禾谷镰孢FgCYP51B蛋白F511L突变对分生孢子产量及其对烯唑醇敏感性的影响

Phe511是禾谷镰孢甾醇-14α-脱甲基酶FgCYP51B活性口袋中的一个重要氨基酸。本研究中,我们探究了FgCYP51B蛋白中该位点突变后对禾谷镰孢主要生物学表型的影响,并通过分子对接探讨了可能的原因。结果表明禾谷镰孢FgCYP51B-F511L突变体在菌落形态、生长速率等表型上与野生型菌株PH-1没有明显差异。但是F511L突变导致分生孢子的产量严重降低,对烯唑醇的敏感性增强。分子对接发现突变后亮氨酸的长侧链使得烯唑醇的甲基发生扭转,侧链朝向发生改变,更有利于与蛋白受体形成较强的疏水作用,这可能是导致FgCYP51B-F511L突变体对烯唑醇敏感性增强的原因。     

Expression and homology modelling of sterol 14α‐demethylase of Magnaporthe grisea and its interaction with azoles

BACKGROUND: Magnaporthe grisea (Hebert) ME Barr infection is one of the most serious diseases for cultivated rice in the world. Sterol 14α‐demethylase (CYP51) is an important drug target for microbial pathogenic infections. To exploit specific and effective fungicides for M. grisea better, the authors have analysed the characteristics of interaction between sterol 14α‐demethylase from M. grisea (MGCYP51) and azoles. MGCYP51 with truncation of N‐terminal residues was cloned and expressed in E. coli, difference binding spectra of MGCYP51 induced by addition of four commercial azoles were determined and molecular modelling of MGCYP51 based on the crystal structure of Mycobacterium tuberculosis Lehmann & Newman and docking with the azoles were performed. RESULTS: The affinity of the azoles for MGCYP51 was positively correlated with their hydrophobicity. Amino acid residues Tyr112, Phe120, Phe220, His308 and Phe497 of MGCYP51, forming a large hydrophobic cavity, are the key residues interacting with azole fungicides. Furthermore, Phe220 and Phe497 are fungus and species specific respectively. CONCLUSION: The results suggest that the more potent azole fungicides for MGCYP51 should possess more hydrophobic groups interacting with residues Phe220 and Phe497. Copyright © 2009 Society of Chemical Industry     

Transfer of the β‐tubulin gene of Botrytis cinerea with resistance to carbendazim into Fusarium graminearum

BACKGROUND: Resistance to carbendazim and other benzimidazole fungicides in Botrytis cinerea (Pers. ex Fr.) and most other fungi is usually conferred by mutation(s) in a single chromosomal β‐tubulin gene, often with several allelic mutations. In Fusarium graminearum Schwade, however, carbendazim resistance is not associated with a mutation in the corresponding β‐tubulin gene. RESULTS: The β‐tubulin gene conferring carbendazim resistance in B. cinerea was cloned and connected with two homologous arms of the β‐tubulin gene of F. graminearum by using a double‐joint polymerase chain reaction (PCR). This fragment was transferred into F. graminearum via homologous double crossover at the site where the β‐tubulin gene of F. graminearum is normally located (the β‐tubulin gene of F. graminearum had been deleted). The transformants were confirmed and tested for their sensitivity to carbendazim. CONCLUSION: The β‐tubulin gene conferring carbendazim resistance in B. cinerea could not express this resistance in F. graminearum, as transformants were still very sensitive to carbendazim. Copyright © 2010 Society of Chemical Industry     

禾谷镰孢菌FgCYP51B蛋白Y137H影响分生孢子及子囊孢子发育

<正>小麦赤霉病(Fusarium Head Blight,FHB),主要是由镰刀菌属真菌引起的一种流行性毁灭性病害。在世界各小麦种植区尤其是温暖潮湿和半潮湿地区发生最为严重~([1,2])。它不仅能够降低小麦产量,其染病麦粒中还会产生对人畜健康严重危害的真菌毒素~([3])。在不同的地区由于气候因素和地理环境的不同,分布着不同的致病菌株。在我国,     

