgress a gene or genes of interest (e.g. Rpi-amr1e) from one species into a related species lacking the gene or genes of interest and typically involves multiple generations of backcrossing of the progeny with the related species and selection at each generation of progeny comprising the gene or genes of interest. Such interspecific hybridization, introgression, and backcrossing methods are well known in the art and can be used in the methods of the present invention. See “Principals of Cultivar Development.” Fehr, 1993. Macmillan Publishing Company, New York; and “Fundamentals of Plant Genetics and Breeding,” Welsh, 1981, John Wiley & Sons, Inc., New York.
In methods of the present invention for introducing at least one Rpi-amr gene of present invention into a plant lacking in its genome the at least one Rpi-amr gene, either the first plant or the second plant can be the pollen donor plant. For example, if the first plant is the pollen donor plant, then the second plant is the pollen-recipient plant. Likewise, if the second plant is the pollen donor plant, then the first plant is the pollen-recipient plant. Following the crossing, the pollen-recipient plant is grown under conditions favorable for the growth and development of the plant and for a sufficient period of time for seed to mature or to achieve an otherwise desirable growth stage for use in a subsequent in vitro germination procedure such as, for example, embryo rescue that is described below. The seed can then be harvested and those seed comprising the Rpi-amr gene(s) identified by any method known in the art including, for example, the methods for identifying a solanaceous plant that displays newly conferred or enhanced resistance to a plant disease caused by at least one race of a Phytophthora sp. that are described elsewhere herein. In certain embodiments, the first plant is a Solanum americanum plant comprising the Rpi-amr gene(s) and the second plant is Solanum americanum plant lacking the Rpi-amr gene(s). In preferred embodiments, the first plant is a Solanum americanum plant comprising the Rpi-amr gene(s) or other solanaceous plant species comprising in its genome the Rpi-amr gene(s) and the second plant is a solanaceous plant species other than Solanum americanum. Preferred solanaceous plants are potato, tomato, eggplant, pepper, tobacco, and petunia.
It is recognized, however, that in certain embodiments of the invention involving interspecific hybridizations, it may be advantageous to harvest the seed resulting from such interspecific hybridizations at an immature growth stage and then to germinate the immature seeds in culture (i.e. in vitro), whereby the seeds are allowed germinate in culture using methods known in art as “embryo rescue” methods. See Reed (2005) “Embryo Rescue,” in Plant Development and Biotechnology, Trigiano and Gray, eds. (PDF). CRC Press, Boca Raton, pp. 235-239; and Sharma et al. (1996) Euphytica 89: 325-337. It is further recognized that “embryo rescue methods are typically used when mature seeds produced by an interspecific cross display little or no germination, whereby few or no interspecific hybrid plants are produced.
The methods of the present invention find use in producing plants with enhanced resistance to a plant disease caused by at least one race of at least one Phytophthora sp. Typically, the methods of the present invention will enhance or increase the resistance of the subject plant to the plant disease by at least 25%, 50%, 75%, 100%, 150%, 200%, 250%, 500% or more when compared to the resistance of a control plant to the same race or races of Phytophthora sp. Unless stated otherwise or apparent from the context of a use, a control plant for the present invention is a plant that does not comprise the heterologous polynucleotide and/or Rpi-amr1e nucleotide sequence of the present invention. Preferably, the control plant is essentially identical (e.g. same species, subspecies, and variety) to the plant comprising the heterologous polynucleotide of the present invention except the control does not comprise the heterologous polynucleotide or Rpi-amr nucleotide sequence. In some embodiments, the control will comprise a heterologous polynucleotide but not comprise the one or more Rpi-amr nucleotide sequences that are in a heterologous polynucleotide of the present invention.
Additionally, the present invention provides transformed plants, seeds, and plant cells produced by the methods of present invention and/or comprising a heterologous polynucleotide of the present invention. Also provided are progeny plants and seeds thereof comprising a heterologous polynucleotide of the present invention. The present invention also provides fruits, seeds, tubers, leaves, stems, roots, and other plant parts produced by the transformed plants and/or progeny plants of the invention as well as food products and other agricultural products comprising, or produced or derived from, the plants or any part or parts thereof including, but not limited to, fruits, tubers, leaves, stems, roots, and seed. Other agricultural products include, for example, smoking products produced from tobacco leaves (e.g. cigarettes, cigars, and pipe and chewing tobacco) and food and industrial starch products produced from potato tubers. It is recognized that such food products can be consumed or used by humans and other animals including, but not limited to, pets (e.g. dogs and cats), livestock (e.g. pigs, cows, chickens, turkeys, and ducks), and animals produced in freshwater and marine aquaculture systems (e.g. fish, shrimp, prawns, crayfish, and lobsters).
Non-limiting examples of the compositions and methods of the present invention are as follows:
1. A nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 31, 32, 33, 34, 35, 46, 51, 56, or 61;
(b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, or 63, and optionally, wherein the nucleotide sequence is not naturally occurring;
(c) the nucleotide sequence set forth in SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 23, 25, 26, 27, 36, 37, 38, 39, 40, 49, 50, 54, 55, 59, 60, 64, or 65;
(d) a nucleotide sequence having at least 90% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 49, 50, 51, 54, 55, 56, 59, 60, 61, 64, and 65, wherein the nucleic acid molecule is capable of conferring resistance to a plant disease caused by at least one race of at least one Phytophthora sp. to a plant comprising the nucleic acid molecule and optionally, wherein the nucleotide sequence is not naturally occurring; and
(e) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to at least one of the amino acid sequences set forth in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, and 63, wherein the nucleic acid molecule is capable of conferring resistance to a plant disease caused by at least one race of at least one Phytophthora sp. to a plant comprising the nucleic acid molecule and optionally, wherein the nucleotide sequence is not naturally occurring.
2. The nucleic acid molecule of embodiment 1, wherein the nucleic acid molecule is an isolated nucleic acid molecule.
3. An expression cassette comprising the nucleic acid molecule of embodiment 1 or 2 and an operably linked heterologous promoter.
4. A vector comprising the nucleic acid molecule of embodiment 1 or 2 or the expression cassette of embodiment 3.
5. A vector of embodiment 4, further comprising an additional R gene.
6. A host cell transformed with the nucleic acid molecule of embodiment 1 or 2, the expression cassette of embodiment 3, or the vector of embodiment 4 or 5.
7. The host cell of embodiment 6, wherein the host cell is a plant cell, a bacterium, a fungal cell, or an animal cell.
8. The host cell of embodiment 6 or 7, wherein the host cell is a solanaceous plant cell.
9. A plant or plant cell comprising the nucleic acid molecule of embodiment 1 or 2, the expression cassette of embodiment 3, or the vector of embodiment 4 or 5.
10. The plant or plant cell of embodiment 9, wherein the plant is a solanaceous plant and the plant cell is a solanaceous plant cell.
11. The plant of embodiment 10, wherein the solanaceous plant is not Solanum americanum and/or Solanum nigrescens, or wherein the solanaceous plant is selected from the group consisting of potato, tomato, eggplant, pepper, tobacco, and petunia.
12. A plant comprising stably incorporated in its genome a heterologous polynucleotide comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 31, 32, 33, 34, 35, 46, 51, 56, or 61;
(b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, or 63:
(c) the nucleotide sequence set forth in SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 23, 25, 26, 27, 36, 37, 38, 39, 40, 49, 50, 54, 55, 59, 60, 64, or 65;
(d) a nucleotide sequence having at least 90% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 49, 50, 51, 54, 55, 56, 59, 60, 61, 64, and 65, wherein the nucleic acid molecule is capable of conferring resistance to a plant disease caused by at least one race of at least one Phytophthora sp. to a plant comprising the nucleic acid molecule; and
(e) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to at least one of the amino acid sequences set forth in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, and 63, wherein the nucleic acid molecule is capable of conferring resistance to a plant disease caused by at least one race of at least one Phytophthora sp. to a plant comprising the nucleic acid molecule.
13. The plant of embodiment 12, wherein the heterologous polynucleotide comprises the nucleotide sequence of any one of (b)-(e) and further comprises a promoter operably linked for the expression of the nucleotide sequence in a plant.
14. The plant of embodiment 13, wherein the promoter is selected from the group consisting of pathogen-inducible, constitutive, tissue-preferred, wound-inducible, and chemical-regulated promoters.
15. The plant of embodiment any one of embodiments 12-14, wherein the plant is a solanaceous plant.
16. The plant of embodiment any one of embodiments 12-15, wherein the solanaceous plant is selected from the group consisting of potato, tomato, eggplant, pepper, tobacco, and petunia.
17. The plant of any one of embodiments 12-16, wherein the plant comprises enhanced resistance to a plant disease caused by at least one race of at least one Phytophthora sp., relative to a control plant.
18. The plant of embodiment 17, wherein the plant comprises enhanced resistance to late blight caused by at least one race of Phytophthora infestans, relative to a control plant.
