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Saccharomycopsis yeasts are natural organic sulfur auxotrophs due to lack of genes required for the uptake and assimilation of sulfate/sulfite. Starvation for methionine induces a shift to a predatory, mycoparasitic life strategy that is unique amongst ascomycetous yeasts. Similar to fungal plant pathogens that separated from Saccharomycopsis more than 400 million years ago, a specialized infection structure called penetration peg is used for prey cell invasion. Penetration pegs are highly enriched with chitin. Here we demonstrate that an ancient and conserved MAP kinase signaling pathway regulates penetration peg formation and successful predation in the predator yeast S. schoenii. Deletion of the MAP kinase gene SsKIL1, a homolog of the Saccharomyces cerevisiae ScKSS1/ScFUS3 and the rice blast Magnaporthe oryzae MoPMK1 genes, as well as deletion of the transcription factor SsSTE12 generate non-pathogenic mutants that fail to form penetration pegs. Comparative global transcriptome analyses using RNAseq indicate loss of the SsKil1-SsSte12-dependent predation response in the mutant strains, while a methionine starvation response is still executed. Within the promoter sequences of genes upregulated during predation we identified a cis-regulatory element similar to the ScSte12 pheromone response element. Our results indicate that, re-routing MAP-kinase signaling by re-wiring Ste12 transcriptional control towards predation specific genes contributed to the parallel evolution of this predacious behaviour in predator yeasts. Consequently, we found that SsSTE12 is dispensable for mating. Author summary: Saccharomycopsis yeasts represent a unique yeast genus of predator yeasts that attack and kill fungal prey cells. This necrotrophic mycoparasitism is initiated upon starvation, preferably for organic sulphur/methionine as predator yeasts are methionine auxotrophs. We have characterized morphological and cellular features of predation using time lapse fluorescence microscopy. This indicated prey sensing, chitin enrichment of penetration pegs used in the attack of prey cells and coordination of predation with the cell cycle resulting in cessation of daughter cell growth and a block in mitosis during predation. A predator yeast attack from the onset of penetration peg formation to the disappearance of the nuclear GFP-signal in the Saccharomyces cerevisiae prey took ~25 minutes. We identified the MAP kinase KIL1 and its putative target STE12 as key virulence genes that govern penetration peg formation. Comparative transcriptomics of mutant strains with the wild type identified specific hunger and predation responses and associated genes. Predation genes were found to harbor a conserved DNA element in their promoters. These findings can aid in deepening our understanding of necrotrophic predator-prey interactions. [ABSTRACT FROM AUTHOR]

BRRI31R is one of the Bangladesh's most promising restorer lines due to its abundant pollen producing capacity, strong restoring ability, good combining ability, high outcrossing rate and genetically diverse from cytoplasmic male sterile (CMS) line. But the drawback of this line is that it is highly susceptible to bacterial blight (BB) disease of rice caused by Xanthomonas oryzae pv. oryzae. The present study highlighted the pyramiding of effective BB resistance genes (xa5, xa13 and Xa21) into the background of BRRI31R, through marker–assisted backcrossing (MABC). Backcross progenies were confirmed and advanced based on the foreground selection of target genes. Pyramided lines were used for pathogenicity test against five Bangladeshi Xanthomonas oryzae (BXo) races (BXo93, BXo220, BXo822, BXo826, BXo887) and confirmed the dominant fertility restore genes, Rf3 and Rf4 and further validated against SNP markers for more confirmation of target resistance genes. All pyramided restorer lines consisted of Xa4 (in built), xa5, xa13, Xa21, and Chalk5 with two fertility restorer genes, Rf3, Rf4. and these restorer lines showed intermediate amylose content (<25%). Restorer lines BRRI31R-MASP3 and BRRI31R-MASP4 showed high levels of resistance against five virulent BXo races and SNP genotyping revealed that these lines also contained a blast resistance gene Pita races. Gene pyramided restorer lines, BRRI31R-MASP3 and BRRI31R-MASP4 can directly be used as a male parent for the development of new BB resistant hybrid rice variety or could be used as a replacement of restorer line of BRRI hybrid dhan5 and 7 to enhance the quality of hybrid seeds as well as rice production in Bangladesh. [ABSTRACT FROM AUTHOR]

