The Molecular Variability Of Two Filovirus And The Effect Of Thermotherapy For Gene Expression In In-Vito Pear Plants

In this study we analysed Cherry green ring mottle virus(CGRMV) isolates CP gene sequences and Apple chlorotic leaf spot virus(ACLSV) isolates 3' UTR and partial CP gene sequences of pears from Canada and China in order to know CGRMV and ACLSV molecular variability characters and the phylogenetic relationship,which established the foundation to prevent virus spread.One of most effective measures for controlling these virus diseases is utilizing certified healthy propagation materials. Thermotherapy is the most widely used technique for the production of virus-free plant materials.Higher virus elimination efficiency and the virus distribution features in in vitro pear plants during thermotherapy were analyzed in this study.The results showed that high temperature decrease the virus titers on meristerm tip,and have no effect on the base,whether it is relative to the host defence mechanism is still unknown.In vitro pear plants infected with ACLSV and Apple stem grooving virus(ASGV) were further used as materials for heat treatment and for construction of cDNA libraries and screening the genes related to host-virus interaction under thermotherapy.The virus life cycle and the host genes expression were further analyzed which will be helpful to know the mechanism of elimination of viruses from hosts.Seeking results are listed as followings:1.A survey was conducted to evaluate the sanitary status of stone fruit tree collections in the Canadian Clonal Genebank(CCG),at the Greenhouse and Processing Crops Research Center(GPCRC) in Harrow Ontario(Canada).Samples collected from clones of 110 cultivars,including 28 sweet cherry,36 sour cherry,12 hybrids and 34 plum accessions were bud-grafted onto indicator seedlings of P.serrulata 'Kwanzan' for virus indexing,symptoms with epinasty and/or rusty necrotic fragments of midrib, suggestive of the response of Kwanzan to infection by CGRMV,were observed on indicator plants inoculated with samples from 8 clones[1 sweet cherry,1 cherry plum (P.besseyi×P.hortulana) and 6 plum].Amplicons of the expected size of 948 bp were consistently produced from 8 samples showing symptoms of CGRMV infection by One-step RT-PCR.To our knowledge,this is the first report of CGRMV infecting plum in North America.The phylogentic analyses 8 Canadian CGRMV isolates of sequences and NCBI available ones(including their origin and species when available),clustered the Canadian isolates in two groups that could be indicative for two different introduction events.2.A survey of apple(Malus domestica) and pear(Pyrus communis) viruses was carried out at the Canadian Clonal Genebank(CCG) during the fall/winter of 2007 and spring of 2008.In total,438 accessions of apple and 122 of pear were tested using randomly collected samples of leaves and/or dormant cuttings and processed using Double Antibody Sandwich - Enzyme Linked Immunosorbent Assay(DAS- ELISA). All samples were tested by ELISA for the presence of ACLSV,Apple stem pitting virus(ASPV),ASGV and Apple mosaic virus(ApMV).Specific infection rates of viruses of apples were ACLSV(48.1%),ASGV(10.0%),ASPV(6.6%) and ApMV (7.1%),and of pears were ACLSV(42.6%),ASGV(0%),AS PV(91.8%) and ApMV(90.1%).Seventeen of the apple accessions were also tested by multiplex RT-PCR and the results proved that RT-PCR is more sensitive than ELISA3.One-step RT-PCR and TC-RT-PCR using primers of CLS6860 and CLS7536 were used to amplify 22 ACLSV isolates in Canada and 24 ACLSV isolates in China.The PCR products were purified,cloned and sequences.The size of cloned fragment was 680nt corresponding to 3' CP gene and 3' NCR region.The nucleotide homology was 84%～100%among the 22 ACLSV isolates whereas the homology of amino acid was 91%～100%.The homology of nucleotide was 100%among the isolates of MAL0427, MAL0270,MAL0537,MAL0375,MAL0577,MAL0844,MAL0976,MAL0107, PYR0206,PYR0112,and PYR0129.The phylogenetic tree analysis at the nucleotide and amino acid level clustered the Canadian isolates in two groups.One group including of Malus0170,Malus0422 and Malus 0908 ACLSV isolates,belonged to the P205 type,the others belonged to B6 type.Meanwhile the sequence datas obtained from the 24 ACLSV isolates in China in the study were compared with previously reported sequences of ACLSV from different sources.The homology and pylogenetic tree results showed that the 24 isolates were divided into 2 groups,the homology of nucleotide and deduced amino acid sequences of 3' gene were 86.3～87.5%and 94.0～96.4%,respectively.The similarities of nucleotide and deduced amino acid sequences among XSJ,SMJ,QX,PL,CL,QYS,GY and C-AP were 95～99%and 98～99%,respectively,which belonged to the same group. Meanwhile among the 16 isolates,12 isolates of ZY,JS1,ZSZ,HL1,JQ,FS,SY,BY, 61-7-7,CX,CS and C-P,had 99～100%homology and 4 isolates of XG,HL2,C-XA,C-HBP,had 95～99%homology,which belonged to the same group of two different branches.The molecular variability of ACLSV within 9 samples was analyzed by the PCR-SSCP.The results showed that the SSCP profile of ZY,ZSZ,HL1,FS,JS1,JQ was of the same type,the SSCP profile of C-X A and HL2 was of the another type.Sequence of JQ and ZY isolates were analyzed,it showed that ACLSV isolates were composed of a population of genetically related variants,the predominat haplotype had very tiny variation with other haplotypes within each population,only 1～10 bases variation was observed and the similarity was more than 99%among them.The sequence analysis showed that the homology of the predominant sequence is 100%between JQ and ZY isolates.4.Comparison of the results visualized by either chemical stain or X-ray film exposure showed that relatively more intense signals and less background were produced by X-ray film exposure than chemical stain for both ASGV and ACLSV.Therefore,the X-ray film exposure is more suitable for the detection of viruses with a very low titer in plant tissues.These results indicated that both ASGV and ACLSV were present in the whole plants,but the highest viral RNA titer was found in phloem parenchyma of vascular bundles in all cross sections.Tissue-printing hybridization(TPH) was used to detect ACLSV and ASGV in thermotherapy-treated in vitro cultured pears at the tip, intermediate stem and base levels,and to evaluate the effect of the tip length on the efficiency of virus elimination.It was found that both ASGV and ACLSV were present at high concentrations in the base and tip of the plants,and were present at much lower concentrations in the intermediate stem.Only tip tissues of 2mm and 0.5 mm long were ACLSV and ASGV-free,respectively,therefore,the use of tips length of 0.5mm and 2mm are recommended for improving the efficiency of virus elimination for pear meristem-tip culture.These results will be helpful for improving the efficiency for virus elimination by thermotherapy.5.Two differentially expressed genes libraries were constructed,a library of up-regulated genes and a library of down-regulated genes in virus-infected in vitro pear(pyrus proflia) were prepared by suppression subtractive hybridization(SSH). Inserts of 1200 randomly selected colonies from the forward-subtractive library and of 1000 colonies from the reverse-subtractive library were identified with the reverse southern dot-blot screening method.Sequencing results showed that 63 genes were showing up-regulated expression and 86 genes were showing down-regulated expression among 149 ESTs under heat-treatment.The function of about 52%gene fragments was unknown,while the rest 48%had putative function clarifying as disease defense,transcription,signal transduction,energy,metabolism and protein synthesis.The results indicated that the process of virus-host interactions was complex and many genes could participate in the process.In this research,we first reported the differentially expressed genes of in vitro pear under thermotherapy, which established the foundation for understanding the molecular mechanisms of virus elimination from in vitro pear plants.