WO2012155112A1 - Résistance à une maladie dans les céréales à médiation par un silençage génique induit par l'hôte - Google Patents

Résistance à une maladie dans les céréales à médiation par un silençage génique induit par l'hôte Download PDF

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Publication number
WO2012155112A1
WO2012155112A1 PCT/US2012/037660 US2012037660W WO2012155112A1 WO 2012155112 A1 WO2012155112 A1 WO 2012155112A1 US 2012037660 W US2012037660 W US 2012037660W WO 2012155112 A1 WO2012155112 A1 WO 2012155112A1
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cereal plant
pathogen
gene
plant
transgenic
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PCT/US2012/037660
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English (en)
Inventor
Richard Michelmore
Jorge Dubcovsky
Manjula GOVINDARAJULU
Dario CANTU
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The Regents Of The University Of California
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Publication of WO2012155112A1 publication Critical patent/WO2012155112A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]

Definitions

  • the present disclosure relates generally to disease-resistant cereals and methods for producing disease-resistant cereals, and more specifically to disease-resistance in cereals mediated by host- induced gene silencing ("HIGS").
  • HGS host- induced gene silencing
  • the disclosure provides transgenic cereals expressing RNA interference (RNAi) constructs designed to silence critical genes in pathogens that cause diseases in cereals.
  • RNAi RNA interference
  • the disclosure provides a transgenic cereal plant capable of host- induced gene silencing of a pathogen
  • the plant includes an expressable RNA interference construct encoding a small, interfering RNA molecule (siRNA) capable of down- regulating or suppressing the expression of at least one gene of a pathogen that is capable of infecting the cereal plant.
  • the plant expresses the siRNA.
  • the siRNA Upon infection by the pathogen, the siRNA is capable of propagating across or crossing the haustorial interface of the pathogen and down-regulating or suppressing expression of the target pathogen gene.
  • Figure 1 depicts detached wheat leaves of lines D6301 and Bobwhite after VIGS treatment and PST infection showing reduced disease after VIGS of ERG11 and ERG2.
  • Figure 2 depicts Stable T 2 transgenics of wheat cv. Bobwhite generated to target EGR2 & ERG11 simultaneously & tested for reaction to stripe rust, Puccinia striiformis.
  • Figure 3 depicts an overview of the HIGS effect.
  • Figure 4 depicts other opportunities for HIGS in crop plants.
  • Figure 5 depicts the coding sequences of for two Puccinia striiformis f. sp. tritici genes (ERG2 and ERG 11) that may be targeted by RNAi.
  • Figure 6 depicts the plasmid map for virus-induced gene silencing (VIGS) plasmid, pa42.
  • Figure 7 depicts the plasmid map for virus-induced gene silencing (VIGS) plasmid
  • Figure 8 depicts the plasmid map for virus-induced gene silencing (VIGS) plasmid, pSL038-l.
  • VIP virus-induced gene silencing
  • Figure 9 depicts the plasmid map for virus-induced gene silencing (VIGS) plasmid, pSL039B-l.
  • VIP virus-induced gene silencing
  • Figure 10 depicts the DNA sequence for the pa42 plasmid.
  • Figure 11 depicts the DNA sequence for the pp.42spl plasmid.
  • Figure 12 depicts the DNA sequence for the pSL038-l plasmid.
  • Figure 13 depicts the DNA sequence for the pSL039B- 1 plasmid.
  • Figure 14 depicts a region of the targeted coding sequence from rust pathogen used for virus-induced gene silencing (VIGS) of pathogen ATPase, ERG2, and ERG11.
  • VIPGS virus-induced gene silencing
  • RNAi-mediated approach that is durable and complements more conventional approaches for providing disease-resistance in cereals.
  • RNA interference RNA interference constructs designed to silence genes in the pathogen that are critical to the three major rust diseases of wheat.
  • Small, interfering RNAs synthesized in the plant may be delivered across the haustorial interface to the pathogen.
  • the objective is to trigger the activation of the silencing machinery in the pathogen to down regulate the expression of genes that are important for pathogen viability or virulence.
  • Such silencing will impair or delay fungal development and growth thereby reducing disease incidence and severity in wheat. Cereal plants with such silencing will also exhibit resistant to pathogens compared to wild type cereal plants. Initial experiments are being conducted with stripe rust. Additional tests will be conducted for leaf and stem rusts.
  • siRNAs small interfering RNAs
  • HIGS Host- Induced Gene Silencing
  • siRNAs can be targeted to vital, conserved genes in the pathogen, HIGS should be race non-specific and durable.
  • SiRNAs can be targeted to specific RNA sequences; therefore, it is likely to be totally benign in the plant with no pleiotropic effects, unless there are generic consequences to making large amounts of siRNAs or by chance there are similar sequences to the trigger sequences in the plant genome (this can be avoided if the genome of the host plant is available). Delivery from the host of a concatenated cassette of transgenic siRNAs against multiple diseases would result in a highly effective single locus that could be easily deployed in breeding programs. Because no new proteins are made, biosafety and health concerns are reduced and should be minimal.
  • RNAi using stable transgenic wheat plants (Fu et al., 2007 & 2009) and transient silencing using Virus Induced Gene Silencing (VIGS) have been used to silence resistance genes in wheat and lettuce (Scofield et al. 2005, Wroblewski et al., 2007). VIGS in itself does not affect the rust interaction, as was shown in studies demonstrating VIGS inactivation of wheat genes involved in resistance to P. graminis (Scofield et al., 2005;
  • RNAi is probably functional in Puccinia species.
  • siRNAs corresponding to genes of Plasmodium berghei encoding cysteine proteases resulted in substantial accumulation of hemoglobin, which is reminiscent of the effect observed upon treating P. falciparum with cysteine protease inhibitors (Mohmmed et al., 2003).
  • the greatest uncertainty in this strategy was whether siRNAs would move across the rust haustorial interface. There are clearly a large number of biochemical exchanges in both directions between plant and pathogen.
  • RNAi can be propagate across or cross the haustorial interface in both directions between a parasitic plant and its host in the case of Triphysaria versicola (a relative of Striga) and lettuce (Tomilov et al., 2008); this is, however, a very different type of interface from that with rusts because it involves phloem connections between the two plants.
  • a method for producing a disease-resistant cereal employs the following overall approach:
  • Potential targets include genes encoding proteins that are known targets of fungicides, which in several cases participate in pathways that are not present in plants.
