Rice OsAAA-ATPase1 is Induced during Blast Infection in a Salicylic Acid-Dependent Manner, and Promotes Blast Fungus Resistance.

Fatty acids (FAs) have been implicated in signaling roles in plant defense responses. We previously reported that mutation or RNAi-knockdown (OsSSI2-kd) of the rice OsSSI2 gene, encoding a stearoyl acyl carrier protein FA desaturase (SACPD), remarkably enhanced resistance to blast fungus Magnaporthe oryzae and the leaf-blight bacterium Xanthomonas oryzae pv. oryzae (Xoo). Transcriptomic analysis identified six AAA-ATPase family genes (hereafter OsAAA-ATPase1-6) upregulated in the OsSSI2-kd plants, in addition to other well-known defense-related genes. Here, we report the functional analysis of OsAAA-ATPase1 in rice's defense response to M. oryzae. Recombinant OsAAA-ATPase1 synthesized in Escherichia coli showed ATPase activity. OsAAA-ATPase1 transcription was induced by exogenous treatment with a functional analogue of salicylic acid (SA), benzothiadiazole (BTH), but not by other plant hormones tested. The transcription of OsAAA-ATPase1 was also highly induced in response to M. oryzae infection in an SA-dependent manner, as gene induction was significantly attenuated in a transgenic rice line expressing a bacterial gene (nahG) encoding salicylate hydroxylase. Overexpression of OsAAA-ATPase1 significantly enhanced pathogenesis-related gene expression and the resistance to M. oryzae; conversely, RNAi-mediated suppression of this gene compromised this resistance. These results suggest that OsAAA-APTase1 plays an important role in SA-mediated defense responses against blast fungus M. oryzae.

MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae.

Autophagy is essential for appressorium-mediated plant infection by Magnaporthe oryzae, the causal agent of rice blast disease and a major threat to global food security. The regulatory mechanism of pathogenicity-associated autophagy, however, remains largely unknown. Here, we report the identification and functional characterization of a plausible ortholog of yeast SNT2 in M. oryzae, which we term MoSNT2. Deletion mutants of MoSNT2 are compromised in autophagy homeostasis and display severe defects in autophagy-dependent fungal cell death and pathogenicity. These mutants are also impaired in infection structure development, conidiation, oxidative stress tolerance and cell wall integrity. MoSnt2 recognizes histone H3 acetylation through its PHD1 domain and thereby recruits the histone deacetylase complex, resulting in deacetylation of H3. MoSnt2 binds to promoters of autophagy genes MoATG6, 15, 16, and 22 to regulate their expression. In addition, MoTor controls MoSNT2 expression to regulate MoTor signaling which leads to autophagy and rice infection. Our study provides evidence of a direct link between MoSnt2 and MoTor signaling and defines a novel epigenetic mechanism by which MoSNT2 regulates infection-associated autophagy and plant infection by the rice blast fungus. ABBREVIATIONS: M. oryzae: Magnaporthe oryzae; S. cerevisiae: Saccharomyces cerevisiae; F. oxysporum: Fusarium oxysporum; U. maydis: Ustilago maydis; Compl.: complemented strains of ΔMosnt2 expressing MoSNT2-GFP; ATG: autophagy-related; HDAC: histone deacetylase complex; Tor: target of rapamycin kinase; MTOR: mechanistic target of rapamycin kinase in mammals; MoSnt2: DNA binding SaNT domain protein in M. oryzae; MoTor: target of rapamycin kinase in M. oryzae; MoAtg8: autophagy-related protein 8 in M. oryzae; MoHos2: hda one similar protein in M. oryzae; MoeIf4G: eukaryotic translation initiation factor 4 G in M. oryzae; MoRs2: ribosomal protein S2 in M. oryzae; MoRs3: ribosomal protein S3 in M. oryzae; MoIcl1: isocitrate lyase in M. oryzae; MoSet1: histone H3K4 methyltransferase in M. oryzae; Asd4: ascus development 4; Abl1: AMP-activated protein kinase ß subunit-like protein; Tig1: TBL1-like gene required for invasive growth; Rpd3: reduced potassium dependency; KAT8: lysine (K) acetyltransferase 8; PHD: plant homeodomain; ELM2: Egl-27 and MTA1 homology 2; GFP: green fluorescent protein; YFP: yellow fluorescent protein; YFPCTF: C-terminal fragment of YFP; YFPNTF: N-terminal fragment of YFP; GST: glutathione S-transferase; bp: base pairs; DEGs: differentially expressed genes; CM: complete medium; MM-N: minimum medium minus nitrogen; CFW: calcofluor white; CR: congo red; DAPI: 4', 6-diamidino-2-phenylindole; BiFC: bimolecular fluorescence complementation; RT: reverse transcription; PCR: polymerase chain reaction; qPCR: quantitative polymerase chain reaction; RNAi: RNA interference; ChIP: chromatin immunoprecipitation.

