Distinct role of HAMP and HAMP-like linker domains in regulating the activity of Hik1p, a hybrid histidine kinase 3 from Magnaporthe oryzae.

Nik1 orthologs or group III hybrid histidine kinases (HHK3) represent a unique cytoplasmic osmosensor that act upstream of HOG/p38 MAPK pathway in fungi. It is an important molecular target for developing new antifungal agents against human pathogens. HHK3 orthologs contain a linear array of alternative HAMP and HAMP-like linker domains (poly-HAMP) in the N-terminal region. HAMP domains are quite common in prokaryotic histidine kinases where it mostly functions as signal transducer mediating conformational changes in the kinase domains. In contrast, poly-HAMP in HHK3 acts as a sensor and signal transducer to regulate histidine kinase activity. However, the mechanistic detail of this is poorly understood. Interestingly, recent studies indicate that the poly-HAMP-mediated regulation of the kinase activity varies among the orthologs. Hik1 is an important HHK3 ortholog from fungus Magnaporthe oryzae. In this paper, we aimed to decipher the role HAMP and HAMP-like linker domains in regulating the activity of Hik1p. We show that Hik1p acts as a bona fide osmosensor and negatively regulates the downstream HOG/p38 MAPK pathway in Saccharomyces cerevisiae. Our data suggest a differential role of the HAMP domains in the functionality of Hik1p. Most interestingly, the deletion of individual domains in poly-HAMP resulted in distinct active forms of Hik1p and thereby indicating that the poly-HAMP domain, instead of acting as on-off switch, regulates the histidine kinase activity by transition through multiple conformational states.

A Magnaporthe Chitinase Interacts with a Rice Jacalin-Related Lectin to Promote Host Colonization.

The genome of rice blast fungus (Magnaporthe oryzae) encodes 15 glycoside hydrolase 18 family chitinases. In this study, we characterized the function of an M. oryzae extracellular chitinase, MoChi1, and its interaction with a host protein, OsMBL1, a jacalin-related Mannose-Binding Lectin (MBL) in rice (Oryza sativa). Deletion of MoChi1 resulted in reduced aerial hyphal formation and reduced virulence in rice by activating the expression of defense-responsive genes. We confirmed MoChi1 interaction with rice OsMBL1 in vitro and in vivo. OsMBL1 was induced by pathogen-associated molecular patterns and M. oryzae infection. Overexpression of OsMBL1 led to activation of rice defense-responsive genes and a chitin-induced reactive oxygen species burst, thereby enhancing resistance to M. oryzae Knockdown of OsMBL1 enhances susceptibility of rice plants to M. oryzae Furthermore, MoChi1 suppressed chitin-induced reactive oxygen species in rice cells and competed with OsMBL1 for chitin binding. Taken together, our study reveals a mechanism in which MoChi1 targets a host lectin to suppress rice immunity.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1.

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (ß3-ß4 loop to α0 helix) and movement of a 'shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a 'closed' state compared with its 'open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

Two distinct nucleic acid binding surfaces of Cdc5 regulate development.

Cell division cycle 5 (Cdc5) is a highly conserved nucleic acid binding protein among eukaryotes and plays critical roles in development. Cdc5 can simultaneously bind to DNA and RNA by its N-terminal DNA-binding domain (DBD), but molecular mechanisms describing its nucleic acid recognition and the regulation of development through its nucleic acid binding remain unclear. Herein, we present a crystal structure of the N-terminal DBD of MoCdc5 (MoCdc5-DBD) from the rice blast fungus Magnaporthe oryzae. Residue K100 of MoCdc5 is on the periphery of a positively charged groove that is formed by K42, K45, R47, and N92 and is evolutionally conserved. Mutation of K100 significantly reduces the affinity of MoCdc5-DBD to a Cdc5-binding element but not to a conventional myeloblastosis (Myb) domain-binding element, suggesting that K100 is a key residue of the high binding affinity to Cdc5-binding element. Another conserved residue (R31) is located close to the U6 RNA in the structure of the spliceosome, and its mutation dramatically reduces the binding capacity of MoCdc5-DBD for U6 RNA. Importantly, mutations in these key residues, including R31, K42, and K100 in AtCDC5, an Arabidopsis thaliana ortholog of MoCdc5, greatly impair the functions of AtCDC5, resulting in pleiotropic development defects and reduced levels of primary microRNA transcripts. Taken together, our findings suggest that Cdc5-DBD binds nucleic acids with two distinct binding surfaces, one for DNA and another for RNA, which together contribute to establishing the regulation mechanism of Cdc5 on development through nucleic acid binding.

