Towards synthetic disease resistance genes: R3a mutants with expanded effector recognition

MARÍA EUGENIA SEGRETIN; MARINA PAIS; MARINA FRANCESCHETTI; MARK J BANFIELD; SOPHIEN KAMOUN

Buenos Aires

Simposio; 32nd New Phytologist Symposium: Plant interactions with other organisms: molecules, ecology and evolution; 2013

New Phytologist

The late blight pathogen Phytophthora infestans is responsible for the most significant global disease of potato. Sustainable strategies to manage late blight rely on the use of broad-spectrum resistance genes, which recognize P. infestans effector proteins with avirulence activity. R3a, an NBS-LRR resistance protein discovered in Solanum demissum, can trigger the hypersentive response (HR) upon the recognition of the P. infestans avirulence effector AVR3aKI. However, AVR3aEM, the product of another Avr3a allele that is predominant in pathogen populations, is able to overcome this resistance. To date, all the characterized P. infestans isolates in nature carry at least one of these AVR3a proteins. Also, gene silencing of Avr3a resulted in significantly reduced virulence of P. infestans, indicating that this effector is important for pathogen fitness. The main goal of this work is to extend R3a recognition specificity to AVR3aEM. To accomplish this, we screened a library of R3a mutant variants obtained by random mutagenesis and identified mutants, called R3a+, that trigger a strong HR upon the recognition of both forms of AVR3a. We determined that single amino acid mutations were responsible for the new recognition specificity. Further characterization of these single-mutation clones showed that at least one of them recognizes AVR3aKI more efficiently than R3a and that R3a has a dominant negative effect on the recognition of AVR3aEM by some of the single-site R3a+ mutants. Moreover, some R3a+ clones could also recognize PcAVR3a4, an AVR3a homologue from Phytophthora capsici. Finally, the library of R3a mutant variants we generated represents a permanent resource to screen for new resistance specificities against AVR3a homologs from various pathogen species, and this work highlights how knowledge on pathogen effector diversity can be exploited to develop synthetic resistance genes with expanded recognition patterns. Our approach has the potential to generate non-GM solutions to the late blight problem using genome editing or targeted variation. This strategy, which we term ?next-generation disease resistance breeding?, is applicable to a multitude of pathosystems.