Inverse correlation between mRNA levels in GFP-silenced transgenic Nicotiana benthamiana and resistance to Potato virus X engineered to contain GFP sequence

Nicotiana benthamiana was transformed with a green fluorescent protein (GFP) gene driven by cauliflower mosaic virus 35S promoter. A GFP-silenced line and a nonsilenced line were selected after ultraviolet irradiation. GFP short-interfering RNAs (siRNAs) were detected in the silenced line but not in the nonsilenced line. T₁ progeny of the silenced line varied in GFP suppression patterns and were grouped into three types (I, II, III) based on the GFP suppression pattern. With Northern blot analysis, different levels of GFP mRNA accumulated, from a very low level in type I and II to an intermediate level in type III, in contrast to a much higher level in the nonsilenced line. Plants were also inoculated with Potato virus X engineered to contain the GFP sequence to evaluate the levels of virus resistance. None to a few GFP spots were observed on inoculated leaves in types I and II, whereas numerous spots and systemic infection appeared in type III. These results showed that virus resistance was inversely correlated with the levels of mRNA, suggesting that the strength of RNA silencing determines the extent of virus resistance.     

蛋白激发子PevD1诱导本生烟植保素辣椒醇的产生和积累

诱导抗病性是实现植物病害绿色防控的重要途径,大丽轮枝菌蛋白激发子PevDl能激活植物免疫系统,提高本生烟对烟草花叶病毒(TMV)和烟草野火病病原菌Pseudomonas syringae pv.tabaci、棉花对大丽轮枝菌Verti-cillium dahliae的抗病性,但分子机制不清晰.前期转录组测序(RNA-S...     

Degree of RNA silencing and the ability of a viral suppressor vary depending on the cell species in a protoplast system

RNA silencing is a sequence-specific defense mechanism against viruses. As a counterdefense, viruses evolved silencing suppressors to interfere with host silencing. In analyses using protoplasts prepared from cultured cells (BY-2) and mesophyll cells of Nicotiana tabacum and N. benthamiana, viral suppressors differentially functioned in different cell types. This phenomenon has not been discussed in earlier papers on protoplast systems and RNA silencing. In investigations of the cellular activities of viral suppressors and their role in the RNA-silencing pathway, assays with host protoplasts offer many advantages and can complement other in planta assays such as Agrobacterium-mediated transient expression.     

Involvement of nitric oxide generation in hypersensitive cell death induced by elicitin in tobacco cell suspension culture

Recent studies suggest that nitric oxide (NO), an important signaling and defense molecule in mammals, plays a key role in activating disease resistance in plants. We characterized NO production by tobacco Bright Yellow-2 cells pharmacologically after treatment with INF1, the major elicitin secreted by the late blight pathogen Phytophthora infestans, prepared from Escherichia coli. NO production rapidly occurred within 1h and reached a maximum level 3–6h after the addition of INF1. Carboxy-PTIO, a NO-specific scavenger, abolished INF1-induced NO production in a dose-dependent manner. Pretreatment of protein synthesis inhibitor cycloheximide and protein kinase inhibitor K252a blocked NO production 3–12h after INF1 treatment, indicating that NO production requires de novo protein synthesis and protein phosphorylation. In an investigation of the relations between NO generation and several defense responses induced by INF1, carboxy-PTIO completely suppressed activation of a 41-kDa protein kinase and cell death by INF1. Carboxy-PTIO also suppressed the induction of hypersensitive-related (hsr) genes HSR515 and HSR203J, the expression of which is strongly correlated with the hypersensitive response in plants. The results suggest that NO plays a crucial role in the induction of hypersensitive cell death.     

