WO2012155109A1

WO2012155109A1 - Host-induced gene silencing in vegetable species to provide durable disease resistance

Info

Publication number: WO2012155109A1
Application number: PCT/US2012/037651
Authority: WO
Inventors: Richard Michelmore; Manjula GOVINDARAJULU
Original assignee: The Regents Of The University Of California
Priority date: 2011-05-11
Filing date: 2012-05-11
Publication date: 2012-11-15

Abstract

The present disclosure provides transgenic vegetable plants, such as lettuce and spinach, expressing RNA interference (RNAi) constructs designed to silence critical genes in pathogens that cause diseases in vegetable plants.

Description

HOST-INDUCED GENE SILENCING IN VEGETABLE SPECIES TO PROVIDE

DURABLE DISEASE RESISTANCE

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0001] This invention was made with government support under Contract No. 2008-35300- 04447 awarded by the United States Department of Agriculture. The government has certain rights in the invention.

BACKGROUND

1. Field

[0002] The present disclosure relates generally to disease-resistant vegetable species and methods for producing disease-resistant vegetable species, and more specifically to disease- resistance in vegetable species mediated by host-induced gene silencing ("HIGS").

2. Description of Related Art

[0003] Several vegetable species are important commercial crops. As with most, agriculture disease-causing pathogens have a marked negative impact on cultivation of vegetable species.

[0004] For example, lettuce is an important crop species and ranks as one of the top ten most valuable crops in the US with an annual value of over $2 billion (Anon., 2007). Lettuce is grown as extensive monocultures, often with several crops per year. Such intensive production makes the crop susceptible to major epidemics and lettuce suffers from several economically important pests and diseases (Davis et al., 1997). These are currently controlled by a combination of genetic resistance, cultural practices, and chemical protection. Genetic resistance is available for some, but not all, diseases. Chemical protection is used extensively to provide a disease-free crop; however, several of these compounds are being withdrawn from agricultural use due to environmental concerns over their safety or have been rendered ineffective by changes in pathogen virulence (Schettini et al., 1991; Brown et al., 2004). Therefore, there is a considerable need for new disease resistant cultivars.

[0005] Oomycete pathogens such as the biotrophic downy mildews (DMs), and the

Phytophthora and Pythium species, chronically cause significant crop losses throughout the world and can cause catastrophic disease epidemics. The DM of lettuce (caused by Bremia lactucae) and spinach (caused by Peronospora farinosa) are significant production constraints of these highly valuable vegetable crops in the US and other regions of the world. Much breeding effort is currently focused on introgressing new genes from wild species in response to changes in pathogen virulence. At least 27 major Dm {Downy mildew) genes or resistance (R) factors are now known that provide resistance against specific isolates of B. lactucae in a gene-for-gene manner in lettuce (Farrara et al., 1987; Bonnier et al., 1994; Maisonneuve et al., 1994; Jeuken & Lindhout, 2002). Dm genes provide high levels of resistance but they have only remained effective for limited periods of time due to changes in pathogen virulence.

[0006] Several other diseases are problematic in lettuce. These include bacterial corky root caused by Sphingomonas suberifaciens, and fungal diseases such as lettuce drop caused by Sclerotinia minor and S. sclerotiorum, powdery mildew caused by Erysiphe cichoracearum, lettuce anthracnose caused by Microdochium panattonianum, and wilts caused by Fusarium oxysporum and Verticillium dahliae. There are also several viral diseases of varying importance such as lettuce mosaic, lettuce big vein, lettuce infectious yellows, beet western yellows, tomato spotted wilt virus, and tomato bushy stunt. Several of these pathogens, including M.

panattonianum, E. cichoracearum, F. oxysporum and V. dahliae represent possible targets for transgene-based disease resistance in lettuce.

[0007] Spinach is also an economically important vegetable crop in the United States, grown on approximately 30,000 ha with an annual value of over $200 million. The genetics of resistance against DM and other diseases in spinach is less well-characterized than in lettuce because fewer resources have been invested and spinach is more difficult to study genetically. Therefore there is great potential and need for transgene-based disease resistance in spinach.