19. The plant of any one of embodiments 12-18, wherein the plant is a potato or tomato plant.
20. A method for enhancing the resistance of a plant to a plant disease caused by at least one race of at least one Phytophthora sp., the method comprising modifying at least one plant cell to comprise a heterologous polynucleotide, the heterologous polynucleotide comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 31, 32, 33, 34, 35, 46, 51, 56, or 61;
(b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, or 63:
(c) the nucleotide sequence set forth in SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 23, 25, 26, 27, 36, 37, 38, 39, 40, 49, 50, 54, 55, 59, 60, 64, or 65;
(d) a nucleotide sequence having at least 90% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 49, 50, 51, 54, 55, 56, 59, 60, 61, 64, and 65, wherein the nucleic acid molecule is capable of conferring resistance to a plant disease caused by at least one race of at least one Phytophthora sp. to a plant comprising the nucleic acid molecule; and
(e) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to at least one of the amino acid sequences set forth in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, and 63, wherein the nucleic acid molecule is capable of conferring resistance to a plant disease caused by at least one race of at least one Phytophthora sp. to a plant comprising the nucleic acid molecule.
21. The method of embodiment 20, wherein the heterologous polynucleotide is stably incorporated into the genome of the plant cell.
22. The method of embodiment 20 or 21, wherein the plant cell is regenerated into a plant comprising in its genome the heterologous polynucleotide.
23. The method of any one of embodiments 20-22, wherein modifying at least one plant cell to comprise a heterologous polynucleotide comprises introducing the heterologous polynucleotide into at least one plant cell.
24. The method of any one of embodiments 20-23, wherein the heterologous polynucleotide comprises the nucleotide sequence of any one of (b)-(e) and further comprises a promoter operably linked for the expression of the nucleotide sequence in a plant.
25. The method of embodiment 24, wherein the promoter is selected from the group consisting of pathogen-inducible, constitutive, tissue-preferred, wound-inducible, and chemical-regulated promoters.
26. The method of any one of embodiments 20-22, wherein modifying at least one plant cell to comprise a heterologous polynucleotide comprises using genome editing to modify the nucleotide sequences of a native or non-native gene in the genome of the plant cell to comprise the nucleotide sequence of any one of (a)-(e).
27. The method of embodiment 26, wherein the modifying further comprise introducing a nucleic acid molecule into the plant cell, wherein the nucleic acid molecule comprises a nucleotide sequence comprising at least a part of the nucleotide sequence of any one of (a)-(e).
28. The method of embodiment 27, wherein at least a portion of the at least a part of the nucleotide sequence of the native or non-native gene is replaced with at least a part of the nucleotide sequence of the nucleic acid molecule.
29. The method of any one of embodiments 22-28, wherein the plant comprising the heterologous polynucleotide comprises enhanced resistance to a plant disease caused by at least one race of at least one Phytophthora sp., relative to a control plant.
30. The method of any one of embodiments 22-29, wherein the plant comprising the heterologous polynucleotide comprises enhanced resistance to late blight caused by at least two races of Phytophthora infestans, relative to a control plant.
31. The method of any one of embodiments 20-30, wherein the plant is a potato or a tomato plant.
32. A plant produced by the method of any one of embodiments 20-31.
33. A fruit, tuber, leaf, or seed of the plant of any one of embodiments 9-19 and 32, wherein the fruit, tuber, leaf or seed comprises the heterologous polynucleotide.
34. A method of limiting a plant disease caused by at least one race of at least one Phytophthora sp. in agricultural crop production, the method comprising planting a seedling, tuber, or seed of the plant of any one of embodiments 9-19 and 32 and growing the seedling, tuber, or seed under conditions favorable for the growth and development of a plant resulting therefrom, wherein the seedling, tuber, or seed comprises the nucleic acid molecule, expression cassette, vector, or heterologous polynucleotide.
35. The method of embodiment 34, further comprising harvesting at least one fruit, tuber, leaf and/or seed from the plant.
36. A method for identifying a solanaceous plant that displays newly conferred or enhanced resistance to a plant disease caused by at least one race of at least one Phytophthora sp., the method comprising detecting in the plant, or in at least one part or cell thereof, the presence of an Rpi-amr nucleotide sequence.
37. The method of embodiment 36, wherein the plant disease is late blight caused by at least one race of Phytophthora infestans.
38. The method of embodiment 36 or 37, wherein the solanaceous plant is a potato or tomato plant.
39. The method of any one of embodiments 36-38, wherein the presence of the Rpi-amr nucleotide sequence is detected by detecting at least one marker within the Rpi-amr nucleotide sequence.
40. The method of any one of embodiments 36-39, wherein the Rpi-amr nucleotide sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NOS: 11, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 49, 50, 51, 54, 55, 56, 59, 60, 61, 64, and 65.
41. The method of any one of embodiments 36-39, wherein detecting the presence of the Rpi-amr nucleotide sequence comprises a member selected from the group consisting of PCR amplification, nucleic acid sequencing, nucleic acid hybridization, and an immunological assay for the detection of the R protein encoded by the Rpi-amr nucleotide sequence.
42. A solanaceous plant identified by the method of any one of embodiments 36-41.
43. The solanaceous plant of embodiment 42, wherein the solanaceous plant is not Solanum americanum and/or Solanum nigrescens.
44. A fruit, tuber, leaf, or seed of the solanaceous plant of embodiment 42 or 43.
45. A plant or plant cell comprising: (i) at least one of an Rpi-amr1e, an allele of Rpi-amr1e, Rpi-amr7d, and Rpi-amr8c, wherein the plant is not a Solanum americanum plant and the plant cell is not a Solanum americanum plant cell or (ii) Rpi-amr6b, wherein the plant is not a Solanum nigrescens plant and the plant cell is not a Solanum nigrescens plant cell.
46. The plant or plant cell of embodiment 45, wherein the plant is a solanaceous plant and the plant cell is a solanaceous plant cell.
47. A method for introducing at least one Rpi-amr gene into a plant, the method comprising:
(a) crossing a first plant comprising in its genome at least one copy of at least one Rpi-amr gene with a second plant lacking in its genome the at least one Rpi-amr gene, whereby at least one progeny plant is produced; and
(b) selecting at least one progeny plant comprising in its genome the at least one Rpi-amr gene.
48. The method of embodiment 47, wherein the first plant is Solanum americanum plant and the second plant is not a Solanum americanum plant or wherein the first plant is Solanum nigrescens plant and the second plant is not a Solanum nigrescens plant.
49. The method of embodiment 47 or 48, wherein the second plant is a Solanum tuberosum plant or a Solanum lycopersicum plant.
50. The method of any one of embodiments 47-49, wherein at least one Rpi-amr gene comprises a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 31, 32, 33, 34, 35, 46, 51, 56, or 61:
(b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, or 63;
(c) the nucleotide sequence set forth in SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 23, 25, 26, 27, 36, 37, 38, 39, 40, 49, 50, 54, 55, 59, 60, 64, or 65;
(d) a nucleotide sequence having at least 90% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 49, 50, 51, 54, 55, 56, 59, 60, 61, 64, and 65, wherein the nucleic acid molecule is capable of conferring resistance to a plant disease caused by at least one race of at least one Phytophthora sp. to a plant comprising the nucleic acid molecule; and
(e) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to at least one of the amino acid sequences set forth in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, and 63, wherein the nucleic acid molecule is capable of conferring resistance to a plant disease caused by at least one race of at least one Phytophthora sp. to a plant comprising the nucleic acid molecule.
51. The method of any one of embodiments 47-50, wherein selecting at least one progeny plant comprises detecting in the progeny plant, or in at least one part or cell thereof, the presence of an Rpi-amr nucleotide sequence using the method according to any one of embodiments 36-41.
52. The method of any one of embodiments 47-51, further comprising (i) backcrossing at least one selected progeny plant of (b) to a solanaceous plant that is of the same species and genotype as second solanaceous plant or of the same species as the second solanaceous plant and lacking in its genome the at least one Rpi-amr gene, whereby at least one progeny plant is produced from the backcrossing; and (ii) selecting at least one progeny plant comprising in its genome the at least one Rpi-amr gene that is produced from the backcrossing of (i).
53. A progeny plant according to any one of embodiments 47-52.
54. The progeny plant of embodiment 53, wherein the solanaceous plant is not Solanum americanum and/or Solanum nigrescens.
55. A fruit, tuber, leaf, or seed of the solanaceous plant of embodiment 53 or 54.
56. Use of the plant, fruit, tuber, leaf or seed of any one of embodiments 9-19, 32, 33, 42-46, and 53-55 in agriculture.
57. A human or animal food product comprising, or produced using, the plant, fruit, tuber, leaf and/or seed of any one of embodiments 9-19, 32, 33, 42-46, and 53-54.
58. A polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, or 63:
(b) the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 49, 50, 51, 54, 55, 56, 59, 60, 61, 64, or 65; and
(c) an amino acid sequence having at least 90% sequence identity to at least one of the amino acid sequences set forth in SEQ ID NOS: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, and 63, wherein a polypeptide comprising the amino acid sequence is capable of conferring resistance to a plant disease caused by at least one race of at least one Phytophthora sp. to a plant comprising the polypeptide.
Additional embodiments of the methods and compositions of the present invention are described elsewhere herein.
