Molecular Identification Of Six Begomoviruses In Fujian And Selection The Interactive Proteins Of RaMoV NSP

Geminiviruses have circular single-stranded DNA genome encapsidated in twinned icosahedral particles. They have caused significant yield loss to many crops worldwide. Several begomoviruses have been reported infecting crops and weeds in south China since 1990s. The current study reported the identification results of begomoviruses in several crops and weeds in Fujian province.Eight leaf samples of Nicotiana tabacum showing stunting symptoms were collected in Fuzhou city of Fujian province in China. The partial DNA-A fragments (500 nucleotide) amplified by PA/PB shared % nucleotide sequence identity. Isolate F3 were selected for determining the complete genome sequence and the complete F3 DNA-A (F3A) was 2739 nts (EF125190). RCA was used to searching the DNA-B molecule and F3 DNA-B (F3B) was determined to be 2720 nts (FJ874926). The complete nucleotide of F3A had the highest sequence identity (95.1%) with an isolate of Ramie mosaic virus (RaMoV, EU596959) from Hainan province. The molecular data showed that F3 was an isolate of RaMoV. This is the first report about RaMoV naturally infencted in N. tabacum. We constructed the infectious clones of F3A and F3B to test the infectivity of F3 in host plants. F3A alone could systemically infect tested plants N. benthamiana, N. tabacum, N. tabacum Xanth, N. glutinosa, N. tabacum cv. Samsun NN/nn and Solanum lycopersicum without inducing symptoms, while F3A+B can induce typical geminiviruses infected symptoms in N. benthamiana, N. tabacum and N. tabacum cv. Samsun NN/nn.Two viruses F11 and F12 was obtained from the same N. tabacum sample YC7 showing leaf curl, enation, vain sticking and stunting symptoms in Fujian province of China. The partial DNA-A sequence (500 nucleotide) amplified by PA/PB showed sample YC7 was mixed infected by viruses F11 and F12. F11 and F12 were determined to be 2741 nts (FJ869907) and 2754 nts (FJ869908), respectively. The complete nucleotide of F11 had the highest sequence identity (97.3%) with PaLCuGuV-[CN:Gd2:02] (AJ558122) , an isolate of Papaya leaf curl Guangdong virus (PaLCuGuV) from Guangdong province. The complete nucleotide of F12 shared the highest sequence identity (90.1%) with AYVVTW-[TW:Tai:99] (AF307861) , an isolate of Ageratum yellow vein virus (AYVV) from Taiwan. The DNAβmolecule associated with the F12 was found with primersβ01/02 (F12β). F12βwas 1344 nts (FJ869909) and it shared the highest sequence identity (96.5%) with AYVB-[TW:CHu:02] (AJ542495) , an isolate of Ageratum yellow vein betasatellite. The molecular data showed that F11 and F12 were another isolate of PaLCuGuV and AYVV, respectively. N. tabacum and N. glutinosa showed leaf curl, enation, vain sticking and stunting symptoms by whitefly transmission when sample as viral source. PCR results showed that these infected plants contained F11, F12 and F12β。Four virus isolates Fp1-Fp4 were isolated from Ipomoea purpurea leaves showing slightly yellow mosaic and crinkled symptoms in Fuzhou city of Fujian province. The 500bp fragments of the isolates amplified by the PA/PB had 97.5% nucleotide sequence identities, suggesting that they were different isolates of the same virus species. Isolate Fp1 was selected for further sequence analysis. Fp1 was 2828 nts, with the typical genomic organization of begomoviral DNA-A (FJ515896). The whole Fp1A sequence showed the highest nucleotide sequence identity (92.1%) with SPLCV-[CN-Js:08] (FJ176701), an isolate of Sweet potato leaf curl virus (SPLCV) from Jiangsu Province of China. The result confirmed that Fp1 was an isolate of SPLCV from Fujian province. To our knowledge, this is the first report of the natural occurrence of SPLCV in I. purpurea. We constructed the infectious clone of Fp1 and test their infectivity in N. benthamiana. The results indicated that Fp1 could not infect