Apoplastic ascorbate oxidases (AOs) play a critical role in reactive oxygen species (ROS)-mediated innate host immunity by regulating the apoplast redox state. To date, little is known about how apoplastic effectors of the rice blast fungus Magnaporthe oryzae modulate the apoplast redox state of rice to subvert plant immunity. In this study, we demonstrated that M. oryzae MoAo1 is an AO that plays a role in virulence by modulating the apoplast redox status of rice cells. We showed that MoAo1 inhibits the activity of rice OsAO3 and OsAO4, which also regulate the apoplast redox status and plant immunity. In addition, we found that MoAo1, OsAO3, and OsAO4 all exhibit polymorphic variations whose varied interactions orchestrate pathogen virulence and rice immunity. Taken together, our results reveal a critical role for extracellular redox enzymes during rice blast infection and shed light on the importance of the apoplast redox state and its regulation in plant-pathogen interactions.

Many pathogens infect hosts through specific organs, such as Ustilaginoidea virens, which infects rice panicles. Here, we show that a microbe-associated molecular pattern (MAMP), Ser-Thr-rich Glycosyl-phosphatidyl-inositol-anchored protein (SGP1) from U. virens, induces immune responses in rice leaves but not panicles. SGP1 is widely distributed among fungi and acts as a proteinaceous, thermostable elicitor of BAK1-dependent defense responses in N. benthamiana. Plants specifically recognize a 22 amino acid peptide (SGP1 N terminus peptide 22, SNP22) in its N-terminus that induces cell death, oxidative burst, and defense-related gene expression. Exposure to SNP22 enhances rice immunity signaling and resistance to infection by multiple fungal and bacterial pathogens. Interestingly, while SGP1 can activate immune responses in leaves, SGP1 is required for U. virens infection of rice panicles in vivo, showing it contributes to the virulence of a panicle adapted pathogen.

The production of reactive oxygen species (ROS) is a ubiquitous defense response in plants. Adapted pathogens evolved mechanisms to counteract the deleterious effects of host-derived ROS and promote infection. How plant pathogens regulate this elaborate response against ROS burst remains unclear. Using the rice blast fungus Magnaporthe oryzae, we uncovered a self-balancing circuit controlling response to ROS in planta and virulence. During infection, ROS induces phosphorylation of the high osmolarity glycerol pathway kinase MoOsm1 and its nuclear translocation. There, MoOsm1 phosphorylates transcription factor MoAtf1 and dissociates MoAtf1-MoTup1 complex. This releases MoTup1-mediated transcriptional repression on oxidoreduction-pathway genes and activates the transcription of MoPtp1/2 protein phosphatases. In turn, MoPtp1/2 dephosphorylate MoOsm1, restoring the circuit to its initial state. Balanced interactions among proteins centered on MoOsm1 provide a means to counter host-derived ROS. Our findings thereby reveal new insights into how M. oryzae utilizes a phosphor-regulatory circuitry to face plant immunity during infection.

Genome dynamics of pathogenic organisms are driven by pathogen and host co-evolution, in which pathogen genomes are shaped to overcome stresses imposed by hosts with various genetic backgrounds through generation of a variety of isolates. This same principle applies to the rice blast pathogen Magnaporthe oryzae and the rice host; however, genetic variations among different isolates of M. oryzae remain largely unknown, particularly at genome and transcriptome levels. Here, we applied genomic and transcriptomic analytical tools to investigate M. oryzae isolate 98-06 that is the most aggressive in infection of susceptible rice cultivars. A unique 1.4 Mb of genomic sequences was found in isolate 98-06 in comparison to reference strain 70-15. Genome-wide expression profiling revealed the presence of two critical expression patterns of M. oryzae based on 64 known pathogenicity-related (PaR) genes. In addition, 134 candidate effectors with various segregation patterns were identified. Five tested proteins could suppress BAX-mediated programmed cell death in Nicotiana benthamiana leaves. Characterization of isolate-specific effector candidates Iug6 and Iug9 and PaR candidate Iug18 revealed that they have a role in fungal propagation and pathogenicity. Moreover, Iug6 and Iug9 are located exclusively in the biotrophic interfacial complex (BIC) and their overexpression leads to suppression of defense-related gene expression in rice, suggesting that they might participate in biotrophy by inhibiting the SA and ET pathways within the host. Thus, our studies identify novel effector and PaR proteins involved in pathogenicity of the highly aggressive M. oryzae field isolate 98-06, and reveal molecular and genomic dynamics in the evolution of M. oryzae and rice host interactions.