Nitric oxide donor seed priming enhances defense responses and induces resistance against pearl millet downy mildew disease

Nitric oxide (NO) donors Nitroso-R-Salt, 2-Nitroso-1-Naphthol and Sodium Nitro Prusside (SNP) were evaluated for their effectiveness in protecting pearl millet [(Pennisetum glaucum L.) R. Br.] plants against downy mildew disease caused by Sclerospora graminicola [(Sacc). Schroet]. Optimization experiments with NO donors showed no adverse effect either on the host or pathogen. Aqueous SNP seed treatment with or without polyethylene glycol (PEG) priming was the most effective in inducing the host resistance against downy mildew both under greenhouse and field conditions. Potassium Ferrocyanide, a structural analog of NO donor lacking NO moiety failed to protect the pearl millet plants from downy mildew indicating a role for NO in induced host resistance. Spatio-temporal studies corroborated that the protection offered by NO donor treatment was systemic in nature and a minimum of 3-day time gap between the inducer treatment and subsequent pathogen inoculation was necessary for maximum resistance development. Disease protection ability of NO donors was also validated as durable in nature. Conversely, prior-treatment with NO scavenger 2-4-carboxyphenyl-4,4,5,5 tetrazoline-1-oxyl-3-oxide potassium salt (C-PTIO) rendered the pearl millet plants relatively susceptible for pathogen infection. Expression of primary defense responses like hypersensitive response, lignin deposition and defense related enzyme phenylalanine ammonialyase −EC 4.3.1.5 (PAL) were enhanced by NO donor treatments.     

Analysis of gene sequences for the nucleocapsid protein from <Emphasis Type="Italic">Tomato spotted wilt virus</Emphasis> for promoting RNA-mediated cross-protection using the <Emphasis Type="Italic">Potato virus X</Emphasis> vector system

Virus interactions between Tomato spotted wilt virus (TSWV) and Potato virus X (PVX) containing the nucleocapsid protein (N) gene sequences were examined to evaluate the capacity of the N gene sequences from TSWV to promote RNA-mediated cross-protection. Plants simultaneously inoculated with TSWV and PVX containing the 3 96bp of the N gene were highly resistant to TSWV infection, whereas no such resistance was observed in plants inoculated with TSWV and PVX containing the 5 96bp. These results suggest that the 3 portion of the N gene has a higher capacity for promoting RNA-mediated cross-protection of TSWV.     

Nitric oxide-overproducing transformants of Pseudomonas fluorescens with enhanced biocontrol of tomato bacterial wilt

The relation between nitric oxide (NO) production and the protective ability of Pseudomonas fluorescens T5 against bacterial wilt disease in tomato was examined. The endogenous nitric oxide reductase gene of T5 was disrupted by homologous recombination using a suicide plasmid. Three disruptants were obtained, and all had higher levels of NO production. Infection with Ralstonia solanacearum was reduced in tomato plants treated with the NO-overproducing transformants compared with the wild type. These results suggest that the modification of pseudomonads to increase their level of NO production is a new approach to enhancing their biocontrol efficacy.     

Depolymerization of the actin cytoskeleton induces defense responses in tobacco plants

Tobacco leaf sections were treated with actin inhibitors, i.e., cytochalasins, to determine the effects of actin depolymerization on tobacco defense responses. Inoculation of the leaf sections with the pathogen Erysiphe cichoracearum, depolymerized the actin cytoskeleton, priming the cells for a hypersensitive response-like cell death. Further, expression of the acidic PR1 and PR2 genes were induced in cytochalasin-treated leaf sections. The intensity of the cytochalasin effects on the defense responses was closely correlated with the extent of actin depolymerization. This suggests that plant cells may perceive perturbation of the actin cytoskeleton, and this stimulus may trigger plant defense responses.     

