Succinyl-proteome profiling of Pyricularia oryzae, a devastating phytopathogenic fungus that causes rice blast disease.

Pyricularia oryzae is the pathogen for rice blast disease, which is a devastating threat to rice production worldwide. Lysine succinylation, a newly identified post-translational modification, is associated with various cellular processes. Here, liquid chromatography tandem-mass spectrometry combined with a high-efficiency succinyl-lysine antibody was used to identify the succinylated peptides in P. oryzae. In total, 2109 lysine succinylation sites in 714 proteins were identified. Ten conserved succinylation sequence patterns were identified, among which, K*******Ksuc, and K**Ksuc, were two most preferred ones. The frequency of lysine succinylation sites, however, greatly varied among organisms, including plants, animals, and microbes. Interestingly, the numbers of succinylation site in each protein of P. oryzae were significantly greater than that of most previous published organisms. Gene ontology and KEGG analysis showed that these succinylated peptides are associated with a wide range of cellular functions, from metabolic processes to stimuli responses. Further analyses determined that lysine succinylation occurs on several key enzymes of the tricarboxylic acid cycle and glycolysis pathway, indicating that succinylation may play important roles in the regulation of basal metabolism in P. oryzae. Furthermore, more than 40 pathogenicity-related proteins were identified as succinylated proteins, suggesting an involvement of succinylation in pathogenicity. Our results provide the first comprehensive view of the P. oryzae succinylome and may aid to find potential pathogenicity-related proteins to control the rice blast disease. Significance Plant pathogens represent a great threat to world food security, and enormous reduction in the global yield of rice was caused by P. oryzae infection. Here, the succinylated proteins in P. oryzae were identified. Furthermore, comparison of succinylation sites among various species, indicating that different degrees of succinylation may be involved in the regulation of basal metabolism. This data facilitates our understanding of the metabolic pathways and proteins that are associated with pathogenicity.

MoImd4 mediates crosstalk between MoPdeH-cAMP signalling and purine metabolism to govern growth and pathogenicity in 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 in the rice blast fungus Magnaporthe oryzae. To explore the underlying mechanism, we identified MoImd4 as an inosine-5'-monophosphate dehydrogenase (IMPDH) homologue that interacts with MoPdeH. Targeted deletion of MoIMD4 resulted in reduced de novo purine biosynthesis and growth, as well as attenuated pathogenicity, which were suppressed by exogenous xanthosine monophosphate (XMP). Treatment with mycophenolic acid (MPA), which specifically inhibits MoImd4 activity, resulted in reduced growth and virulence attenuation. Intriguingly, further analysis showed that MoImd4 promotes the phosphodiesterase activity of MoPdeH, thereby decreasing intracellular cAMP levels, and MoPdeH also promotes the IMPDH activity of MoImd4. Our studies revealed the presence of a novel crosstalk between cAMP regulation and purine biosynthesis in M. oryzae, and indicated that such a link is also important in the pathogenesis of M. oryzae.

Role of lysine residues of the Magnaporthe oryzae effector AvrPiz-t in effector- and PAMP-triggered immunity.

Magnaporthe oryzae is an important fungal pathogen of both rice and wheat. However, how M. oryzae effectors modulate plant immunity is not fully understood. Previous studies have shown that the M. oryzae effector AvrPiz-t targets the host ubiquitin-proteasome system to manipulate plant defence. In return, two rice ubiquitin E3 ligases, APIP6 and APIP10, ubiquitinate AvrPiz-t for degradation. To determine how lysine residues contribute to the stability and function of AvrPiz-t, we generated double (K1,2R-AvrPiz-t), triple (K1,2,3R-AvrPiz-t) and lysine-free (LF-AvrPiz-t) mutants by mutating lysines into arginines in AvrPiz-t. LF-AvrPiz-t showed the highest protein accumulation when transiently expressed in rice protoplasts. When co-expressed with APIP10 in Nicotiana benthamiana, LF-AvrPiz-t was more stable than AvrPiz-t and was less able to degrade APIP10. The avirulence of LF-AvrPiz-t on Piz-t:HA plants was less than that of AvrPiz-t, which led to resistance reduction and lower accumulation of the Piz-t:HA protein after inoculation with the LF-AvrPiz-t-carrying isolate. Chitin- and flg22-induced production of reactive oxygen species (ROS) was higher in LF-AvrPiz-t than in AvrPiz-t transgenic plants. In addition, LF-AvrPiz-t transgenic plants were less susceptible than AvrPiz-t transgenic plants to a virulent isolate. Furthermore, both AvrPiz-t and LF-AvrPiz-t interacted with OsRac1, but the suppression of OsRac1-mediated ROS generation by LF-AvrPiz-t was significantly lower than that by AvrPiz-t. Together, these results suggest that the lysine residues of AvrPiz-t are required for its avirulence and virulence functions in rice.

