A novel hairpin library-based approach to identify NBS–LRR genes required for effector-triggered hypersensitive response in Nicotiana benthamiana

Cyril Brendolise, Mirco Montefiori, Romain Dinis, Nemo Peeters, Roy D. Storey, Erik H. Rikkerink

PTI and ETI are the two major defence mechanisms in plants. ETI is triggered by the detection of pathogen effectors, or their activity, in the plant cell and most of the time involves internal receptors known as resistance (R) genes. An increasing number of R genes responsible for recognition of specific effectors have been characterised over the years; however, methods to identify R genes are often challenging and cannot always be translated to crop plants. We present a novel method to identify R genes responsible for the recognition of specific effectors that trigger a hypersensitive response (HR) in Nicotiana benthamiana. This method is based on the genome-wide identification of most of the potential R genes of N. benthamiana and a systematic silencing of these potential R genes in a simple transient expression assay. A hairpin-RNAi library was constructed covering 345 R gene candidates of N. benthamiana. This library was then validated using several previously described R genes. Our approach indeed confirmed that Prf, NRC2a/b and NRC3 are required for the HR that is mediated in N. benthamiana by Pto/avrPto (prf, NRC2a/b and NRC3) and by Cf4/avr4 (NRC2a/b and NRC3). We also confirmed that NRG1, in association with N, is required for the Tobacco Mosaic Virus (TMV)-mediated HR in N. benthamiana. We present a novel approach combining bioinformatics, multiple-gene silencing and transient expression assay screening to rapidly identify one-to-one relationships between pathogen effectors and host R genes in N. benthamiana. This approach allowed the identification of previously described R genes responsible for detection of avirulence determinants from Pseudomonas, Cladosporium and TMV, demonstrating that the method could be applied to any effectors/proteins originating from a broad range of plant pathogens that trigger an HR in N. benthamiana. Moreover, with the increasing availability of genome sequences from model and crop plants and pathogens, this approach could be implemented in other plants, accelerating the process of identification and characterization of novel resistance genes.