The Regulatory Mechanism For The Autoinhibition And Activation And The Subcellular Localization Of Tomato Resistance Protein Sw-5b

The main innate immunity is controlled by resistance (R) proteins. The recognition of a specific pathogen effector by a corresponding R protein can initiate a cascade of defense responses. NB-LRR proteins take up the largest class of R proteins. They are multidomain proteins that possess an N-terminal domain, a central NB-ARC domain, and a C-terminal leucine-rich repeat (LRR) domain. Plant NB-LRR proteins are roughly classified by their N-terminal structure into two categories: CC-NB-LRR with a coiled-coil (CC) domain and TIR-NB-LRR with a Toll/interleukin-1 receptor (TIR) domain. It is important for plants to evolve mechanisms to minimize the unnecessary R gene expression under non-infection conditions and then to maximize defense responses via activation of R gene expression or R protein activity in the face of pathogen invasion.Tomato immune receptor protein Sw-5b is a CC-NB-LRR type R protein with an extended N-terminal domain (NTD). However, how the extra N-terminal sequence coordinates with CC and NB-LRR regulate these resistance proteins need to be elucidated. Furthermore, an increasing number of studies show that the subcellular localization of resistance proteins play a pivotal role in disease resistant signal pathways, while the subcellular localization of R protein in plants does not have a unified conclusion. We used Sw-5b and Tomato spotted wilt tospovirus (TSWV) movement protein NSm as a model .This study focuses on two issues and has carried out as following:1. Multilayered regulatory mechanism for the autoinhibition and activation of tomato resistance protein Sw-5bWe systematically dissected the distinct function of each subdomain of Sw-5b and the relationship between them, which was a critical step to understand the mechanism of its regulation function. It was found that the NB-LRR segment retains the function of inducing HR in the presence of NSm, and specifically recognizing NSm, as the full-length Sw-5b does. These results based on homology-based modeling and site directed mutation suggest that the conserved NB-ARC domain acts as the molecular switch for autoinhibition/activation of the Sw-5b NB-LRR segment. The NB-ARC domain induced a constitutive HR, albeit weak, in the absence of NSm. However, coexpression of NB-ARC with LRR did not induce HR. By introducing constitutive mutations S594A, D642E and D857V into the NB-LRR, CC-NB-LRR and NTD-CC-NB-LRR backbone except NTD-CC-NB-LRRD857V was unable to induce HR, the self-activating activity of other mutants completely appeared or weak. Co-IP analysis showed that the LRR physically associated with NB-ARC; CC physically interacte with LRR but not with NB-ARC; NTD physically interacted with both CC and NB-LRR. Collectively, these data suggest that the three layers of negative regulation include LRR, CC and NTD to maintain Sw-5b in a deep autoinhibited state in the absence of the viral elicitor NSm. Moreover, NTD-CC-NB-LRR and its derived mutants, as NB-LRR did, induced strong HR in the presence of NSm,whereas CC-NB-LRR and its derived mutants only induced a weak HR. These results indicate that the release of the inhibitory effects of LRR on NB-ARC and the further release of the inhibitory effects of CC on NB-LRR by NTD in the presence of NSm, thereby fully activating Sw-5b. Furthermore, this layer of autoinhibition and inhibition of NB-LRR segment is not sufficient to confer resistance against virus infection. The full-length Sw-5b transgenic plants conferred resistance against TSWV infection in either the 35S promoter-or native promoter-derived. We propose that, to resolve the negative regulation of CC on NB-LRR, Sw-5b evolved an extra N-terminal domain to coordinate with CC, and consequently, Sw-5b developed a multilayered regulatory mechanism to control autoinhibition and activation. Importantly, this multilayered regulatory mechanism of NTD?CC and NB-LRR is essential for Sw-5b to confer resistance against tospovirus.2. The subcelluar localization and its localization signal of NB-LRR receptor Sw-5bTo determine the subcelluar localization of Sw-5b, we fused the Sw-5b protein with YFP at the N-terminus. No obvious differences was found in cell death induced by untagged Sw-5b and YFP-Sw-5b proteins in the presence of the NSm. Confocal microscopy shows that YFP fluorescence was observed in the cytoplasm and the nucleus for YFP-Sw-5b. To test if the subcellular localization of Sw-5b is of importance for its function, enforced Sw-5b subcellular localization, by tagging with a nuclear localization sequence (NLS) or a nuclear export sequence (NES) shows that Sw-5b activity in cell death is enhanced in cytoplasm but suppressed in nucleus. The nuclear localized of Sw-5b is sufficient to mediate disease resistance against TSWV and the cytoplasm pools alone cannot initiating disease resistance. To manipulated the nucleocytoplasmic distribution of the NSm by the similar way,showing that recognition of the NSm takes place in the cytoplasm. The Sw-5b P-loop mutation suggests that nucleotide-bound conformation is not required for nuclear localization. SGT1-silenced did not affect Sw-5b nucleocytoplasmic distribution. Furthermore, YFP-NTD1 (51.7 kDa) displayed similar nucleocytoplasmic distributions with YFP-Sw-5b. YFP-NTD2 (42 kDa) was almost completely absent from the nucleolus and predominantly present in nucleocytoplasmic. YFP-NB-LRR (109 kDa)can be observed in the layer of cytoplasm surrounding the nucleus. On the basis of these finding,we suggests Sw-5b-mediated cell death and Sw-5b -mediated resistance are independent of each other.