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.

Kinetic Characterization of the C-H Activation Step for the Lipoxygenase from the Pathogenic Fungus Magnaporthe oryzae: Impact of N-Linked Glycosylation.

Lipoxygenases from pathogenic fungi belong to the lipoxygenase family of enzymes, which catalyze C-H activation of polyunsaturated fatty acids to form a diverse set of cell-signaling hydroperoxides. While the lipoxygenase catalytic domains are structurally and functionally similar, these fungal enzymes are decorated with N-linked glycans. The impact of N-linked glycans on the structure and function of these enzymes remains largely unknown. One exemplary system is MoLOX, a lipoxygenase from the fungus Magnaporthe oryzae, that is emerging as an important target for the devastating rice blast disease. Herein, we demonstrate that hydrogen transfer, associated with C-H cleavage of the substrate linoleic acid by MoLOX, is rate-determining and occurs by a hydrogen tunneling mechanism. Using the differential enthalpic barrier for hydrogen and deuterium transfer, ΔEa, as a kinetic reporter of tunneling efficiency, a disproportionate increase in the activation energy for deuterium transfer is observed upon treatment of MoLOX with a peptide:N-glycosidase that cleaves N-linked carbohydrates from the protein. This increased ΔEa is consistent with an impairment of substrate positioning in the enzyme-substrate complex for both the tunneling ready state and the ground state. These results provide new insight into the functional consequences of N-linked glycosylation on lipoxygenase C-H activation and have important implications for MoLOX inhibitor design.

Disruption of putative short-chain acyl-CoA dehydrogenases compromised free radical scavenging, conidiogenesis, and pathogenesis of Magnaporthe oryzae.

Short-chain acyl-CoA dehydrogenase (Scad) mediated ß-oxidation serves as the fastest route for generating essential energies required to support the survival of organisms under stress or starvation. In this study, we identified three putative SCAD genes in the genome of the globally destructive rice blast pathogen Magnaporthe oryzae, named as MoSCAD1, MoSCAD2, and MoSCAD3. To elucidate their function, we deployed targeted gene deletion strategy to investigate individual and the combined influence of MoSCAD genes on growth, stress tolerance, conidiation and pathogenicity of the rice blast fungus. First, localization and co-localization results obtained from this study showed that MoScad1 localizes to the endoplasmic reticulum (ER), MoScad2 localizes exclusively to the mitochondria while MoScad3 partially localizes to the mitochondria and peroxisome at all developmental stages of M. oryzae. Results obtained from this investigation showed that the deletion of MoSCAD1 and MoSCAD2 caused a minimal but significant reduction in the growth of ΔMoscad1 and ΔMoscad2 strains, while, growth characteristics exhibited by the ΔMoscad3 strain was similar to the wild-type strain. Furthermore, we observed that deletion of MoSCAD2 resulted in drastic reduction in conidiation, delayed conidia germination, triggered the development of abnormal appressorium and suppressed host penetration and colonization efficiencies of the ΔMoscad1 strain. This study provides first material evidence confirming the possible existence of ER ß-oxidation pathway in M. oryzae. We also infer that mitochondria ß-oxidation rather than peroxisomal and ER ß-oxidation play an essential role in the vegetative growth, conidiation, appressorial morphogenesis and progression of pathogenesis in M. oryzae.

Secreted protein MoHrip2 is required for full virulence of Magnaporthe oryzae and modulation of rice immunity.

