Effects of turnip mosaic virus and turnip yellow mosaic virus on the survival, growth and reproduction of wild cabbage (Brassica oleracea)

Wild plants of Brassica oleracea (wild cabbage) are commonly infected with turnip mosaic poty virus (TuMV), turnip yellow mosaic tymovirus (TYMV) and several other viruses. A field experiment in which plants were inoculated either with TuMV or TYMV showed that virus infection significantly reduced survival, growth and reproduction. Relative to water inoculated-controls, plants infected with TYMV had greater mortality, were shorter, had a smaller leaf area and number, showed a greater amount of damage from herbivory and chlorosis, were less likely to flower and produced fewer pods and lower total seed output. Plants infected with TuMV did not appear to be adversely affected at first; however, mortality after 18 months was higher than control plants. Although TuMV infection had no effect on the number of plants flowering, the infected plants did produce fewer pods and a lower total seed output. We conclude that both viruses can significantly affect vegetative and reproductive performance of wild cabbage and hence that introgression of virus resistance (particularly when conferred by a major gene or a transgene) from a crop might increase plant fitness in natural populations of this species. Ecological risk assessments of virus resistance transgenes must do more than survey adult plants in natural populations for the presence of the target virus. Failure to detect the virus could be due to high mortality on infection with the virus.     

The prevalence and spatial distribution of viruses in natural populations of Brassica oleracea

We report a survey of four viruses (beet western yellows luteovirus (BWYV), cauliflower mosaic caulimovirus (CaMV), turnip mosaic potyvirus (TuMV), turnip yellow mosaic tymovirus (TYMV)) in five natural populations of Brassica oleracea in Dorset (UK). All four viruses were common; 43% of plants were infected with BWYV, 60% with CaMV, 43% with TuMV and 18% with TYMV. For each virus there were significant differences in the proportion of infected plants among populations, which were not completely explained by differences in the age of plants. Multiple virus infections were prevalent, with 54% of plants having two or more virus types. There were statistically significant associations between pairs of viruses. The CaMV was positively associated with the other three viruses, and BWYV was also positively associated with TuMV. There was no detectable association between BWYV and TYMV, whereas TuMV and TYMV were negatively associated. We suggest these associations result from BWYV, CaMV and TuMV having aphid vectors in common, as aphids are attracted to plants that already have a virus infection. Infected plants were distributed randomly or were very weakly aggregated within populations. The implications of widespread multiple virus infections in natural plant populations are discussed with respect to the release of transgenic plants expressing virus-derived genes.     

Multiple artificial microRNAs targeting conserved motifs of the replicase gene confer robust transgenic resistance to negative-sense single-stranded RNA plant virus

MicroRNAs (miRNAs) regulate the abundance of target mRNAs by guiding cleavage at sequence complementary regions. In this study, artificial miRNAs (amiRNAs) targeting conserved motifs of the L (replicase) gene of Watermelon silver mottle virus (WSMoV) were constructed using Arabidopsis pre-miRNA159a as the backbone. The constructs included six single amiRNAs targeting motifs A, B1, B2, C, D of E, and two triple amiRNAs targeting motifs AB1E or B2DC. Processing of pre-amiRNAs was confirmed by agro-infiltration, and transgenic Nicotiana benthamiana plants expressing each amiRNA were generated. Single amiRNA transgenic lines expressing amiR-LB2 or amiR-LD showed resistance to WSMoV by delaying symptom development. Triple amiRNA lines expressing amiR-LB2, amiR-LD and amiR-LC provided complete resistance against WSMoV, with no indication of infection 28 days after inoculation. Resistance levels were positively correlated with amiRNA expression levels in these single and triple amiRNA lines. The triple amiR-LAB1E line did not provide resistance to WSMoV. Similarly, the poorly expressed amiR-LC and amiR-LE lines did not provide resistance to WSMoV. The amiR-LA- and amiR-LB1-expressing lines were susceptible to WSMoV, and their additional susceptibility to the heterologous Turnip mosaic virus harbouring individual target sequences indicated that these two amiRNAs have no effect in vivo. Transgenic lines expressing amiR-LB2 exhibited delayed symptoms after challenge with Peanut bud necrosis virus having a single mismatch in the target site. Overall, our results indicate that two amiRNAs, amiR-LB2 and amiR-LD, of the six designed amiRNAs confer moderate resistance against WSMoV, and the triple construct including the two amiRNAs provides complete resistance.     

