Search

Australian grapevine viroid (AGVd) is found in only three countries in the world. Here, the genetic diversity and phylogenetic relationships of AGVd isolates from three different grape varieties (Thomson Seedless, Jingchuan and Zaoyu) in China were studied. A hundred of independent cDNA clones from each of the three isolates, in total of 300, were sequenced. We identified 29 sequence variants including two predominant ones in Thomson Seedless, and 48 each including a unique predominant one in Jingchuan and Zaoyu. In silico structure analysis revealed that base changes were clustered in the left terminal domain of the predicted secondary structure in all three isolates. Further, these changes were shown to affect their secondary structures to varying degrees. Genetic diversity and phylogenetic analysis of four predominant sequence variants from this study, plus four others from Australia and Tunisia, revealed obvious regional disparity and variety-specificity in AGVd.

For this study, 68 plum samples were collected from 12 provinces of China. Low molecular weight RNAs were extracted and used for dot-blot, reverse transcription-polymerase chain reaction (RT-PCR), return-polyacrylamide gel electrophoresis, and biological indexing using cucumber. Results showed that 15 out of the 68 plum samples were positive for Hop stunt viroid (HSVd). Four positive samples were selected for cloning and sequence analysis. Results indicated that most HSVd sequences from the plum in China had 1-3 nucleotide changes from the closest HSVd in GenBank. In addition, the sample PB21 (cv. 'Friar') collected from Hebei province had one sequence with a 15-nt duplication (named HSVd D-15) at the 244/245 position in the lower central region. By biological indexing, cucumber seedlings, an indicator plant for HSVd, were inoculated with RNAs directly extracted from the original plum source (PB21, cv. 'Friar'). Nucleotide sequencing analysis of the progeny showed that HSVd, but not HSVd-D15, was recovered from inoculated cucumbers. Although very unlikely, the possibility that this extra sequence was the result of a PCR artifact cannot be completely ruled out.

In fission yeast (Schizosaccharomyces pombe), pac1 gene was cloned with 99.3% nucleotide sequence similarity with published pac1 in GenBank. In pET-5α expression system, the expression product of cloned pac1 in E. coli showed activity to degrade the double-strand RNA. Harboring the binary vector pBI121, which contains pac1 gene, Agrobacterium tumefaciens strain LBA4404 was used to transform the wheat immature embryos precultured 7–10 d. After preregeneration, regeneration and selection culture stage, totally 41 G418 resistant plants were obtained, in which 25 lines were proved to integrate with transgene and express transgene normally by PCR, Dot blot, RT-PCR and ELISA detection. Antivirus test carried out on 25 positive lines with high dose of Barley yellow dwarf virus-GPV revealed that 12 lines had resistance to BVDV-GPV in low level, another 12 lines had resistance to BVDV-GPV in middle level, and 1 line showed resistance to BVDV-GPV in high level. However, both low and middle level of resistance plants showed no symptoms when infected by viruses at low dose, which suggested the dose-dependent effect of the resistance mediated by pac1 to BYDV-GPV.

The complete nucleotide sequence of the ssRNA genome of a Chinese GPV isolate of barley yellow dwarf virus (BYDV) was determined. It comprised 5673 nucleotides, and the deduced genome organization resembled that of members of the genus Polerovirus. It was most closely related to cereal yellow dwarf virus-RPV (77% nt identity over the entire genome; coat protein amino acid identity 79%). The GPV isolate also differs in vector specificity from other BYDV strains. Biological properties, phylogenetic analyses and detailed sequence comparisons suggest that GPV should be considered a member of a new species within the genus, and the name Wheat yellow dwarf virus-GPV is proposed.

RNA interference (RNAi) is a phenomenon that the double-stranded RNA (dsRNA) intermediates the degradation of complementary mRNA found in many organisms. This is a specifically mechanism involved in kinds of proteins to complete the interference function. Structure of siRNA affects which strand will be assembled into RISC. Another role of siRNA is directing RITS complex to bind with homologue chromosome, and then induces heterochromatinization. Although systemic silence induced by dsRNA is observed in Caenorhabditis elegans and plants, this progress is probably transmembrane protein-dependent, and mostly, the systemic silencing is controlled by multi-factors.

An expression vector expected to induce RNA interference against Barley yellow dwarf virus (BYDV), which expressed a composite hpRNA with the dsRNA stem homologous of BYDV GPV replicase gene and the antisense RNA loop homologous of coat protein gene, was designed without marker gene. The vector was transferred into callus cells from wheat (Triticum aestivum L.) immature embryos by particle bombardment. To select the positive transformants as early as possible, a rapid PCR, which does not need extract wheat DNA instead of few leaves, was used at regeneration stage of plantlets. Totally 21 plants proved to contain alien sequence. Antivirus test with high dose infected virus revealed that, 9 plants showed low level of resistance to BVDV, 6 plants showed moderate resistance and 6 plants showed high level of resistance. Interestingly, both low and moderate levels of resistance plants were no symptoms when infected by viruses at low dose. It suggests the dose- dependent effect of the resistance mediated by hpRNA to BYDV-GPV.

Resistance to barley yellow dwarf virus (BYDV) has been described in lines derived from crosses between wheat and Thinopyrum intermedium. The resistance in one such line, Zhong 4, originating in China, was expressed against two different serotypes of BYDV as measured by accumulation of virus in infected seedlings. F1 hybrids between wheat (2n = 42) and Zhong 4 (2n = 56) had 49 chromosomes and intermediate levels of virus when compared to the parent lines. Cytological and molecular hybridization studies suggested that the BYDV resistance in Zhong 4 is carried on a set of seven pairs of nonwheat chromosomes that derived from a combination of the E and X genomes present in Th. intermedium.Key words: Barley yellow dwarf virus, wheat, ribosomal DNA, Agropyron intermedium, Thinopyrum intermedium, virus resistance.