The polygonal shape of cells in proliferating epithelia is a result of the tensile forces of the cytoskeletal cortex and packing geometry set by the cell cycle. In the larval Drosophila epidermis, two cell populations, histoblasts and larval epithelial cells, compete for space as they grow on a limited body surface. They do so in the absence of cell divisions. We report a striking morphological transition of histoblasts during larval development, where they change from a tensed network configuration with straight cell outlines at the level of adherens junctions to a highly folded morphology. The apical surface of histoblasts shrinks while their growing adherens junctions fold, forming deep lobules. Volume increase of growing histoblasts is accommodated basally, compensating for the shrinking apical area. The folded geometry of apical junctions resembles elastic buckling, and we show that the imbalance between the shrinkage of the apical domain of histoblasts and the continuous growth of junctions triggers buckling. Our model is supported by laser dissections and optical tweezer experiments together with computer simulations. Our analysis pinpoints the ability of histoblasts to store mechanical energy to a much greater extent than most other epithelial cell types investigated so far, while retaining the ability to dissipate stress on the hours time scale. Finally, we propose a possible mechanism for size regulation of histoblast apical size through the lateral pressure of the epidermis, driven by the growth of cells on a limited surface. Buckling effectively compacts histoblasts at their apical plane and may serve to avoid physical harm to these adult epidermis precursors during larval life. Our work indicates that in growing nondividing cells, compressive forces, instead of tension, may drive cell morphology. During the development of the larval Drosophila epidermis, histoblasts and epithelial cells compete for space as they grow and undergo morphological changes. This study shows that compressive forces play a key role in controlling the shape of histoblasts, leading to the buckling of their apical junctions and changing the morphology of the epidermis from straight to highly folded outlines. [ABSTRACT FROM AUTHOR]

Rice blast disease, caused by the fungus Magnaporthe oryzae, poses a severe threat to rice production, particularly in Asia where rice is a staple food. Concerns over fungicide resistance and environmental impact have sparked interest in exploring natural fungicides as potential alternatives. This study aimed to identify highly potent natural fungicides against M. oryzae to combat rice blast disease, using advanced molecular dynamics techniques. Four key proteins (CATALASE PEROXIDASES 2, HYBRID PKS-NRPS SYNTHETASE TAS1, MANGANESE LIPOXYGENASE, and PRE-MRNA-SPLICING FACTOR CEF1) involved in M. oryzae's infection process were identified. A list of 30 plant metabolites with documented antifungal properties was compiled for evaluation as potential fungicides. Molecular docking studies revealed that 2-Coumaroylquinic acid, Myricetin, Rosmarinic Acid, and Quercetin exhibited superior binding affinities compared to reference fungicides (Azoxystrobin and Tricyclazole). High throughput molecular dynamics simulations were performed, analyzing parameters like RMSD, RMSF, Rg, SASA, hydrogen bonds, contact analysis, Gibbs free energy, and cluster analysis. The results revealed stable interactions between the selected metabolites and the target proteins, involving important hydrogen bonds and contacts. The SwissADME server analysis indicated that the metabolites possess fungicide properties, making them effective and safe fungicides with low toxicity to the environment and living beings. Additionally, bioactivity assays confirmed their biological activity as nuclear receptor ligands and enzyme inhibitors. Overall, this study offers valuable insights into potential natural fungicides for combating rice blast disease, with 2-Coumaroylquinic acid, Myricetin, Rosmarinic Acid, and Quercetin standing out as promising and environmentally friendly alternatives to conventional fungicides. These findings have significant implications for developing crop protection strategies and enhancing global food security, particularly in rice-dependent regions. [ABSTRACT FROM AUTHOR]