Unless expressly stated or apparent from the context of usage, the methods and compositions of the present invention can be used with any plant species including, for example, monocotyledonous plants, dicotyledonous plants, and conifers. Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g. B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), triticale (×Triticosecale or Triticum×Secale) sorghum (Sorghum bicolor, Sorghum vulgare), teff (Eragrostis tef), millet (e.g. pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), switchgrass (Panicum virgatum), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossvpium hirsutum), strawberry (e.g. Fragaria×ananassa, Fragaria vesca, Fragaria moschata, Fragaria virginiana, Fragaria chiloensis), sweet potato (Ipomoea batatus), yam (Dioscorea spp., D. rotundata, D. cayenensis, D. alata, D. polystachwya, D. bulbifera, D. esculenta, D. dumetorum, D. trifida), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), oil palm (e.g. Elaeis guineensis, Elaeis oleifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), date (Phoenix dactylifera), cultivated forms of Beta vulgaris (sugar beets, garden beets, chard or spinach beet, mangelwurzel or fodder beet), sugarcane (Saccharum spp.), oat (Avena sativa), barley (Hordeum vulgare), cannabis (Cannabis sativa, C. indica. C. ruderalis), poplar (Populus spp.), eucalyptus (Eucalyptus spp.), Arabidopsis thaliana. Arabidopsis rhizogenes, Nicotiana benthamiana, Brachypodium distachyon vegetables, ornamentals, and conifers and other trees. In specific embodiments, plants of the present invention are crop plants (e.g. potato, tobacco, tomato, maize, sorghum, wheat, millet, rice, barley, oats, sugarcane, alfalfa, soybean, peanut, sunflower, cotton, safflower, Brassica spp., lettuce, strawberry, apple, citrus, etc.).
Vegetables include tomatoes (Lycopersicon esculentum), eggplant (also known as “aubergine” or “brinjal”) (Solanum melongena), pepper (Capsicum annuum), lettuce (e.g. Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), chickpeas (Cicer arietinum), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. Fruit trees and related plants include, for example, apples, pears, peaches, plums, oranges, grapefruits, limes, pomelos, palms, and bananas. Nut trees and related plants include, for example, almonds, cashews, walnuts, pistachios, macadamia nuts, filberts, hazelnuts, and pecans.
In specific embodiments, the plants of the present invention are crop plants such as, for example, maize (corn), soybean, wheat, rice, cotton, alfalfa, sunflower, canola (Brassica spp., particularly Brassica napus, Brassica rapa, Brassica juncea), rapeseed (Brassica napus), sorghum, millet, barley, triticale, safflower, peanut, sugarcane, tobacco, potato, tomato, and pepper.
Preferred plants of the invention are solanaceous plants. As used herein, the term “solanaceous plant” refers to a plant that is a member of the Solanaceae family. Such solanaceous plants include, for example, domesticated and non-domesticated members of Solanaceae family. Solanaceous plants of the present invention include, but are not limited to, potato (Solanum tuberosum), eggplant (Solanum melongena), petunia (Petunia spp., e.g. Petunia×hybrida or Petunia hybrida), tomatillo (Physalis philadelphica), Cape gooseberry (Physalis peruviana), Physalis sp., woody nightshade (Solanum dulcamara), garden huckleberry (Solanum scabrum), gboma eggplant (Solanum macrocarpon), pepper (Capsicum spp; e.g. Capsicum annuum, C. baccaltum, C. chinense, C. frutescens, C. pubescens, and the like), tomato (Solanum lycopersicum or Lycopersicon esculentum), tobacco (Nicotiana spp., e.g. N. tabacum. N. benthamiana), Solanum americanum, Solanum nigrescens Solanum demissum, Solanum stolonferum, Solanum papita, Solanum bulbocastanum, Solanum edinense, Solanum schenckii, Solanum hjertingii, Solanum venturi, Solanum mochiquense, Solanum chacoense, and Solanum pimpinellifolium. In preferred embodiments of the methods and compositions of the present invention, the solanaceous plants are solanaceous plants grown in agriculture including, but not limited to, potato, tomato, tomatillo, Cape gooseberry, eggplant, pepper, tobacco, and petunia In more preferred embodiments, the solanaceous plants are potato and tomato. In even more preferred embodiments, the preferred plant is potato. In certain other embodiments of the methods and compositions disclosed herein, the preferred solanaceous plants are all solanaceous plants except for Solanum americanum and/or Solanum nigrescens. In yet other embodiments of the methods and compositions disclosed herein, the preferred plants are all plants except for Solanum americanum and/or Solanum nigrescens.
The term “solanaceous plant” is intended to encompass solanaceous plants at any stage of maturity or development, as well as any cells, tissues or organs (plant parts) taken or derived from any such plant unless otherwise clearly indicated by context. Solanaceous plant parts include, but are not limited to, fruits, stems, tubers, roots, flowers, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, and the like. As used herein, the term “tuber” is intended to mean a whole tuber or any part thereof such as, for example, a slice or a portion of potato tuber comprising one or more buds (i.e. “eyes”) suitable for planting in a field to produce a potato plant. The present invention also includes seeds produced by the solanaceous plants of the present invention.
The composition and methods of the present invention find us in producing plants with enhanced resistance to at least one race of at least one Phytophthora sp. In preferred embodiments of the invention, the Phytophthora sp. is Phytophthora infestans. In other embodiments, the Phytophthora sp. is a Phytophthora sp. that is capable of causing a plant disease on at least one plant. For the present invention, Phytophthora spp. include, but are not limited to, Phytophthora infestans, Phytophthora parasitica. Phytophthora ramorum, Phytophthora ipomoeae, Phytophthora mirabilis, Phytophthora capsici, Phytophthora porri, Phytophthora sojae, Phytophthora palmivora, and Phytophthora phaseoli.
In one embodiment of the invention, the nucleotide sequences encoding R proteins have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the entire nucleotide sequence set forth in at least one of SEQ ID NOS: 1, 4, 7, 10, 13, 16, 19, 22, 31, 32, 33, 34, 35, 46, 51, 56, and 61 or to a fragment thereof. In another embodiment of the invention, the nucleotide sequences encoding R proteins have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the entire nucleotide sequence set forth in at least one of SEQ ID NOS: 3, 6, 9, 12, 15, 18, 21, 23, 25, 26, 27, 36, 37, 38, 39, 40, 49, 50, 54, 55, 59, 60, 64, and 65 or to a fragment thereof.
The present invention encompasses isolated or substantially purified polynucleotide (also referred to herein as “nucleic acid molecule”, “nucleic acid” and the like) or protein (also referred to herein as “polypeptide”) compositions. An “isolated” or “purified” polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an “isolated” polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e. sequences located at the 5′ and 3′ ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
Fragments and variants of the disclosed polynucleotides and proteins encoded thereby are also encompassed by the present invention. By “fragment” is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of polynucleotides comprising coding sequences may encode protein fragments that retain biological activity of the full-length or native protein. Alternatively, fragments of a polynucleotide that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the invention.
In certain embodiments of the invention, the fragments and variants of the disclosed polynucleotides and proteins encoded thereby are those that are capable of conferring to a plant resistance to a plant disease caused by at least one race of at least one Phytophthora sp. Preferably, a polynucleotide comprising a fragment of a native R polynucleotide of the present invention is capable of conferring resistance to a plant disease caused by at least one race of at least one Phytophthora sp. to a plant comprising the polynucleotide. Likewise, a protein or polypeptide comprising a native R protein of the present invention is preferably capable of conferring resistance to a plant disease caused by at least one race of at least one Phytophthora sp. to a plant comprising the protein or polypeptide.
Polynucleotides that are fragments of a native R polynucleotide comprise at least 16, 20, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000 contiguous nucleotides, or up to the number of nucleotides present in a full-length R polynucleotide disclosed herein.
“Variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a polynucleotide having deletions (i.e. truncations) at the 5′ and/or 3′ end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the R proteins of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode an R protein of the invention. Generally, variants of a particular polynucleotide of the invention will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein. In certain embodiments of the invention, variants of a particular polynucleotide of the invention will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 49, 50, 51, 54, 55, 56, 59, 60, 61, 64, and 65, and optionally comprise a non-naturally occurring nucleotide sequence that differs from the nucleotide sequence set forth in SEQ ID NO: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 49, 50, 51, 54, 55, 56, 59, 60, 61, 64, and/or 65 by at least one nucleotide modification selected from the group consisting of the substitution of at least one nucleotide, the addition of at least one nucleotide, and the deletion of at least one nucleotide. It is understood that the addition of at least one nucleotide can be the addition of one or more nucleotides within a nucleotide sequence of the present invention (e.g. SEQ ID NO: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 49, 50, 51, 54, 55, 56, 59, 60, 61, 64, or 65), the addition of one or more nucleotides to the 5′ end of a nucleotide sequence of the present invention, and/or the addition of one or more nucleotides to the 3′ end of a nucleotide sequence of the present invention.
Variants of a particular polynucleotide of the invention (i.e. the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, a polynucleotide that encodes a polypeptide with a given percent sequence identity to at least one polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, and 63 is disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In certain embodiments of the invention, variants of a particular polypeptide of the invention will have at least about 60%, 65%, 70%, 750/6, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of the amino acid sequences set forth in SEQ ID NOS: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, or 63, and optionally comprises a non-naturally occurring amino acid sequence that differs from at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, and 63 by at least one amino acid modification selected from the group consisting of the substitution of at least one amino acid, the addition of at least one amino acid, and the deletion of at least one amino acid. It is understood that the addition of at least one amino acid can be the addition of one or more amino acids within an amino acid sequence of the present invention (e.g. SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, or 63), the addition of one or more amino acids to the N-terminal end of an amino acid sequence of the present invention, and/or the addition of one or more amino acids to the C-terminal end of an amino acid sequence of the present invention.
