Identifying Factors Interacted With CGMMV Sturctual Proteins And Functional Analysis Of SHSP In Watermelon

Cucumber green mottle mosaic virus(CGMMV),a member of the genus Tobamovirus in the family Virgaviridae,threantening cucurbits crops,has been listed as an important quarantine object in China.In this study,we constructed a yeast two-hybrid cDNA library of watermelon stem and leaf tissue infected with CGMMV and screened the host factors intercated with CGMMV coat protein(CP)and helicase(HEL)domain of replicase protein which will pave the way for understanding molecular pathogenesis of CGMMV and provide a new idea for anti-CGMMV breeding.Major results as follows:(1)Six host factors interacted with CGMMV CP and two host factors interacted with the HEL domain of replicase protein.First,a yeast two-hybrid cDNA library of watermelon stem and leaf tissue infected with CGMMV was successfully established.The cDNA library was screened by using the CP of CGMMV and the HEL domain of the replicase protein as baits and six host proteins interacted with CP and two host proteins interacted with HEL were obtained after one-to-one interaction verification in yeast.The host proteins interacted with CP include Translationally Controlled Tumor Protein,12-oxophytodienoate reductase 2,thioredoxin h,Membrane-anchored ubiquitin-fold protein,Eukaryotic initiation factor 4B and 4A-11;host proteins interacted with the HEL domain include Small heat shock protein and methionine sulfoxide reductase;(2)Verified the interaction between sHSP and CGMMV HEL domain of replication protein in vivo and vitro.The sHSP(NbsHSP)gene of Nicotiana benthamiana was cloned with the highest homology and similar function to watermelon sHSP(ClsHSP).The interactions between the NbsHSP/ ClsHSP and the replication protein p129,methyltransferase(MT)domain or Internal Region(IR)of p129 were verified in yeast cells,respectively.The results showed that NbsHSP/ ClsHSP could interact with CGMMV p129 and its HEL domain but not interact with MT or IR domain of p129.These results suggest that ClsHSP and NbsHSP interact with the HEL domain of CGMMV replication protein specifically.In order to avoid the false positive of yeast two-hybrid,we used GST Pull-down and Co-Immunoprecipitation methods further verify the interaction between ClsHSP,NbsHSP and HEL domain.The results of several methods were consistent,indicating that sHSP was indeed interacted with and the HEL domain of CGMMV replication protein.(3)Clarified the upregulation of host sHSP inhibited CGMMV RNA accumulation and delayed the symptom appearance.By qRT-PCR analysis,we found that the initial infection of CGMMV could up-regulate the expression of sHSPs,and the expression of sHSP(Cla017945)interacted with HEL was up-regulated the most obvious.The expression of sHSPs restored to normal level when the system leaves showed the viral symptom.The subcellular localization experiments revealed that the granules aggregated by sHSPs in the cytoplasm in the healthy cells disappeared in the cells infected with CGMMV.The distribution pattern of sHSP was consistent with the infected cells when co-expressed with CGMMV HEL,indicating that CGMMV HEL altered the distribution of sHSP in cells by interacting with it.In order to further analyze the role of sHSP in CGMMV infection,we obtained transgenic N.benthamiana plants that silenced or overexpressed NbsHSP.The transgenic N.benthamiana plants inoculated with CGMMV revealed that overexpression NbsHSP was significantly delayed the disease development and CGMMV RNA accumulation was much less than that in the wild N.benthamiana plants.We hypothesize that sHSP may be a primary defense response factor involved in host response to CGMMV infection,and the CGMMV infection is promoted by its HEL interacting with sHSP to disrupt the host's defense response.This study showed that sHSP interacted with HEL might participate in the host's defense response,and whether sHSP can be used as a new target for future anti-CGMMV breeding needs further research.