Grisea Mutant Research Articles

Transmission of the G143A QoI‐resistance point mutation through anastomosis in Magnaporthe grisea

Soon after the introduction of Qo inhibitor fungicides in 1996, the point mutation leading to the amino acid exchange glycine to alanine at the 143 position of the mitochondrial cytochrome b gene was identified as the main cause of resistance. The present study describes the role of anastomosis in the transmission of the G143A mutation in Magnaporthe grisea. Two M. grisea mutants were co-cultivated on oatmeal agar and also co-inoculated on barley leaves. The mutants differed by the presence of the G143A mutation in one isolate and a disrupted AOX gene by insertion of a hygromycin gene in the other (M-145). Specific resistant (r) or sensitive (s) phenotypes of 409 monosporic cultures were determined on media amended with either hygromycin (H) or azoxystrobin (S) plus SHAM. The phenotypes identified reflected not only the phenotypes of mutants M-145 and G143A but also the wild-type parent phenotype HsSs and a new HrSr isolate. Identification of the M. grisea phenotypes HrSr and HsSs suggests that anastomosis occurred during co-cultivation and co-inoculation of the mutants M-145 and G143A, allowing the transfer of the G143A point mutation from the QoI-resistant isolate to the susceptible isolate.

A Differential Genome-Wide Transcriptome Analysis: Impact of Cellular Copper on Complex Biological Processes like Aging and Development

The regulation of cellular copper homeostasis is crucial in biology. Impairments lead to severe dysfunctions and are known to affect aging and development. Previously, a loss-of-function mutation in the gene encoding the copper-sensing and copper-regulated transcription factor GRISEA of the filamentous fungus Podospora anserina was reported to lead to cellular copper depletion and a pleiotropic phenotype with hypopigmentation of the mycelium and the ascospores, affected fertility and increased lifespan by approximately 60% when compared to the wild type. This phenotype is linked to a switch from a copper-dependent standard to an alternative respiration leading to both a reduced generation of reactive oxygen species (ROS) and of adenosine triphosphate (ATP). We performed a genome-wide comparative transcriptome analysis of a wild-type strain and the copper-depleted grisea mutant. We unambiguously assigned 9,700 sequences of the transcriptome in both strains to the more than 10,600 predicted and annotated open reading frames of the P. anserina genome indicating 90% coverage of the transcriptome. 4,752 of the transcripts differed significantly in abundance with 1,156 transcripts differing at least 3-fold. Selected genes were investigated by qRT-PCR analyses. Apart from this general characterization we analyzed the data with special emphasis on molecular pathways related to the grisea mutation taking advantage of the available complete genomic sequence of P. anserina. This analysis verified but also corrected conclusions from earlier data obtained by single gene analysis, identified new candidates of factors as part of the cellular copper homeostasis system including target genes of transcription factor GRISEA, and provides a rich reference source of quantitative data for further in detail investigations. Overall, the present study demonstrates the importance of systems biology approaches also in cases were mutations in single genes are analyzed to explain the underlying mechanisms controlling complex biological processes like aging and development.

Caracterización molecular y genética de aislamientos patógenos de Pyricularia grisea del trigo ( Triticum aestivum Lam.) y triticale ( x Triticosecale Wittmack) en la Provincia de Paraná, Brasil

Isolates of Pyricularia grisea from wheat (Triticum aestivum Lam.) and triticale (x Triticosecale Wittmack) spikes with blast symptoms were analyzed by classical (VCG) and molecular (RAPD) techniques. P. grisea mutants, unable to use sodium nitrate (nit) as nitrogen source, were obtained with potassium chlorate. For vegetative compatibility (VCG) tests, genetically complementary nit mutant pairs were inoculated in a medium with sodium nitrate as a single nitrogen source. P. grisea isolates were divided into two vegetative compatibility groups and two RAPD groups. Since vegetative compatible strains may mutually exchange genetic and cytoplasmatic material, the contribution of the parasexual cycle in the genetic variability of Brazilian P. grisea isolates is discussed.

