Characterization of virus-derived small interfering RNAs in Apple stem grooving virus-infected in vitro-cultured Pyrus pyrifolia shoot tips in response to high temperature treatment.

BACKGROUND: Heat treatment (known as thermotherapy) together with in vitro culture of shoot meristem tips is a commonly used technology to obtain virus-free germplasm for the effective control of virus diseases in fruit trees. RNA silencing as an antiviral defense mechanism has been implicated in this process. To understand if high temperature-mediated acceleration of the host antiviral gene silencing system in the meristem tip facilitates virus-derived small interfering RNAs (vsiRNA) accumulation to reduce the viral RNA titer in the fruit tree meristem tip cells, we used the Apple stem grooving virus (ASGV)-Pyrus pyrifolia pathosystem to explore the possible roles of vsiRNA in thermotherapy. RESULTS: At first we determined the full-length genome sequence of the ASGV-Js2 isolate and then profiled vsiRNAs in the meristem tip of in vitro-grown pear (cv. 'Jinshui no. 2') shoots infected by ASGV-Js2 and cultured at 24 and 37 °C. A total of 7,495 and 7,949 small RNA reads were obtained from the tips of pear shoots cultured at 24 and 37 °C, respectively. Mapping of the vsiRNAs to the ASGV-Js2 genome revealed that they were unevenly distributed along the ASGV-Js2 genome, and that 21- and 22-nt vsiRNAs preferentially accumulated at both temperatures. The 5'-terminal nucleotides of ASGV-specific siRNAs in the tips cultured under different temperatures had a similar distribution pattern, and the nucleotide U was the most frequent. RT-qPCR analyses suggested that viral genome accumulation was drastically compromised at 37 °C compared to 24 °C, which was accompanied with the elevated levels of vsiRNAs at 37 °C. As plant Dicer-like proteins (DCLs), Argonaute proteins (AGOs), and RNA-dependent RNA polymerases (RDRs) are implicated in vsiRNA biogenesis, we also cloned the partial sequences of PpDCL2,4, PpAGO1,2,4 and PpRDR1 genes, and found their expression levels were up-regulated in the ASGV-infected pear shoots at 37 °C. CONCLUSIONS: Collectively, these results showed that upon high temperature treatment, the ASGV-infected meristem shoot tips up-regulated the expression of key genes in the RNA silencing pathway, induced the biogenesis of vsiRNAs and inhibited viral RNA accumulation. This study represents the first report on the characterization of the vsiRNA population in pear plants infected by ASGV-Js2, in response to high temperature treatment.

Divergent molecular variants of Grapevine virus B (GVB) from corky bark (CB)-affected and CB-negative LN33 hybrid grapevines.

Analysis of two Grapevine virus B (GVB)-infected LN33 hybrid grapevines revealed that a plant exhibiting clear symptoms of corky bark (CB) disease was infected with two molecular variants of the virus, whereas a plant exhibiting no disease symptoms was infected with only one variant. Sequence results indicated that the single variant in the CB-negative grapevine was also one of the two present in the CB-affected hybrid. Plant extracts from these two grapevines were used to successfully transmit the virus to N. benthamiana. After further cloning and sequencing, two clearly divergent variants were identified. Comparative molecular analysis of the variants, named here GVB 953-1 and GVB-H1, respectively, transmitted from CB-affected and consistently CB-negative plants, revealed short genomic regions, most of them highly divergent, that encoded amino acid sequences, containing significant amino acid substitutions altering the net charges of their respective proteins. Interestingly, a comparison of these variants to genome sequence data of GVB variants GVB Italy and GVB 94/971 available from the GenBank, revealed that these significant amino acid substitutions were the same for, and unique to, the variant pairs GVB 953-1/GVB Italy and GVB-H1/GVB 94/971. This despite the variants of each pair being otherwise clearly different at nucleotide and amino acid levels. In addition, both sets of variants differed substantially in their respective 3'-non-translated (3'NTR) regions. The relevance of these findings is discussed.

The Coat Protein of Citrus Yellow Vein Clearing Virus Interacts with Viral Movement Proteins and Serves as an RNA Silencing Suppressor.

Citrus yellow vein clearing virus is a newly accepted member of the genus Mandarivirus in the family Alphaflexiviridae. The triple gene block proteins (TGBp1, TGBp2 and TGBp3) encoded by plant viruses in this family function on facilitating virus movement. However, the protein function of citrus yellow vein clearing virus (CYVCV) have never been explored. Here, we showed in both yeast two-hybrid (Y2H) and bimolecular fluorescence (BiFC) assays that the coat protein (CP), TGBp1 and TGBp2 of CYVCV are self-interacting. Its CP also interacts with all three TGB proteins, and TGBp1 and TGBp2 interact with each other but not with TGBp3. Furthermore, the viral CP colocalizes with TGBp1 and TGBp3 at the plasmodesmata (PD) of epidermal cells of Nicotiana benthamiana leaves, and TGBp1 can translocate TGBp2 from granular-like structures embedded within ER networks to the PD. The results suggest that these proteins could coexist at the PD of epidermal cells of N. benthamiana. Using Agrobacterium infiltration-mediated RNA silencing assays, we show that CYVCV CP is a strong RNA silencing suppressor (RSS) triggered by positive-sense green fluorescent protein (GFP) RNA. The presented results provide insights for further revealing the mechanism of the viral movement and suppression of RNA silencing.