Gene-for-gene coevolution between Albugo candida and Arabidopsis; mining non-host resistance genes for white rust control in Brassicaceae crops

Plant disease results in substantial losses in crop production, and imposes great costs on farmers. For example, control of potato blight costs UK farmers ~ £60M/year, and Asian soybean rust costs Brazilian farmers ~$800M/year. We aim to provide resistance genes that enable disease to be controlled by genetics rather than chemistry.

To provide reliable genetic solutions, we need a better understanding of how plants and their pathogens interact. Albugo species cause white rust (WR) disease in crucifer plants, including Brassica crops (eg broccoli and oilseed rape), and the model plant Arabidopsis. WR-infected plants become extremely susceptible to other diseases; we wish to understand the basic mechanisms by which this happens. Pathogens deliver molecules called effectors to host cells that interfere with host immune mechanisms. Plant resistance (R) genes recognize such effectors and then activate immunity. To overcome R genes, pathogens must evade detection by mutations in genes that encode effectors. We aim to identify the WR effector repertoire, and the best way to identify effectors is to find those that are recognized by R genes.

Plant breeders often use R genes from wild relatives by crossing them into crop varieties. However, single R genes can be rapidly overcome by resistance-breaking pathogen races. We aim to clone multiple WR resistance (WRR) genes from the model plant Arabidopsis that act against WR strains that infect Brassica or other crucifer crops. By transforming crops with multiple independently acting WRR genes, the risk is reduced that a single mutation in the pathogen will create a resistance-breaking strain. In India, Australia and Canada, Brassica juncea is an important oilseed crop, fungicides are expensive for poor farmers and there are insufficient sources of WRR. Arabidopsis genes have already been identified against B. juncea strains of WR; we aim to discover and deploy additional such genes. The oilseed Camelina sativa has been engineered to produce 25% of its seed oil as "heart-healthy" polyunsaturated fatty acids identical to those in fish oil, but C. sativa is susceptible to UK WR strains. We will survey Arabidopsis natural genetic variation to identify and clone additional WRR genes against these strains, and verify their efficacy in C. sativa, prior to building a multigene stack to protect C. sativa against known UK strains of WR.

We used a genetic trick to identify variation in Arabidopsis for WRR genes that act against B juncea strain of WR. However, this trick did not work to identify variation for resistance to B. oleracea (broccoli, cauliflower, Brussels sprouts) strains of WR. We will test a different trick that enables us to mutate all candidate R genes that might confer WRR to the B. oleracea strains, and thus identify new WRR genes against these strains. Such genes have the potential to provide an excellent source of resistance against B. oleracea-infecting WR strains.

To identify effectors from WR that are recognized by WRR genes, we can transiently co-express a WRR gene with a set of various effector candidate genes in tobacco leaves using Agrobacterium, and if there is recognition, activation of defence results in cell death in the infiltrated part of the leaf. We can thus identify which effector is recognized by which WRR gene. Such knowledge is essential to ensure that different WRR genes really do recognize different effectors, and also as a prelude to investigating how each effector suppresses host immunity in future experiments

These studies will provide multigene stacks that should provide durable resistance. Success with this approach using the Arabidopsis model system to facilitate isolation of multiple distinct WRR genes, will validate conceptually similar approaches to cloning multiple R genes from wild relatives of wheat or potato, to protect the crop against rusts or late blight.

We will use genetics to identify multiple new Arabidopsis resistance genes against white rust (WR) of crucifers- especially Brassica juncea-infecting Ac2V- and introduce them in multi-gene stacks to protect Brassica and Camelina sativa crops. We will assign function to many orphan Arabidopsis NB-LRR-encoding R genes, identify corresponding WR recognized effectors in tobacco transient assays, and obtain new basic insights into crucifer/Albugo candida coevolution.