MGOS: A Resource for Studying Magnaporthe grisea and Oryza sativa Interactions

The MGOS (Magnaporthe grisea Oryza sativa) web-based database contains data from Oryza sativa and Magnaporthe grisea interaction experiments in which M. grisea is the fungal pathogen that causes the rice blast disease. In order to study the interactions, a consortium of fungal and rice geneticists was formed to construct a comprehensive set of experiments that would elucidate information about the gene expression of both rice and M. grisea during the infection cycle. These experiments included constructing and sequencing cDNA and robust long-serial analysis gene expression libraries from both host and pathogen during different stages of infection in both resistant and susceptible interactions, generating >50,000 M. grisea mutants and applying them to susceptible rice strains to test for pathogenicity, and constructing a dual O. sativa-M. grisea microarray. MGOS was developed as a central web-based repository for all the experimental data along with the rice and M. grisea genomic sequence. Community-based annotation is available for the M. grisea genes to aid in the study of the interactions.

Oxy2 as a transcriptional activator gene for copper uptake in Cryptococcus neoformans.

Cryptococcus neoformans is subject to oxidative attack by host immune cells; consequently, oxidant-resistant mechanisms may be important in pathogenesis. Mutations at the OXY2 locus confer decreased laccase and increased sensitivity to hyperbaric oxygen in the background of the oxyl mutation, but, alone, do not confer sensitivity to oxidants. Because metal deficiency can potentiate or ameliorate sensitivity to oxidants, and because the melanin-synthesizing laccase contains copper, we investigated copper acquisition in an oxy2 mutant. We found that its external Cu/Fe reductase activity was lower than that of wild type, and although copper deprivation induced the reductase in the wild type, it did not do so in oxy2. Oxy2 is sensitive to copper chelation but resistant to high copper, suggesting that copper transport is decreased. The strain expresses large amounts of alternate oxidase in response to Cu-chelation, perhaps in response to defective, Cu-deprived cytochrome oxidase, and is resistant to the oxidant, plumbagin, under this condition, perhaps due to the high alternate oxidase. These phenotypes are similar to those of the mac1- mutant of Saccharomyces cerevisiae and the melanin-deficient grisea mutant of Podospora anserina, in which homologous transcriptional activators for the reductase and copper transporter genes are mutated. They constitute physiologic evidence that oxy2 is mutated in a homologous copper-related transcriptional activator of C. neoformans.

CPMK2, an SLT2-homologous mitogen-activated protein (MAP) kinase, is essential for pathogenesis of Claviceps purpurea on rye: evidence for a second conserved pathogenesis-related MAP kinase cascade in phytopathogenic fungi.

Cpmk2, encoding a mitogen-activated protein (MAP) kinase from the ascomycete Claviceps purpurea, is an orthologue of SLT2 from Saccharomyces cerevisiae, the first isolated from a biotrophic, non-appressorium-forming pathogen. Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue. Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered. As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen, Magnaporthe grisea, that have been deprived of their MAP kinase gene mps1, the ability of cpmk2 to complement the defects of delta mps1 was investigated. Interestingly, the C. purpurea gene, under the control of its own promoter, was able to complement the M. grisea mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves. This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2/MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity.

Copper homeostasis and aging in the fungal model system Podospora anserina: differential expression of PaCtr3 encoding a copper transporter

Lifespan extension of Podospora anserina mutant grisea is caused by a loss-of-function mutation in the nuclear gene Grisea. This gene encodes the copper regulated transcription factor GRISEA recently shown to be involved in the expression of PaSod2 encoding the mitochondrial manganese superoxide dismutase. Here we report the identification and characterization of a second target gene. This gene, PaCtr3, encodes a functional homologue of the Saccharomyces cerevisiae high affinity copper permease yCTR3. PaCtr3 is not expressed in the grisea mutant confirming the assumption that the extension of lifespan is primarily caused by cellular copper limitation and a switch from a cytochrome oxidase (COX)-dependent to and alternative oxidase (AOX)-dependent respiration. Transcript levels of PaCtr3 and PaSod2 respond to copper, iron, manganese and zinc. Transcription of PaCtr3 was found to be down-regulated during senescence of wild-type cultures suggesting that the intracellular copper concentration is raised in old cultures. A two hybrid analysis suggested that GRISEA acts as a homodimer. In accordance, an inverted repeat was identified as a putative binding sequence in the promoter region of PaCtr3 and of PaSod2. Finally, the expression of PaCtr3 in transformants of the grisea mutant led to lifespan shortening. This effect correlates with the activity of the copper-dependent COX demonstrating a strong link between copper-uptake, respiration and lifespan.