Rice blast, caused by rice blast fungus (Magnaporthe oryzae), is a global threat to food security, with up to 50% yield losses. Panicle blast is a severe form of rice blast, and disease responses vary between cultivars with different genotypes. Reactive oxygen species (ROS)-mediated signaling reactions and the phenylpropanoid pathway are important defense mechanisms involved in recognizing and resisting against fungal infection. To understand rice-M. oryzae interactions in resistant and susceptible cultivars, we determined dynamic changes in the activities of five defense-related enzymes in resistant cultivar jingsui 18 and susceptible cultivar jinyuan 899 infected with M. oryzae from 4 to 25 days after infection. We then performed untargeted metabolomics analyses to profile the metabolomes of the cultivars under infected and non-infected conditions. Dynamic changes in the activities of five defense-related enzymes were closely related to panicle blast resistance in rice. Metabolome data analysis identified 634 differentially accumulated metabolites (DAMs) between resistant and susceptible cultivars following infection, potentially explaining differences in disease response between varieties. The most enriched DAMs were associated with lipids and lipid-like molecules, phenylpropanoids and polyketides, organoheterocyclic compounds, organic acids and derivatives, and lignans, neolignans, and related compounds. Multiple metabolic pathways are involved in resistance to panicle blast in rice, including biosynthesis of other secondary metabolites, amino acid metabolism, lipid metabolism, phenylpropanoid biosynthesis, arachidonic acid metabolism, arginine biosynthesis, tyrosine metabolism, tryptophan metabolism, tyrosine and tryptophan biosynthesis, lysine biosynthesis, and oxidative phosphorylation. [ABSTRACT FROM AUTHOR]

Autophagy and Cell wall integrity (CWI) signaling are critical stress-responsive processes during fungal infection of host plants. In the rice blast fungus Magnaporthe oryzae, autophagy-related (ATG) proteins phosphorylate CWI kinases to regulate virulence; however, how autophagy interplays with CWI signaling to coordinate such regulation remains unknown. Here, we have identified the phosphorylation of ATG protein MoAtg4 as an important process in the coordination between autophagy and CWI in M. oryzae. The ATG kinase MoAtg1 phosphorylates MoAtg4 to inhibit the deconjugation and recycling of the key ATG protein MoAtg8. At the same time, MoMkk1, a core kinase of CWI, also phosphorylates MoAtg4 to attenuate the C-terminal cleavage of MoAtg8. Significantly, these two phosphorylation events maintain proper autophagy levels to coordinate the development and pathogenicity of the rice blast fungus. Author summary: Autophagy is a conserved cellular degradation and recycling process important in maintaining proper cellular homeostasis whereas the cell wall integrity (CWI) signaling pathway is also a conserved regulatory pathway in stress response. CWI signaling not only operates alone but also functions through crosstalk with autophagy during fungal infection of host plants. Despite the importance, little is known about how autophagy and CWI signaling coordinate their regulatory roles. Here, we have found that autophagy-related (ATG) protein MoAtg4 phosphorylation mediates the coordination between autophagy and CWI signaling, which underlies the development and pathogenicity of Magnaporthe oryzae. Both ATG protein MoAtg1 and CWI kinase MoMkk1 phosphorylate MoAtg4, which inhibits the deconjugation and C-terminal cleavage of MoAtg8, respectively, to maintain proper autophagy levels. Our study revealed a novel coordination between autophagy and CWI signaling and how the two pathways coordinate to regulate the development and pathogenicity of the blast fungus. [ABSTRACT FROM AUTHOR]

