MgSlt2, a cellular integrity MAP kinase gene of the fungal wheat pathogen Mycosphaerella graminicola, is dispensable for penetration but essential for invasive growth

Among expressed sequence tag libraries of Mycosphaerella graminicola isolate IPO323, we identified a full-length cDNA clone with high homology to the mitogen-activated protein (MAP) kinase Slt2 in Saccharomyces cerevisiae. This MAP kinase consists of a 1242-bp open reading frame, and encodes a 414-amino-acid protein. We designated this homolog MgSlt2, generated MgSlt2 knockout strains in M. graminicola isolate IPO323, and found several altered phenotypes in vitro as well as in planta. In yeast glucose broth, MgSlt2 disruptants showed a defective polarized growth in the tip cells upon aging, causing substantial local enlargements culminating in large swollen cells containing two to four nuclei. The MgSlt2 disruptants showed a significantly increased sensitivity to several fungicides, including miconazole (2x), bifonazole (>4x), imazalil (5x), and cyproconazole (10x), and were hypersensitive to glucanase. Unlike the wild type, MgSlt2 disruptants did not produce aerial mycelia and did not melanize on potato dextrose agar. Although cytological analysis in planta showed normal penetration of wheat stomata by the germ tubes of the MgSlt2 disruptants, subsequently formed hyphal filaments frequently were unable to branch out and establish invasive growth resulting in highly reduced virulence, and prevented pycnidia formation. Therefore, we conclude that MgSlt2 is a new pathogenicity factor in M. graminicola.     

Gene discovery and gene expression in the rice blast fungus, Magnaporthe grisea: analysis of expressed sequence tags

Over 28,000 expressed sequence tags (ESTs) were produced from cDNA libraries representing a variety of growth conditions and cell types. Several Magnaporthe grisea strains were used to produce the libraries, including a nonpathogenic strain bearing a mutation in the PMK1 mitogen-activated protein kinase. Approximately 23,000 of the ESTs could be clustered into 3,050 contigs, leaving 5,127 singleton sequences. The estimate of 8,177 unique sequences indicates that over half of the genes of the fungus are represented in the ESTs. Analysis of EST frequency reveals growth and cell type-specific patterns of gene expression. This analysis establishes criteria for identification of fungal genes involved in pathogenesis. A large fraction of the genes represented by ESTs have no known function or described homologs. Manual annotation of the most abundant cDNAs with no known homologs allowed us to identify a family of metallothionein proteins present in M. grisea, Neurospora crassa, and Fusarium graminearum. In addition, multiply represented ESTs permitted the identification of alternatively spliced mRNA species. Alternative splicing was rare, and in most cases, the alternate mRNA forms were unspliced, although alternative 5' splice sites were also observed.     

Intraspecific comparison and annotation of two complete mitochondrial genome sequences from the plant pathogenic fungus Mycosphaerella graminicola.

The mitochondrial genomes of two isolates of the wheat pathogen Mycosphaerella graminicola were sequenced completely and compared to identify polymorphic regions. This organism is of interest because it is phylogenetically distant from other fungi with sequenced mitochondrial genomes and it has shown discordant patterns of nuclear and mitochondrial diversity. The mitochondrial genome of M. graminicola is a circular molecule of approximately 43,960bp containing the typical genes coding for 14 proteins related to oxidative phosphorylation, one RNA polymerase, two rRNA genes and a set of 27 tRNAs. The mitochondrial DNA of M. graminicola lacks the gene encoding the putative ribosomal protein (rps5-like), commonly found in fungal mitochondrial genomes. Most of the tRNA genes were clustered with a gene order conserved with many other ascomycetes. A sample of 35 additional strains representing the known global mt diversity was partially sequenced to measure overall mitochondrial variability within the species. Little variation was found, confirming previous RFLP-based findings of low mitochondrial diversity. The mitochondrial sequence of M. graminicola is the first reported from the family Mycosphaerellaceae or the order Capnodiales. The sequence also provides a tool to better understand the development of fungicide resistance and the conflicting pattern of high nuclear and low mitochondrial diversity in global populations of this fungus.     

Expressed sequence tags from phytophthora sojae reveal genes specific to development and infection

Six unique expressed sequence tag (EST) libraries were generated from four developmental stages of Phytophthora sojae P6497. RNA was extracted from mycelia, swimming zoospores, germinating cysts, and soybean (Glycine max (L.) Merr.) cv. Harosoy tissues heavily infected with P. sojae. Three libraries were created from mycelia growing on defined medium, complex medium, and nutrient-limited medium. The 26,943 high-quality sequences obtained clustered into 7,863 unigenes composed of 2,845 contigs and 5,018 singletons. The total number of P. sojae unigenes matching sequences in the genome assembly was 7,412 (94%). Of these unigenes, 7,088 (90%) matched gene models predicted from the P. sojae sequence assembly, but only 2,047 (26%) matched P. ramorum gene models. Analysis of EST frequency from different growth conditions and morphological stages revealed genes that were specific to or highly represented in particular growth conditions and life stages. Additionally, our results indicate that, during infection, the pathogen derives most of its carbon and energy via glycolysis of sugars in the plant. Sequences identified with putative roles in pathogenesis included avirulence homologs possessing the RxLR motif, elicitins, and hydrolytic enzymes. This large collection of P. sojae ESTs will serve as a valuable public genomic resource.     

