拟南芥转录因子IDD4不同突变体对灰葡萄孢菌的抗性分析

为明确IDD家族IDD4基因在拟南芥Arabidopsis thaliana抵抗灰葡萄孢菌Botrytis cinerea侵染过程中的作用,通过统计病情指数检测拟南芥野生型（wild type,WT）植株、过表达植株IDD4-OE和缺失突变体idd4植株感染灰葡萄孢菌情况,利用组织染色检测叶片细胞死亡和H₂O₂的积累情况,采用实时荧光定量PCR（real-time quantitative-PT-PCR,qRT-PCR）技术分析灰葡萄孢菌肌动蛋白基因Bc. ACTIN在3种植株叶片中的表达情况,并施加0.1 mmol/L外源水杨酸（salicylic acid,SA）后测定IDD4-OE植株的病情指数。结果显示,不同株系对灰葡萄孢菌的抗性由高到低依次为idd4＞WT＞IDD4-OE,IDD4-OE植株中病原菌感染部位的寄主细胞死亡程度比idd4植株严重。染色结果表明,病原菌侵染拟南芥后4 h,接种部位已有H₂O₂积累。qRT-PCR反应结果显示,Bc. ACTIN在IDD4-OE中比在idd4植株中的表达水平更高,表明灰葡萄孢菌在IDD4-OE植株中的繁殖速率更快。对IDD4-OE植株外源施加SA后,其病情指数、Bc. ACTIN表达量与WT植株间均无显著差异,说明SA能将感病植株的抗性提高至WT植株的水平,表明IDD4作为负调控因子参与了拟南芥对灰葡萄孢菌的抗性调控,SA在其中发挥着重要作用。     

Bioluminescence reporter assay system to monitor Arabidopsis MPK3 gene expression in response to infection by Botrytis cinerea

The Arabidopsis MPK3 gene product participates in disease resistance mediated by the MAP kinase cascade. The expression of the MPK3 gene is induced by pathogen inoculation and treatment with chemicals such as salicylic acid (SA) and methyl jasmonate (JA), but the detailed expression pattern of the MPK3 gene has been largely unknown. To investigate MPK3 gene expression in response to disease stress, we fused the MPK3 promoter to the firefly luciferase gene to create a real-time monitoring system for regulated gene expression in planta. The results of an in vivo reporter assay using transgenic Arabidopsis plants harboring MPK3::Fluc showed that the MPK3 promoter activity was induced by treatment with chemicals such as SA and benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), that induce defense gene expression. Inoculation with the fungal pathogen Botrytis cinerea resulted in systemic induction of MPK3::Fluc.     

Soybean plants expressing an active oligomeric oxalate oxidase from the wheat gf-2.8 (germin) gene are resistant to the oxalate-secreting pathogen Sclerotina sclerotiorum

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum (Lib.) De Bary is a serious fungal disease of soybean. Senescing petals provide a starting nutrient source for the invasion of healthy tissue by the advancing oxalic acid secreting fungal hyphae. Since oxalic acid is a major pathogenicity factor of SSR, transgenic soybean capable of degrading oxalic acid may be resistant to the pathogen. Transgenic soybean plants were produced byAgrobacterium -mediated transformation with the wheat germin gene (gf-2.8) encoding an oligomeric protein, oxalate oxidase (OxO), which oxidizes oxalic acid to carbon dioxide and hydrogen peroxide (H₂O₂). Transgenic soybean homozygous for 35S- gf-2.8 produced an approx. 130 kDa protein indistinguishable from wheat germin, and with OxO activity. OxO activity was prominent in cell walls proximal to the site of pathogen attack. The transgenics had greatly reduced disease progression and lesion length following cotyledon and stem inoculation with S. sclerotiorum indicating that the germin gene product conferred resistance to SSR. This is the first report of plant resistance to the fungal pathogen S. sclerotiorum in transgenic plants expressing OxO.     

