WO2012155112A1

WO2012155112A1 - Disease-resistance in cereals mediated by host-induced gene silencing

Info

Publication number: WO2012155112A1
Application number: PCT/US2012/037660
Authority: WO
Inventors: Richard Michelmore; Jorge Dubcovsky; Manjula GOVINDARAJULU; Dario CANTU
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 cereals expressing RNA interference (RNAi) constructs designed to silence critical genes in pathogens that cause diseases in cereals.

Description

DISEASE-RESISTANCE IN CEREALS MEDIATED BY HOST-INDUCED GENE

SILENCING

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 cereals and methods for producing disease-resistant cereals, and more specifically to disease-resistance in cereals mediated by host- induced gene silencing ("HIGS").

2. Description of Related Art

[0003] Wheat and other cereals suffer from several serious diseases (Prescott et al., 1986). Of particular importance are the potentially devastating rust diseases caused by fungi belonging to the Uredinales. Three rusts are especially troublesome to wheat: Puccinia triticina, P.

graminisf. sp. tritici, and P. striiformis f. sp. tritici that cause leaf, stem, and stripe rusts respectively (Mcintosh et al., 1995). Control of these diseases has mainly been through the use of genetically resistant cultivars, although the efficacy and durability of such resistance has been variable. There have been repeated boom-and-bust cycles of the release and increase of cultivars carrying major genes for resistance followed by the evolution of virulent variants of the pathogen and the consequent crash of the cultivars (Suneson, 1960).

[0004] The threat from wheat rusts has been brought into heightened focus by the recent emergence and spread of the UG99 race of the wheat stem rust, P. graminis

(globalrust.org/traction/permalink/Multimedia398). So-called because it was first identified in Uganda in 1999, this race has spread from Uganda to Ethiopia and more recently to Yemen and Iran and is heading for the large wheat producing areas in Central and South Asia (Hovm0ller et al., 2010). Approximately 80% of the wheat cultivars grown in the at-risk areas are susceptible to UG99. Epidemics can result in near-total crop loss. [0005] There are sources of major and minor gene resistance to UG99 and other races of rust. Introgression of these genes into cultivated wheat clearly is a very high priority. However, some of them are in marginally sexually compatible genepools and their introgression into cultivated wheat is a slow and labor-intensive process. Also, there is no guarantee as to the level of sustained resistance that they will provide. Moreover, the optimal strategy for gene deployment to provide the most durable resistance is still unclear.

BRIEF SUMMARY

[0006] In one embodiment, the disclosure provides transgenic cereals expressing RNA interference (RNAi) constructs designed to silence critical genes in pathogens that cause diseases in cereals.

[0007] In one particular embodiment, 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. Further, the plant expresses 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.

DESCRIPTION OF THE FIGURES

[0008] 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.

[0009] Figure 2 depicts Stable T₂ transgenics of wheat cv. Bobwhite generated to target EGR2 & ERG11 simultaneously & tested for reaction to stripe rust, Puccinia striiformis.

[0010] Figure 3 depicts an overview of the HIGS effect.

[0011] Figure 4 depicts other opportunities for HIGS in crop plants.

[0012] 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. [0013] Figure 6 depicts the plasmid map for virus-induced gene silencing (VIGS) plasmid, pa42.

[0014] Figure 7 depicts the plasmid map for virus-induced gene silencing (VIGS) plasmid,

[0015] Figure 8 depicts the plasmid map for virus-induced gene silencing (VIGS) plasmid, pSL038-l.

[0016] Figure 9 depicts the plasmid map for virus-induced gene silencing (VIGS) plasmid, pSL039B-l.

[0017] Figure 10 depicts the DNA sequence for the pa42 plasmid.

[0018] Figure 11 depicts the DNA sequence for the pp.42spl plasmid.

[0019] Figure 12 depicts the DNA sequence for the pSL038-l plasmid.

[0020] Figure 13 depicts the DNA sequence for the pSL039B- 1 plasmid.

[0021] 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.

DETAILED DESCRIPTION

[0022] 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.

[0023] Described herein is an RNAi-mediated approach that is durable and complements more conventional approaches for providing disease-resistance in cereals.

[0024] The present disclosure provides transgenic wheat expressing RNA interference (RNAi) 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.

[0025] 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.

