Materiales corrosivos

ASPV defends against it hosts’ defenses

To live in host cells or to escape from host immunity, plant viruses involved a series of defense strategies. Here we investigated ASPV population structures and molecular diversity of ASPV pear isolates based on its function important gene CP and TGB in China, so as to infer the evolutionary mechanisms of ASPV. Our study showed that mutations (including insertions or deletions), purifying selection, and recombination were important factors driving ASPV evolutions in China or maybe even in the world. And also ASPV defends against it hosts by encoding a VSR, which was its CP. Surprisingly, the great molecular diversity of ASPV CP isolates did not affect its VSR activity and pathogenicity. But how ASPV CP protected its VSR function and how the VSR function need to be further studied.

Our results indicate that ASPV has great molecular diversity among its hosts (pear, apple, Korla pear), probably because when a virus adapts to a new host, it was primarily manifested as amino acid substitutions, which could allow more efficient virus entry into the new host, block interactions with host proteins or promote escape from both the new and the old host’s immune responses (Moya et al 2004; Boulila 2010; Bandín and Dopazo 2011). However, interactions between ASPV and its hosts have been not studied yet and further studies of these interactions will need to be explored. Molecular diversity of a virus may result in dramatic changes in the biological properties of the virus, which included the appearance of resistance-breaking strains or the acquisition of broader host ranges. Here we showed that ASPV molecular diversity not only induced different biological properties on its herbaceous host N. occidentails but also resulted in antigenic variation of different ASPV CP isolates, which leaded to their differences in serological reactivity among rCPs of different ASPV isolates. However, unfortunately we did not furture expore how the molecular variation affected those results, which will need more studies.

VsiRNAs derived from ASPV detected by deep suquencing and VSR activity of ASPV CP indicated that RNA silencing of pear plant play a role in defending against ASPV. The pear genome sequence were recently reported (Wu et al 2013), which provides a great advantage to further study the RNA silencing components of pear plant defending against ASPV.

Arabidopsis thaliana defends against TRV

Plants have developed a series of mechanisms to defend themselves against viruses. Here we have investigated which elements of the Arabidopsis RNA silencing machinery are required to defend against TRV (Chapter 4). Our results are summerized in Figure 5-1 (Fig. 5-1).

Firstly, we have shown that VIGS intensity of dcl2/dcl3/dcl4 plants correlated positively with temperature, which propably indicated that DCL1 also could target TRV dsRNAs. This result was inconsistent with previous results that atDCL1 was not involved in TRV-PDS VIGS (Deleris et al 2006). Explaination for this contradiction was that probably the expression level of DCL1 correlated positively with temperature: at low temperature the expression level of DCL1 was not enough to involve in targeting TRV dsRNAs, as the temperature became high DCL1 expression level was increased.

Secondly, we have shown that TRV susceptibility, recovery and VIGS appear to be separable phenomena, with AGO2 and AGO4 playing important roles in initial susceptibility to TRV, AGO1 playing an important role in VIGS. These results suggest the existence of distinct RISC complexes that target different RNA populations within the cell and over time, which was cleary showed in Figure 5-1: AGO2 and AGO4 containing RISC complexes specially targeted TRV viral RNA, however, AGO1 containing RISC complexes specially targeted endogenous PDS mRNA. However, in our results it seemed as yet unidentified players mediating recovery, but our results indicated either the involvement of RNA silencing components in recovery is highly redundant or additional mechanisms was involved in TRV-GFP recovery. A recent study showed that TRV induced nonrecovery symptoms on N. tabacum plants that Coilin gene was knocked down, whereas TRV infected wildtype N. tabacum plants went recovery (Shaw et al 2014). Coilin is a key component of Cajal bodies (CBs) and also the scaffolding protein essential for CB formation and function (Collier et al 2006). CBs are distinct nuclear bodies physically and functionally associated with the nucleolus. In addition to their traditional function in coordinating maturation of certain nuclear RNAs, CBs participate in cell cycle regulation, development and regulation of stress responses. CBs have been identified in many orgnisms including Arabidopsis and Arabidopsis also encoed Coilin protein (Collier et al 2006). It is not clear whether Coilin plays a role in an as-yet unknown RNA silencing mechanism or whether it triggers a separate defense response that restricts virus accumulation concomitantly with RNA silencing (Ghoshal et al 2015). Thus, the interplay

of RNA silencing and other plant defense responses may regulate the establishment of symptom recovery in some plant-virus interactions, which need to be further tested.

Thirdly, results in our study indicated that homologous viral RNAs were targeted by two ways, either by slicing activity or by translationl repression and the part of translationl repressed TRV RNAs went to the PBs structure. Probably translational repression of viral RNAs likely plays an important role in restricting viral RNAs accumulation and that PB function plays an important role in clearing viral RNA from the cell. The fact that PBs are induced only at later stages of infection again suggests a temporal aspect to virus resistance. If we accept that an increase in PBs is indicative of increased translational repression, then the lack of PB induction at early time points would suggest that RNA silencing defenses against TRV are largely mediated through slicing activity, most likely mediated by AGO2, followed by translational repression mediated by additional AGO proteins. Decapping complexes are not thought to participate directly in translational repression, but rather in the processing of repressed mRNAs or producing aberrant RNAs and thus DCP2 would necessarily act downstream of AGO proteins.

Finally, the its1 mutant here was the only mutant described with an increased VIGS intensity and this mutant could be useful in functional genomics approaches in Arabidopsis in the furture. In the hypomorphic DCP2 mutant its1 plants, increased accumulation of TRV-GFP and TRV-PDS viral RNAs suggests that the decapping/PB pathway may be an important mechanism for degrading viral RNA. However, increased TRV-GFP RNAs accumulated in its1 plants was not associated with increased GFP protein, which indicated that much of the “excess” viral RNA represents aberrant or non-translating RNA.

Fig. 5-1 A model for RNA silencing in Arabidopsis plants against TRV

Once Arabidopsis was infected with TRV, TRV Replicative intermediate dsRNAs were recognized by DCL2, DCL3 and DCL4, and were cut into vsiRNAs (if exogenous factors were inserted into TRV vector, for example, PDS, there would be also PDS derived siRNAs). However, at high temperature (26 ℃), DCL1 may be involved in defending against TRV-PDS. TRV derived siRNAs were loaded into AGO2 and AGO4 containing slicing complex, specially targeting TRV viral RNA, however, PDS derived siRNAs were loaded into AGO1 containing slicing complex, specially targeting endogenous

PDS mRNA. Results in our study indicated that homologous RNAs were targeted by two ways, either by

slicing or by translationl repression. Our results indicated that those translationl repression RNAs went to P-bodies. In its1 mutant, temporary storaged TRV viral RNA in P-bodies went back to RNA silencing