Yi Zhen Deng

A multidrug resistance transporter in Magnaporthe is required for host penetration and for survival during oxidative stress.

In prokaryotes and eukaryotes, multidrug resistance (MDR) transporters use energy-dependent efflux action to regulate the intracellular levels of antibiotic or xenobiotic compounds. Using mutational analysis of ABC3, we define an important role for such MDR-based efflux during the host penetration step of Magnaporthe grisea pathogenesis. Mutants lacking ABC3 were completely nonpathogenic but were surprisingly capable of penetrating thin cellophane membranes to some extent. The inability of abc3Δ to penetrate the host surface was most likely a consequence of excessive buildup of peroxide and accumulation of an inhibitory metabolite(s) within the mutant appressoria. Treatment with antioxidants partially suppressed the host penetration defects in the abc3Δ mutant. abc3Δ was highly sensitive to oxidative stress and was unable to survive the host environment and invasive growth conditions. ABC3 transcript levels were redox-regulated, and on host surfaces, the activation of ABC3 occurred during initial stages of blast disease establishment. An Abc3-green fluorescent protein fusion localized to the plasma membrane in early appressoria (and in penetration hyphae) but became predominantly vacuolar during appressorial maturity. We propose that ABC3 function helps Magnaporthe to cope with cytotoxicity and oxidative stress within the appressoria during early stages of infection-related morphogenesis and likely imparts defense against certain antagonistic and xenobiotic conditions encountered during pathogenic development.

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 Moatg8Δ 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.

Sorting nexin Snx41 is essential for conidiation and mediates glutathione-based antioxidant defense during invasive growth in Magnaporthe oryzae

The sorting nexins Atg20/Snx42 and Snx41 regulate membrane traffic and endosomal protein sorting and are essential for Cvt and/or pexophagy in yeast. Previously, we showed that macroautophagy is necessary for conidiation in the rice-blast fungus Magnaporthe oryzae. Here, we analyzed the physiological function(s) of selective autophagy in Magnaporthe through targeted deletion of MGG_12832, an ortholog of yeast SNX41 and ATG20/SNX42. Loss of MGG_12832 (hereafter SNX41) abolished conidia formation and pathogenesis in M. oryzae. Snx41-GFP localized as dynamic puncta or short tubules that are partially associated with autophagosomes and/or autophagic vacuoles. PX domain, but not macroautophagy per se, was required for such localization of Snx41-GFP in Magnaporthe. Although not required for nonselective autophagy, Snx41 was essential for pexophagy in Magnaporthe. We identified Oxp1, an ATP-dependent oxoprolinase in the gamma-glutamyl cycle, as a binding partner and potential retrieval target of Snx41-dependent protein sorting. The substrate of Oxp1, 5-oxoproline, could partially restore conidiation in the snx41Δ. Exogenous glutathione, a product of the gamma-glutamyl cycle, significantly restored pathogenicity in the snx41Δ mutant, likely through counteracting the oxidative stress imposed by the host. We propose that the gamma-glutamyl cycle and glutathione biosynthesis are subject to regulation by Snx41-dependent vesicular trafficking, and mediate antioxidant defense crucial for in planta growth and pathogenic differentiation of Magnaporthe at the onset of blast disease in rice.

Atg24-assisted mitophagy in the foot cells is necessary for proper asexual differentiation in Magnaporthe oryzae

Macroautophagy-mediated glycogen catabolism is required for asexual differentiation in the blast fungus, Magnaporthe oryzae. However, the function(s) of selective subtypes of autophagy has not been studied therein. Here, we report that mitophagy, selective autophagic delivery of mitochondria to the vacuoles for degradation, occurs during early stages of Magnaporthe conidiation. Specifically, mitophagy was evident in the foot cells while being undetectable in aerial hyphae and/or conidiophores. We show that loss of MoAtg24, a sorting nexin related to yeast Snx4, disrupts mitophagy and consequently leads to highly reduced conidiation, suggesting that mitophagy in the foot cells plays an important role during asexual development in Magnaporthe. Ectopic expression of yeast ScATG32 partially suppressed the conidiation initiation defects associated with MoATG24 deletion. MoAtg24 was neither required for pexophagy nor for macroautophagy, or for MoAtg8 localization per se, but directly associated with and likely recruited mitochondria to the autophagic structures during mitophagy. Lastly, MoAtg24 was also required for oxidative stress response in Magnaporthe.

