| Geminiviruses occur in the world and have caused significant yield losses to many crops. In China, several economically important crops, such as tobacco, tomato, squash and papaya, have suffered from begomovirus infection in Guangxi, Yunnan and Hainan provinces since 1990s. Recently, Ageratum yellow vein virus (AYVV) and Cotton leaf curl Multan virus (CLCuMV) were found to be associated with satellite DNAβ as well as a nanovirus-like small DNA component, referred to as DNA1. DNA1 is distributing widely but its function is not clear. In order to know the occurrence in China, role in disease development and impact to geminivirus evolution, study was carried out on DNAl moleucles in geminivirus samples collected from different parts of Yunnan province.DNAl molecules were identified from Tobacco curl shoot virus (TbCSV) isolates Y35 and Y115 using PCR and Southern blot, it is a circular single-stranded DNA molecule, and only associated with TbCSV containing DNAβ. Immunotrapping PCR test showed DNA1 could be encapsidated in virus particles and transmitted by whitefly.DNAl molecules were identified only in some virus samples collected from tobacco, tomato and Ageratum among ~300 geminivirus samples collected from tobacco, tomato, Ageratum, Siegesbeckia and Malvastrum in Yunnan. All these isolates associated with DNAl were also found to be associated with DNAβ, but not all the isolated associated with DNAβ were accompanied by DNAl. Analysis of the complete nucleotide sequences of the twenty-eight DNAl molecules revealed that DNAl is approximately half the size of the genomic DNA, and has little sequence homology to either DNA-A or DNA-B of beomoviruses. DNA1 contains nanovirus-conserved nonanucleotide TAGTATTAC and a stem-loop structure similar to geminiviruses, encodes a protein with molecular weight of about 36-37 kDa in virion-sense which resembles a replication-associated protein of nanoviruses, contains an adenine rich (A-rich) region immediately downstream of the Rep gene. This A-rich region is approximately 153-169 nucleotides in length with an A content of between 52.3 % and 58.4 %. Sequence comparisons among 28 DNAl molecules showed they had 66.5-99.7 % nucleotide identity and consisted of four main types, each associated with one of the four Chinese begomovirus species (TbCSV, Tobacco leaf curl Yunnanvirus, Tomato yellow leaf curl China virus and an uncertain helper virus). The Rep sequences of DNA1 components were highly conserved with more than 88.3 % amino acid sequence identity among the 28 DNA1 molecules and 81.0-92.1 % amino acid sequence identities were found when compared with other reported DNAl molecules. The mixed infection of DNA1 molecules was found in some samples indicating one begomovirus can have different DNA1 molecules.Infectious clones of TbCSV-Y35 DNA1 and Tobacco leaf curl Yunnan virus (TLCYNV)-Y132 DNAl were constructed and their interactions with TbCSV/DNAβ were investigated. Infectivity showed TbCSV DNA1 alone could not systemically infect Nicotiana benthamiana and it need helper virus for movement. TbCSV DNA1 could attenuate leaf curling symptoms induced by TbCSV or TbCSV and DNAβ in the early stage of symptom development and induced leaf cluster at a later stage of symptom development in N. benthamiana plants. Southern blot analysis revealed that TbCSV DNA1 could reduce viral DNA accumulation. Viral DNA accumulation was not reduced when plants were co-inoculated with TbCSV DNAβ, but the TbCSV DNAβ level was dramatically reduced in the presence of TbCSV DNA1. The leaf disc assay demonstrated that TbCSV DNAl could replicate autonomously. Infectivity tests showed that heterogenous TLCYNV-Y132 DNAl had no effect on the symptoms induced by TbCSV or TbCSV and DNAβ in N. benthamiana plants, although TLCYNV-Y132 DNA1 could replicate in the TbCSV infected plants. |
PDF Full Text Request