| Rice is one of the most important crop plants in China. Viral diseases are a majorthreat to rice production. In the last decade, several rice virus diseases had broken outand caused great harm to agricultural production in China. In these virus diseases,southern rice black streaked dwarf disease, caused by Southern Rice black streakeddwarf virus(SRBSDV), a newly identified species in the genus Fijivirus, becameparticularly important for it had brought huge loss to people in recent years. Thebiology and molecular biology about it were studied by many domestic and overseasscholars. Some biological or molecular biological characteristics of SRBSDV, such asgeographical distribution, symptom, property of pathogen, cytopathology and varietyresistance, genome structure, virus-encoded proteins, were made clear, while thefunctions of most virus-encoded proteins, distribution and change of the virus in thehost, pathogenic mechanism of the virus, and molecular mechanisms of plantresistance and susceptibility to SRBSDV have not been known. Therefore, the studies,which on the surveillance of rice virus diseases, the interaction between viral proteinsand host proteins, the distribution and change of the virus in the host, have importantsignificance for elucidating these questiones.Firstly, in2009-2012, the occurrence and epidemiology of rice virus diseases inFujian, Hubei, Jiangxi, Hunan, Hainan provinces were investigated and the diagnostictests of rice virus diseases were taken for these provinces. Based on these, the virusvarieties, distributive characteristics and epidemiology of rice virus diseases in theseprovinces were analysed.The results were as below: in Hubei province, theoccurrence of southern rice black streaked dwarf disease on rice an maize was firstreported and the existence of rice stripe disease was confirmed; The rice virusdiseases outbreak in domestic had almost all occurred in Fujian province, and thevirus varieties were SRBSDV, RBSDV(Rice black streaked dwarf virus), RSV(Ricestripe virus), RRSV(Rice ragged stunt virus), RDV(Rice dwarf virus), RGSV(Ricegrassy stunt virus); Hunan and Jiangxi provinces were the most seriously regionswhere rice virus diseases, caused by SRBSDV or/and RRSV, were pandemic in recent years; the overwintering virus sources in Hainan were the important primary source ofSRBSDV and RRSV in southern China.Secondly, the genome of SRBSDV Hubei isolate was determined by randomprimer amplification method, and based on the genome, the full-length of13viralgenes were amplified and expressed in cells of Nicotiana benthamiana, respectively.And then the subcellular localizations of virus-encoded proteins were observed byconfocal fluorescent microscopy. The results showed: the genome of SRBSDV Hubeiisolate was29122bp and the accession numbers of segment10to1wereHM585270to HM585879; P1, P4, Pns52, P91could localize in the cytoplasm andform polymers as small mobile particles(P1, P4) or large mobile particles(Pns52, P91);P2, P8, P3and P10could localize both in the cell nucleus and in the cytoplasm asmobile particles; P51, Pns6, Pns71, Pns72and Pns92could localize in the cellcytoplasm and/or on the cell membrane and all form network structures attached oncytoskeletons, Pns6and P51could also form small granular polymers in the cellcytoplasm.Thirdly, the distribution and change of the virus in host were analysed byRT-PCR, electronic microscopy and Real-time PCR. The results showed: SRBSDVonly existed in the phloem cells of rice plants and was a phloem-limited viruse;SRBSDV could exist in every part of rice seeds from infected rice plants at differentstages, but it was not a seed-transmitted virus; The virus volume in roots was morethan leaf sheaths and leaves at young seedling stage, but at tillering stage the virusvolume were a roughly similar in roots, leaf sheaths, leaves and stems, while atflowering and filling stage the virus volumes in different parts was various and that ofmidstems was the most.The results further illustrate that the virus volume was indynamic changes in different part of infected rice plants at different growth stages.Finally, in order to study the interaction between the virus and host plant, Pns6ofSRBSDV was used as bait protein to screen rice Library by yeast two-hybridsystem(YTHS). And24rice proteins were obtained for their possibly interacting wtihPns6.8proteins were selected for further experiments. Constructed YTHS and BiFCvectors of the8proteins and identified their interactions with Pns6by YTHS and BiFC. The results showed that Pns6could interact with5proteins of them as40Sribosomal protein S9-2, ubiquitin-conjugating enzyme, AP2domain containingprotein, zinc finger protein, eukaryotic translation elongation factor1A. Then on thisbasis, eukaryotic translation elongation factor1A from Oryza sativa (OseEF1A) wasselected to study the significance of its interaction with Pns6for virus infection and totest the universality of the interactions between OseEF1A and rice viruses. The resultsshowed:1, SRBSDV Pns6could interact with itslf and with Pns91in yeast, OseEF1Acould co-localize with Pns6, but not Pns91when the two corresponding proteins wereexpressed together in epidermal cells of N. benthamiana. However, when the threeproteins were expressed together, co-localization of OseEF1A and Pns91could beobserved;2, RRSV Pns10and Pns6could also interact with OseEF1A in yeast and inepidermal cells of N. Benthamiana. When OseEF1A and Pns6or Pns10were expressedtogether, co-localization could be observed. It is interesting to note that both RRSV andSRBSDV use viroplasm-associated proteins to interact with OseEF1A. Therefore, theinteraction of viroplasm-associated proteins with OseEF-1A may sequester the riceprotein to sites not accessible by its cellular partners;3, In yeasts, OseEF1A also couldinteract with proteins from other rice viruses such as Pns11of RGDV, NS3of RSV,NS1, NS3, PC3of RGSV and AC4proteins from Clerodendrum yellow mosaic Chinavirus and Tomato yellow leaf curl Guangdong virus. These results confirmed theuniversality of plant virus utilizing eEF1A. |
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