Replication And Expression Of Rice Stripe Virus 0(RSV) In Rice Protoplast

A rice protoplast system that can be infected by RSV has been established. Following polyethyleneglycol (PEG) mediated, rice protoplasts were inoculated with purified RSV ribonucleoproteins (RNPs). After extracting total protein of protoplasts that are infected for various times during the course of infection, two proteins, coat protein (CP) and disease-specific protein (SP), encoded by viral complementary RNA3 (vcRNA3) and viral RNA4 (vRNA4) respectively, could be detected. Using the rabbit antiserum directed towards the CP and SP as probes, we researched the infection cycle of RSV and expression periods of SP in rice protoplasts by ELISA and Western-blot. The time course experiments showed: (1) RSV in protoplasts begins to replicate up to I 6h after inoculation. The replication reached its peak after 38h. (2): SP began to express up to 1 2h after inoculation, and the expression reached its peak after 32h. At the same time, total RNAs were extracted from protoplast samples collected at various time, blotted onto PVDF membrane, and hybridized to DIG-labeled riboprobes specific of gene coding region of the vcRNA3 and vRNA4 strands. Northern blot experiments were performed on total RNA extracted from protoplast samples, and the in vivo synthesis of the mRNA of vcRNA3 and vRNA4 (CP- mRNA and SP-mRNA) was observed. The time course analyses of the amount of CP- mRNA and SP-mRNA in infected rice cell collected at various time was detected by Northern blot, whose results showed that the accumulation of CP-mRNA and SP-mRNA could be detected after 8h, and the accumulation of CP-mRNA could reach its peak about 30h, and then begins to decrease after that. But it still had a higher level. However, the accumulation of SP-mRNA could reach its peak at about 24h, then drops obviously. Intact chloroplasts from infected rice leaves that expressing different symptoms were made after being infected by RSV. Then total protein of chloroplasts was extracted separately. The change of the accumulation of CP and SP from protoplasts in different severity of symptom were detected by Western-blot to analyze the relationship between the changes and the severity of symptom when using antiserum of CP and SP as probes. The result showed that CP and SP exist in chloroplasts of infected rice, and that the concentration of CP and SP inside chloroplasts is positively related to severity of symptom. In vitro, the research on transmission system of trans-membrane was done by inoculating purified RSV NCPs to the chloroplasts that were purified from healthy and intact rice plant.According the result of our experiment we concluded that: in vitro, the RSV particles are able to enter intact chloroplasts rapidly.