Genetic Diversity Analysis And Pathogenicity-associated Genes Determination Of Prunus Necrotic Ringspot Virus

Prunus necrotic ringspot virus (PNRSV) is a worldwide viral pathogen that infects many stone fruit trees including peach, cherry, plum, apricot, almond and nectarine as well as some ornamentals such as roses and lily. Although the morphology and genetic diversity of PNRSV were much more studied, the genetic diversity of Chinese and Canadian PNRSV isolates was seldom studied. Thus, some other molecular features, such as pathogenicity determiants of PNRSV, was still not clear. In this study, we firstly studied the genetic diversity of PNRSV isolates deriving from China and Canada, and developed integrated-PCR for detection and group differentiation of the virus. Furthermore, the complete genome of two PNRSV isolates Chr3and Pch12were cloned and sequenced, and the infectious cDNA clones of Chr3and Pchl2were successfully constructed. Eventually, the pathogenicity determinants of PNRSV were identified.1. Prunus necrotic ringspot virus (PNRSV) was detected by RT-PCR from different Prunus species widely grown in China, and its infection rate was about31.3%. The sequences corresponding to the coat protein (CP) gene of10PNRSV isolates obtained in this study showed88.4%-100%nucleotide sequence identity and89.8%-100%amino acid sequence identity. Phylogenetic analysis of CP sequences showed that all PNRSV isolates from China clustered into three groups represented by PV96, PV32and PE5, and their distribution frequencies were29.4%,58.8%and11.8%, respectively. Based upon CP sequences, an integrated RT-PCR assay was developed for the specific identification of PNRSV isolates in the three groups.2. To study PNRSV in Canada, a total of69samples including32from cherry and37from peach, were randomly collected from three separate plots in a research farm in the Niagara Fruit Belt, Ontario. Enzyme-linked immunosorbent assay (ELISA) showed that18out of32cherry trees and13out of37peach samples were infected by PNRSV with an average incidence of44.9%. The presence of PNRSV in ELISA-positive samples was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The near full-length genomic RNA3segment encoding the5’-proximal movement protein (MP) and the3’-proximal coat protein (CP) of the31samples was cloned and sequenced. Phylogenetic analysis of MP and CP genes suggest these31isolates belong to two groups, PV96and PV32, with the former as the predominant group. Throughout the2011growing season, no obvious phenotypic differences were observed between PNRSV- positive and negative trees. To our best knowledge, this study represents the first characterization of PNRSV in Canada.3. The near-full-length genome sequences of RNA1,2and3of PNRSV were produced using a degenerate primer RNAs-R, complementary to the3’untranslated terminal region (UTR) of RNA1,2and3of PNRSV, and one of three5’-UTR primers RNA1-F, RNA2-F and RNA3-F (based on GenBank accession numbers AF278534, AF278535, and S78312, respectively). The5’or3’terminal cDNAs of RNA1,2and3were obtained using a5’or3’RACE kit (Invitrogen). The full-length genomic cDNA sequences of isolates Chr3and Pch12were deposited in the GenBank database with accession numbers (JN416771through JN416776). The molecular features of the PNRSV isolates Chr3and Pch12was identified and analysed.4. A binary plant expression vector pCass4-Rz was used as the backbone plasmid to integrate full-length genomic cDNAs of PNRSV. The full-length cDNAs of genomic segments RNA1, RNA2, and RNA3of PNRSV isolates Chr3and Pch12were successfully cloned into plasmid pCass4-Rz, respectively. The full-length cDNAs were placed between the35S promoter and the ribozyme sequence cassette (Rz). To test infectivity of the Chr3-and Pch12-derived clones, cucumber cotyledons (cv. Straight Eight) were co-infiltrated with a mixture of Agrobacterium cultures containing three T-DNA constructs integrated with the corresponding full-length cDNAs of genomic RNAs1,2and3. At18days post inoculation (dpi), plants were examined for symptoms induced by the virus. Plants infiltrated with Chr3clones showed yellowish and mild necrosis symptoms on the third and fourth true leaves of plants, whereas Pch12clones apparently induced more severe symptoms on cucumber such as severe ringspot and necrosis on all leaves including cotyledons and very small stature. DAS-ELISA and RT-PCR confirmed the presence of the PNRSV virus in all the cucumber plants infiltrated with the Chr3or Pch12clones.To further test if these clones are also infectious on their natural hosts, three mixed T-DNA constructs were biolistically introduced into peach (cv. Loring or GF305) and cherry (cv. Bing) seedlings. Cherry plants inoculated with Pch12showed dwarfism symptoms, necrosis on newly emerging leaves and the death of the young shoot tip at18dpi. In contrast, isolate Chr3induced milder symptoms on all inoculated cherry plants, and the identified symptoms included irregularly shaped necrosis spots on new emerging leaves and slightly smaller stature. DAS-ELISA and RT-PCR confirmed all those cherry plants bombarded with Pch12-or Chr3-derived clones were infected by PNRSV. On peach, about one fourth of GF305seedlings inoculated with Pch12showed severe chlorotic ringspot symptoms on new emerging leaves, which quickly became necrotic and dropped off to form ’shot holes’ at14dpi, and about one third of inoculated Loring peach seedlings displayed mild mosaic symptoms on newly emerging leaves at20dpi. Along with growth of peach seedlings, the symptoms gradually disappeared. All peach seedlings initially showing symptoms were PNRSV positive by DAS-ELISA and RT-PCR. No symptoms were discernible on any Loring or GF305peach seedlings bombarded with Chr3-derived clones. No PNRSV infection was evident either in the newly emerging leaves or in the leaves bombarded with Chr3cDNAs. The results showed the cDNA clones of Chr3and Pch12were infectious on those experimental and natural hosts.Taken together, the above data suggest that the Chr3and Pch12-derived full-length cDNA clones are infectious on both experimental and original natural hosts but possess different pathological properties with Pch12being more aggressive on all the three tested plant species.5. We engineered hybrid viruses by exchanging genomic segments (RNA1, RNA2or RNA3) or swapping different regions of the segments between Chr3and Pch12, and the infectivity assays showed both1C region in RNA1and2M region in RNA2contributed to the pathogenicity discrepancy. To further identify the amino acids in1C and2M regions conferring the severe pathogenicity of Pch12, we constructed seven single and double amino acid(s) substitution mutants using Pch12as a background. Of the mutants tested, the amino acid mutated at the position279from ’K’ to ’N’ in2M region resulted in the pathogenicity change of Pch12from ’severe’ to ’mild’. However, it was not observed that amino acid(s) substitution in the1C region would change the pathogenicity of Pch12. Gain-of-function experiments demonstrate that both the1C region and K279of Pch12were required for severe pathogenicity. Our results suggest that PNRSV RNA1and2codetermine viral pathogenicity to adapt to alternating natural Prunus hosts likely through mediating viral accumulation.