Respiration, copper availability and SOD activity in P. anserina strains with different lifespan.

P. anserina mutants with impairments in complex IV (COX) of the respiratory chain are characterized by an increase in lifespan. Examples are the nuclear grisea mutant with a moderate lifespan extension (60%) and the immortal extranuclear ex1 mutant. Here we report data demonstrating that in mutant ex1 the level of the alternative oxidase (PaAOX) is significantly higher than in mutant grisea. PaAOX levels appear to be reversely dependent on COX activity. The activity profile of superoxide dismutases in the ex1 mutant resembles the profile in senescent wild-type cultures with a high cytoplasmic copper/zinc superoxide dismutase (PaSOD1) and a low mitochondrial manganese superoxide dismutase (PaSOD2) activity. In the grisea mutant, PaSOD1 activity is only detectable in cultures grown in copper-supplemented medium. The two copper-regulated genes PaCtr3 (coding for a high affinity copper transporter) and PaSod2 are not expressed in the two mutants grown in standard medium. The repression of these genes as well as the activity of PaSOD1 is dependent on the availability of cellular copper, which appears to be high in COX-deficient strains such as mutant ex1 and in the senescent wild-type strain. In the wild-type, changes in the cellular localization of copper and in the delivery of this metal to different proteins appear to occur during senescence. Collectively, the data explain the characteristic lifespan of the investigated strains as the result of differences in energy transduction and in the machinery protecting against oxidative stress.

Definition of tissue-specific and general requirements for plant infection in a phytopathogenic fungus.

Although plant diseases are usually characterized by the part of the plant that is affected (e.g., leaf spots, root rots, wilts), surprisingly little is known about the factors that condition the ability of pathogens to colonize different plant tissues. Here we demonstrate that the leaf blast pathogen Magnaporthe grisea also can infect plant roots, and we exploit this finding to distinguish tissue-specific and general requirements for plant infection. Tests of a M. grisea mutant collection identified some mutants that were defective specifically in infection of either leaves or roots, and others such as the map kinase mutant pmk1 that were generally defective in pathogenicity. Conservation of a functional PMK1-related MAP kinase in the root pathogen Gaeumannomyces graminis was also demonstrated. Exploitation of the ability of M. grisea to infect distinct plant tissues thus represents a powerful tool for the comprehensive dissection of genetic determinants of tissue specificity and global requirements for plant infection.

The Second Naphthol Reductase of Fungal Melanin Biosynthesis inMagnaporthe grisea: TETRAHYDROXYNAPHTHALENE REDUCTASE

Mutants of Magnaporthe grisea harboring a defective gene for 1,3, 8-trihydroxynaphthalene reductase retain the capability to produce scytalone, thus suggesting the existence of a second naphthol reductase that can catalyze the reduction of 1,3,6, 8-tetrahydroxynaphthalene to scytalone within the fungal melanin biosynthetic pathway. The second naphthol reductase gene was cloned from M. grisea by identification of cDNA fragments with weak homology to the cDNA of trihydroxynaphthalene reductase. The amino acid sequence for the second naphthol reductase is 46% identical to that of trihydroxynaphthalene reductase. The second naphthol reductase was produced in Esherichia coli and purified to homogeneity. Substrate competition experiments indicate that the second reductase prefers tetrahydroxynaphthalene over trihydroxynaphthalene by a factor of 310; trihydroxynaphthalene reductase prefers trihydroxynaphthalene over tetrahydroxynaphthalene by a factor of 4.2. On the basis of the 1300-fold difference in substrate specificities between the two reductases, the second reductase is designated tetrahydroxynaphthalene reductase. Tetrahydroxynaphthalene reductase has a 200-fold larger K(i) for the fungicide tricyclazole than that of trihydroxynaphthalene reductase, and this accounts for the latter enzyme being the primary physiological target of the fungicide. M. grisea mutants lacking activities for both trihydroxynaphthalene and tetrahydroxynaphthalene reductases do not produce scytalone, indicating that there are no other metabolic routes to scytalone.

