Functional Scanning Of Apple Geminivirus Encoded Proteins And Identification Of New Viruses In Melon

Apple geminivirus(AGV)is a recently identified geminivirus which is isolated from the apple tree in China.We carried out functional scanning of apple geminivirus proteins to identify symptom determinants and suppressor of posttranscriptional gene silencing(PTGS).Our results indicated that AGV V2 is an important virulence factor which localized to nucleus and cytoplasm.AGV V2 is a suppressor of PTGS and induces severe symptom of crinkle and necrosis when expressing from potato virus X(PVX).AGV C1 is also a virulence determinant which elicits systemic necrosis symptom when expressed from PVX vector.The AGV C4-GFP is targeted to cytoplasm,plasma membrane,nucleus and chloroplast with complex localization.The inoculation of PVX-C4 on N.benthamiana induced severe upward leaf curling which implied that AGV C4 also functions as a symptom determinant,and mutation analyses suggested that the acylated residues on Gly2 and Cys8 play important roles in its sub-cellular localization and symptom development.Three RNA viruses—Cucumis melo cryptic virus(CmCV),Cucumis melo amalgavirus 1(CmAV1),and melon necrotic spot virus(MNSV)—were identified from a melon(Cucumis melo)sample showing yellow vein symptom by analysis of transcriptome dataset.CmCV has two dsRNA genome segments;dsRNA-1 is 1592 bp in size,containing a conserved RNA-dependent RNA polymerase(RdRp),and dsRNA-2 is 1715 bp in size,and encodes a coat protein(CP).The sequence alignment and phylogenetic analyses of the CmCV RdRp and CP indicated CmCV clusters with approved or putative deltapartitiviruses in well-supported monophyletic clade.The RdRp of CmCV shared an amino acid sequence identity of 60.7%with the closest RdRp of beet cryptic virus 3,and is<57%identical to other partitiviruses.CmAV1 is a nonsegmented dsRNA virus with a genome of 3424 bp,including two partially overlapping open reading frames(ORFs)encoding a putative CP and RdRp.The sequence alignment and phylogenetic analyses of CmAV1 RdRp revealed that it belongs to the genus Amalgavirus in the family Amalgaviridae.The RdRp of CmAV1 shares 57.7%of its amino acid sequence identity with the most closely related RdRp of Phalaenopsis equestris amalgavirus 1,and is<47%identical to the other reported amalgaviruses.These analyses suggest that CmCV and CmAV1 are novel species in the genera Amalgavirus and Deltapartitivirus,respectively.These findings enrich our understanding of new plant dsRNA virus species.