Rice gall dwarf disease is one of the most serious diseases in rice in Southern China, Thailand, Korea and other Southeastern Asian countries (2, 11, 13). It was first discovered in Thailand in 1979 (11). It has erupted several times in Southern China since 1980s, and is still represents one of the main rice diseases in these regions. The symptoms include stunting of the plants, gall formation along leaf blades and sheaths, and dark green discoloration (2, 11).
Rice gall dwarf virus (RGDV), the pathogen of this disease, is grouped in the genus Phytoreovirus (11, 12) in the family of Roeviridae (12). The virus multiplies in leafhopper vectors such as Nephotettix nigropictus and N. cinticeps. It is transmitted in a persistent manner and restricted within the phloem cells. RGDV, as well as Rice dwarf virus (RDV) and Wound tumor virus (WTV), is an icosahedral double-shelled particle approximately 65-70 nm in diameter that contains 12 genome segments of dsRNA (4, 10, 12), which have been named S1 to S12 according to their electrophoretic mobility in polyacrylamide gels.
RNAs of all 12 genome segments of RDV and WTV have been sequenced (8, 9, 15). But in the case of RGDV, only the nucleotide sequences of genome segments S2 (8), S3 (15), S5 (3), S8 (10), S9 (4), S10 (10), and S11 (9) of Thailand isolate have been reported.
In China, this disease broke out first in Xinyin County, Guangdong Province. Fortunately, it was successfully controlled by controlling its principal vectors at the early stage of rice growth. But in recent years, the presence of RGDV has also been discovered in other Counties, such as GaoZhou and DeQin, in Guangdong Province.
To determine the epidemiology of the rice gall dwarf disease, an investigation was carried out in China. This disease was found to be present in other two counties of Guangdong Province, BoLuo and CongHua, which revealed that RGDV had been continuing to spread. How RGDV has been introduced to these areas remains unknown since the leafhopper could not migrate for long distances and raised the additional question as to why is RGDV sporadic in Thailand while epidemic in South China? Previous reports showed that some differences were found between isolates from China and from Thailand in their principal vectors and artificial inoculation hosts (2). To understand the relationships between Chinese and Thailand isolates, the structure and function of RGDV isolate, and viral transmission in South China, the RGDV segment S8, encoding the outer major capsid protein (Pns8) from five different locations in China, was sequenced and Pns8 of one isolate (XY) was analyzed in this study. To our knowledge, this is the first report on sequence analysis and genetic comparison of segment 8 from difference RGDV isolates, and the expression of the capsid protein.
The genomic segment 8 of five isolates from China were amplified and sequenced successfully. The results showed that they were all 1578 bp in length and each contained one long open reading frame which extended for 1301 nt from 21 nt, and encoded the RGDV major outer capsid protein. The nucleotide sequence data reported in this paper were deposited in Genbank with accession numbers of AY999077 (BL), AY999078 (CH), AY999079 (DQ), AY999080 (GZ) and AY999081 (XY), respectively. The genomic segment 8 and the coding region of these five Chinese isolates shared 94.8% ~95.6% and 95.0%~96.0% nucleotide sequence identities with that of the Thailand isolate respectively, 97.3%~98.8% and 97.3%~99.1% within these five isolates respectively (Table 1). The variations of nucleotide sequence between Chinese isolates and the Thailand isolate were greater than that within the Chinese isolates. Variation seemed to be related to the geographic distance at the nucleotide level. But at the amino acid level, the situation changed. The deduced amino acid (Pns8) of GZ isolate was identical to that of the Thailand isolate, while the variability's of Pns8 within these five Chinese isolates ranged from 0.5% to 2.1% (Table 2). The residue variation position appeared to be random. It is of note that XY and GZ, the two neighbor isolates, still had a variation of 2 residues. The sequence divergence resulted in apparent changes in the theoretical isoelectric point (pI) of the deduced peptides. The pIs of the deduced peptides of both BL and DQ isolates were 7.62, while the others were 6.74. The effect of the changes remained unknown since no remarkable phenotype variability was observed within the five Chinese isolates.
Table 1. Comparison of the full-length and coding region sequences of genomic segment 8 of RGDV five Chinese isolates (BL, CH, DQ, GZ, XY) and Thailand isolate (TL)
Table 2. Divergence of the deduced amino acids of the outer main capsid protein gene of RGDV five Chinese isolates (BL, CH, DQ, GZ, XY) and Thailand isolate (TL)
Two phylogenetic trees were built and shown in rectangular cladogram on the basis of the nucleotide sequences of the S8 and its coding region (Fig. 1). The two phylogenetic trees were similar and all the five Chinese isolates were grouped together, which indicated that they were more closely related to each other than to Thailand isolate. It is interesting that the two isolates, BL and CH, first reported in this paper, were clustered with XY isolate in one clade. This suggested that RGDV in BoLuo County and CongHua County might be newly introduced from Xinyi County, the original region, where the disease occurred first in China. The phylogenetic tree could not be constructed on the basis of amino acid sequences by maximum parsimony method because there were insufficient parsimony-informative sites.
Segment 8 cDNA encoding Pns8 of XY isolate was amplified and digested with EcoRI/SalI, ligated into linear vector pET-28b (+), and the recombinant pET.CP vector was obtained. Sequencing data showed that protein expression codons were completely correct. After E. coli Rossetta (DE3) Ⅱ cells containing pET.CP were grown at 37 ℃ and the OD600 reached 0.6, followed by further induction culture with IPTG for 3, 5, 7 hours at 42 respectively, the ℃ total cell fractions from growing 3-7 hours showed that the recombinant fusion protein was produced. This was further confirmed by SDS-PAGE analysis (Fig. 2). Molecular mass (51kDa) of the protein band was in good agreement with the size deduced from the whole amino acid sequence. On the contrary, no similar protein bands were detected in 3 control samples (Fig. 2). This preliminarily indicated that Pns8 was successfully expressed in E. coli Rossetta (DE3) Ⅱ.
Figure 2. Analysis of prokaryotic expression of recombinant RGDV pET.CP plasmid with SDS-PAGE. 1, E.coli. Rossetta(DE3) without induction; 2 Ⅱ, E.coli. Rossetta(DE3)Ⅱ induced with IPTG; 3, E.coli. Rossetta(DE3) containing Ⅱ pET.CP without induction; 4, Protein Marker; 5-8, E.coli. Rossetta (DE3) containing pET.CP was induced with IPTG Ⅱ for 1, 3, 5 and 7 hours, respectively.