Characterization of the recombinant forms arising from a Potato virus X chimeric virus infection under RNA silencing pressure

Recombination is a frequent phenomenon in RNA viruses whose net result is largely influenced by selective pressures. RNA silencing in plants acts as a defense mechanism against viruses and can be used to engineer virus resistance. Here, we have investigated the influence of RNA silencing as a selective pressure to favor recombinants of PVX-HCT, a chimeric Potato virus X (PVX) vector carrying the helper-component proteinase (HC-Pro) gene from Plum pox virus (PPV). All the plants from two lines expressing a silenced HC-Pro transgene were completely resistant to PPV. However a significant proportion became infected with PVX-HCT. Analysis of viral RNAs accumulating in silenced plants revealed that PVX-HCT escaped silencing-based resistance by removal of the HC-Pro sequences that represented preferential targets for transgene-promoted silencing. The virus vector also tended to lose the HC-Pro insert when infecting transgenic plants containing a nonsilenced HC-Pro transgene or wild-type (wt) Nicotiana benthamiana plants. Nevertheless, loss of HC-Pro sequences was faster in nonsilenced transgenic plants than in wt plants, suggesting the transgene plays a role in promoting a higher selective pressure in favor of recombinant virus versions. These results indicate that the outcome of recombination processes depends on the strength of selection pressures applied to the virus.     

Tomato chlorotic mottle virus is a target of RNA silencing but the presence of specific short interfering RNAs does not guarantee resistance in transgenic plants

Tomato chlorotic mottle virus (ToCMoV) is a begomovirus found widespread in tomato fields in Brazil. ToCMoV isolate BA-Se1 (ToCMoV-[BA-Se1]) was shown to trigger the plant RNA silencing surveillance in different host plants and, coinciding with a decrease in viral DNA levels, small interfering RNAs (siRNAs) specific to ToCMoV-[BA-Se1] accumulated in infected plants. Although not homogeneously distributed, the siRNA population in both infected Nicotiana benthamiana and tomato plants represented the entire DNA-A and DNA-B genomes. We determined that in N. benthamiana, the primary targets corresponded to the 5' end of AC1 and the embedded AC4, the intergenic region and 5' end of AV1 and overlapping central part of AC5. Subsequently, transgenic N. benthamiana plants were generated that were preprogrammed to express double-stranded RNA corresponding to this most targeted portion of the virus genome by using an intron-hairpin construct. These plants were shown to indeed produce ToCMoV-specific siRNAs. When challenge inoculated, most transgenic lines showed significant delays in symptom development, and two lines had immune plants. Interestingly, the levels of transgene-produced siRNAs were similar in resistant and susceptible siblings of the same line. This indicates that, in contrast to RNA viruses, the mere presence of transgene siRNAs corresponding to DNA virus sequences does not guarantee virus resistance and that other factors may play a role in determining RNA-mediated resistance to DNA viruses.     

Transgenic <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> plants resistant to cucumber green mottle mosaic virus based on RNA silencing

RNA silencing technology was used to confer resistance to cucumber green mottle mosaic virus (CGMMV). Nicotiana benthamiana was transformed with a transgene designed to produce an inverted repeat RNA containing CGMMV-coat protein gene (CP) sequences, which were separated by an intron sequence, under the control of the cauliflower mosaic virus 35S promoter. We attempted to confirm the resistance of seven independent transgenic lines; five lines showed resistance to CGMMV infection. The systemic spread of virus was prevented after the inoculation of CGMMV, and the CP-specific short interfering RNA (siRNA) was detected in resistant lines. Thus, the resistance against CGMMV through RNA silencing is strong and efficient.     

Silencing of Plum pox virus 5′UTR/P1 sequence confers resistance to a wide range of PPV strains

An effective disease-control strategy should protect the host from the major economically important and geographically widespread variants of a pathogen. Plum pox virus (PPV) is the causal agent of sharka, the most devastating viral disease of Prunus species. We have shown previously that the hairpin RNA expression driven by h-UTR/P1, h-P1/HCPro, h-HCPro and h-HCPro/P3 constructs, derived from the PPV-M ISPaVe44 isolate, confers resistance to the homologous virus in Nicotiana benthamiana plants. Since the production of transgenic stone fruits and their evaluation for PPV resistance would take several years, the ISPaVe44-resistant plant lines were used to evaluate which construct would be the best candidate to be transferred to Prunus elite cultivars. To do that, nine PPV isolates of the D, M, Rec, EA and C strains originally collected from five Prunus species in different geographical areas, were typed by sequencing and used to challenge the transgenic N. benthamiana lines; 464 out of 464 virus-inoculated plants of lines h-UTR/P1, h-HCPro and h-HCPro/P3 showed complete and long-lasting resistance to the seven PPV isolates of D, M and Rec strains. Moreover, the h-UTR/P1 plants were also fully resistant to PPV-C and -EA isolates. Our data suggest that the h-UTR/P1 construct is of particular practical interest to obtain stone fruit plants resistant to the sharka disease.     

