We isolated an MgATG1gene encoding a serine/threonine protein kinase from the rice blast fungusMagnaporthe grisea. In the ΔMgatg1mutant, in which the MgATG1gene had been deleted, autophagy was blocked; the mutant also showed fewer lipid droplets in its conidia, lower turgor pressure of the appressorium, and such defects in morphogenesis as delayed initiation and slower germination of conidia. As a result of lower turgor pressure of the appressorium, the ΔMgatg1mutant lost its ability to penetrate and infect the two host plants, namely, rice and barley. However, normal values of the parameters and infective abilities were restored on reintroducing an intact copy of the MgATG1gene into the mutant. Autophagy is thus necessary for turnover of organic matter during the formation of conidia and appressoria and for normal development and pathogenicity inM. grisea.

Because of great challenges and workload in deleting genes on a large scale, the functions of most genes in pathogenic fungi are still unclear. In this study, we developed a high-throughput gene knockout system using a novel yeast-Escherichia-Agrobacterium shuttle vector, pKO1B, in the rice blast fungus Magnaporthe oryzae. Using this method, we deleted 104 fungal-specific Zn₂Cys₆ transcription factor (TF) genes in M. oryzae. We then analyzed the phenotypes of these mutants with regard to growth, asexual and infection-related development, pathogenesis, and 9 abiotic stresses. The resulting data provide new insights into how this rice pathogen of global significance regulates important traits in the infection cycle through Zn₂Cys₆TF genes. A large variation in biological functions of Zn₂Cys₆TF genes was observed under the conditions tested. Sixty-one of 104 Zn₂Cys₆ TF genes were found to be required for fungal development. In-depth analysis of TF genes revealed that TF genes involved in pathogenicity frequently tend to function in multiple development stages, and disclosed many highly conserved but unidentified functional TF genes of importance in the fungal kingdom. We further found that the virulence-required TF genes GPF1 and CNF2 have similar regulation mechanisms in the gene expression involved in pathogenicity. These experimental validations clearly demonstrated the value of a high-throughput gene knockout system in understanding the biological functions of genes on a genome scale in fungi, and provided a solid foundation for elucidating the gene expression network that regulates the development and pathogenicity of M. oryzae. Magnaporthe oryzae is not only the fungus causing the rice blast disease, which leads to 20–30% losses in rice production, but also a primary model pathosystem for understanding host-pathogen interactions. However, there is no high-throughput gene knockout system constructed, and little is known about most of the genes in this fungus. We developed a high-throughput gene knockout system, and using this system, we obtained null mutants of 104 fungal-specific Zn₂Cys₆ transcription factor (TF) genes by screening 8741 primary transformants in M. oryzae. We analyzed the functions of these TF genes in development, pathogenesis, and stress responses under 9 conditions. We found that 61 Zn₂Cys₆ TF genes play indispensable and diversified roles in fungal development and pathogenicity. CNF1 is the first reported TF gene that strongly and negatively regulates asexual development in the rice blast fungus, and CCA1, CNF1, CNF2, CONx1, GPF1, GTA1, MoCOD1 and PCF1 are required for pathogenicity. We further found via RNA-seq that GPF1 and CNF2 have similar mechanisms in gene expression regulation related to pathogenicity. The resulting data provide new insights into how Zn₂Cys₆ TF genes regulate important traits during the infection cycle of this rice blast pathogen.

Atg4 is a unique cysteine protease responsible for the cleavage of the carboxyl terminus of Atg8 during the formation of autophagosomes in yeast. Here we report that MoAtg4, an Atg4 homologue in Ma...

Plant pathogenic fungi utilize a series of complex infection structures, in particular the appressorium, to gain entry to and colonize plant tissue. As a consequence of the accumulation of huge quantities of glycerol in the cell the appressorium generates immense intracellular turgor pressure allowing the penetration peg of the appressorium to penetrate the leaf cuticle. Autophagic processes are ubiquitous in eukaryotic cells and facilitate the bulk degradation of macromolecules and organelles. The study of autophagic processes has been extended from the model yeast Saccharomyces cerevisiae to pathogenic fungi such as the rice blast fungus Magnaporthe oryzae. Significantly, null mutants for the expression of M. oryzae autophagy gene homologs lose their pathogenicity for infection of host plants. Clarification of the functions and network of interactions between the proteins expressed by M. oryzae autophagy genes will lead to a better understanding of the role of autophagy in fungal pathogenesis and help in the development of new strategies for disease control.