Functional Analysis Of The V3 Protein Encoded By Tomato Yellow Leaf Curl Virus And The Mechanistic Study Of The Interaction Between Turnip Mosaic Virus-Encoded Proteins And AtRIN13

Begomovirus is the largest genus of single stranded DNA virus in plant viruses,and Potyviruses is the largest genus of positive stranded RNA virus in plant viruses.The viruses in these two genus have caused global devastating diseases and led to large economic losses.To better understand the pathogenicity of plant viruses,plant resistance to viruses,and plant-virus interaction,tomato yellow leaf curl virus and turnip mosaic virus were studied in this study.As for TYLCV,we identified a new isoloate,TYLCV-BJ,and constructed its infectious clone.By analyzing the sequences of TYLCV,we found that there exist a lot of additional open reading frames(ORFs)in TYLCV genome.One of the ORFs,which we named V3,was highly conserved among 26 begomoviruses.The V3 promoter could drive GUS and GFP expression.We constructed a mutant infectious clone,TYLCV-mV3,and found it displayed a weaker virulence compared to wild type TYLCV in inoculated Nicotiana benthamiana and tomato plants.Furthermore,transgenic overexpression of V3 could complement the virulence of TYLCV-mV3.We also used a potato X virus(PVX)-based recombinant vector to express V3,which could increase the accumulation of PVX in infected plants at 30 days post inoculation.Further study revealed that V3 is located in the Golgi apparatus,endoplasmic reticulum and plasmodesmata.V3 could move rapidly along the microfilament in cells,and V3 could also move between cells and partially complement the movement of a movement-deficient clone TuMV-GFP-P3N-PIPO-m1.In addition,V3 functions as an RNA silencing suppressor,which could inhibit both post-transcriptional gene silencing(PTGS)and transcriptional gene silencing(TGS).Here,our study firstly described the V3 protein encoded by TYLCV,which is required for full infection of TYLCV.The results imply that TYLCV may encode many neglected small proteins besides the canonical 6 proteins described to date,and this study provides new overview for identifying the functions of these small proteins and may expand the knowledge of interaction mechanisms between the Geminiviruses and host plants.In a previous report,AtRIN13 could enhance the resistance of Arabidopsis to bacteria.Here,with inoculating TuMV into the AtRIN13-overexpressing plants or rin13 mutant plants,we observed that AtRIN13 played an anti-viral role in TuMV infection.We found that over-expression of AtRIN13 led to the high accumulation of salicylic acid(SA)and activated the expression of pathogenesis-related genes.Furthermore,we found there existed the interactions between AtRIN13 and NIb,CI encoded by TuMV through assays of Y2 H,BiFC and co localization.In addition,AtRIN13 could reduce the protein accumulation of NIb and CI in plants.Furthermore,our data showed that AtRIN13 was an intrinsic disordered protein and interacted with 20 S proteasome subunits AtPAG1/AtPBA1,and guided the degradation of CI through the ubiquitin independent 20 S proteasome pathway.These results elucidate that AtRIN13 can not only induce SA resistance pathway to inhibit virus infection,but also exploit 20 S proteasome pathway to degrade viral protein,thus reveal the dual mechanism of AtRIN13 resistance to plant virus infection.