We describe in this study punchless, a nonpathogenic mutant from the rice blast fungus M. grisea, obtained by plasmid-mediated insertional mutagenesis. As do most fungal plant pathogens, M. grisea differentiates an infection structure specialized for host penetration called the appressorium. We show that punchless differentiates appressoria that fail to breach either the leaf epidermis or artificial membranes such as cellophane. Cytological analysis of punchless appressoria shows that they have a cellular structure, turgor, and glycogen content similar to those of wild type before penetration, but that they are unable to differentiate penetration pegs. The inactivated gene, PLS1, encodes a putative integral membrane protein of 225 aa (Pls1p). A functional Pls1p-green fluorescent protein fusion protein was detected only in appressoria and was localized in plasma membranes and vacuoles. Pls1p is structurally related to the tetraspanin family. In animals, these proteins are components of membrane signaling complexes controlling cell differentiation, motility, and adhesion. We conclude that PLS1 controls an appressorial function essential for the penetration of the fungus into host leaves. P lant pathogenic fungi are a major threat for crop production worldwide. Several of these fungal pathogens depend on the formation of a specialized cell, called an appressorium, for the successful penetration of host plant surfaces (1, 2). This penetration process is typical of fungal leaf spots such as rice blast caused by the filamentous ascomycete Magnaporthe grisea (3). This disease is disseminated by water-splashed conidia (ref. 4; Fig. 1A). After their adhesion to the host leaf by means of a strong glue (ref. 5; Fig. 1B), conidia germinate and differentiate appressoria at the tips of their germ tubes (Fig. 1C). This developmental process is induced by chemical and physical stimuli from the host surface such as hardness, hydrophobicity, or cutin monomers (2, 3). This dome-shaped, thick-walled, and melanized cell (Fig. 1D) promotes the differentiation of a specialized hypha called the penetration peg that is involved in penetration of plant surfaces (ref. 6; pp in Fig. 1E). The differentiation of appressoria and penetration pegs is the result of complex morphogenetic processes that involve major developmental and metabolic changes (2,3,6). Inside the epidermal plant cell, the penetration peg differentiates into a bulbous infection hypha that invades the whole cell and colonizes the leaf.To isolate genes involved in this infection process, we generated a collection of M. grisea pathogenicity mutants by using plasmid-mediated insertional mutagenesis. This strategy was successful for the cloning of pathogenicity genes in Colletotrichum lindemuthianum (7), Ustilago maydis,** and M. grisea (8,9). In this study, we isolated punchless, a nonpathogenic mutant defective in penetration of plant surfaces. We show that the PLS1 gene is inactivated in punchless and encodes a putative membrane protein related to the tetraspanin family (10...

Oomycetes represent some of the most devastating plant and animal pathogens. Typical examples are Phytophthora infestans, which causes potato and tomato late blight, and Saprolegnia parasitica, responsible for fish diseases. Despite the economical and environmental importance of oomycete diseases, their control is difficult, particularly in the aquaculture industry. Carbohydrate synthases are vital for hyphal growth and represent interesting targets for tackling the pathogens. The existence of 2 different chitin synthase genes (SmChs1 and SmChs2) in Saprolegnia monoica was demonstrated using bioinformatics and molecular biology approaches. The function of SmCHS2 was unequivocally demonstrated by showing its catalytic activity in vitro after expression in Pichia pastoris. The recombinant SmCHS1 protein did not exhibit any activity in vitro, suggesting that it requires other partners or effectors to be active, or that it is involved in a different process than chitin biosynthesis. Both proteins contained N-terminal Microtubule Interacting and Trafficking domains, which have never been reported in any other known carbohydrate synthases. These domains are involved in protein recycling by endocytosis. Enzyme kinetics revealed that Saprolegnia chitin synthases are competitively inhibited by nikkomycin Z and quantitative PCR showed that their expression is higher in presence of the inhibitor. The use of nikkomycin Z combined with microscopy showed that chitin synthases are active essentially at the hyphal tips, which burst in the presence of the inhibitor, leading to cell death. S. parasitica was more sensitive to nikkomycin Z than S. monoica. In conclusion, chitin synthases with species-specific characteristics are involved in tip growth in Saprolegnia species and chitin is vital for the micro-organisms despite its very low abundance in the cell walls. Chitin is most likely synthesized transiently at the apex of the cells before cellulose, the major cell wall component in oomycetes. Our results provide important fundamental information on cell wall biogenesis in economically important species, and demonstrate the potential of targeting oomycete chitin synthases for disease control.

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