“Variant” protein is intended to mean a protein derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and/or C-terminal end of the native protein; deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of an R protein will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%°, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein (e.g. the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, or 63) as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymollette. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington. D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal.
Thus, the genes and polynucleotides of the invention include both the naturally occurring sequences as well as mutant and other variant forms. Likewise, the proteins of the invention encompass naturally occurring proteins as well as variations and modified forms thereof. More preferably, such variants confer to a plant or part thereof comprising the variant enhanced resistance a plant disease caused by at least one race of at least one Phytophthora sp. In some embodiments, the mutations that will be made in the DNA encoding the variant will not place the sequence out of reading frame. Optimally, the mutations will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.
The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by assays that are disclosed herein below.
Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.
The polynucleotides of the invention can be used to isolate corresponding sequences from other organisms, particularly other plants. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire sequences set forth herein or to variants and fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. “Orthologs” is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, isolated polynucleotides that encode R proteins having at least 60% amino acid sequence identity to a full-length amino acid sequence of at least one of the R proteins disclosed herein or otherwise known in the art, or to variants or fragments thereof, are encompassed by the present invention.
In one embodiment, the orthologs of the present invention have coding sequences comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater nucleotide sequence identity to at least one nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 49, 50, 51, 54, 55, 56, 59, 60, 61, 64, and 65 and/or encode proteins comprising least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater amino acid sequence identity to at least one amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 2, 5, 8, 11, 14, 17, 20, 24, 28, 29, 30, 41, 42, 43, 44, 45, 47, 48, 52, 53, 57, 58, 62, and 63.
In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.
In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic DNA fragments or cDNA fragments (i.e. genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the polynucleotides of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
For example, an entire polynucleotide disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding polynucleotide and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among the sequence of the gene or cDNA of interest sequences and are optimally at least about 10 nucleotides in length, and most optimally at least about 20 nucleotides in length. Such probes may be used to amplify corresponding polynucleotides for the particular gene of interest from a chosen plant by PCR. This technique may be used to isolate additional coding sequences from a desired plant or as a diagnostic assay to determine the presence of coding sequences in a plant. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology Hybridization with Nucleic Acid Probes, Part 1, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
It is recognized that the R protein coding sequences of the present invention encompass polynucleotide molecules comprising a nucleotide sequence that is sufficiently identical to the nucleotide sequence of any one or more of SEQ ID NOS: 1 and 3. The term “sufficiently identical” is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g. with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 45%, 55%, or 65% identity, preferably 75% identity, more preferably 85%, 90%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently identical.
To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e. percent identity=number of identical positions/total number of positions (e.g. overlapping positions)×100). In one embodiment, the two sequences are the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the polynucleotide molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g. XBLAST and NBLAST) can be used. BLAST, Gapped BLAST, and PSI-Blast, XBLAST and NBLAST are available on the World Wide Web at ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Alignment may also be performed manually by inspection.
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the full-length sequences of the invention and using multiple alignment by mean of the algorithm Clustal W (Nucleic Acid Research, 22(22):4673-4680, 1994) using the program AlignX included in the software package Vector NTI Suite Version 7 (InforMax, Inc., Bethesda, Md., USA) using the default parameters; or any equivalent program thereof. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by CLUSTALW (Version 1.83) using default parameters (available at the European Bioinformatics Institute website on the World Wide Web at ebi.ac.uk/Tools/clustalw/index).
The use of the term “polynucleotide” is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides, can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
The heterologous polynucleotides or polynucleotide constructs comprising R protein coding regions can be provided in expression cassettes for expression in the plant or other organism or non-human host cell of interest. The cassette will include 5′ and 3′ regulatory sequences operably linked to the R protein coding region. “Operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide or gene of interest and a regulatory sequence (i.e. a promoter) is functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the R protein coding region to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.
The expression cassette will include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region (i.e. a promoter), a R protein coding region of the invention, and a transcriptional and translational termination region (i.e. termination region) functional in plants or other organism or non-human host cell. The regulatory regions (i.e. promoters, transcriptional regulatory regions, and translational termination regions) and/or the R protein coding region or of the invention may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the R protein coding region of the invention may be heterologous to the host cell or to each other.
As used herein, “heterologous” in reference to a nucleic acid molecule, polynucleotide, nucleotide sequence, or polynucleotide construct is a nucleic acid molecule, polynucleotide, nucleotide sequence, or polynucleotide construct that originates from a foreign species, or, if from the same species, is modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
As used herein, a “native gene” is intended to mean a gene that is a naturally-occurring gene in its natural or native position in the genome of a plant. Such a native gene has not been genetically engineered or otherwise modified in nucleotide sequence and/or position in the genome the plant through human intervention, nor has such a native gene been introduced into the genome of the plant via artificial methods such as, for example, plant transformation.
As used herein, a “non-native gene” is intended to mean a gene that has been introduced into a plant by artificial means and/or comprises a nucleotide sequence that is not naturally occurring in the plant. Non-native genes include, for example, a gene (e.g. an R gene) that is introduced into the plant by a plant transformation method. Additionally, when a native gene in the genome of a plant is modified, for example by a genome-editing method, to comprise a nucleotide sequence that is different (i.e. non-identical) from the nucleotide sequence of native gene, the modified gene is a non-native gene.
The present invention provides host cells comprising at least of the nucleic acid molecules, expression cassettes, and vectors of the present invention. In preferred embodiments of the invention, a host cells is plant cell. In other embodiments, a host cell is selected from the group consisting of a bacterium, a fungal cell, and an animal cell. In certain embodiments, a host cell is non-human animal cell. However, in some other embodiments, the host cell is an in-vitro cultured human cell.
While it may be optimal to express the R protein using heterologous promoters, the native promoter of the corresponding R gene may be used.
The termination region may be native with the transcriptional initiation region, may be native with the operably linked R protein coding region of interest, may be native with the plant host, or may be derived from another source (i.e. foreign or heterologous to the promoter, the R protein of interest, and/or the plant host), or any combination thereof. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) (ell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.
Where appropriate, the polynucleotides may be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
The expression cassettes may additionally contain 5′ leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); poty virus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.
In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g. transitions and transversions, may be involved.
A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in plants. Such constitutive promoters include, for example, the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.
Tissue-preferred promoters can be utilized to target enhanced expression of the R protein coding sequences within a particular plant tissue. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997)Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.
Generally, it will be beneficial to express the gene from an inducible promoter, particularly from a pathogen-inducible promoter. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g. PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116. See also WO 99/43819, herein incorporated by reference.
Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al. (1988)Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386 (nematode-inducible); and the references cited therein. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).
Additionally, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter may be used in the heterologous polynucleotides of the invention. Such wound-inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology 14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76); MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like, herein incorporated by reference.
Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.
The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004). J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and yellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al. (2004) J. Cell Science 117:943-54). For additional selectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992)Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference.
The above list of selectable marker genes is not intended to be limiting. Any selectable marker gene can be used in the present invention.
Numerous plant transformation vectors and methods for transforming plants are available. See, for example, An, G. et al. (1986) Plant Pysiol., 81:301-305; Fry, J., et al. (1987) Plant Cell Rep. 6:321-325; Block. M. (1988) Theor. Appl Genet. 76:767-774; Hinchee, et al. (1990) Stadler. Genet. Symp. 203212.203-212; Cousins, et al. (1991) Aust. J. Plant Physiol. 18:481-494; Chee, P. P. and Slightom, J. L. (1992) Gene. 118:255-260; Christou, et al. (1992) Trends. Biotechnol. 10:239-246; D'Halluin, et al. (1992) Bio/Technol. 10:309-314; Dhir, et al. (1992) Plant Physiol. 99:81-88; Casas et al. (1993) Proc. Nat. Acad Sci. USA 90:11212-11216; Christou, P. (1993) In Vitro Cell. Dev. Biol.-Plant; 29P:119-124; Davies, et al. (1993) Plant Cell Rep. 12:180-183; Dong, J. A. and Mchughen, A. (1993) Plant Sci. 91:139-148; Franklin. C. I. and Trieu, T. N. (1993) Plant. Physiol. 102:167; Golovkin, et al. (1993) Plant Sci. 90:41-52; Guo Chin Sci. Bull. 38:2072-2078; Asano, et al. (1994) Plant Cell Rep. 13; Ayeres N. M. and Park, W. D. (1994) Crit. Rev. Plant. Sci. 13:219-239; Barcelo, et al. (1994) Plant. J. 5:583-592; Becker, et al. (1994) Plant. J. 5:299-307; Borkowska et al. (1994) Acta. Physiol Plant. 16:225-230; Christou, P. (1994) Agro. Food. Ind. Hi Tech. 5: 17-27; Eapen et al. (1994) Plant Cell Rep. 13:582-586; Hartman, et al. (1994) Bio-Technology 12: 919923; Ritala, et al. (1994) Plant. Mol. Biol. 24:317-325; and Wan, Y. C. and Lemaux, P. G. (1994) Plant Physiol. 104:3748.
