Molecular Identification Of Pear Chlorotic Leaf Spot-associated Virus And Functional Analysis Of Its P5 Protein

Pear chlorotic leaf spot-associated virus(PCLSaV)is a newly identified pear-infecting virus in the genus Emaravirus by our laboratory.The genome of PCLSaV composed of at least five negative-sense RNA(-ss RNA)segments.In order to further clarify the molecular characteristics of PCLSaV and establish an efficient and stable RT-PCR detection technology,in this study,the complete genome sequence of two PCLSaV isolates were determined,the virions were purified and observed under electronic microscopy,and primers used for reverse transcription and the PCR detection of PCLSaV were designed and tested.Meanwhile,the biological functions of the virus-specific P5 protein were analyzed.The main findings are as follows:1.Two pear samples(HB and NJ)collected from Hubei Province and Jiangsu Province were subjected to RNA sequencing.After sequence assembly and blastx analysis,partial sequences of PCLSaV genomic RNAs1-5 were identified.The gaps in the sequence of the PCLSaV isolate NJ(PCLSaV-NJ)were filled by RT-PCR,and their complete genome sequences were determined.Sequence analysis showed that the lengths of the viral genomic RNAs 1-5 varied among the viral isolates.However,the size of each protein was identical among different isolates.The nucleotide sequences of RNA2 and RNA5 were more variable among PCLSaV isolates with the lowest identities of 90.0% and 93.5%,respectively.The simultaneous amplification of the viral genomic RNAs using primers specific for each of the viral RNAs 1-5 further confirmed that these RNA strands are belong to PCLSaV genomic RNAs.The particles of PCLSaV were succesfuly extracted from the diseased pear leaves,and it was found that the sizes of the PCLSaV virus particles changed greatly,with a diameter of 23-172 nm,which was different from the sizes(80-120 nm)of other emarviruses.2.Random primers(pd(N)6),and primers 3C and 5H designed based on the conserved sequence at the end of PCLSaV were used to synthesize c DNA.RT-PCR and nested RTPCR assays using specific primers designed based on viral RNA1,RNA3 and RNA5 showed that nested RT-PCR using primer 3C for c DNA synthesis and RNA5 specific primers for the PCR reaction had relatively high effieciency for the virus detection.RTPCR detection of PCLSaV in different leaves and tissues of one-year old shoots of pears showed that PCLSaV could be effectively detected in pear leaves with visible chlorotic leaf spots,but were less detected in phloem tissue.Moreover,178 leaf samples of pear from different locations were RT-PCR tested for PCLSaV.Out of 110 pear samples showing symptoms of chlorotic spots or ring spots,75 were PCLSaV-positive(68.2%),and only 1out of 68 asymptomatic samples was PCLSaV positive,further indicating taht PCLSaV infection can induce obvious disease of pear trees.3.The silencing supression activity of the PCLSaV-encoded proteins was analyzed.It was found that the virus encoded specific protein P5 could partially supress local RNA silencing and strongly suppress systemic RNA silencing.P5 also could cause cell necrosis and the accumulation of reactive oxygen in Nicotiana benthamiana leaves,and increase the expression levels of PR protein genes.The subcellular localization assays showed that P5 localized at the endoplasmic reticulum,plasmodesmata,and in the nucleus as granular structures with variable sizes.It is found that the P5 could induce the formation of endoplasmic reticulum vesicles.Bimolecular fluorescence complementation assays showed that the P5 were self-interactable,which occurred at the same locations in subcellular localization assays.4.Six P5 mutants with deletions of nuclear localization signal,signal peptide sites and truncated fragments of the viral P5 were constructed.It was found that the mutant without nuclear localization signal or signal peptide lost self-interaction ability,but not for the ability of local RNA silencing supression and inducing necrosis of N.benthamiana cells,suggesting that nuclear localization signal and signal peptide were not determinant for the funtions.The amino acids at position 20-90 aa were found to be necessary fo RNA silencing supression and and necrosis induction of N.benthamiana cells.