Methylmalonate-semialdehyde dehydrogenase mediated metabolite homeostasis essentially regulate conidiation, polarized germination and pathogenesis in Magnaporthe oryzae.

Plants generate multitude of aldehydes under abiotic and biotic stress conditions. Ample demonstrations have shown that rice-derived aldehydes enhance the resistance of rice against the rice-blast fungus Magnaporthe oryzae. However, how the fungal pathogen nullifies the inhibitory effects of host aldehydes to establish compatible interaction remains unknown. Here we identified and evaluated the in vivo transcriptional activities of M. oryzae aldehyde dehydrogenase (ALDH) genes. Transcriptional analysis of M. oryzae ALDH genes revealed that the acetylating enzyme Methylmalonate-Semialdehyde Dehydrogenase (MoMsdh/MoMmsdh) elevated activities during host invasion and colonization of the fungus. We further examined the pathophysiological importance of MoMSDH by deploying integrated functional genetics, and biochemical approaches. MoMSDH deletion mutant ΔMomsdh exhibited germination defect, hyper-branching of germ tube and failed to form appressoria on hydrophobic and hydrophilic surface. The MoMSDH disruption caused accumulation of small branch-chain amino acids, pyridoxine and AMP/cAMP in the ΔMomsdh mutant and altered Spitzenkörper organization in the conidia. We concluded that MoMSDH contribute significantly to the pathogenesis of M. oryzae by regulating the mobilization of Spitzenkörper during germ tube morphogenesis, appressoria formation by acting as metabolic switch regulating small branch-chain amino acids, inositol, pyridoxine and AMP/cAMP homeostasis.

PKA activity is essential for relieving the suppression of hyphal growth and appressorium formation by MoSfl1 in Magnaporthe oryzae.

In the rice blast fungus Magnaporthe oryzae, the cAMP-PKA pathway regulates surface recognition, appressorium turgor generation, and invasive growth. However, deletion of CPKA failed to block appressorium formation and responses to exogenous cAMP. In this study, we generated and characterized the cpk2 and cpkA cpk2 mutants and spontaneous suppressors of cpkA cpk2 in M. oryzae. Our results demonstrate that CPKA and CPK2 have specific and overlapping functions, and PKA activity is essential for appressorium formation and plant infection. Unlike the single mutants, the cpkA cpk2 mutant was significantly reduced in growth and rarely produced conidia. It failed to form appressoria although the intracellular cAMP level and phosphorylation of Pmk1 MAP kinase were increased. The double mutant also was defective in plant penetration and Mps1 activation. Interestingly, it often produced fast-growing spontaneous suppressors that formed appressoria but were still non-pathogenic. Two suppressor strains of cpkA cpk2 had deletion and insertion mutations in the MoSFL1 transcription factor gene. Deletion of MoSFL1 or its C-terminal 93-aa (MoSFL1ΔCT) was confirmed to suppress the defects of cpkA cpk2 in hyphal growth but not appressorium formation or pathogenesis. We also isolated 30 spontaneous suppressors of the cpkA cpk2 mutant in Fusarium graminearum and identified mutations in 29 of them in FgSFL1. Affinity purification and co-IP assays showed that this C-terminal region of MoSfl1 was essential for its interaction with the conserved Cyc8-Tup1 transcriptional co-repressor, which was reduced by cAMP treatment. Furthermore, the S211D mutation at the conserved PKA-phosphorylation site in MoSFL1 partially suppressed the defects of cpkA cpk2. Overall, our results indicate that PKA activity is essential for appressorium formation and proper activation of Pmk1 or Mps1 in M. oryzae, and phosphorylation of MoSfl1 by PKA relieves its interaction with the Cyc8-Tup1 co-repressor and suppression of genes important for hyphal growth.