禾谷镰刀菌拮抗菌Bacillus amyloliquefaciens AX-3的分离鉴定及拮抗物质的鉴定

禾谷镰刀菌Fusarium graminearum可引起小麦赤霉病,该病严重威胁我国小麦的质量安全。本研究从生物防治的角度出发,分离筛选拮抗菌,研究拮抗菌的拮抗特性和拮抗能力。试验采用系列稀释法和平板对峙法从小麦样品中分离筛选获得禾谷镰刀菌拮抗菌AX-3,根据16S rDNA基因、gyrB基因序列构建的系统发育进化树以及生理生化特性结果,菌株AX-3被鉴定为解淀粉芽胞杆菌Bacillus amyloliquefaciens。通过平板对峙法测定拮抗谱,结果显示AX-3对多种镰刀菌属病原真菌都具有良好的拮抗特性,抑菌圈半径为17~32 mm,表明AX-3具有广谱的拮抗性能。采用柱层析及制备液相色谱法对AX-3中的拮抗物质进行分离纯化,然后使用高分辨质谱及核磁共振的方法对拮抗物质进行结构鉴定表明,解淀粉芽胞杆菌AX-3产生的一种抗菌物质为macrolactin A。     

Application of cycleave PCR to the detection of a point mutation (F167Y) in the β2‐tubulin gene of Fusarium graminearum

BACKGROUND: Resistance of Fusarium graminearum to the benzimidazole fungicide carbendazim is caused by point mutations in the β₂‐tubulin gene (FGSG_06611.3). The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field isolates in China. It is important to find a suitable method for rapid detection and quantification of this point mutation in the F. graminearum populations. RESULTS: A pair of primers, Codon167F/Codon167R, were designed to amplify a fragment containing the mutation site, and two cycling probes labelled with different fluorescent reporters were used to detect whether the mutation was present. A cycleave real‐time PCR method was developed for rapid determination of the frequency of this point mutation in 282 F. graminearum perithecia collected from different fields in Jiangsu Province, China. The mutation frequency in ascospores from the perithecia to carbendazim by a spore germination assay was 6.0%, while the frequency of the point mutation at codon 167 by the cycleave real‐time PCR assay was 3.9%. CONCLUSION: The cycleave real‐time PCR method is suitable for accurate detection of the codon 167 point mutation. The frequency of this mutation in the β₂‐tubulin gene represents the resistance frequency in F. graminearum populations to carbendazim. Copyright © 2011 Society of Chemical Industry     

A comparative molecular field analysis study of obtusifoliol 14α-methyl demethylase inhibitors

This report describes the development of a Comparative Molecular Field Analysis (CoMFA) model from a set of obtusifoliol 14α-methyl demethylase (DM) inhibitors to aid in the design of herbicides targeting sterol biosynthesis. CoMFA is a three-dimensional (3-D) quantitative structure–activity relationship (QSAR) method that is useful in the probing of receptor binding sites when experimental structure data are unavailable. Conformational analysis and SAR of some rigid and active analogs were used to build the initial model using the active analog hypothesis. The model was subsequently used to design compounds that retain the active site shape requirements, but incorporate physical properties that favor soil-applied herbicidal action. In addition, a second-generation CoMFA model incorporating the newly designed inhibitors was developed and represents the current understanding of the DM binding site. This model was derived from a pharmacophore developed from two methods, the active analog approach as well as from the Catalyst program. The fact that two independent methods produced a similar pharmacophore strengthens the validity of the model. © 1999 Society of Chemical Industry     

The Y123H substitution perturbs FvCYP51B function and confers prochloraz resistance in laboratory mutants of Fusarium verticillioides

