The MAT1 locus is required for microconidia-mediated sexual fertility in the rice blast fungus.

Rice blast fungus (Pyricularia oryzae) is a heterothallic ascomycete that causes the most destructive disease in cultivated rice worldwide. This fungus reproduces sexually and asexually, and its mating type is determined by the MAT1 locus, MAT1-1 or MAT1-2. Interestingly, most rice-infecting field isolates show a loss of female fertility, but the MAT1 locus is highly conserved in female-sterile isolates. In this study, we performed a functional analysis of MAT1 using the CRISPR/Cas9 system in female- and male-fertile isolates and female-sterile (male-fertile) isolates. Consistent with a previous report, MAT1 was essential for sexual reproduction but not for asexual reproduction. Meanwhile, deletion mutants of MAT1-1-1, MAT1-1-2, and MAT1-1-3 exhibited phenotypes different from those of other previously described isolates, suggesting that the function of MAT1-1 genes and/or their target genes in sexual reproduction differs among strains or isolates. The MAT1 genes, excluding MAT1-2-6, retained their functions even in female-sterile isolates, and deletion mutants lead to loss or reduction of male fertility. Although MAT1 deletion did not affect microconidia (spermatia) production, microconidia derived from the mutants could not induce perithecia formation. These results indicated that MAT1 is required for microconidia-mediated male fertility in addition to female fertility in P. oryzae .

Dysfunctional Pro1 leads to female sterility in rice blast fungi.

Although sexual reproduction is widespread in eukaryotes, some fungal species can only reproduce asexually. In the rice blast fungus Pyricularia (Magnaporthe) oryzae, several isolates from the region of origin retain mating ability, but most isolates are female sterile. Therefore, female fertility may have been lost during its spread from the origin. Here, we show that functional mutations of Pro1, a global transcriptional regulator of mating-related genes in filamentous fungi, is one cause of loss of female fertility in this fungus. We identified the mutation of Pro1 by backcrossing analysis between female-fertile and female-sterile isolates. The dysfunctional Pro1 did not affect the infection processes but conidial release was increased. Furthermore, various mutations in Pro1 were detected in geographically distant P. oryzae, including pandemic isolates of wheat blast fungus. These results provide the first evidence that loss of female fertility may be advantageous to the life cycle of some plant pathogenic fungi.

BSR1, a Rice Receptor-like Cytoplasmic Kinase, Positively Regulates Defense Responses to Herbivory.

Crops experience herbivory by arthropods and microbial infections. In the interaction between plants and chewing herbivores, lepidopteran larval oral secretions (OS) and plant-derived damage-associated molecular patterns (DAMPs) trigger plant defense responses. However, the mechanisms underlying anti-herbivore defense, especially in monocots, have not been elucidated. The receptor-like cytoplasmic kinase Broad-Spectrum Resistance 1 (BSR1) of Oryza sativa L. (rice) mediates cytoplasmic defense signaling in response to microbial pathogens and enhances disease resistance when overexpressed. Here, we investigated whether BSR1 contributes to anti-herbivore defense responses. BSR1 knockout suppressed rice responses triggered by OS from the chewing herbivore Mythimna loreyi Duponchel (Lepidoptera: Noctuidae) and peptidic DAMPs OsPeps, including the activation of genes required for biosynthesis of diterpenoid phytoalexins (DPs). BSR1-overexpressing rice plants exhibited hyperactivation of DP accumulation and ethylene signaling after treatment with simulated herbivory and acquired enhanced resistance to larval feeding. As the biological significance of herbivory-induced accumulation of rice DPs remains unexplained, their physiological activities in M. loreyi were analyzed. The addition of momilactone B, a rice DP, to the artificial diet suppressed the growth of M. loreyi larvae. Altogether, this study revealed that BSR1 and herbivory-induced rice DPs are involved in the defense against chewing insects, in addition to pathogens.

Genome editing using a versatile vector-based CRISPR/Cas9 system in Fusarium species.