Arginine is a semi-essential amino acid that affects physiological and biochemical functions. The CPA2 gene in yeast encodes a large subunit of arginine-specific carbamoyl phosphate synthetase (CPS) and is involved in arginine biosynthesis. Here, an ortholog of yeast CPA2 was identified in the rice blast fungus Magnaporthe oryzae, and was named MoCPA2. MoCpa2 is an 1180-amino acid protein which contains an ATP grasp domain and two CPSase domains. Targeted deletion of MoCPA2 supported its role in de novo arginine biosynthesis in M. oryzae as mutant phenotypes were complemented by arginine but not ornithine. The ΔMocpa2 mutant exhibited defects in asexual development and pathogenicity but not appressorium formation. Further examination revealed that the invasive hyphae of the ΔMocpa2 mutant were restricted mainly to the primary infected cells. In addition, the ΔMocpa2 mutant was unable to induce a plant defense response and had the ability to scavenge ROS during pathogen-plant interactions. Structure analysis revealed that the ATP grasp domain and each CPS domain were indispensable for the proper localization and full function of MoCpa2. In summary, our results indicate that MoCpa2 plays an important role in arginine biosynthesis, and affects growth, conidiogenesis, and pathogenicity. These results suggest that research into metabolism and processes that mediate amino acid synthesis are valuable for understanding M. oryzae pathogenesis.

The emergence of fungicide resistance severely threatens crop production by limiting the availability and application of established fungicides. Therefore, it is urgent to identify new fungicidal targets for controlling plant diseases. Here, we characterized the function of a conserved homoserine O-acetyltransferase (HOA) from the rice blast fungus Magnaporthe oryzae that could serve as the candidate antifungal target. Deletion of the MoMET2 and MoCYS2 genes encoding HOAs perturbed the biosynthesis of methionine and S-adenyl methionine, a methyl group donor for epigenetic modifications, and severely attenuated the development and virulence of M. oryzae. The ∆Momet2 mutant is significantly increased in 5-methylcytosine (5mC) modification that represses the expression of genes required for pathogenicity, including MoGLIK and MoCDH-CYT. We further showed that host-induced gene silencing (HIGS) targeting MoMET2 and MoCYS2 effectively controls rice blasts. Our studies revealed the importance of HOA in the development and virulence of M. oryzae, which suggests the potential feasibility of HOA as new targets for novel anti-rice blast measurements.

The rice blast fungus Magnaporthe oryzae forms specialized infectious structures called appressoria that breach host cells to initiate infection. Previous studies demonstrated that the regulator of G-protein signaling (RGS)-like protein MoRgs7 undergoes endocytosis upon fungal sensing of hydrophobic environmental cues to activate cAMP signaling required for appressorium formation. However, the mechanism by which MoRgs7 internalizes and its fate remains undetermined. We here show that MoSep1, a conserved protein kinase of Mitotic Exit Network (MEN), phosphorylates MoRgs7 to regulate its function. MoRgs7 phosphorylation determines its interaction with MoCrn1, a coronin-like actin-binding protein homolog that also modulates the internalization of MoRgs7. Importantly, the endocytic transport of MoRgs7 is critical for its GTPase-activating protein (GAP) function important in cAMP signaling. Together, our findings revealed a novel mechanism by which M. oryzae activates MoRgs7-mediated hydrophobic cue-sensing signal transduction involving protein phosphorylation and endocytic transport to govern appressorium formation and fungal pathogenicity.

