植物病毒卫星RNA干扰其辅助病毒致病性的分子机理
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摘要
植物RNA病毒常常伴随有小的卫星RNA。卫星RNA是一类小的非编码RNA,基因组大小为200-1500nt,通常不编码蛋白,完全依赖于辅助病毒来完成复制、包被、移动和传播,且和其辅助病毒的基因组不存在序列同源性。部分卫星RNA可以影响辅助病毒在寄主植物上诱发的症状,多数为减轻,少数会加重寄主症状。传统理论认为卫星RNA是通过与辅助病毒竞争复制酶,减少了辅助病毒的复制,从而减轻了寄主症状。然而,传统理论不能解释卫星RNA在减轻辅助病毒诱导症状的同时,部分辅助病毒复制量不受影响的现象。由此推测,卫星RNA可能存在其它途径来减轻辅助病毒诱导的症状。
RNA沉默(或称为RNAi)是一种广泛存在于真核生物中,由小RNA介导的、以序列特异性的方式抑制或破坏靶标基因表达,在DNA或转录水平上的调控机制。RNA沉默可以调控基因的表达、修饰DNA和特异染色体区域组蛋白的表观遗传、防御转座子和病毒等核酸的入侵。病毒为了成功侵染寄主,也进化了相应的反防卫机制。大部分是通过编码基因沉默抑制子(viral suppressor of RNA silencing, VSR)蛋白结合病毒的siRNA,阻止RNA沉默复合体(RNA-induced silencing complex, RISC)的形成,抑制病毒基因组的降解。
基于RNA沉默具有调控植物正常生长发育和防御病毒的功能、VSR具有干扰RNA沉默反防卫机制、多数卫星RNA在复制过程中伴随着巨量的卫星RNA siRNA产生的三个依据,本文提出了以下假说来解释卫星RNA减轻病毒对寄主植物所诱导的症状。1)病毒通过其产生的VSR干扰寄主植物的miRNA代谢途径,从而致病；2)卫星RNA通过其产生的巨量siRNA捕获并饱和大部分的VSR,减少VSR干扰寄主植物的miRNA代谢途径,减轻病毒对寄主植物诱导的症状。
本文通过对烟草、辅助病毒CMV和Y-卫星RNA(Y-Satellite RNA, Y-Sat)组成的模式系统进行研究,验证了以上假说。模式系统选用CMV亚组Ⅱ的病毒株Q-CMV作为负对照。Y-Sat被选用于本研究,是因为卫星RNA的研究中以对CMV卫星RNA研究最多,而Y-Sat在CMV卫星RNA中具有代表性,可以导致本氏烟和普通烟等的斑驳黄花,方便了对病毒侵染过程的检测。
验证实验分为两个方面,1)应用GUS报告基因监测在有无卫星RNA伴随侵染时P19和2b两种VSR的活性；2)应用miRl68表达量易受VSR诱导的特性,监测在有无卫星RNA伴随侵染时VSR对miR168的影响。验证实验获得了以下几个方面的结果：
1. hpGUS可以有效的沉默GUS,而TBSV的P19和SD-CMV编码的2b两个VSR构件都可以干扰hpGUS介导的GUS沉默。实验采用农杆菌浸润法将GUS+hpGUS、GUS+hpGUS+P19或2b等构件转入本氏烟中进行瞬时表达,比较有无VSR共浸润时GUS蛋白量的变化。并通过GUS染色和GUS的相对酶活性测定分析了P19和2b的活性。实验成功模拟了植物内源的小RNA代谢途径,检验了P19和2b对hpGUS介导的GUS沉默的影响；实验结果与以前报道的VSR可以干扰hpRNA介导RNA沉默的实验结果相吻合。