Resistance of transformed and non-transformed oilseed rape cv. HM-81 to the infection with cauliflower mosaic,turnip yellow mosaic and turnip mosaic viruses

Resistance of transformed and non-transformed spring oilseed rape cv. HM-81 to the infection with cauliflower mosaic virus (CaMV), turnip yellow mosaic virus (TYMV) and turnip mosaic virus (TuMV) was studied, to determine the influence of transformation on susceptibility of plants to viruses. For experiments the non-segregating R 1 generation of primary transformant HM-81-JZ and control plants of cv. HM-81 were used. The primary transformant was obtained by inoculation of stems withAgrobacterium rhizogenes 15834. All transformed plants of R 1 generation had typically „transformed“ phenotype. No significant differences were revealed in the resistance of both transformed and non-transformed plants to each virus, as proved by qualitative and quantitative ELISA and visual evaluation of symptoms. Transformed plants infected with turnip yellow mosaic virus showed significantly lower reduction of green mass yield than non-transformed. In the case of CaMV and TuMV infection reduction of yield of transformed and non-transformed plants was almost the same.     

Artificial MicroRNAs highly accessible to targets confer efficient virus resistance in plants

Short-hairpin RNAs based on microRNA (miRNA) precursors to express the artificial miRNAs (amiRNAs) can specifically induce gene silencing and confer virus resistance in plants. The efficacy of RNA silencing depends not only on the nature of amiRNAs but also on the local structures of the target mRNAs. However, the lack of tools to accurately and reliably predict secondary structures within long RNAs makes it very hard to predict the secondary structures of a viral genome RNA in the natural infection conditions in vivo. In this study, we used an experimental approach to dissect how the endogenous silencing machinery acts on the 3′ untranslated region (UTR) of the Cucumber mosaic virus (CMV) genome. Transiently expressed 3′UTR RNAs were degraded by site-specific cleavage. By comparing the natural cleavage hotspots within the 3′UTR of the CMV-infected wild-type Arabidopsis to those of the triple dcl2/3/4 mutant, we acquired true small RNA programmed RNA-induced silencing complex (siRISC)-mediated cleavage sites to design valid amiRNAs. We showed that the tRNA-like structure within the 3′UTR impeded target site access and restricted amiRNA-RISC-mediated cleavage of the target viral RNA. Moreover, target recognition in the less-structured area also influenced siRISC catalysis, thereby conferring different degrees of resistance to CMV infection. Transgenic plants expressing the designed amiRNAs that target the putative RISC accessible target sites conferred high resistance to the CMV challenge from both CMV subgroup strains. Our work suggests that the experimental approach is credible for studying the course of RISC target recognition to engineer effective gene silencing and virus resistance in plants by amiRNAs.     

Viral virulence protein suppresses RNA silencing-mediated defense but upregulates the role of microrna in host gene expression

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are processed by the ribonuclease Dicer from distinct precursors, double-stranded RNA (dsRNA) and hairpin RNAs, respectively, although either may guide RNA silencing via a similar complex. The siRNA pathway is antiviral, whereas an emerging role for miRNAs is in the control of development. Here, we describe a virulence factor encoded by turnip yellow mosaic virus, p69, which suppresses the siRNA pathway but promotes the miRNA pathway in Arabidopsis thaliana. p69 suppression of the siRNA pathway is upstream of dsRNA and is as effective as genetic mutations in A. thaliana genes involved in dsRNA production. Possibly as a consequence of p69 suppression, p69-expressing plants contained elevated levels of a Dicer mRNA and of miRNAs as well as a correspondingly enhanced miRNA-guided cleavage of two host mRNAs. Because p69-expressing plants exhibited disease-like symptoms in the absence of viral infection, our findings suggest a novel mechanism for viral virulence by promoting the miRNA-guided inhibition of host gene expression.     