Systemic resistance to bacterial leaf speck pathogen in Arabidopsis thaliana induced by the culture filtrate of a plant growth-promoting fungus (PGPF) Phoma sp. GS8-1

The culture filtrate (CF) from the plant growth-promoting fungus Phoma sp. GS8-1 was found to induce systemic resistance in Arabidopsis thaliana against the bacterial leaf speck pathogen Pseudomonas syringae pv. tomato DC3000 (Pst), and the underlying mechanism was studied. Roots of A. thaliana were treated with CF from GS8-1, and plants expressed a clear resistance to subsequent Pst infection; disease severity was reduced, and proliferation of pathogen was suppressed. Various mutants of A. thaliana were used to test whether the CF induced resistance through one of the known signaling pathways: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). The CF was fully protective against Pst in Arabidopsis mutants jar1 and ein2 similar to wild-type plants. However, its efficacy was reduced in plants containing transgene NahG. Examination of systemic gene expression revealed that CF modulates the expression of SA-inducible PR-1, PR-2 and PR-5 genes, the JA/ET-inducible ChitB gene, and the ET-inducible Hel gene. Moreover, the expression of these genes was further enhanced upon subsequent stimulation after attack by Pst. Our data suggest that in addition to a partial requirement for SA, the signals JA and ET may also play a role in defense signaling in Arabidopsis.     

Local and systemic resistance to fungal pathogens triggered by an AVR9-mediated hypersensitive response in tomato and oilseed rape carrying the Cf-9 resistance gene

Tomato and transgenic oilseed rape plants expressing the Cf-9 resistance gene develop a hypersensitive response (HR) after injection of the corresponding Avr9 gene product. It was investigated whether induction of a HR conferred resistance to different fungal pathogens in tomato and oilseed rape. Induction of an AVR9 mediated HR at the pathogen infection site delayed the development of the biotrophs Oidium lycopersicum in tomato and Erysiphe polygoni in oilseed rape, but enhanced the development of the necrotrophs Botrytis cinerea and Alternaria solani in tomato and Sclerotinia sclerotiorum in oilseed rape. Interestingly, delayed fungal disease development was observed in plant tissues surrounding the HR lesion regardless of whether a necrotrophic or biotrophic pathogen was used. In tomato, AVR9 injection induced systemic expression of PR1, PR2 and PR3 defence genes but did not induce systemic resistance to O. lycopersicum, B. cinerea or A. solani. In oilseed rape, AVR9 injection temporarily induced systemic resistance to Leptosphaeria maculans and E. polygoni, but did not induce detectable systemic expression of PR1, PR2 or Cxc750. These results give new insights into the potential uses of an induced HR to engineer disease resistance.     

Inhibitors of N-linked glycosylation induce systemic acquired resistance in cucumber

Localized treatment of cucumber (Cucumis sativus L. cv. Wisonsin) cotyledons with inhibitors of N-glycosylation such as tunicamycin or amphomycin resulted in systemic acquired resistance in the first leaf to the fungal pathogen Colletotrichum lagenarium. Resistance was maximal as early as 2 days after application and best results were observed when the inhibitor was used at 100 μ . The same treatment also induced salicylic acid accumulation as well as the expression of chitinase and a PR1-like protein. The systemic effect is not caused by the transport of tunicamycin, since tunicamycin was not detected in the leaves. Within 2 h after application tunicamycin inhibited N-glycosylation, but not protein synthesis as indicated by labelling experiments. The amount of large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase decreased after tunicamycin treatment and after pathogen inoculation and the expression of BiP, a protein localized in the endoplasmic reticulum was enhanced. The activation of defense reactions seems to be dependent and sensitive to N-linked glycosylation.     

Amino acid substitution in the coat protein of Melon necrotic spot virus causes loss of binding to the surface of Olpidium bornovanus zoospores

Melon necrotic spot virus (MNSV) is transmitted by the fungus Olpidium bornovanus. In this study, we used immunofluorescence microscopy to detect MNSV particles over the entire surface of the O. bornovanus zoospore; MNSV particles were not detected on the related fungus O. virulentus, which cannot transmit MNSV. The amino acid substitution Ile → Phe at position 300 in the MNSV coat protein resulted in loss of both specific binding and fungal transmission, while virion assembly and biological aspects were unaffected. Taken together, these results suggest that the MNSV coat protein acts as a ligand to the O. bornovanus zoospore as part of a fungal-vector transmission system.     

大丽轮枝菌效应蛋白VdSRP2的功能分析

为明确效应蛋白VdSRP2在大丽轮枝菌Verticillium dahliae中的生物学功能,从大丽轮枝菌落叶型菌株V592中克隆VdSRP2基因并进行生物信息学分析,利用酵母转化酶分泌系统对其信号肽活性进行测定,采用实时荧光定量PCR(real-time fluorescence quantitative PCR,qPCR)技术分析VdSRP2基因在大丽轮枝菌中的表达模式,并以V592菌株为材料获得VdSRP2基因的敲除突变体和过表达体菌株,通过表型分析和致病性测定确定VdSRP2基因的生物学功能。结果显示,VdSRP2基因编码232个氨基酸,含有5个半胱氨酸残基,N-端信号肽具有分泌活性,为真菌的典型效应蛋白;VdSRP2基因主要在大丽轮枝菌菌丝和微菌核中表达,其中经棉花根系诱导培养24 h时表达量最高;与野生型菌株V592相比,VdSRP2基因敲除导致大丽轮枝菌的产孢量和孢子萌发率显著下降,不能形成微菌核,对棉花的致病力明显减弱;但VdSRP2基因敲除不影响大丽轮枝菌的穿透能力;VdSRP2基因在本氏烟和棉花叶片瞬时表达不会诱导细胞死亡。表明VdSRP2是大丽轮枝菌微菌核形成必需...     