2. Y-Sat影响了VSR干扰RNA沉默的功能。在野生型本氏烟上进行Q-CMV和Q-CMV+Y-Sat感染实验,然后瞬时表达GUS、hpGUS和P19或2b等构件。在只有Q-CMV感染时,P19和2b都可以干扰hpGUS介导的GUS沉默；在Q-CMV+Y-Sat感染的样品中,P19和2b对GUS的沉默却被削弱了；在只有GUS构件时,存在GUS基因的共抑制现象(另一种RNA沉默形式)；在GUS+P19或GUS+2b共浸润的样品中,共抑制现象明显减少,而在有Y-Sat感染时,即使有P19或2b构件的存在,共抑制水平仍然很高。通过对GUS mRNA的Northern印迹分析,GUS mRNA的量与GUS蛋白的量正相关,证实了沉默发生在RNA水平。上述结果证明了Y-Sat能影响VSR干扰RNA沉默的功能,说明VSR对植物内源小RNA介导的沉默途径的影响能被卫星RNA削弱。
3. Y-Sat导致的斑驳黄化对实验体系无明显影响。CMV+Y-Sat共同侵染的本氏烟植株出现了斑驳黄化症状,影响了植物的正常光合作用,也可能对整个实验体系产生干扰。为了排除Y-Sat导致的斑驳黄化对实验体系的影响,实验用CHLI突变体mCHLI转化本氏烟,获得不再出现斑驳黄化植株。应用mCHLI转基因本氏烟F2代,进行了Q-CMV或Q-CMV+Y-Sat的侵染,然后瞬时表达GUS、.hpGUS和P19或2b、GFP等构件。实验结果表明,VSR影响了hpGUS介导的GUS沉默,在有Q-CMV+Y-Sat共侵染的样品中,Y-Sat干扰了VSR的功能。实验结果与在野生型本氏烟中所得结果一致,说明了Y-Sat导致的斑驳黄化对实验体系无明显影响。
4.在Y-Sat感染且有GUS+hpGUS+VSR共浸润的样品中,GUS仍被高度沉默的现象不是由于hpGUS产生更多siRNA所致。在用GUS、hpGUS和P19或2b进行共浸润时,Q-CMV+Y-Sat共同感染比Q-CMV单独感染的样品产生了更高水平的GUS沉默。这种现象的出现有可能是由于Y-Sat干扰了VSR的功能,也有可能是由于hpGUS产生了更多的siRNA,从而介导了更多的GUS沉默。为了排除后一种可能性,实验设计了hpGUS siRNA的Northern印迹检测分析。结果显示,在感染Q-CMV+Y-Sat样品中,hpGUS siRNA的量并没有增加,排除了hpGUS产生更多siRNA介导更多的GUS沉默的可能性。
5.Y-卫星RNA siRNA捕获了VSR,削弱VSR对hpGUS介导的GUS沉默途径的影响。实验应用P19抗体进行RNA-免疫捕获分析,先对植株感染Q-CMV或Q-CMV+Y-Sat处理,再进行GUS、hpGUS、P19共浸润,后采用P19抗体沉淀P19蛋白复合体,并从沉淀复合体中分离RNA,通过Northern印迹分析与P19结合的hpGUS siRNA、Y-Sat siRNA量。结果显示,在Q-CMV感染的样品中,有部分hpGUS siRNA与P19结合；而在Q-CMV+Y-Sat感染的样品中,与P19结合的hpGUS siRNA较少,大部分的P19和Y-Sat siRNA结合在一起。结果说明P19是通过结合hpGUS siRNA来干扰hpGUS介导的GUS沉默；Y-Sat siRNA通过饱和VSR,增加游离的hpGUS siRNA,介导了较高水平的GUS沉默。实验结果进一步表明卫星RNA是通过产生的大量siRNA捕获VSR,从而削弱VSR对寄主小RNA介导的对内源基因调控的干扰。
6. Y-Sat可以削弱CMV编码的2b对miR168的诱导。miRNA在植物生长发育过程中起着至关重要的作用。有报道证实了VSR可以诱导miR168的表达量,miR168负调控AGO1mRNA,影响了植物miRNA代谢途径中关键蛋白AGO1的量。为了证实VSR对植物内源miRNA的影响是病毒致病的主导因素,且卫星RNA可以减少VSR对miRNA代谢的影响,本实验以miR168的表达量为指示,检测了感染CMV亚组Ⅱ(Q-CMV)和CMV亚组Ⅰ(Fny-CMV)及与Y-Sat的本氏烟样品中miR168的表达量。