ER retention receptor, MoERR1 is required for fungal development and pathogenicity in the rice blast fungus, Magnaporthe oryzae.

ER retention receptor is a seven trans-membrane protein that plays pivotal roles in function and integrity of endoplasmic reticulum (ER). Insertional mutagenesis of Magnaporthe oryzae identified MoERR1 as a pathogenicity gene encoding putative ER retention receptor orthologous to ERD2 in Saccharomyces cerevisiae. Search through the genome identified that M. oryzae possesses another ortholog of ERD2, which is designated as MoERR2. When MoERR1 and MoERR2 were tagged with GFP, both were localized to ER. Targeted disruption of MoERR1 showed pleiotropic effects on phenotypes, while deletion of MoERR2 had no effect on phenotypes we examined. The disruption mutant of MoERR1 showed growth retardation and produced significantly reduced number of conidia with aberrant morphology. Appressoria from the mutant were unable to penetrate into plant tissues presumably due to defect in cell wall integrity, thereby rendering the mutant non-pathogenic. The MoERR1 mutant also appeared to display abnormal ER structure and mis-regulation of genes involved in chaperone function and unfolded protein response under ER stress condition. Taken together, these results suggest that MoERR1 is a ER retention receptor required for function and integrity of ER, and that MoERR1-mediated ER functionalities are essential for fungal development and pathogenesis.

Phosphodiesterase MoPdeH targets MoMck1 of the conserved mitogen-activated protein (MAP) kinase signalling pathway to regulate cell wall integrity in rice blast fungus Magnaporthe oryzae.

In the rice blast fungus Magnaporthe oryzae, the high-affinity cyclic adenosine monophosphate (cAMP) phosphodiesterase MoPdeH is important not only for cAMP signalling and pathogenicity, but also for cell wall integrity (CWI) maintenance through an unknown mechanism. By utilizing affinity purification, we found that MoPdeH interacts with MoMck1, one of the components of the mitogen-activated protein (MAP) kinase cascade that regulates CWI. Overexpression of MoMCK1 suppressed defects in autolysis and pathogenicity of the ΔMopdeH mutant, although partially, suggesting that MoPdeH plays a critical role in CWI maintenance mediated by the MAP kinase pathway. We found that MoMck1 and two other MAP kinase cascade components, MoMkk1 and MoMps1, modulate intracellular cAMP levels by regulating the expression of MoPDEH through a feedback loop. In addition, disruption of MoMKK1 resulted in less aerial hyphal formation, defective asexual development and attenuated pathogenicity. Moreover, MoMkk1 plays a role in the response to osmotic stress via regulation of MoOsm1 phosphorylation levels, whereas endoplasmic reticulum (ER) stress enhances MoMps1 phosphorylation and loss of the MAP kinase cascade component affects the unfolded protein response (UPR) pathway. Taken together, our findings demonstrate that MoPdeH functions upstream of the MoMck1-MoMkk1-MoMps1 MAP kinase pathway to regulate CWI, and that MoPdeH also mediates crosstalk between the cAMP signalling pathway, the osmotic sensing high osmolarity glycerol (HOG) pathway and the dithiothreitol (DTT)-induced UPR pathway in M. oryzae.

Anacardic acid induces apoptosis-like cell death in the rice blast fungus Magnaporthe oryzae.

Anacardic acid (6-pentadecylsalicylic acid), extracted from cashew nut shell liquid, is a natural phenolic lipid well known for its strong antibacterial, antioxidant, and anticancer activities. Its effect has been well studied in bacterial and mammalian systems but remains largely unexplored in fungi. The present study identifies antifungal, cytotoxic, and antioxidant activities of anacardic acid in the rice blast fungus Magnaporthe oryzae. It was found that anacardic acid causes inhibition of conidial germination and mycelial growth in this ascomycetous fungus. Phosphatidylserine externalization, chromatin condensation, DNA degradation, and loss of mitochondrial membrane potential suggest that growth inhibition of fungus is mainly caused by apoptosis-like cell death. Broad-spectrum caspase inhibitor Z-VAD-FMK treatment indicated that anacardic acid induces caspase-independent apoptosis in M. oryzae. Expression of a predicted ortholog of apoptosis-inducing factor (AIF) was upregulated during the process of apoptosis, suggesting the possibility of mitochondria dependent apoptosis via activation of apoptosis-inducing factor. Anacardic acid treatment leads to decrease in reactive oxygen species rather than increase in reactive oxygen species (ROS) accumulation normally observed during apoptosis, confirming the antioxidant properties of anacardic acid as suggested by earlier reports. Our study also shows that anacardic acid renders the fungus highly sensitive to DNA damaging agents like ethyl methanesulfonate (EMS). Treatment of rice leaves with anacardic acid prevents M. oryzae from infecting the plant without affecting the leaf, suggesting that anacardic acid can be an effective antifungal agent.