Viruses infecting winter oilseed rape (Brassica napus ssp. oleifera)

Turnip mosaic virus (TuMV) and cauliflower mosaic virus (CaMV) have been found infecting field crops of winter oilseed rape (Brassica napus ssp. oleifera) in South Warwickshire. Other viruses found include broccoli necrotic yellows virus (BNYV) and a member of the beet western yellows virus group. Systemic leaf symptoms caused by TuMV varied within and between cultivars; the three predominant reaction types were classified as necrotic, mosaic and immune. Some recently introduced cultivars of oilseed rape were more severely affected by TuMV infection than older cultivars. Reactions to CaMV were less varied and immunity was not found. The seed yield from TuMV and CaMV-infected plants was less than that of healthy control plants. This effect was due to infected plants producing either fewer seeds, smaller seeds or both. Germination of seeds from infected plants was unaffected if sown soon after harvest. After storage for one year the germination of seed from a virus infected plant was significantly less than that of seed from a virus-free plant. All commercial cultivars tested were experimentally susceptible to turnip yellow mosaic virus (TYMV) and some American strains of cucumber mosaic virus (CMV).     

MicroRNAs with analogous target complementarities perform with highly variable efficacies in Arabidopsis

In plants, the silencing efficacy of microRNAs (miRNAs) is thought to be predominantly determined by the degree of complementarity to their target genes. Here, silencing efficacy was determined for Arabidopsis miR159 and four artificial miRNAs (amiRNAs) that all target MYB33/MYB65 with analogous complementarities. As determined through complementation of a loss-of-function mir159 mutant, the amiRNAs displayed highly variable efficacies, none of which was as strong as endogenous miR159. This was despite amiRNA expression levels being many fold-higher than miR159 in wild-type. The results highlight the variable nature of miRNA silencing efficacy in plants, where it appears that factors additional to complementarity strongly impact silencing.     

萝卜优异种质对芜菁花叶病毒抗性的遗传分析

为了深入认识萝卜优异种质对芜菁花叶病毒(TuMV)的抗性遗传机制,本研究以高抗TuMV的自交系Q07-12(P_1)和高感自交系KB07-10(P_2)为亲本,构建了包括P_1、P_2、F_1、B_1、B_2及F_2的6个群体,采用数量性状主基因+多基因混合遗传的六世代联合分析法,对其抗性遗传规律进行了研究。结果表明,萝卜对TuMV的抗性受两对主基因控制,并符合"加性-显性-上位性主基因+加性-显性-上位性多基因"模型,其中主基因遗传率在B_1、B_2及F_2群体中分别为89%、55%和95%,多基因的遗传率分别为0、40.9%和0,环境因素对抗性强弱的影响较小。此结果表明了该优异种质在萝卜抗病毒遗传改良中的利用价值,为萝卜抗病育种和抗病基因的挖掘提供了理论依据。     

Selection for immunity in swede (Brassica napus) to infection by turnip mosaic virus

Fully heritable immunity to turnip mosaic virus (TuMV) was found in swede (Brassica napus) cvs Ruta Otofte and Bangholm. Attempts to ‘break’ this immunity were unsuccessful. The reactions of two immune lines of cv. Ruta Otofte to cauliflower mosaic virus, clubroot (Plasmodiophora brassicae) and powdery mildew (Erysiphe cruciferarum) were unchanged compared with the unselected parent line. A symptomless response to TuMV inoculation (possibly immunity) was also found in cv. Acme but not in the clubroot resistant cv. Marian. The percentage of plants which gave a symptomless response to TuMV inoculation differed greatly between cultivars and some seed stocks.     

Artificial microRNA-mediated virus resistance in plants

RNA silencing in plants is a natural defense system against foreign genetic elements including viruses. This natural antiviral mechanism has been adopted to develop virus-resistant plants through expression of virus-derived double-stranded RNAs or hairpin RNAs, which in turn are processed into small interfering RNAs (siRNAs) by the host's RNA silencing machinery. While these virus-specific siRNAs were shown to be a hallmark of the acquired virus resistance, the functionality of another set of the RNA silencing-related small RNAs, microRNAs (miRNAs), in engineering plant virus resistance has not been extensively explored. Here we show that expression of an artificial miRNA, targeting sequences encoding the silencing suppressor 2b of Cucumber mosaic virus (CMV), can efficiently inhibit 2b gene expression and protein suppressor function in transient expression assays and confer on transgenic tobacco plants effective resistance to CMV infection. Moreover, the resistance level conferred by the transgenic miRNA is well correlated to the miRNA expression level. Comparison of the anti-CMV effect of the artificial miRNA to that of a short hairpin RNA-derived small RNA targeting the same site revealed that the miRNA approach is superior to the approach using short hairpin RNA both in transient assays and in transgenic plants. Together, our data demonstrate that expression of virus-specific artificial miRNAs is an effective and predictable new approach to engineering resistance to CMV and, possibly, to other plant viruses as well.     