N6-methyladenosine RNA methylation is involved in virulence of the rice blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae).

N6-methyladenosine (m6A) RNA methylation is a conserved modification of RNA in eukaryotes. Pyricularia oryzae, a filamentous phytopathogenic fungus, is the cause of a destructive rice blast disease that can lead to significant declines in rice production. Here, we characterized the function of m6A RNA methylation in the development and virulence of P. oryzae by studying four genes with functional genomics. We found that PoIme4 is an N6-adenosine-methyltransferase, and deletion of PoIME4 led to decreased levels of m6A RNA methylation. PoYth1 and PoYth2 are two m6A-binding proteins, and deletion of PoYTH2 led to reduced conidiation. Co-localization experiments showed that PoAlkb1 (an mRNA:m6A demethylase) and PoYth1 were co-localized with PoDcp1 in the processing bodies involved in mRNA decay. Virulence tests showed that PoIME4, PoALKB1, PoYTH1 and PoYTH2 were involved in virulence on rice in P. oryzae. Therefore, these experimental evidences provide new and important information about the roles of m6A RNA methylation in fungal asexual reproduction and pathogenicity.

Iron- and Reactive Oxygen Species-Dependent Ferroptotic Cell Death in Rice-Magnaporthe oryzae Interactions.

Hypersensitive response (HR) cell death is the most effective plant immune response restricting fungal pathogen invasion. Here, we report that incompatible rice (Oryza sativa) Magnaporthe oryzae interactions induce iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death in rice cells. Ferric ions and ROS (i.e., H2O2) accumulated in tissues undergoing HR cell death of rice leaf sheath tissues during avirulent M. oryzae infection. By contrast, iron did not accumulate in rice cells during virulent M. oryzae infection or treatment with the fungal elicitor chitin. Avirulent M. oryzae infection in ΔOs-nadp-me2-3 mutant rice did not trigger iron and ROS accumulation and suppressed HR cell death, suggesting that NADP-malic enzyme2 is required for ferroptotic cell death in rice. The small-molecule ferroptosis inhibitors deferoxamine, ferrostatin-1, and cytochalasin E and the NADPH oxidase inhibitor diphenyleneiodonium suppressed iron-dependent ROS accumulation and lipid peroxidation to completely attenuate HR cell death in rice sheaths during avirulent M. oryzae infection. By contrast, the small-molecule inducer erastin triggered iron-dependent ROS accumulation and glutathione depletion, which ultimately led to HR cell death in rice in response to virulent M. oryzae These combined results demonstrate that iron- and ROS-dependent signaling cascades are involved in the ferroptotic cell death pathway in rice to disrupt M. oryzae infection.

Heat-Shock Proteins MoSsb1, MoSsz1, and MoZuo1 Attenuate MoMkk1-Mediated Cell-Wall Integrity Signaling and Are Important for Growth and Pathogenicity of Magnaporthe oryzae.

The mitogen-activated protein kinase (MAPK) MoMkk1 governs the cell-wall integrity (CWI) pathway in rice blast fungus Magnaporthe oryzae. To understand the underlying mechanism, we have identified MoSsb1 as one of the MoMkk1-interacting proteins. MoSsb1 is a stress-seventy subfamily B (Ssb) protein homolog, sharing high amino acid sequence homology with the 70-kDa heat shock proteins (Hsp70s). Hsp70 are a family of conserved and ubiquitously expressed chaperones that regulate protein biogenesis by promoting protein folding, preventing protein aggregation, and controlling protein degradation. We found that MoSsb1 regulates the synthesis of nascent polypeptide chains and this regulation is achieved by being in complex with other members of Hsp70s MoSsz1 and 40-kDa Hsp40 MoZuo1. MoSsb1 is important for the growth, conidiation, and full virulence of the blast fungus and this role is also shared by MoSsz1 and MoZuo1. Importantly, MoSsb1, MoSsz1, and MoZuo1 are all involved in the regulation of the CWI MAPK pathway by modulating MoMkk1 biosynthesis. Our studies reveal novel insights into how MoSsb1, MoSsz1, and MoZuo1 affect CWI signaling that is involved in regulating growth, differentiation, and virulence of M. oryzae and highlight the conserved functional mechanisms of heat-shock proteins in pathogenic fungi.