The methods of the invention involve introducing a heterologous polynucleotide or polynucleotide construct into a plant. By “introducing” is intended presenting to the plant the heterologous polynucleotide or polynucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a heterologous polynucleotide or polynucleotide construct to a plant, only that the heterologous polynucleotide or polynucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing heterologous polynucleotides or polynucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
By “stable transformation” is intended that the heterologous polynucleotide or polynucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by progeny thereof. By “transient transformation” is intended that a heterologous polynucleotide or polynucleotide construct introduced into a plant does not integrate into the genome of the plant. It is recognized that stable and transient transformation methods comprise introducing one or more nucleic acid molecules (e.g. DNA), particularly one or more recombinant nucleic acid molecules (e.g. recombinant DNA) into a plant, plant cell, or other host cell or organism.
For the transformation of plants and plant cells, the nucleotide sequences of the invention are inserted using standard techniques into any vector known in the art that is suitable for expression of the nucleotide sequences in a plant or plant cell. The selection of the vector depends on the preferred transformation technique and the target plant species to be transformed.
Methodologies for constructing plant expression cassettes and introducing foreign nucleic acids into plants are generally known in the art and have been previously described. For example, foreign DNA can be introduced into plants, using tumor-inducing (Ti) plasmid vectors. Other methods utilized for foreign DNA delivery involve the use of PEG mediated protoplast transformation, electroporation, microinjection whiskers, and biolistics or microprojectile bombardment for direct DNA uptake. Such methods are known in the art. (U.S. Pat. No. 5,405,765 to Vasil et al.; Bilang et al. (1991) Gene 100: 247-250; Scheid et al., (1991) Mol. Gen. Genet., 228: 104-112; Guerche et al., (1987) Plant Science 52: 111-116; Neuhause et al., (1987) Theor. Appl Genet. 75: 30-36; Klein et al., (1987) Nature 327: 70-73; Howell et al., (1980) Science 208:1265; Horsch et al., (1985) Science 227: 1229-1231; DeBlock et al., (1989) Plant Physiology 91: 694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988) and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989). The method of transformation depends upon the plant cell to be transformed, stability of vectors used, expression level of gene products and other parameters.
Other suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection as Crossway et al. (1986) Biotechniques 4:320-334, electroporation as described by Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation as described by Townsend et al., U.S. Pat. No. 5,563,055, Zhao et al., U.S. Pat. No. 5,981,840, direct gene transfer as described by Paszkowski et al. (1984) EMBO J. 3:2717-2722, and ballistic particle acceleration as described in, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No. 5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lecl transformation (WO 00/28058). Also see, Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.
The polynucleotides of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a heterologous polynucleotide or polynucleotide construct of the invention within a viral DNA or RNA molecule. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931; herein incorporated by reference.
If desired, the modified viruses or modified viral nucleic acids can be prepared in formulations. Such formulations are prepared in a known manner (see e.g. for review U.S. Pat. No. 3,060,084, EP-A 707 445 (for liquid concentrates), Browning, “Agglomeration”, Chemical Engineering, Dec. 4, 1967, 147-48. Perry's Chemical Engineer's Handbook, 4th Ed., McGraw-Hill, New York, 1963, pages 8-57 and et seq. WO 91/13546, U.S. Pat. Nos. 4,172,714, 4,144,050, 3,920,442, 5,180,587, 5,232,701, 5,208,030, GB 2,095,558, U.S. Pat. No. 3,299,566, Klingman, Weed Control as a Science, John Wiley and Sons, Inc., New York, 1961, Hance et al. Weed Control Handbook, 8th Ed., Blackwell Scientific Publications, Oxford, 1989 and Mollet, H., Grubemann, A., Formulation technology, Wiley VCH Verlag GmbH, Weinheim (Germany), 2001, 2. D. A. Knowles, Chemistry and Technology of Agrochemical Formulations, Kluwer Academic Publishers, Dordrecht, 1998 (ISBN 0-7514-0443-8), for example by extending the active compound with auxiliaries suitable for the formulation of agrochemicals, such as solvents and/or carriers, if desired emulsifiers, surfactants and dispersants, preservatives, antifoaming agents, anti-freezing agents, for seed treatment formulation also optionally colorants and/or binders and/or gelling agents.
In specific embodiments, the polynucleotides, polynucleotide constructs, and expression cassettes of the invention can be provided to a plant using a variety of transient transformation methods known in the art. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986)Mol Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) PNAS Sci. 91: 2176-2180 and Hush et al. (1994) J. Cell Science 107:775-784, all of which are herein incorporated by reference. Alternatively, the polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and Agrobacterium tumefaciens-mediated transient expression as described elsewhere herein.
The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example. McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as “transgenic seed”) having a heterologous polynucleotide or polynucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.
Any methods known in the art for modifying DNA in the genome of a plant can be used to modify genomic nucleotide sequences in planta, for example, to create or insert a resistance gene or even to replace or modify an endogenous resistance gene or allele thereof. Such methods include, but are not limited to, genome-editing (or gene-editing) techniques, such as, for example, methods involving targeted mutagenesis, homologous recombination, and mutation breeding. Targeted mutagenesis or similar techniques are disclosed in U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972, 5,871,984, and 8,106,259; all of which are herein incorporated in their entirety by reference. Methods for gene modification or gene replacement comprising homologous recombination can involve inducing double breaks in DNA using zinc-finger nucleases (ZFN), TAL (transcription activator-like) effector nucleases (TALEN), Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease (CRISPR/Cas nuclease), or homing endonucleases that have been engineered endonucleases to make double-strand breaks at specific recognition sequences in the genome of a plant, other organism, or host cell. See, for example, Durai et al., (2005) Nucleic Acids Res 33:5978-90; Mani et al. (2005) Biochem Biophys Res Comm 335:447-57; U.S. Pat. Nos. 7,163,824, 7,001,768, and 6,453,242; Amould et al. (2006) J Mol Biol 355:443-58; Ashworth et al., (2006) Nature 441:656-9; Doyon et al. (2006) J Am Chem Soc 128:2477-84; Rosen et al., (2006) Nucleic Acids Res 34:4791-800; and Smith et al., (2006) Nucleic Acids Res 34:e149; U.S. Pat. App. Pub. No. 2009/0133152; and U.S. Pat. App. Pub. No. 2007/0117128; all of which are herein incorporated in their entirety by reference.
Unless stated otherwise or apparent from the context of a use, the term “gene replacement” is intended to mean the replacement of any portion of a first polynucleotide molecule or nucleic acid molecule (e.g. a chromosome) that involves homologous recombination with a second polynucleotide molecule or nucleic acid molecule using a genome-editing technique as disclosed elsewhere herein, whereby at least a part of the nucleotide sequence of the first polynucleotide molecule or nucleic acid molecule is replaced with the second polynucleotide molecule or nucleic acid molecule. It is recognized that such gene replacement can result in additions, deletions, and/or modifications in the nucleotide sequence of the first polynucleotide molecule or nucleic acid molecule and can involve the replacement of an entire gene or genes, the replacement of any part or parts of one gene, or the replacement of non-gene sequences in the first polynucleotide molecule or nucleic acid molecule.
TAL effector nucleases (TALENs) can be used to make double-strand breaks at specific recognition sequences in the genome of a plant for gene modification or gene replacement through homologous recombination. TAL effector nucleases are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a plant or other organism. TAL effector nucleases are created by fusing a native or engineered transcription activator-like (TAL) effector, or functional part thereof, to the catalytic domain of an endonuclease, such as, for example, FokI. The unique, modular TAL effector DNA binding domain allows for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA binding domains of the TAL effector nucleases can be engineered to recognize specific DNA target sites and thus, used to make double-strand breaks at desired target sequences. See, WO 2010/079430; Morbitzer et al. (2010) PNAS 10.1073/pnas. 1013133107; Scholze & Boch (2010) Virulence 1:428-432; Christian et al. Genetics (2010) 186:757-761; Li el al. (2010) Nuc. Acids Res. (2010) doi:10.1093/nar/gkq704; and Miller et al. (2011) Nature Biotechnology 29:143-148; all of which are herein incorporated by reference.
The CRISPR/Cas nuclease system can also be used to make double-strand breaks at specific recognition sequences in the genome of a plant for gene modification or gene replacement through homologous recombination. The CRISPR/Cas nuclease is an RNA-guided (simple guide RNA, sgRNA in short) DNA endonuclease system performing sequence-specific double-stranded breaks in a DNA segment homologous to the designed RNA. It is possible to design the specificity of the sequence (Cho S. W. et al., Nat. Biotechnol. 31:230-232, 2013; Cong L. et al., Science 339:819-823, 2013; Mali P. et al., Science 339:823-826, 2013; Feng Z. et al., Cell Research: 1-4, 2013).
In addition, a ZFN can be used to make double-strand breaks at specific recognition sequences in the genome of a plant for gene modification or gene replacement through homologous recombination. The Zinc Finger Nuclease (ZFN) is a fusion protein comprising the part of the FokI restriction endonuclease protein responsible for DNA cleavage and a zinc finger protein which recognizes specific, designed genomic sequences and cleaves the double-stranded DNA at those sequences, thereby producing free DNA ends (Urnov F. D. et al., Nat Rev Genet. 11:636-46, 2010; Carroll D., Genetics. 188:773-82, 2011).
Breaking DNA using site specific nucleases, such as, for example, those described herein above, can increase the rate of homologous recombination in the region of the breakage. Thus, coupling of such effectors as described above with nucleases enables the generation of targeted changes in genomes which include additions, deletions and other modifications.