Regulation of elicitin-induced ethylene production in suspension-cultured tobacco BY-2 cells

INF1 elicitin, a proteinaceous elicitor produced by Phytophthora infestans, induces a hypersensitive response in tobacco BY-2 cells. In response to elicitin, tobacco cells produce both reactive oxygen species (ROS) and ethylene (ET). To investigate the regulation of elicitin-induced ET production, we pharmacologically analyzed the effects of several chemicals on ET production. Inhibitors of ROS generation or ROS chelators efficiently inhibited ET production, whereas simultaneous treatment of a superoxide anion-generating system with salicylhydroxamic acid recovered ET production. In an in vitro experiment, superoxide anion was necessary and sufficient for conversion of 1-aminocyclopropane-1-carboxylate (ACC) to ET because ET was produced from ACC solely in the presence of the superoxide-generating chemical KO₂. ET production was also inhibited by lipoxygenase (LOX) inhibitors, indicating a possible involvement of LOX-mediated generation of superoxide anion and ET production itself. Furthermore, elicitin-induced ET production was completely inhibited by the protein synthesis inhibitor cycloheximide but recovered after exogenous application of ACC, indicating that de novo protein synthesis is required for ACC accumulation, leading to ET production. We also investigated the effects of several phytohormones on elicitor-induced ET production and discuss their role in the defense response.     

Harpin-elicited hypersensitive cell death and pathogen resistance require the NDR1 and EDS1 genes

Plants sprayed with harpin, a bacterial protein that induces hypersensitive cell death (HCD), develop systemic acquired resistance (SAR) without macroscopic necrosis. HCD sometimes accompanies the development of resistance conferred by resistance (R) genes. In Arabidopsis, some R genes require one or both of the signalling components NDR1 and EDS1 for function. This study addresses whether HCD, NDR1 and EDS1 are required for induction of SAR by harpin. When Arabidopsis and tobacco leaves were sprayed with harpin, microscopic hypersensitive response (micro-HR) lesions developed. Systemic expression of PR genes and the development of resistance were accompanied by micro-HR, except in the ndr1-1 mutant, in which harpin induced micro-HR without the development of resistance or expression of the PR-1 gene. Cell death and resistance did not occur following treatment with harpin in plants that could not accumulate salicylic acid. Harpin also failed to induce resistance in Arabidopsis eds1-1 mutants. Therefore, harpin-induced resistance seems to develop concomitantly with cell death and resistance requires NDR1 and EDS1.     

心叶烟NPR1基因全长cDNA序列的克隆及表达分析

 NPR1(non-expressor of pathogenesis-related gene 1)基因在拟南芥系统获得抗性中起着关键作用,可调控拟南芥植株广谱抗性的发生。本文报道了从心叶烟中克隆NPR1同源基因(NgNPR1)及其表达特性的研究结果。NgNPR1 cDNA全长2253 bp,编码588个氨基酸。将NgNPR1基因组全长与cDNA序列进行比对发现,NgNPR1基因组DNA含有4个外显子和3个内含子。Southern杂交分析表明,在心叶烟基因组中NgNPR1为单拷贝基因。采用绿色荧光蛋白在洋葱表皮瞬时表达的试验,证明了NgNPR1蛋白在水杨酸诱导时会从细胞质转运到细胞核中。Northern杂交分析发现,NgNPR1基因可以被与植物抗病相关的信号分子如水杨酸、茉莉酸甲酯、过氧化氢和乙烯所诱导。进一步研究发现,植物病原物如赤星病菌、青枯病菌和烟草花叶病毒对心叶烟植株的侵染也会使NgNPR1表达量增加。这些结果表明,NgNPR1基因在心叶烟植株抵御病原物侵染过程中可能起着重要作用。     

Tomato spotted wilt virus triggers specific and shared defense mechanisms in hypersensitive and susceptible Solanaceae hosts

In situ and in vitro techniques were employed to investigate the metabolic changes caused by Tomato spotted wilt virus in hypersensitive and susceptible hosts; Petunia hybrida and Nicotiana tabacum, respectively. In petunia, H₂O₂ accumulation preceded increased peroxidase and shikimate dehydrogenase activity at local lesion sites. In systemic tobacco plants, peroxidase activity was induced prior to symptom onset and the activity of shikimate dehydrogenase was disrupted upon viral infection. Taken together, our data suggest that reactive oxygen species-based mechanisms of defense are shared by hypersensitive and susceptible hosts, although downstream components and regulatory mechanisms are distinct.     

