Mechanistic Insights Into Tomato SUVH1-4 In Tomato Yellow Leaf Curl Virus(TYLCV) Infection And Functional Identification Of The TYLCV C7 Protein

The viruses in the family of Geminiviridae contain one or two single-stranded circular DNA of 2.5-3.0 kb as virus genomes,with a total gene length of about 2.5-5.2 kb.These viruses are distributed widely and cause huge losses to the production and development of cassava,cotton,tomato and other crops.Begomovirus is the largest genus of the family Geminiviridae,the viruses in the genus cause devastating crop diseases.Due to the lack of effective treatments for viruses,the prevalence of viral diseases has greatly impacted the agricultural system.Therefore,it is urgent to study the pathogenesis of plant viruses and put forward effective control measures.This study focuses on the in-depth understanding of the interactions between tomato yellow leaf curl virus(TYLCV)and host plants,and the molecular biology of TYLCV.The main results are described as followings:Through the detection of gene expression in tomato plants infected with TYLCV,it was found that SlSUVH1,SISUVH2,SlSUVH3,and SlSUVH4 were all up-regulated.In order to study the role of these four genes in TYLCV infection,the subcellular localizations of four proteins were observed.Confocal imagines showed that SlSUVH1,SlSUVH2,SlSUVH3 and SlSUVH4 were all localized in the nucleus.Using overexpression and virus induced gene silencing-based system to overexpress SUVH1-4 or to silence SUVH1-4,we found that SlSUVH1 and SlSUVH3 positively regulated the TYLCV infection,while SISUVH2 and SlSUVH4 played anti-viral roles during the TYLCV infection.Using yeast two-hybrid assay,bimolecular fluorescence complementation assay,co-localization and co-immunoprecipitation assays,we confirmed that SlSUVH1,SlSUVH2,SlSUVH3 and SlSUVH4 interacted with the TYLCV-encoded C2 protein.These results preliminarily elucidated the roles of SlSUVH1,SlSUVH2,SlSUVH3,SlSUVH4 in the TYLCV infection,and provided evidence for further studying the underlying mechanisms.In this study,a novel C7 protein was identified through mass spectrometry analysis of TYLCV-infected N.benthamiana samples,and the function of C7 was analyzed in detail.Firstly,the transcription initiation site of C7 was identified by 5’RACE,which proved that C7 was geneated during viral transcription.The mutation of C7 negatively affected viral infection in two different hosts,Nicotiana benthamiana and Solanum lycopersicum,demonstrating that C7 has a biological function and is crucial for viral infection.TYLCV C7 was found to localize in the nucleus and cytoplasm,and the virus infection did not affect its subcellular localization patterns.The interactions between C7 and TYLCV-encoded V2 and C2 were determined by yeast two-hybrid test,bimolecular fluorescence complementation test and co-localization assays.C7 interacted with C2 in the nucleus,while C7 interacted with V2 in the cytoplasm and they formed bright granules when they were co-expressed together,Using the potato X virus(PVX)-based recombinant vector,we found that overexpression of C7 was able to induce more severe mosaic disease and promote more accumulations of the viral coat proteins in the late infection stage.C7 was also found to inhibit GFP-induced RNA silencing.In conclusion,these data revealed that the C7 protein encoded by TYLCV is a pathogenic factor and RNA silencing inhibitor,which played a critical role in TYLCV infection.Above all,SlSUVH1 and SISUVH3 promoted TYLCV infection,SlSUVH2 and SlSUVH4 inhibited TYLCV infection,and SlSUVH1-4 interacted with the C2 protein encoded by TYLCV.At the same time,a new protein C7 encoded by TYLCV was also functionally identified.The results showed that TYLCV C7 was a pathogenic factor and inhibited plant PTGS.