The poly(A) binding protein is internalized in virus-induced vesicles or redistributed to the nucleolus during turnip mosaic virus infection

Poly(A) binding protein 2 (PABP2) of Arabidopsis thaliana was previously shown to interact with VPg-Pro of turnip mosaic virus (TuMV) and may consequently play an important role during infection. Subcellular fractionation experiments revealed that PABP2 was predominantly a cytoplasmic soluble protein in healthy plants. However, in TuMV-infected plants, a subpopulation of PABP2 was membrane associated or was localized in the nucleus. Confocal microscopy experiments indicated that PABP2 was partially retargeted to the nucleolus in the presence of TuMV VPg-Pro. In addition, the membrane association of PABP2 during TuMV infection resulted from the internalization of the host protein in 6K-VPg-Pro-induced vesicles, as shown by a combination of confocal microscopy and sucrose gradient fractionation experiments. This redistribution of an important translation initiation factor to the nucleolus and to membrane structure likely underlies two important processes of the TuMV replication cycle.     

Molecular Evolution of a Viral Non-Coding Sequence under the Selective Pressure of amiRNA-Mediated Silencing

Plant microRNAs (miRNA) guide cleavage of target mRNAs by DICER-like proteins, thereby reducing mRNA abundance. Native precursor miRNAs can be redesigned to target RNAs of interest, and one application of such artificial microRNA (amiRNA) technology is to generate plants resistant to pathogenic viruses. Transgenic Arabidopsis plants expressing amiRNAs designed to target the genome of two unrelated viruses were resistant, in a highly specific manner, to the appropriate virus. Here, we pursued two different goals. First, we confirmed that the 21-nt target site of viral RNAs is both necessary and sufficient for resistance. Second, we studied the evolutionary stability of amiRNA-mediated resistance against a genetically plastic RNA virus, TuMV. To dissociate selective pressures acting upon protein function from those acting at the RNA level, we constructed a chimeric TuMV harboring a 21-nt, amiRNA target site in a non-essential region. In the first set of experiments designed to assess the likelihood of resistance breakdown, we explored the effect of single nucleotide mutation within the target 21-nt on the ability of mutant viruses to successfully infect amiRNA-expressing plants. We found non-equivalency of the target nucleotides, which can be divided into three categories depending on their impact in virus pathogenicity. In the second set of experiments, we investigated the evolution of the virus mutants in amiRNA-expressing plants. The most common outcome was the deletion of the target. However, when the 21-nt target was retained, viruses accumulated additional substitutions on it, further reducing the binding/cleavage ability of the amiRNA. The pattern of substitutions within the viral target was largely dominated by G to A and C to U transitions.     

A Turnip yellow mosaic virus infection system in Arabidopsis suspension cell culture

Turnip yellow mosaic virus (TYMV) is a positive-strand RNA virus able to infect Arabidopsis thaliana. To establish a TYMV infection system in Arabidopsis cell culture, TYMV replicons with the capsid protein gene replaced by a reporter gene expressing the Sh ble protein conferring zeocin resistance were used to transfect Arabidopsis cells. Zeocin-resistant Arabidopsis calli were used to generate a suspension cell culture. Detection of viral proteins and RNAs after 18 months in culture demonstrated persistent replication of the replicon. The Arabidopsis cell culture yielded soluble, active replication complexes, providing a useful tool to study host factors involved in TYMV replication.     