[0008] Accordingly, vegetable species lines having durable and effective resistance to disease-causing pathogens are desirable.

BRIEF SUMMARY

[0009] In one embodiment, the disclosure provides transgenic vegetable species expressing RNA interference (RNAi) constructs designed to silence critical genes in pathogens that cause diseases in vegetable species. [0010] In one particular embodiment, the disclosure provides a transgenic vegetable 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 vegetable plant. Further, the plant expresses the siRNA. Upon infection by the pathogen, the siRNA is capable down-regulating or suppressing expression of the target pathogen gene.

DESCRIPTION OF THE FIGURES

[0011] Figure 1 depicts silencing screening results for stable transgenic lines (T screened with GUS staining assay.

[0012] Figure 2 depicts analysis results for leaf disks of resistant or partially resistant T₂ plants from families that exhibited 1:2:1 segregation at the seedling stage.

[0013] Figure 3 depicts parts of the targeted coding sequences for RNAi sequences used for generating stable lettuce plants.

[0014] Figure 4 depicts resistance exhibited in T₃ lettuce plants to downy mildew.

DETAILED DESCRIPTION

[0015] The following description sets forth exemplary methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

[0016] Described herein is an RNAi-mediated approach that is durable and complements more conventional approaches for providing disease-resistance in vegetable species.

[0017] The present disclosure provides transgenic vegetable plants expressing RNA interference (RNAi) constructs designed to silence genes in the pathogen that are critical to the development of disease in vegetable plants. Small, interfering RNAs synthesized in the plant may be delivered 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 pathogen development and growth thereby reducing disease incidence and severity in the vegetable plant. Vegetable plants with such silencing will also exhibit resistant to pathogens compared to wild type vegetable plants.

Host-induced gene silencing

[0018] Gene silencing using small interfering RNAs (siRNAs) produced in the host plant offers the opportunity to knock out the expression of key genes in a pathogen (Host- Induced Gene Silencing; HIGS). Because 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.

[0019] RNAi using stable transgenic plants have been used to silence resistance genes in lettuce (Wroblewski et al., 2007). Gene silencing has been induced experimentally in transgenic Phytophthora spp. (Ah-Fong et al., 2008), which is an oomycete that is related to B. lactucae and other downy mildews.

[0020] Movement of siRNAs from host to pathogen or parasite has been demonstrated in several cases. Transgenic Arabidopsis expressing siRNAs for a conserved root knot nematode (RKN) secretory peptide were resistant to four major RKN species (Huang et al., 2006).

Similarly, intravenous injections of siRNAs corresponding to genes of Plasmodium berghei encoding cysteine proteases (berghepain-1 & 2) into mice 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). Prior to our work, the greatest unknown in the HIGS strategy was whether siRNAs would move across the haustorial interface and generate a change in disease susceptibility. There are clearly a large number of biochemical exchanges in both directions between plant and pathogen. However, there were few data on what and how such exchanges occur in compatible interactions. We have demonstrated phenotypically that RNAi can be propagated 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 fungal and oomycete pathogens because it involves phloem connections between the two plants.

[0021] Currently there are two published manuscripts relating to HIGS of fungal diseases in cereals. The first manuscript describes modification of the reaction of barley to powdery mildew (Nowara et al., 2010) and the second describes RNAi of genes in stripe rust on wheat (Yin et al., 2011). However, neither manuscript provides clear data on changes in disease resistance. Also, both manuscripts rely only on transient expression data. Finally, neither manuscript has data from stable transgenics. At present, there are no reports of HIGS against oomycete or fungal pathogens in vegetable crop species.

Overall Strategy

[0022] In one embodiment, a method for producing a disease-resistant vegetable plant employs the following overall approach:

1) A) Selection of essential pathogen gene targets and B) generation of constructs to silence them using siRNA expressed in planta.

2) Generation of stable transgenic lettuce plants expressing RNAi constructs.

3) Testing of inoculated transgenics for reduced transcript levels of the targeted pathogen genes and reduced susceptibility to B. lactucae.