Fusarium species include important filamentous fungal pathogens that can infect plants, animals, and humans. Meanwhile, some nonpathogenic Fusarium species are promising biocontrol agents against plant pathogens. Here, we developed a genome editing technology using a vector-based CRISPR/Cas9 system for Fusarium oxysporum f. sp. lycopersici (Fol). This optimized CRISPR/Cas9 system, harboring an endogenous U6 small nuclear RNA promoter for the expression of single-guide RNA and an endogenous H2B nuclear localization signal for the localization of Cas9, enabled efficient targeted gene knock-out, including in the accessory chromosomal regions in Fol. We further demonstrated single crossover-mediated targeted base editing and endogenous gene tagging. This system was also applicable for genome editing in F. oxysporum f. sp. spinaciae and F. commune without any modifications, suggesting that this CRISPR/Cas9 vector has a potential application for a broad range of researches on other Fusarium species.

Screening of tenuazonic acid production-inducing compounds and identification of NPD938 as a regulator of fungal secondary metabolism.

The control of secondary metabolism in fungi is essential for the regulation of various cellular functions. In this study, we searched the RIKEN Natural Products Depository (NPDepo) chemical library for inducers of tenuazonic acid (TeA) production in the rice blast fungus Pyricularia oryzae and identified NPD938. NPD938 transcriptionally induced TeA production. We explored the mode of action of NPD938 and observed that this compound enhanced TeA production via LAE1, a global regulator of fungal secondary metabolism. NPD938 could also induce production of terpendoles and pyridoxatins in Tolypocladium album RK99-F33. Terpendole production was induced transcriptionally. We identified the pyridoxatin biosynthetic gene cluster among transcriptionally induced secondary metabolite biosynthetic gene clusters. Therefore, NPD938 is useful for the control of fungal secondary metabolism.

Dihydropyriculol produced by Pyricularia oryzae inhibits the growth of Streptomyces griseus.

Dihydropyriculol is a major secondary metabolite of Pyricularia oryzae. However, the biological activity of dihydropyriculol has not been reported. Here, we showed that dihydropyriculol has inhibitory activity against Streptomyces griseus. Localization analysis of dihydropyriculol revealed that dihydropyriculol could reach to S. griseus under confrontation culture. These results suggest that dihydropyriculol can be used as a chemical weapon against S. griseus.

Controlling the production of phytotoxin pyriculol in Pyricularia oryzae by aldehyde reductase.

Pyricularia oryzae is one of the most devastating plant pathogens in the world. This fungus produces several secondary metabolites including the phytotoxin pyriculols, which are classified into 2 types: aldehyde form (pyriculol and pyriculariol) and alcohol form (dihydropyriculol and dihydropyriculariol). Although interconversion between the aldehyde form and alcohol form has been predicted, and the PYC10 gene for the oxidation of alcohol form to aldehyde is known, the gene responsible for the reduction of aldehyde to alcohol form is unknown. Furthermore, previous studies have predicted that alcohol analogs are biosynthesized via aldehyde analogs. Herein, we demonstrated that an aldo/keto reductase PYC7 is responsible for the reduction of aldehyde to alcohol congeners. The results indicate that aldehyde analogs are biosynthesized via alcohol analogs, contradicting the previous prediction. The results suggest that P. oryzae controls the amount of pyriculol analogs using two oxidoreductases, PYC7 and PYC10, thereby controlling the bioactivity of the phytotoxin.

Overexpressed BSR1-Mediated Enhancement of Disease Resistance Depends on the MAMP-Recognition System.

Plant plasma membrane-localized receptors recognize microbe-associated molecular patterns (MAMPs) and activate immune responses via various signaling pathways. Receptor-like cytoplasmic kinases (RLCKs) are considered key signaling factors in plant immunity. BROAD-SPECTRUM RESISTANCE 1 (BSR1), a rice RLCK, plays a significant role in disease resistance. Overexpression of BSR1 confers strong resistance against fungal and bacterial pathogens. Our recent study revealed that MAMP-triggered immune responses are mediated by BSR1 in wild-type rice and are hyperactivated in BSR1-overexpressing rice. It was suggested that hyperactivated immune responses were responsible for the enhancement of broad-spectrum disease resistance; however, this remained to be experimentally validated. In this study, we verified the above hypothesis by disrupting the MAMP-recognition system in BSR1-overexpressing rice. To this end, we knocked out OsCERK1, which encodes a well-characterized MAMP-receptor-like protein kinase. In the background of BSR1 overaccumulation, the knockout of OsCERK1 nearly abolished the enhancement of blast resistance. This finding indicates that overexpressed BSR1-mediated enhancement of disease resistance depends on the MAMP-triggered immune system, corroborating our previously suggested model.

Induction of secondary metabolite production by hygromycin B and identification of the 1233A biosynthetic gene cluster with a self-resistance gene.

