A novel molecular tracing system for rice and citrus pathogenic Xanthomonas – XANTHracing

The originality and novelty of this project reside on the identification, characterization, sequencing of CRISPR loci (Clustered Regularly Interspaced Short Palindromic Repeats) and VNTR (variable number of tandem repeats) in a large collection of strains of Xanthomonas oryzae and Xanthomonas citri. The CRISPR and VNTR are largely unexplored and unexploited in phytopathogenic bacteria in general and in the Xanthomonas genus in particular. The Xanthomonas genus is represented by gram-negative bacteria associated with various plants and having a high degree of host specificity. Xanthomonas can cause diseases in more than 400 different host plants belonging to both monocots and dicots. The genus Xanthomonas is an excellent model for understanding the evolutionary and epidemiological aspects of host-bacteria interaction. The access to genomic resources such as Xanthomonas genome sequences that have not been yet exploited for developing CRISPR and MLVA typing is of great importance in designing the genetic tools that we need within the project. Epidemiological surveillance of plant pathogens is essential for disease control and the protection of the plants The increasing importance of bacterial phytopathogens such as X. oryzae and X. citri and emergence of new virulent strains in agriculture and their subsequently negative impact on yields and on the global economy reveal the urgent need to develop new, original and robust molecular typing system. The tracing of bacterial strains and appearance of new ermerging and virulent isolates require discriminating and precise tools for their identification. For this we propose to develop a new molecular typing system at large scale which is fast, sensitive, robust, reproducible and universally applicable. We propose to develop the system on two species of Xanthomonas (citri and oryzae) as a model study and proof of concept. To achieve our objective, we propose 1) to analyse and sequence the CRISPR loci of a large set of strains. In the future this will permit the development of a oligonucleotide CRISPR array, 2) a VNTR analysis. This study focuses on Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight, and Xanthomonas citri pv. citri (Xcc), the causal agent of citus canker. These pathogens are quarantine organisms that represent a significant threat for agriculture and global food security. About 200 strains representative of the genetic and geographical diversity within each of the two Xanthomonas species have been selected and will be analyzed. Typing methods based on CRISPR and VNTR will be developed and evaluated for Xoo and Xcc strain characterization, diagnosis and epidemiological surveillance on a large scale. The reliability, reproducibility and discriminating power will be tested and a comparison of results obtained in different laboratories will be conducted. The data acquired will be used to develop the first molecular typing tool for phytopathogenic bacteria based on CRISPR/VNTR and ready to use at a large geographical scale. Databases will be developed and put online to make them easily accessible. This information will allow defining sampling strategies for future field experiments, quarantine decisions and also the selection of representative strains for testing varietal resistance. This will allow the development of breeding strategies for sustainable plant protection. The project will also provide fundamental knowledge for future research on the function of CRISPR loci in Xanthomonas organisms. The project involves three partners representing institutes involved in fundamental and applied research (IRD, CIRAD and the University of Orsay). Researchers involved in this project are all well recognized as experts in their field (microbiology, plant pathology, phylogeny, genetics, genomics, epidemiology) with a broad expertise in genetics, genomics and epidemiology of pathogenic bacteria with particular expertise on the Xanthomonas genus.