[0026] 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;

www.ag.purdue.edu/agry/Pages/scofield.aspx). Fungi are also capable of RNAi-mediated gene silencing; the majority of fungi possess the RNAi machinery and RNAi has been used for functional characterization of fungal genes (Nakayashiki et al. 2006; Nakayashiki & Nguyen 2008; Nakayashiki 2005). BLAST searches with sequences of genes encoding RNAi pathway proteins in Neurospora crassa revealed the presence of homologous genes in P. graminis (D. Cantu & J. Dubcovsky, unpublished). Therefore, RNAi is probably functional in Puccinia species. [0027] 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). 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. However, there were few reports on what and how such exchanges occur in compatible interactions, previously, the inventors have demonstrated that 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.

[0028] Currently there are two published manuscripts relating to HIGS 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 (both manuscripts are lack detailed information and designed to document HIGS.) Also both manuscripts rely only on transient expression data. Finally, neither manuscript has data from stable transgenics.

The Overall Strategy

[0029] In one embodiment, a method for producing a disease-resistant cereal employs the following overall approach:

1) A) Select essential pathogen gene targets, sequence them in the three rusts, and B) design constructs to silence them using siRNA expressed in wheat cells.

2) Conduct transient VIGS assays to demonstrate whether siRNAs can cross the haustorial interface and alter rust interaction phenotypes. 3) Generate stable transgenic wheat plants expressing RNAi constructs.

4) Test inoculated transgenics for reduced transcript levels of the targeted pathogen genes and reduced susceptibility to three wheat rusts.

5) Disseminate transgenics for further testing and conversion to other cultivars. [0030] The first four steps of the strategy are discussed in more detail, below.

1) A) Target Gene Selection

[0031] 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 Puccinia spp., so RNAi will be effective against numerous isolates and possibly multiple species.

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

[0032] Sequences are available from multiple species. Several potential targets are being considered, for example:

[0033] 1) Genes involved in sterol biosynthesis: 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).

[0034] 2) Genes involved in cell wall biosynthesis: 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).

[0035] 3) Genes encoding structural proteins: β-tubulin is the target of many highly effective fungicides (e.g., methyl benzimidazole carbamates and benzamides). However, β- tubulins are highly conserved proteins. We would have to ensure that there is insufficient sequence similarity at the nucleotide level to wheat β-tubulin encoding pathogen genes targeted for RNAi.

[0036] 4) Genes encoding signal transduction proteins: MAP/histidine kinase that is involved in osmotic signal transduction is the target of Phenyl Pyrroles fungicides (PP- fungicides).

[0037] 5) Additional genes: Many more targets will become available as more genome sequences of Puccinia spp. become available and the molecular basis of pathogenicity becomes better understood.

1) B) Design and Generation of Constructs

[0038] Based on the above criteria we have selected approximately 15 candidate targets. We obtained sequences for each target focusing initially on stripe rust because it is easier to work with locally. We sequenced nearly the entire genome of P. striiformis using an Illumina Genome Analyzer II. 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 wheat ESTs and in the collection of 454 reads from hexaploid wheat (5x coverage; www.cerealsdb.uk.net).

[0039] Although 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.

(2005) to inactivate wheat genes including Lr21 involved in resistance to P. graminis.

Constructs are being made to test individual conserved segments as well as concatemers of target gene sequences. [0040] For the production of the constructs for stable transformation, segments from two or three genes may be cloned in tandem, in antisense and sense orientations in each RNAi construct. When possible, 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. To develop PCR products including multiple gene segment targets, 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.

2) VIGS Induction

[0041] For each plant to be inoculated, we linearize 0.7 μg of plasmid DNA for each of the three genomic RNAs of BSMV (pa and pSL038-l are linearized with Mlul and ρβ with Spel). The linearized plasmids are used for in vitro transcription of viral RNAs using the mMessage mMachine™ T7 in vitro transcription kit (Ambion). Equal amounts of each of the in vitro transcription reactions for the α, β and γ RNAs are combined and applied to the second leaf of 7- day-old seedlings by rub inoculation. At least 20 plants are inoculated for each construct.

[0042] 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.

3) Generating Stable Transgenics

[0043] 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 μg 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 T₂ progeny lines. 4) Disease Assays

[0044] 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").

[0045] For PST 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

temperature. Rust severity is evaluated two to three weeks after inoculation using a 0 to 9 scale of infection type (IT) (Line et al., 1970): 0 to 3 (resistant, none to trace level sporulation), 4 to 6 (intermediate, light to moderate sporulation), 7 to 9 (susceptible, abundant sporulation). If differences are quantitative, the percentage of leaf surface covered with PST pustules is quantified using the digital image analysis program "pd" available at

plantpathology.ucdavis.edu/faculty/Epstein (Fu et al., 2009).

