Genetic Diversity Of Potato Spindle Tuber Viroid (PSTVd) And Its Induced Gene Silencing

Viroids can infect various plants and thereby causing important losses in agriculture. As there is no evidence for RNA translation, their pathogenic effects must result from direct interaction with host components. Viroids thus offer a unique model for exploring host-pathogen interactions that depend strictly on the pathogen's RNA sequence and structure. RNA silencing (RNAi) is a conserved defense mechanism plants and other eukaryotes use to protect their genomes against aberrant nucleic acids. A hallmark of this defense response is the production of 21～24 nt viral small RNAs via mechanisms that remain to be fully understand. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors, as counter-defense. The occurrence of viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA silencing systems.In this study, the PSTVd samples collected from Heilongjiang of the main potato area were cloned and sequenced. The genetic stability of PSTVd was analyzed. The gene silencing induced by PSTVd and the interaction between viroid and host were deeply studied. This study also tried to disclose siPSTVd are mostly from structured PSTVd(+) RNAs or generated from dsRNA intermediates during viral replication. Further analysis on the accumulation levels of the endogenous tomato and potato miRNA pathway can be modulated by PSTVd infection or not. The results were as follows:1. The diversity of the viroidospherePSTVd samples of PKS 1-6 plantlet in vitro supplied by Keshan Potato Research Institute was cloned and sequenced. Compared with severe strains(KF 440-2, RG1) and intermediate strain (PSTVd int) reported in Genebank, PKS 1-6 had more differences in sequence(identity were only 97.21%, 97.77% and 98.61%, respectively), whereas PKS 1-6 was more constant in sequence with mild strains(KF-5, GI 333357 and GB X76844) (identity were 99.16%, 97.72% and 99.44%, respectively). From sequence alignment, PKS 1-6 isolate can be defined as mild strain. To inoculate PKS 1-6 isolate to N. benthamiana plants that transgenically express GFP(line GFP 16C) and analysis the sequence of 16C samples infected PSTVd, three of four PKS 1-6 progenies in 16C had mutations, one maintain parental sequence. The mutant region located in the CCR(at position 81, 82 and 267), Var (at position 127 and 136), and Path(at position 56). For sequence analyses of PSTVd samples collected Fujin potato field, five in six clones had the identical sequence with PKS 1-6, and only PEF-14 variant had 2 mutants at position 4(TL) and 296(Path). But for sequence analyses of PSTVd from Taikang, four in eleven clones were identical compared with PKS 1-6.A total of 22 clones were sequenced. Among them, 12 mutations had been observed, whereas in them 10 clones had the idential sequence as PKS 1-6. In all clones, the number of mutant nucletides was between 0～3, and the homology was between 99.16%～100%, and the lowest free energy difference was between -159.10%～-173.6% kcal/mol. The mutation mainly located in CCR and Var(variant frequent was 38.1% and 28.9%, respectively), followed by Path region(variant frequent was 14.3%) and TL and TR(variant frequent were 9.5%).2. PSTVd did not function as an RNA silencing suppressor at the cellar and whole plant levelsWe used transgenical N. benthamiana 16C to test PSTVd suppressor activity. When 16C plants were co-infiltrated by PSTVd recombinant vectors containing sense, antisense monomeric and dimeric PSTVd, respectively, together with vector containing GFP.The GFP fluorescence of local leaves was observed with dim intensity for co-infiltration with PSTVd construct than control, indicating that PSTVd induced the silencing in a certain degree. The fact that PSTVd could induce gene silencing and produced sense and antisense siPSTVd were confirmed by siRNA gel plot. To exclude the possibility that the PSTVd sequence can induce the GFP suppress for the sequence identity, we align the sequence of PSTVd and GFP. The identity was only 47.6% and longest identical sequence was only 9 bp, so the alignment results exclude the possibility. Meanwhile, we devised the experiment that we co-infiltrated hp pdsi and PSTVd vector to N.B. and observed that PSTVd vector promote silencing of plant pds gene. From the hybridization results of si35S-promoter, the level of PSTVd and control had no significant difference. This result addressed the phenomena of inducing silencing was not reduced the 35s-promoter. From northern blot of mGFP, the mGFP quantity level of PSTVd and GFP co-infiltration turned less then the control, that indicate the transcription of GFP can be affected by PSTVd, and mGFP decreased , so the fluorescence turned weak. From these results, we speculated the PSTVd can be inducer of gene silencing in the transcriptional level for the methylation.3. siPSTVd were derived predominantly from structured RNA of PSTVdFrom the results of northern blot for siPSTVd by the infiltration the vector with the dimeric replicated PSTVd and non-full length and non-replicated PSTVd to N.B., we observed that the specific PSTVd siRNA could be detected for both of them. That indicated the structured PSTVd could be the source of siPSTVd. The profile obtained in the siPSTVd showed that the majority of PSTVd siRNAs were of the (+)polarity. This observation is consistent with the notion that genomic PSTVd RNA folds into a highly structured conformation, which is then cleaved by DCL in vivo.4. PSTVd siRNAs do not modulate miRNA pathwayBoth polarities of siPSTVd could be detected for positive samples inoculated in tomato (Rutgers) and potato, which mean gene silencing was induced after the infection of PSTVd. No siPSTVd were produced for mock treatment. For the same membrane, the mi159 and mi167 blotting were done. The results showed no significantly different for the miRNA level for positive and negative samples. Our analyses showed PSTVd is unlikely to affect the pathway of endogenous miRNAs, at least for the main miRNA of both mi159 and mi167. Studying the strategies that viroids infect a plant also helps to address basic molecular and cellular processes. It's significant to understand the structure, function and pathway of pathogenicity. Meanwhile, it will provide important principle value for disclosing the mechanism of gene silencing and interaction between viroid host and pathogen, and also provide the reference of defence to viroid.