MoHrip2, identified from Magnaporthe oryzae as an elicitor, can activate plant defense responses either in the form of recombinant protein in vitro or ectopic expressed protein in rice. However, its intrinsic function in the infective interaction of M. oryzae-rice is largely unknown. Here, we found that mohrip2 expression was significantly induced at stages of fungal penetration and colonization. Meanwhile, the induced MoHrip2 mainly accumulated in the rice apoplast by outlining the entire invasive hyphae during infection, and its secretion was via the conventional endoplasmic reticulum (ER)-to-Golgi pathway, demonstrating the nature of MoHrip2 as an apoplastic effector. What's more, the disease facilitating function of MoHrip2 was revealed by the significantly compromised virulence of Δmohrip2 mutants on rice seedlings and even on the wounded rice leaves. Inoculations of these mutant strains on rice leaf sheaths showed a reduction in penetration and subsequent expansion of fungal growth, which is probably due to activated host immunity including the expression of certain defense-related genes and the production of certain phytoalexins. Altogether, these results demonstrated the necessity of MoHrip2 in suppression of host immunity and the full virulence of M. oryzae.

The inhibitor of apoptosis protein MoBir1 is involved in the suppression of hydrogen peroxide-induced fungal cell death, reactive oxygen species generation, and pathogenicity of rice blast fungus.

The inhibitor of apoptosis protein (IAP) family has been identified in a variety of organisms. All IAPs contain one to three baculoviral IAP repeat (BIR) domains, which are required for anti-apoptotic activity. Here, we identified a type II BIR domain-containing protein, MoBir1, in the rice blast fungus Magnaporthe oryzae. Expression of the MoBIR1 gene in Saccharomyces cerevisiae suppressed hydrogen peroxide-induced cell death and delayed yeast cell chronological aging. Delayed aging was found to require the carboxyl terminus of MoBir1. M. oryzae transformants overexpressing the MoBIR1 gene demonstrated increased growth rate and biomass, delayed mycelial aging, and enhanced resistance to hydrogen peroxide but reduced reactive oxygen species generation and virulence. Moreover, MoBIR1-overexpressing transformants exhibited anti-apoptotic activity. However, MoBIR1 silencing resulted in no obvious phenotypic changes, compared with the wild-type M. oryzae strain Guy11. Our findings broaden the knowledge on fungal type II BIR domain-containing proteins.

Overexpression of Magnaporthe Oryzae Systemic Defense Trigger 1 (MoSDT1) Confers Improved Rice Blast Resistance in Rice.

The effector proteins secreted by a pathogen not only promote virulence and infection of the pathogen, but also trigger plant defense response. Therefore, these proteins could be used as important genetic resources for transgenic improvement of plant disease resistance. Magnaporthe oryzae systemic defense trigger 1 (MoSDT1) is an effector protein. In this study, we compared the agronomic traits and blast disease resistance between wild type (WT) and MoSDT1 overexpressing lines in rice. Under control conditions, MoSDT1 transgenic lines increased the number of tillers without affecting kernel morphology. In addition, MoSDT1 transgenic lines conferred improved blast resistance, with significant effects on the activation of callose deposition, reactive oxygen species (ROS) accumulation and cell death. On the one hand, overexpression of MoSDT1 could delay biotrophy-necrotrophy switch through regulating the expression of biotrophy-associated secreted protein 4 (BAS4) and Magnaporthe oryzaecell death inducing protein 1 (MoCDIP1), and activate plant defense response by regulating the expression of Bsr-d1, MYBS1, WRKY45, peroxidase (POD), heat shock protein 90 (HSP90), allenoxide synthase 2 (AOS2), phenylalanine ammonia lyase (PAL), pathogenesis-related protein 1a (PR1a) in rice. On the other hand, overexpression of MoSDT1 could increase the accumulation of some defense-related primary metabolites such as two aromatic amino acids (L-tyrosine and L-tryptohan), 1-aminocyclopropane carboxylic acid, which could be converted to ethylene, vanillic acid and L-saccharopine. Taken together, overexpression of MoSDT1 confers improved rice blast resistance in rice, through modulation of callose deposition, ROS accumulation, the expression of defense-related genes, and the accumulation of some primary metabolites.

