Study On Pathogenic Mechanism Of Two Geminiviruses In Fujian Province

Whitefly-transmitted geminiviruses (family Geminiviridae) have circular single-standed genomes in twinned quasi-isometric particles and are responsible for major crop losses worldwide. Recent years, there are also reports of geminiviruses in south of China, and the threat of the disease is spreading. In this study, we identified the begomoviruses from weed in Fujian province, and investigated the role of the viruses in pathogenecity preliminarily.Kudzu (Pueraria montana) exhibiting characteristic genimivirus symptoms-vein yellowing was collected in fields located in Fuzhou, China. Using the degenerate PCR primers (PA/PB), small fragments of approximately 500 bp could be amplified in all of the three samples (Yg1, Yg2, Yg3). The sequencing results of PCR products (Acc. No. FJ539016, FJ539017 and FJ539018) sharing high sequence identity above 98.8% showed they were duplicate clones, then Yg3 sample was selected for further research.The complete sequence of Yg3 DNA-A was obtained by a pair of back-to-back primers. Yg3 DNA-A was 2729 nt in length (Acc. No. FJ539014), which shared the highest identity (92.9%) with KuMV-[VN:Hoa:05] DNA-A (Acc. No. DQ641690). Similar to those of other begomoviruses, the Yg3 DNA-A molecule contained four complementary sense ORFs (AC1, AC2, AC3 and AC4) and two viral sense ORFs (AV1 and AV2), separated by an intergenic region (IR) between AV2 and AC1. And the stem-loop sequence in the putative intergenic region contained the conserved nonanucleotide sequence TAATATTAC.The satellite DNA-βcomponent was not detected in the samples by PCR using primers specific for the molecules. Then we used the TempliPhiTM Kit find out that the Yg3 accompanied with another 2677 nt fragment (Acc. No. FJ539015), which shared the highest identity (81.7%) with KuMV-[VN:Hoa:05] DNA-B (Acc. No. DQ641691). Therefore, Yg3 is a Kudzu mosaic virus islate from China, so the name Kudzu mosaic virus-Yg3 (KuMV-Yg3) isolate is proposed. The Yg3 DNA-B molecule possessed two major ORFs: BC1 on the complementary sense strand and BV1 on the viral sense strand, and also contained the conserved nonanucleotide sequence TAATATTAC.In order to investigate the role of Kudzu mosaic virus in pathogenecity, infectious clones of DNA-A and DNA-B of KuMV-Yg3 were constructed. Infectivity assay showed that neither DNA-A nor DNA-B could couse any symptoms, while DNA-A plus DNA-B could infect Nicotiana benthamiana plants with yellow chlorosis spots in leaves.Additional, we constructed the infectious clones of DNA-A (Acc. No. EF527823) and DNA-β(Acc. No. EF527824) of Ageratum yellow vein virus-F2 (AYVV-F2) and another defective DNA-β(ΔDNA-β, Acc. No. FJ605171) preserved in our laboratory. These might help us to study the role of Ageratum yellow vein virus in pathogenecity. Infectivity assay showed that either AYVV-F2 DNA-A plus DNA-βor AYVV-F2 DNA-A plusΔDNA-βcould systemically infect Nicotiana benthamiana, Nicotiana tobacum plants with slightly yellow vein symptoms.