The rice blast fungus Magnaporthe oryzae differentiates specialized cells called appressoria that are required for fungal penetration into host leaves. In this study, we identified the novel basic leucine zipper (bZIP) transcription factor BIP1 (B-ZIP Involved in Pathogenesis-1) that is essential for pathogenicity. BIP1 is required for the infection of plant leaves, even if they are wounded, but not for appressorium-mediated penetration of artificial cellophane membranes. This phenotype suggests that BIP1 is not implicated in the differentiation of the penetration peg but is necessary for the initial establishment of the fungus within plant cells. BIP1 expression was restricted to the appressorium by both transcriptional and post-transcriptional control. Genome-wide transcriptome analysis showed that 40 genes were down regulated in a BIP1 deletion mutant. Most of these genes were specifically expressed in the appressorium. They encode proteins with pathogenesis-related functions such as enzymes involved in secondary metabolism including those encoded by the ACE1 gene cluster, small secreted proteins such as SLP2, BAS3, BAS4, and AVR-Pi9 effectors, as well as plant cuticle and cell wall degrading enzymes. Interestingly, this BIP1 network is different from other known infection-related regulatory networks, highlighting the complexity of gene expression control during plant-fungal interactions. Promoters of BIP1-regulated genes shared a GCN4/bZIP-binding DNA motif (TGACTC) binding in vitro to BIP1. Mutation of this motif in the promoter of MGG_08381.7 from the ACE1 gene cluster abolished its appressorium-specific expression, showing that BIP1 behaves as a transcriptional activator. In summary, our findings demonstrate that BIP1 is critical for the expression of early invasion-related genes in appressoria. These genes are likely needed for biotrophic invasion of the first infected host cell, but not for the penetration process itself. Through these mechanisms, the blast fungus strategically anticipates the host plant environment and responses during appressorium-mediated penetration. Author summary: The identification of gene regulatory networks controlling pathogenicity is a major research goal for understanding plant infection and for developing new strategies for disease control. Rice is the staple food for half the world's population, but its cultivation is threatened by the rice blast fungus Magnaporthe oryzae that causes severe yield losses. This fungus can breach intact plant leaves using specialized cells called appressoria. Here, we have identified in a pathogenicity mutant screen using random insertional mutagenesis, the novel M. oryzae bZIP transcription factor BIP1 that is essential for the infection. BIP1 is not implicated in the development of appressoria or the subsequent penetration of host leaves, but is necessary for the initial establishment of the fungus within plant cells. BIP1 orchestrates the expression of a unique set of early invasion-related genes within appressoria, encoding secreted effectors, enzymes, secondary metabolism-related enzymes, and signaling membrane receptors. Our experimental data suggest that BIP1 controls their expression by interacting directly with a TGACTC motif present in their promoters. Remarkably distinct from other known pathogenicity networks, the BIP1 regulatory network underscores the intricate control of fungal gene expression during infection. BIP1 seems to prepare M. oryzae for early biotrophic growth during appressorium-mediated penetration. [ABSTRACT FROM AUTHOR]

Fungal pathogens are one of the major reasons for biotic stress on rice (Oryza sativa L.), causing severe productivity losses every year. Breeding for host resistance is a mainstay of rice disease management, but conventional development of commercial resistant varieties is often slow. In contrast, the development of disease resistance by targeted genome manipulation has the potential to deliver resistant varieties more rapidly. The present study reports the first cloning of a synthetic maize chitinase 1 gene and its insertion in rice cv. (Basmati 385) via Agrobacterium-mediated transformation to confer resistance to the rice blast pathogen, Pyricularia oryzae. Several factors for transformation were optimized; we found that 4-week-old calli and an infection time of 15 minutes with Agrobacterium before colonization on co-cultivation media were the best-suited conditions. Moreover, 300 μM of acetosyringone in co-cultivation media for two days was exceptional in achieving the highest callus transformation frequency. Transgenic lines were analyzed using molecular and functional techniques. Successful integration of the gene into rice lines was confirmed by polymerase chain reaction with primer sets specific to chitinase and hpt genes. Furthermore, real-time PCR analysis of transformants indicated a strong association between transgene expression and elevated levels of resistance to rice blast. Functional validation of the integrated gene was performed by a detached leaf bioassay, which validated the efficacy of chitinase-mediated resistance in all transgenic Basmati 385 plants with variable levels of enhanced resistance against the P. oryzae. We concluded that overexpression of the maize chitinase 1 gene in Basmati 385 improved resistance against the pathogen. These findings will add new options to resistant germplasm resources for disease resistance breeding. The maize chitinase 1 gene demonstrated potential for genetic improvement of rice varieties against biotic stresses in future transformation programs. [ABSTRACT FROM AUTHOR]