[0023] The steps of the strategy are discussed in more detail, below.

1 ) A) Target Gene Selection

[0024] Gene targets have been selected based on several criteria:

They are key genes in the pathogen for either viability or virulence. 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. They are not functionally redundant.

They are highly conserved within B. lactucae, so RNAi will be effective against numerous isolates.

They are not present in lettuce or their sequences should be highly diverged from lettuce sequences.

[0025] Sequences are available from multiple species. Several potential targets are being considered, for example:

[0026] 1) Genes involved in cell wall biosynthesis, such as cellulose synthase.

[0027] 2) Genes encoding structural protein such as β -tubulin, actin, highly abundant and stage specific mRNAs (e.g. , HAM34).

[0028] 3) Genes involved in protein synthesis, such as elongation factor.

[0029] 4) Genes involved in toxin secretion, such as Nep 1-like proteins, NLP2 and NLP3.

[0030] 5) Additional genes: More targets will become available as more genome sequence of B. lactucae becomes accessible and the molecular basis of pathogenicity becomes better understood.

1) B) Design and Generation of Constructs

[0031] Based on the above criteria we initially selected 7 candidate targets. 300 to 400 bp regions were selected from conserved regions of each gene to act as triggers for RNAi. These were checked by BLAST analysis to confirm the absence of stretches of 21 nt or more of identical sequence in the lettuce genome. Inverted-repeat silencing constructs that include an inverted repeat of the beta-glucuronidase reporter gene (GUS) (Wroblewski et al., 2007) were designed to silence the above list of target genes. In brief, using basic cloning protocols resulted in a 400 bp fragment of the beta-glucuronidase reporter gene (GUS) in a sense orientation, a sense-oriented fragment of target gene, a 788 bp fragment of intron-3 from the gene encoding pyruvate orthophosphate dikinase (pdk) of Flaveria trinervia (Rosche et al., 1995), followed by the same target gene fragment in antisense orientation and an antisense version of the same GUS fragment within the silencing vector.

2) Generation of Stable Transgenic Lettuce Plants

[0032] Lettuce is readily transformed using Agrobacterium tumefaciens (Michelmore et al., 1987). Therefore, RNAi constructs can be easily tested using stable transgenics. For the production of the constructs for stable transformation, segments from each gene were cloned in tandem, in antisense and sense orientations into each RNAi construct as described above.

Silencing constructs were transformed into LBA4404 strain of Agrobacterium tumefaciens (Hoekema et al, 1984).

[0033] Approximately 15 primary stable transformants (Tis) of L. sativa cv. Cobham Green were generated for each silencing construct using A. tumefaciens -mediated transformation by the UC Davis Plant Transformation Facility. Each Ti plant was assayed for the function of the silencing constructs using A. tumefaciens-mediated transient assays (Schob et al., 1997;

Wroblewski et al., 2007). A. tumefaciens strain C58C1 containing pTFS40 construct encodes a functional GUS gene. This A. tumefaciens strain was suspended in water (OD₆oo 0.5-0.6) and leaves were infiltrated when plants were at the 2 to 3 leaf stage. Leaves were collected 4 dpi and histochemical staining for GUS activity was performed (Jefferson et al., 1987). Absence of GUS activity was taken as indicative that the silencing construct was functional. The frequency of plants exhibiting silencing varied from 0 to 38% (Figure 1). All Tis that exhibited silencing were selfed to generate T₂ seed. Tis that did not show silencing (positive for GUS staining) were also taken to T₂ seed as controls.

3) Testing of Transgenics: Disease Assays

[0034] The following example illustrates certain embodiments of the methods described herein and/or may be useful in better understanding the provided methods and systems.

