Construction Of Infectious Clones And Identification Of Pathogenic Factors Of SLCCNV And MNSV

Cucumis.melo is one of the important economic crops in China.In recent years,the new emerging virus disease has seriously affected the development of the melon industry.Squash leaf curl China virus?SLCCNV?and melon necrotic spot virus?MNSV?are the newly reported virus species on melons in China.In order to elucidate the pathogenic mechanism of the two viruses,the infectious clones of SLCCNV and MNSV were constructed.The viral pathogenic factors and pathogenic mechanism were analyzed to lay the foundation for prevention and control of the viruses.The main progresses were as follows:1?SLCCNV was first identified on melon,the infection clones of SLCCNV-HN was constructed and the virus transmission test by Bemisia tabaci was carried out.The full-length sequence of SLCCNV-HN was determined and phylogenetic tree analysis was carried out,the results showed that the SLCCNV-HN melon isolate,the SLCCNV-Hn pumpkin isolate,SLCCNV-Hn61 and SLCCNV-SY zucchini isolates were clustered one group and formed independent branches.The plants infected by the infectious clones of SLCCNV-HN were dwarfed and curled at 20 days post inoculation?dpi?.Then the virus transmission test by Bemisia tabaci was carried out,it was found that the plants inoculated by Bemisia tabaci showed the same symptoms as those the wild-type strains.2?It was clarified that the SLCCNV AC5 gene was a pathogenic factors of the SLCCNV,and AC5 was also the virus silencing suppressor and the silencing activity was related to the nuclear localization signal.The Nicotiana benthamiana?N.benthamiana?plants inoculated with the potato virus X?PVX?heterologous expression system expressing the AC5 gene showed typical necrotic symptoms,the results showed that AC5 triggered the hypersensitive reaction in N.benthamiana.The DAB staining test confirmed that the allergic necrosis reaction was related to the accumulation of H₂O₂.In addition,based on the infectious clones of SLCCNV-HN,we further confirmed that AC5 gene is a pathogenic factors of the SLCCNV by site-directed mutagenesis of AC5 gene.The transient expression vector of AC5protein was co-infiltrated with p35S-GFP to inoculate 16c N.benthamiana,there were obvious green fluorescence signals in the infiltrated area at 5 dpi,the results indicated that the SLCCNV AC5 protein is an RNA silencing suppressor of the post-transcriptional gene silencing and induced by single-stranded GFP?S-PTGS?.The wild N.benthamiana was co-infiltrated with p35s-dsGFP,p35S-GFP and p35S-AC5,and no green fluorescence signal in the infiltrating region was detected at 5dpi,which indicated that AC5 did not inhibit the silencing induced by double-stranded GFP?IR-PTGS?.It is also found that AC5 can inhibit the system transportation of GFP by the movement of silencing signal of GFP system.Based on bio-informatics,the nuclear localization region of AC5 was predicted to be 102-115aa,we constructed the deletion mutant of AC5 nuclear localization signal.After co-infiltrating 16c plants,it was found that the deletion mutant of nuclear localization signal lost its silencing activity,the results showed that the nuclear localization signal of AC5 protein was related to the activity of silencing suppressors.3?It was confirmed that p42 was a pathogenic factors of MNSV and interacted with host factor 3-Hydroxyisobutyrate dehydrogenase-like 1?3HID1?.The N.benthamiana was inoculated with the PVX heterologous expression system expressing the p42 gene,the result showed that p42 was a pathogenic factor.In addition,we constructed the infectious clone of MNSV and the infectious clone vector carrying GFP by overlap PCR.Based on the infectious clone of MNSV,we further found the R-domain and S-domain of p42 were pathogenic domains.Furthermore,the cDNA library of melon infected by MNSV was constructed and six host factors interacting with p42 were screened.The interaction between 3HID1 gene and p42 was verified by yeast two-hybrid,BIFC and co-location.The results provide a basis for exploring the molecular pathogenesis of the interaction between p42 and host factors.