The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance.

Plant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain proteins (NB-LRRs) are immune sensors which recognize pathogen-derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR-independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR-Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo- and hetero-complexes and interact through their coiled-coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR-Pia, neither RGA4 nor RGA5 is re-localized to the nucleus. These results establish a model for the interaction of hetero-pairs of NB-LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor.

Disruption and molecular characterization of calpains-related (MoCAPN1, MoCAPN3 and MoCAPN4) genes in Magnaporthe oryzae.

Calpains are intracellular, cysteine proteases found in plants, animals and fungi functioning as signal transduction components in different cellular pathways including sporulation and alkaline adaptation in fungi. Calpains-related MoCAPN1 (MGG_14872), MoCAPN3 (MGG_15810) and MoCAPN4 (MGG_04818) genes from Magnaporthe oryzae genome which are 2604, 3513 and 771-bp in length and encoding identical proteins of 867, 1170 and 256 amino acids were functionally characterized for different phenotypes through gene disruption method. All the mutants except those for MoCAPN1 showed normal phenotypes. In pathogenicity test, the mutants did not lead to any visible changes in phenotypes causing similar blast lesions on blast susceptible rice and barley leaves as those of the Guy-11 strain suggesting no major role in pathogenicity. Germ tubes formation, appressorium formation, mycelium radial growth and mating with 2539 strain were indistinguishable among the mutants and Guy-11 strains. Cell wall integrity (congo red) test, stress response under chemical pressure (ZnSO4, CuSO4 and CdCl2), osmotic and oxidative (NaCl and H2O2) stress response, growth response on glucose and nitrogen deficient media resulted in similar results in the mutants and Guy-11 strains. However, mutants for ΔMoCAPN1 gene produced reduced (0.57±0.15B and 0.54±0.05B) conidia compared to that (1.69±0.13A) of the Guy-11 strain showing its involvement in conidiation.

Temporal analysis of the magnaporthe oryzae proteome during conidial germination and cyclic AMP (cAMP)-mediated appressorium formation.

Rice blast disease caused by Magnaporthe oryzae is one of the most serious threats to global rice production. During the earliest stages of rice infection, M. oryzae conidia germinate on the leaf surface and form a specialized infection structure termed the appressorium. The development of the appressorium represents the first critical stage of infectious development. A total of 3200 unique proteins were identified by nanoLC-MS/MS in a temporal study of conidial germination and cAMP-induced appressorium formation in M. oryzae. Using spectral counting based label free quantification, observed changes in relative protein abundance during the developmental process revealed changes in the cell wall biosynthetic machinery, transport functions, and production of extracellular proteins in developing appressoria. One hundred and sixty-six up-regulated and 208 down-regulated proteins were identified in response to cAMP treatment. Proteomic analysis of a cAMP-dependent protein kinase A mutant that is compromised in the ability to form appressoria identified proteins whose developmental regulation is dependent on cAMP signaling. Selected reaction monitoring was used for absolute quantification of four regulated proteins to validate the global proteomics data and confirmed the germination or appressorium specific regulation of these proteins. Finally, a comparison of the proteome and transcriptome was performed and revealed little correlation between transcript and protein regulation. A subset of regulated proteins were identified whose transcripts show similar regulation patterns and include many of the most strongly regulated proteins indicating a central role in appressorium formation. A temporal quantitative RT-PCR analysis confirmed a strong correlation between transcript and protein abundance for some but not all genes. Collectively, the data presented here provide the first comprehensive view of the M. oryzae proteome during early infection-related development and highlight biological processes important for pathogenicity.