结果显示,Q-CMV和Fny-CMV都可以诱导miRl68的表达量；当有Y-Sat共同感染时,miR168的表达量明显减少,感染植株症状减轻；Fny-CMV可以引起相对于Q-CMV而言更高的miR168累积,感染植株也产生了更严重的症状。结果说明Y-Sat可以削弱CMV编码的2b对miR168的诱导,且miR168的表达量高低与植物发病程度紧密相关。实验用半定量RT-PCR检测CMVRNA4a,发现Y-Sat在削弱2b对miR168诱导的同时,编码2b蛋白CMV RNA4a的量有轻微的减少,导致miR168表达量减少的原因部分可能是由于Y-Sat共同感染时2b量的减少。
7. Y-Sat显著的抑制了P1/HcPro对miR168的诱导。本文应用了由烟草蚀纹病毒(Tobacco etch virus, TEV)编码的VSR P1/HcPro转基因普通烟(N. tabacum)体系对miR168的表达量进行了检测分析。实验结果显示,在P1/HcPro转基因普通烟中,miR168被明显的诱导,同时出现茎基部弯曲生长的表现型；在Q-CMV+Y-Sat共同感染转基因普通烟中,miR168的表达水平显著降低,茎基部弯曲生长的表现型基本消失。实验结果说明,植物病毒VSR通过影响植物内源的miRNA代谢途径而致病,而卫星RNA通过干扰VSR,削弱病毒的致病性,减轻病毒诱导的症状。
以上7个结论,从小RNA的水平验证了植物病毒卫星RNA弱化辅助病毒致病机理的假说,证明了1)病毒通过其产生的VSR干扰寄主植物的miRNA代谢途径,从而致病；2)卫星RNA通过其产生的巨量siRNA捕获并饱和大部分的VSR,减少VSR干扰寄主植物的miRNA代谢途径,减轻病毒对寄主植物诱导的症状。
卫星RNA主要存在于植物病毒,DI RNA主要存在于动物病毒。假说在植物和病毒卫星RNA上面得到了部分验证,作者推断假说还适用于动物和病毒DI RNA。部分动物病毒编码的VSR,也具有结合小RNA的特征,动物病毒的DI RNA,在复制过程可能也会产生大量的siRNA,饱和VSR,干扰VSR的致病性。
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.
RNA沉默(或称为RNAi)是一种广泛存在于真核生物中,由小RNA介导的、以序列特异性的方式抑制或破坏靶标基因表达,在DNA或转录水平上的调控机制。RNA沉默可以调控基因的表达、修饰DNA和特异染色体区域组蛋白的表观遗传、防御转座子和病毒等核酸的入侵。病毒为了成功侵染寄主,也进化了相应的反防卫机制。大部分是通过编码基因沉默抑制子(viral suppressor of RNA silencing, VSR)蛋白结合病毒的siRNA,阻止RNA沉默复合体(RNA-induced silencing complex, RISC)的形成,抑制病毒基因组的降解。
基于RNA沉默具有调控植物正常生长发育和防御病毒的功能、VSR具有干扰RNA沉默反防卫机制、多数卫星RNA在复制过程中伴随着巨量的卫星RNA siRNA产生的三个依据,本文提出了以下假说来解释卫星RNA减轻病毒对寄主植物所诱导的症状。1)病毒通过其产生的VSR干扰寄主植物的miRNA代谢途径,从而致病；2)卫星RNA通过其产生的巨量siRNA捕获并饱和大部分的VSR,减少VSR干扰寄主植物的miRNA代谢途径,减轻病毒对寄主植物诱导的症状。
本文通过对烟草、辅助病毒CMV和Y-卫星RNA(Y-Satellite RNA, Y-Sat)组成的模式系统进行研究,验证了以上假说。模式系统选用CMV亚组Ⅱ的病毒株Q-CMV作为负对照。Y-Sat被选用于本研究,是因为卫星RNA的研究中以对CMV卫星RNA研究最多,而Y-Sat在CMV卫星RNA中具有代表性,可以导致本氏烟和普通烟等的斑驳黄花,方便了对病毒侵染过程的检测。
