Phylogenetically conserved TIR-NLR proteins induce transcriptional reprogramming

Nucleotide-binding leucine-rich repeat (NLR) proteins play fundamental roles in plant innate immunity by detecting pathogen invasion and activating immune signaling, ultimately resulting in hypersensitive cell death. The subclass of Toll/Interleukin 1-type (TIR) NLRs is widely distributed across dicot plants. Activated TIR-NLRs (TNLs) rely on the downstream proteins EDS1, PAD4, SAG101, and RPW8-type CCR-NLRs to mediate immune signaling. Here, we studied the molecular evolution of a clade of highly conserved TIR-NLRs (con-TNLs) that are widely distributed across dicot plant species. We then used con-TNL orthologs from Arabidopsis thaliana, Nicotiana benthamiana, and Spinacia oleracea, to study their function in immune signaling using in planta expression and transcriptomics. We examined the ability of con-TNLs to induce immune readouts by transiently expressing autoactive variants in N. benthamiana leaves. Interestingly, while none of the wild-type or autoactive con-TNLs showed visible hypersensitive cell death when expressed in N. benthamiana, both wild-type and autoactive N. benthamiana and A. thaliana con-TNLs induced hypersensitive cell death after expression in N. tabacum. However, RNAseq analyses of N. benthamiana leaves revealed transcriptional reprogramming caused by autoactive con-TNLs compared to the wild-type proteins. While the S. oleracea con-TNL did not induce any major transcriptional changes, several differentially expressed genes (DEGs) were detected in the A. thaliana con-TNL, and to a lesser extent, in the N. benthamiana con-TNLs. DEGs had a similar expression pattern to that induced by the autoactive ROQ1, a well-studied TIR-NLR which recognizes a bacterial effector and triggers cell death. Our data suggest that autoactive con-TNLs are able to signal and induce transcriptional reprogramming comparable to classical TNLs when expressed transiently in N. benthamiana, even though they do not induce a visible hypersensitive response.