Identification of Botrytis cinerea genes up-regulated during infection and controlled by the Galpha subunit BCG1 using suppression subtractive hybridization (SSH)

The Galpha subunit BCG1 plays an important role during the infection of host plants by Botrytis cinerea. Delta bcg1 mutants are able to conidiate, penetrate host leaves, and produce small primary lesions. However, in contrast to the wild type, the mutants completely stop invasion of plant tissue at this stage; secondary lesions have never been observed. Suppression subtractive hybridization (SSH) was used to identify fungal genes whose expression on the host plant is specifically affected in bcg1 mutants. Among the 22 differentially expressed genes, we found those which were predicted to encode proteases, enzymes involved in secondary metabolism, and others encoding cell wall-degrading enzymes. All these genes are highly expressed during infection in the wild type but not in the mutant. However, the genes are expressed in both the wild type and the mutant under certain conditions in vitro. Most of the BCG1-controlled genes are still expressed in adenylate cyclase (bac) mutants in planta, suggesting that BCG1 is involved in at least one additional signaling cascade in addition to the cAMP-depending pathway. In a second SSH approach, 1,500 clones were screened for those that are specifically induced by the wild type during the infection of bean leaves. Of the 22 BCG1-controlled genes, 11 also were found in the in planta SSH library. Therefore, SSH technology can be successfully applied to identify target genes of signaling pathways and differentially expressed genes in planta.     

Deletion of the SNP1 trypsin protease from Stagonospora nodorum reveals another major protease expressed during infection

The wheat fungal pathogen Stagonospora nodorum produces an extracellular trypsin-like protease, SNP1, during early stages of hyphal growth on the surface of host leaves and during penetration. Variation of SNP1 mRNA levels and enzyme activity during infection, were correlated with levels of aggressiveness of three wild-type isolates. SNP1 was deleted in two wild-type isolates using a gene replacement strategy. SNP1-deleted mutants completely lacked trypsin activity in vitro and on inoculated wheat leaves, but were not reduced in pathogenicity. SNP1-deleted mutants still have 50% of the total alkaline protease activity of wild-type. This residual activity comes from a previously undetected alkaline protease with subtilisin-like substrate and inhibitor specificities, which is produced in vitro and on host leaves. We hypothesize that this subtilisin protease may act in concert with SNP1 and may compensate for the loss of trypsin protease activity in the SNP1-deletion mutants.     

灰葡萄孢丝裂原活化蛋白激酶编码基因bmp1和bmp3的功能

【背景】植物病原真菌丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)信号途径参与病菌有性生殖、细胞壁完整、菌丝侵染、致病力、胁迫响应等过程,灰葡萄孢MAPK信号途径参与病菌生长发育、致病力以及胁迫响应,但MAPK信号途径基因在灰葡萄孢中的功能尚未完全阐明,该信号途径对灰葡萄孢的生长发育和致病力的调控机制尚不明确。【目的】明确灰葡萄孢MAPK编码基因bmp1、bmp3在病菌生长发育、致病力以及氧化胁迫响应过程的功能,为进一步阐明MAPK信号途径调控灰葡萄孢生长发育和致病力的分子机制奠定基础。【方法】利用RNAi技术构建灰葡萄孢MAPK编码基因bmp1和bmp3的RNAi突变体,并以野生型BC22菌株为对照,对bmp1和bmp3基因的RNAi突变体的表型、致病力以及对氧化胁迫的敏感性进行分析。【结果】灰葡萄孢bmp1和bmp3基因的RNAi突变体其菌落形态、菌丝形态均与野生型BC22菌株没有明显差别;bmp1基因的RNAi突变体生长速率明显减慢,分生孢子产量明显降低;bmp3基因的RNAi突变体的生长速率与野生型BC22菌株没有明显差别,不能产生分生孢子。bmp1和bmp3基因的RNAi突变体在番茄果实的表面均不能产生明显的致病症状,而且不能穿透玻璃纸。bmp1基因的RNAi突变体在含有H_2O_2的培养基上受抑制的程度显著低于野生型,而在含甲萘醌的培养基上受抑制的程度显著高于野生型;bmp3基因的RNAi突变体在含有H_2O_2和甲萘醌的培养基受抑制的程度均显著高于野生型。【结论】灰葡萄孢bmp1基因正调控病菌生长、分生孢子形成、致病力和穿透能力,参与调控病菌对氧化胁迫的响应;灰葡萄孢bmp3基因正调控病菌分生孢子形成、致病力、穿透能力以及对氧化胁迫的响应。     