Intraspecific comparison and annotation of two complete mitochondrial genome sequences from the plant pathogenic fungus Mycosphaerella graminicola

The mitochondrial genomes of two isolates of the wheat pathogen Mycosphaerella graminicola were sequenced completely and compared to identify polymorphic regions. This organism is of interest because it is phylogenetically distant from other fungi with sequenced mitochondrial genomes and it has shown discordant patterns of nuclear and mitochondrial diversity. The mitochondrial genome of M. graminicola is a circular molecule of approximately 43,960bp containing the typical genes coding for 14 proteins related to oxidative phosphorylation, one RNA polymerase, two rRNA genes and a set of 27 tRNAs. The mitochondrial DNA of M. graminicola lacks the gene encoding the putative ribosomal protein (rps5-like), commonly found in fungal mitochondrial genomes. Most of the tRNA genes were clustered with a gene order conserved with many other ascomycetes. A sample of 35 additional strains representing the known global mt diversity was partially sequenced to measure overall mitochondrial variability within the species. Little variation was found, confirming previous RFLP-based findings of low mitochondrial diversity. The mitochondrial sequence of M. graminicola is the first reported from the family Mycosphaerellaceae or the order Capnodiales. The sequence also provides a tool to better understand the development of fungicide resistance and the conflicting pattern of high nuclear and low mitochondrial diversity in global populations of this fungus.     

Differential expression of mepA, mepC and smtE during growth and development of Microbotryum violaceum

Many fungi require a dimorphic switch from budding to filamentous growth to cause infection. Although the control of dimorphism has been elucidated for organisms such as Saccharomyces cerevisiae and Ustilago maydis, almost nothing is known about the control of mating and dimorphism in Microbotryum violaceum. M. violaceum mepA, mepC and smtE are homologs of genes whose encoded products act as, or interact with, components of the MAPK and cAMP-PKA pathways, conserved pathways that regulate mating and dimorphism in other fungi. A comparison of gene expression under various in vitro conditions was superimposed on a comparison of in vitro vs. in planta expression to yield a more complete picture of the expression of these genes in M. violaceum during fungal development. For the most part the expression of these genes was highest on low ammonium, intermediate for mated and in planta, and lowest on rich medium. As expected, under conditions of low ammonium, expression of the M. violaceum ammonium permease genes mepA and mepC mirrors that of S. cerevisiae MEP2 and U. maydis ump2. An intriguing possibility is that MepA is a sensor to signal when conditions are conducive for mating. The upregulation of smtE, which encodes a PAK kinase, suggests that the MAPK pathway regulates, at least partially, mating and might be linked to ammonium sensing/transport in M. violaceum.     

Tri1 in Fusarium graminearum encodes a P450 oxygenase

Gibberella zeae (asexual state Fusarium graminearum) is a major causal agent of wheat head blight and maize ear rot in North America and is responsible for contamination of grain with deoxynivalenol and related trichothecene mycotoxins. To identify additional trichothecene biosynthetic genes, cDNA libraries were prepared from fungal cultures under trichothecene-inducing conditions in culture and in planta. A gene designated LH1 that was highly expressed under these conditions exhibited only moderate (59%) similarity to known trichothecene biosynthetic cytochrome P450s. To determine the function of LH1, gene disruptants were produced and assessed for trichothecene production. Gene disruptants no longer produced 15-acetyldeoxynivalenol, which is oxygenated at carbon 7 (C-7) and C-8, but rather accumulated calonectrin and 3-deacetylcalonectrin, which are not oxygenated at either C-7 or C-8. These results indicate that gene LH1 encodes a cytochrome P450 responsible for oxygenation at one or both of these positions. Despite the relatively low level of DNA and amino acid sequence similarity between the two genes, LH1 from G. zeae is the probable homologue of Tri1, which encodes a cytochrome P450 required for C-8 oxygenation in F. sporotrichioides.     

氮源受限条件下植物病原真菌氮调控基因表达特性

研究证实植物病害的发生往往是由于植物病原真菌分泌的效应子诱导引起的,在此过程中,调控效应基因表达能够了解病原菌的侵染过程。细胞的营养状况据推测对于效应基因的表达起着重要的作用。已有研究表明在氮胁迫条件下相同效应基因的诱导作用在植株体内和体外是一致的,表明氮源缺乏的环境在植物体进化的早期就已经存在了。文章阐述了在氮受限条件下真菌致病系统中效应基因调控机制及其已经发现的氮调节基因特异性表达研究结果,通过对比几个病原菌中氮调控基因的功能,比较寄主植物体内和体外在氮限制条件下基因的诱导效应,从而揭示出氮的有效性在寄主植物病害发展过程中起到重要作用。     

Comparative bioinformatic analysis of genes expressed in common bean (Phaseolus vulgaris L.) seedlings.

