Conidial Morphogenesis and Septin-Mediated Plant Infection Require Smo1, a Ras GTPase-Activating Protein in Magnaporthe oryzae.

The pathogenic life cycle of the rice blast fungus Magnaporthe oryzae involves a series of morphogenetic changes, essential for its ability to cause disease. The smo mutation was identified > 25 years ago, and affects the shape and development of diverse cell types in M. oryzae, including conidia, appressoria, and asci. All attempts to clone the SMO1 gene by map-based cloning or complementation have failed over many years. Here, we report the identification of SMO1 by a combination of bulk segregant analysis and comparative genome analysis. SMO1 encodes a GTPase-activating protein, which regulates Ras signaling during infection-related development. Targeted deletion of SMO1 results in abnormal, nonadherent conidia, impaired in their production of spore tip mucilage. Smo1 mutants also develop smaller appressoria, with a severely reduced capacity to infect rice plants. SMO1 is necessary for the organization of microtubules and for septin-dependent remodeling of the F-actin cytoskeleton at the appressorium pore. Smo1 physically interacts with components of the Ras2 signaling complex, and a range of other signaling and cytoskeletal components, including the four core septins. SMO1 is therefore necessary for the regulation of RAS activation required for conidial morphogenesis and septin-mediated plant infection.

Molecular and phenotypic characterization of Sebacina vermifera strains associated with orchids, and the description of Piriformospora williamsii sp. nov.

Sebacinales was described in 2004 and is currently recognized as the earliest diverging lineage of mycorrhizal Basidiomycota. In addition, recent research has demonstrated that no other known fungal order harbours a broader spectrum of mycorrhizal types. Yet because of the character poor morphology of these inconspicuous fungi, a reliable systematic framework for Sebacinales is still out of reach. In order to increase the body of comparative data on Sebacinales, we followed a polyphasic approach using a sampling of seven diverse Sebacinales strains, including several isolates of Australian orchid mycorrhizae, Piriformospora indica, and a multinucleate rhizoctonia isolated from a pot culture of Glomus fasciculatum (Williams 1985) with clover. We performed molecular phylogenetic analyses from candidate barcoding regions [rDNA: internal transcribed spacer (ITS)1-5.8-ITS2, 28S; translation elongation factor 1-α (TEF)], enzymatic profiling, genome size estimation by quantitative polymerase chain reaction (PCR), and karyotype analysis using pulsed field gel electrophoresis. Here, we report significant differences in the physiological and molecular parameters inferred from these morphologically very similar strains. Particularly, our results indicate that intron sequences of the TEF gene are useful markers for Sebacinales at the species level. As a first taxonomic consequence, we describe Piriformospora williamsii as a new member of the so far monotypic genus Piriformospora and show that this genus contains still undescribed species that were recently discovered as endophytes of field-collected specimens of Anthyllis, Medicago, and Lolium in Germany.

Endophytic life strategies decoded by genome and transcriptome analyses of the mutualistic root symbiont Piriformospora indica.

Recent sequencing projects have provided deep insight into fungal lifestyle-associated genomic adaptations. Here we report on the 25 Mb genome of the mutualistic root symbiont Piriformospora indica (Sebacinales, Basidiomycota) and provide a global characterization of fungal transcriptional responses associated with the colonization of living and dead barley roots. Extensive comparative analysis of the P. indica genome with other Basidiomycota and Ascomycota fungi that have diverse lifestyle strategies identified features typically associated with both, biotrophism and saprotrophism. The tightly controlled expression of the lifestyle-associated gene sets during the onset of the symbiosis, revealed by microarray analysis, argues for a biphasic root colonization strategy of P. indica. This is supported by a cytological study that shows an early biotrophic growth followed by a cell death-associated phase. About 10% of the fungal genes induced during the biotrophic colonization encoded putative small secreted proteins (SSP), including several lectin-like proteins and members of a P. indica-specific gene family (DELD) with a conserved novel seven-amino acids motif at the C-terminus. Similar to effectors found in other filamentous organisms, the occurrence of the DELDs correlated with the presence of transposable elements in gene-poor repeat-rich regions of the genome. This is the first in depth genomic study describing a mutualistic symbiont with a biphasic lifestyle. Our findings provide a significant advance in understanding development of biotrophic plant symbionts and suggest a series of incremental shifts along the continuum from saprotrophy towards biotrophy in the evolution of mycorrhizal association from decomposer fungi.

Karyotype analysis, genome organization, and stable genetic transformation of the root colonizing fungus Piriformospora indica.

Piriformospora indica (Basidiomycota, Sebacinales) is a root colonizing fungus which is able to increase biomass and yield of crop plants and to induce local and systemic resistance to fungal diseases and tolerance to abiotic stress. A prerequisite for the elucidation of the mode of action of this novel kind of symbiosis is knowledge of the genome organization as well as the development of tools to study and modify gene functions. Here we provide data on the karyotype and genetic transformation strategies. The fungus was shown to possess at least six chromosomes and a genome size of about 15.4-24Mb. Sequences of the genes encoding the elongation factor 1-alpha (TEF) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used for genome size estimation through real-time PCR analysis. Chromosomal location investigated by Southern blot and expression analysis suggested that TEF and GAPDH are single-copy genes with strong and constitutive promoters. A genetic transformation system was established using a fragment of the TEF promoter region for construction of vectors carrying the selectable marker hygromycin B phosphotransferase. Results demonstrate that P. indica can be stably transformed by random genomic integration of foreign DNA and that it posses a relative small genome as compared to other members of the Basidiomycota.