We found that the protein synthesis inhibitor hygromycin B induced the production of secondary metabolites, including lucilactaene, NG-391, fusarubin, 1233A, and 1233B, in the filamentous fungus, Fusarium sp. RK97-94. We identified the biosynthetic gene cluster for 1233A, an HMG-CoA synthase inhibitor. The biosynthetic gene cluster consisted of four genes, one of which was involved in conferring self-resistance to 1233A.

Biosynthetic gene cluster identification and biological activity of lucilactaene from Fusarium sp. RK97-94.

We identified the biosynthetic gene cluster for lucilactaene, a cell cycle inhibitor from a filamentous fungus Fusarium sp. RK 97-94. The luc1 knockout strain accumulated demethylated analogs, indicating the involvement of Luc1 methyltransferase in lucilactaene biosynthesis. Lucilactaene showed potent antimalarial activity. Our data suggested that methylation and ether ring formation are essential for its potent antimalarial activity.

Extremely Low Concentrations of Acetic Acid Stimulate Cell Differentiation in Rice Blast Fungus.

Metabolic switching and rewiring play a dynamic role in programmed cell differentiation. Many pathogenic microbes need to survive in nutrient-deficient conditions and use the glyoxylate cycle, an anaplerotic pathway of the tricarboxylic acid cycle, to produce carbohydrates. The plant pathogenic fungus Magnaporthe oryzae (Pyricularia oryzae) has a unique chitin deacetylase, Cbp1. The spatiotemporal activity of this protein is required for modification of the M. oryzae wall and for cell differentiation into the specialized infection structure (appressorium). Here we show that acetic acid, another product released by the Cbp1-catalyzed conversion of chitin into chitosan, induces appressorium formation. An extremely low concentration (fM) of acetic acid restored cell differentiation in a Δcbp1 mutant possibly through the glyoxylate cycle.

Broad-Spectrum Disease Resistance Conferred by the Overexpression of Rice RLCK BSR1 Results from an Enhanced Immune Response to Multiple MAMPs.

Plants activate their immune system through intracellular signaling pathways after perceiving microbe-associated molecular patterns (MAMPs). Receptor-like cytoplasmic kinases mediate the intracellular signaling downstream of pattern-recognition receptors. BROAD-SPECTRUM RESISTANCE 1 (BSR1), a rice (Oryza sativa) receptor-like cytoplasmic kinase subfamily-VII protein, contributes to chitin-triggered immune responses. It is valuable for agriculture because its overexpression confers strong disease resistance to fungal and bacterial pathogens. However, it remains unclear how overexpressed BSR1 reinforces plant immunity. Here we analyzed immune responses using rice suspension-cultured cells and sliced leaf blades overexpressing BSR1. BSR1 overexpression enhances MAMP-triggered production of hydrogen peroxide (H2O2) and transcriptional activation of the defense-related gene in cultured cells and leaf strips. Furthermore, the co-cultivation of leaves with conidia of the blast fungus revealed that BSR1 overexpression allowed host plants to produce detectable oxidative bursts against compatible pathogens. BSR1 was also involved in the immune responses triggered by peptidoglycan and lipopolysaccharide. Thus, we concluded that the hyperactivation of MAMP-triggered immune responses confers BSR1-mediated robust resistance to broad-spectrum pathogens.

Chloramphenicol inhibits eukaryotic Ser/Thr phosphatase and infection-specific cell differentiation in the rice blast fungus.

Chloramphenicol (Cm) is a broad-spectrum classic antibiotic active against prokaryotic organisms. However, Cm has severe side effects in eukaryotes of which the cause remains unknown. The plant pathogenic fungus Magnaporthe oryzae, which causes rice blast, forms an appressorium to infect the host cell via single-cell differentiation. Chloramphenicol specifically inhibits appressorium formation, which indicates that Cm has a novel molecular target (or targets) in the rice blast fungus. Application of the T7 phage display method inferred that MoDullard, a Ser/Thr-protein phosphatase, may be a target of Cm. In animals Dullard functions in cell differentiation and protein synthesis, but in fungi its role is poorly understood. In vivo and in vitro analyses showed that MoDullard is required for appressorium formation, and that Cm can bind to and inhibit MoDullard function. Given that human phosphatase CTDSP1 complemented the MoDullard function during appressorium formation by M. oryzae, CTDSP1 may be a novel molecular target of Cm in eukaryotes.