Plum pox potyvirus resistance associated to transgene silencing that can be stabilized after different number of plant generations

Nicotiana benthamiana plants were transformed with a fragment of the plum pox potyvirus (PPV) genome that encodes the nuclear inclusion a (NIa) and b (NIb) proteins and the N-terminus of the capsid protein (NIa–NIb–CP*). Lines transformed with this PPV genomic fragment harboring mutations in the GDD replicase-motif were also obtained. Plants of NIaΔV lines that carry a GDD to VDD mutation in the PPV transgene, were immune to PPV infection. The resistance was highly specific, since it was only partially overcome by a PPV strain different to that from which the transgene was derived, and no resistance was observed after inoculation with a second potyvirus. PPV was not able to replicate in protoplasts isolated from NIaΔV transgenic plants, indicating that the resistance was functional at the single cell level. Only a fraction of plants from lines transformed with the NIa–NIb–CP* fragment harboring a GDD to ADD mutation (NIaΔA lines), were resistant to PPV infection. This same phenotype was observed in plants expressing the wild-type construction (NIaΔ), although the progeny of some non-infected plants seemed to be completely resistant to PPV, independently of the allelic status of the parental plant. In all cases, the resistance phenotype correlated positively with low levels of transgene mRNA accumulation, suggesting that it was mainly due to a gene silencing mechanism. Our results show that, although the transgene was not silenced in all R1 plants from some individual lines, a stable silenced status could be reached in the following generations.     

CMV抑制PVYN CP基因沉默及其介导的抗病性

以前曾报道用RNA介导的抗病毒策略,获得了高度抗病的表达马铃薯Y病毒坏死株系外壳蛋白基因(PVY^N CP)的转基因烟草,并对T1、T2代转基因植株进行了遗传和抗病性分析。此次以T,代转基因植株为试验材料,在筛选高度抗病植株并证明其抗病性是基于转基因沉默的基础上,采用Northern杂交的方法,证明CMV侵染抑制了转基因植株中PVY^N CP基因的沉默,而且CMV对PVY^N CP基因沉默的抑制部位是发生在接种后的新生叶上,接种叶及其下部叶片中PVY^N CP基因沉默则未受到影响。采用ELISA方法对CMV PVY^N复合接种的转基因植株进行PVY^N检测,结果表明,接种叶及下部叶没有检测到PVY^N,植株叶片对PVY^N表现为抗病。而在CMV接种后植株新生叶中则检测出了高滴度的PVY^N,植株叶片对PVY^N表现为感病。该文报道了在表达PVY^N CP基因的RNA介导抗性转基因植株中,异源病毒侵染抑制了转基因的沉默,并导致转基因植株的抗病性丧失。     

Evidence that RNA silencing-mediated resistance to beet necrotic yellow vein virus is less effective in roots than in leaves

In plants, RNA silencing is part of a defense mechanism against virus infection but there is little information as to whether RNA silencing-mediated resistance functions similarly in roots and leaves. We have obtained transgenic Nicotiana benthamiana plants encoding the coat protein readthrough domain open reading frame (54 kDa) of Beet necrotic yellow vein virus (BNYVV), which either showed a highly resistant or a recovery phenotype following foliar rub-inoculation with BNYVV. These phenotypes were associated with an RNA silencing mechanism. Roots of the resistant plants that were immune to foliar rub-inoculation with BNYVV could be infected by viruliferous zoospores of the vector fungus Polymyxa betae, although virus multiplication was greatly limited. In addition, virus titer was reduced in symptomless leaves of the plants showing the recovery phenotype, but it was high in roots of the same plants. Compared with leaves of silenced plants, higher levels of transgene mRNAs and lower levels of transgene-derived small interfering RNAs (siRNAs) accumulated in roots. Similarly, in nontransgenic plants inoculated with BNYVV, accumulation level of viral RNA-derived siRNAs in roots was lower than in leaves. These results indicate that the RNA silencing-mediated resistance to BNYVV is less effective in roots than in leaves.     