Tempo and mode of plant RNA virus escape from RNA interference-mediated resistance

A biotechnological application of artificial microRNAs (amiRs) is the generation of plants that are resistant to virus infection. This resistance has proven to be highly effective and sequence specific. However, before these transgenic plants can be deployed in the field, it is important to evaluate the likelihood of the emergence of resistance-breaking mutants. Two issues are of particular interest: (i) whether such mutants can arise in nontransgenic plants that may act as reservoirs and (ii) whether a suboptimal expression level of the transgene, resulting in subinhibitory concentrations of the amiR, would favor the emergence of escape mutants. To address the first issue, we experimentally evolved independent lineages of Turnip mosaic virus (TuMV) (family Potyviridae) in fully susceptible wild-type Arabidopsis thaliana plants and then simulated the spillover of the evolving virus to fully resistant A. thaliana transgenic plants. To address the second issue, the evolution phase took place with transgenic plants that expressed the amiR at subinhibitory concentrations. Our results show that TuMV populations replicating in susceptible hosts accumulated resistance-breaking alleles that resulted in the overcoming of the resistance of fully resistant plants. The rate at which resistance was broken was 7 times higher for TuMV populations that experienced subinhibitory concentrations of the antiviral amiR. A molecular characterization of escape alleles showed that they all contained at least one nucleotide substitution in the target sequence, generally a transition of the G-to-A and C-to-U types, with many instances of convergent molecular evolution. To better understand the viral population dynamics taking place within each host, as well as to evaluate relevant population genetic parameters, we performed in silico simulations of the experiments. Together, our results contribute to the rational management of amiR-based antiviral resistance in plants.     

NbALD1 mediates resistance to turnip mosaic virus by regulating the accumulation of salicylic acid and the ethylene pathway in Nicotiana benthamiana

AGD2-LIKE DEFENCE RESPONSE PROTEIN 1 (ALD1) triggers plant defence against bacterial and fungal pathogens by regulating the salicylic acid (SA) pathway and an unknown SA-independent pathway. We now show that Nicotiana benthamiana ALD1 is involved in defence against a virus and that the ethylene pathway also participates in ALD1-mediated resistance. NbALD1 was up-regulated in plants infected with turnip mosaic virus (TuMV). Silencing of NbALD1 facilitated TuMV infection, while overexpression of NbALD1 or exogenous application of pipecolic acid (Pip), the downstream product of ALD1, enhanced resistance to TuMV. The SA content was lower in NbALD1-silenced plants and higher where NbALD1 was overexpressed or following Pip treatments. SA mediated resistance to TuMV and was required for NbALD1-mediated resistance. However, on NahG plants (in which SA cannot accumulate), Pip treatment still alleviated susceptibility to TuMV, further demonstrating the presence of an SA-independent resistance pathway. The ethylene precursor, 1-aminocyclopropanecarboxylic acid (ACC), accumulated in NbALD1-silenced plants but was reduced in plants overexpressing NbALD1 or treated with Pip. Silencing of ACS1, a key gene in the ethylene pathway, alleviated the susceptibility of NbALD1-silenced plants to TuMV, while exogenous application of ACC compromised the resistance of Pip-treated or NbALD1 transgenic plants. The results indicate that NbALD1 mediates resistance to TuMV by positively regulating the resistant SA pathway and negatively regulating the susceptible ethylene pathway.     

A role for inositol hexakisphosphate in the maintenance of basal resistance to plant pathogens

Phytic acid (myo-inositol hexakisphosphate, InsP6) is an important phosphate store and signal molecule in plants. However, low-phytate plants are being developed to minimize the negative health effects of dietary InsP6 and pollution caused by undigested InsP6 in animal waste. InsP6 levels were diminished in transgenic potato plants constitutively expressing an antisense gene sequence for myo-inositol 3-phosphate synthase (IPS, catalysing the first step in InsP6 biosynthesis) or Escherichia coli polyphosphate kinase. These plants were less resistant to the avirulent pathogen potato virus Y and the virulent pathogen tobacco mosaic virus (TMV). In Arabidopsis thaliana, mutation of the gene for the enzyme catalysing the final step of InsP6 biosynthesis (InsP5 2-kinase) also diminished InsP6 levels and enhanced susceptibility to TMV and to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae. Arabidopsis thaliana has three IPS genes (AtIPS1-3). Mutant atips2 plants were depleted in InsP6 and were hypersusceptible to TMV, turnip mosaic virus, cucumber mosaic virus and cauliflower mosaic virus as well as to the fungus Botrytis cinerea and to P. syringae. Mutant atips2 and atipk1 plants were as hypersusceptible to infection as plants unable to accumulate salicylic acid (SA) but their increased susceptibility was not due to reduced levels of SA. In contrast, mutant atips1 plants, which were also depleted in InsP6, were not compromised in resistance to pathogens, suggesting that a specific pool of InsP6 regulates defence against phytopathogens.