Moss-Erwinia pathosystem reveals possible similarities in pathogenesis and pathogen defense in vascular and nonvascular plants

Vascular plants have various inducible resistance mechanisms as defense against pathogens. Mosses, small nonvascular plants (subkingdom Bryophyta), have been little studied in regard to their pathogens or modes of defense. Data here show that Erwinia carotovora, a bacterial plant pathogen that causes softrot in many dicotyledonous plants, can also cause soft rot symptoms in the moss Physcomitrella patens. Infection of moss by E. carotovora required pathogenicity factors similar to those required to infect vascular plants and, again as in vascular plants, salicylic acid (SA) induced moss to inhibit tissue maceration by Erwinia. These data reveal that SA-dependent defense pathways may have evolved before differentiation of vascular and nonvascular plants.     

Electrophoretic and serological comparisons of pathogenesis-related (b) proteins from different plant species

Preparations of pathogenesis-related (b) proteins (PRs) from differentNicotiana species, tomato,Gynura aurantiaca, bean, and cowpea were compared to each other and to bean chitinase and a constitutive apple agglutinin by electrophoresis in polyacrylamide gels both in the absence and in the presence of SDS, and by serological double diffusion analysis using antisera against tobacco PRs and bean chitinase. PRs from different plant genera displayed a similar but not identical range of relative mobilities in both native and SDS gels, whereas bean chitinase and apple agglutinin were clearly different. None of the antisera reacted with any of the PR preparations from plant genera other than the one from which the antigen(s) had been derived. Whilst PRs within the genusNicotiana are serologically related and can be identical, PRs from different plant genera seem to be sufficiently different to be considered as genus-specific.     

FgLEU1在禾谷镰刀菌亮氨酸生物合成及产毒致病中起关键作用

为挖掘新型药剂的潜在靶标,利用靶向基因敲除和互补技术研究赤霉病病原菌禾谷镰刀菌Fusarium graminearum中必需氨基酸亮氨酸合成酶编码基因FgLEU1的功能,并测定禾谷镰刀菌的生物学表型。结果表明,FgLEU1编码亮氨酸合成途径中的3-异丙基苹果酸脱水酶,其敲除突变体表现亮氨酸营养缺陷。生物学表型测定结果显示,与野生型菌株相比,FgLEU1敲除突变体的产孢量和孢子萌发率显著下降,产孢量仅为野生型菌株的20.96%,培养4 h后孢子萌发率下降了49.45%,且合成脱氧雪腐镰刀烯醇（呕吐毒素）能力丧失,在麦穗上的致病力下降,仅能侵染接种小穗,赤霉病症状不能扩展。外源添加一定量的亮氨酸、FgLeu1催化产物或导入含启动子的全长FgLEU1基因可以恢复敲除突变体表型缺陷。表明FgLEU1基因在禾谷镰刀菌亮氨酸合成、菌丝孢子形成及产毒致病过程中发挥着重要作用,可作为新型安全杀菌剂的潜在研发靶标,用于持续有效控制麦类赤霉病和镰刀菌毒素。     

Mating type gene MAT1-2-1 is common among Japanese isolates of Verticillium dahliae

Mating type genes of Verticillium dahliae, a wilt pathogen affecting many plant species, were identified to examine sexual recombination between Japanese pathotypes. We amplified a DNA sequence encoding high mobility group (HMG) box from V. dahliae using PCR. A cloned genomic DNA fragment included a sequence homologous to MAT1-2-1 gene. Despite that sequence's presence in all V. dahliae isolates we used, MAT1-1-1 (an opposite mating type gene) was never amplified. We concluded that V. dahliae is potentially heterothallic. Furthermore, sexual bias practically obviates sexual recombination between Japanese pathotypes. This report describes, for the first time, a mating type gene of phytopathogenic Verticillium.