The interplay between plant and pathogen is a dynamic process, with the host's innate defense mechanisms serving a crucial role in preventing infection. In response to many plant pathogen infections, host cells generate the key regulatory molecule, reactive oxygen species (ROS), to limit the spread of the invading organism. In this study, we reveal the effects of fungal peroxisome dynamics on host ROS homeostasis, during the rice blast fungus Magnaporthe oryzae infection. The elongation of the peroxisome appears contingent upon ROS and links to the accumulation of ROS within the host and the infectious growth of the pathogen. Importantly, we identify a peroxisomal 3-ketoacyl-CoA thiolase, MoKat2, responsible for the elongation of the peroxisome during the infection. In response to host-derived ROS, the homodimer of MoKat2 undergoes dissociation to bind peroxisome membranes for peroxisome elongation. This process, in turn, inhibits the accumulation of host ROS, which is necessary for successful infection. Overall, our study is the first to highlight the intricate relationship between fungal organelle dynamics and ROS-mediated host immunity, extending the fundamental knowledge of pathogen-host interaction.

Previously, it was found that Nep1Mo (a Nep1-like protein from Magnaporthe oryzae) could trigger a variety of plant responses, including stomatal closure, hypersensitive cell death (HCD), and defence-related gene expression, in Nicotiana benthamiana. In this study, it was found that Nep1Mo-induced cell death could be inhibited by the virus-induced gene silencing of NbALY916 in N. benthamiana. NbALY916-silenced plants showed impaired Nep1Mo-induced stomatal closure, decreased Nep1Mo-induced production of hydrogen peroxide (H2O2) and nitric oxide (NO) in guard cells, and reduced Nep1Mo-induced resistance against Phytophthora nicotianae. It also found that the deletion of AtALY4, an orthologue of NbALY916 in Arabidopsis thaliana, impaired Nep1Mo-triggered stomatal closure, HCD, and defence-related gene expression. The compromised stomatal closure observed in the NbALY916-silenced plants and AtALY4 mutants was inhibited by the application of H2O2 and sodium nitroprusside (an NO donor), and both Nep1Mo and H2O2 stimulated guard cell NO synthesis. Conversely, NO-induced stomatal closure was found not to require H2O2 synthesis; and NO treatment did not induce H2O2 production in guard cells. Taken together, these results demonstrate that the NbAlY916/AtAlY4-H2O2-NO pathway mediates multiple Nep1Mo-triggered responses, including stomatal closure, HCD, and defence-related gene expression.

Regulator of G-protein signaling (RGS) proteins primarily function as GTPase-accelerating proteins (GAPs) to promote GTP hydrolysis of Gα subunits, thereby regulating G-protein mediated signal transduction. RGS proteins could also contain additional domains such as GoLoco to inhibit GDP dissociation. The rice blast fungus Magnaporthe oryzae encodes eight RGS and RGS-like proteins (MoRgs1 to MoRgs8) that have shared and distinct functions in growth, appressorium formation and pathogenicity. Interestingly, MoRgs7 and MoRgs8 contain a C-terminal seven-transmembrane domain (7-TM) motif typical of G-protein coupled receptor (GPCR) proteins, in addition to the conserved RGS domain. We found that MoRgs7, but not MoRgs8, couples with Gα MoMagA to undergo endocytic transport from the plasma membrane to the endosome upon sensing of surface hydrophobicity. We also found that MoRgs7 can interact with hydrophobic surfaces via a hydrophobic interaction, leading to the perception of environmental hydrophobiccues. Moreover, we found that MoRgs7-MoMagA endocytosis is regulated by actin patch-associated protein MoCrn1, linking it to cAMP signaling. Our studies provided evidence suggesting that MoRgs7 could also function in a GPCR-like manner to sense environmental signals and it, together with additional proteins of diverse functions, promotes cAMP signaling required for developmental processes underlying appressorium function and pathogenicity.

