Andrew J. Foster

The transcription factor Con7p is a central regulator of infection‐related morphogenesis in the rice blast fungus Magnaporthe grisea

A strain harbouring an insertion within the promoter of the CON7 gene of Magnaporthe grisea was isolated. This gene was previously shown to be essential for appressorium formation and growth in planta and is predicted to encode a transcription factor. Microarray‐based gene expression analysis was used to identify several genes whose transcription during germination depends on Con7p. These include the pathogenicity factor‐encoding gene PTH11 and several other genes which like PTH11 are predicted to encode G protein‐coupled receptors. Microarray analysis also revealed several Con7p‐dependent genes which may encode factors determining cell wall structure or function, either through the synthesis/degradation of cell wall components or by association with the cell exterior. One Con7p‐dependent gene predicted to encode a class VII chitin synthase was deleted, leading to dramatic consequences on the pathogenic development of the resultant strain. Within the con7– mutant, a 29% reduction in chitin content of germinated spores was found and the mutant was hypersensitive to the chitin synthase inhibitor nikkomycin Z. A green fluorescent protein‐tagged Con7p was found to have nuclear localization within spores. Taken together, these observations suggest that Con7p encodes a transcription factor required for the transcription of several genes which participate in disease‐related morphogenesis in M. grisea.

Ferricrocin synthesis in Magnaporthe grisea and its role in pathogenicity in rice

SUMMARY Iron is an essential element for the growth of nearly all organisms. In order to overcome the problem of its low bioavailability, microorganisms (including fungi) secrete siderophores, high-affinity iron chelators. As the acquisition of iron is also a key step in infection processes, siderophores have been considered as potential virulence factors in several host-pathogen interactions. Most fungi produce siderophores of the hydroxamate-type, which are synthesized by non-ribosomal peptide synthetases (NRPSs). Magnaporthe grisea, the causal agent of rice blast disease, produces ferricrocin as intracellular storage siderophore and excretes coprogens. In the M. grisea genome we identified SSM1, an NRPS gene, and a gene encoding an l-ornithine N5-monooxygenase (OMO1) that is clustered with SSM1 and responsible for catalysing the first step in siderophore biosynthesis, the N(5) hydroxylation of ornithine. Disruption of SSM1 confirmed that the gene encodes ferricrocin synthetase. Pathogenicity of these mutants towards rice was reduced, suggesting a role of this siderophore in pathogenicity of M. grisea.

MAP kinase signalling pathway components and targets conserved between the distantly related plant pathogenic fungi Mycosphaerella graminicola and Magnaporthe grisea.

Mycosphaerella graminicola is a dimorphic fungus which causes Septoria tritici leaf blotch. This report describes the examination of the role of several components of the Pmk1p/Fus3p mitogen-activated protein kinase (MAPK) signalling pathway in the development of this species. The genes encoding the MAPK kinase kinase MgSte11p and the MAPK kinase MgSte7p were found to be indispensible for pathogenicity while the deletion of the gene encoding the proposed scaffold protein MgSte50p led to a reduction in virulence. These phenotypes were attributed to a reduced ability to form filaments on the plant surface which prevented penetration. A delayed disease progression was observed on deletion of the gene MGSTE12. The MGSTE7, MGSTE50 and MGSTE12 genes were able to complement mutants of Magnaporthe grisea lacking the orthologous genes. Interactions between the My. graminicola signalling components were also investigated. Furthermore genes whose MgSte12p/Mst12p dependence is conserved between My. graminicola and Ma. grisea were identified.

The Magnaporthe grisea class VII chitin synthase is required for normal appressorial development and function.