In Silico Identification of Potential Inhibitor Against a Fungal Histone Deacetylase, RPD3 from Magnaporthe Oryzae.

Histone acetylation and deacetylation play an essential role in the epigenetic regulation of gene expression. Histone deacetylases (HDAC) are a group of zinc-binding metalloenzymes that catalyze the removal of acetyl moieties from lysine residues from histone tails. These enzymes are well known for their wide spread biological effects in eukaryotes. In rice blast fungus, Magnaporthe oryzae, MoRPD3 (an ortholog of Saccharomyces cerevisiae Rpd3) was shown to be required for growth and development. Thus in this study, the class I HDAC, MoRpd3 is considered as a potential drug target, and its 3D structure was modelled and validated. Based on the model, a total of 1880 compounds were virtually screened (molecular docking) against MoRpd3 and the activities of the compounds were assessed by docking scores. The in silico screening suggested that [2-[[4-(2-methoxyethyl) phenoxy] methyl] phenyl] boronic acid (-8.7 kcal/mol) and [4-[[4-(2-methoxyethyl) phenoxy] methyl] phenyl] boronic acid (-8.5 kcal/mol) are effective in comparison to trichostatin A (-7.9 kcal/mol), a well-known general HDAC inhibitor. The in vitro studies for inhibition of appressorium formation by [2-[[4-(2-methoxyethyl) phenoxy] methyl] phenyl] boronic acid has resulted in the maximum inhibition at lower concentrations (1 µM), while the trichostatin A exhibited similar levels of inhibition at 1.5 µM. These findings thus suggest that 3D quantitative structure activity relationship studies on [2-[[4-(2-methoxyethyl) phenoxy] methyl] phenyl] boronic acid compound can further guide the design of more potential and specific HDAC inhibitors.

Role of the Histone Acetyltransferase Rtt109 in Development and Pathogenicity of the Rice Blast Fungus.

Acetylation of histone H3 lysine 56 (H3K56) by the fungal-specific histone acetyltransferase Rtt109 plays important roles in maintaining genome integrity and surviving DNA damage. Here, we investigated the implications of Rtt109-mediated response to DNA damage on development and pathogenesis of the rice blast fungus Magnaporthe oryzae (anamorph: Pyricularia oryzae). The ortholog of Rtt109 in M. oryzae (MoRtt109) was found via sequence homology and its functionality was confirmed by phenotypic complementation of the Saccharomyces cerevisiae Rtt109 deletion strain. Targeted deletion of MoRtt109 resulted in a significant reduction in acetylation of H3K56 and rendered the fungus defective in hyphal growth and asexual reproduction. Furthermore, the deletion mutant displayed hypersensitivity to genotoxic agents, confirming the conserved importance of Rtt109 in genome integrity maintenance and genotoxic stress tolerance. Elevated expression of DNA repair genes and the results of the comet assay were consistent with constitutive endogenous DNA damage. Although the conidia produced from the mutant were not impaired in germination and appressorium morphogenesis, the mutant was significantly less pathogenic on rice leaves. Transcriptomic analysis provided insight into the factors underlying phenotypic defects that are associated with deficiency of H3K56 acetylation. Overall, our results indicate that MoRtt109 is a conserved histone acetyltransferase that affects proliferation and asexual fecundity of M. oryzae through maintenance of genome integrity and response to DNA damage.

The ß-1,3-glucanosyltransferases (Gels) affect the structure of the rice blast fungal cell wall during appressorium-mediated plant infection.

