Characterization Of Singaling Domain Of Immune Receptor Sw-5b NLR To Induce Hypersentive Response-Programed Cell Death

Plants have evolved cell surface immune receptor proteins(PRRs)and intracellular immune receptor proteins(NLRs)to recognize pathogenic effectors and thereby initiate an innate immune systems to combat pathogen infection.NLR-like receptor protein is the largest class of resistance protein,and its structure mainly includes N-terminal domain,nucleic acid binding domain(NB)and leucine-rich repeat domain(LRR)NLRs are roughly classified by their N-terminal structure into two categories:CC-NB-LRR and TIR-NB-LRR.The protein product of most R genes mediates the recognition of elicitors produced by pathogens and activates downstream signaling pathways after recognition,resulting in a strong host response.Although the mechanisms by which effector recognition is linked to the activation of defense signaling is poorly understood,many studies have helped to identify the domains of the R protein involved in recognition or signaling functions.In general,the LRR domain at the C-terminus of NLR proteins is considered to be a major determinant of specific recognition of pathogen effectors in resistance responses,but there has been controversy over the regions responsible for downstream signaling in plant NLR proteins,and previous studies have shown that the initiation of downstream resistance signals is primarily mediated by the NIR domain of the NLR protein,TIR or CC,and the dimerization of the TIR/CC domain is decisive for the induction of downstream resistance signals.In the present study,we used the tomato immunoreceptor protein Sw-5b belonging to the CC-NB-LRR class as a model to investigate the signaling mechanism of allergic death and the mechanism of dimerization of its domains.In addition,we performed a full-length genome analysis of a Chinese Lettuce Chlorosis virus(LCV)isolate from China and analyzed its sequence:1.Characterization of singaling domain of immune receptor Sw-5b NLR to induce hypersentive response-programed cell deathIn order to identify which domain of Sw-5b alone can induce cell death when the elicitor NSm is absent,we transiently expressed Sw-5b NTD,CC,NB,ARC and LRR in N.benthamiana and found that the NB domain of tomato Sw-5b NLR can induce a downstream cell death signal and produce a hypersensitive response-programmed cell death(HR-PCD)in the expressed tissue.Agrobacterium concentration gradient experiments showed that NB-induced HR-PCD was directly proportional to the expression level of NB protein.Real-time quantitative PCR revealed that expression of the NB domain induced expression of pathogenesis related proteins(PRs)in the resistance pathway.Virus-induced gene silencing(VIGS)revealed that NB-domain-induced HR-PCD is dependent on SGT1 protein,which plays an important role in plant disease resistance.This further indicates that Sw-5b NB domain-induced HR-PCD is an active disease-resistant defense response.The deletion mutation assay and the three-dimensional structural homology modeling of the NB domain revealed that the N-terminal and C-terminal regions of the NB domain are critical for the induction of HR-PCD.In addition,site-directed mutagenesis analysis revealed that the K568R mutant in the NB domain P-loop motif was no longer induced by HR-PCD.Co-IP results showed that the NB domain could be dimerized in plants,while the NBP-loop K568R mutant cannot be dimerized.Co-expression of the NBP-loop K568R mutant with NBWT did not interfere with NBWT-induced HR-PCD.In addition,we also found that the NB domain lacks 20 amino acids(NBC?20)at the C-terminus and does not induce HR-PCD,but in the Co-IP experiment,the NB deletion mutant is still capable of dimerization.More importantly,co-expression of NBC?20 and NBWT significantly interfered with NB-induced HR-PCD,indicating that NBC?20 and NBWT of HR-PCD could not be induced to form dimers,which interfered with the conduction and stimulation of downstream allergic death.Therefore,we conclude that Sw-5b NB dimerization or multimerization is very important for signaling and exciting downstream HR-PCD signals.In addition,laser scanning confocal microscopy(LSCM)analysis showed that YFP-NB was localized in the cytoplasm and nucleus.By locating Sw-5b to the nucleus and cytoplasm,it was shown that Sw-5b NB domain-induced HR-PCD requires the NB distribution in the cytoplasm.In addition,the study found that accessory protein NRCs are involved in the NB-mediated HR-PCD.In summary,we have analyzed a novel regulatory mechanism for induction of allergic death in plant immune receptor proteins in this study,and found that the non-N-terminal domain of Sw-5b NLR protein induces HR-PCD.The dimerization of NB domains plays an important role in downstream resistance signaling,and the helper protein NRC is required for NB-induced cell death.Our findings further extend the understanding of the molecular regulatory mechanisms of plant NLR activation defense signaling.2.Full-length genome analysis and sequence analysis of a Chinese lettuce chlorotic virus isolate from NanjingPrevious studies have reported that resisitance of Sw-5b against tomato spotted wilt virus is lost after infecting a tomato carrying a Sw-5b resistance gene.In this study,we isolated and identified the isolate of Lettuce Chlorosis Virus(LCV),named LCV-NJ,and determined the full-length genomic sequence of LCV-NJ.LCV-NJ genome consists of two RNAs;RNA1 containing 8165 nucleotides and RNA2 containing 8454 nucleotides.Based on ORF finder prediction,LCV-NJ RNA1 was found to contain four open reading frames(ORFs)with a genomic structure similar to that of LCV-California.The amino acid sequence homology between LCV-NJ and LCV-California RNA1 was predicted using the MegAlign program of DNASTAR software.The results showed that the amino acid sequence homology of ORFs 1a and 1b of the two isolates was 92%and 99%,respectively.ORF2 and ORF3 of LCV-NJ have only 63%and 71%homology with LCV-California.In addition,after nucleotide alignment analysis,it was found that the 5' non-coding region(5'UTR)of LCV-NJ RNA1 is 100%identical to LCV-California 5' UTR,but the 3' UTR of LCV-NJ RNA1 contains the insertion of three different sequences of 5 nt,5 nt and 15 nt,respectively.In addition,LCV-NJ RNA2 contains only 9 ORFs compared to the 10 ORFs of LCV-California RNA2,and 173 nucleotide sequences in the 3' end region of LCV-NJ RNA2 are deleted,resulting in ORF 10 lost.A 70 nt fragment was inserted upstream of the LCV-NJ ORF2,and the TMHMM analysis revealed that the LCV-NJ ORF2 encodes a traNSmembrane protein P4 that is different from the LCV-California ORF2.The ORF1 of RNA-NJ lacks six amino acids,which results in an extra 18 nucleotides of the RNA2 5'UTR of LCV-NJ.In addition,the sequence between ORF1 and ORF 3-9 of LCV-NJ RNA2 and the LCV-California isolate is highly conserved with 91%-98%amino acid sequence homology.These results indicate that LCV-NJ/LCV-California has undergone genomic recombination and/or rearrangement in RNA1 and RNA2.This genetic recombination or mutation may be due to the virus adapting to different regions and better infecting host plants.Our isolated and identified Crinivirus LCV-NJ,which lays an important foundation for further study of the mechanism of Sw-5b resistance to tomato spotted wilt virus.