RNA silencing against viruses: molecular arms race between Cucumber mosaic virus and its host

Plants have developed RNA silencing as an antiviral defense mechanism. To escape from the plant host’s defenses, viruses have countered their host’s antiviral silencing by producing RNA silencing suppressor proteins (RSSs). Although the mode of action of the majority of viral RSSs has been found to be through double-stranded RNA-binding, viruses have different strategies to counteract the host’s antiviral silencing pathways. The 2b protein of Cucumber mosaic virus, which is one of the most extensively studied viral RSSs, is reviewed here to provide insights on the molecular arms race between viruses and their host plants.     

Enhancement of resistance to soft rot (Pectobacterium carotovorum subsp. carotovorum) in transgenic Brassica rapa

Brassica rapa (chinese cabbage) is one of the main vegetable crops grown in Asian countries. Pectobacterium carotovorum subsp. carotovorum causes severe economic loss in this crop as well as in other Brassica crops through soft rot disease. Cysteine proteases like bromelain, papain or ficin show toxic effects to herbivorous insects and pathogenic bacteria. They have been known to be critical factors in plant defence mechanisms. The current study investigated the effect of bromelain gene (BL1) of pineapple (Ananas comosus L. Merrill) on enhancing resistance to soft rot in transgenic Brassica rapa ‘Seoulbaechu’. Three homozygous T₂ lines were inoculated with Pectobacterium carotovorum subsp. carotovorum and BL8-2 line showed the lowest rate of infected leaves (RIL) in both wound inoculation and non-wound inoculation, when the non-infected line showed 100 % RIL in both cases. The highest expression of BL1 gene was also observed in BL8-2 homozygous line. Thus, the over-expressed BL1 gene conferred enhanced resistance to soft rot in Brassica rapa.     

Turnip crinkle virus with nonviral gene cancels the effect of silencing suppressors of P19 and 2b in Arabidopsis thaliana

In plants, green fluorescent protein (GFP) has become a preferred molecular marker for gene expression and cellular localization, and plant viral vectors are valuable tools for heterologous gene expression. Some plant viruses have been used for expression of GFP, and the activities of these viruses are barely affected by the extra GFP gene. In contrast, the packaging and the length of Turnip crinkle virus (TCV) genome is strictly limited when foreign genes are inserted into the coding sequences of TCV genome. In this report, we removed the silencing suppressor p38 from TCV, and constructed GFP derivatives of TCV. Then the resulting TCV mutants were used to infect Arabidopsis plants containing mutations in key silencing pathway genes, including triple dcl2/dcl3/dcl4, dcl2, dcl4 and ago mutant plants. Our results demonstrate that the activity of TCV is affected by nonviral GFP insert in Arabidopsis plants, and RNA silencing appears not play an important role. AGOs appear to be more efficient at slicing RNAs of viral origin, especially AGO2 and AGO7. Although the viral suppressors of RNA silencing (VSRs) P19 and 2b can enhance the accumulation of viral RNAs, neither P19 nor 2b can significantly increase the expression of TCV mutants with nonviral genes. TCV is an example of an RNA virus that is recalcitrant to add nonviral gene sequences.     

Effect of passage through the gut of Eocanthecona furcellata on Spodoptera litura multiple nucleopolyhedrovirus infectivity and its subsequent dissemination

