The Role Of RNA Silencing In Satellite RNA-mediated Attenuation Of Virus Symptoms In Plants

Plant RNA viruses are often associated with subviral RNA agents known as satellite RNAs (satRNAs), which are small non-coding RNAs and share little or no sequence homology with their associated virus (helper virus). The genomes of satRNAs are around200-1500nucleotides (nt), which depend on their helper viruses for replication, encapsidation, systemic movement and transmission. The majority of satRNAs attenuate symptoms induced by the helper viruses, while only a few satRNAs have been shown to exacerbate disease phenotypes by inducing their own symptoms in infected host plants. However, how satRNAs attenuate symptoms remains unclear. It has been proposed that satRNAs may reduce helper virus accumulation by competing for RNA replicase or increasing antiviral silencing against the helper virus, thereby reducing symptoms in the host plant. However, symptom reduction by satRNAs is often not associated with a corresponding reduction in helper virus accumulation. This suggests that some other factors or mechanisms may be involved.RNA silencing is a sequence-specific RNA degradation process induced by double-stranded RNA (dsRNA) or self-complementary hairpin RNA (hpRNA). This dsRNA or hpRNA is processed by Dicer, an RNase Ⅲ-like endoribonuclease, to generate small RNAs (sRNAs), which are loaded onto Argonaute (AGO) proteins to form the RNA-induced silencing complex (RISC). RISC is guided by these sRNAs to bind and degrade cognate mRNAs or other single-stranded RNAs. RNA silencing in plants has been well established as an anti-viral defence mechanism. Viral infection is associated with the accumulation of virus-derived small interfering RNAs (siRNAs), which in turn direct the degradation of viral genome RNAs. To overcome this host defence mechanism, viruses have evolved a counter-defense strategy by encoding suppressors of RNA silencing (VSRs). A predominant mode of action by VSRs is to bind double-stranded sRNAs, thereby preventing the formation of RISC, which is essential for the silencing of viral genomes by the host. VSRs are key symptom determinants of plant viruses, possibly because they interfere with the host sRNA pathways especially the microRNA (miRNA) pathway. miRNAs play critical roles in such processing as cell division, leaf formation and flower development in plants. VSRs affect the expression and function of miRNAs, thereby have the potential to cause developmental abnormalities and hence disease-associated symptoms. A typical example is the enhanced accumulation of miR168caused by viral infection through the function of VSRs. The miR168negatively regulates the expression of AGO1, a key component of RISC. The induction of miR168reduces the expression of AGO1protein, which can subsequently interfere with normal plant development and decrease antiviral RNA silencing, both of which can increase viral disease symptoms.A recently observed characteristic of satRNAs is that their replication is associated with extremely high amount of satRNA-derived siRNAs (sat-siRNAs). This characteristic, together with the evidence that VSRs interfere with host small RNA function and hence plant development, shows that sat-siRNAs and VSRs may be involved in the symptom attenuation mechanism. In this thesis, a RNA silencing-based hypothesis for satRNA-mediated symptom attenuation was proposed and validated. It is based on that symptom reduction by satRNAs is due to sequestration of VSRs by sat-siRNAs, which minimizes the VSR’s interference with host miRNA function.Firstly, whether satRNA infection would minimize the effect of VSRs on host siRNA-directed gene silencing was tested. Using a β-glucuronidase (GUS) report gene system in conjunction with Agrobacterium-infiltration (Agro-infiltration) assays, the interaction between two VSRs (P19and2b) and Y-satellite RNA (Y-Sat) of Cucumber mosaic virus (CMV) was examined. To facilitate the experiments, a satRNA-free CMV isolate (Q-CMV) and transgenic Nicotiana. benthamiana plants resistant to Y-Sat-induced yellowing symptoms were developed. Results showed that ⅰ) GUS expression was effectively silenced by a hpRNA construct (hpGUS) but this silencing was suppressed by co-Agro-infiltration with P19or2b constructs; ⅱ) Upon Y-Sat infection, strong hpGUS-induced GUS silencing occurred despite the presence of P19 or2b, indicating that the function of the VSRs was interfered with by Y-Sat infection; iii) Agro-infiltrated GUS underwent sense co-suppression, which was inhibited by P19or2b co-infiltration, but upon Y-Sat infection, the co-suppression was enhanced despite the presence of P19or2b, again indicating that the function of the VSRs was interfered with by Y-Sat infection. Taken