Subeelialar Localization And Functional Study Of Rice Stripe Virus Pe2Protin

Rice stripe disease is one of the most serious rice diseases and causes significant yield losses in rice. This disease is caused by rice stripe virus (RSV) which is transmitted by the small brown planthopper (Laodelphax striatellus) in a circulative, propagative manner.RSV is the typical member of the genus Tenuivirus. Its genome consists of four single stranded-RNAs that encode seven proteins. Pc2protein is encoded by viral complementary RNA2(vcRNA2) and its predicted molecular weight is94kD. It is difficult to study Pc2protein because of its large molecular weight, complex modification after translation and possible cytocity to cell. Up to now, only its nucleotide sequence and detection in host cells have been reported. It was previously reported that Pc2-N could be detected in both rice and insect infected by RSV, which suggested that it was an important, multifunctional protein and possibly play an important role in the transmission of disease and inducion of symptom in host plants. This article firstly studies its subcellular location, function and interaction with host cell, which foundation for studying its role in the infection, symptom formation and transmission.Chapter1of this thesis is a general introduction of RSV including its classification, genome structure and the function of encoded protein. The function of membrane proteins in plant and two-hybrid system are also introduced in this chapter.In chapter2, the processing and intracellular localization of Pc2in the insect cells were studied. Computer aided prediction revealed the presence of four transmembrane domains (TM1, TM2, TM3and TM4) and two signal peptide cleavage sites (scl and sc2) in the Pc2. The total RNA was extracted from rice leaf infected by RSV and used for the amplication of pc2gene fragment by RT-PCR. The enhanced green fluorescent protein (eGFP) was fused to the Pc2and expressed in Sf9cells by transfection. The fusion proteins were detected by anti-GFP monoclonal antibodies. The results showed that the Pc2was not cleaved at the proposed sc14((between the amino acid18and19), but partially cleaved at the sc2(between the amino acid381and382) to produce two proteins named Pc2-N and Pc2-C, respectively. Pc2-N or Pc2-C was able to transport to the Endoplasmic reticulum (ER) membranes independently, suggesting the presence of ER-targeting signal in both Pc2-N and Pc2-C. Further mutagenesis studies revealed that Pc2contained three ER-targeting domains, including TM2, TM3and TM4, which were further confirmed by subcellular fractionation and dissociation experiments. The results led us to propose a model for the topology of the Pc2in which an internal signal peptide TM3immediately followed a cleavage site, and two ER-targeting regions (TM2and TM4) were present. The Pc2may be translated as a precursor and then processed into two mature proteins Pc2-N and Pc2-C. Both Pc2-N and Pc2-C contain ER-targeting signal and are able to transport to the ER independently.In chapter3, the membrane fusion activity of Pc2protein has been studied. Sequence analysis revealed that RSV NSvc2was similar to the membrane glycoproteins of several members in the family Bunyaviridae including including a consensus class Ⅱ fusion peptide and carboxyl terminal transmembrane domain, which suggested that Pc2might induce cell membrane fusion as these glycoproteins. To conveniently study the membrane fusion activity of the NSvc2, we used Bac-to-Bac baculovirus expression system to construct cell surface display vectors for expressing Nsvc2on the insect cell surface as the membrane glycoproteins of the enveloped viruses. The gp64signal peptide of Autographa californica multiple nucleopolyhedrosis virus (AcMNPV) and the transmembrane(TM) and cytoplasmic terminal domains(CTD) of vesicular stomatitis virus (VSV) glycoprotein were inserted into transfer vector pFastBacI under the control of polyhedron promoter. Then the gene encoding intended proteins were cloned between the gp64sigal peptide and transmembrane region of VSV. The constructed recombinant transfer plasmids were inserted into the genome of AcMNPV by transoposition to achieve recombinant baculovirus Ac-X-VSV. The confocal microscope results of the infected cells showed showed that using baculovirus surface display system constructed in this study, eGFP was displayed on the cell surface. Furthermore, RSV capsid protein (CP), Pc2and its truncated proteins were also successfully transported onto cell surface. So, this system was a powerful tool to display the target proteins on the insect cell surface. When induced by low pH, the membrane fusion was not observed in the cells that expressed Pc2. Furthermore, the membrane fusion was also not detected when co-expressing Pc2and CP on insect cell surface. Thus, RSV Pc2is probably different from the phlebovirus counterparts, which could suggest different functions.In chapter4, the rice cDNA library was constructed and used Pc2as a bait to screen the cDNA library. The total RNA was extracted from rice and mRNA was isolated. The mRNA was used as template for the construction of yeast-two hybrid cDNA library using the technology of homologous recombination. The rice cDNA library was detected and the results showed that it had a titer o f4x10’CFU/mL, and the most of insert fragments were between500bp to2000bp in size, which indicated that the cDNA library was qualified for the yeast two hybrid screening. The bait plasmids pGBK-Pc2-N and pGBK-Pc2-C was successfully constructed by fusing the encoding fragments of Pc2-N and Pc2-C to the vector pGBKT7, respectively. The results of the toxicity and self activating effect on yeast strain AH109showed that Pc2-N had neither toxicity nor self activating effect on AH109, while Pc2-C was toxic to yeast strain AH109and thus was not suitable to yeast two-hybrid. Then rice cDNA library was screened by the Pc2-N as a bait to identify protein (s) that interacted with Pc2-N and eight candidates positive clones were obtained. BLAST results showed that these cDNAs encoded five proteins, which mainly were the proteins involved in photosystem and stress. This suggested that Pc2had an important effect on the symptom of host plant.