Infectivity of Spodoptera litura multiple nucleopolyhedrovirus (SpltMNPV) was compared between before (P0) and after (P1–P4) passage in subsequent generations through the gut of Eocanthecona furcellata. Viable virus was detected in E. furcellata feces up to 6 days after feeding on infected S. litura larvae. NPV mortality ranged between 93% and 10% when test larvae were exposed to polyhedra voided in feces collected after 1 and 6 days post-infected meal, respectively. The mean number of polyhedral occlusion bodies (POBs) in excreta and their infectivity (%) at all passages did not vary significantly. The comparison of observed LD₅₀ and ST₅₀ values among all passages did not reveal significant differences owing to their overlapping confidence limits. The gut-passed virus did not show a detrimental effect on survival rate, longevity, fecundity and percent egg hatchability of E. furcellata in the subsequent three generations. A field trial was also conducted to estimate virus dissemination through feces of predators that were fed upon prey infected with polyhedra before passage, after passage and healthy (control) prey and subsequently released on cabbage plants. An additional viral mortality up to the magnitude of 13–17% was noticed in the former two treatments. However, within these two treatments the viral mortality did not vary significantly. It was concluded that E. furcellata disseminated the virus through their feces into the ecosystem without any adverse effect to it and infectivity of the SpltMNPV is not altered after passage through the gut of the predator.     

Exogenous Silicon Protects <Emphasis Type="Italic">Brassica napus</Emphasis> Plants from Salinity-Induced Oxidative Stress Through the Modulation of AsA-GSH Pathway,Thiol-Dependent Antioxidant Enzymes and Glyoxalase Systems

Although silicon (Si) has showed its potential role in mitigating abiotic stress-induced damages in many plant species its role in coordinated induction of antioxidant defense is yet to be elucidated. Therefore, we studied rapeseed (Brassica napus) seedlings applied with exogenous Si for changes occurring in antioxidant defense and glyoxalase systems. Seedlings (12-day-old) grown semi-hydroponically were exposed to Si (silicon dioxide, SiO₂; 1?mM) solely and in combination with NaCl (100 and 200?mM) for 48?h. Salinity created oxidative damage by increasing H₂O₂ and malondialdehyde (MDA) contents resulting in disruption of antioxidant defense system and in arousing methylglyoxal (MG) toxicity by the down-regulation of glyoxalase enzyme activities. Exogenous Si treatment showed reduction of both H₂O₂ and MDA contents and up-regulation of antioxidant components including the activities of related enzymes (APX, MDHAR, DHAR, GR, GST, GPX and CAT) and the contents of AsA and GSH. Enhanced activities of glyoxalase I (Gly I) and glyoxalase II (Gly II) detoxified the toxic MG. Thus, this study clearly indicates that Si improved plant tolerance to salinity stress through enhancement of both antioxidant defense and glyoxalase systems that led to reduced oxidative damage and MG toxicity.     

转录后基因沉默

 转录后基因沉默(PTGS)普遍存在于生物界,如植物的共抑制、源于病原的RNA介导的病毒抗性、真菌的基因沉默和动物的RNA干扰等。这类现象有许多共同特点,如都是以向细胞内引入核酸(转基因、双链RNA或病毒RNA)为诱因,依赖RNA的RNA聚合酶(RdRP)活性与基因沉默密切相关,在发生基因沉默的细胞中大多存在特定长度的小分子RNA (21~25 nt),PTGS导致细胞质内mRNA的特异性降解,不同生物的PTGS相关基因及产物具有很高的相似性,基因沉默能够在细胞间传播并能以表型遗传的方式传递给下一代等。这说明各种生物的转录后基因沉默可能有相似的遗传起源,是一种抵抗外来核酸(如病毒和转座子)入侵的共同的防御机制     

Development of an effective screening method for partial resistance to Alternaria brassicicola (dark leaf spot) in Brassica rapa