N. benthamiana.Six samples of Emilia sonchifolia (Fz1-Fz5) and Crassocephalum crepidioides (Fz6) leaves showing conspicuous yellow veins were collected in Zhangzhou city of Fujian province. Total DNA was extracted from leaves of these plants and tested by rolling circle amplification (RCA). Amplification products of Fz1-Fz6 were digested by the restriction enzyme BamH I, EcoR I, Kpn I, Sac I, Xba I and Sal I, respectively. Restriction product (2.7 kb for EcoR I; 1.3 kb for Xba I) of Fz1-Fz6 were sequenced and the partial sequences indicated that these plants were infected by the same virus. Fz1 was used for further analysis. The complete Fz1 comprised 2725 nts (EU377539) and its associated DNAβcomprised 1337 nts (FJ869906). The complete Fz1 sequence was most closely related to Vernonia yellow vein virus (VeYVV-[IN:Mad:05], AM182232), with 76.7% nucleotide sequence identity. In line with the demarcation criteria for identifying begomovirus species, Fz1 is considered as a distinct begomovirus, for which the name Emilia yellow vein virus (EmYVV) is proposed. We also constructed the infectious clones of Fz1 to test the pathogenicity. Results showed that Fz1DNA-A alone could infected the N. benthamiana with little leaf curl symptom. Fz1 DNA-A + DNAβcould induce sever leaf curl symptom.Virus isolates Fz7-Fz9 were obtained from Clerodendrum cyrtophyllum Turcz plants showing yellow mosaic in Fujian, China. Total 500bp fragments were amplified with the degenerate primers PA/PB, and they shared 99.54% nucleotide sequence identity. The complete Fz7 DNA-A sequence is 2776 nts (FJ011668) and shares the highest nucleotide sequence identity (82%) with ClGMV. The molecular data showed that Fz7-Fz9 were isolates of a new begomovirus for which the name Clerodendrum yellow mosaic China virus (ClYMCNV) is proposed. DNA-B molecule was identified in Fz7 with 2739 nts (FJ011669). The results confirmed ClYMCNV is a novel bipartite begomovirus. Infectious clone of Fz7 was constructed to test the pathogenicity of ClYMCNV next.RaMoV AV1, AV2, AC1, AC2, AC3, AC4, BV1 and BC1 genes were amplified by PCR. Then, BV1 was inserted into yeast two-hybrid system bait vector pGBKT7 and AV1, AV2, AC1, AC2, AC3, AC4, BV1 and BC1 was inserted into prey vector pGADT7. In order to detect the self-activation of the eight proteins and toxicity to yeast cell AH109, all the recombinants were transformed into AH109 individually, and the transformants were plated on the different synthetic dropout nutrient medium. The results showed: all the genes don't self-activate and have no toxictiy to yeast cell; In order to detect BV1self-interaction, BV1 and other seven proteins, pGBK-BV1 and all pGAD-X were transformed into AH109, individually. Results showed that interaction of BV1 with AV2, BV1 with AC3, BV1 with BC1 were detected.The results of detection showed that the titer of primary"N. benthamiana"cDNA library were 3.1×107 cfu/mL and the titer of amplified library were 3.4×107 cfu/mL; The recombination rates were both above 95%; The size of most inserts were 900-1000bp in the cDNA library. The cDNA library is available for mating screen. In order to detect the self-activation of BV1 and toxicity to yeast cell Y187, the transformants Y187 (pGBKT7-BV1) were plated on the different synthetic dropout nutrient medium. The result showed that BV1 don't self-activate and have no toxictiy to Y187 yeast cell. Then, the Y187 (pGBKT7-BV1) and AH109 (pGADT7-cDNA) were mated and the products were plated on the different synthetic dropout nutrient medium. Results showed that eight positive clones were acquired by screening"N. benthamiana"cDNA library using RaMoV BV1 as bait protein; According to the function annotation of homologous sequences and reports about the protein function, only three positive clones are meaningful. The mechanism and function of the interaction between viral proteins and three host proteins SnRK1 and DXR were predicted.