验证实验分为两个方面,1)应用GUS报告基因监测在有无卫星RNA伴随侵染时P19和2b两种VSR的活性；2)应用miRl68表达量易受VSR诱导的特性,监测在有无卫星RNA伴随侵染时VSR对miR168的影响。验证实验获得了以下几个方面的结果：
1. hpGUS可以有效的沉默GUS,而TBSV的P19和SD-CMV编码的2b两个VSR构件都可以干扰hpGUS介导的GUS沉默。实验采用农杆菌浸润法将GUS+hpGUS、GUS+hpGUS+P19或2b等构件转入本氏烟中进行瞬时表达,比较有无VSR共浸润时GUS蛋白量的变化。并通过GUS染色和GUS的相对酶活性测定分析了P19和2b的活性。实验成功模拟了植物内源的小RNA代谢途径,检验了P19和2b对hpGUS介导的GUS沉默的影响；实验结果与以前报道的VSR可以干扰hpRNA介导RNA沉默的实验结果相吻合。
2. Y-Sat影响了VSR干扰RNA沉默的功能。在野生型本氏烟上进行Q-CMV和Q-CMV+Y-Sat感染实验,然后瞬时表达GUS、hpGUS和P19或2b等构件。在只有Q-CMV感染时,P19和2b都可以干扰hpGUS介导的GUS沉默；在Q-CMV+Y-Sat感染的样品中,P19和2b对GUS的沉默却被削弱了；在只有GUS构件时,存在GUS基因的共抑制现象(另一种RNA沉默形式)；在GUS+P19或GUS+2b共浸润的样品中,共抑制现象明显减少,而在有Y-Sat感染时,即使有P19或2b构件的存在,共抑制水平仍然很高。通过对GUS mRNA的Northern印迹分析,GUS mRNA的量与GUS蛋白的量正相关,证实了沉默发生在RNA水平。上述结果证明了Y-Sat能影响VSR干扰RNA沉默的功能,说明VSR对植物内源小RNA介导的沉默途径的影响能被卫星RNA削弱。
3. Y-Sat导致的斑驳黄化对实验体系无明显影响。CMV+Y-Sat共同侵染的本氏烟植株出现了斑驳黄化症状,影响了植物的正常光合作用,也可能对整个实验体系产生干扰。为了排除Y-Sat导致的斑驳黄化对实验体系的影响,实验用CHLI突变体mCHLI转化本氏烟,获得不再出现斑驳黄化植株。应用mCHLI转基因本氏烟F2代,进行了Q-CMV或Q-CMV+Y-Sat的侵染,然后瞬时表达GUS、.hpGUS和P19或2b、GFP等构件。实验结果表明,VSR影响了hpGUS介导的GUS沉默,在有Q-CMV+Y-Sat共侵染的样品中,Y-Sat干扰了VSR的功能。实验结果与在野生型本氏烟中所得结果一致,说明了Y-Sat导致的斑驳黄化对实验体系无明显影响。
4.在Y-Sat感染且有GUS+hpGUS+VSR共浸润的样品中,GUS仍被高度沉默的现象不是由于hpGUS产生更多siRNA所致。在用GUS、hpGUS和P19或2b进行共浸润时,Q-CMV+Y-Sat共同感染比Q-CMV单独感染的样品产生了更高水平的GUS沉默。这种现象的出现有可能是由于Y-Sat干扰了VSR的功能,也有可能是由于hpGUS产生了更多的siRNA,从而介导了更多的GUS沉默。为了排除后一种可能性,实验设计了hpGUS siRNA的Northern印迹检测分析。结果显示,在感染Q-CMV+Y-Sat样品中,hpGUS siRNA的量并没有增加,排除了hpGUS产生更多siRNA介导更多的GUS沉默的可能性。
