Similarities and Differences of Autophagy in Mammals, Plants, and Microbes.

Autophagy, a highly conserved metabolic process in eukaryotes, is a widespread degradation/recycling system. However, there are significant differences (as well as similarities) between autophagy in animals, plants, and microorganisms such as yeast. While the overall process of autophagy is similar between different organisms, the molecular mechanisms and the pathways regulating autophagy are different, which is manifested in the diversity and specificity of the genes involved. In general, the autophagy system is much more complicated in mammals than in yeast. In addition, there are some differences in the types of autophagy present in animals, plants, and microorganisms. For example, there is a unique type of selective autophagy called the cytoplasm-to-vacuole targeting (Cvt) pathway in yeast, and a special kind of autophagy, chloroplast autophagy, exists in plants. In conclusion, although autophagy is highly conserved in eukaryotes, there are still many differences between autophagy of animals, plants, and microorganisms.

F-box proteins MoFwd1, MoCdc4 and MoFbx15 regulate development and pathogenicity in the rice blast fungus Magnaporthe oryzae.

The Skp1-Cul1-F-box-protein (SCF) ubiquitin ligases are important parts of the ubiquitin system controlling many cellular biological processes in eukaryotes. However, the roles of SCF ubiquitin ligases remain unclear in phytopathogenic Magnaporthe oryzae. Here, we cloned 24 F-box proteins and confirmed that 17 proteins could interact with MoSkp1, showing their potential to participate in SCF complexes. To determine their functions, null mutants of 21 F-box-containing genes were created. Among them, the F-box proteins MoFwd1, MoCdc4 and MoFbx15 were found to be required for growth, development and full virulence. Fluorescent-microscopy observations demonstrated that both MoFbx15 and MoCdc4 were localized to the nucleus, compared with MoFwd1, which was distributed in the cytosol. MoCdc4 and MoFwd1 bound to MoSkp1 via the F-box domain, the deletion of which abrogated their function. Race tube and qRT-PCR assays confirmed that MoFwd1 was involved in circadian rhythm by regulating transcription and protein stability of the core circadian clock regulator MoFRQ. Moreover, MoFWD1 also orchestrates conidial germination by influencing conidial amino acids pools and oxidative stress release. Overall, our results indicate that SCF ubiquitin ligases play indispensable roles in development and pathogenicity in M. oryzae.

Autophagy in plant pathogenic fungi.

Autophagy is a conserved cellular process that degrades cytoplasmic constituents in vacuoles. Plant pathogenic fungi develop special infection structures and/or secrete a range of enzymes to invade their plant hosts. It has been demonstrated that monitoring autophagy processes can be extremely useful in visualizing the sequence of events leading to pathogenicity of plant pathogenic fungi. In this review, we introduce the molecular mechanisms involved in autophagy. In addition, we explore the relationship between autophagy and pathogenicity in plant pathogenic fungi. Finally, we discuss the various experimental strategies available for use in the study of autophagy in plant pathogenic fungi.

VPS9 domain-containing proteins are essential for autophagy and endocytosis in Pyricularia oryzae.

Pyricularia oryzae is the causal pathogen of rice blast disease. Autophagy has been shown to play important roles in P. oryzae development and plant infection. The P. oryzae endosomal system is highly dynamic and has been shown to be associated with conidiogenesis and pathogenicity as well. To date, the crosstalk between autophagy and endocytosis has not been explored in P. oryzae. Here, we identified three P. oryzae VPS9 domain-containing proteins, PoVps9, PoMuk1 and PoVrl1. We found that PoVps9 and PoMuk1 are localized to vesicles and are each co-localized with PoVps21, a recognized marker of early endosomes. Deletion of PoVPS9 resulted in severe defects in endocytosis and autophagosome degradation and impaired the localization of PoVps21 to endosomes. Additionally, deletion of the PoMUK1 gene in the ΔPovps9 mutant background exhibited more severe defects in development, autophagy and endocytosis compared with the ΔPovps9 mutant. Pull-down assay showed that PoVps9 interacts with PoVps21, PoRab11 and PoRab1, which have been verified to participate in endocytosis. Furthermore, yeast two-hybrid and co-immunoprecipitation assays confirmed that PoVps9 directly interacts with the GDP form of PoVps21. Thus, PoVps9 is a key protein involved in autophagy and in endocytosis.