The nucleic acid molecules, expression cassettes, vectors, and heterologous polynucleotides of the present invention may be used for transformation and/or genome editing of any plant species, including, but not limited to, monocots and dicots.
As used herein, the term “plant” includes seeds, plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, tubers, propagules, leaves, flowers, branches, fruits, roots, root tips, anthers, and the like. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides. As used herein, “progeny” and “progeny plant” comprise any subsequent generation of a plant whether resulting from sexual reproduction and/or asexual propagation, unless it is expressly stated otherwise or is apparent from the context of usage.
As used herein, the terms “transgenic plant” and “transformed plant” are equivalent terms that refer to a “plant” as described above, wherein the plant comprises a heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct that is introduced into a plant by, for example, any of the stable and transient transformation methods disclosed elsewhere herein or otherwise known in the art. Such transgenic plants and transformed plants also refer, for example, the plant into which the heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct was first introduced and also any of its progeny plants that comprise the heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct.
In certain embodiments of the invention, the methods involve the planting of seedlings and/or tubers and then growing such seedlings and tubers so as to produce plants derived therefrom and optionally harvesting from the plants a plant part or parts. As used herein, a “seedling” refers to a less than fully mature plant that is typically grown in greenhouse or other controlled- or semi-controlled (e.g. a cold frame) environmental conditions before planting or replanting outdoors or in a greenhouse for the production a harvestable plant part, such as, for example, a tomato fruit, a potato tuber or a tobacco leaf. As used herein, a “tuber” refers to an entire tuber or part or parts thereof, unless stated otherwise or apparent from the context of use. A preferred tuber of the present invention is a potato tuber.
In the methods of the invention involving planting a tuber, a part of tuber preferably comprises a sufficient portion of the tuber whereby the part is capable of growing into a plant under favorable conditions for the growth and development of a plant derived from the tuber. It is recognized that such favorable conditions for the growth and development of crop plants, particularly solanaceous crop plants, are generally known in the art.
In some embodiments of the present invention, a plant cell is transformed with a heterologous polynucleotide encoding an R protein of the present invention. The term “expression” as used herein refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product. The “expression” or “production” of a protein or polypeptide from a DNA molecule refers to the transcription and translation of the coding sequence to produce the protein or polypeptide, while the “expression” or “production” of a protein or polypeptide from an RNA molecule refers to the translation of the RNA coding sequence to produce the protein or polypeptide. Examples of heterologous polynucleotides and nucleic acid molecules that encode R proteins are described elsewhere herein.
The use of the terms “DNA” or “RNA” herein is not intended to limit the present invention to polynucleotide molecules comprising DNA or RNA. Those of ordinary skill in the art will recognize that the methods and compositions of the invention encompass polynucleotide molecules comprised of deoxyribonucleotides (i.e. DNA), ribonucleotides (i.e. RNA) or combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues including, but not limited to, nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). The polynucleotide molecules of the invention also encompass all forms of polynucleotide molecules including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like. Furthermore, it is understood by those of ordinary skill in the art that the nucleotide sequences disclosed herein also encompasses the complement of that exemplified nucleotide sequence.
The invention is drawn to compositions and methods for enhancing the resistance of a plant to plant disease, particularly to compositions and methods for enhancing the resistance of a plant to a plant disease caused by at least one race of at least one Phytophthora sp. By “disease resistance” is intended that the plants avoid the disease symptoms that are the outcome of plant-pathogen interactions. That is, pathogens are prevented from causing plant diseases and the associated disease symptoms, or alternatively, the disease symptoms caused by the pathogen is minimized or lessened.
The following examples are offered by way of illustration and not by way of limitation.
Recently, the cloning of Rpi-amr3i from a Mexican accession of Solanum americanum has been reported (Witek et al. (2016) Nat. Biotechnol. 34: 656; see also WO 2016/182881; both of which are herein incorporated by reference). In an attempt to identify additional S americanum genes for resistance to Phytophthora infestans, we investigated a set of S. americanum (2n) accessions, obtained from seed collections (Table 1), for their immune response towards P. infestans.
Pathogen susceptibility was assessed in detached leaf assays (DLAs), using three highly virulent P. infestans isolates (06_3928A, 88069 and EC1). Accession 954750186 was susceptible to all tested isolates (supporting mycelial growth and sporulation (Witek et al. ((2016) Nat Biotechnol 34: 656)). All other accessions remained fully resistant, with no visible sign of infection or only small sites of hypersensitive response (HR) in the form of local cell death at the site of P. infestans inoculation.
To determine the genetic basis of S. americanum resistance, we crossed seven resistant accessions, namely 954750184, sn27, Veg422, A14750006, SOLA 425, Wang 2058 and A14750130 as male parents to the susceptible line 954750186. Resistant F1 plants were self-pollinated, and we tested 60 to 100 plants per F2 for the response to 06_3928A and 88069 and found that the progeny of six crosses segregated in a ratio suggesting the presence of a single (semi) dominant resistance gene (fitting 3:1 or 2:1). One cross showed 15:1 segregation, suggesting the presence of two or more unlinked R genes (Table 1).
We initially focused on F2 and F3 populations derived from crosses of the resistant parent 954750184. Bulked susceptible (BS) Genomic DNA samples were created from 94 of the most susceptible F2 and F3 plants. We subsequently subjected BS gDNA, as well as from resistant (R) and susceptible parent (S) to Illumina-based RenSeq (76 bp PE reads), and additionally RAD-seq experiments. Additionally, we performed Whole Genome Shotgun sequencing (WGS) on R and S samples with Illumina HiSeq 90 bp PE reads. We used our previously published in silico trait mapping pipelines (Jupe et al. ((2013) Plant J. 76: 530) and Witek et al. ((2016) Nat Biotechnol 34: 656)) to perform single nucleotide polymorphism (SNP) calling and detection of polymorphisms linked to disease resistance. Screening a set of markers derived from these analyses on DNA of 94 susceptible F2 and F3 plants identified 12 markers linked with resistance response that flank the R locus between 7.5 cM to one side and 4.3 cM to the other side. While four markers were found to co-segregate with the resistance, two were found to be located around 1 cM on either side; CAPS marker RAD_3 (BslI) distal and the PCR-marker CLC_I (WGS_I1) to the proximal side (
The 118 informative recombinants (homozygous susceptible to one side and heterozygous to the other) were further genotyped using the eight linked markers (
Comparison of the linkage map with the potato reference genome identified the homogeneous CNL-3 NLR gene sub-family to be within the cosegregating locus. This cluster comprises 18 members on potato reference chromosome 11. Marker WGS_2 was designed on a S. americanum WGS data derived NLR sequence, orthologous to the CNL-3 cluster. WGS_2 was then used to probe for two BAC clones (outsourced to BioS&T; Quebec, Canada; see on the World Wide Web: biost.com). While the co-segregating marker WGS_2 was present on both derived BAC clones 5G and 12H, a further co-segregating marker WGS_3 was only present on 12H. Differences between both BAC clones were further identified through the HindIII digestion pattern. Both were subsequently sequenced on the PacBio RS platform and assembled into single contigs of 125,327 bp (5G) and 144.006 bp (12H) and further assembled to a single contig of 192,456 bp.
Prediction of open reading frames identified 11 potential coding sequences, nine of which were NLRs, as identified by mapping of R parent RenSeq reads as well as NLR-parser analysis (Steuernagel et al. ((2015) Bioinformatics 31: 1665,
We cloned the open reading frames of the 7 candidate NLRs into a binary expression vector under control of a 35S promoter and transformed into Agrobacterium. These constructs were transiently expressed in N. benthamiana detached leaves, which were subsequently inoculated with the P. infestans isolate 88069 as described in Witek et al. ((2016) Nat Biotechnol 34: 656). P. infestans growth was observed 6 days post inoculation on GFP-infiltrated control leaves and all other constructs, except for the Rpi-amr3i control and the candidate gene Rpi-amr1e. 35S:Rpi-acmr1e infiltrated leaves showed no to small HR at 6 days post inoculation (dpi) (
We created stable transgenic plants with Rpi-amr1e constructs under native regulatory elements in the tetraploid cultivar Maris Piper using the transformation method described in Kumar et al. ((1996) Plant J. 9:147). Transgenic plants showed resistance against P. infestans race 88069 (
Genotyping of 10-20 susceptible F2 plants from populations derived from resistant accessions sn27, Veg422, A14750006, SOLA 425, Wang 2058 and A14750130 showed that resistance is linked to the Rpi-amr1 locus. To test whether Rpi-amr1e orthologs confer resistance, we performed SMRT RenSeq on resistant accessions and assembled NLRs as described in Witek et al. ((2016) Nat Biotechnol 34: 656). We next mapped all assembled contigs to coding sequence of Rpi-amr1e allowing for 10% mismatches and gaps and selected the closest, transcribed orthologs (Table 2 for % amino acid sequence identity), as identified by mapping the cDNA RenSeq reads. In three resistant parents, namely Veg422, A14750130 and Wang 2058, identified genes showed 100% identity on amino acid level to Rpi-amr1e, while the remaining accessions had above 94% identity to functional Rpi-amr1e (Table 2). We cloned polymorphic genes under control of 35S promoter (sn27 and A14750006) or under native regulatory elements (SOLA425) into binary expression vector. These constructs were transiently expressed in N. benthamiana detached leaves and inoculated with P. infestans isolate 88069 (24 hours post infiltration) and assessed for resistance at 6 dpi. All tested genes confer enhanced resistance to P. infestans, similar to Rpi-amr1e, when compared to GFP control infiltration.
