Mitochondrial ß-oxidation regulates organellar integrity and is necessary for conidial germination and invasive growth in Magnaporthe oryzae.

Fatty acids stored as triglycerides, an important source of cellular energy, are catabolized through ß-oxidation pathways predicted to occur both in peroxisomes and mitochondria in filamentous fungi. Here, we characterize the function of Enoyl-CoA hydratase Ech1, a mitochondrial ß-oxidation enzyme, in the model phytopathogen Magnaporthe oryzae. Ech1 was found to be essential for conidial germination and viability of older hyphae. Unlike wild-type Magnaporthe, the ech1Δ failed to utilize C14 fatty acid and was partially impeded in growth on C16 and C18 fatty acids. Surprisingly, loss of ß-oxidation led to significantly altered mitochondrial morphology and integrity with ech1Δ showing predominantly vesicular/punctate mitochondria in contrast to the fused tubular network in wild-type Magnaporthe. The ech1Δ appressoria were aberrant and displayed reduced melanization. Importantly, we show that the significantly reduced ability of ech1Δ to penetrate the host and establish therein is a direct consequence of enhanced sensitivity of the mutant to oxidative stress, as the defects could be remarkably reversed through exogenous antioxidants. Overall, our comparative analyses reveal that peroxisomal lipid catabolism is essential for appressorial function of host penetration, whereas mitochondrial ß-oxidation primarily contributes to conidial viability and maintenance of redox homeostasis during host colonization by Magnaporthe.

Autophagy-assisted glycogen catabolism regulates asexual differentiation in Magnaporthe oryzae.

Autophagy, a conserved pathway for bulk cellular degradation and recycling in eukaryotes, regulates proper turnover of organelles, membranes and certain proteins. Such regulated degradation is important for cell growth and development particularly during environmental stress conditions, which act as key inducers of autophagy. We found that autophagy and MoATG8 were significantly induced during asexual development in Magnaporthe oryzae. An RFP-tagged MoAtg8 showed specific localization and enrichment in aerial hyphae, conidiophores and conidia. We confirmed that loss of MoATG8 results in dramatically reduced ability to form conidia, the asexual spores that propagate rice-blast disease. Exogenous supply of glucose or sucrose significantly suppressed the conidiation defects in a MoATG8-deletion mutant. Comparative proteomics based identification and characterization of Gph1, a glycogen phosphorylase that catalyzes glycogen breakdown, indicated that autophagy-assisted glycogen homeostasis is likely important for proper aerial growth and conidiation in Magnaporthe. Loss of Gph1, or addition of G6P significantly restored conidiation in the Moatg8Delta mutant. Overproduction of Gph1 led to reduced conidiation in wild-type Magnaporthe strain. We propose that glycogen autophagy actively responds to and regulates carbon utilization required for cell growth and differentiation during asexual development in Magnaporthe.

Methods for functional analysis of macroautophagy in filamentous fungi.

Autophagy is a bulk degradative process responsible for the turnover of membranes, organelles, and proteins in eukaryotic cells. Genetic and molecular regulation of autophagy has been independently elucidated in budding yeast and mammalian cells. In filamentous fungi, autophagy is required for several important physiological functions, such as asexual and sexual differentiation, pathogenic development, starvation stress and programmed cell death during heteroincompatibility. Here, we detail biochemical and microscopy methods useful for measuring the rate of induction of autophagy in filamentous fungi, and we summarize the methods that have been routinely used for monitoring macroautophagy in both yeast and filamentous fungi. The role of autophagy in carbohydrate catabolism and cell survival is discussed along with the specific functions of macroautophagy in fungal development and pathogenesis.

Host invasion during rice-blast disease requires carnitine-dependent transport of peroxisomal acetyl-CoA.