  • RNAi will be effective against numerous isolates and possibly multiple species.
  • Sequences are available from multiple species. Several potential targets are being considered, for example:
  • Rusts are susceptible to multiple fungicides that inhibit enzymes involved in sterol biosynthesis.
  • Lanosterol 14-alpha-demethylase, C-8 sterol isomerase, and squalene epoxidase are the targets of DMI-fungicides (demethylation inhibitors), amine fungicides, and of allyamines in medical fungicides and thiocarbamates, respectively (Kuck et al., 2008).
  • Chitin synthases are the target of polyoxin fungicides. These are encoded by multigene families in P. graminis as in other fungi but there are conserved domains (Broeker et al., 2006). RNAi can down-regulate multiple gene family members if the trigger sequence is designed to conserved regions (e.g. Wroblewski et al., 2007; F. Piston et al., unpublished).
  • ⁇ -tubulin is the target of many highly effective fungicides (e.g., methyl benzimidazole carbamates and benzamides).
  • ⁇ - tubulins are highly conserved proteins. We would have to ensure that there is insufficient sequence similarity at the nucleotide level to wheat ⁇ -tubulin encoding pathogen genes targeted for RNAi.
  • MAP/histidine kinase that is involved in osmotic signal transduction is the target of Phenyl Pyrroles fungicides (PP- fungicides).
  • RNAi in stable transgenic plants is possibly a more reliable method for down-regulating genes than VIGS, generating transgenic lines of wheat is complex and time- consuming. Therefore, VIGS is being used to test for the efficacy of each gene.
  • the constructs for VIGS use the same barley stripe mosaic virus (BSMV) vector as used by Scofield et al.
  • BSMV barley stripe mosaic virus
  • Constructs are being made to test individual conserved segments as well as concatemers of target gene sequences.
  • segments from two or three genes may be cloned in tandem, in antisense and sense orientations in each RNAi construct.
  • the cloned segments will be selected to have stretches of more than 21 nucleotides of perfect identity between the homologous genes in multiple rust species.
  • a 30-bp overlapping region will be included in the reverse primer of the first gene and in the forward primer of the second gene. We have already used this strategy to generate a construct simultaneously targeting the ERG2 and ERG11 genes in ergosterol biosynthetic pathway of stripe rust.
  • Plants are challenged with rust -eight days after BSMV inoculation. Plants inoculated with control constructs carrying a fragment of PDS show photobleaching after 10 days.
  • the small RNAs from inoculated plants will be profiled using high-throughput sequencing of small RNAs. We have found this to be highly informative as to the types and amounts of siRNAs generated by different constructs (T. Wroblewski, R. Michelmore, unpublished). This will provide quantitative data on the expression of small RNAs of plant and pathogen origin and may provide evidence for siRNAs produced in the plant crossing the haustorial interface.
  • Embryonic calli of hexaploid spring cultivar Bobwhite are bombarded using a 1 : 1 molar ratio of the pANDA construct and UBI::BAR selectable marker plasmid (15.5 ⁇ g total) coated onto 1000 nm gold particles (Seashell, La Jolla, CA), according to the manufacturer's instructions.
  • Transformants are selected as previously described (Okubara et al., 2002, Uauy et ah, 2006). Transformants were selfed to generate T ⁇ progeny lines; T ⁇ progeny lines were also selfed in order to generate T 2 progeny lines. 4) Disease Assays
  • Stable transgenics and plants exhibiting VIGS are/will be tested for resistance to each of the three major rust disease of wheat either at the 1 to 4 leaf stage ("seedling inoculation") or after flag leaves are fully emerged (“adult-plant inoculation”).
  • plants are placed in a dew chamber without light at 10°C for 24 h and inoculated with a mixture of urediniospores and talcum powder that is dusted on the leaf tissue. Two days after inoculation plants are then moved to growth chambers at room
  • Example 1 Illumina sequencing of a highly virulent PST race
  • PST Puccinia striiformis f. sp. tritici
  • the assembled contigs provide an estimated coverage of at least 88% of the stripe rust genome.
  • PST genes we used an ab initio gene prediction program and identified 22,815 putative coding sequences and applied a comparative approach based on genomic sequence from the three wheat rust species to improve gene annotation. This sequencing effort is described in a manuscript submitted to PLoS One. Sequences and assemblies will be publicly available through GenBank. This sequence information is enabling the cloning of PST genes to generate the RNAi constructs employed in some of the following experiments.
  • VIGS assay In order to efficiently test silencing of rust genes in wheat, we used a VIGS assay (Scofield et al., 2005). We are currently using rust susceptible wheat lines D6301, and Bobwhite for these VIGS assays. Six targets from PST have so far been cloned and three have been tested using VIGS assays. The tested targets include two genes, ERG2 and ERG11, encoding enzymes in the ergosterol biosynthesis pathway, a C-8 sterol isomerase and a lanosterol 14-alpha- demethylase, respectively and a plasma membrane ATPasel that is highly expressed in Puccinia spp. haustoria.
  • RNAi construct containing 185bp fragment of the phytoene desaturase (PDS) and empty vector control was used to provide a phenotypic readout of VIGS.
  • Seven day-old wheat seedlings were infected with BSMV by rub inoculation. Plants were challenged with PST urediniospores eight days after BSMV inoculation. Plants inoculated with control construct carrying a fragment of PDS showed photobleaching after 10 days.
  • ERGl encodes a squalene epoxidase. This will be tested using VIGS along with lljll and llo3 that were recently shown to be down-regulated by HIGS (Yin et al. 2011; DOI: 10.1094). Additional target genes are being selected, cloned and tested using VIGS.
  • RNAi constructs in the pANDA expression vector targeting ERG2, ERGl 1, 12j 12 and 12o3 were produced and used to generate stable transgenics. Five independent
  • RNAi constructs carrying the ERGl 1/2 RNAi construct were identified. The heritability, sequence and expression of the RNAi construct were confirmed. T 2 plants from these transgenics are growing and we will be able to test their susceptibility to PST within the next few months.
  • T 2 plants will be screened using multiple and highly virulent PST races.
  • new pANDA constructs will be constructed to target PST ERGl and in P. graminis f. sp. tritici (stem rust; PGT) ERGl, ERGl and ERG11.
  • PGT seed rust
  • ERGl, ERGl and ERG 11 sequences selected share stretches of identity with P. triticina and the susceptibility of the transgenic wheat lines carrying this RNAi construct will be tested for their reaction to both stem and leaf rust.