We found Arabidopsis transgressive segregant lines susceptible to race Ac2V. Many Arabidopsis lines resist WR races Em2 and Nc2 via WRR4, and Nc2/Em2-susceptible lines lack WRR4. We identified WRR8 and WRR9 against Ac2V in WRR4-lacking accessions Sf-2 and Hi-0. We will screen many, diverse Arabidopsis accessions; Nc2/Em2 susceptible lines will be inspected for their WRR4, WRR8 and WRR9 sequences at the 1001 genomes database. Those lacking these 3 WRR genes will be crossed to Ac2V susceptible lines and corresponding new R genes cloned, accelerated by RenSeq (R gene enrichment sequencing). Stacks carrying multiple new WRR genes against Ac2V will be assembled and used for B. juncea transformation. C. sativa is susceptible to Arabidopsis-infecting strains of WR; we will identify additional WRR genes to combine in stacks for protection of C. sativa, a future source of heart-healthy oils. B. oleracea-infecting WR strain AcBoT is resisted by all Arabidopsis. Using CrispR/Cas9 mutagenesis, we will mutate all TIR-NB-LRR R genes of Arabidopsis to discover which confer AcBoT resistance; these genes will then be tested in B. oleracea to verify function against AcBoT.

We sequenced multiple A. candida strains and compared them to identify effector candidates that might be recognized by specific WRR genes. Transient co-expression in tobacco of WRR4 with effector candidates revealed CCG28 as recognized by WRR4. As additional WRR genes are cloned, using this validated method, we will find new recognized effectors

The PI will take the lead on managing the pathways to impact, which will be discussed at regular project meetings.

The research in this proposal focuses on defining the set of genes in Arabidopsis that confer resistance to races of white rust (Albugo candida) that cause disease in Brassica and Camelina crops, and on identifying the effectors recognized by these WRR genes. We will assemble gene stacks carrying 3 or more distinct white rust resistance (WRR) genes to maximize durability of each gene. We have support from researchers working on Brassica juncea and Albugo in Canada (Dr. Hossein Borhan), India (Prof. Deepak Pental) and Australia (Prof. Martin Barbetti). They will test any lines we create in transgenic B. juncea for resistance against local strains of A. candida. This will extend the set of genes that can be tested within the scope of a CGAT proposal (decision pending) jointly with Prof. Pental and Prof. Eric Holub at U. Warwick. We have the support of Prof. Johnathan Napier at Rothamsted who is leading development of GM Camelina sativa engineered to accumulate elevated levels of heart-healthy oils as an oilseed crop.

Communications and Public Engagement
The output of this proposal will be disseminated to a broad audience through primary research articles and review articles in academic journals, and at international and UK conferences.

I already engage broadly with the public around the value of GM field trials for potato late blight resistance. I am an outspoken advocate of GM solutions to crop problems (see http://www.guardian.co.uk/environment/2011/jul/21/gm-debate and http://news.bbc.co.uk/1/hi/sci/tech/8789279.stm ) and I am a director of www.ISAAA.org. This project will provide an additional basis for public engagement.
We explained how and why we conducted a GM potato field trial at www.tsl.ac.uk/gmtrial. We anticipate that when we have Brassica and Camelina lines carrying multiple WRR genes we will undertake a similar set of GM trials and public engagement activities to explain the rationale.

Exploitation and Application
TSL aims to bring to public use efficiently, any discoveries we make that could elevate disease resistance in crops. When we discover new WRR genes, we will file for intellectual property protection either with Plant Biosciences Ltd at JIC or with the 2Blades foundation (www.2blades.org). 2Blades is a charity dedicated to solving important crop disease problems. As a member of the 2Blades science advisory board, I am well placed to help push from discovery to deployment. We are thus in a good position to ensure that any discoveries are translated as rapidly as possible to transgenic plant lines with elevated disease resistance, prior to market acceptance and commercialization.

Training
TSL/JIC/UEA offers extensive opportunities for professional and scientific training. The PDRAs will use these resources to facilitate professional and technical development. In particular the PDRAs will receive extensive training in Illumina methods for R gene enrichment sequencing (RenSeq), and in associated bioinformatics competence such as assembly and analysis of contigs from short reads. They will also be trained in drafting manuscripts for publication. We aim to submit at least two manuscripts to international journals based on the outcomes of this research. One of the postdocs will also receive strong training in the new technology of using CrispR/Cas9 targeted mutagenesis in plants.

The PI and PDRAs will participate in both formal and informal seminar series within TSL/JIC/UEA, as well as lab meetings. The PDRAs will be given opportunities to attend national and international conferences (e.g. as given above) and present their work via poster and oral presentations. They are also likely to help supervise PhD student and masters or rotation student participants in the project.