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.

MoMip11, a MoRgs7-interacting protein, functions as a scaffolding protein to regulate cAMP signaling and pathogenicity in the rice blast fungus Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae has eight regulators of G-protein signaling (RGS) and RGS-like proteins (MoRgs1 to MoRgs8) that exhibit both distinct and shared regulatory functions in the growth, differentiation and pathogenicity of the fungus. We found MoRgs7 with a unique RGS-seven transmembrane (7-TM) domain motif is localized to the highly dynamic tubule-vesicular compartments during early appressorium differentiation followed by gradually degradation. To explore whether this involves an active signal perception of MoRgs7, we identified a Gbeta-like/RACK1 protein homolog in M. oryzae MoMip11 that interacts with MoRgs7. Interestingly, MoMip11 selectively interacted with several components of the cAMP regulatory pathway, including Gα MoMagA and the high-affinity phosphodiesterase MoPdeH. We further showed that MoMip11 promotes MoMagA activation and suppresses MoPdeH activity thereby upregulating intracellular cAMP levels. Moreover, MoMip11 is required for the response to multiple stresses, a role also shared by Gbeta-like/RACK1 adaptor proteins. In summary, we revealed a unique mechanism by which MoMip11 links MoRgs7 and G-proteins to reugulate cAMP signaling, stress responses and pathogenicity of M. oryzae. Our studies revealed the multitude of regulatory networks that govern growth, development and pathogenicity in this important causal agent of rice blast.

New findings on phosphodiesterases, MoPdeH and MoPdeL, in Magnaporthe oryzae revealed by structural analysis.

The cyclic adenosine monophosphate (cAMP) signalling pathway mediates signal communication and sensing during infection-related morphogenesis in eukaryotes. Many studies have implicated cAMP as a critical mediator of appressorium development in the rice blast fungus, Magnaporthe oryzae. The cAMP phosphodiesterases, MoPdeH and MoPdeL, as key regulators of intracellular cAMP levels, play pleiotropic roles in cell wall integrity, cellular morphology, appressorium formation and infectious growth in M. oryzae. Here, we analysed the roles of domains of MoPdeH and MoPdeL separately or in chimeras. The results indicated that the HD and EAL domains of MoPdeH are indispensable for its phosphodiesterase activity and function. Replacement of the MoPdeH HD domain with the L1 and L2 domains of MoPdeL, either singly or together, resulted in decreased cAMP hydrolysis activity of MoPdeH. All of the transformants exhibited phenotypes similar to that of the ΔMopdeH mutant, but also revealed that EAL and L1 play additional roles in conidiation, and that L1 is involved in infectious growth. We further found that the intracellular cAMP level is important for surface signal recognition and hyphal autolysis. The intracellular cAMP level negatively regulates Mps1-MAPK and positively regulates Pmk1-MAPK in the rice blast fungus. Our results provide new information to better understand the cAMP signalling pathway in the development, differentiation and plant infection of the fungus.

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.

Reaction of wheat plants and alternative hosts to Magnaporthe oryzae / Reação de plantas de trigo e hospedeiros alternativos a Magnaporthe oryzae

Blast disease, caused by the fungus Magnaporthe oryzae, has a major impact on wheat farming. The study of plant responses to pathogens has improved the management of this disease. Moreover, it is important to identify potential host plants in the crops' vicinity and to understand reactions caused by plant-pathogen interactions. The objective of this study was to assess the histopathology of wheat plants, Digitaria insularis and Digitaria sanguinalis inoculated with M. oryzae isolates obtained either rice or wheat plants. Thirty-three days after sowing, greenhouse-grown plants of all three species were inoculated with each M. oryzae isolate. The observed effects (48 hours after inoculation) differed depending on the particular interaction between each pathogen isolate-plant species pair. For instance, wheat and D. sanguinalis had the weakest defensive response against spore germination, production of melanized appressoria, and appressorial penetration, with average values above 87, 90, and 43%, respectively, for these events in these plants. Furthermore, germination and appressoria melanization were more aggressive in the rice isolate than in the wheat isolate. Additionally, evidence for a defensive response (such as cell death) was observed in wheat plants inoculated with rice isolates. However, such a response was absent in plants inoculated using wheat isolates, presumably because pathogen recognition failed.(AU)

