| Geminiviruses are a large class of plant viruses with a single-stranded circular DNA genome.In addition,the serious epidemic caused by the new species of geminivirus in the 20th century has had a serious impact on the production of crops such as cotton,cereals,beans and tomatoes.With the development of high-throughput sequencing,researchers have also detected new geminivirus species from woody plants such as citrus,mulberry,and apple.Citrus chlorotic dwarf-associated virus（CCDa V）is a single-component DNA virus isolated from citrus,which has caused huge potential risk economic losses to the citrus industry in China.CCDa V has been recently classified as a new genus of Citlodavirus in the Geminiviridae family.Some proteins encoded by geminiviruses are essential for the interaction between virus and host plants.However,the current research on geminiviruses mainly focuses on isolation,identification and genome sequencing,while the biological characteristics and pathogenesis are still unclear.In this paper,based on the screening and identification of CCDa V pathogenicity-related proteins,the regulatory mechanism of WRKY transcription factor 1 on cell death caused by pathogenicity-related proteins in N.benthamiana was preliminarily studied.The main results are as follows:1.Identification of CCDa V pathogenicity-related proteinsIn order to screen and identify the pathogenicity-related proteins of CCDa V,the full-length sequences of six proteins encoded by CCDa V were constructed into Potato virus X（PVX）clones containing GFP.Symptom observation showed that only Nicotiana benthamiana infiltrated with RepA-GFP and Rep-GFP showed leaf necrosis symptoms on the fifth day of infiltration.Further experiments with 3,3’-diaminoaniline（DAB）and electrolyte leakage showed that RepA and Rep could induce H2O2 accumulation and increase ion permeability,which confirmed that the protein encoded by CCDa V could cause HR-type cell necrosis in N.benthamiana.In addition,this study excluded the possible effect of GFP on RepA and Rep-induced necrosis of N.benthamiana leaves by using PVX vector to express RepA and Rep.Enzyme linked immunosorbent assay（ELISA）showed that the difference in PVX content was not related to the HR-type cell necrosis symptoms caused by RepA in N.benthamiana.The six ORFs of CCDa V were further constructed into the binary recombinant vector p Nm GFPer,and it was found that the expression of RepA and Rep showed local necrosis symptoms on N.benthamiana.In summary,Rep and RepA in the six ORFs of CCDa V are pathogenicity-related proteins and can induce HR-type cell necrosis in N.benthamiana.2.Identification of key regions of cell death in RepA-induced hypersensitivity（HR）In order to explore the key domains of RepA-induced HR-type cell necrosis in N.benthamiana,the sequence and domain of RepA were analyzed by bioinformatics,and eight RepA deletion mutants（DM1-DM8）were constructed and transiently expressed in N.benthamiana by Agrobacterium-mediated.The results showed that the rolling circle replication motifs（aa16-20,aa57-63 and aa103-108）of RepA were necessary to induce HR response in N.benthamiana.In addition,the results of subcellular localization experiments showed that RepA and deletion mutants containing rolling circle replication motifs(RepADM1,RepADM7-DM8)were localized in the nucleus of N.benthamiana epidermis,while deletion mutants without rolling circle replication motifs(RepADM2-DM6)were mainly localized in the cytoplasm.By combining the localization results and symptoms,it was speculated that the nuclear localization of RepA may be related to its induced HR response in N.benthamiana.In addition,PSORT online software predicted that no basic amino acids consistent with the nuclear localization signal sequence were found,suggesting the presence of atypical nuclear localization signals.3.RepA-induced plant resistance responseIn order to further determine the association between RepA-induced HR and the immune response of N.benthamiana,the expression levels of key genes（Nb PR1,Nb PR2,Nb PR3,Nb PR4 and Nb PR5,Nb NPR1）in the salicylic acid（SA）and jasmonic acid（JA）pathways were determined by RT-q PCR.The results showed that the expression levels were significantly increased,but there was no change in the expression of Nb ERF1 in the ethylene（ET）pathway.In order to explore whether the HR response induced by RepA can stimulate plant resistance to other types of viruses,Tomato yellow leaf curl virus（TYLCV）infectious clones were co-infiltrated into N.benthamiana with Agrobacterium containing 35s-GFP or RepA-GFP,respectively.The results showed that RepA could alleviate the leaf curl symptoms of N.benthamiana caused by TYLCV.In order to verify whether the virus-encoded protein can inhibit the HR response,other proteins（V1,V2,V3,V4 and Rep）encoded by CCDa V were co-expressed with RepA protein in N.benthamiana.The results showed that other CCDa V proteins could not inhibit the HR-type cell death response induced by RepA.4.Regulation of WRKY1 on RepA-induced HR cell deathIn order to screen the host factors of HR induced by RepA,Tobacco rattle virus（TRV）vector was used to silence these（WRKY1,NPR1,AOX,COI,CTR,NDR1,RAR1,NTF6 and MEK2）signal cascade components in N.benthamiana.At 9 days post-infiltration（dpi）of successful silencing,RepA-GFP was inoculated into the silenced N.benthamiana plants,and the phenotype was observed at 3 dpi.The results showed that there was no necrosis symptom in N.benthamiana inoculated with RepA after successful silencing of WRKY1.The expression level of Nb WRKY1 in N.benthamiana leaves infiltrated with RepA-GFP was detected by RT-q PCR.In addition,this study found that there was no direct interaction between RepA and Nb WRKY1 through yeast two-hybrid experiments.Furthermroe,firefly luciferase complementation imaging（LCI）and bimolecular fluorescence complementation（Bi FC）assay also showed RepA could not interact with WRKY1. |