Identification and characterization of in planta-expressed secreted effector proteins from Magnaporthe oryzae that induce cell death in rice.

Interactions between rice and Magnaporthe oryzae involve the recognition of cellular components and the exchange of complex molecular signals from both partners. How these interactions occur in rice cells is still elusive. We employed robust-long serial analysis of gene expression, massively parallel signature sequencing, and sequencing by synthesis to examine transcriptome profiles of infected rice leaves. A total of 6,413 in planta-expressed fungal genes, including 851 genes encoding predicted effector proteins, were identified. We used a protoplast transient expression system to assess 42 of the predicted effector proteins for the ability to induce plant cell death. Ectopic expression assays identified five novel effectors that induced host cell death only when they contained the signal peptide for secretion to the extracellular space. Four of them induced cell death in Nicotiana benthamiana. Although the five effectors are highly diverse in their sequences, the physiological basis of cell death induced by each was similar. This study demonstrates that our integrative genomic approach is effective for the identification of in planta-expressed cell death-inducing effectors from M. oryzae that may play an important role facilitating colonization and fungal growth during infection.

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.

Two phosphodiesterase genes, PDEL and PDEH, regulate development and pathogenicity by modulating intracellular cyclic AMP levels in Magnaporthe oryzae.

Cyclic AMP (cAMP) signaling plays an important role in regulating multiple cellular responses, such as growth, morphogenesis, and/or pathogenicity of eukaryotic organisms such as fungi. As a second messenger, cAMP is important in the activation of downstream effector molecules. The balance of intracellular cAMP levels depends on biosynthesis by adenylyl cyclases (ACs) and hydrolysis by cAMP phosphodiesterases (PDEases). The rice blast fungus Magnaporthe oryzae contains a high-affinity (PdeH/Pde2) and a low-affinity (PdeL/Pde1) PDEases, and a previous study showed that PdeH has a major role in asexual differentiation and pathogenicity. Here, we show that PdeL is required for asexual development and conidial morphology, and it also plays a minor role in regulating cAMP signaling. This is in contrast to PdeH whose mutation resulted in major defects in conidial morphology, cell wall integrity, and surface hydrophobicity, as well as a significant reduction in pathogenicity. Consistent with both PdeH and PdeL functioning in cAMP signaling, disruption of PDEH only partially rescued the mutant phenotype of ΔmagB and Δpka1. Further studies suggest that PdeH might function through a feedback mechanism to regulate the expression of pathogenicity factor Mpg1 during surface hydrophobicity and pathogenic development. Moreover, microarray data revealed new insights into the underlying cAMP regulatory mechanisms that may help to identify potential pathogenicity factors for the development of new disease management strategies.

Potassium and sodium uptake systems in fungi. The transporter diversity of Magnaporthe oryzae.

In this study, we report an inventory of the K(+) uptake systems in 62 fungal species for which the complete genome sequences are available. This inventory reveals that three types of K(+) uptake systems, TRK and HAK transporters and ACU ATPases, are widely present in several combinations across fungal species. PAT ATPases are less frequently present and are exceptional in Ascomycota. The genome of Magnaporthe oryzae contains four TRK, one HAK, and two ACU genes. The study of the expression of these genes at high K(+), K(+) starvation, and in infected rice leaves revealed that the expression of four genes, ACU1, ACU2, HAK1, and TRK1 is much lower than that of TRK2, TRK3, and TRK4, except under K(+) starvation. The two ACU ATPases were cloned and functionally identified as high-affinity K(+) or Na(+) uptake systems. These two ATPases endow Saccharomyces cerevisiae with the capacity to grow for several generations in low Na(+) concentrations when K(+) was absent, which produces a dramatic increase of cellular Na(+)/K(+) ratio.

Cyanide, a coproduct of plant hormone ethylene biosynthesis, contributes to the resistance of rice to blast fungus.