In lower eukaryotes, beta-oxidation of fatty acids is restricted primarily to the peroxisomes and the resultant acetyl-CoA molecules (and the chain-shortened fatty acids) are transported via the cytosol into the mitochondria for further breakdown and usage. Using a loss-of-function mutation in the Magnaporthe grisea PEROXIN6 orthologue, we define an essential role for peroxisomal acetyl-CoA during the host invasion step of the rice-blast disease. We show that an Mgpex6Delta strain lacks functional peroxisomes and is incapable of beta-oxidation of long-chain fatty acids. The Mgpex6Delta mutant lacked appressorial melanin and host penetration, and was completely non-pathogenic. We further show that a peroxisome-associated carnitine acetyl-transferase (Crat1) activity is essential for such appressorial function in Magnaporthe. CRAT1-minus appressoria showed reduced melanization, but were surprisingly incapable of elaborating penetration pegs or infection hyphae. Exogenous addition of excess glucose during infection stage caused partial remediation of the pathogenicity defects in the crat1Delta strain. Moreover, Mgpex6Delta and crat1Delta mycelia showed increased sensitivity to Calcofluor white, suggesting that weakened cell wall biosynthesis in a glucose-deficient environment leads to appressorial dysfunction in these mutants. Interestingly, CRAT1 was itself essential for growth on acetate and long-chain fatty acids. Thus, carnitine-dependent metabolic activities associated with the peroxisomes, cooperatively facilitate the appressorial function of host invasion during rice-blast infections.

Chemical- and irradiation-induced mutants of indica rice IR64 for forward and reverse genetics.

IR64, the most widely grown indica rice in South and Southeast Asia, possesses many positive agronomic characteristics (e.g., wide adaptability, high yield potential, tolerance to multiple diseases and pests, and good eating quality,) that make it an ideal genotype for identifying mutational changes in traits of agronomic importance. We have produced a large collection of chemical and irradiation-induced IR64 mutants with different genetic lesions that are amenable to both forward and reverse genetics. About 60,000 IR64 mutants have been generated by mutagenesis using chemicals (diepoxybutane and ethylmethanesulfonate) and irradiation (fast neutron and gamma ray). More than 38,000 independent lines have been advanced to M4 generation enabling evaluation of quantitative traits by replicated trials. Morphological variations at vegetative and reproductive stages, including plant architecture, growth habit, pigmentation and various physiological characters, are commonly observed in the four mutagenized populations. Conditional mutants such as gain or loss of resistance to blast, bacterial blight, and tungro disease have been identified at frequencies ranging from 0.01% to 0.1%. Results from pilot experiments indicate that the mutant collections are suitable for reverse genetics through PCR-detection of deletions and TILLING. Furthermore, deletions can be detected using oligomer chips suggesting a general technique to pinpoint deletions when genome-wide oligomer chips are broadly available. M4 mutant seeds are available for users for screening of altered response to multiple stresses. So far, more than 15,000 mutant lines have been distributed. To facilitate broad usage of the mutants, a mutant database has been constructed in the International Rice Information System (IRIS; http: //www.iris.irri.org) to document the phenotypes and gene function discovered by users.

Woronin body function in Magnaporthe grisea is essential for efficient pathogenesis and for survival during nitrogen starvation stress.

The Woronin body is a peroxisome-derived dense-core vesicle that is specific to several genera of filamentous ascomycetes, where it has been shown to seal septal pores in response to cellular damage. The Hexagonal peroxisome (Hex1) protein was recently identified as a major constituent of the Woronin body and shown to be responsible for self-assembly of the dense core of this organelle. Using a mutation in the Magnaporthe grisea HEX1 ortholog, we define a dual and essential function for Woronin bodies during the pathogenic phase of the rice blast fungus. We show that the Woronin body is initially required for proper development and function of appressoria (infection structures) and subsequently necessary for survival of infectious fungal hyphae during invasive growth and host colonization. Fungal mycelia lacking HEX1 function were unable to survive nitrogen starvation in vitro, suggesting that in planta growth defects are a consequence of the mutant's inability to cope with nutritional stress. Thus, Woronin body function provides the blast fungus with an important defense against the antagonistic and nutrient-limiting environment encountered within the host plant.