RNA silencing is a conserved mechanism found ubiquitously in eukaryotic organisms.It has been used to regulate gene expression and development.In addition,RNA silencing serves as an important mechanism in plants' defense against invasive nucleic acids,such as viruses,transposons,and transgenes.As a counter-defense,most plants,and some animal viruses,encode RNA silencing suppressors to interfere at one or several points of the silencing pathway.In this study,we showed that Pns12 of RGDV (Rice gall dwarf virus) exhibits silencing suppressor activity on the reporter green fluorescent protein in transgenic Nicotiana benthamiana line 16c.Pns12 of RGDV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA.Expression of Pns12 also enhanced Potato virus X pathogenicity in N.benthamiana.Collectively,these results suggested that RGDV Pns12 functions as a virus suppressor of RNA silencing,which might target an upstream step of dsRNA formation in the RNA silencing pathway.Furthermore,we showed that Pns12 is localized mainly in the nucleus of N.benthamiana leaf cells.
In order to preserve virus for identifying the resistance of rice varieties against rice black-streaked dwarf disease, a simple and reliable method was developed, through which virus-free small brown planthopper (SBPH) acquired rice black-streaked dwarf virus (RBSDV) from frozen infected leaves and the virus was transmitted to healthy rice plants. The experimental results showed that SBPH could obtain RBSDV from frozen infected rice leaves and the virus could be transmitted to a susceptible rice variety. For the ability to acquire RBSDV and transmit the virus to healthy plants by SBPH, there was no significant difference between frozen infected leaves and in vitro infected leaves. The novel method could be applied to identification of rice variety resistance to rice black-streaked dwarf disease, facilitating the breeding process for rice black-streaked dwarf disease resistance.
The major viral diseases that occur on rice plants in Zhejiang Province, eastern China, are stripe and rice black-streaked dwarf diseases. Rice stripe disease is only caused by rice stripe tenuivirus(RSV), while rice black-streaked dwarf disease can be caused by rice black-streaked dwarf fijivirus(RBSDV) and/or southern rice black-streaked dwarf fijivirus(SRBSDV). Here we review the characterization of these viruses, methods for their detection, and extensive surveys showing their occurrence and spread in the province.
Rice black-streaked dwarf virus (RBSDV) is a recognized member of the genus Fijivirus,family Reoviridae.Its genome has ten double-stranded RNA (dsRNA) segments (S1-S10),in which the fifth genome segment (S5) contains two open reading frames (ORFs) with a partially overlapping region.The second ORF of RBSDV S5 encodes a viral nonstructural protein named p5b with unknown function.To reveal the function of p5b,its gene was ligated into the bait plasmid pGBKT7 and an expression library containing rice cDNAs was constructed using plasmid pGADT7 for yeast two-hybrid assay.The bait protein p5b was detected in yeast by western blot,and the result of an auto-activation test showed that p5b could not autonomously activate the expression of reporter genes in yeast.Then the bait protein p5b was used for screening the cDNA expression libraries of rice.Gene fragments of some pivotal enzymes involved in photosynthesis,respiration and other important metabolic processes,were identified to interact with p5b in yeast,suggesting that these interactions may play roles in symptom development in infected plants.
Rice ragged stunt virus(RRSV) is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein(CP) gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21(DE3) using the pMAL-C2 X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody(MAb) against RRSV was obtained by fusing mouse myeloma cells(Sp 2/0) with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay(ACP-ELISA), a dot enzyme-linked immunosorbent assay(dot-ELISA), and immunocapture-RT-PCR(IC-RT-PCR) assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1:40 960, 1:1 280 and 1:655 360(w/v, g mL-1), respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1:12 800 and 1:1 600(an individual planthopper μL-1), respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.
LIU HuanSONG Xi-jiaoNI Yue-qunLU Li-naZHOU Xue-pingWU Jian-xiang