A brusone é uma doença causada pelo fungo Magnaporthe oryzae e de grande impacto para a triticultura. O estudo das respostas da planta aos patógenos tem auxiliado no manejo das doenças, e a identificação das plantas hospedeiras nas proximidades da lavoura é importante, assim como conhecer as reações de interação com o patógeno. O objetivo deste estudo foi determinar os eventos histológicos de plantas de trigo Digitaria insularis e Digitaria sanguinalis inoculadas com isolados de M. oryzae de plantas de arroz e trigo. Aos 33 dias da semeadura, as plantas cultivadas em estufa foram inoculadas com dois isolados de M. oryzae. O efeito observado nas plantas, 48 horas após a inoculação, mostrou interações diferentes para cada isolado do patógeno e entre as espécies de plantas. As plantas de trigo e D. sanguinalis foram os materiais com menores respostas de defesa à germinação, de produção de apressórios melanizados e penetração do apressório, com valores acima de 87, 90 e 43%, respectivamente. Entre os isolados do patógeno, o mais agressivo para germinação e produção de apressório melanizado foi o proveniente de plantas de arroz. A resposta de defesa da planta, como a morte celular, foi observada nas plantas de trigo inoculadas com isolado proveniente de plantas de arroz, enquanto não houve resposta de defesa da planta quando inoculada com isolado obtido de plantas de trigo, provavelmente por não ocorrer o reconhecimento do patógeno.(AU)

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.

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.

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.

Combined GC- and UHPLC-HR-MS Based Metabolomics to Analyze Durable Anti-fungal Resistance Processes in Cereals.

Introduction of durable resistance genes in crops is an important strategy to prevent yield loss caused by pathogens. The durable multi-pathogen resistance gene Lr34 originating from wheat is widely used in breeding, and is functionally transferable to barley and rice. The molecular resistance mechanism of Lr34, encoding for an adenosine triphosphate-binding cassette transporter, is not known yet. To understand the molecular function and the defense response of durable disease resistance in cereals, the metabolic response of Lr34 was investigated in, except for the Lr34 gene, genetically identical lines of barley, rice and wheat. A broad range of compounds including primary, secondary and lipophilic metabolites were analyzed by a combination of gas (GC) and liquid chromatography (LC) mass spectrometry (MS) based methods. Data from metabolomics correlated well with transcriptomics data for plant defense responses such as the formation of anti-fungal hordatines or the components of the glyoxylate cycle. Induction of the glyoxylate cycle found in transgenic Lr34 rice grown in the greenhouse was confirmed in field-grown natural Lr34 wheat. Constitutively active plant defense responses were observed in the different cereals.

Identification and characterization of suppressors of plant cell death (SPD) effectors from Magnaporthe oryzae.

Phytopathogenic microorganisms, including the fungal pathogen Magnaporthe oryzae, secrete a myriad of effector proteins to facilitate infection. Utilizing the transient expression of candidate effectors in the leaves of the model plant Nicotiana benthamiana, we identified 11 suppressors of plant cell death (SPD) effectors from M. oryzae that were able to block the host cell death reaction induced by Nep1. Ten of these 11 were also able to suppress BAX-mediated plant cell death. Five of the 11 SPD genes have been identified previously as either essential for the pathogenicity of M. oryzae, secreted into the plant during disease development, or as suppressors or homologues of other characterized suppressors. In addition, of the remaining six, we showed that SPD8 (previously identified as BAS162) was localized to the rice cytoplasm in invaded and surrounding uninvaded cells during biotrophic invasion. Sequence analysis of the 11 SPD genes across 43 re-sequenced M. oryzae genomes revealed that SPD2, SPD4 and SPD7 have nucleotide polymorphisms amongst the isolates. SPD4 exhibited the highest level of nucleotide diversity of any currently known effector from M. oryzae in addition to the presence/absence polymorphisms, suggesting that this gene is potentially undergoing selection to avoid recognition by the host. Taken together, we have identified a series of effectors, some of which were previously unknown or whose function was unknown, that probably act at different stages of the infection process and contribute to the virulence of M. oryzae.

Structure-function analyses of the Pth11 receptor reveal an important role for CFEM motif and redox regulation in rice blast.