The Cucumber vein yellowing virus silencing suppressor P1b can functionally replace HCPro in Plum pox virus infection in a host-specific manner

Plant viruses of the genera Potyvirus and Ipomovirus (Potyviridae family) use unrelated RNA silencing suppressors (RSS) to counteract antiviral RNA silencing responses. HCPro is the RSS of Potyvirus spp., and its activity is enhanced by the upstream P1 protein. Distinctively, the ipomovirus Cucumber vein yellowing virus (CVYV) lacks HCPro but contains two P1 copies in tandem (P1aP1b), the second of which functions as RSS. Using chimeras based on the potyvirus Plum pox virus (PPV), we found that P1b can functionally replace HCPro in potyviral infections of Nicotiana plants. Interestingly, P1a, the CVYV protein homologous to potyviral P1, disrupted the silencing suppression activity of P1b and reduced the infection efficiency of PPV in Nicotiana benthamiana. Testing the influence of RSS in host specificity, we found that a P1b-expressing chimera poorly infected PPV's natural host, Prunus persica. Conversely, P1b conferred on PPV chimeras the ability to replicate locally in cucumber, CVYV's natural host. The deleterious effect of P1a on PPV infection is host dependent, because the P1aP1b-expressing PPV chimera accumulated in cucumber to higher levels than PPV expressing P1b alone. These results demonstrate that a potyvirus can use different RSS, and that particular RSS and upstream P1-like proteins contribute to defining the virus host range.     

Long-distance movement,virulence, and RNA silencing suppression controlled by a single protein in hordei- and potyviruses: complementary functions between virus families

RNA silencing is a natural defense mechanism against genetic stress factors, including viruses. A mutant hordeivirus (Barley stripe mosaic virus [BSMV]) lacking the gammab gene was confined to inoculated leaves in Nicotiana benthamiana, but systemic infection was observed in transgenic N. benthamiana expressing the potyviral silencing suppressor protein HCpro, suggesting that the gammab protein may be a long-distance movement factor and have antisilencing activity. This was shown for gammab proteins of both BSMV and Poa semilatent virus (PSLV), a related hordeivirus. Besides the functions in RNA silencing suppression, gammab and HCpro had analogous effects on symptoms induced by the hordeiviruses. Severe BSMV-induced symptoms were correlated with high HCpro concentrations in the HCpro-transgenic plants, and substitution of the gammab cistron of BSMV with that of PSLV led to greatly increased symptom severity and an altered pattern of viral gene expression. The efficient systemic infection with the chimera was followed by the development of dark green islands (localized recovery from infection) in leaves and exemption of new developing leaves from infection. Recovery and the accumulation of short RNAs diagnostic of RNA silencing in the recovered tissues in wild-type N. benthamiana were suppressed in HCpro-transgenic plants. These results provide evidence that potyviral HCpro and hordeivirus gammab proteins contribute to systemic viral infection, symptom severity, and RNA silencing suppression. HCpro's ability to suppress the recovery of plants from viral infection emphasizes recovery as a manifestation of RNA silencing.     

Targeting of genomic and negative‐sense strands of viral RNA contributes to antiviral resistance mediated by artificial miRNAs and promotes the emergence of complex viral populations

Technology based on artificial small RNAs, including artificial microRNAs (amiRNAs), exploits natural RNA silencing mechanisms to achieve silencing of endogenous genes or pathogens. This technology has been successfully employed to generate resistance against different eukaryotic viruses. However, information about viral RNA molecules effectively targeted by these small RNAs is rather conflicting, and factors contributing to the selection of virus mutants escaping the antiviral activity of virus‐specific small RNAs have not been studied in detail. In this work, we transformed Nicotiana benthamiana plants with amiRNA constructs designed against the potyvirus plum pox virus (PPV), a positive‐sense RNA virus, and obtained lines highly resistant to PPV infection and others showing partial resistance. These lines have allowed us to verify that amiRNA directed against genomic RNA is more efficient than amiRNA targeting its complementary strand. However, we also provide evidence that the negative‐sense RNA strand is cleaved by the amiRNA‐guided RNA silencing machinery. Our results show that the selection pressure posed by the amiRNA action on both viral RNA strands causes an evolutionary explosion that results in the emergence of a broad range of virus variants, which can further expand in the presence, and even in the absence, of antiviral challenges.     

假马鞭曲叶病毒和中国胜红蓟黄脉病毒C4蛋白是RNA沉默的抑制子

为了研究中国胜红蓟黄脉病毒(Ageratum yellow vein Chin virus,AYVCNV)和假马鞭曲叶病毒(Stachytarpheta leafcurl virus,StaLCV)C4蛋白的功能,利用烟草脆裂病毒(Tobacco rattle virus,TRV)载体在本氏烟(Nicotianabenthamiana)中分别表达了这两种病毒的C4蛋白,结果发现它们均能在本氏烟中引起类似于病毒侵染的症状,推测AYVCNV和StaLCV的C4蛋白是病毒的致病因子;在RNA沉默的抑制试验中,AYVCNV和StaLCV的C4蛋白均能够在表达gfp基因的转基因本氏烟(16c)上抑制由gfp基因正义链引起的基因沉默的建立,证明它们都是RNA沉默的抑制子。