The fungal wall is pivotal for cell shape and function, and in interfacial protection during host infection and environmental challenge. Here, we provide the first description of the carbohydrate composition and structure of the cell wall of the rice blast fungus Magnaporthe oryzae. We focus on the family of glucan elongation proteins (Gels) and characterize five putative ß-1,3-glucan glucanosyltransferases that each carry the Glycoside Hydrolase 72 signature. We generated targeted deletion mutants of all Gel isoforms, that is, the GH72+ , which carry a putative carbohydrate-binding module, and the GH72- Gels, without this motif. We reveal that M. oryzae GH72+ GELs are expressed in spores and during both infective and vegetative growth, but each individual Gel enzymes are dispensable for pathogenicity. Further, we demonstrated that a Δgel1Δgel3Δgel4 null mutant has a modified cell wall in which 1,3-glucans have a higher degree of polymerization and are less branched than the wild-type strain. The mutant showed significant differences in global patterns of gene expression, a hyper-branching phenotype and no sporulation, and thus was unable to cause rice blast lesions (except via wounded tissues). We conclude that Gel proteins play significant roles in structural modification of the fungal cell wall during appressorium-mediated plant infection.

Effects of UV-B radiation on the infectivity of Magnaporthe oryzae and rice disease-resistant physiology in Yuanyang terraces.

The traditional rice variety "Baijiaolaojing" was planted in Yuanyang terraces (1600 m altitude) under field conditions. The effects of enhanced UV-B radiation (0 kJ m-2, 2.5 kJ m-2, 5.0 kJ m-2 and 7.5 kJ m-2) on the rice-Magnaporthe oryzae system were studied with respect to the Magnaporthe oryzae infection, the disease-resistance physiology of the rice and the rice blast disease condition. The results showed that under enhanced UV-B radiation, the infectivity of Magnaporthe oryzae was decreased, which could significantly inhibit its growth and sporulation. The activities of rice leaf disease-resistance-related enzymes (phenylalanine ammonia-lyase, lipoxygenase, chitinase and ß-1,3-glucanase) were significantly increased under enhanced UV-B radiation. Following inoculation with Magnaporthe oryzae, levels of disease-resistance-related substances in the rice leaves were significantly increased. Among the results, it was found that leaves after UV-B radiation had a more significant resistance response. The level of UV-B irradiation showed a parabolic relationship with the rice blast index (r2 = 0.85, P < 0.01; in the control group, r2 = 0.88, P < 0.01). The disease index decreased with increase in irradiation. The DI was at a minimum with enhanced UV-B irradiance of 4 kJ m-2; thereafter, it increased with increasing irradiation. The enhanced UV-B radiation had a direct impact on the growth of rice and Magnaporthe oryzae, and it indirectly changed the rice-Magnaporthe oryzae system. UV-B radiation could reduce the harmful impact of rice blast.

Natural products as sources of new fungicides (IV): Synthesis and biological evaluation of isobutyrophenone analogs as potential inhibitors of class-II fructose-1,6-bisphosphate aldolase.

Several recently identified antifungal compounds share the backbone structure of acetophenones. The aim of the present study was to develop new isobutyrophenone analogs as new antifungal agents. A series of new 2,4-dihydroxy-5-methyl isobutyrophenone derivatives were prepared and characterized by 1H, 13C NMR and MS spectroscopic data. These products were evaluated for in vitro antifungal activities against seven plant fungal pathogens by the mycelial growth inhibitory rate assay. Compounds 3, 4a, 5a, 5b, 5e, 5f and 5g showed a broad-spectrum high antifungal activity. On the other hand, for the first time, these compounds were also assayed as potential inhibitors against Class II fructose-1,6-bisphosphate aldolase (Fba) from the rice blast fungus, Magnaporthe grisea. Compounds 5e and 5g were found to exhibit the inhibition constants (Ki) for 15.12 and 14.27 µM, respectively, as the strongest competitive inhibitors against Fba activity. The possible binding-modes of compounds 5e and 5g were further analyzed by molecular docking algorithms. The results strongly suggested that compound 5g could be a promising lead for the discovery of new fungicides via targeting Class II Fba.