MoRad6-mediated ubiquitination pathways are essential for development and pathogenicity in Magnaporthe oryzae.

The ubiquitin system modulates protein functions through targeting substrates for ubiquitination. Here, E2 conjugating enzyme MoRad6-related ubiquitination pathways are identified and analyzed in Magnaporthe oryzae, the causal agent of rice blast disease. Disruption of MoRad6 leads to severe defects in growth, sporulation, conidial germination, appressorium formation, and plant infection. To depict the functions of MoRad6, three putative ubiquitin ligases, MoRad18, MoBre1 and MoUbr1, are also characterized. Deletion of MoRad18 causes minor phenotypic changes, while MoBre1 is required for growth, conidiation and pathogenicity in M. oryzae. Defects in ΔMobre1 likely resulted from the reduction in di- and tri-methylation level of Histone 3 lysine 4 (H3K4). Notably, MoUbr1 is crucial for conidial adhesion and germination, possibly by degrading components of cAMP/PKA and mitogen-activated protein kinase (MAPK) Pmk1 signaling pathways via the N-end rule pathway. Germination failure of ΔMoubr1 conidia could be rescued by elevation of cAMP level or enhanced Pmk1 phosphorylation resulting from further deletion of MoIra1, the M. oryzae homolog of yeast Ira1/2. These reveal vital effects of cAMP/PKA and MAPK Pmk1 signaling on conidial germination in M. oryzae. Altogether, our results suggest that MoRad6-mediated ubiquitination pathways are essential for the infection-related development and pathogenicity of M. oryzae.

The small GTPase MoYpt7 is required for membrane fusion in autophagy and pathogenicity of Magnaporthe oryzae.

Rab GTPases are required for vesicle-vacuolar fusion during vacuolar biogenesis in fungi. To date, little is known about the biological functions of the Rab small GTPase components in Magnaporthe oryzae. In this study, we investigated MoYpt7 of M. oryzae, a homologue of the small Ras-like GTPase Ypt7 in Saccharomyces cerevisiae. Cellular localization assays showed that MoYpt7 was predominantly localized to vacuolar membranes. Using a targeted gene disruption strategy, a ΔMoYPT7 mutant was generated that exhibited defects in mycelial growth and production of conidia. The conidia of the ΔMoYPT7 mutant were malformed and defective in the formation of appressoria. Consequently, the ΔMoYPT7 mutant failed to cause disease in rice and barley. Furthermore, the ΔMoYPT7 mutant showed impairment in autophagy, breached cell wall integrity, and higher sensitivity to both calcium and heavy metal stress. Transformants constitutively expressing an active MoYPT7 allele (MoYPT7-CA, Gln67Leu) exhibited distinct phenotypes from the ΔMoYPT7 mutant. Expression of MoYPT7-CA in MoYpt7 reduced pathogenicity and produced more appressoria-forming single-septum conidia. These results indicate that MoYPT7 is required for fungal morphogenesis, vacuole fusion, autophagy, stress resistance and pathogenicity in M. oryzae.

A VASt-domain protein regulates autophagy, membrane tension, and sterol homeostasis in rice blast fungus.