The plant pathogenic fungus Magnaporthe grisea is able to enter its host via appressorium-mediated penetration. Earlier investigations have shown that these infection structures are rich in the cell wall polysaccharide chitin. Previously, we have described how the transcription of a class VII chitin synthase-encoding gene CHS7 is completely dependent on the putative transcription factor Con7p during the germination of conidia, and how con7(-) mutants are unable to form appressoria under any conditions tested. Because of the pleiotropic effects of the con7(-) mutation, we examined the consequences of the targeted deletion of CHS7. The chs7(-) mutants generated were unable to form appressoria on artificial surfaces, except following the application of the exogenous inducers 1,16-hexadecanediol and cyclic adenosine monophosphate. The appressoria formed had a reduced chitin content and were often found to be smaller and misshapen compared with the wild-type. chs7(-) mutants were significantly reduced in their ability to enter rice plants, but growth in planta was not affected. Reverse transcriptase-polymerase chain reaction analysis demonstrated that CHS7 transcription was strongly induced on germination of spores, and a green fluorescent protein-tagged Chs7p protein was found to be produced abundantly during infection-related morphogenesis. Together, these data suggest that the class VII chitin synthase Chs7p of M. grisea is required for normal appressorium formation and function.

Siderophore synthesis in Magnaporthe grisea is essential for vegetative growth, conidiation and resistance to oxidative stress

The plant pathogenic fungus Magnaporthe grisea excretes siderophores of the coprogen-type for iron acquisition and uses ferricrocin for intracellular iron storage. In the present report we characterize mutants with defects in extracellular siderophore biosynthesis. Deletion of the M. grisea SSM2 gene, which encodes a non-ribosomal peptide synthetase, resulted in a loss of the production of all coprogens. The mutant strains had a reduced growth rate, produced fewer conidia and were more sensitive to oxidative stress. Ferricrocin production was not affected. Upon deletion of M. grisea OMO1, a gene predicted to encode an L-ornithine-N(5)-monooxygenase, no siderophores of any type were detected, the strain was aconidial, growth rate was reduced and sensitivity to oxidative stress was increased. Abundance of several proteins was affected in the mutants. The Deltassm2 and Deltaomo1 mutant phenotypes were complemented by supplementation of the medium with siderophores or reintroduction of the respective genes.

Histidine kinases mediate differentiation, stress response, and pathogenicity in Magnaporthe oryzae

The aim of this study is a functional characterization of 10 putative histidine kinases (HIKs)‐encoding genes in the phytopathogenic fungus Magnaporthe oryzae. Two HIKs were found to be required for pathogenicity in the fungus. It was found that the mutant strains ΔMohik5 and ΔMohik8 show abnormal conidial morphology and furthermore ΔMohik5 is unable to form appressoria. Both HIKs MoHik5p and MoHik8p appear to be essential for pathogenicity since the mutants fail to infect rice plants. MoSln1p and MoHik1p were previously reported to be components of the HOG pathway in M. oryzae. The ΔMosln1 mutant is more susceptible to salt stress compared to ΔMohik1, whereas ΔMohik1 appears to be stronger affected by osmotic or sugar stress. In contrast to yeast, the HOG signaling cascade in phytopathogenic fungi apparently comprises more elements. Furthermore, vegetative growth of the mutants ΔMohik5 and ΔMohik9 was found to be sensitive to hypoxia‐inducing NaNO2‐treatment. Additionally, it was monitored that NaNO2‐treatment resulted in MoHog1p phosphorylation. As a consequence we assume a first simplified model for hypoxia signaling in M. oryzae including the HOG pathway and the HIKs MoHik5p and MoHik9p.

The role of the Tra1p transcription factor of Magnaporthe oryzae in spore adhesion and pathogenic development.

Transcription factors play a critical regulatory role in development by binding DNA and initiating alterations in gene transcription. The transcript of the putative Magnaporthe oryzae transcription factor-encoding gene TRA1 accumulates during germination and this accumulation was previously found to depend on the transcription factor Con7p. In the current work tra1⁻ mutants were generated and these strains were found to exhibit a reduced attachment, germination, appressorium formation and virulence. Adhesion to artificial and plant surfaces was affected, and FITC-labelled concanavalin A, a lectin which inhibits attachment of Magnaporthe spores, showed a reduced affinity for mutant spore tip where it normally preferentially binds. We used microarray analysis to identify Tra1p-dependent genes from two different sources: aerial structures and conidia. Mutation of 11 Tra1p-dependent genes showed that the predicted transcription factor encoding gene TDG2 is required for normal adhesion and virulence, that the genes TDG7 and TDG4 are required for normal sporulation and that TDG6 is required for wild-type levels of spore adhesion.

