R3a resistance protein mutants with extended effector recognition specificity

SEGRETIN M.E. AND S. KAMOUN

Tolouse, Francia

Otro; Oomycete Molecular Genetics Network Meeting; 2010

Oomycete Molecular Genetics Network

Phytophthora infestans is one of the most devastating pathogens affecting potato production worldwide. One strategy to generate resistant cultivars is the introduction of resistance genes that are able to recognize P. infestans effector proteins with avirulence activities. R3a, a resistance protein discovered in potato, can trigger an hypersentive response upon the recognition of the avirulence effector AVR3aKI from P. infestans but cannot recognize AVR3aEM, the product of another allele that is predominant in pathogen populations. To date, all the characterized P. infestans strains in nature carry at least one of these AVR3a proteins. The objective of this work is to extend R3a recognition specificity to AVR3aEM. To accomplish this, we generated a library of R3a mutant variants obtained by random mutagenesis. The mutated molecules were cloned in a T-DNA binary vector and transformed into Agrobacterium tumefaciens. We screened the mutant clones by co-agroinfiltration with AVR3aEM in Nicotiana benthamiana plants, and evaluated the presence of HR-like phenotypes after 5 days. Of approximately 2200 evaluated clones, 20 triggered different degrees of HR-like responses and were subjected to new rounds of infiltrations to confirm the results. In parallel, the candidate clones were co-infiltrated with AVR3aKI and with a negative control plasmid to investigate the conservation of the original R3a recognition specificity and also to eliminate auto-active R3a mutants. In total, 17 clones were selected for further analyses, including sequencing and the construction of chimeric clones to investigate which mutations are responsible of the observed phenotypes. This works highlights how knowledge of pathogen effectors can be exploited for engineering novel resistance genes.