Fusarium verticillioides reduces corn yield and contaminates infected kernels with the toxin fumonisin, which is harmful to humans and animals. Previous research has demonstrated that F. verticillioides can be controlled by the azole fungicide prochloraz. Currently, prochloraz is used as a foliar spray to control maize disease in China, which will increase the risk of resistance. Although F. verticillioides resistance to prochloraz has not been reported in the field, possible resistance risk and mechanisms resulting in prochloraz resistance were explored in the laboratory. Four prochloraz‐resistant strains of F. verticillioides were generated by successive selection on fungicide‐amended media. The mycelial growth rates of the mutants were inversely related to the level of resistance. All four mutants were cross‐resistant to the triazole fungicides triadimefon, tebuconazole and difenoconazole, but not to the multisite fungicide chlorothalonil or to the MAP/histidine‐kinase inhibitor fungicide fludioxonil. Based on the Y123H mutation in FvCYP51B, the four resistant mutants were subdivided into two genotypes: PCZ‐R1 mutants with wildtype FvCYP51B and PCZ‐R2 mutants with substitution Y123H in FvCYP51B. Wildtype FvCYP51B complemented the function of native ScCYP51 in Saccharomyces cerevisiae YUG37::erg11, whereas Y123H‐mutated FvCYP51B did not. For the PCZ‐R1 mutants, induced expression of FvCYP51A increased resistance to prochloraz. For the PCZ‐R2 mutants, disruption of FvCYP51B function by the Y123H substitution caused constitutive up‐regulation of FvCYP51A expression and thus resistance to prochloraz.     

The mechanism of resistance to sterol 14α demethylation inhibitors in a mutant (Erg 40) of Ustilago maydis

Resistance to DMI fungicides is a problem in both agriculture and medicine. Several mechanisms of resistance exist, but, as yet, few have been characterised in field resistant strains of plant pathogens. One approach to evaluating the role of mutations in the sterol 14α demethylase (14DM) target site requires cloning this gene and confirming its identity by complementation in an appropriate mutant. The azole‐resistant mutant, Erg 40, of Ustilago maydis which is totally blocked at the 14α demethylation step in sterol biosynthesis seems to be suitable for such expression studies. Transformation of Erg 40 with a plasmid containing the yeast 14α demethylase (CYP51A1) gene removed the block in sterol biosynthesis and generated azole‐sensitive transformants. Detailed analysis of these transformants failed to detect the presence of the yeast gene and suggested, instead, that changes in sterol biosynthesis resulted simply from the transformation protocol and not from the incorporation of extracellular DNA. Subsequent sequence analysis has revealed a mutation in the 14α demethylase gene of Erg 40. The results suggest that azole resistance in Erg 40 is not simply controlled by this mutation but involves some additional regulatory function, and consequently Erg 40 is not suitable for complementation studies with CYP51A1 genes. © 2000 Society of Chemical Industry     

Phylogenetic position and nucleotide diversity of Fusarium oxysporum f. sp. vanillae worldwide based on translation elongation factor 1α sequences

Fusarium oxysporum f. sp. vanillae is considered the most important fungus affecting vanilla crops around the world, causing rot on vanilla roots and stems. Previous studies showed that the ability to infect vanilla plants is a polyphyletic trait among strains of the Fusarium oxysporum species complex (FOSC). The same studies proposed a single origin for F. oxysporum f. sp. vanillae isolates sampled from Mexico, the centre of origin and distribution of vanilla. The aim of this work was to test the hypothesis of the monophyletic origin of a wider sample of isolates of F. oxysporum f. sp. vanillae infecting Mexican vanilla and estimate nucleotide diversity of pathogen isolates from the main vanilla‐producing countries. Sequence data for the TEF1α gene from 106 isolates was assembled. The phylogenetic analyses suggest that some Mexican isolates of F. oxysporum f. sp. vanillae belong in two well‐supported clades, mixed with isolates from Madagascar, Indonesia, Réunion and Comoros. The phylogenetic position of other Indonesian and Mexican isolates is unresolved. Estimations of nucleotide diversity showed that the population from Mexico is genetically more diverse than the other three populations from Madagascar, Indonesia and Réunion. The results support a polyphyletic origin of vanilla‐infecting isolates of F. oxysporum worldwide, and also reject the proposition that Mexican isolates have a single origin. The phylogenetic optimizations over the strict consensus tree of the ability to infect vanilla plants suggest that pathogenic strains around the world are the product of multiple shifts of pathogenesis and dispersion events.     

