Maize TIR-only Proteins ZmTIR1 and ZmTIR2, but not ZmTIR3 Confer Auto-active Hypersensitive Response Likely by Forming Condensation

Nucleotide binding, leucine-rich-repeat (NLR) proteins are the major intracellular receptors for defending against pathogen infection. The recognition between NLRs and pathogen secreted effectors often triggers a localized programmed cell death termed hypersensitive response (HR). Despite significant progresses have been achieved in understanding canonical NLRs with the N-terminal Toll/interleukin-1 receptor (TIR) domains, the molecular mechanisms underlying TIR-only proteins in plant immune responses remain unclear. In this study, we identified six TIR-containing proteins in maize, including three TIR-only proteins. Functional analysis showed that ZmTIR1 and ZmTIR2, but not ZmTIR3, confer autoactive HR when transiently expressed in N. benthamiana. The autoactivity conferred by ZmTIR1 and ZmTIR2 depends on EDS1-PAD4-RNL module and their putative NADase activities. Interestingly, ZmTIR1 and ZmTIR2 predominantly localize in the punctate dots and likely form condensation, while ZmTIR3 mainly localizes in the cytoplasm and the nucleus. Two specific amino acids in the BB-loop region were identified to be required for ZmTIR1- and ZmTIR2-mediated condensation formation and auto-HR. Furthermore, ZmTIR and ZmTIR2 are induced by Cochliobolus heterostrophus, the causal agent of southern leaf blight (SLB) in maize, and knock-down the expression of ZmTIR1 or ZmTIR2 decreased the resistance to SLB in maize. Our study reveals a novel mechanism of monocot TIR-only proteins in maize immune responses.