MoSET1 (Histone H3K4 Methyltransferase in Magnaporthe oryzae) Regulates Global Gene Expression during Infection-Related Morphogenesis.

Here we report the genetic analyses of histone lysine methyltransferase (KMT) genes in the phytopathogenic fungus Magnaporthe oryzae. Eight putative M. oryzae KMT genes were targeted for gene disruption by homologous recombination. Phenotypic assays revealed that the eight KMTs were involved in various infection processes at varying degrees. Moset1 disruptants (Δmoset1) impaired in histone H3 lysine 4 methylation (H3K4me) showed the most severe defects in infection-related morphogenesis, including conidiation and appressorium formation. Consequently, Δmoset1 lost pathogenicity on wheat host plants, thus indicating that H3K4me is an important epigenetic mark for infection-related gene expression in M. oryzae. Interestingly, appressorium formation was greatly restored in the Δmoset1 mutants by exogenous addition of cAMP or of the cutin monomer, 16-hydroxypalmitic acid. The Δmoset1 mutants were still infectious on the super-susceptible barley cultivar Nigrate. These results suggested that MoSET1 plays roles in various aspects of infection, including signal perception and overcoming host-specific resistance. However, since Δmoset1 was also impaired in vegetative growth, the impact of MoSET1 on gene regulation was not infection specific. ChIP-seq analysis of H3K4 di- and tri-methylation (H3K4me2/me3) and MoSET1 protein during infection-related morphogenesis, together with RNA-seq analysis of the Δmoset1 mutant, led to the following conclusions: 1) Approximately 5% of M. oryzae genes showed significant changes in H3K4-me2 or -me3 abundance during infection-related morphogenesis. 2) In general, H3K4-me2 and -me3 abundance was positively associated with active transcription. 3) Lack of MoSET1 methyltransferase, however, resulted in up-regulation of a significant portion of the M. oryzae genes in the vegetative mycelia (1,491 genes), and during infection-related morphogenesis (1,385 genes), indicating that MoSET1 has a role in gene repression either directly or more likely indirectly. 4) Among the 4,077 differentially expressed genes (DEGs) between mycelia and germination tubes, 1,201 and 882 genes were up- and down-regulated, respectively, in a Moset1-dependent manner. 5) The Moset1-dependent DEGs were enriched in several gene categories such as signal transduction, transport, RNA processing, and translation.

Crystal Structure of Manganese Lipoxygenase of the Rice Blast Fungus Magnaporthe oryzae.

Lipoxygenases (LOX) are non-heme metal enzymes, which oxidize polyunsaturated fatty acids to hydroperoxides. All LOX belong to the same gene family, and they are widely distributed. LOX of animals, plants, and prokaryotes contain iron as the catalytic metal, whereas fungi express LOX with iron or with manganese. Little is known about metal selection by LOX and the adjustment of the redox potentials of their protein-bound catalytic metals. Thirteen three-dimensional structures of animal, plant, and prokaryotic FeLOX are available, but none of MnLOX. The MnLOX of the most important plant pathogen, the rice blast fungusMagnaporthe oryzae(Mo), was expressed inPichia pastoris.Mo-MnLOX was deglycosylated, purified to homogeneity, and subjected to crystal screening and x-ray diffraction. The structure was solved by sulfur and manganese single wavelength anomalous dispersion to a resolution of 2.0 Å. The manganese coordinating sphere is similar to iron ligands of coral 8R-LOX and soybean LOX-1 but is not overlapping. The Asn-473 is positioned on a short loop (Asn-Gln-Gly-Glu-Pro) instead of an α-helix and forms hydrogen bonds with Gln-281. Comparison with FeLOX suggests that Phe-332 and Phe-525 might contribute to the unique suprafacial hydrogen abstraction and oxygenation mechanism of Mo-MnLOX by controlling oxygen access to the pentadiene radical. Modeling suggests that Arg-525 is positioned close to Arg-182 of 8R-LOX, and both residues likely tether the carboxylate group of the substrate. An oxygen channel could not be identified. We conclude that Mo-MnLOX illustrates a partly unique variation of the structural theme of FeLOX.