入侵害虫松树蜂气味结合蛋白与其相关信息化学物质的分子对接

为探究入侵我国的重大林业检疫性害虫松树蜂Sirex noctilio的气味结合蛋白(odorant binding protein,OBP)与其相关信息化学物质的结合模式和结合能力,采用Swiss-model在线服务器对松树蜂触角中高表达的4种OBP序列进行同源模建,采用Procheck、Verify_3D和ERRAT程序对模建质量进行评价;并应用Autodock软件对OBP模型和相关信息化学物质进行分子对接分析。结果显示,在松树蜂触角中高表达的4种蛋白为SnocOBP4、SnocOBP6、SnocOBP9和SnocOBP12,其同源模建所得模型质量较好:均满足拉氏构象图中氨基酸位于最佳合理区的数量大于90%的条件;三维结构与一级结构的兼容性评分大于0.2;所得ERRAT值分别为85.71%、95.10%、98.15%和91.82%。分子对接显示,与松树挥发物α-蒎烯和β-蒎烯结合最好的蛋白是SnocOBP12,结合能分别为-5.36 kJ/mol和-5.48 kJ/mol;与雌性信息素成分顺-9-二十九烯结合最好的蛋白是SnocOBP4,结合能为-5.42 kJ/mol;与雄性信息素成分顺-3-癸烯醇和顺-4-癸烯醇结合最好的蛋白是SnocOBP9,结合能分别为-4.37 kJ/mol和-4.45 k J/mol;而3-蒈烯、顺-7-二十七烯和反-2,4-癸二烯醛等气味分子与SnocOBP6的结合能较低,结合能最低的是顺-7-二十七烯,为-6.08 k J/mol。雄蜂主要信息素成分顺-3-癸烯醇与其同分异构体反-3-癸烯醇相比,与SnocOBP9的结合能较低;但是反-3-癸烯醇能以较强的氢键竞争SnocOBP9结合位点。研究表明这4种松树蜂OBP与相应植物挥发物或雌雄信息素具有较好的亲和力,参与了松树蜂对不同信息化学物质的识别过程。     

Differential metabolic response of barley genotypes,varying in resistance,to trichothecene‐producing and ‐nonproducing (tri5−) isolates of Fusarium graminearum

Resistance of barley to Fusarium graminearum was studied using a pair each of resistant and susceptible black and yellow barley lines. The spikelets were inoculated with a trichothecene‐producing isolate, a trichothecene‐nonproducing isolate (tri5^?), or a mock solution. Spikelets were collected 72 h after inoculation and metabolites were analysed using a LC‐hybrid MS system. Metabolite abundances were used to identify the constitutive (RRC) and induced resistance‐related metabolites (RRI). The pathogen virulence factor, DON, and its plant detoxification product, DON‐3‐O‐glucoside (D3G), were also identified and designated as resistance‐indicator (RI) metabolites. The RRC, RRI and RI metabolites were putatively identified. Jasmonic acid was significantly induced in barley following inoculation with a trichothecene‐producing isolate, but not with a tri5^? isolate. The former isolate reduced the induction of both the number and amount of RR metabolites. The metabolites cinnamic acid, sinapoyl alcohol, coniferin, catechin and naringin were identified only in response to the inoculation with a tri5^? mutant. The abundances of p‐coumaric acid, coniferaldehyde and sinapaldehyde increased more in response to the tri5^? mutant than to the trichothecene‐producing isolate. The total amount of DON synthesized and its conversion to D3G varied greatly between the resistant and susceptible black barley, but not in yellow barley. Interestingly, an increase in the amount of total DON produced was associated with a decrease in the conversion of DON to D3G. The roles of RRC, RRI and RI metabolites in plant defence and their further use as potential biomarkers in screening are discussed.