G protein signaling mediates developmental processes and pathogenesis of Alternaria alternata

A G protein alpha subunit gene (AGA1) has been cloned and characterized from a toxigenic and necrotrophic Alternaria alternata pathogen. Targeted disruption of AGA1 in the apple pathotype of A. alternata gave rise to mutants that differed in colony and conidial morphology as well as sporulation. The conidia of wild type and deltaAGA1 mutants showed equal germination on cellulose membranes. However, wild-type germ tubes formed readily from different points around the conidia, grew randomly, and were often branched, whereas those of the mutants formed only at one or both ends of the conidia and tended to grow in straight paths. Targeted disruption of AGA1 also resulted in reduction of pathogenicity on apple leaves, although the mutant produced host-specific AM-toxin, a fungal secondary metabolite associated with pathogenicity of the pathogen, at levels similar to the wild-type strain. Measurement of the intracellular cAMP levels of the mutant revealed that it was consistently higher than that of the wild type, indicating that AGA1 negatively regulates cAMP levels similar to mammalian Galphai systems. These results indicate that the signal transduction pathway represented by AGA1 appears to be involved in developmental pathways leading to sporulation and pathogenesis of A. alternata.     

The Galpha subunit BCG1, the phospholipase C (BcPLC1) and the calcineurin phosphatase co-ordinately regulate gene expression in the grey mould fungus Botrytis cinerea

The Gα subunit BCG1 is essential for pathogenicity of the grey mould fungus Botrytis cinerea . Several processes such as the transition from primary infection to secondary invasive growth and the production of the phytotoxin botrydial are regulated by BCG1 via a cAMP-independent pathway. Our recent finding that the botrydial biosynthesis genes belong to the group of Ca²⁺/calcineurin-dependent genes suggested for the first time a connection between this Gα subunit and the calcineurin signalling pathway. To investigate whether this co-regulation of genes by BCG1 and calcineurin is a common feature, a cDNA macroarray approach was used to compare the gene expression pattern of the wild-type and the Δ bcg1 mutant, non-treated or treated with the calcineurin inhibitor cyclosporin A. We identified three sets of genes whose expression was regulated either by both BCG1 and calcineurin, or only by one of them. Among the BCG1/calcineurin-co-regulated genes, we found a new gene cluster coding for a yet unknown polyketide secondary metabolite. Furthermore, we show for the first time in a phytopathogenic fungus that the phospholipase C (BcPLC1) is a component of the BCG1- and calcineurin-dependent signalling pathway as several BCG1- and calcineurin-dependent genes were downregulated in bcplc1 knock-down mutants.     

The Fus3/Kss1 MAP kinase homolog Amk1 regulates the expression of genes encoding hydrolytic enzymes in Alternaria brassicicola

Mitogen-activated protein (MAP) kinases have been shown to be required for virulence in diverse phytopathogenic fungi. To study its role in pathogenicity, we disrupted the Amk1 MAP kinase gene, a homolog of the Fus3/Kss1 MAP kinases in Saccharomyces cerevisiae, in the necrotrophic Brassica pathogen, Alternaria brassicicola. The amk1 disruption mutants showed null pathogenicity on intact host plants. However, amk1 mutants were able to colonize host plants when they were inoculated on a physically damaged host surface, or when they were inoculated along with nutrient supplements. On intact plants, mutants expressed extremely low amounts of several hydrolytic enzyme genes that were induced over 10-fold in the wild-type during infection. These genes were also dramatically induced in the mutants on wounded plants. These results imply a correlation between virulence and the expression level of specific hydrolytic enzyme genes plus the presence of an unidentified pathway controlling these genes in addition to or in conjunction with the Amk1 pathway.     

