Molecular Characterization Of ASPV And The Primary Study On The Interaction Between The Viarl TGBp And Host Proteins

Apple stem pitting virus(ASPV) naturally infects pear and apple. In some cultivated pear plants, the infection of the virus can induce vein yellowing and fruit pitting. However, it is usually symptomless in cultivated apple trees. ASPV is the type species of the genus Foveavirus, in the family Betaflexiviridae. The positive sense and single-stranded genomic RNA of the virus consists of about 9300 nucleotides(nts) and contained five open reading frames(ORFs). ORF1 encodes a replicase-complex polyprotein(Rep); ORF2~4 compose a triple gene block(TGB) and encode movement-related proteins. ORF5 encodes a coat protein(CP). In this study, we sequenced complete CP gene and TGB genes of ASPV isolates from pear trees and determined the genome sequence of a pear isolate LYC from Pyrus bretschneideri cv. Laiyang Cili. Meanwhile, interactions within and between TGB proteins were tested by Y2 H and the viral TGB proteins were localized at subcellular levels. Results are listed as follows:1. The presence of ASPV in 51 pear samples and 4 apple samples collected from four provinces in China were detected by RT-PCR. Results showed that 35 pear samples and 4 apple samples were positive for ASPV. The CP gene of ASPV isolates from from 17 pear samples and 2 apple samples, and the TGB genes of ASPV isolates from 16 pear samples and 2 apple samples.was seqenced Sequences analyses showed that the virus isolates were molecular divergent, and the CP gene sequences of tested isolates shared similarities ranging from 63.8% to 99.8% at nucleotide(nt) level and 69.8% to 99.8% at amino acid(aa) level. The sequences of the whole TGB and TGB1, 2 and 3 of tested isolates shared nt similarities of 74.8-99.7%, 74.4-99.6%, 71.4-99.4% and 74.2-100%, respectively. The proteins encoded by TGB1, TGB2 and TGB3 shared aa similarities of were 71.3-100%, 73.3-100% and 65.0-100%, respectively. Phylogenetic analyzes based on their CP and TGB gene sequence showed that all isolates clustered into four phylogroups.2. The genomic sequence of an ASPV isolate LYC from Pyrus bretschneidericv. Tse-Li was determined, was and consisted of 9273 nt without ploy(A). ASPV-LYC had the same genomic structure with those of other reported ASPV isolates. The whole genome of ASPV-LYC and other reported ASPV isolates shared similarities ranging from 69.9% to 78.3 %, and ASPV-LYC was mostly identical to a recently reported Apple green crinkle associated virus(AGCa V)isolate K10 by sharing 79.9 % similarity. Its CP gene also showed a relatively higher identity with K10 by sharing 77.4 % nt and 83.2 % aa similarities. Multiple sequence alignments showed that the CP of APSV isolates LYC had several co-variation amino acid sites with isolate K10, whilst its Rep shared co-variation sites with ASPV-KL1 and ASPV-KL9. In the phylogenetic trees based on the nucleotide sequences of whole genome, intergenic non-coding region II(IG-NCR II) and acid amino sequences of CP, LYC was clustered into the same branch with K10. However, LYC clustered into the same branch with KL1 and KL9 in the acid amino sequences-based phylogenetic tree on Rep, and clustered into the same branch with ASPV-Hannover in the IG-NCR I-basedphylogenetic tree. A recombination event was detected in the ORF1 of LYC by using RDP4 sofeware, with Palampur and KL9 as major and minor parents3.The interactions within and between TGB proteins of ASPV isolate HN-HB6-10 were tested by Y2 H assays. Results showed that TGBp1 could interact with itself, but not with other TGB proteins. Meanwhile, TGBp1 and TGBp3 could interact with AT4/1 protein from Nicotiana occidentalis plants. Each of TGB proteins were fused with YFP and infratially expressed in Nicotiana benthamiana leaves. Observation under a confocal microscopy showed that the cell wall, plasmalemma and vesicle in cytoplasm of leaves inoculated YFP-TGBp1 had YFP fluorescence signals, and spots with strong fluorescence signals appeared on cell walls, suggesting that TGBp1 of ASPV might be located in cell wall, plasmodesmata and cytoplasm in tobacco leaves. In YFP-TGBp2 inoculated leaves, YFP signals presented in nucleus,endoplasmic reticulum and cell wall, indicating that TGBp2 might be located in nucleus, endoplasmic reticulum and cell wall in tobacco leaves.