Functional analysis of genomically linked NLR proteins in plant innate immunity

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The translocation of nuclear localization signal (NLS)-containing proteins through nuclear pore complexes (NPC) depends on transport adapters of the importin-α family that recognize and directly bind NLSs to recruit their cargo proteins to the nuclear transport machinery. The Arabidopsis thaliana genome encodes for nine importin-α isoforms. IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal resistance and contributes to autoimmunity of the nucleotide-binding leucine-rich repeat (NLR) immune receptor mutant snc1 (suppressor of npr1-1, constitutive1). Plant NLRs generally function as intracellular immune receptors that detect pathogen secreted effectors to induce immunity, typically involving a hypersensitive cell death response (HR). Most Arabidopsis genes encoding NLRs are genomically linked in pairs or cluster in chromosomal regions, suggesting cooperative functions. NLRs that function in pairs or networks show specialization towards effector sensing and downstream signaling. The work presented here shows that, among the nine α-importins in Arabidopsis, IMP-α3/MOS6 is the main nuclear transport receptor of both the auto-active and wild-type NLR protein SNC1. In addition, IMP-α3/MOS6 is selectively required for basal resistance to mildly virulent Pseudomonas syringae pv. tomato (Pst) DC3000 bacteria lacking the type-III effector proteins AvrPto and AvrPtoB (ΔAvrPto/PtoB), suggesting functional specialization in nuclear import of immunity related cargos. The truncated NLR protein TIR-NB13 (TN13) was identified as an additional interaction partner that selectively associates with IMP-α3/MOS6 in planta. Significantly, the gene encoding TN13 is directly linked to a full length TIR-NB-LRR (TNL) gene that was named TNL LINKED TO TN13 (TLT13). Both TN13 and TLT13 are required for resistance of Arabidopsis to Pst DC3000 (ΔAvrPto/PtoB). TN13 and TLT13 co-localize to the membrane of the endoplasmic reticulum via N-terminal transmembrane domains and interact with each other in transient expression assays in Nicotiana benthamiana. In contrast to TN13, TLT13 induces a cell death response upon transient expression in N. benthamiana that depends on known functional motifs in the TIR and NB domains, the downstream defense regulators NbEDS1a and NbSAG101b, as well as the NbNRG1 family of helper NLRs. The interaction of TN13 and TLT13 with phylogenetically related NLRs, encoded by a segmentally duplicated chromosomal region, suggests that TN13 and TLT13 are part of a larger NLR network.