A COmbination of systems Biology and experimental high-throughput approaches to engIneer durable Resistance against plAnt viruses in crops – COBRA

COBRA is built on five transdisciplinary workpackages (WPs). The objective of WP1 is to build an exhaustive and comprehensive database of plant-virus interaction networks for the three crops of interest. Such a database facilitates identification of new host susceptibility genes (S-genes) that could serve as resistance genes when mutated. In WP2, the biological relevance of some of the interactors unraveled in WP1 or by COBRA partners will be validated, demonstrating that they are not only plant/virus interacting factors but they are susceptibility factors encoded by bona fide S-genes. The goal of WP3 is to screen for alleles of resistance for the S-genes identified above. Alleles of resistance to viral infection will be searched among mutagenised tomato, barley and peach collections. Germplasm collections of those three species will also be surveyed as well as core collections representative of their diversity in order to implement marker assisted selection and association studies for resistance to crop viruses. Any valuable plant material will be immediately transferred to crop breeding programs. First, COBRA focuses on the search for variants of strong candidate S-genes already validated or patented by the project’s partners. The high throughput platforms set-up for the few above candidates will then serve to screen for variants of new candidates highlighted in WP1. In the fourth WP, we plan to decipher two independent host pathways that poty and potexviruses are hijacking in order to complete their viral infectious cycles. Extra biological information will be generated in order to optimize the search for virus resistant variants. Finally, WP5 is aimed at selecting among the above interactors, host proteins as candidates-of-choice for gene pyramiding. The purpose here is to provide knowledge on the viral factor(s) that determine(s) the breaking of the resistance and to evaluate the durability of the resistance prior to transfer to crop species

The COBRA database is set up, it links together interactomics, transcriptomics and genomics data and allows to easily find information on genes involved in crop/virus interactions. In parallel, new candidate genes were identified by Yeast two hybrid systems or by forward genetic approaches. All those potential S-genes are currently being validated in loss-of-function mutants. In the last two years, two new S-genes (cPGK2 and CSN5a) have been identified. However, they cannot be used straightforward by breeders because knock-out mutants are either not viable or plants are severely stunted. Effort is needed to optimise the search and/or the use of variants, in particular by the identification of domain(s) of plant-virus interaction. For this purpose, mutations were introduced into the wildtype sequences and transferred to the knockout mutants. They are evaluated for plant growth and resistance to viruses. In parallel, direct interaction between the cPGK2 or CSN5A proteins with a specific viral protein is being tested. The identification of a plant susceptibility factor in a pathosystem is very likely to be transferable to crop species. Indeed, since most of the fruit tree, tomato and barley cultivars are susceptible to their respective viral pathogens, we postulate that susceptibility alleles are predominant in the current breeding germplam. We thus screened for mutation in host factors such as CSN5, cPGK2, PDIL. In fruit trees, we identified allelic series of those three S-genes, they are being tested for resistance to sharka. In the meantime, we screened for point mutation in the tomato CSN5 gene. Three microtom lines were identified and self-pollinated to obtain homozygous lines. Among those three lines, one is presenting a premature STOP codon. They are being tested for resistance to PepMoV and other viruses infecting this cultivar.

A major drawback of using genetic resistance to protect crops against viral infections is that, in many cases, it is easily overcome by the emergence of resistance-breaking strains. In the cases of recessive resistance that derive from incompatibility between an essential host factor and counterpart viral elements, the virus can escape by introducing mutations either restoring compatibility with host factor, facilitating productive interactions with an alternative host factor. Thus, it is recommendable to select resistance sources affecting essential but distinct virus factors with strong operative constraints or/and acting on various steps of the virus life cycle and then combine them in a single genotype. Therefore, besides completing all activities developed in the first year, our main focus for the next period will be to assess the extent of protection of candidate resistance traits to different viruses and to predict the durability of these new resistance genes, in combination or not. It is expected to provide strategies to exploit S-genes as an alternative in crop breeding for viral disease resistance.

Communications (posters):
Arnoux X., Delmas F., German S., Walter J., Michon T. and Decroocq V. Targeting CSN5 domains to promote resistance to Plum Pox Virus in Arabidopsis thaliana. Rencontres de Virologie Végétale, Aussois, Janv 2014.
Tricon D., Faivre d’Arcier J., Eyquard J-P., Decroocq V. Search for natural variants of resistance genes to Sharka disease in Prunus species. Rencontres de Virologie Végétale, Aussois, Janv 2014.

Plant viruses cause an estimated 50 billion € loss worldwide per year. Viral diseases represent one of
the most limiting factors in European crop production having negative effects on the quantity and quality
of foodstuffs. The scope of COBRA is to intensify applied and fundamental research on plant/virus
interactions in order to diversify targets for resistance gene pyramiding strategies and to match the
research outputs with industry and breeding applications. COBRA benefits from multidisciplinary
research teams involving genomics, bio-informatics, population genetics, molecular biology, virology and
plant breeding. It focuses on three major crops, barley, tomato and stone fruit trees. The originality of
COBRA is to test the generic mode of interference of plant viruses, from annual plants to perennials,
from dicotyledons to monocotyledons in order to implement complex and durable resistance.
COBRA is built on five transdisciplinary workpackages (WPs). The objective of WP1 is to build an
exhaustive and comprehensive database of plant-virus interaction networks for the three crops of
interest. Such a database facilitates identification of new host susceptibility genes (S-genes) that could
serve as resistance genes when mutated. In WP2, the biological relevance of some of the interactors
unraveled in WP1 or by COBRA partners will be validated, demonstrating that they are not only
plant/virus interacting factors but they are susceptibility factors encoded by bona fide S-genes. The goal
of WP3 is to screen for alleles of resistance for the S-genes identified above. Alleles of resistance to
viral infection will be searched among mutagenised tomato, barley and peach collections. Germplasm
collections of those three species will also be surveyed as well as core collections representative of their
diversity in order to implement marker assisted selection and association studies for resistance to crop
viruses. Any valuable plant material will be immediately transferred to crop breeding programs. In year
1, COBRA focuses on the search for variants of strong candidate S-genes already validated or patented
by the project’s partners. The high throughput platforms set-up for the few above candidates will then
serve to screen for variants of candidates validated in WP2 (year 2) before targeting hundreds of
candidates highlighted in WP1 (year 3). In the fourth WP, we plan to decipher two independent host
pathways that poty and potexviruses are hijacking in order to complete their viral infectious cycles. Extra
biological information will be generated in order to optimize in WP3 the search for virus resistant
variants. Finally, WP5 is aimed at selecting among the above interactors, host proteins as
candidates-of-choice for gene pyramiding. The purpose here is to provide knowledge on the viral
factor(s) that determine(s) the breaking of the resistance provided by candidate genes identified in the
above WPs and to evaluate the durability of the resistance.