We mapped cDNA RenSeq data to BAC contig with TopHat splice junction mapper for RNA-Seq reads (Trapnell et al. (2009) Bioinformatics 25:1105-1111) and detected two dominant splice variants for Rpi-amr1e gene (SEQ ID NO: 22). The most abundant version, supported by over 80% of cDNA reads, consists of 4 exons (SEQ ID NO: 23) and encodes a protein of 1013 amino acids (SEQ ID NO: 24). The remaining cDNA reads show that several other splice variants corresponding to various forms of 3′ truncation of SEQ ID NO: 23 are possible. We confirmed this by 3′ rapid amplification of cDNA ends (RACE) PCR and observed the following CDS sequences: SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27 coding for 1004 amino acids (SEQ ID NO: 28), 925 amino acids (SEQ ID NO: 29) and 868 amino acids (SEQ ID NO: 30) proteins, respectively.
We used the transformation method as described previously to construct stable transgenic potato plants (cv. Maris Piper) carrying the Rpi-amr1e gene (SEQ ID NO: 23). We recovered 10 transgenic lines where presence of Rpi-amr1e was confirmed by PCR with gene-specific primers. In DLAs, nine lines showed resistance against P. infestans isolate 88069 (
In addition to alleles of Rpi-amr1e disclosed above in Example 6 (accession sn27, also referred to herein as SP1032; accession SOLA425, also referred to herein as SP2307; and accession A14750006, also referred to herein as SP1123), we found three more populations, derived from resistant parents 954750174 (also referred to herein as SP2272), A14750130 (SP3400) and 954750172 (SP3408) where resistance co-segregates with the Rpi-amr1e locus. To test if Rpi-amr1e alleles were involved in this resistance, we performed SMRT RenSeq and looked for the closest transcribed homolog of Rpi-amr1e as described in Example 6. The gene from SP3408 showed less than 92.5% identity to Rpi-amr1e. The remaining two candidate sequences were more diverged and showed 89.3% identify on amino acid level to Rpi-amr1e; however, they were 1000/identical to each other. We cloned two new Rpi-amr1e alleles and also three previously reported (SP1032, SOLA425 and SP1123) under their native regulatory elements into a binary vector as described earlier; SP1032 (SEQ ID NO: 31, SP1123 (SEQ ID NO: 32, SP2272 (SEQ ID NO: 33, SP2307 (SEQ ID NO: 34, SP3408 (SEQ ID NO: 35. In transient complementation assays, all genes conferred resistance against P. infestans isolate 88069 (
We annotated coding sequences of the functional Rpi-amr1e alleles using AUGUSTUS gene prediction software (Stanke et al. (2008) Bioinformatics 24: 637-644) and also by alignments with the coding sequence of Rpi-amr1e (SEQ ID NO: 23). The predicted CDS sequences for accessions SP1032 (SEQ ID NO: 36), SP1123 (SEQ ID NO: 37), SP2272 (SEQ ID NO: 38), SP2307 (SEQ ID NO: 39) and SP3408 (SEQ ID NO: 40) encode 986 amino acids (SEQ ID NO: 41), 987 amino acids (SEQ ID NO: 42), 976 amino acids (SEQ ID NO: 43), 986 amino acids (SEQ ID NO: 44) and SP3408 (SEQ ID NO: 45) proteins, respectively.
We investigated the immune response towards P. infestans in S. nigrescens (2n) accession A14750423 (also referred to herein as SP3409; country of origin, Mauritius). In detached leaf assays (DLAs) with the highly virulent P. infestans isolates (06_3928A, 88069, EC1 and NL07434). Plants of the accession SP3409 remained fully resistant (R parent), with no obvious signs of infection or only small sites of hypersensitive response (HR) in the form of local cell death at the site of P. infestans inoculation.
To determine the genetic basis of resistance, we crossed the resistant line SP3409 as a male parent to the susceptible line SP2271 (S parent, reported in Witek et al. (2016) Nat. Biotechnol. 34: 656-660) as a female parent. Heterozygous F1 progeny showed no segregation for resistance to P. infestans isolate 06_3928A and EC1 (6-8 plants were tested for each F1), and were allowed to self-pollinate to generate F2 progenies. We tested 90 F2 progeny for resistance to the P. infestans isolate 88069 and found F2 progenies segregate in a 3:1, suggesting the presence of a single dominant resistance gene, which we named Rpi-amr6. Hence this F2 population was selected for R gene identification.
We successfully applied a previously described method to clone R genes without construction of BAC libraries using a Solanum NLR bait library (Witek et al. (2016) Nat. Biotechnol. 34: 656-660). To define the complement of NLRs from resistant SP3409 parental line, we captured 3-4 kb gDNA fragments and sequenced in two SMRT cells. This resulted in more than 32 k reads of inserts (ROI). De novo assembly of ROI with Geneious and analysis with NLR-parser (Steuernagel et al. (2015) Bioinformatics. 31: 1665-1667) identified 287 full length and 555 partial NLRs. To identify linked candidate NLRs we performed Illumina RenSeq on gDNA from 42 susceptible individuals from F2 plants (bulked susceptible, BS) as described (Jupe et al. ((2013) Plant J. 76: 530-544; Witek et al. (2016) Nat. Biotechnol. 34: 656-660). The Illumina MiSeq run generated 744,943; 2,824,501; 678,099 and 1,597,558 paired-end reads for resistant (R) parent, susceptible (S) parent, bulk susceptible and cDNA of resistant parent respectively. After performing initial QC, we mapped the MiSeq data (R, S parents, and BS) to assembly of PacBio data of R parent. We used our previously published in silico trait mapping pipelines (Jupe et al. ((2013) Plant J. 76: 530) and Witek et al. (2016) Nat. Biotechnol. 34: 656-660)) to perform SNP (calling and detection of polymorphisms linked to disease resistance. Briefly, we called homozygous SNPs between S and R parents, and looked for contigs which showed absence of R specific allele (less than 5% R allele in BS). Transcriptionally active NLRs and their intron/exon structure were annotated with cDNA RenSeq reads as described previously (Andolfo et al. (2014) BMC Plant Biol. 14:120; Witek et al. (2016) Nat. Biotechnol. 34: 656-660). These identified five candidate NLRs for Rpi-amr6. We further confirmed co-segregation of these sequences using gene specific markers (data not shown).
We cloned the open reading frames of the candidate NLRs for Rpi-amr6 into a binary expression vector under native regulatory elements and then introduced the vectors into Agrobacterium tumefaciens strain AGL-1. These constructs were transiently expressed in N. benthamiana leaves which were detached 24 hours later and inoculated with the P. infestans isolates 88069 and US-23 as described in Witek et al. ((2016) Nat. Biotechnol. 34: 656-660). At 6 dpi restriction of P. infestans growth was observed with candidate construct Rpi-amr6b (SEQ ID NO: 46) and with the Rpi-amr3 positive control construct, while symptoms of P. infestans infection were visible on GFP-infiltrated control leaves and remaining candidate genes (
A construct carrying the Rpi-amr6b gene under its native regulatory elements (SEQ ID NO: 1) in pICSLUS0001 binary vector (Witek et al. (2016) Nat Biotechnol 34: 656-660) was used to create stable transgenic potato plants in cv. Maris Piper background using transformation method for potato as described in Kumar et al. ((1996) Plant J, 9: 147-158). We recovered 5 stable transgenic lines showing presence of transgene in PCR test with Rpi-amr6b specific primers. One line exhibited enhanced resistance to P. infestans race 88069 (
Rpi-amr6b comprises a 5,131 bp open reading frame (ORF). The mapping pattern of cDNA data suggests that Rpi-amr6b undergoes alternative splicing, and two splice forms can be distinguished. The dominant (i.e. most abundant) transcript variant consists of 5 exons (SEQ ID NO: 49) encoding a protein of 961 amino acids (SEQ ID NO: 47). A longer transcript (SEQ ID NO: 50) was also detected that encodes a protein of 978 amino acids (SEQ ID NO: 48). Both proteins contain typical characteristics of a CC-NB-LRR class resistance protein, including coiled-coil domain (CC; amino acids 4-114), nucleotide binding domain (NB-ARC; amino acids 153-437) and two potential leucine-reach repeats (LRR; amino acids 683-800).
To determine chromosomal location of Rpi-amr6b, we used an enrichment-based genotyping, named GenSeq (Chen et al. “Identification and rapid mapping of a gene conferring broad-spectrum late blight resistance in the diploid potato species Solanum verrucosum through DNA capture technologies,” Theor. Appl. Genet., submitted 2017) with 19,716 biotinylated RNA baits targeting 1,143 conserved ortholog set (COS) (Lindqvist-Kreuze et al. (2013) BMC Genetics, 14: 51-51) and 837 single copy genes identified in the potato reference genome (DM) (Potato Genome Sequencing Consortium (2011) Nature 475:189-195). Targeted enrichment and Illumina sequencing was performed as described above, which generated 536,335; 1,200,699 and 658,651 paired-end reads for gDNA of R parent (SP3409), S parent (SP2271), and BS, respectively. After performing initial QC, we mapped the MiSeq data to the potato DM sequence. To find the linked region, we annotated homozygous SNPs between S and R parents that were present/absent in BS (less than 5% R allele; Jupe et al. (2013) Plant J. 76: 530-544). Based on these SNPs, we developed genetic markers and confirmed that Rpi-amr6b maps to the 78 Mb region of chromosome 1 in the DM sequence, which is different from the position of Rpi-amr1e which maps to the top of chromosome 11 (around 6 Mb in the DM reference genome).