Gene cluster of Pseudomonas syringae pv. "phaseolicola" controls pathogenicity of bean plants and hypersensitivity of nonhost plants

Loss of the ability of Pseudomonas syringae pv. "phaseolicola" NPS3121 to elicit a hypersensitive response on tobacco and other nonhost plants was associated with loss of pathogenicity on the susceptible host bean. Eight independent, prototrophic transposon Tn5 insertion mutants which had lost the ability to elicit a hypersensitive response on tobacco plants were identified. Six of these mutants no longer produced disease lesions on primary leaves of the susceptible bean cultivar Red Kidney and failed to elicit a hypersensitive response on the resistant bean cultivar Red Mexican and on the nonhost plants tomato, cowpea, and soybean. The two remaining mutants had reduced pathogenicity on Red Kidney bean and elicited variable hypersensitive responses on the other plants tested. Southern blot analysis indicated that each mutant carried a single independent Tn5 insertion in one of three EcoRI fragments of about 17, 7, and 5 kilobases. Marker exchange mutagenesis further supported the conclusion that the pleiotropic mutant phenotype was not associated with multiple Tn5 insertions. A genomic library of the wild-type strain was constructed in the cosmid vector pLAFR3. A recombinant plasmid, designated pPL6, that carried P. syringae pv. "phaseolicola" genomic sequences was identified by colony hybridization. This plasmid restored the wild-type phenotype to all but one mutant, suggesting that genes affected by the insertions were clustered. Structural analysis of pPL6 and the wild-type genome indicated that the 17- and 5-kilobase EcoRI fragments were contiguous in the strain NPS3121 genome.     

农杆菌介导的小孢拟盘多毛孢遗传转化及突变体筛选

本研究通过农杆菌EHA105介导的方法,以含潮霉素抗性基因和GFP基因的双元载体pCAMBgfp为转化载体,对小孢拟盘多毛孢Pestalotiopsis microspora KFRD-2菌株进行遗传转化。基于潮霉素及GFP荧光抗性进行转化子的初步筛选,随后,进一步对转化子的菌落特征、菌丝生长速率、产孢量、荧光稳定性及致病力进行验证。结果获得阳性转化子100余株,转化效率达200个转化子/10⁶个孢子。大部分转化子与野生型菌株无明显形态及致病力差异。同时,获得了14株菌丝形态、产孢量或致病力与野生型存在明显差异的突变株,可用于小孢拟盘多毛孢关键致病基因的挖掘验证及致病机理等研究。     

Isolation and characterization of protease overproducing mutants of Trichoderma harzianum

Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of fungal hydrolytic enzymes is considered as the main mechanism involved in the antagonistic process. Strain Trichoderma harzianum T334 is a potential biocontrol agent against plant pathogenic fungi with the ability to produce low levels of proteases constitutively. To improve its fungal antagonistic capacity, mutagenetic program was undertaken for the construction of protease overproducing derivates. The mutant strains were obtained by means of UV-irradiation and were selected for p-fluorophenyl-alanine resistance or altered colony morphology. It was revealed by means of specific chromogenic protease substrates that both trypsin-like and chymotrypsin-like protease secretion was elevated in most of the mutant strains. The profiles of isoenzymes were different between the mutants and the wild-type strain, when examined by gel filtration chromatography. Certain mutants proved to be better antagonists against plant pathogens in in vitro antagonism experiments. This study suggests the possibility of using mutants with improved constitutive extracellular protease secretion against plant pathogenic fungi.     