[0035] The T₂s generated from cv. Cobham Green were challenged with B. lactucae to assay for resistance due to HIGS. Cv. Cobham Green expresses no known Dm genes for resistance and is susceptible to most isolates of B. lactucae, including isolates SF5 and CA1309. A suspension of conidia of isolate SF5 was sprayed onto seven-day-old T₂ seedlings (25 seedlings/T₂ family) as well as non-transgenic Cobham Green as a control. The seedlings were scored two weeks later for resistance using a three-point scale: 0 to 25% = resistant, none to trace amounts of sporulation; 25 to 75% = partially resistant, light to moderate sporulation and yellowing of the leaves; 75 to 100% = susceptible, abundant sporulation with necrotic lesions. Four and two T₂ families with silencing of the HAM34 and cellulose synthase genes respectively were resistant or partially resistant, which is indicative of HIGS (Figure 2). Two of these T₂ families from each of the silenced genes (HAM24 and cellulose synthase) segregated 1:2: 1 for resistance reflecting the segregation of the trans gene. Seedlings that showed less than 75% sporulation were grown further and assayed for GUS at the 2-to-3 leaf stage as described above (Figure 2). At this stage, additional tests for resistance were also carried out using isolates SF5 and CA1309 inoculated onto leaf discs (Figure 2). Results paralleled those observed with seedlings; the same plants that were resistant at the seedling stage were resistant in the leaf disc assay (Figure 2). The T₂ seedlings that exhibited GUS activity, and therefore no silencing, were susceptible to both isolates of B. lactucae. The two T₂ families segregating for HAM34 and cellulose synthase RNAi constructs (Figure 2) were selfed to produce T₃ seeds (four families total). So far, six T₃ families for HAM34 have been tested for resistance against B. lactucae as above. A suspension of isolate SF5 conidia was sprayed onto seven-day-old T₃ seedlings (20 seedlings/T₃ family) as well as non-transgenic cv. Cobham Green as a control. Some plants in all of six T₃ families for HAM4 silencing gene showed complete resistance to B. lactucae (Figure 4). These seedlings were grown further and assayed for GUS at the 2-to-3 leaf stage as described above.

Simultaneously, detached leaf discs were inoculated with SF5 to test for resistance. Results paralleled those observed with seedlings; the same plants that were resistant at the seedling stage were resistant in the leaf disc assay and also were negative for GUS staining and therefore exhibited silencing (Figure 4). Demonstrating silencing of the B. lactucae genes is impossible in lines exhibiting such high levels of resistance because there is no pathogen material to analyze. Therefore, we have observed highly effective HIGS at the disease phenotype level in stable transgenics with two out of the six B. lactucae genes tested so far. REFERENCES

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[0038] Bonnier, F. J. K., Reinink, K., & Groenwold, R. (1994). Genetic analysis of Lactuca accessions with new major gene resistance to lettuce downy mildew. Phytopathology 84: 462- 468.

[0039] Brown, S., Koike, S.T., Ochoa, O.E., Laemmlen, F. and Michelmore, R.W. (2004). Insensitivity to the fungicide, Fosetyl- Aluminum, in California isolates of lettuce downy mildew, Bremia lactucae. Plant Dis. 46:1059-1069.

[0040] Davis, R.M., Subbarao, K.V., Raid, R.N. and Kurtz, E.A. (1997). Compendium of lettuce diseases. APS Press, St. Paul, MN.

[0041] Farrara, B.F., Ilott, T.W., & Michelmore, R.W. (1987). Genetic analysis of factors for resistance to downy mildew {Bremia lactucae) in species of lettuce {Lactuca sativa and L.

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[0042] Hoekema A, de Pater BS, Fellinger AJ, Hooykaas PJ, Schilperoort RA (1984). The limited host range of an Agrobacterium tumefaciens strain extended by a cytokinin gene from a wide host range T-region. EMBO J 3:3043-3047.

[0043] Huang, G, Allen, R, Davis, EL, Baum, TJ, Hussey, RS (2006). Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root- knot nematode parasitism gene. Proc Natl Acad Sci USA 103:14302-14306. [0044] Jefferson RA, Kavanagh TA, Bevan MV (1987). GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901-3907.

[0045] Jeuken, M., & Lindhout, P. (2002). Lactuca saligna, a non-host for lettuce downy mildew {Bremia lactucae), harbors a new race-specific Dm gene and three QTLs for resistance. Theoretical and Applied Genetics 105: 384-391.