The ArfGAP protein MoGlo3 regulates the development and pathogenicity of Magnaporthe oryzae.

The ADP ribosylation factor (Arf) and the coat protein complex I (COPI) are involved in vesicle transport. Together with GTPase-activating proteins (ArfGAPs) and guanine exchange factors (ArfGEFs) that regulate the activity of Arf, they govern vesicle formation, COPI trafficking and the maintenance of the Golgi complex. In an ongoing effort to study the role of membrane trafficking in pathogenesis of the rice blast fungus Magnaporthe oryzae, we identified MoGlo3 as an ArfGAP protein that is homologous to Glo3p of the budding yeast Saccharomyces cerevisiae. As suspected, MoGlo3 partially complements the function of yeast Glo3p. Consistent with findings in S. cerevisiae, MoGlo3 is localized to the Golgi, and that the localization is dependent on the conserved BoCCS domain. We found that MoGlo3 is highly expressed during conidiation and early infection stages and is required for vegetative growth, conidial production and sexual development. We further found that the ΔMoglo3 mutant is defective in endocytosis, scavenging of the reactive oxygen species, and in the response to endoplasmic reticulum (ER) stress. The combined effects result in failed appressorium function and decreased pathogenicity. Moreover, we provided evidence showing that the domains including the GAP, BoCCS and GRM are all important for normal MoGlo3 functions. Our studies further illustrate the importance of normal membrane trafficking in the physiology and pathogenicity of the rice blast fungus.

The D-lactate dehydrogenase MoDLD1 is essential for growth and infection-related development in Magnaporthe oryzae.

Rice blast disease caused by Magnaporthe oryzae is initiated by the attachment of conidia to plant surfaces. Germ tubes emerging from conidia develop melanized appressoria to physically penetrate the host surface. Previous studies revealed that appressorium development requires the breakdown of storage lipids and glycogen that occur in peroxisomes and the cytosol respectively, culminating in production of pyruvate. However, the downstream product(s) entering the mitochondria for further oxidation is unclear. In this study, we aimed to investigate the molecular basis underlying the metabolic flux towards the mitochondria associated with the infectious-related development in M. oryzae. We showed that D-lactate is a key intermediate metabolite of the mobilization of lipids and glycogen, and its oxidative conversion to pyruvate is catalysed by a mitochondrial D-lactate dehydrogenase MoDLD1. Deletion of MoDLD1 caused defects in conidiogenesis and appressorium formation, and subsequently the loss of fungal pathogenicity. Further analyses demonstrated that MoDLD1 activity is involved in the maintenance of redox homeostasis during conidial germination. Thus, MoDLD1 is a critical modulator that channels metabolite flow to the mitochondrion coupling cellular redox state, and contributes to development and virulence of M. oryzae.

The cyclin dependent kinase subunit Cks1 is required for infection-associated development of the rice blast fungus Magnaporthe oryzae.

Cell cycle regulation is pivotal for proper cell division and cellular differentiation in eukaryotic cells. The central regulators that govern eukaryotic cell cycle progression are cyclin-dependent kinases (CDKs) and their partners. Here, we report that Magnaporthe oryzae CKS1 encodes a cyclin-dependent kinase subunit, which plays a significant role in regulation of plant infection. We demonstrate that CKS1 is a functional homolog of CKS1/SUC1 and can physically interact with the CDK protein Cdc28, and Som1, a downstream regulator of the cyclic AMP-dependent Protein Kinase A pathway. CKS1 deletion mutants are severely impaired in hyphal growth, sexual reproduction, melanin pigmentation and conidiogenesis. Cks1 mutants are able to form appressoria from hyphal tips, but these are unable to re-polarize, and rice infection is impaired. CKS1 also affects chitin and glucan synthase activity during cell wall differentiation and fungal hydrophobin function. CKS1, therefore, encodes a conserved CDK-binding partner, essential for appressorium-mediated plant infection by the rice blast fungus.