In our screening, S. americanum accession A54750014 (also referred to herein as SP1101) showed strong resistance to P. infestans isolate 88069 in DLA assay. All 60 plants in F2 progeny derived from the cross with susceptible SP2271 were resistant, suggesting presence of two or more resistant genes. To separate these genes, we back-crossed F1 plants to susceptible SP2271 (BC1) followed by another backcross to SP2271 (BC2). Resistant BC2F1 plants were self-pollinated to generate BC2F2 and segregation ratio was tested on 90 plants in a population. An additional 600 plants for selected population were sown and phenotyped in DLA for P. infestans response. gDNA from 133 susceptible plants was isolated and the RenSeq pipeline performed as described above to identify linked candidate genes. Additionally, with the standard SNP calling pipeline described above, we performed RenSeq and SNP calling on bulked resistance (BR) sample (BR, 20 R plants from segregation population) to identify fixed susceptible loci. Briefly, for homozygous SNPs between S and R parents that were absent in BS (criteria as described above) we counted the allele ratio from BR data. SNPs showing less than 5% of R allele were annotated as fixed and excluded from further analysis. This revealed eight NLR-encoding candidate genes which were cloned and tested in transient assay as described above. Candidate Rpi-amr7d (SEQ ID NO: 51) conferred resistance in transient assay (
Rpi-amr7d comprises a 5131 bp ORF. Mapping of cDNA reads suggests that Rpi-amr7d undergoes alternative splicing and two splice forms can be distinguished. The dominant transcript variant consists of 5 exons (SEQ ID NO: 54) encoding a protein of 961 amino acids (SEQ ID NO: 52). An additional transcript (SEQ ID NO: 55) was detected that encoded a protein of 978 amino acids (SEQ ID NO: 53). Both proteins contain typical characteristics of a CC-NB-LRR class resistance protein, including coiled-coil domain (CC; amino acids 2-120), nucleotide binding domain (NB-ARC; amino acids 154-432) and two potential leucine-reach repeats (LRR; amino acids, 683-800).
S. americanum accession SOLA 226 (also referred to herein as SP2300) showed strong resistance to P. infestans isolate 88069 in DLA assay. In an F2 population derived from cross between resistant SP2300 and susceptible SP2271 plants, we observed a 15:1 segregation ratio (resistant to susceptible), suggesting presence of two unlinked dominant resistant genes. We showed that resistance was co-segregating with previously cloned Rpi-amr3 gene (Witek et al. (2016) Nat. Biotechnol. 34: 656-660; see also WO 2016/182881 patent application). Using Rpi-amr3 gene-specific markers we screened F2 population and selected resistant plants which lacked Rpi-amr3. Plants were self-pollinated and resulting F3 populations screened with P. infestans isolate 88069 to detect families segregating in ratio 3:1 (resistant to susceptible). From one of the populations segregating 3:1, 600 plants were phenotyped using the DLA for P. infestans response, gDNA from 114 susceptible (BS) and 20 resistant (BR) plants was isolated, and the RenSeq pipeline performed as described above to identify linked candidate genes. Additionally, to standard SNP calling pipeline described above, we performed RenSeq and SNP calling on a BR resistance sample to identify fixed susceptible loci. Briefly, for homozygous SNPs between S and R parents that were absent in BS (criteria as described above) we counted the allele ratio from BR data. SNPs showing less than 5% of R allele were annotated as fixed and excluded from further analysis. This resulted in 10 NLR-encoding candidate genes which were cloned and tested in transient assays as described above. Candidate Rpi-amr8c (SEQ ID NO: 56) conferred resistance in a transient assay (
Rpi-amr8c comprises a 5125 bp ORF. Mapping of cDNA data suggests that Rpi-amr8c undergoes alternative splicing, and two splice forms can be distinguished. The dominant transcript variant consists of 5 exons (SEQ ID NO: 59) encoding a protein of 960 amino acids (SEQ ID NO: 58). An additional transcript (SEQ ID NO: 60) was detected that encodes a protein of 986 amino acids (SEQ ID NO: 57). Both proteins contain typical characteristics of a CC-NB-LRR class resistance protein, including coiled-coil domain (CC: amino acids 2-120), nucleotide binding domain (NB-ARC; amino acids 153-431) and two potential leucine-reach repeats (LRR; amino acids 683-800).
S. americanum accession SOLA 425 (also referred to herein as SP2307), showed strong resistance to P. infestans isolate 88069 in the DLA assay. In an F2 population derived from a cross between resistant SP2307 and susceptible SP2271 plants, we observed a segregation ratio 9:1 (resistant to susceptible), suggesting the presence of more than one resistance gene. Resistant F2 plants were self-pollinated, and the resulting F3 populations were screened with P. infestans isolate 88069 to detect families segregating in ratio 3:1 (resistant to susceptible). From one population showing 3:1 segregation, 600 plants were phenotyped in DLA for P. infestans response, gDNA from 117 susceptible (BS) and 20 resistant (BR) plants was isolated and the RenSeq pipeline performed as described above to identify linked candidate genes. In addition to standard SNP calling as described above, we performed RenSeq and SNP calling on a BR sample to identify fixed susceptible loci. Briefly, for homozygous SNPs between S and R parents that were absent in BS (criteria as described above), we counted allele ratios from BR data SNPs showing less than 5% of R allele were annotated as fixed and excluded from further analysis. This resulted in 10 NLR-encoding genes which were cloned and tested in transient assay as described above. Candidate Rpi-amr9d (SEQ ID NO: 61) conferred resistance in a transient assay (
Rpi-amr9d comprises a 7357 bp ORF. Mapping of cDNA data suggests that the gene Rpi-amr9d undergoes alternative splicing and two splice forms can be distinguished. The dominant transcript variant consists of 5 exons (SEQ ID NO: 64) encoding a protein of 986 amino acids (SEQ ID NO: 63). An additional transcript (SEQ ID NO: 65) was detected that encodes a protein of 1011 amino acids (SEQ ID NO: 62). Both proteins contain typical characteristics of a CC-NB-LRR class resistance protein, including coiled-coil domain (SEQ ID NO: 62, amino acids 2-145; SEQ ID NO: 63, amino acids 2-120), nucleotide binding domain (SEQ ID NO: 62, amino acids 179-457; SEQ ID NO: 63, amino acids 154-432 amino acids) and two potential leucine-reach repeats (SEQ ID NO: 62, amino acids 683-800; SEQ ID NO: 63, amino acids 683-986). Alignment of the nucleotide sequence of Rpi-amr9d with homolog of Rpi-amr1e cloned from resistant line SP2307, namely Rpi-amr1_2307 (see Examples 6 and 8, above), showed that these two genes are 100% identical.
We aligned full-length amino acid sequences (Table 3) of all Rpi-amr1e functional homologs and also Rpi-amr6b. Rpi-amr7d and Rpi-amr8c and generated a phylogenetic tree (
Our data show that there is an extensive allelic variation for a functional Rpi-amr1e in S. americanum, with up to 12% differences between different alleles. Thus, it is highly probable that various alleles can recognize unrelated P. infestans effectors. This was shown for a barley mildew resistance locus (MIA), where diverse alleles of Mia immune receptor recognize sequence-unrelated avirulence genes of the cognate pathogen (Lu et al. (2016) PNAS 18:E6486-E6495).
The article “a” and “an” are used herein to refer to one or more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one or more element.
Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
This application is the U.S. National Stage of International Application PCT/US2017/066691, filed Dec. 15, 2017, which designates the U.S. and was published by the International Bureau in English on Jun. 21, 2018, and which claims the benefit of U.S. Provisional Patent Application No. 62/435,451, filed Dec. 16, 2016, which is hereby incorporated herein in its entirety by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/066691 | 12/15/2017 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/112356 | 6/21/2018 | WO | A |
| Number | Date | Country |
|---|---|---|
| 2009-013468 | Jan 2009 | WO |
| 2013-009935 | Jan 2013 | WO |
| 2016-182881 | Nov 2016 | WO |
| Entry |
|---|
| Uniprot M0ZPC5_SOLTU (2013, https://www.genome.jp/dbget-bin/www_bget?uniprot: M0ZPC5_SOLTU). |
| McHale et al, 2006, Genome Biol., vol. 7, article 212. |
| Witek et al, 2020, https://doi.org/10.1101/2020.05.15.095497. |
| Guo et al, 2004, Proc. Natl. Acad. Sci. USA 101: 9205-9210; p. 9209. |
| Kamil Witek, et al. “Accelerated Cloning of a potato late blight-resistance gene using RenSeq and SMRT Sequencing,” Nature Biotechnology, vol. 34, No. 6, Apr. 25, 2016, pp. 656-660 (XP055285612). |
| International Search Report and Written Opinion dated Mar. 16, 2018 in PCT/US2017/066691. |
| Number | Date | Country | |
|---|---|---|---|
| 20190359998 A1 | Nov 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62435451 | Dec 2016 | US |