Defence signalling pathways in cereals.

The combination of mutational and molecular studies has shed light on the role of reactive oxygen intermediates and programmed cell death in cereal disease resistance mechanisms. Rice Rac1 and barley Rar1 represent conserved disease resistance signalling genes, which may have related functions in animals. The analysis of non-pathogenic Magnaporthe grisea mutants may provide novel tools to study host defence pathways.

PaGrg1, a glucose-repressible gene of Podospora anserina that is differentially expressed during lifespan.

A loss-of-function mutation in Grisea, a gene of Podospora anserina that was previously shown to code for a copper-modulated transcription factor, leads to a significant increase in lifespan. In an attempt to identify and to isolate potential target genes controlled by GRISEA, a RT-differential-display screen was performed. This approach resulted in the identification of a gene, PaGrg1, that is differentially expressed in the wild-type and in the long-lived grisea mutant. In the mutant, transcript levels of PaGrg1 were found to be much lower than in the wild-type even if copper was added to the growth medium in amounts that revert the phenotype of this copper-uptake mutant to wild-type characteristics. PaGrg1 is a discontinuous gene with a single intron and encodes a protein of 71 amino acids sharing a high degree of sequence identity (65%) with the developmentally regulated, catabolite-repressed grg-1 gene of Neurospora crassa. Transcription of PaGrg1 increases upon carbon starvation indicating that PaGRG1 represents a putative stress protein. Transcript levels of PaGrg1 were found to increase during aging in both the wild-type strain and the long-lived mutant. However, even in the senescent stage of grisea, they are much lower than in juvenile cultures of the wild-type strain. The data suggest that threshold transcript levels of PaGrg1, and/ or additional unidentified genes which are controlled by GRISEA and which are subject to catabolite repression, are significantly involved in lifespan control. This conclusion is supported by the finding that, in contrast to the wild-type, the lifespan of grisea does not increase when cultures are grown on non-repressible carbon sources.

Deletion of Two Endo-β-1,4-Xylanase Genes Reveals Additional Isozymes Secreted by the Rice Blast Fungus

Fungal pathogens secrete hydrolases during infection of plant tissues capable of fragmenting the primary cell wall polysaccharides of the host. Magnaporthe grisea, the fungal pathogen that causes blast disease of graminaceous monocots, secretes two distinct endo-β-1,4-D-xylanases when grown on xylan-rich rice cell walls as the carbon source. We have previously reported the cloning of the genes encoding these two xylanases, XYL1 and XYL2 (formerly XYN22 and XYN33, respectively; see S.-C. Wu, S. Kauffmann, A. G. Darvill, and P. Albersheim, Mol. Plant-Microbe Interact. 8:506–514, 1995). We now present three M. grisea mutants created by selective deletion of XYL1 and/or XYL2. The xyl1 mutant grows as well as the parent in culture medium when rice cell walls or xylan is the sole carbon source. Under the same conditions, the xyl2 mutant grows slightly slower than the parent, whereas the xyl1/xyl2 double mutant exhibits a 50% reduction in accumulation of total mycelial mass. Under conditions idealized for infection, all three mutants infect host plants as efficiently as the parent, indicating neither XYL1 nor XYL2 is required by M. grisea for infection. Endoxylanase assays showed that, at the stationary stage of growth in culture when the accumulation of total xylanase activity is at its maximum, the xyl1 mutant retains ≈ 88%, xyl2, 39%, and xyl1/xyl2, 19% of the endoxylanase activity of the parent. Partial protein purification of xylanases secreted by the xyl1/xyl2 double mutant revealed four distinct endoxylanase activities. One of the xylanases, XYL3, is present in the culture filtrate of both the parent and the mutant strains, and, like XYL1, has been identified as a member of the Family G xylanases. The other three xylanases are not found in the culture filtrates of the parent or of the xyl1 mutant. Thus, M. grisea appears capable of secreting additional xylanases and does so when XYL2, a member of the Family F glycanases, has been eliminated.