In order to develop a method to measure resistance to Alternaria brassicicola (cause of dark leaf spot disease) in Brassica rapa, the effects of inoculum concentration, leaf stage, leaf age and incubation temperature of inoculation on infection were studied under controlled conditions using several B. rapa genotypes. Three inoculation methods (cotyledon, detached leaf and seedling inoculation) were evaluated for this purpose. The detached leaf inoculation test was the most suitable for screening B. rapa genotypes because clear symptoms were observed on the leaves in less than 24 h, and there was a significant positive correlation between the results from the detached leaf inoculation test and the seedling inoculation test, an established method considered to yield reliable results. In addition, it was very easy to screen plants for resistance on a large scale and to maintain standard physical conditions using detached leaves. For successful infection, inoculum concentration should be adjusted to 5 × 10⁴ conidia ml⁻¹, and incubation temperature should be between 20 °C and 25 °C. The 3rd/4th true leaves from 30 day-old plants were optimal for inoculation. In a screening test using 52 cultivars of B. rapa, the detached leaf test effectively discriminated between various levels of partial resistance among cultivars. As a result, we identified two cultivars, viz Saori and Edononatsu, as highly resistant and five cultivars, viz Tokinashi Taisai, Yajima Kabu, Purara, Norin-F₁-Bekana and Tateiwa Kabu, as having borderline resistance.     

Using an Arabidopsis oligonucleotide microarray to analyze the secretory pathway genes' Response to TuMV infection in Brassica rapa

Previous evidence suggested the Turnip mosaic virus viral ribonuclear protein complex, comprising the viral RNA, moved to the plasmodesmata via the secretory pathway. We performed a global analysis of gene expression in Brassica rapa and identified a complex array of changes affecting pathogen response. Thirty-eight secretory pathway genes were identified. The quantitative real-time polymerase chain reaction results were consistent with microarray analysis. Combined with previous studies, we speculated that the secretory pathway participates in viral replication and movement. Exploring the precise function of the genes identified in this analysis will offer new insights into viral defense in B. rapa.     

Sensitivity of Rhizoctonia solani causing rice sheath blight to fluxapyroxad in China

Fluxapyroxad,3-(difluoromethyl)-1-methyl-N-(3’,4’,5’-trifluorobiphenyl-2-yl) pyrazole-4-carboxamide (C₁₈H₁₂F₅N₃O, Fig. 1), is a SDHI fungicide, which is a new active ingredient that interferes with succinate ubiquinone reductase in the electron transport chain of fungi. Between 2008 and 2010, a total of 128 isolates of Rhizoctonia solani from Anhui Province of China were characterized for the baseline sensitivity to fluxapyroxad. The isolates collected between 2008 and 2010 all showed similar sensitivity to fluxapyroxad. Baseline sensitivity was distributed as unimodal curves with an average EC₅₀ value of 0.054?±?0.014 μg ml^?1. However, EC₅₀ values of boscalid for inhibition of mycelial growth of R. solani isolates ranged from 1.89 to 2.68 μg/ml and the average (±SE) EC₅₀ value was 2.212?±?0.228 μg/ml, indicating that the R. solani isolates were less sensitive to boscalid than that of fluxapyroxad. Fluxapyroxad exhibited excellent protective and curative activity against rice sheath blight and provided 82.6–94.2 % protective or curative control efficacy. In field trials, control efficacy of fluxapyroxad at 100 g a.i/ha 15 days and 30 day after second application was 83.4–88.0 %, suggesting excellent activity against sheath blight. Control efficacy of boscalid at a dosage of 600 g a.i/ha 15 days and 30 day after second application was about 51.7–57.0 %. There was a significant difference in the efficacy between fluxapyroxad and boscalid or jinggangmycin. These results suggested that fluxapyroxad is a good alternative fungicide to jinggangmycin for the control of rice sheath blight.     

Accumulation of short interfering RNAs characteristic of RNA silencing precedes recovery of tomato plants from severe symptoms of <Emphasis Type="Italic">Potato spindle tuber viroid</Emphasis> infection

Tomato plants infected with Potato spindle tuber viroid (PSTVd) had severe leaf curling and vein necrosis. The disease symptoms began to diminish during the late stages of infection, however, and almost healthy-looking leaves began to appear on the upper portion of the plants. PSTVd concentrations reached their highest levels in leaves with severe symptoms and decreased in upper leaves recovering from severe symptoms. PSTVd-specific short interfering RNAs (siRNAs), characteristic of RNA silencing, accumulated in all leaves in which PSTVd reached a detectable level, suggesting that recovery from severe disease was induced by RNA silencing via sequence-specific degradation of PSTVd.     