Single crossover-mediated targeted nucleotide substitution and knock-in strategies with CRISPR/Cas9 system in the rice blast fungus.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing has become a promising approach for efficient and versatile genetic engineering in various organisms; however, simple and precise nucleotide modification methods in filamentous fungi have been restricted to double crossover type homologous recombination (HR). In this study, we developed a novel genome editing strategy via single crossover-mediated HR in the model filamentous fungus Pyricularia (Magnaporthe) oryzae. This method includes the CRISPR/Cas9 system and a donor vector harboring a single homology arm with point mutations at the CRISPR/Cas9 cleavage site. Using this strategy, we demonstrated highly efficient and freely programmable base substitutions within the desired genomic locus, and target gene disrupted mutants were also obtained via a shortened (100-1000 bp) single homology arm. We further demonstrated that this method allowed a one-step GFP gene knock-in at the C-terminus of the targeted gene. Since the genomic recombination does not require an intact protospacer-adjacent motif within the donor construct and any additional modifications of host components, this method can be used in various filamentous fungi for CRISPR/Cas9-based basic and applied biological analyses.

Identification, characterization, and structural analyses of a fungal endo-ß-1,2-glucanase reveal a new glycoside hydrolase family.

endo-ß-1,2-Glucanase (SGL) is an enzyme that hydrolyzes ß-1,2-glucans, which play important physiological roles in some bacteria as a cyclic form. To date, no eukaryotic SGL has been identified. We purified an SGL from Talaromyces funiculosus (TfSGL), a soil fungus, to homogeneity and then cloned the complementary DNA encoding the enzyme. TfSGL shows no significant sequence similarity to any known glycoside hydrolase (GH) families, but shows significant similarity to certain eukaryotic proteins with unknown functions. The recombinant TfSGL (TfSGLr) specifically hydrolyzed linear and cyclic ß-1,2-glucans to sophorose (Glc-ß-1,2-Glc) as a main product. TfSGLr hydrolyzed reducing-end-modified ß-1,2-gluco-oligosaccharides to release a sophoroside with the modified moiety. These results indicate that TfSGL is an endo-type enzyme that preferably releases sophorose from the reducing end of substrates. Stereochemical analysis demonstrated that TfSGL is an inverting enzyme. The overall structure of TfSGLr includes an (α/α)6 toroid fold. The substrate-binding mode was revealed by the structure of a Michaelis complex of an inactive TfSGLr mutant with a ß-1,2-glucoheptasaccharide. Mutational analysis and action pattern analysis of ß-1,2-gluco-oligosaccharide derivatives revealed an unprecedented catalytic mechanism for substrate hydrolysis. Glu-262 (general acid) indirectly protonates the anomeric oxygen at subsite -1 via the 3-hydroxy group of the Glc moiety at subsite +2, and Asp-446 (general base) activates the nucleophilic water via another water. TfSGLr is apparently different from a GH144 SGL in the reaction and substrate recognition mechanism based on structural comparison. Overall, we propose that TfSGL and closely-related enzymes can be classified into a new family, GH162.

Svp26 facilitates ER exit of mannosyltransferases Mnt2 and Mnt3 in Saccharomyces cerevisiae.

After being translocated into the ER lumen, membrane and secretory proteins are transported from the ER to the early Golgi by COPII vesicles. Incorporation of these cargo proteins into COPII vesicles are facilitated either by direct interaction of cargo proteins with COPII coat proteins or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in yeast Saccharomyces cerevisiae. ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that efficient incorporation of Mnt2 and Mnt3 into COPII vesicles is also dependent on the function of Svp26. Mnt2 and Mnt3 are Golgi-localized α-1,3-mannosyltransferases with type II membrane topology involved in protein O-glycosylation. Immunoisolation of the yeast Golgi subcompartments quantitatively showed that Mnt2 and Mnt3 are more abundant in the early Golgi fraction than in the late Golgi fraction. Subcellular fractionation and fluorescence microscopy showed that deletion of the SVP26 gene results in the accumulation of Mnt2 and Mnt3 in ER. Using an in vitro COPII vesicle formation assay, we further demonstrate that Svp26 facilitates incorporation of Mnt2 and Mnt3 into COPII vesicles. Finally, we showed that Mnt2 and Mnt3 were co-immunoprecipitated with Svp26 from digitonin-solubilized membranes. These results indicate that Svp26 functions as an ER exit adaptor protein of Mnt2 and Mnt3.

