Interaction Study Of Immune Receptor Sw-5b With Tomato Spotted Wilt Virus Movement Protein NSm And With Plant Proteins Involved In Downstream Defense Signalling

Tomato spotted wilt virus (TSWV) is one of the most devastating plant viruses with multi-segment negative sense strand,which has caused enormous losses to agronomic crop production throughout the world.Disease resistance genes play an important role in breeding TSWV-resistant varieties.Among them,the disease resistance gene Sw-5b on tomato is highly resistant to TSWV.Sw-5b is derived from Peruvian tomato containing NB-LRR (nucleotide-binding,leucine-rich repeat) structure,it recognizes the movement protein NSm of TSWV to induce resistance response.The disease resistant protein with NB-LRR structure is also called immune receptor protein.In addition to the NB-LRR structure,Sw-5b also has an additional SD domain (Solanaceae domain) at the N-terminus.So far,how the immune receptor protein Sw-5b recognizes TSWV NSm and how it initiate downstream defense response remains poorly understood.To address these two problems,this study used the membrane protein yeast two-hybrid system to investigate the direct interaction and recognition between Sw-5b and TSWV NSm.At the same time,the yeast two-hybrid screen library was used to screen downstream resistance-related interaction proteins of the key domains of Sw-5b.In this study,the membrane protein yeast two-hybrid system based on split ubiquitin was firstly used.TS WV NSm was fused with the Cub terminusof split ubiquitin,and Sw-5b with the NubG terminus of split ubiquitin,the results showed that tomato immune receptor protein Sw-5b can directly interact with TSWV NSm.For Sw-5b resistance,when the two single amino acids C118 or T120 of the virus movement protein NSm are mutated to Y or N,the NSm mutants can break down the resistance of Sw-5b.Consistent with this,the split ubiquitin membrane protein yeast two-hybrid results showed that the immune receptor protein Sw-5b no longer interacts with NSmC118Y and NSmT120N mutans.Sw-5b contains n SD domain at the N-terminus and an NB-LRR structure at the C-terminus.In this study,the SD domain and NB-LRR domain were further fused to the the NubG end of ubiquitin,the results showed that both the SD and the NB-LRR domain can interact with TSWV NSm,but the interaction strength is significantly weaker than the full-length Sw-5b.Therefore,these results indicate that both the N-terminal SD domain and the NB-LRR domain of Sw-5b can interact with TSWV NSm.Sw-5b has integrated two domains to recognize viral NSm in the same receptor,and the two domains have an additive effect.This dual recognition mechanism significantly enhance the sensitivity to detect the TSWV NSm.In order to identify the downstream resistance signal components initiated by Sw-5b,the SD and NB domains of Sw-5b were used as baits to screen N.benthamiana yeast two-hybrid library.SD is a special domain at the N-terminal of Sw-5b.In this study,Sw-5b SD was first constructed into the bait vector pGBKT7.The bait vector pGBKT7-SD does not have self-activation.Through the yeast library mating screening,the 8.1 million diploid was screened in the N.benthamiana yeast two-hybrid library,and 8 SD interacting proteins were obtained after yeast recovery verification.The Sw-5b NB domain can directly induce death in plants,so it is a key signaling domain.Thus,Sw-5b NB was constructed into the bait vector pGBKT7.The pGBKT7-NB does not have self-activation.Through the mating and screening of yeast library,the 3.5 million diploid was screened in the N.benthamiana yeast two-hybrid library,and 7 proteins NB interacting were obtained.The Sw-5b SD domain interacting protein VPS37 (SD-IP8) obtained above was used to generate VPS37 knock out N.benthamiana by CRISPR/Cas9 technology.By designing two targets sgRNA1 and sgRNA2 that attack VPS37,a CRISPR/ Cas9-VPS37-g1 & g2 vector was constructed.Through Agrobacterium-mediated leaf disc transgene and regeneration,13 independent VPS37 knockout lines of wild-type N.benthamiana and Sw5b-transgenic N.benthamiana were finally obtained.Sequencing analysis of the Sw5b transgenic N.benthamiana knockout mutants showed that sgRNA1 had a high cleavage success rate,and 8 of 13 lines had base additions,deletions,or mutations;while sgRNA2 had only 4 of 13 lines that had base additions,deletions or mutations.In addition,it was found that the knock out lines which successfully cleaved in sgRNA1,also successfully cut in sgRNA2.Among them,1-2 and 1-8 knockout lines had base deletions at the target sites where sgRNAl and sgRNA2 acted at the same time,and also protein changes occurred at the target sites,thus successfully achieved gene knockout lines.The establishment of VPS37 gene knockout mutant lines laid a base for exploring the function of VPS37 in the Sw-5b-mediated defense response.In the end of this study,the Sw-5b NB domain interacting protein GATA24 transcription factor screened in the N.benthamiana yeast two-hybrid library was further investigated.Bimolecular Fluorescent Complementation (BiFC) and Luciferase Complementation Assay both showed that the Sw-5b NB domain does interact with transcription factor GATA24 in plants.On this basis,a yeast two-hybrid assay was carried out between different domains of Sw-5b,including SD domain,CC domain,NB domain and LRR domain,with GATA transcription factor.We found that transcription factor GATA24 not only interacts with NB domain,but also interact with the Sw-5b SD domain.These findings laid an important foundation for the analysis of how Sw-5b initiates downstream resistance mechanisms.