Rice response to simultaneous bacterial blight and drought stress during compatible and incompatible interactions

Plant response to one type of stress can be affected by simultaneous exposure to a second stress, for example when abiotic and biotic stresses occur together. Ten rice genotypes comprising those with bacterial blight (BB) resistance (R) genes, drought quantitative trait loci (QTLs) plus a BB R gene, and BB susceptible genotypes, were subjected to mild and moderate drought stress and plants were inoculated with two Xoo strains (PXO99 and PXO145) to simulate the challenges rice crops face under simultaneous stress of drought and BB. Plant height and dry shoot biomass were significantly reduced by drought stress treatments. The BB disease lesion lengths varied according to rice genotypes and PXO99 Xoo multiplication and spread in planta was higher compared to that of PXO145, which generally decreased under mild drought stress. Rice genotype IRBB7 (Xa7) showed less Xoo spread and a reduced Xoo multiplication under drought stress compared to the well-watered control with PXO145. In contrast, in genotypes with a different BB R gene and/or drought QTLs [IRBB4 (Xa4), IR87705–6-9-B (Xa4 + qDYT _2.2 ), IR87707–445-B-B-B (Xa4 + qDYT _2.2  + qDYT _4.1 ) and IR87707–446-B-B-B (Xa4 + qDYT _2.2  + qDYT _4.1 )], Xoo multiplication and spread in planta was higher with drought stress. This study has shown that drought stress affected rice response to the BB pathogen and the response varied according to the rice genotype. It is concluded that evaluating rice varieties under combined abiotic and biotic stresses will be the best strategy to determine biotic stress resistance durability under climate change.     

高温激活RNA沉默介导的心叶烟抗病毒防御反应

 在真核细胞内,RNA沉默是一种保守的抵抗病毒及外源基因等寄生分子的防御机制。温度可以明显地影响植物体与病毒之间的相互作用。在常温状态下,常常会伴随病毒病的爆发,但在较高温度下,症状则会减轻,甚至有些感病植株新生叶片可以较快地恢复健康。目前这类现象的内在机制还不完全清楚。本文通过定量分析不同温度下马铃薯X病毒(Potato virus X,PVX)和马铃薯Y病毒(Potato virus Y,PVY)侵染的心叶烟(Nicotiana glutinosa)植株体内病毒蛋白含量,结合症状观察,确定30℃左右是PVX和PVY在心叶烟上引起症状消失即发生系统性RNA沉默的临界温度,此时的感病植株上部新生展开叶片呈现无症状态,下部已发病叶片症状不变。在常温时,由病毒激发的RNA沉默会受到抑制,病毒来源的小分子干涉RNA (small interfering RNAs,siRNAs)含量极少或根本检测不到,植株对于病毒的敏感性较强。当温度达到30或32℃时,感病植株下部症状保持叶片中,病毒起源的siRNAs含量明显增加,而上部新生健康叶片中却检测不到siRNAs的存在。这说明高温激活了RNA沉默介导的植物抗病毒防御反应,导致病毒症状减轻甚至消失。     

<Emphasis Type="Italic">Alternaria</Emphasis> species associated with early blight epidemics on tomato and other <Emphasis Type="Italic">Solanaceae</Emphasis> crops in northwestern Algeria