Sterols are a class of lipids critical for fundamental biological processes and membrane dynamics. These molecules are synthesized in the endoplasmic reticulum (ER) and are transported bi-directionally between the ER and plasma membrane (PM). However, the trafficking mechanism of sterols and their relationship with macroautophagy/autophagy are still poorly understood in the rice blast fungus Magnaporthe oryzae. Here, we identified the VAD1 Analog of StAR-related lipid transfer (VASt) domain-containing protein MoVast1 via co-immunoprecipitation in M. oryzae. Loss of MoVAST1 resulted in conidial defects, impaired appressorium development, and reduced pathogenicity. The MoTor (target of rapamycin in M. oryzae) activity is inhibited because MoVast1 deletion leads to high levels of sterol accumulation in the PM. Site-directed mutagenesis showed that the 902 T site is essential for localization and function of MoVast1. Through filipin or Flipper-TR staining, autophagic flux detection, MoAtg8 lipidation, and drug sensitivity assays, we uncovered that MoVast1 acts as a novel autophagy inhibition factor that monitors tension in the PM by regulating the sterol content, which in turn modulates the activity of MoTor. Lipidomics and transcriptomics analyses further confirmed that MoVast1 is an important regulator of lipid metabolism and the autophagy pathway. Our results revealed and characterized a novel sterol transfer protein important for M. oryzae pathogenicity.Abbreviations: AmB: amphotericin B; ATMT: Agrobacterium tumefaciens-mediated transformation; CM: complete medium; dpi: days post-inoculation; ER: endoplasmic reticulum; Flipper-TR: fluorescent lipid tension reporter; GO: Gene ontology; hpi: hours post-inoculation; IH: invasive hyphae; KEGG: kyoto encyclopedia of genes and genomes; MoTor: target of rapamycin in Magnaporthe oryzae; PalmC: palmitoylcarnitine; PM: plasma membrane; SD-N: synthetic defined medium without amino acids and ammonium sulfate; TOR: target of rapamycin; VASt: VAD1 Analog of StAR-related lipid transfer; YFP, yellow fluorescent protein.

MoFap7, a ribosome assembly factor, is required for fungal development and plant colonization of Magnaporthe oryzae.

Fap7, an important ribosome assembly factor, plays a vital role in pre-40S small ribosomal subunit synthesis in Saccharomyces cerevisiae via its ATPase activity. Currently, the biological functions of its homologs in filamentous fungi remain elusive. Here, MoFap7, a homologous protein of ScFap7, was identified in the rice blast fungus Magnaporthe oryzae, which is a devastating fungal pathogen in rice and threatens food security worldwide. ΔMofap7 mutants exhibited defects in growth and development, conidial morphology, appressorium formation and infection, and were sensitive to oxidative stress. In addition, site-directed mutagenesis analysis confirmed that the conserved Walker A motif and Walker B motif in MoFap7 are essential for the biological functions of M. oryzae. We further analyzed the regulation mechanism of MoFap7 in pathogenicity. MoFap7 was found to interact with MoMst50, a regulator functioning in the MAPK Pmk1 signaling pathway, that participates in modulating plant penetration and cell-to-cell invasion by regulating the phosphorylation of MoPmk1. Moreover, MoFap7 interacted with the GTPases MoCdc42 and MoRac1 to control growth and conidiogenesis. Taken together, the results of this study provide novel insights into MoFap7-mediated orchestration of the development and pathogenesis of filamentous fungi.

Current opinions on autophagy in pathogenicity of fungi.

The interaction between pathogens and their host plants is a ubiquitous process. Some plant fungal pathogens can form a specific infection structure, such as an appressorium, which is formed by the accumulation of a large amount of glycerin and thereby the creation of an extremely high intracellular turgor pressure, which allows the penetration peg of the appressorium to puncture the leaf cuticle of the host. Previous studies have shown that autophagy energizes the accumulation of pressure by appressoria, which induces its pathogenesis. Similar to other eukaryotic organisms, autophagy processes are highly conserved pathways that play important roles in filamentous fungal pathogenicity. This review aims to demonstrate how the autophagy process affects the pathogenicity of plant pathogens.

The casein kinase MoYck1 regulates development, autophagy, and virulence in the rice blast fungus.

Casein kinases are serine/threonine protein kinases that are evolutionarily conserved in yeast and humans and are involved in a range of important cellular processes. However, the biological functions of casein kinases in the fungus Magnaporthe oryzae, the causal agent of destructive rice blast disease, are not characterized. Here, two casein kinases, MoYCK1 and MoHRR25, were identified and targeted for replacement, but only MoYCK1 was further characterized due to the possible nonviability of the MoHRR25 deletion mutant. Disruption of MoYCK1 caused pleiotropic defects in growth, conidiation, conidial germination, and appressorium formation and penetration, therefore resulting in reduced virulence in rice seedlings and barley leaves. Notably, the MoYCK1 deletion triggered quick lipidation of MoAtg8 and degradation of the autophagic marker protein GFP-MoAtg8 under nitrogen starvation conditions, in contrast to the wild type, indicating that autophagy activity was negatively regulated by MoYck1. Furthermore, we found that HOPS (homotypic fusion and vacuolar protein sorting) subunit MoVps41, a putative substrate of MoYck1, was co-located with MoAtg8 and positively required for the degradation of MoAtg8-PE and GFP-MoAtg8. In addition, MoYCK1 is also involved in the response to ionic hyperosmotic and heavy metal cation stresses. Taken together, our results revealed crucial roles of the casein kinase MoYck1 in regulating development, autophagy and virulence in M. oryzae.

Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae.

The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48 h at 22 °C could lead to a relatively highest frequency of transformation, and 200 µM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast-Escherichia-Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae.

Crosstalk between SNF1 pathway and the peroxisome-mediated lipid metabolism in Magnaporthe oryzae.

The SNF1/AMPK pathway has a central role in response to nutrient stress in yeast and mammals. Previous studies on SNF1 function in phytopathogenic fungi mostly focused on the catalytic subunit Snf1 and its contribution to the derepression of cell wall degrading enzymes (CWDEs). However, the MoSnf1 in Magnaporthe oryzae was reported not to be involved in CWDEs regulation. The mechanism how MoSnf1 functions as a virulence determinant remains unclear. In this report, we demonstrate that MoSnf1 retains the ability to respond to nutrient-free environment via its participation in peroxisomal maintenance and lipid metabolism. Observation of GFP-tagged peroxisomal targeting signal-1 (PTS1) revealed that the peroxisomes of ΔMosnf1 were enlarged in mycelia and tended to be degraded before conidial germination, leading to the sharp decline of peroxisomal amount during appressorial development, which might impart the mutant great retard in lipid droplets mobilization and degradation. Consequently, ΔMosnf1 exhibited inability to maintain normal appressorial cell wall porosity and turgor pressure, which are key players in epidermal infection process. Exogenous glucose could partially restore the appressorial function and virulence of ΔMosnf1. Toward a further understanding of SNF1 pathway, the ß-subunit MoSip2, γ-subunit MoSnf4, and two putative Snf1-activating kinases, MoSak1 and MoTos3, were additionally identified and characterized. Here we show the null mutants ΔMosip2 and ΔMosnf4 performed multiple disorders as ΔMosnf1 did, suggesting the complex integrity is essential for M. oryzae SNF1 kinase function. And the upstream kinases, MoSak1 and MoTos3, play unequal roles in SNF1 activation with a clear preference to MoSak1 over MoTos3. Meanwhile, the mutant lacking both of them exhibited a severe phenotype comparable to ΔMosnf1, uncovering a cooperative relationship between MoSak1 and MoTos3. Taken together, our data indicate that the SNF1 pathway is required for fungal development and facilitates pathogenicity by its contribution to peroxisomal maintenance and lipid metabolism in M. oryzae.

MoMon1 is required for vacuolar assembly, conidiogenesis and pathogenicity in the rice blast fungus Magnaporthe oryzae.

Mon1 protein is involved in cytoplasm-to-vacuole trafficking, vacuolar morphology and autophagy, and is required for homotypic vacuole fusion in Saccharomyces cerevisiae. Here we identify MoMON1 from Magnaporthe oryzae as an ortholog of S. cerevisiae MON1, essential for the morphology of the vacuole and vesicle fusion. Target gene deletion of MoMON1 resulted in accumulation of small punctuate vacuoles in the hypha and hypersensitivity to monensin, an antibiotic that blocks intracellular protein transport. The ΔMomon1 mutant exhibited significantly reduced aerial hyphal development and poor conidiation. Conidia of ΔMomon1 were able to differentiate appressoria. However, ΔMomon1 was non-pathogenic on rice leaves, even after wound inoculation. In addition, ΔMomon1 was slightly hypersensitive to Congo red and SDS, but not to cell wall degrading enzymes, suggesting significant alterations in its cell wall. The autophagy process was blocked in the ΔMomon1 mutant. Taken together, our results suggest that MoMON1 has an essential function in vacuolar assembly, autophagy, fungal development and pathogenicity in M. oryzae.