Genetics of phytopathology: Secondary metabolites as virulence determinants of fungal plant pathogens

The kingdom fungi comprises a highly diverse array of species. Where species diversification has been studied in detail, fungal species can be 6 times as numerous as those of flowering plants. On this basis, since approximately 270,000 flowering plants are known today, it is estimated that more than 1.5 million fungal species may exist (Carlile and Watkinson 1994; Hawksworth 2001)). Fungi are able to colonize a broad range of substrata; they may live as saprophytes, associated with plants as mycorrhiza and as pathogens of plants, animals and microorganisms (Dix and Webster 1995). As saprophytes, fungi contribute to the ecological balance of the environment by degrading organic materials, such as decaying plant material, dung and organic pollutants. In mycorrhiza, fungi provide phosphate for the associated plant while they gain carbohydrates in exchange (Smith and Read 1997). However, many plant-associated fungi do not provide any mutual benefit for the colonized hosts. Whereas biotrophic fungi rely on the living host for nutrient provision, others (necrotrophs) often kill and exploit the surrounding tissue. The high biodiversity of fungal species is also reflected by exceedingly rich diversity of metabolism and the corresponding metabolites that affect the attacked plant. A large group of pathogens known as necrotrophs secrete phytotoxic secondary metabolites to kill the host tissue and to avoid initiation of defense responses. Secondary metabolites have been defined as metabolic products not essential for growth and without obvious function for the producing cell during its life cycle (Aharonowitz and Demain 1980). Secondary metabolite production depends both on the genotype of the organism and the environmental conditions under which growth takes place, for example the composition of the medium used to culture the organism (Weinberg 1974; Bu’Lock 1975). So far, several thousand sec-

Functions of the Magnaporthe oryzae Flb3p and Flb4p transcription factors in the regulation of conidiation

The Magnaporthe oryzae genes FLB3 and FLB4, orthologues of the Aspergillus nidulans regulators of conidiation FlbC and FlbD, were inactivated. These genes encode C2H2 zinc finger and Myb-like transcription factors, respectively, in A. nidulans. Analysis of the resultant mutants demonstrated that FLB4 is essential for spore formation and that strains lacking this gene are fluffy in their colony morphology due to an inability to complete conidiophore formation. Meanwhile, FLB3 is required for normal levels of aerial mycelium formation. We identified genes dependent on both transcription factors using microarray analysis. This analysis revealed that the transcription of several genes encoding proteins implicated in sporulation in Magnaporthe oryzae and other filamentous fungi are affected by FLB3 or FLB4 inactivation. Furthermore, the microarray analysis indicates that Flb3p may effectively reprogramme the cell metabolically by repressing transcription of genes encoding biosynthetic enzymes and inducing transcription of genes encoding catabolic enzymes. Additionally, qRT-PCR was employed and showed that FLB3 and FLB4 transcripts are enriched in synchronously sporulating cultures, as were the transcripts of other genes that are necessary for normal conidiation, consistent with a role for their gene products in this process.

Identification of Fungicide Targets in Pathogenic Fungi

The rapid emergence of fungicide resistance has brought a strong demand for plant protectants with a new mode of action. One of the challenges for modern plant pathology research is the identification of genes and gene products essential for the establishment of pathogenic interactions between host and pathogen. Genome-wide technologies, such as transcriptomics, metabolomics, and proteomics represent a new opportunity to identify such targets in phytopathogenic fungi. This chapter reviews genome-wide approaches that have emerged in the post-genomics era, e.g. comparative genomics and gene expression profiling experiments such as microarray-based transcriptional profiling, serial analysis of gene expression, and massively parallel signature sequencing. It also discusses prospects for exploiting these modern technologies for the development of plant protectants and system biology.