[0046] Maisonneuve, B., Anderson, P., & Michelmore, R. W. (1994). Rapid mapping of two genes for resistance to downy mildew derived from Lactuca serriola to existing clusters of resistance genes. Theoretical and Applied Genetics 89:96-104.

[0047] Mohmmed, A, Dasaradhi, PVN, Bhatnagar, RK, Chauhan, VS, Malhotra, P (2003). In vivo gene silencing in Plasmodium berghei— a mouse malaria model. Biochem Biophys Res Comm 309:506-511.

[0048] Nowara D, Gay A, Lacomme C, Shaw J, Ridout C, Douchkov D, Hensel G, Kumlehn J, Schweizer P (2003). HIGS: Host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell 22:3130-3141.

[0049] Rosche E, Westhoff P (1995). Genomic structure and expression of the pyruvate, orthophosphate dikinase gene of the dicotyledonous C4 plant Flaveria trinervia (Asteraceae). Plant Mol Biol 29:663-678.

[0050] Schob H, Kunz C, Meins F Jr (1997). Silencing of transgenes introduced into leaves by agroinfiltration: a simple, rapid method for investigating sequence requirements for gene silencing. Mol Gen Genet 256:581-585.

[0051] Schettini, T.M., Legg, E.J. and Michelmore, R.W. (1991). Insensitivity to metalaxyl in California populations of Bremia lactucae and resistance of California lettuce cultivars to downy mildew. Phytopathology 81:64-70.

[0052] Tomilov, A, Tomilova, N, Wroblewski, T, Michelmore, RW Yoder, J (2008). Trans- specific gene silencing between host and parasitic plants. Plant J 51:803-818. [0053] Wroblewski T, Piskurewicz U, Tomczak A, Ochoa O, Michelmore RW (2007). Silencing of the major family of NBS-LRR-encoding genes in lettuce results in the loss of multiple resistance specificities. Plant 751:803-18.

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Claims

CLAIMS What is claimed is:

1. A transgenic vegetable plant capable of host-induced gene silencing of a pathogen, the plant comprising: 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 vegetable plant; wherein the vegetable plant expresses the siRNA.

2. The transgenic vegetable plant of claim 1, wherein the transgenic vegetable plant exhibits resistance to infection by the pathogen as compared to a wild type vegetable plant.

3. The transgenic vegetable plant of claims 1 or 2, wherein the pathogen is an oomycete microbe or a fungus.

4. The transgenic vegetable plant of claim 3, wherein the pathogen is selected from the group consisting of Phytophthora sp., Pythium sp., Bremia lactucae, and Peronospora farinosa.

5. The transgenic vegetable plant of claim 3, wherein the pathogen is selected from the group consisting of Microdochium panattonianum, Erysiphe cichoracearum, Fusarium oxysporum, and Verticillium dahliae.

6. The transgenic vegetable plant of and of claims 1 to 5, wherein the siRNA is capable of propagating across or crossing the haustorial interface of the pathogen.

7. The transgenic vegetable plant of any of claims 1 to 6, wherein the at least one gene of the pathogen is selected from the group consisting of a gene involved in cell wall biosynthesis, a gene encoding a structural protein, a gene involved in protein synthesis, and a protein involved in toxin secretion.

8. The transgenic vegetable plant of claim 7, wherein the at least one gene of the pathogen is selected from the group consisting of cellulose synthase, B-tubulin, actin, HAM34, elongation factor, NLP2, and NLP3.

9. The transgenic vegetable plant of any of claims 1 to 8, wherein the vegetable plant is a lettuce plant.

10. The transgenic vegetable plant of any of claims 1 to 8, wherein the vegetable plant is a spinach plant.

11. The transgenic vegetable plant of any of claims 1 to 10, wherein the transgenic vegetable plant is a T₂ or later progeny of a Ti primary transformant that comprises 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 vegetable plant.

12. The transgenic vegetable plant of claim 11, wherein the T₂ or later progeny of the Ti primary transformant is a T₃ or later progeny of the Ti primary transformant.