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Abstract

La présente invention concerne des céréales transgéniques exprimant des constructions d'ARN interférence (ARNi) conçus pour silencer des gènes critiques dans des pathogènes qui provoquent des maladies dans les céréales.
PCT/US2012/037660 2011-05-11 2012-05-11 Résistance à une maladie dans les céréales à médiation par un silençage génique induit par l'hôte WO2012155112A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014126845A1 (fr) * 2013-02-14 2014-08-21 Washington State University Génération de plantes cultivées résistant à la maladie de la rouille des graminées par réduction au silence de gènes pathogènes spécifiques
WO2015004174A1 (fr) * 2013-07-10 2015-01-15 Basf Se Arni inhibant l'expression des gènes cyp51 pouvant être utilisés en vue de la lutte contre les champignons et les oomycètes phytopathogènes
CN108220304A (zh) * 2018-02-02 2018-06-29 山东农业大学 小麦条锈菌pstg_06371基因在条锈病防治中的应用和抗条锈菌小麦的培育方法
WO2020035619A1 (fr) 2018-08-17 2020-02-20 Centre National De La Recherche Scientifique (Cnrs) Procédés de lutte biologique à base d'arn pour protéger des plantes contre des bactéries pathogènes et/ou favoriser des effets bénéfiques de bactéries symbiotiques et commensales
EP3967745A1 (fr) 2020-09-11 2022-03-16 Immunrise Production à base de chlorelle de petits arns enchâssées dans des vésicules extracellulaires pour des applications de lutte biologique

