Molecular variability of Tobacco vein banding mosaic virus populations
Introduction
The genus Potyvirus (family Potyviridae) consists of more than 200 virus species and is the largest one of plant-infecting viruses (Fauquet et al., 2005). These viruses have a single-stranded positive-sense RNA genome that encodes a large polyprotein, which is subsequently cleaved into ten mature proteins by three virus-encoded proteinases (Riechmann et al., 1992). PIPO, a frame-shift protein encoded by overlapping open reading frame pipo in the P3-encoding region, has been reported to play roles in the infection and pathogenesis of potyviruses (Chung et al., 2008). Potyviruses have highly variable genome due to lack of proof-reading activity in their RNA dependent RNA polymerases, which make them good models for virus variability and evolution studies (García-Arenal et al., 2003). Understanding the genetic structure and the factors that contribute to the evolution of potyviruses is not only an important aspect of evolutionary biology but also an issue for virus control.
Tobacco vein banding mosaic virus (TVBMV) is a distinct species of the genus Potyvirus (Chang et al., 1994, Habera et al., 1994, Yu et al., 2007). It was once a major threat to tobacco production in North America and Taiwan, but only of minor importance to that in mainland China (Tian et al., 2007). However, in recent years TVBMV has become one of the most prevalent viruses infecting tobacco in several provinces including Shandong in the east coast, Henan in the central, and Yunnan in the south west of China (Tian et al., 2007, Chen et al., 2009, Wang et al., 2010). The synergism of TVBMV with Potato virus X (PVX; the genus Potexvirus), which is also widespread in the tobacco fields (Chen et al., 2009), will cause more severe disease and increase the tobacco losses.
As other potyviruses, the genome of TVBMV is error-prone and highly variable. Our previous results have shown that, based on their coat protein (CP) gene, TVBMV isolates are clustered to three groups correlated with their geographical origins (Tian et al., 2007), and TVBMV has unique RITC motif in its helper component-proteinase (HC-Pro) instead of KITC for most other potyviruses (Yu et al., 2007, Wang et al., 2010). The recent increasing incidence of TVBMV in tobacco fields in China (Chen et al., 2009) suggests the accumulation of TVBMV genetic variations. However, the details and the mechanism underlying the evolution of TVBMV are unknown. In this study, we sequenced and analyzed the genes encoding the helper component-proteinase (HC-Pro), the third protein (P3), the first 6K protein (6K1), coat ptoein (CP) and PIPO of 40 TVBMV isolates collected from major tobacco-growing areas in China. The results showed that the TVBMV isolates can be divided into two groups based on P3, pipo, 6K1genes and the concatenated sequences, and three groups on HC-Pro and CP genes; the isolates of Yunnan were genetically different from those of other regions; no recombination occurred between TVBMV isolates from different geographical regions.
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Virus samples
Tobacco leaves with vein banding and/or mosaic symptoms were collected from main tobacco-growing provinces, including Gansu, Hunan, Heilongjiang, Liaoning, Shaanxi, Shandong and Yunnan, in China from 2007 to 2009. The samples were subjected to indirect PTA-ELISA detection (Lan et al., 2007) and those infected with TVBMV will be either used for extraction of total RNA or stored at −80 °C for future use. The geographical locations and the details of the collected TVBMV isolates are listed in
Identities between TVBMV isolates
We obtained 40 TVBMV isolates from Gansu, Shaanxi, Shandong and Yunnan provinces from 2007 to 2009 (Supplementary Fig. 1). Interestingly no TVBMV isolate was obtained from Heilongjiang and Liaoning provinces of northeastern China, or Hunan province of southern China. Sequencing the HC-Pro, P3/6K1 and CP genes resulted in sequences of 1374 nt for HC-Pro gene, 1200 nt for P3/6K1 genes and 816 nt for CP gene. The overlapping pipo gene that locates within P3 gene was 180 nt.
Analyses of the resultant nt
Discussion
Studies of the population structures of several potyviruses showed some details of genetic variations at regional or global scales (Glasa et al., 2004, Moreno et al., 2004, Tomitaka and Ohshima, 2006, Ogawa et al., 2008, Ohshima et al., 2002, Seo et al., 2009, Tomimura et al., 2003). In this research, we analyzed the genetic variability of TVBMV populations using the sequences encoding HC-Pro, P3 (and PIPO), 6K1, and CP, which amounted to one-third of the complete genome. Our results showed
Acknowledgements
We are grateful to Drs. Zhaokui Guo of Heilongjiang Tobacco Institute, Yuanhua Wu of Shenyang Agricultural University, Yunfeng Wu of Northwest A & F University, and Risheng Hu of Hunan Tobacco Company for their help with sample collections. This study was supported by grants from Natural Science Foundations of China (NSFC, 30971895, 31011130031), Shandong Provincial Natural Science Foundation (Z2007D04), New Century Excellent Young Talents in the Universities (NCET-07-0520), Special Research
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These authors contributed equally.