Eukaryotic cells respond to environmental stimuli when cell surface receptors are bound by environmental ligands. The binding initiates a signal transduction cascade that results in the appropriate intracellular responses. Studies have shown that endocytosis is critical for receptor internalization and signaling activation. In the rice blast fungus Magnaporthe oryzae, a non-canonical G-protein coupled receptor, Pth11, and membrane sensors MoMsb2 and MoSho1 are thought to function upstream of G-protein/cAMP signaling and the Pmk1 MAPK pathway to regulate appressorium formation and pathogenesis. However, little is known about how these receptors or sensors are internalized and transported into intracellular compartments. We found that the MoEnd3 protein is important for endocytic transport and that the ΔMoend3 mutant exhibited defects in efficient internalization of Pth11 and MoSho1. The ΔMoend3 mutant was also defective in Pmk1 phosphorylation, autophagy, appressorium formation and function. Intriguingly, restoring Pmk1 phosphorylation levels in ΔMoend3 suppressed most of these defects. Moreover, we demonstrated that MoEnd3 is subject to regulation by MoArk1 through protein phosphorylation. We also found that MoEnd3 has additional functions in facilitating the secretion of effectors, including Avr-Pia and AvrPiz-t that suppress rice immunity. Taken together, our findings suggest that MoEnd3 plays a critical role in mediating receptor endocytosis that is critical for the signal transduction-regulated development and virulence of M. oryzae.

The cell wall provides a crucial barrier to stress imposed by the external environment. In the rice blast fungus Magnaporthe oryzae, this stress response is mediated by the cell wall integrity (CWI) pathway, consisting of a well-characterized protein phosphorylation cascade. However, other regulators that maintain CWI phosphorylation homeostasis, such as protein phosphatases (PPases), remain unclear. Here, we identified two PPases, MoPtc1 and MoPtc2, that function as negative regulators of the CWI pathway. MoPtc1 and MoPtc2 interact with MoMkk1, one of the key components of the CWI pathway, and are crucial for the vegetative growth, conidial formation, and virulence of M. oryzae. We also demonstrate that both MoPtc1 and MoPtc2 dephosphorylate MoMkk1 in vivo and in vitro, and that CWI stress leads to enhanced interaction between MoPtc1 and MoMkk1. CWI stress abolishes the interaction between MoPtc2 and MoMkk1, providing a means of deactivation for CWI signalling. Our studies reveal that CWI signalling in M. oryzae is a highly coordinated regulatory mechanism vital for stress response and pathogenicity.

Dynamins are large superfamily GTPase proteins that are involved in various cellular processes including budding of transport vesicles, division of organelles, cytokinesis, and pathogen resistance. Here, we characterized several dynamin-related proteins from the rice blast fungus Magnaporthe oryzae and found that MoDnm1 is required for normal functions, including vegetative growth, conidiogenesis, and full pathogenicity. In addition, we found that MoDnm1 co-localizes with peroxisomes and mitochondria, which is consistent with the conserved role of dynamin proteins. Importantly, MoDnm1-dependent peroxisomal and mitochondrial fission involves functions of mitochondrial fission protein MoFis1 and WD-40 repeat protein MoMdv1. These two proteins display similar cellular functions and subcellular localizations as MoDnm1, and are also required for full pathogenicity. Further studies showed that MoDnm1, MoFis1 and MoMdv1 are in complex to regulate not only peroxisomal and mitochondrial fission, pexophagy and mitophagy progression, but also appressorium function and host penetration. In summary, our studies provide new insights into how MoDnm1 interacts with its partner proteins to mediate peroxisomal and mitochondrial functions and how such regulatory events may link to differentiation and pathogenicity in the rice blast fungus.