A vacuolar glucoamylase, Sga1, participates in glycogen autophagy for proper asexual differentiation in Magnaporthe oryzae

Nutrient limitation acts as a trigger for the synthesis of glycogen, which serves as a carbon and energy reserve during starvation. Recently, we reported that an autophagy-deficient mutant (atg8Δ) shows severe reduction in aerial hyphal growth and conidiation in the rice-blast fungus Magnaporthe oryzae, and proposed that autophagy plays an important role in facilitating glycogen homeostasis to ensure proper asexual differentiation in Magnaporthe. Here, we identify and characterize a vacuolar glucoamylase function (Sga1) that hydrolyses glycogen to meet the energy requirements during asexual development in Magnaporthe. Loss of SGA1 resulted in significant reduction in conidiation compared to the wild-type Magnaporthe strain. More importantly, an sga1Δ atg8Δ double deletion mutant showed further reduction in conidiation compared to the atg8Δ mutant in Magnaporthe. Forced localization of GFP-Sga1 to the cytoplasm (through removal of the predicted signal peptide) led to increased conidiation in wild type and the sga1Δ, but more interestingly, significantly restored conidiation in the atg8Δ mutant. Our results indicate that autophagy and Sga1 act cooperatively in vacuolar glycogen breakdown, which is essential for conidia formation but dispensable for pathogenicity in Magnaporthe.

Role of Macroautophagy in Nutrient Homeostasis During Fungal Development and Pathogenesis

Macroautophagy is a non-selective, bulk degradation process conserved in eukaryotes. Response to starvation stress and/or regulation of nutrient breakdown/utilization is the major intracellular function of macroautophagy. Recent studies have revealed requirement for autophagy in diverse functions such as nutrient homeostasis, organelle degradation and programmed cell death in filamentous fungal pathogens, for proper morphogenesis and differentiation during critical steps of infection. In this review, we aim to summarize the physiological functions of autophagy in fungal virulence, with an emphasis on nutrient homeostasis in opportunistic human fungal pathogens and in the rice-blast fungus, Magnaporthe oryzae. We briefly summarize the role of autophagy on the host side: for resistance to, or subversion by, the pathogens.

Twilight, a Novel Circadian-Regulated Gene, Integrates Phototropism with Nutrient and Redox Homeostasis during Fungal Development

Phototropic regulation of circadian clock is important for environmental adaptation, organismal growth and differentiation. Light plays a critical role in fungal development and virulence. However, it is unclear what governs the intracellular metabolic response to such dark-light rhythms in fungi. Here, we describe a novel circadian-regulated Twilight (TWL) function essential for phototropic induction of asexual development and pathogenesis in the rice-blast fungus Magnaporthe oryzae. The TWL transcript oscillates during circadian cycles and peaks at subjective twilight. GFP-Twl remains acetylated and cytosolic in the dark, whereas light-induced phosphorylation (by the carbon sensor Snf1 kinase) drives it into the nucleus. The mRNA level of the transcription/repair factor TFB5, was significantly down regulated in the twl∆ mutant. Overexpression of TFB5 significantly suppressed the conidiation defects in the twl∆ mutant. Furthermore, Tfb5-GFP translocates to the nucleus during the phototropic response and under redox stress, while it failed to do so in the twl∆ mutant. Thus, we provide mechanistic insight into Twl-based regulation of nutrient and redox homeostasis in response to light during pathogen adaptation to the host milieu in the rice blast pathosystem.

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.

The role of snx41-based pexophagy in magnaporthe development.

Pexophagy, the degradation of peroxisomes via selective autophagy, depends on Atg20/Snx42 function in Saccharomyces cerevisiae. Besides its role in selective autophagy, Atg20/Snx42 is also involved in an autophagy-independent endosomal retrieval trafficking, in cooperation with two other sorting nexins, Snx41 and Snx4. Recently, we reported that the sorting nexin MoSnx41, which showed high sequence similarity to yeast Snx41 and Snx42/Atg20 proteins, regulates the gamma-glutamyl cycle and GSH production and is essential for conidiation and pathogenicity in Magnaporthe oryzae. Pexophagy was also found to be defective in Mosnx41Δ mutant. These findings indicate that MoSnx41 likely serves combined functions of Snx42/Atg20 and Snx41 in M. oryzae.. In this study, we performed complementation analyses and demonstrate that MoSnx41 alone serves the dual function of protein sorting (ScSnx41) and pexophagy (ScSnx42/Atg20). To study the potential biological function of pexophagy in fungal pathogenic life cycle, we created deletion mutants of potential pexophagy-specific genes, and characterized them in terms of pexophagy, conidiation and pathogenesis. We identified Pex14 as an essential protein for pexophagy in M. oryzae. Overall, our results show that pexophagy per se is not essential for asexual development or virulence in M. oryzae.