The role of syringic acid in the interaction between oil palm and Ganoderma boninense,the causal agent of basal stem rot

Novel inoculation and assessment methods for Ganoderma boninense infection of oil palm are reported. The involvement of phenolic acids in the interaction was examined. HPLC was used to monitor changes in the concentrations of three specific phenolics: syringic acid (SA), caffeic acid and 4‐hydroxybenzoic acid, identified as the main compounds that accumulated. The work reported here focuses on SA, the most antifungal of the molecules detected. The oil palm cv. AVROS, reported by local planters to be less susceptible than others, showed higher accumulation of SA than cvs Ekona and Calabar. Accumulation was promoted by addition of chitosan to the plant growing medium. By the end of the time‐course, the concentration of SA decreased in the oil palm tissues inoculated with G. boninense, suggesting possible metabolism by the pathogen. This loss was, however, not detected in tissues treated with chitosan alone and was greatly reduced when G. boninense was combined with this polymer. In vitro studies on antifungal activity of SA were done using concentrations ranging from 50 to 110 μg mL^?1, those typically recorded in oil palm roots. SA was found to be antifungal (EC₅₀ 90–100 μg mL^?1). The concentration of SA detected in root tissues, especially in the presence of chitosan, could inhibit growth of G. boninense. The pathogen was shown to degrade SA in vitro. However, at the highest concentration tested, metabolism was greatly delayed, only occurring after a lag phase in pathogen growth. Accumulation of phenolic acids, especially SA, may prove a useful trait in breeding resistant oil palm cultivars.     

Induced resistance in tomato plants to the toxin-dependent necrotrophic pathogen Alternaria alternata

A necrotrophic pathogen, the tomato pathotype of Alternaria alternata (Aa) causes Alternaria stem canker on tomato. Its pathogenicity depends on the production of host-specific AAL-toxin. Pre-inoculation with nonpathogenic Aa or pretreatment an elicitor prepared from Aa reduced disease symptoms by the pathogen. Salicylic acid (SA)- and jasmonic acid (JA)-dependent defense responses in tomato are not involved in the resistance to the pathogen induced by nonpathogenic Aa. The results suggest that an alternative and unknown signaling pathway independent of SA- and JA-signaling might modulate the induced resistance by activating the expression of the multiple defense genes.     

Expression of the tobacco β-1,3-glucanase gene, PR-2d, following induction of SAR with Peronospora tabacina

Systemic acquired resistance (SAR) is induced following inoculation of Peronospora tabacina sporangia into the stems of Nicotiana tabacum plants highly susceptible to the pathogen. Previous results have shown that accumulation of acidic β-1,3-glucanases (PR-2's) following induction of SAR by P. tabacina may contribute to resistance to P. tabacina. We showed that up-regulation of the PR-2 gene, PR-2d, following stem inoculation with P. tabacina, is associated with SAR. Studies using plants transformed with GUS constructs containing the full length promoter from PR-2d or promoter deletions, provided evidence that a previously characterized regulatory element that is involved in response to salicylic acid (SA), may be involved in regulation of PR-2d following induction of SAR with P. tabacina. This work provides evidence that regulation of PR-2 genes during P. tabacina-induced SAR may be similar to regulation of these genes during infection of N-gene tobacco by TMV or following exogenous application of SA, and provides further support for the role of SA in regulation of genes during P. tabacina-induced SAR.     