Magnaporthe oryzae causes rice blasts posing serious threats to food security worldwide. During infection, M. oryzae utilizes several transmembrane receptor proteins that sense cell surface cues to induce highly specialized infectious structures called appressoria. However, little is known about the mechanisms of intracellular receptor tracking and their function. Here, we described that disrupting the coat protein complex II (COPII) cargo protein MoErv14 severely affects appressorium formation and pathogenicity as the ΔMoerv14 mutant is defective not only in cAMP production but also in the phosphorylation of the mitogen-activated protein kinase (MAPK) MoPmk1. Studies also showed that either externally supplementing cAMP or maintaining MoPmk1 phosphorylation suppresses the observed defects in the ΔMoerv14 strain. Importantly, MoErv14 is found to regulate the transport of MoPth11, a membrane receptor functioning upstream of G-protein/cAMP signaling, and MoWish and MoSho1 function upstream of the Pmk1-MAPK pathway. In summary, our studies elucidate the mechanism by which the COPII protein MoErv14 plays an important function in regulating the transport of receptors involved in the appressorium formation and virulence of the blast fungus.

Previous studies have shown that methionine from root exudates affects the rhizosphere bacterial population involved in soil nitrogen fixation. A transgenic line of Zigongdongdou soybean cultivar (ZD91) that expresses Arabidopsis cystathionine γ-synthase resulting in an increased methionine production was examined for its influence to the rhizosphere bacterial population. Using 16S rRNA gene-based pyrosequencing analysis of the V4 region and DNA extracted from bacterial consortia collected from the rhizosphere of soybean plants grown in an agricultural field at the pod-setting stage, we characterized the populational structure of the bacterial community involved. In total, 87,267 sequences (approximately 10,908 per sample) were analyzed. We found that Acidobacteria, Proteobacteria, Bacteroidetes, Actinobacteria, Chloroflexi, Planctomycetes, Gemmatimonadetes, Firmicutes, and Verrucomicrobia constitute the dominant taxonomic groups in either the ZD91 transgenic line or parental cultivar ZD, and that there was no statistically significant difference in the rhizosphere bacterial community structure between the two cultivars.

Protein phosphatases are critical regulators in eukaryotic cells. For example, the budding yeast Saccharomyces cerevisiae dual specificity protein phosphatase (DSP) ScYvh1 regulates growth, sporulation, and glycogen accumulation. Despite such importance, functions of Yvh1 proteins in filamentous fungi are not well understood. In this study, we characterized putative protein phosphatase MoYvh1, an Yvh1 homolog in the rice blast fungus Magnaporthe oryzae. Deletion of the MoYVH1 gene resulted in significant reductions in vegetative growth, conidial production, and virulence. The ΔMoyvh1 mutant also displayed defects in cell-wall integrity and was hyposensitive to the exogenous osmotic stress. Further examination revealed that the ΔMoyvh1 mutant had defects in appressorium function and invasive hyphae growth, resulting attenuated pathogenicity. Interestingly, we found that MoYvh1 affects the scavenging of host-derived reactive oxygen species that promotes M. oryzae infection. Finally, overexpression of the phosphodiesterase MoPDEH suppressed the defects in conidia formation and pathogenicity of the ΔMoyvh1 mutant, suggesting MoYvh1 could regulate MoPDEH for its function. Our study reveals not only the importance of MoYvh1 proteins in growth, differentiation, and virulence of the rice blast fungus but, also, a genetic link between MoYvh1 and MoPDEH-cAMP signaling in this fungus.