The interaction of Magnaporthe oryzae, the rice blast fungus, and rice begins when M. oryzae establishes contact with the host plant surface. On perception of appropriate surface signals, M. oryzae forms appressoria and initiates host invasion. Pth11, an important G-protein-coupled receptor necessary for appressorium formation in M. oryzae, contains seven transmembrane regions and a CFEM (common in several fungal extracellular membrane proteins) domain with the characteristic eight cysteine residues. We focused on gaining further insight into the role of the CFEM domain in the putative surface sensing/response function of Pth11. Increased/constitutive expression of CFEM resulted in precocious, albeit defective, appressoria formation in wild-type M. oryzae. The Pth11C63A/C65A mutant, probably with disrupted disulfide bonds in the CFEM, showed delayed appressorium formation and reduced virulence. Furthermore, the accumulation of reactive oxygen species (ROS) was found to be altered in the pth11Δ strain. Strikingly, antioxidant treatment induced appressorium formation in pth11Δ. The Gα subunit MagB and the mitogen-activated protein (MAP) kinase Pmk1 were required for the formation of antioxidant-induced appressoria. We conclude that the CFEM domain of Pth11 is required for proper development of the appressoria, appressoria-like structures and pathogenicity. Highly regulated ROS homeostasis is important for Pth11-mediated appressorium formation in M. oryzae.

Activation of ethylene signaling pathways enhances disease resistance by regulating ROS and phytoalexin production in rice.

Ethylene plays diverse roles in plant growth, development and stress responses. However, the roles of ethylene signaling in immune responses remain largely unknown. In this study, we showed that the blast fungus Magnaporthe oryzae infection activated ethylene biosynthesis in rice. Resistant rice cultivars accumulated higher levels of ethylene than susceptible ones. Ethylene signaling components OsEIN2 and the downstream transcription factor OsEIL1 positively regulated disease resistance. Mutation of OsEIN2 led to enhanced disease susceptibility. Whole-genome transcription analysis revealed that responsive genes of ethylene, jasmonates (JAs) and reactive oxygen species (ROS) signaling as well as phytoalexin biosynthesis genes were remarkably induced. Transcription of OsrbohA/B, which encode NADPH oxidases, and OsOPRs, the JA biosynthesis genes, were induced by M. oryzae infection. Furthermore, we demonstrated that OsEIL1 binds to the promoters of OsrbohA/OsrbohB and OsOPR4 to activate their expression. These data suggest that OsEIN2-mediated OsrbohA/OsrbohB and OsOPR transcription may play essential roles in ROS generation, JA biosynthesis and the subsequent phytoalexin accumulation. Therefore, the involvement of ethylene signaling in disease resistance is probably by activation of ROS and phytoalexin production in rice during M. oryzae infection.

Calpains are involved in asexual and sexual development, cell wall integrity and pathogenicity of the rice blast fungus.

Calpains are ubiquitous and well-conserved proteins that belong to the calcium-dependent, non-lysosomal cysteine protease family. In this study, 8 putative calpains were identified using Pfam domain analysis and BlastP searches in M. oryzae. Three single gene deletion mutants (ΔMocapn7, ΔMocapn9 and ΔMocapn14) and two double gene deletion mutants (ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7) were obtained using the high-throughput gene knockout system. The calpain disruption mutants showed defects in colony characteristics, conidiation, sexual reproduction and cell wall integrity. The mycelia of the ΔMocapn7, ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7 mutants showed reduced pathogenicity on rice and barley.

Antifungal Activity of Eucalyptus Oil against Rice Blast Fungi and the Possible Mechanism of Gene Expression Pattern.

Eucalyptus oil possesses a wide spectrum of biological activity, including anti-microbial, fungicidal, herbicidal, acaricidal and nematicidal properties. We studied anti-fungal activities of the leaf oil extracted from Eucalyptus. grandis × E. urophylla. Eleven plant pathogenic fungi were tested based on the mycelium growth rates with negative control. The results showed that Eucalyptus oil has broad-spectrum inhibitory effects toward these fungi. Remarkable morphological and structural alterations of hypha have been observed for Magnaporthe grisea after the treatment. The mRNA genome array of M. grisea was used to detect genes that were differentially expressed in the test strains treated by the Eucalyptus oil than the normal strains. The results showed 1919 genes were significantly affected, among which 1109 were down-regulated and 810 were up-regulated (p < 0.05, absolute fold change >2). According to gene ontology annotation analysis, these differentially expressed genes may cause abnormal structures and physiological function disorders, which may reduce the fungus growth. These results show the oil has potential for use in the biological control of plant disease as a green biopesticide.