together, these results indicated that satRNA infection interferes with the function of VSRs and reduces their suppressor effect on small RNA-induced silencing of the host. This is consistent with the hypothesis that satRNAs reduce helper virus-caused symptoms by minimizing the effect of helper virus-encoded VSRs on host miRNA or siRNA-mediated gene regulation.In order to rule out the possibility if the increased GUS silencing by Y-Sat infection in the presence of VSRs was due to increased amounts of GUS siRNAs, the effect of Y-Sat infection on the accumulation of hpGUS-derived siRNA accumulation was conducted by Northern blot hybridization. Results showed that Y-Sat infection did not affect the processing of hpGUS RNA or the accumulation of hpGUS siRNA, which indicated that the effect of Y-Sat infection on GUS silencing was due to interference with the VSR function.To demonstrate that satRNA-derived siRNAs indeed bind VSRs and sequester the VSRs from interfering with host sRNA function, RNA-immunoprecipitation was deployed by using P19antibodies to examine the siRNAs associated with P19in the presence or absence of Y-Sat infection. It showed that Y-Sat infection reduced the amount of hpGUS-derived siRNA bound to the P19VSR, and instead, P19was bound with abundant Y-Sat-derived siRNAs. This result indicated that the increased hpGUS-induced silencing by Y-Sat infection in the presence of VSRs was due to saturation of the VSRs by Y-Sat siRNAs, releasing the hpGUS-derived siRNAs available for directing GUS silencing. These results supported the hypothesis that satRNAs reduce the effect of helper virus-encoded VSRs on host sRNA-mediated gene regulation by sequestering the VSRs with Y-Sat siRNAs.An ultimate demonstration of the hypothesis is to show that the interference of host miRNA or siRNA function is solely or predominantly responsible for helper virus-induced symptoms, and this interference of host sRNA function is diminished upon satRNAs infection. For this purpose, the effect of Y-Sat infection on the accumulation of miR168was examined. miR168is a primary regulator in the host miRNA and siRNA pathways and a key indicator of VSR function. The results showed that infection of N. benthamiana by both subgroup Ⅰ (Fny-CMV) and subgroup Ⅱ (Q-CMV) CMV strains induced miR168accumulation, and this induction was downregulated in the presence of Y-Sat. These results indicated that Y-Sat reduces the helper virus CMV2b-caused induction of miR168. Interestingly, the level of miR168induction was correlated with the severity of CMV-induced symptoms, which implies that the interference of host sRNA pathways by VSRs are indeed responsible for virus-caused symptoms, and Y-Sat-mediated symptom attenuation is indeed due to minimized interference of host sRNA pathways by VSRs.In addition to CMV-infected N. benthamiana plants, the effect of Y-Sat infection on miR168accumulation in Pl/HcPro (VSR of Tobacco etch virus, TEV) transgenic N. tabacum plants were also investigated. The results showed that Pl/HcPro strongly induced miR168expression, and this induction was largely reversed upon CMV Y-Sat infection. Moreover, the tilted phenotype associated with transgenic Pl/HcPro plants was also largely corrected by CMV Y-Sat infection. These results suggested that satRNA infection generally minimizes the VSR-caused interference of host miRNA and siRNA function in plants, and this diminished effect on host sRNA function accounts for reduced symptoms caused by helper viruses.In summary, this thesis provides several pieces of experimental evidence to demonstrate an RNA silencing-based hypothesis proposed to answer such a longstanding question as how viral satRNAs attenuate disease symptoms caused by their helper viruses in plants:ⅰ) Agrobacterium infiltration of viral-infected N. benthamiana leaves showed that the Tombusvirus-encoded P19VSR and the CMV-encoded2b VSR both effectively suppressed hpRNA-induced silencing, but this suppression was released upon infection with the CMV Y-Sat; ⅱ) RNA immunoprecipitation showed that, in the presence of Y-Sat, P19was saturated with Y-Sat-derived siRNAs, resulting in reduced amount of hpRNA-derived GUS siRNAs bound to P19; ⅲ) Northern blot hybridization analyses of CMV-infected N. benthamiana plants or P1/HcPro-expressing transgenic N. tabacum plants showed that Y-Sat infection minimized the induction of miR168expression caused either by CMV infection or by overexpression of Pl/HcPro VSR. Taken together, these results support the hypothesis that satRNAs attenuate symptoms by sequestering helper virus-encoded VSRs through abundant satRNA-derived siRNAs, preventing the VSRs from interfering with host siRNA or miRNA-directed gene regulation and hence improving plant development.