GTP-binding protein (G-protein) and regulator of G-protein signaling (RGS) mediated signal transduction are critical in the growth and virulence of the rice blast pathogen Magnaporthe oryzae. We have previously reported that there are eight RGS and RGS-like proteins named MoRgs1 to MoRgs8 playing distinct and shared regulatory functions in M. oryzae and that MoRgs1 has a more prominent role compared to others in the fungus. To further explore the unique regulatory mechanism of MoRgs1, we screened a M. oryzae cDNA library for genes encoding MoRgs1-interacting proteins and identified MoCkb2, one of the two regulatory subunits of the casein kinase (CK) 2 MoCk2. We found that MoCkb2 and the sole catalytic subunit MoCka1 are required for the phosphorylation of MoRgs1 at the plasma membrane (PM) and late endosome (LE). We further found that an endoplasmic reticulum (ER) membrane protein complex (EMC) subunit, MoEmc2, modulates the phosphorylation of MoRgs1 by MoCk2. Interestingly, this phosphorylation is also essential for the GTPase-activating protein (GAP) function of MoRgs1. The balance among MoRgs1, MoCk2, and MoEmc2 ensures normal operation of the G-protein MoMagA-cAMP signaling required for appressorium formation and pathogenicity of the fungus. This has been the first report that an EMC subunit is directly linked to G-protein signaling through modulation of an RGS-casein kinase interaction.

Fatty acid β-oxidation is critical for fatty acid degradation and cellular development. In the rice blast fungus Magnaporthe oryzae, fatty acid β-oxidation is reported to be important mainly for turgor generation in the appressorium. However, the role of fatty acid β-oxidation during invasive hyphal growth is rarely documented. We demonstrated that blocking peroxisomal fatty acid β-oxidation impaired lipid droplet (LD) degradation and infectious growth of M. oryzae. We found that the key regulator of pathogenesis, MoMsn2, which we identified previously, is involved in fatty acid β-oxidation by targeting MoDCI1 (encoding dienoyl-coenzyme A [CoA] isomerase), which is also important for LD degradation and infectious growth. Cytological observations revealed that MoMsn2 accumulated from the cytosol to the nucleus during early infection or upon treatment with oleate. We determined that the low-density lipoprotein receptor-related protein MoLrp1, which is also involved in fatty acid β-oxidation and infectious growth, plays a critical role in the accumulation of MoMsn2 from the cytosol to the nucleus by activating the cyclic AMP signaling pathway. Our results provide new insights into the importance of fatty acid oxidation during invasive hyphal growth, which is modulated by MoMsn2 and its related signaling pathways in M. oryzae.

Genetically modified (GM) crops have brought various economic benefits but may also have adversely affected soil microorganisms. To examine whether transgenic high-methionine soybean ZD91 alters the bacterial community structure in the rhizosphere, we performed a 2-year follow-up study using the transgenic high-methionine soybean cultivar ZD91 and wild type cultivar ZD. The community composition and the relative abundance of bacteria in rhizosphere soil were determined by sequencing of the 16S rRNA amplicon. Our results indicated that transgenic soybean ZD91 had no significantly effects on rhizosphere bacterial communities. Instead, the plant growth stage and year appeared to have a stronger effect on bacterial communities. Our findings therefore provided reliable scientific evidence for potential commercial cultivation of cultivar ZD91.

The use of transgenic plants in agriculture provides many economic benefits, but it also raises concerns over the potential impact of transgenic plants on the environment. We here examined the impact of transgenic high-methionine soybean ZD91 on the arbuscular mycorrhizal (AM) fungal community structure in rhizosphere soil. Our investigations based on clone libraries were conducted in field trials at four growth stages of the crops each year from 2012 to 2013. A total of 155 operational taxonomic units (OTUs) of AM fungi were identified based on the sequences of small subunit ribosomal RNA (SSU rRNA) genes. There were no significant differences found in AM fungal diversity in rhizosphere soil during the same growth stage between transgenic soybean ZD91 and its non-transgenic parental soybean ZD. In addition, plant growth stage and year had the strongest effect on the AM fungal community structure while the genetically modified (GM) trait studied was the least explanatory factor. In conclusion, we found no indication that transgenic soybean ZD91 cultivation poses a risk for AM fungal communities in agricultural soils.