The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.     

cDNA microarray analysis of Helicobacter pylori-mediated alteration of gene expression in gastric cancer cells

Helicobacter pylori infection stimulates several intracellular signaling pathways and is accompanied by increased gene expression in gastric epithelial cells. High-density cDNA microarray was used to characterize the mRNA expression profile of genes in human gastric cancer cells (MKN45, AGS) cocultured with H. pylori. Coculture with cag pathogenicity island (PAI)-positive H. pylori (wild-type) significantly up-regulated mRNA expression in 8 of 2304 genes tested. In 6 (interleukin-8, I(kappaB)alpha, A20, ERF-1, keratin K7, glutathione peroxidase) of the 8 genes, up-regulation was confirmed by RT-PCR. In coculture with isogenic cagE-negative mutant ((Delta)cagE), which encodes a type IV secretion system with other genes in the cag PAI, no significant up-regulation was found. We further analyzed the role of A20. Transfection of expression vector encoding A20 resulted in an inhibition of H. pylori-mediated NF-kappaB activation, indicating that H. pylori-mediated A20 expression could be a negative regulator of NF-kappaB activation. Taken together, these results indicate the importance of microarray technology as a tool for analyzing the complex interplay between H. pylori and the host.     

Saccharomyces cerevisiae SSD1 orthologues are essential for host infection by the ascomycete plant pathogens Colletotrichum lagenarium and Magnaporthe grisea

Fungal plant pathogens have evolved diverse strategies to overcome the multilayered plant defence responses that confront them upon host invasion. Here we show that pathogenicity of the cucumber anthracnose fungus, Colletotrichum lagenarium, and the rice blast fungus, Magnaporthe grisea, requires a gene orthologous to Saccharomyces cerevisiae SSD1, a regulator of cell wall assembly. Screening for C. lagenarium insertional mutants deficient in pathogenicity led to the identification of ClaSSD1. Following targeted gene replacement, appressoria of classd1 mutants retained the potential for penetration but were unable to penetrate into host epidermal cells. Transmission electron microscopy suggested that appressorial penetration by classd1 mutants was restricted by plant cell wall-associated defence responses, which were observed less frequently with the wild-type strain. Interestingly, on non-host onion epidermis classd1 mutants induced papilla formation faster and more abundantly than the wild type. Similarly, colonization of rice leaves by M. grisea was severely reduced after deletion of the orthologous MgSSD1 gene and attempted infection by the mutants was accompanied by the accumulation of reactive oxygen species within the host cell. These results suggest that appropriate assembly of the fungal cell wall as regulated by SSD1 allows these pathogens to establish infection by avoiding the induction of host defence responses.     

Identification of a second Listeria secA gene associated with protein secretion and the rough phenotype

We describe the identification and characterization of a second secA gene in Listeria monocytogenes. This gene, termed secA2, is involved in smooth-rough phenotypic variation and secA2 expression contributes to bacterial virulence. Spontaneous rough (R-) variants of L. monocytogenes grow in chains and form rough colonies on solid media. A subset of R-variants, classified here as type I, also shows reduced secretion of an autolysin, p60. We find that disruptions and in frame deletions in secA2 confer phenotypes identical to those of spontaneous type I R-variants. Additionally, the secA2 genes from two spontaneous type I R-variants encoded truncated SecA2 proteins. Mutations were not found in the secA2 genes from the remaining five independent R-variants, four of which showed a distinct (type II) rough morphology and secreted wild-type levels of p60. Expression of an epitope-tagged SecA2 in the DeltasecA2 strain and a spontaneous R-variant restored normal cell septation and smooth colony morphology. These data suggest that mutations in both secA2 and other genes contribute to smooth-rough phase variation in L. monocytogenes. Expression of the full-length SecA2 also promotes secretion of p60 and a set of additional L. monocytogenes proteins. We hypothesize that SecA2-dependent protein secretion plays a role in the colonization of environmental and host surfaces.