Chitin-deacetylase activity induces appressorium differentiation in the rice blast fungus Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae differentiates a specialized infection structure called an appressorium to invade rice cells. In this report, we show that CBP1, which encodes a chitin-deacetylase, is involved in the induction phase of appressorium differentiation. We demonstrate that the enzymatic activity of Cbp1 is critical for appressorium formation. M. oryzae has six CDA homologues in addition to Cbp1, but none of these are indispensable for appressorium formation. We observed chitosan localization at the fungal cell wall using OGA488. This observation suggests that Cbp1-catalysed conversion of chitin into chitosan occurs at the cell wall of germ tubes during appressorium differentiation by M. oryzae. Taken together, our results provide evidence that the chitin deacetylase activity of Cbp1 is necessary for appressorium formation.

Identification and characterization of a novel gene encoding the NBS1 protein in Pyricularia oryzae.

The ascomycete Pyricularia oryzae (teleomorph: Magnaporthe oryzae) causes one of the most serious diseases known as rice blast. The Nijmegen breakage syndrome protein (NBS1) is essential for DNA repair; thus, we studied the P. oryzae NBS1 homolog (PoNBS1). A PoNBS1 null mutant exhibited high sensitivity to DNA damage-inducing agents. The mutant also exhibited the retarded hyphal growth, and induced abnormal conidial germination and shape, but showed normal appressorium formation. The phenotypes of the null mutant were complemented by introducing the cDNA of PoNBS1 driven by a TrpC promoter of Aspergillus nidulans. In addition, the null mutant similarly complemented with the PoNBS1 cDNA lacking the FHA domain that had a normal phenotype except for hyphal growth. These results suggest that PoNBS1 is involved in DNA repair and normal development in P. oryzae. Moreover, the FHA domain of PoNBS1 participates in normal hyphal growth.

The role of catalase-peroxidase secreted by Magnaporthe oryzae during early infection of rice cells.

The biological role of a secretory catalase of the rice blast fungus Magnaporthe oryzae was studied. The internal amino acid sequences of the partially purified catalase in the culture filtrate enabled us to identify its encoding gene as a catalase-peroxidase gene, CPXB, among four putative genes for catalase or catalase-peroxidase in M. oryzae. Knockout of the gene drastically reduced the level of catalase activity in the culture filtrate and supernatant of conidial suspension (SCS), and increased the sensitivity to exogenously added H2O2 compared with control strains, suggesting that CPXB is the major gene encoding the secretory catalase and confers resistance to H2O2 in hyphae. In the mutant, the rate of appressoria that induced accumulation of H2O2 in epidermal cells of the leaf sheath increased and infection at early stages was delayed; however, the formation of lesions in the leaf blade was not affected compared with the control strain. These phenotypes were complimented by reintroducing the putative coding regions of CPXB driven by a constitutive promoter. These results suggest that CPXB plays a role in fungal defense against H2O2 accumulated in epidermal cells of rice at the early stage of infection but not in pathogenicity of M. oryzae.

Expression specificity of CBP1 is regulated by transcriptional repression during vegetative growth of Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae produces appressoria during the infection of a host. In M. oryzae, the appressorium formation-related gene CBP1 (Chitin Binding Protein 1) is specifically expressed during the early stage of appressorium differentiation. The transcription factor CON7 activates CBP1 expression. However, many aspects of the regulation of CBP1 expression are still unknown. In this report, the CBP1 5' upstream region was analyzed using an egfp reporter. Deletion of the CBP1 5 ' upstream region caused derepression of reporter gene activity during vegetative growth. This result suggests that CBP1 expression is repressed during vegetative growth. The key 5 ' upstream sequences for CBP1 repression were examined. Furthermore, cis- and trans-acting elements of the negative regulatory region were investigated. Here, we discuss the transcriptional regulatory mechanism of CBP1.

Construction of a binary vector for knockout and expression analysis of rice blast fungus genes.

We developed an efficient method to analyze gene function and expression of the rice blast fungus. We constructed a GATEWAY binary vector, which generates a gene-targeted disruptant carrying a green fluorescent protein gene under the native promoter of the target gene. Using this method, the knockout efficiency and expression patterns of two hypothetical genes were determined.

Targeted gene disruption of the neuronal calcium sensor 1 homologue in rice blast fungus, Magnaporthe grisea.

We isolated a neuronal calcium sensor 1/frequenin-like gene, Mg-NCS-1, from Magnaporthe grisea and evaluated the phenotypes of null-mutants of the gene. The putative Mg-NCS-1 protein showed high similarity to the other NCS-1 proteins. The null-mutants had normal growth and pathogenicity similar to the parental strain, but their growth was suppressed in high concentrations of Ca2+ or acidic conditions.

A novel gene, CBP1, encoding a putative extracellular chitin-binding protein, may play an important role in the hydrophobic surface sensing of Magnaporthe grisea during appressorium differentiation.

The conidial germ tube of the rice blast fungus, Magnaporthe grisea, differentiates a specialized cell, an appressorium, required for penetration into the host plant. Formation of the appressorium is also observed on artificial solid substrata such as polycarbonate. A novel emerging germ tube-specific gene, CBP1 (chitin-binding protein), was found in a cDNA subtractive differential library. CBP1 coded for a putative extracellular protein (signal peptide) with two similar chitin-binding domains at both ends of a central domain with homology to fungal chitin deacetylases and with a C-terminus domain rich in Ser/Thr related extracellular matrix protein such as agglutinin. The consensus sequence of the chitin-binding domain found in CBP1 has never been reported in fungi and is similar to the chitin-binding motif in plant lectins and plant chitinases classes I and IV. CBPI was disrupted in order to identify its function. Null mutants of CBP1 failed to differentiate appressoria normally on artificial surface but succeeded in normally differentiating appressoria on the plant leaf surface. Since the null mutant Cbp1- showed abnormal appressorium differentiation only on artificial surfaces and was sensitive to the chemical inducers, CBP1 seemed to play an important role in the recognition of physical factors on solid surfaces.