Clubroot, caused by the protozoan parasite Plasmodiophora brassicae Woronin, is one of the most damaging diseases of Brassica napus worldwide. Resistant plant material is valuable for cultivation in all areas of high incidence of the disease and intensive growth of oilseed rape. We have evaluated clubroot resistance, plant morphology and seed quality in 15 lines of an F₄ generation and selected six lines of F₅ generation of interspecific hybrids obtained from a cross between a male sterile line of B. napus ‘MS8’, selected from resynthesized oilseed rape (B. rapa ssp. chinensis × B. oleracea var. gemmifera) and an ecotype of B. rapa ssp. pekinensis. Clubroot resistance was evaluated using a bioassay with P₁-P₅ pathotypes of P. brassicae (according to the classification of Somé et al. 1996). The resistance to the pathotype P₁ was successfully fixed in the F₅ generation, and improved in some lines in respect to the pathotypes P₂-P₄. The resistance to P₁ and the other tested pathotypes was not linked. Characterization of plant material included recent techniques of FISH and BAC-FISH with a special focus on the analysis of ribosomal DNA (rDNA) of selected individuals. Two hybrid lines combined high levels of resistance with appropriate plant morphology, good seed quality traits and a stable chromosome number and arrangement. Recent techniques of ‘chromosome painting’ provided good insight into chromosome organization in the hybrids obtained, and offered opportunities of further improvement of the breeding process.     

Molecular cloning and virus-induced gene silencing of MiASB in the southern root-knot nematode, Meloidogyne incognita

Root-knot nematodes (Meloidogyne spp.), cause serious damage to agricultural production worldwide. In this study, we designed special primers based on the predicted Mitochondrial ATP synthase b subunit gene (ASB) sequence to clone the same gene in M. incognita (MiASB). The identity between the cloned MiASB and the predicted MiASB was as high as 100 %. Using the tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) system, we delivered MiASB RNAi triggers to the M. incognita feeding site on tomato seedlings, resulting in significantly fewer galls on the seedlings. Sixty days after inoculation with M. incognita, the number of root galls induced on the MiASB silence-treated seedlings was reduced by 64.3 % compared to that on the control seedlings, and reduced by 64.1 % compared to that on untreated control seedlings. This study revealed the MiASB silencing had a positive effect on the control of root-knot nematodes, and MiASB may be associated with the formation of galls caused by the nematode.     

Biochemical changes in the <Emphasis Type="Italic">Brassica juncea-fruticulosa</Emphasis> introgression lines after <Emphasis Type="Italic">Lipaphis erysimi</Emphasis> (Kaltenbach) infestation

Insect damage leads to changes in biochemical profile of plants. Response of three Brassica juncea-fruticulosa introgression lines (already reported resistant to Lipaphis erysimi) in terms of changes in biochemical constituents after aphid infestation was studied along with B. fruticulosa (resistant parent), B. juncea var. PBR ?210 (susceptible parent) and B. rapa ecotype brown sarson BSH-1 (susceptible check). These six genotypes were grown under aphid infested and uninfested conditions and were sampled at peak aphid infestation to analyze the biochemical changes caused by aphid feeding from top 10 cm central twig of plant. A significant reduction in glucosinolates content in aphid infested plants of three introgression lines (I₈, I₇₉ and I₈₂) was observed while opposite was observed in B. fruticulosa, PBR-210 and BSH-1. Exactly opposite trend was observed for total phenols where aphid infestation resulted in significant increase in phenols content in the three introgression lines while a decrease was observed in B. fruticulosa, PBR-210 and BSH-1. A general trend of decline in flavonols, total sugars and free amino acids content was observed after aphid infestation in all the genotypes. Glucosinolates and total phenols served as biochemical bases of resistance in the three introgression lines since there was downregulation of glucosinolates and upregulation of total phenols as against opposite trend observed in BSH-1 and PBR-210.     

