Recognition of a salivary effector by the TNL protein RCSP promotes effector-triggered immunity and systemic resistance in Nicotiana benthamiana

Insects secret chemosensory proteins (CSPs) into plant cells as potential effector proteins during feeding. The molecular mechanisms underlying how CSPs activate plant immunity remain largely unknown. We show that CSPs from six distinct insect orders induce dwarfism when overexpressed in Nicotiana benthamiana. Agrobacterium-mediated transient expression of Nilaparvata lugens CSP11 (NlCSP11) triggered cell death and plant dwarfism, both of which were dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), neuregulin 1 (NRG1) and SENESCENCE-ASSOCIATED GENE 101 (SAG101), indicating the activation of effector-triggered immunity (ETI) in N. benthamiana. Overexpression of NlCSP11 led to stronger systemic resistance against Pseudomonas syringae DC3000 lacking effector HopQ1-1 and tobacco mosaic virus, and induced higher accumulation of salicylic acid (SA) in uninfiltrated leaves compared to another effector XopQ that is recognized by a Toll-interleukin-1 receptor (TIR) domain nucleotide-binding leucine-rich repeat receptor (TNL) called ROQ1 in N. benthamiana. Consistently, NlCSP11-induced dwarfism and systemic resistance, but not cell death, were abolished in N. benthamiana transgenic line expressing the SA-degrading enzyme NahG. Through large-scale virus-induced gene silencing screening, we identified a TNL protein that mediates the recognition of CSPs (RCSP), including aphid effector MP10 that triggers resistance against aphids in N. benthamiana. Co-immunoprecipitation, bimolecular fluorescence complementation and AlphaFold2 prediction unveiled an interaction between NlCSP11 and RCSP. Interestingly, RCSP does not contain the conserved catalytic glutamic acid in the TIR domain, which is required for TNL function. Our findings point to enhanced ETI and systemic resistance by a TNL protein via hyperactivation of the SA pathway. Moreover, RCSP is the first TNL identified to recognize an insect effector.