5.Y-卫星RNA siRNA捕获了VSR,削弱VSR对hpGUS介导的GUS沉默途径的影响。实验应用P19抗体进行RNA-免疫捕获分析,先对植株感染Q-CMV或Q-CMV+Y-Sat处理,再进行GUS、hpGUS、P19共浸润,后采用P19抗体沉淀P19蛋白复合体,并从沉淀复合体中分离RNA,通过Northern印迹分析与P19结合的hpGUS siRNA、Y-Sat siRNA量。结果显示,在Q-CMV感染的样品中,有部分hpGUS siRNA与P19结合；而在Q-CMV+Y-Sat感染的样品中,与P19结合的hpGUS siRNA较少,大部分的P19和Y-Sat siRNA结合在一起。结果说明P19是通过结合hpGUS siRNA来干扰hpGUS介导的GUS沉默；Y-Sat siRNA通过饱和VSR,增加游离的hpGUS siRNA,介导了较高水平的GUS沉默。实验结果进一步表明卫星RNA是通过产生的大量siRNA捕获VSR,从而削弱VSR对寄主小RNA介导的对内源基因调控的干扰。
6. Y-Sat可以削弱CMV编码的2b对miR168的诱导。miRNA在植物生长发育过程中起着至关重要的作用。有报道证实了VSR可以诱导miR168的表达量,miR168负调控AGO1mRNA,影响了植物miRNA代谢途径中关键蛋白AGO1的量。为了证实VSR对植物内源miRNA的影响是病毒致病的主导因素,且卫星RNA可以减少VSR对miRNA代谢的影响,本实验以miR168的表达量为指示,检测了感染CMV亚组Ⅱ(Q-CMV)和CMV亚组Ⅰ(Fny-CMV)及与Y-Sat的本氏烟样品中miR168的表达量。结果显示,Q-CMV和Fny-CMV都可以诱导miRl68的表达量；当有Y-Sat共同感染时,miR168的表达量明显减少,感染植株症状减轻；Fny-CMV可以引起相对于Q-CMV而言更高的miR168累积,感染植株也产生了更严重的症状。结果说明Y-Sat可以削弱CMV编码的2b对miR168的诱导,且miR168的表达量高低与植物发病程度紧密相关。实验用半定量RT-PCR检测CMVRNA4a,发现Y-Sat在削弱2b对miR168诱导的同时,编码2b蛋白CMV RNA4a的量有轻微的减少,导致miR168表达量减少的原因部分可能是由于Y-Sat共同感染时2b量的减少。
7. Y-Sat显著的抑制了P1/HcPro对miR168的诱导。本文应用了由烟草蚀纹病毒(Tobacco etch virus, TEV)编码的VSR P1/HcPro转基因普通烟(N. tabacum)体系对miR168的表达量进行了检测分析。实验结果显示,在P1/HcPro转基因普通烟中,miR168被明显的诱导,同时出现茎基部弯曲生长的表现型；在Q-CMV+Y-Sat共同感染转基因普通烟中,miR168的表达水平显著降低,茎基部弯曲生长的表现型基本消失。实验结果说明,植物病毒VSR通过影响植物内源的miRNA代谢途径而致病,而卫星RNA通过干扰VSR,削弱病毒的致病性,减轻病毒诱导的症状。
以上7个结论,从小RNA的水平验证了植物病毒卫星RNA弱化辅助病毒致病机理的假说,证明了1)病毒通过其产生的VSR干扰寄主植物的miRNA代谢途径,从而致病；2)卫星RNA通过其产生的巨量siRNA捕获并饱和大部分的VSR,减少VSR干扰寄主植物的miRNA代谢途径,减轻病毒对寄主植物诱导的症状。
卫星RNA主要存在于植物病毒,DI RNA主要存在于动物病毒。假说在植物和病毒卫星RNA上面得到了部分验证,作者推断假说还适用于动物和病毒DI RNA。部分动物病毒编码的VSR,也具有结合小RNA的特征,动物病毒的DI RNA,在复制过程可能也会产生大量的siRNA,饱和VSR,干扰VSR的致病性。
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.
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