[0046] Since Bobwhite carries the stripe rust resistance gene Yr9, we use stripe rust race PST113 and PST130, which are virulent on this seedling resistance gene, and that we have successfully used before (Fu et al., 2009; Chen et al., 2010). Bobwhite is also susceptible to stem rust race UG99. Transgenic plants will be evaluated for resistance to stem rust by Dr. Yue Jin at the Cereal Disease Research Unit (MN). Stable transgenic plants will also be evaluated for leaf rust resistance using races virulent on plants expressing Lr26, which is present in Bobwhite, by the US leaf rust specialist Jim Kolmer at the Cereal Disease Research Unit (MN). Both Dr. Jin and Dr. Kolmer have agreed to test our transgenic lines. In addition, lines that show resistance to the races of stripe rust that we have at Davis will be tested for resistance to additional races by Dr. Xianming Chen at Washington State University who has a large the collection of stripe rust races.

[0047] The following examples illustrate certain embodiments of the methods described herein and/or may be useful in better understanding the provided methods and systems. Example 1: Illumina sequencing of a highly virulent PST race

[0048] We have focused initially on Puccinia striiformis f. sp. tritici (PST) because of the relative ease with which it can be analyzed and lack of containment issues. In order to obtain sequences of target genes, we used Illumina sequencing to rapidly access genomic sequence information for PST race 130 (PST- 130), a highly virulent race from the United States (Chen et al., 2010). We obtained nearly 80 million high quality paired-end reads (55 to 62 x coverage) that were assembled into 29,189 contigs spanning a total of 64.8 Mb (including 84 kb of mitochondrial genome) after exclusion of low quality reads and contaminant sequences. The assembled contigs provide an estimated coverage of at least 88% of the stripe rust genome. To facilitate access to 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.

Example 2: Virus induced gene silencing (VIGS)

[0049] 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. Fragments of 300 to 400bp from the targeted genes were cloned into pGEM- TEasy, sequence validated and transferred to pSL038-l (BSMV vector) using the Notl sites flanking the pGEM cloning site. Control 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. We screened VIGS infected wheat plants phenotypically (10 plants/cultivar/gene) three weeks after stripe rust inoculation. The VIGS results from two independent biological replicates suggests that silencing either ERGl or ERG 11 results in partial reduction in stripe rust in comparison to our PDS and empty vector controls that showed heavy sporulation on the D6301 and Bobwhite plants (Figure 1). Similarly, heavy sporulation was visible on ATPasel silenced plants. These results support ERGl and ERG 11 as potential targets for host-induced gene silencing (HIGS) of PST. Further replications using these constructs are currently underway. Silencing will be confirmed by qRT-PCR and sRNAseq.

[0050] Based on these encouraging results, we have cloned an additional gene in the ergosterol pathway, ERGl, which 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.

Example 3: Generation of stable transgenics of wheat

[0051] 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

transformants carrying the ERGl 1/2 RNAi construct were identified. The heritability, sequence and expression of the RNAi construct were confirmed. T₂ plants from these transgenics are growing and we will be able to test their susceptibility to PST within the next few months.

[0052] The T₂ plants will be screened using multiple and highly virulent PST races.

Additionally, new pANDA constructs will be constructed to target PST ERGl and in P. graminis f. sp. tritici (stem rust; PGT) ERGl, ERGl and ERG11. The PGT 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. REFERENCES

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Claims

CLAIMS What is claimed is:

1. A transgenic cereal 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 cereal plant; wherein the cereal plant expresses the siRNA.

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

3. The transgenic cereal plant of claims 1 or 2, wherein the pathogen is a fungus.

4. The transgenic cereal plant of claim 3, wherein the pathogen is selected from the group consisting of Puccinia triticina, Puccinia graminis, and Puccinia striiformis.

5. The transgenic cereal plant of any one of claims 1 to 4, wherein the siRNA is capable of propagating across or crossing the haustorial interface of the pathogen.

6. The transgenic cereal plant of any one of claims 1 to 5, wherein the at least one gene of the pathogen is selected from the group consisting of a gene involved in sterol biosynthesis, a gene involved in cell wall biosynthesis, a gene encoding a structural protein, a gene encoding a signal transduction protein.

7. The transgenic cereal plant of claim 6, wherein the at least one gene of the pathogen is selected from the group consisting of ATPase, ERG 1, ERG2, and ERG11.

8. The transgenic cereal plant of any one of claims 1 to 7, wherein the cereal plant is a wheat plant.

9. The transgenic cereal plant of any one of claims 1 to 8, wherein the transgenic cereal plant is a Ti or later progeny of a T₀ transformant that exhibits 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.

10. The transgenic cereal plant of claim 9, wherein the Ti or later progeny of the T₀ transformant is a T₂ or later progeny of the To transformant.