Citations (4)

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EP1482037A1 (fr) * 2003-05-30 2004-12-01 Wah Hin Alex Yeung L'inhibition de l'expression de genes par l'introduction de précurseurs de petits ARN interférents specialement seléctionnés, tels que les siARN double brin ou d'autres formes de siARN, permettant la formation de siARN in vivo et in vitro
US20060200878A1 (en) * 2004-12-21 2006-09-07 Linda Lutfiyya Recombinant DNA constructs and methods for controlling gene expression
US20090048111A1 (en) * 2002-10-04 2009-02-19 Myriad Genetics, Incorporated Rna interference using a universal target
CA2750997A1 (fr) * 2009-02-03 2010-08-12 Consejo Superior De Investigaciones Cientificas Polynucleotide comprenant des sequences de gliadines de ble et son utilisation pour le silencage par arni

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090048111A1 (en) * 2002-10-04 2009-02-19 Myriad Genetics, Incorporated Rna interference using a universal target
EP1482037A1 (fr) * 2003-05-30 2004-12-01 Wah Hin Alex Yeung L'inhibition de l'expression de genes par l'introduction de précurseurs de petits ARN interférents specialement seléctionnés, tels que les siARN double brin ou d'autres formes de siARN, permettant la formation de siARN in vivo et in vitro
US20060200878A1 (en) * 2004-12-21 2006-09-07 Linda Lutfiyya Recombinant DNA constructs and methods for controlling gene expression
CA2750997A1 (fr) * 2009-02-03 2010-08-12 Consejo Superior De Investigaciones Cientificas Polynucleotide comprenant des sequences de gliadines de ble et son utilisation pour le silencage par arni

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014126845A1 (fr) * 2013-02-14 2014-08-21 Washington State University Génération de plantes cultivées résistant à la maladie de la rouille des graminées par réduction au silence de gènes pathogènes spécifiques
WO2015004174A1 (fr) * 2013-07-10 2015-01-15 Basf Se Arni inhibant l'expression des gènes cyp51 pouvant être utilisés en vue de la lutte contre les champignons et les oomycètes phytopathogènes
US20160215290A1 (en) * 2013-07-10 2016-07-28 Basf Se Rnai for the control of phytopathogenic fungi and oomycetes by inhibiting the expression of cyp51 genes
EP3431601A3 (fr) * 2013-07-10 2019-04-03 Basf Se Arni pour le contrôle de champignons phytopathogènes et d'oomycètes par inhibition de l'expression de gènes cyp51
US11441147B2 (en) 2013-07-10 2022-09-13 Basf Se RNAi for the control of phytopathogenic fungi and oomycetes by inhibiting the expression of CYP51 genes
CN108220304A (zh) * 2018-02-02 2018-06-29 山东农业大学 小麦条锈菌pstg_06371基因在条锈病防治中的应用和抗条锈菌小麦的培育方法
CN108220304B (zh) * 2018-02-02 2020-07-14 山东农业大学 小麦条锈菌pstg_06371基因在条锈病防治中的应用和抗条锈菌小麦的培育方法
WO2020035619A1 (fr) 2018-08-17 2020-02-20 Centre National De La Recherche Scientifique (Cnrs) Procédés de lutte biologique à base d'arn pour protéger des plantes contre des bactéries pathogènes et/ou favoriser des effets bénéfiques de bactéries symbiotiques et commensales
WO2020035620A1 (fr) 2018-08-17 2020-02-20 Centre National De La Recherche Scientifique (Cnrs) Procédés thérapeutiques à base d'arn pour protéger des animaux vis-à-vis de bactéries pathogènes et/ou favoriser les effets bénéfiques de bactéries symbiotiques et commensales
WO2021032794A1 (fr) 2018-08-17 2021-02-25 Centre National De La Recherche Scientifique (Cnrs) Procédés thérapeutiques à base d'arn pour protéger des animaux contre des bactéries pathogènes et/ou favoriser les effets bénéfiques de bactéries symbiotiques et commensales
EP3967745A1 (fr) 2020-09-11 2022-03-16 Immunrise Production à base de chlorelle de petits arns enchâssées dans des vésicules extracellulaires pour des applications de lutte biologique
WO2022053689A2 (fr) 2020-09-11 2022-03-17 Immunrise Production à base de chlorelle de petits arn intégrés dans des vésicules exacellulares à des fins d'application de biorégulation

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