To rapidly and cost-effectively generate gene expression data, we developed an annotated unigene database of common bean (Phaseolus vulgaris L.). In this study, 3 cDNA libraries were constructed from the bean breeding line SEL1308, 1 from young leaf and 2 from seedlings inoculated or not inoculated with the fungal pathogen Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara, which causes anthracnose in common bean. To this date, 5255 single-pass sequences have been included in the database after selection based on sequence quality. These ESTs were trimmed and clustered using the computer programs Phred and CAP3 to form a unigene collection of 3126 unique sequences. Within clusters, 318 single nucleotide polymorphisms (SNPs) and 68 insertions-deletions (indels) were found, indicating the presence of paralogous gene families in our database. Each unigene sequence was analyzed for possible function using their similarity to known genes represented in the GenBank database and classified into 14 categories. Only 314 unigenes showed significant similarities to Phaseolus genomic sequences and P. vulgaris ESTs, which indicates that 90% (2818 unigenes) of our database represent newly discovered common bean genes. In addition, 12% (387 unigenes) were shown to be specific to common bean. This study represents a first step towards the discovery of novel genes in beans and a valuable source of molecular markers for expressed gene tagging and mapping.     

MgMfs1, a major facilitator superfamily transporter from the fungal wheat pathogen Mycosphaerella graminicola, is a strong protectant against natural toxic compounds and fungicides

MgMfs1, a major facilitator superfamily (MFS) gene from the wheat pathogenic fungus Mycosphaerella graminicola, was identified in expressed sequence tag (EST) libraries. The encoded protein has high homology to members of the drug:H(+) antiporter efflux family of MFS transporters with 14 predicted transmembrane spanners (DHA14), implicated in mycotoxin secretion and multidrug resistance. Heterologous expression of MgMfs1 in a hypersensitive Saccharomyces cerevisiae strain resulted in a strong decrease in sensitivity of this organism to a broad range of unrelated synthetic and natural toxic compounds. The sensitivity of MgMfs1 disruption mutants of M. graminicola to most of these compounds was similar when compared to the wild-type but the sensitivity to strobilurin fungicides and the mycotoxin cercosporin was increased. Virulence of the disruption mutants on wheat seedlings was not affected. The results indicate that MgMfs1 is a true multidrug transporter that can function as a determinant of pathogen sensitivity and resistance to fungal toxins and fungicides.     

Phenotypic and genetic analysis of the Triticum monococcum-Mycosphaerella graminicola interaction

Here, the aim was to understand the cellular and genetic basis of the Triticum monococcum-Mycosphaerella graminicola interaction. Testing for 5 yr under UK field conditions revealed that all 24 T. monococcum accessions exposed to a high level of natural inocula were fully resistant to M. graminicola. When the accessions were individually inoculated in the glasshouse using an attached leaf seeding assay and nine previously characterized M. graminicola isolates, fungal sporulation was observed in only three of the 216 interactions examined. Microscopic analyses revealed that M. graminicola infection was arrested at four different stages post-stomatal entry. When the inoculated leaves were detached 30 d post inoculation and incubated at 100% humidity, abundant asexual sporulation occurred within 5 d in a further 61 interactions. An F(2) mapping population generated from a cross between T. monococcum accession MDR002 (susceptible) and MDR043 (resistant) was inoculated with the M. graminicola isolate IPO323. Both resistance and in planta fungal growth were found to be controlled by a single genetic locus designated as TmStb1 which was linked to the microsatellite locus Xbarc174 on chromosome 7A(m). Exploitation of T. monococcum may provide new sources of resistance to septoria tritici blotch disease.     

Expressed sequence tags from the flower pathogen Claviceps purpurea

The ascomycete Claviceps purpurea (ergot) is a biotrophic flower pathogen of rye and other grasses. The deleterious toxic effects of infected rye seeds on humans and grazing animals have been known since the Middle Ages. To gain further insight into the molecular basis of this disease, we generated about 10 000 expressed sequence tags (ESTs)—about 25% originating from axenic fungal culture and about 75% from tissues collected 6–20 days after infection of rye spikes. The pattern of axenic vs. in planta gene expression was compared. About 200 putative plant genes were identified within the in planta library. A high percentage of these were predicted to function in plant defence against the ergot fungus and other pathogens, for example pathogenesis-related proteins. Potential fungal pathogenicity and virulence genes were found via comparison with the pathogen–host interaction database (PHI-base; http://www.phi-base.org ) and with genes known to be highly expressed in the haustoria of the bean rust fungus. Comparative analysis of Claviceps and two other fungal flower pathogens (necrotrophic Fusarium graminearum and biotrophic Ustilago maydis ) highlighted similarities and differences in their lifestyles, for example all three fungi have signalling components and cell wall-degrading enzymes in their arsenal. In summary, the analysis of axenic and in planta ESTs yielded a collection of candidate genes to be evaluated for functional roles in this plant–microbe interaction.