Identification of proteins that bind to the neuroprotective agent neoechinulin A.

Neoechinulin A is an indole alkaloid with several biological activities. We previously reported that this compound protects neuronal PC12 cells from cytotoxicity induced by the peroxynitrite generator 3-morpholinosydnonimine (SIN-1), but the target proteins and precise mechanism of action of neoechinulin A were unclear. Here, we employed a phage display screen to identify proteins that bind directly with neoechinulin A. Our findings identified two proteins, chromogranin B and glutaredoxin 3, as candidate target binding partners for the alkaloid. QCM analyses revealed that neoechinulin A displays high affinity for both chromogranin B and glutaredoxin 3. RNA interference-mediated depletion of chromogranin B decreased the sensitivity of PC12 cells against SIN-1. Our results suggested chromogranin B is a plausible target of neoechinulin A.

Chitin-deacetylase activity induces appressorium differentiation in the rice blast fungus Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae differentiates a specialized infection structure called an appressorium to invade rice cells. In this report, we show that CBP1, which encodes a chitin-deacetylase, is involved in the induction phase of appressorium differentiation. We demonstrate that the enzymatic activity of Cbp1 is critical for appressorium formation. M. oryzae has six CDA homologues in addition to Cbp1, but none of these are indispensable for appressorium formation. We observed chitosan localization at the fungal cell wall using OGA488. This observation suggests that Cbp1-catalysed conversion of chitin into chitosan occurs at the cell wall of germ tubes during appressorium differentiation by M. oryzae. Taken together, our results provide evidence that the chitin deacetylase activity of Cbp1 is necessary for appressorium formation.

The receptor-like cytoplasmic kinase BSR1 mediates chitin-induced defense signaling in rice cells.

Broad-Spectrum Resistance 1 (BSR1) encodes a rice receptor-like cytoplasmic kinase, and enhances disease resistance when overexpressed. Rice plants overexpressing BSR1 are highly resistant to diverse pathogens, including rice blast fungus. However, the mechanism responsible for this resistance has not been fully characterized. To analyze the BSR1 function, BSR1-knockout (BSR1-KO) plants were generated using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Experiments using suspension-cultured cells revealed that defense responses including H2O2 production (i.e. oxidative burst) and expression of defense-related genes induced by autoclaved conidia of the rice blast fungus significantly decreased in BSR1-KO cells. Furthermore, a treatment with chitin oligomers which function as microbe-associated molecular patterns (MAMPs) of the rice blast fungus resulted in considerably suppressed defense responses in BSR1-KO cells. These results suggest that BSR1 is important for the rice innate immunity triggered by the perception of chitin.

Aminooxy-naphthylpropionic acid and its derivatives are inhibitors of auxin biosynthesis targeting l-tryptophan aminotransferase: structure-activity relationships.

We previously reported l-α-aminooxy-phenylpropionic acid (AOPP) to be an inhibitor of auxin biosynthesis, but its precise molecular target was not identified. In this study we found that AOPP targets TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1). We then synthesized 14 novel compounds derived from AOPP to study the structure-activity relationships of TAA1 inhibitors in vitro. The aminooxy and carboxy groups of the compounds were essential for inhibition of TAA1 in vitro. Docking simulation analysis revealed that the inhibitory activity of the compounds was correlated with their binding energy with TAA1. These active compounds reduced the endogenous indole-3-acetic acid (IAA) content upon application to Arabidopsis seedlings. Among the compounds, we selected 2-(aminooxy)-3-(naphthalen-2-yl)propanoic acid (KOK1169/AONP) and analyzed its activities in vitro and in vivo. Arabidopsis seedlings treated with KOK1169 showed typical auxin-deficient phenotypes, which were reversed by exogenous IAA. In vitro and in vivo experiments indicated that KOK1169 is more specific for TAA1 than other enzymes, such as phenylalanine ammonia-lyase. We further tested 41 novel compounds with aminooxy and carboxy groups to which we added protection groups to increase their calculated hydrophobicity. Most of these compounds decreased the endogenous auxin level to a greater degree than the original compounds, and resulted in a maximum reduction of about 90% in the endogenous IAA level in Arabidopsis seedlings. We conclude that the newly developed compounds constitute a class of inhibitors of TAA1. We designated them 'pyruvamine'.