The emergence of fungicide resistance severely threatens crop production by limiting the availability and application of established fungicides. Therefore, it is urgent to identify new fungicidal targets for controlling plant diseases. Here, we characterized the function of a conserved homoserine O-acetyltransferase (HOA) from the rice blast fungus Magnaporthe oryzae that could serve as the candidate antifungal target. Deletion of the MoMET2 and MoCYS2 genes encoding HOAs perturbed the biosynthesis of methionine and S-adenyl methionine, a methyl group donor for epigenetic modifications, and severely attenuated the development and virulence of M. oryzae. The ∆Momet2 mutant is significantly increased in 5-methylcytosine (5mC) modification that represses the expression of genes required for pathogenicity, including MoGLIK and MoCDH-CYT. We further showed that host-induced gene silencing (HIGS) targeting MoMET2 and MoCYS2 effectively controls rice blasts. Our studies revealed the importance of HOA in the development and virulence of M. oryzae, which suggests the potential feasibility of HOA as new targets for novel anti-rice blast measurements. Author summary: Despite the effectiveness of available antifungal agents in combating rice blasts, the emergence of resistance to these agents requires the reexamination of fungal pathogens, plant-fungi interactions, and virulence factors. Characterization of virulence factors is expected to improve the understanding of fungus pathogenesis and to help exploration for an antifungal drug target. We here reported that HOAs of the fungal original could serve as a potential target for antifungal agents. Specifically, we found that homoserine O-acetyltransferases MoMet2 and MoCys2 are important in the biosynthesis of methionine and S-adenyl methionine that are critical for the growth, development, and virulence of M. oryzae. We further demonstrated that MoMet2-mediated 5mC modification regulates gene expression and is required for infection by the rice blast fungus. Additionally, using host-induced gene silencing (HIGS) targeting MoMET2 and MoCYS2 genes provided protection against rice blasts. In all, the studies shed significant new insights in understanding the role of methionine synthesis in fungal pathogenicity and in identifying potential antifungal targets. [ABSTRACT FROM AUTHOR]

Plant pathogens secrete proteins called effectors that target host cellular processes to promote disease. Recently, structural genomics has identified several families of fungal effectors that share a similar three-dimensional structure despite remarkably variable amino-acid sequences and surface properties. To explore the selective forces that underlie the sequence variability of structurally-analogous effectors, we focused on MAX effectors, a structural family of effectors that are major determinants of virulence in the rice blast fungus Pyricularia oryzae. Using structure-informed gene annotation, we identified 58 to 78 MAX effector genes per genome in a set of 120 isolates representing seven host-associated lineages. The expression of MAX effector genes was primarily restricted to the early biotrophic phase of infection and strongly influenced by the host plant. Pangenome analyses of MAX effectors demonstrated extensive presence/absence polymorphism and identified gene loss events possibly involved in host range adaptation. However, gene knock-in experiments did not reveal a strong effect on virulence phenotypes suggesting that other evolutionary mechanisms are the main drivers of MAX effector losses. MAX effectors displayed high levels of standing variation and high rates of non-synonymous substitutions, pointing to widespread positive selection shaping the molecular diversity of MAX effectors. The combination of these analyses with structural data revealed that positive selection acts mostly on residues located in particular structural elements and at specific positions. By providing a comprehensive catalog of amino acid polymorphism, and by identifying the structural determinants of the sequence diversity, our work will inform future studies aimed at elucidating the function and mode of action of MAX effectors. Author summary: Fungal plant pathogens use small secreted proteins, called effectors, to manipulate to their own advantage their host's physiology and immunity. The evolution of these effectors, whether spontaneously or in response to human actions, can lead to epidemics or the emergence of new diseases. It is therefore crucial to understand the mechanisms underlying this evolution. In this article, we report on the evolution of effectors in one of the prime experimental model systems of plant pathology, Pyricularia oryzae, the fungus causing blast diseases in rice, wheat, and other cereals or grasses. We further characterize in this fungus a particular class of effectors, called MAX effectors, using structural models based on experimental protein structures of effectors. We show that this class of effector is produced by the pathogen during the early stages of infection, when plant cells are still alive. By comparing the gene content of isolates infecting different plant species, we show that the MAX effector arsenal is highly variable from one isolate to another. Finally, using the inferential framework of population genetics, we demonstrate that MAX effectors exhibit very high genetic variability and that this results from the action of natural selection. [ABSTRACT FROM AUTHOR]

