To cause plant diseases, pathogenic micro-organisms secrete effector proteins into host tissue to suppress immunity and support pathogen growth. Bacterial pathogens have evolved several distinct secretion systems to target effector proteins, but whether fungi, which cause the major diseases of most crop species, also require different secretory mechanisms is not known. Here we report that the rice blast fungus Magnaporthe oryzae possesses two distinct secretion systems to target effectors during plant infection. Cytoplasmic effectors, which are delivered into host cells, preferentially accumulate in the biotrophic interfacial complex, a novel plant membrane-rich structure associated with invasive hyphae. We show that the biotrophic interfacial complex is associated with a novel form of secretion involving exocyst components and the Sso1 t-SNARE. By contrast, effectors that are secreted from invasive hyphae into the extracellular compartment follow the conventional secretory pathway. We conclude that the blast fungus has evolved distinct secretion systems to facilitate tissue invasion.

Magnaporthe oryzae is the causal agent of rice blast disease, the most devastating disease of cultivated rice (Oryza sativa) and a continuing threat to global food security. To cause disease, the fungus elaborates a specialized infection cell called an appressorium, which breaches the cuticle of the rice leaf, allowing the fungus entry to plant tissue. Here, we show that the exocyst complex localizes to the tips of growing hyphae during vegetative growth, ahead of the Spitzenkörper, and is required for polarized exocytosis. However, during infection-related development, the exocyst specifically assembles in the appressorium at the point of plant infection. The exocyst components Sec3, Sec5, Sec6, Sec8, and Sec15, and exocyst complex proteins Exo70 and Exo84 localize specifically in a ring formation at the appressorium pore. Targeted gene deletion, or conditional mutation, of genes encoding exocyst components leads to impaired plant infection. We demonstrate that organization of the exocyst complex at the appressorium pore is a septin-dependent process, which also requires regulated synthesis of reactive oxygen species by the NoxR-dependent Nox2 NADPH oxidase complex. We conclude that septinmediated assembly of the exocyst is necessary for appressorium repolarization and host cell invasion.

Glycosylphosphatidylinositol-anchored (beta)-1,3-glucanosyltransferases play active roles in fungal cell wall biosynthesis and morphogenesis and have been implicated in virulence on mammals. The role of beta-1,3-glucanosyltransferases in pathogenesis to plants has not been explored so far. Here, we report the cloning and mutational analysis of the gas1 gene encoding a putative beta-1,3-glucanosyltransferase from the vascular wilt fungus Fusarium oxysporum. In contrast to Candida albicans, expression of gas1 in F. oxysporum was independent of ambient pH and of the pH response transcription factor PacC. Gene knockout mutants lacking a functional gas1 allele grew in a way similar to the wildtype strain in submerged culture but exhibited restricted colony growth on solid substrates. The restricted growth phenotype was relieved by the osmotic stabilizer sorbitol, indicating that it may be related to structural alterations in the cell wall. Consistent with this hypothesis, deltagas1 mutants exhibited enhanced resistance to cell wall-degrading enzymes and increased transcript levels of chsV and rho1, encoding a class V chitin synthase and a small monomeric G protein, respectively. The deltagas1 mutants showed dramatically reduced virulence on tomato, both in a root infection assay and in a fruit tissue-invasion model, thus providing the first evidence for an essential role of fungal beta-1,3-glucanosyltransferases during plant infection.

SummaryRho-type GTPases regulate polarized growth in yeast by reorganization of the actin cytoskeleton and through signalling pathways that control the expression of cell wall biosynthetic genes. We report the cloning and functional analysis of rho1 from Fusarium oxysporum, a soilborne fungal pathogen causing vascular wilt on plants and opportunistic infections in humans. F. oxysporum strains carrying either a Drho1 loss-of-function mutation or a rho1 G14V gain-offunction allele were viable, but displayed a severely restricted colony phenotype which was partially relieved by the osmotic stabilizer sorbitol, indicating structural alterations in the cell wall. Consistent with this hypothesis, Drho1 strains showed increased resistance to cell wall-degrading enzymes and staining with Calcofluor white, as well as changes in chitin and glucan synthase gene expression and enzymatic activity. Re-introduction of a functional rho1 allele into the Drho1 mutant fully restored the wild-type phenotype. The Drho1 strain had dramatically reduced virulence on tomato plants, but was as virulent as the wild type on immunodepressed mice. Thus, Rho1 plays a key role during fungal infection of plants, but not of mammalian hosts.