Allelochemical ethyl 2-methyl acetoacetate (EMA) induces oxidative damage and antioxidant responses in Phaeodactylum tricornutum

Ethyl 2-methyl acetoacetate (EMA) is a novel allelochemical exhibiting inhibitory effects on the growth of marine unicellular alga Phaeodactylum tricornutum (P. tricornutum). Oxidative damage and antioxidant responses in P. tricornutum were investigated to elucidate the mechanism involved in EMA inhibition on algal growth. The increase in reactive oxygen species (ROS) levels and malondialdehyde (MDA) contents following exposure to EMA suggested that alga was suffered from oxidative stress and severely damaged. The decrease in cell activity and cellular inclusions suggested that cell growth was greatly inhibited. The activities of the antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxide (GSH-PX) and glutathione S-transferase (GST) increased with the exposure concentration and decreased with the prolongation of exposure time. Cellular ascorbic acid (AsA) and reduced glutathione (GSH) systems were also involved in resisting oxidative stress of EMA by altering the composition of AsA and GSH pools. EMA exposure increased the contents of AsA, GSH, dehydroascorbate (DAsA) and glutathione (GSSG). However, the regeneration rate of AsA/DAsA did not change obviously between treatments and the control, while that of GSH/GSSG decreased significantly under 14 mmol/L EMA exposure on the 3rd day. These results showed that EMA-induced oxidative damage might be responsible for EMA inhibition on P. tricornutum growth and cellular antioxidant enzymes and non-enzymatic antioxidants were improved to counteract the oxidative stress.     

Inhibition of fatty acid biosynthesis in isolated bean and maize chloroplasts by herbicidal phenoxy-phenoxypropionic acid derivatives and structurally related compounds

The effects of nine phenoxy-phenoxypropionic acid derivatives and structurally related compounds on the incorporation of [¹⁴C]-acetate into free fatty acids in isolated bean and maize chloroplasts were studied. The compounds tested were esters and the corresponding free acids, OH-diclofop, a nonherbicidal metabolite of diclofop in plants, and d and l enantiomers of diclofop. Fatty acid biosynthesis in bean chloroplasts was not affected by all compounds. OH-Diclofop had a weak inhibitory effect on fatty acid synthesis in maize chloroplasts, while free acids were stronger inhibitors than the corresponding esters in the same system. Uptake studies with diclofop-methyl and diclofop indicated that the esters showed higher uptake rates in chloroplasts suspension. d-Diclofop (I₅₀, 9 × 10^?8M) was a more potent inhibitor than l-diclofop (I₅₀, 4 × 10^?6M). This agrees with the low herbicidal activity of the l enantiomer in vivo. The results suggest that the mode of action in this type of herbicide may be closely linked with the inhibition of fatty acid biosynthesis. The tolerance of beans could be based on an insensitivity of the target site.     

Environmental effects on growth and sporulation of <Emphasis Type="Italic">Fusarium</Emphasis> spp. causing internal fruit rot in bell pepper

Internal fruit rot in bell pepper (Capsicum annuum L.) is mainly caused by members of the Fusarium lactis species complex (FLASC) and to a lesser extent by Fusarium oxysporum and Fusarium proliferatum. Despite the importance of the disease, there is hardly no information about growth, sporulation and germination dynamics of FLASC. In order to understand the dominance of FLASC as main pathogen of internal fruit rot, the effects of temperature (5 °C – 35 °C), water activity (a_w 0.76–0.96), pH (pH 3 - pH 9) and oxygen concentration (2.5% - 20%) on growth and sporulation of all three Fusarium species were compared. In addition, germination kinetics were also investigated. FLASC showed optimal mycelium growth and sporulation in the narrow range of 25 °C, while both other strains were also tolerant for higher temperatures to 30 °C. FLASC was also characterized by a broad pH optimum from pH 3–7 while F. oxysporum (pH 4–7) and F. proliferatum (pH 5–8) were more demanding concerning pH. In addition, optimal sporulation occurred in the acid region for FLASC (pH 3) whilst neutral and alkaline pH were more favourable for the other species. Germination kinetics revealed that FLASC did not benefit from an earlier and/or faster germination process. A thorough understanding of the growth characteristics and dominance of FLASC as main pathogen for internal fruit rot is inevitable to develop sustainable control measures for the disease.