Analysis Of MaYVV-induced Host Genes Related To Yellowing Vein Symptom In Nicotiana Benthamiana By RNA-Seq

Malvastrum yellow vein virus(MaYVV)is a monopartite begomovirus in family Geminiviridae,which was first reported as a yellow vein disease in the Honghe region of Yunnan Province in 2003.MaYVV commonly occurs in Yunnan,Sichuan infecting weeds such as Malvastrum coromandelianum and Ageratum conyzoides.MaYVV associated with its cognate betasatellite(MaYVB)could cause yellow vein and downward leaf curling symptoms on Nicotiana benthamiana.In order to identify host genes related to the formation of host yellow vein symptom,we successfully performed RNA-Seq of MaYVV/MaYVB infected and non-inoculated control plants and analyzed differentially expressed host genes.The GO enrichment analysis of differentially expressed genes revealed that after infection with the virus,the differentially expressed genes in N.benthamiana were significantly enriched to 136 terms,which were mainly enriched in the process of tRNA metabolism,photosystem II oxygen evolution complex,etc.The KEGG pathway enrichment analysis revealed that the pathways of porphyrin,chlorophyll metabolism and photosynthesis were significantly changed after virus infection,and it was concluded that the abnormal changes in porphyrin and chlorophyll metabolism and photosynthetic pathways may be related to yellow vein symptom induced by MaYVV/MaYVB.Based on the above analysis,12 differentially expressed genes related to chloroplast formation and chlorophyll synthesis were screened for RT-qPCR validation and further study: Nb-0004,Nb-1010,Nb-2010,Nb-4002,Nb-1015,Nb-3001,Nb-2006,Nb-0001,Nb-2021,Nb-9016,Nb-1009,Nb-0013(According to RNA-Seq data,except to Nb-0013,all other genes were down-regulated post MaYVV/MaYVB infection).BLAST analysis of the 12 candidate gene sequences screened in this study revealed that the candidate gene Nb-0001 is ChiI gene,Nb-0004 is Ftshil1 gene,Nb-1015,Nb-1010 is Ftshil4 gene,Nb-3001,Nb-2010 is Ftshil2 gene,Nb-9016 encodes a tobacco mycelium protein-binding protein,Nb-0013 encodes a tobacco transition endoplasmic reticulum ATPase,Nb-2021 and Nb-1009 encodes a chaperone protein ClpB3,and Nb-4002 is a ChiD gene,Nb-2006 encodes a tobacco RuvB-like protein.The 12 candidate genes screened by the gene-silencing technique mediated by Tabacco rattle virus(TRV)were used for functional verification.The results showed that at 14 dpi(day post inoculation),eight silenced genes(Nb-0004,Nb-1010,Nb-2010,Nb-4002,Nb-1015,Nb-3001,Nb-2006,Nb-0001)showed symptoms of albino,shrinkage,malformation,and leaf margin,while silencing of four candidate genes(Nb-9016,Nb-0013,Nb-2021,Nb-1009)did not cause obvious phenotypic changes,similar to negative control.The above results indicate that 8 candidate genes are associated with leaf color formation in N.benthamiana.To clarify the effects of these 12 candidate genes on chlorophyll formation and chloroplast structure in N.benthamiana,the chlorophyll content in N.benthamiana was determined by the acetone method and the ultrastructure of the chloroplast was observed by electron microscopy.The results showed that the down-regulation of the above-mentioned candidate genes resulted in a decrease in chlorophyll content and dysplasia of chloroplasts in N.benthamiana.Down-regulation of Nb-0004 and Nb- 0001 resulted in a decrease in chlorophyll content of 91.21% and 91.46%,respectively,which had a serious impact on the formation of chlorophyll and chloroplasts,while down-regulation of Nb-9016,Nb-0013,Nb-2021,and Nb-1009 genes did not cause significant effects.The above results indicate that these eight candidate genes that caused significant changes in leaf color and shape of N.benthamiana after down-regulation were involved in the chlorophyll synthesis or chloroplast formation process in N.benthamiana,but did not cause significant changes in the phenotype of N.benthamiana after down-regulation.Candidate genes were not involved in chloroplast formation or chlorophyll synthesis in N.benthamiana.To study the effects of candidate genes Nb-0001,Nb-0004,Nb-1010,Nb-1015,Nb-2006,Nb-2010,Nb-3001,and Nb-4002 on virus infection,After 7 days post candidate genes down-regulation,MaYVV/MaYVB was inoculated continuously.After 15 dpi,the phenotype of N.benthamiana was observed,and virus accumulation was analyzed by RT-qPCR.The results showed that down-regulation of the above 8 candidate genes and the inoculation of MaYVV/MaYVB resulted in mild yellow vein symptom and down-regulated of Nb-1015,Nb-0004,Nb-4002,Nb-2010,Nb-0001 Nb-3001andNb-2006,resulted in significantly down-regulation of MaYVV in N.benthamiana inoculated with MaYVV/MaYVB.The expression of MaYVV was significantly up-regulated after down-regulating Nb-1010 in MaYVV/MaYVB-inoculated plantts.The expression of MaYVB was significantly reduced in all the eight treatments.The research in this thesis shows that the infection of MaYVV regulates the expression of chloroplast-related genes in N.benthamiana,and down-regulation of genes related to chloroplasts of N.benthamiana reduces the accumulation of virus.Therefore,this study hypothesized that ChiI gene,Ftshi1 genes,Ftshil2 gene,Ftshil4 gene,ChiD gene and RuvBL2 may be involved in MaYVV/MaYVB symptom induction on N.benthamiana.