Rice (Oryza sativa) plants carrying the Pi-i resistance gene to blast fungus Magnaporthe oryzae restrict invaded fungus in infected tissue via hypersensitive reaction or response (HR), which is accompanied by rapid ethylene production and formation of small HR lesions. Ethylene biosynthesis has been implicated to be important for blast resistance; however, the individual roles of ethylene and cyanide, which are produced from the precursor 1-aminocyclopropane-1-carboxylic acid, remain unevaluated. In this study, we found that Pi-i-mediated resistance was compromised in transgenic rice lines, in which ethylene biosynthetic enzyme genes were silenced and then ethylene production was inhibited. The compromised resistance in transgenic lines was recovered by exogenously applying cyanide but not ethephon, an ethylene-releasing chemical in plant tissue. In a susceptible rice cultivar, treatment with cyanide or 1-aminocyclopropane-1-carboxylic acid induced the resistance to blast fungus in a dose-dependent manner, while ethephon did not have the effect. Cyanide inhibited the growth of blast fungus in vitro and in planta, and application of flavonoids, secondary metabolites that exist ubiquitously in the plant kingdom, enhanced the cyanide-induced inhibition of fungal growth. These results suggested that cyanide, whose production is triggered by HR in infected tissue, contributes to the resistance in rice plants via restriction of fungal growth.

PdeH, a high-affinity cAMP phosphodiesterase, is a key regulator of asexual and pathogenic differentiation in Magnaporthe oryzae.

Cyclic AMP-dependent pathways mediate the communication between external stimuli and the intracellular signaling machinery, thereby influencing important aspects of cellular growth, morphogenesis and differentiation. Crucial to proper function and robustness of these signaling cascades is the strict regulation and maintenance of intracellular levels of cAMP through a fine balance between biosynthesis (by adenylate cyclases) and hydrolysis (by cAMP phosphodiesterases). We functionally characterized gene-deletion mutants of a high-affinity (PdeH) and a low-affinity (PdeL) cAMP phosphodiesterase in order to gain insights into the spatial and temporal regulation of cAMP signaling in the rice-blast fungus Magnaporthe oryzae. In contrast to the expendable PdeL function, the PdeH activity was found to be a key regulator of asexual and pathogenic development in M. oryzae. Loss of PdeH led to increased accumulation of intracellular cAMP during vegetative and infectious growth. Furthermore, the pdeHDelta showed enhanced conidiation (2-3 fold), precocious appressorial development, loss of surface dependency during pathogenesis, and highly reduced in planta growth and host colonization. A pdeHDelta pdeLDelta mutant showed reduced conidiation, exhibited dramatically increased (approximately 10 fold) cAMP levels relative to the wild type, and was completely defective in virulence. Exogenous addition of 8-Br-cAMP to the wild type simulated the pdeHDelta defects in conidiation as well as in planta growth and development. While a fully functional GFP-PdeH was cytosolic but associated dynamically with the plasma membrane and vesicular compartments, the GFP-PdeL localized predominantly to the nucleus. Based on data from cAMP measurements and Real-Time RTPCR, we uncover a PdeH-dependent biphasic regulation of cAMP levels during early and late stages of appressorial development in M. oryzae. We propose that PdeH-mediated sustenance and dynamic regulation of cAMP signaling during M. oryzae development is crucial for successful establishment and spread of the blast disease in rice.

Chemical composition and in vitro antimicrobial activity of the volatile oils from Gliomastix murorum and Pichia guilliermondii, two endophytic fungi in Paris polyphylla var. yunnanensis.

Volatile oils were obtained by hydro-distillation from Gliomastix murorum and Pichia guilliermondii, two endophytic fungi isolated from the traditional Chinese medicinal herb Paris polyphylla var. yunnanensis. The oils were analyzed for their chemical composition by gas chromatography-mass spectrometry (GC-MS). Palmitic acid (15.5%), (E)-9-octadecenoic acid (11.6%), 6-pentyl-5,6-dihydropyran-2-one (9.7%), and (7Z,10Z)-7,10- hexadecadienoic acid (8.3%) were the major compounds of the 40 identified components in G. murorum volatile oil. 1,1,3a,7-Tetramethyl-1a,2,3,3a,4,5,6,7b-octahydro-1H-cyclopropa[a]- naphthalene (25.9%), palmitic acid (15.5%), 1-methyl-2,4-di- (prop-1-en-2-yl)-1- vinylcyclohexane (7.9%), (E)-9-octadecenoic acid (7.3%), and (9E,12E)-ethyl-9,12-octadecadienoate (5.2%) were the major compounds of the 27 identified components in P. guilliermondii volatile oil. The in vitro antimicrobial activity of the volatile oils was also investigated to evaluate their efficacy against six bacteria and one phytopathogenic fungus. The minimum inhibitory concentration (MIC) values of the volatile oils against the test bacteria ranged from 0.20 mg/mL to 1.50 mg/mL. One of the most sensitive bacteria was Xanthomonas vesicatoria with an MIC of 0.20 mg/mL and 0.40 mg/mL for G. murorum and P. guilliermondii, respectively. The mean inhibitory concentration (IC50) of the volatile oils against spore germination of Magnaporthe oryzae was 0.84 mg/mL for G. murorum and 1.56 mg/mL for P. guilliermondii. These results indicated that the volatile oils from the endophytic fungi have strong antimicrobial activity and could be a potential source of antimicrobial ingredients.

