Studies On The Relationship Between Autophagy And The Growth, Development And Pathogenicity Of Rice Blast Fungus Magnaporthe Grisea

Autophagy is a ubiquitous and evolutionarily conserved process that degrades and recycles long-lived proteins and organelles in all eukaryotic cells. It is an intracellular, bulk degradation process in which cytosol and organelles are sequestered within double-membrane vesicles termed autophagosomes that deliver the contents to the lysosome/vacuole for degradation and recycling of the macromolecules. For many years, autophagy has been presumed to be involved in cellular architectural changes that occur during differentiation and development, presumably via its role in organelle and protein turnover. It has been showed by the genetic data in Saccharomyces cerevisiae, Dictyostelium discoideum, and Caenorhabdiitis elegans that certain differentiation and developmental processes, especially in lower eukaryotic organisms, are triggered by environmental stressors that regulate autophagy positively.Magnaporthe grisea, a filamentous ascomycete fungus, is well known as the causal agent of rice blast, the most serious disease of cultivated rice throughout the world, and has been developed as a model organism for the investigation of fungus-host interaction. Appressorium, a specialized cell necessary for infection by the rice blast fungus, generates tremendous intracellular turgor pressure (as much as 8.0 Mpa) to penetrate the leaf cuticle. This enormous turgor in appressorium is a consequence of the accumulation of very large quantities of glycerol in the cell, and potential sources of its biosynthesis are the lipid, glycogen, as well as the sugars, trehalose and mannitol, in the conidium.The differentiation and development from the conidium to the appressorium, and then from the appressorium to the penetration peg or infectious hyphae, require a cell to undergo significant phenotypic changes accompanied by the breakdown and recycling of old cellular components within about 30 h. It is found that appressorium formation involves autophagy, and that the MgATG8 gene arrests death of conidia but also renders the fungus nonpathogenic in the case of M. grisea. This work seeks to find out whether autophagy has a role in the process whereby a hypha forms a conidium that goes on to develop the appressorium and in generating the turgor pressure exerted by the appressorium. An EST (expressed sequence tags) (clone s197: GenBank Accession No. CK828251) for MgATG1 (autophagy-related gene 1) was found in the appressorium of the rice blast fungus. MgATG1 encodes a serine/threonine protein kinase. Considering the importance of the rice blast fungus as a primary model for host-pathogen interaction, the role of autophagy in fungal development, appressorium turgor, and pathogenicity of M. grisea through the turnover of organelle and protein is a very attractive topic for research—and is the subject of this paper wherein we report that autophagy is blocked in the mutant lacking the gene MgATG1, and that the mutant also shows fewer lipid droplets in the conidium, lower turgor pressure of the appressorium, and loss of ability to penetrate its host. Expression of GFP-ATG1 was uniformly detectable in the cytoplasm of conidia, mycelia, and appressoria. MgATG1 restored the corresponding defects in the starvation-sensitive phenotype ofΔatg1 mutant of S. cerevisiae. In mating experiments,Δmgatg1 mutant did not form perithecia.Similarly, an MgATG5 gene was isolated based on PCR, from the rice blast fungus, M. grisea. In theΔmgatg5 mutant, in which MgATG5 gene was deleted by targeted gene replacement, 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 and formation of appressorium. As a result of lower turgor pressure of the appressorium, theΔmgatg5 mutant lost its ability to penetrate and infect the two tested host plants, namely rice and barley. These properties were restored on re-introducing an intact copy of MgATG5 coding sequence into the mutants. Expression of GFP-ATG5 was detectable in the cytoplasm of conidia, mycelia, and appressoria, especially in karyon. MgATG5 restored the corresponding defects in the starvation-sensitive phenotype ofΔatg5 mutant of S. cerevisiae. In mating experiments,Δmgatg5 mutant did not form perithecia.In summary, autophagy is thus necessary for the turnover of organic matter during the formation of conidia and appressoria and for normal development and pathogenicity of M. grisea. MgATG1 and MgATG5 play a significant role in autophagy of M. grisea, which are responsible for fungal morphogenesis and ensure the turnover of organic matter necessary for generating appressorium turgor of required strength. The MgATG1-deleted and MgATG5-deleted mutants show defects in autophagy, the ability to survive starvation, conidiation, conidial germination, lipid turnover, and appressorium turgor. As a result, theΔmgatg1 orΔmgatg5 mutant loses its penetration ability and pathogencity to the two tested host plants, namely rice cv CO-39 and barley ZJ-8.