Enhancement of Postharvest Disease Resistance in Ya Li Pear (Pyrus bretschneideri) Fruit by Salicylic Acid Sprays on the Trees during Fruit Growth

The Ya Li pear (Pyrus bretschneideri) trees were sprayed three times with 2.5 mM salicylic acid (SA) around 30, 60 and 90 days after full flowering. The fruit were harvested at commercial maturity (about 120 days after full flowering), inoculated with Penicillium expansum, and incubated at 20 °C, 95–100% RH. The results showed that resistance to the pathogen of the mature pear fruit was remarkably enhanced by the SA sprays. Disease incidence in the SA-treated fruit was 58.0% or 26.5%, and lesion diameter on SA-treated fruit was 58.4% or 29.0% lower than that in/on fruit without SA treatment (control) on day 12 or 17 after incubation, respectively. The SA spray applied to the trees around 30 days after full flowering notably enhanced accumulation of hydrogen peroxide in the young fruit. Meanwhile, activities of defense enzymes, including peroxidase, phenylalanine ammonia-lyase (PAL), chitinase or β-1,3-glucanase in the young fruit from SA-treated trees was 29.5%, 60.0%, 24.4% or 35.7% higher than that in the control fruit 4 days after the SA spraying. Furthermore, after harvest, activities of PAL, chitinase and β-1,3-glucanase were still significantly higher in the mature pear fruit from the trees sprayed three times with SA than those of the control fruit. Activities of the antioxidant enzymes including catalase and ascorbate peroxidase in the young fruit were significantly reduced by SA spraying. However, the activity of another antioxidant enzyme, glutathione reductase in the young fruit was significantly enhanced by SA spraying. These results suggest that enzymes exerting their functions in different ways may be coordinately regulated by SA in the pear fruit. Our study indicates that treatment of SA sprays on the trees may provide further protection against postharvest disease of Ya Li pear fruit in practice and could be used as an alternative and economical approach to reduce application of chemical fungicides.     

Biological Control of Sclerotinia pseudotuberosa and Other Fungi During Moist Storage of Quercus robur Seeds

The fungal pathogen Sclerotinia pseudotuberosa is a major cause of deterioration during storage of Quercus robur seeds (acorns) and along with other, mainly saprotrophic fungi, contributes to the decline of viability and vigour in the acorn population. Hot-water thermotherapy (HWT; 41 °C for 2.5 h) killed the fungal pathogen S. pseudotuberosa and prolonged the storage life of acorns. The addition of the systemic fungicide benomyl to the HWT and/or the broad-spectrum fungicide thiram as a seed dressing further enhanced the storage life of acorns. Three fungal antagonists, Coniothyrium minitans, Trichoderma sp. (KW3) and Trichoderma virens (G20), were also applied as a film-coating to acorns using a polyvinylacetate sticker achieving ca. 10⁷–10⁸ viable conidia per acorn. The biological treatments provided protection against infection and the spread of infection of S. pseudotuberosa and other fungi on the acorns during storage over several months. All treated and stored acorns grew normally following sowing in seedbeds. The Trichoderma species were more effective than C. minitans, with T. virens being the most effective. T. virens reduced pathogen spread from acorns infected with S. pseudotuberosa to `clean' acorns when T. virens coated infected and 'clean' acorns were mixed together. However, T. virens was less effective than HWT at preventing the proliferation of this pathogen within individual acorns that were infected before coating. A combination of HWT and subsequent coating with T. virens provided a more effective control against both S. pseudotuberosa and saprotrophic fungi than when either treatment was applied alone.     

<Emphasis Type="Italic">Leptosphaeria maculans</Emphasis>, a fungal pathogen of <Emphasis Type="Italic">Brassica napus</Emphasis>, secretes a subtilisin-like serine protease

Leptosphaeria maculans,a fungal pathogen of Brassica napus, secretes large amounts of a 28kDa protein (SP2) in liquid culture. This protein shows high sequence similarity to secreted serine proteases from other ascomycetes and is the major component of culture filtrate with protease activity, as analysed on casein zymogels. The sp2 gene is expressed during infection of B.napuscotyledons when L. maculans hyphae are growing between mesophyll cells, as well as at later stages when the fungus invades the vascular tissue.     

Development of PCR Primers for a New Fusarium oxysporum Pathogenic on Paris Daisy (Argyranthemum frutescens L.)

The inverse PCR technique was applied to clone genomic DNA flanking insertion sites of sequences homologous to the transposable element Fot1 in the genome of a new pathogenic isolate of Fusarium oxysporum obtained from wilted Argyranthemum frutescens (Paris daisy). Based on the genomic flanking regions, a primer was designed which when paired to a second primer matching the Fot1 sequence allowed detection of this pathogen by PCR. The primer pair Mg5/Mg6 could specifically identify nine tested isolates of F. oxysporum from A. frutescens, when fungal genomic DNA was used as template. Moreover, the primer pair Mg5/Mg6 allowed successful detection of the pathogen in stem and root tissue from asymptomatic plants that were artificially inoculated with a representative isolate of F. oxysporum from A. frutescens.