Homeobox transcription factors are required for conidiation and appressorium development in the rice blast fungus Magnaporthe oryzae.

The appropriate development of conidia and appressoria is critical in the disease cycle of many fungal pathogens, including Magnaporthe oryzae. A total of eight genes (MoHOX1 to MoHOX8) encoding putative homeobox transcription factors (TFs) were identified from the M. oryzae genome. Knockout mutants for each MoHOX gene were obtained via homology-dependent gene replacement. Two mutants, DeltaMohox3 and DeltaMohox5, exhibited no difference to wild-type in growth, conidiation, conidium size, conidial germination, appressorium formation, and pathogenicity. However, the DeltaMohox1 showed a dramatic reduction in hyphal growth and increase in melanin pigmentation, compared to those in wild-type. DeltaMohox4 and DeltaMohox6 showed significantly reduced conidium size and hyphal growth, respectively. DeltaMohox8 formed normal appressoria, but failed in pathogenicity, probably due to defects in the development of penetration peg and invasive growth. It is most notable that asexual reproduction was completely abolished in DeltaMohox2, in which no conidia formed. DeltaMohox2 was still pathogenic through hypha-driven appressoria in a manner similar to that of the wild-type. However, DeltaMohox7 was unable to form appressoria either on conidial germ tubes, or at hyphal tips, being non-pathogenic. These factors indicate that M. oryzae is able to cause foliar disease via hyphal appressorium-mediated penetration, and MoHOX7 is mutually required to drive appressorium formation from hyphae and germ tubes. Transcriptional analyses suggest that the functioning of M. oryzae homeobox TFs is mediated through the regulation of gene expression and is affected by cAMP and Ca(2+) signaling and/or MAPK pathways. The divergent roles of this gene set may help reveal how the genome and regulatory pathways evolved within the rice blast pathogen and close relatives.

Functional analysis of an alpha-1,2-mannosidase from Magnaporthe oryzae.

Identification of enzymes that are expressed during host colonization and characterization of their biochemical properties are prerequisite to understanding their role in the pathogen-host interaction. Nine alpha-1,2-mannosidase homologs were identified in the analysis of the Magnaporthe oryzae genome. Endoplasmic reticulum localized alpha-1,2-mannosidases play an important role in protein glycosylation. However, several members of the alpha-1,2-mannosidase gene family are predicted to be secreted. The biological role of such extracellular enzymes in host colonization has not been defined. Here, we characterized a secreted alpha-1,2-mannosidase of M. oryzae, MGG_00994.6, and found that the mature polypeptide is a glycoprotein capable of hydrolyzing alpha-1,2 linked mannobiose. The gene is expressed during growth in vitro and during colonization on rice plants, however, deletion of the gene did not affect pathogenicity. Five other members of the alpha-1,2-mannosidase of M. oryzae were expressed with a pattern similar to MGG_00994.6, suggesting the potential for functional redundancy. These results form the basis for additional studies on the role of this gene family in the rice blast fungus and its interaction with rice.

Under pressure: investigating the biology of plant infection by Magnaporthe oryzae.

The filamentous fungus Magnaporthe oryzae causes rice blast, the most serious disease of cultivated rice. Cellular differentiation of M. oryzae forms an infection structure called the appressorium, which generates enormous cellular turgor that is sufficient to rupture the plant cuticle. Here, we show how functional genomics approaches are providing new insight into the genetic control of plant infection by M. oryzae. We also look ahead to the key questions that need to be addressed to provide a better understanding of the molecular processes that lead to plant disease and the prospects for sustainable control of rice blast.