Isoprothiolane (IPT) resistance has emerged in Magnaporthe oryzae, due to the long-term usage of IPT to control rice blast in China, yet the mechanisms of the resistance remain largely unknown. Through IPT adaptation on PDA medium, we obtained a variety of IPT-resistant mutants. Based on their EC50 values to IPT, the resistant mutants were mainly divided into three distinct categories, i.e., low resistance (LR, 6.5 ≤ EC50 < 13.0 μg/mL), moderate resistance 1 (MR-1, 13.0 ≤ EC50 < 25.0 μg/mL), and moderate resistance 2 (MR-2, 25.0 ≤ EC50 < 35.0 μg/mL). Molecular analysis of MoIRR (Magnaporthe oryzae isoprothiolane resistance related) gene demonstrated that it was associated only with the moderate resistance in MR-2 mutants, indicating that other mechanisms were associated with resistance in LR and MR-1 mutants. In this study, we mainly focused on the characterization of low resistance to IPT in M. oryzae. Mycelial growth and conidial germination were significantly reduced, indicating fitness penalties in LR mutants. Based on the differences of whole genome sequences between parental isolate and LR mutants, we identified a conserved MoVelB gene, encoding the velvet family transcription factor, and genetic transformation of wild type isolate verified that MoVelB gene was associated with the low resistance. Based on molecular analysis, we further demonstrated that the velvet family proteins VelB and VeA were indispensable for IPT toxicity and the deformation of the VelB-VeA-LaeA complex played a vital role for the low IPT-resistance in M. oryzae, most likely through the down-regulation of the secondary metabolism-related genes or CYP450 genes to reduce the toxicity of IPT. Author summary: Isoprothiolane (IPT) resistance has emerged in Magnaporthe oryzae, due to the long-term usage of IPT to control rice blast in China, yet the mechanisms of the resistance remain largely unknown. Here, we explored the mechanisms of low IPT resistance in M. oryzae. Combining the whole genome sequencing and genetic transformation, we identified a conserved MoVelB gene, encoding the velvet family transcription factor to be a determinant for IPT toxicity. We further demonstrated that the deformation of the VelB-VeA-LaeA complex conferred the low IPT-resistance in M. oryzae, most likely through down-regulating the secondary metabolism-related genes or CYP450 genes to reduce the toxicity of IPT. This study improved our understanding of the resistance mechanism as well as the mode of action of IPT which will be helpful for making suitable strategies to manage the emerging resistance of IPT in M. oryzae. [ABSTRACT FROM AUTHOR]

The rice blast fungus Magnaporthe oryzae forms specialized infectious structures called appressoria that breach host cells to initiate infection. Previous studies demonstrated that the regulator of G-protein signaling (RGS)-like protein MoRgs7 undergoes endocytosis upon fungal sensing of hydrophobic environmental cues to activate cAMP signaling required for appressorium formation. However, the mechanism by which MoRgs7 internalizes and its fate remains undetermined. We here show that MoSep1, a conserved protein kinase of Mitotic Exit Network (MEN), phosphorylates MoRgs7 to regulate its function. MoRgs7 phosphorylation determines its interaction with MoCrn1, a coronin-like actin-binding protein homolog that also modulates the internalization of MoRgs7. Importantly, the endocytic transport of MoRgs7 is critical for its GTPase-activating protein (GAP) function important in cAMP signaling. Together, our findings revealed a novel mechanism by which M. oryzae activates MoRgs7-mediated hydrophobic cue-sensing signal transduction involving protein phosphorylation and endocytic transport to govern appressorium formation and fungal pathogenicity. Author summary: During its pathogenic interaction with rice, M. oryzae forms a highly specialized infectious structure called appressorium to initiate infection. Previous studies showed that the seven transmembrane (7-TM) domain-containing regulators of G-protein signaling (RGS)-like protein MoRgs7 is vital for the perception of environmental cues and cAMP-dependent appressorium formation. However, little is known about how MoRgs7 transduces hydrophobic cues and their regulation. In this study, we have identified that MoSep1 phosphorylates MoRgs7 during appressorium formation, and phosphorylation of MoRgs7 leads to a high binding affinity with MoCrn1 and its internalization. Additionally, the internalization of MoRgs7 contributes to the activation of cAMP signaling, further governing appressorium formation and pathogenicity. [ABSTRACT FROM AUTHOR]

Magnaporthe oryzae, the rice blast fungus, is one of the most dangerous rice pathogens, causing considerable crop losses around the world. In order to explore the rice blast-resistant sources, initially performed a large-scale screening of 277 rice accessions. In parallel with field evaluations, fifty-two rice accessions were genotyped for 25 major blast resistance genes utilizing functional/gene-based markers based on their reactivity against rice blast disease. According to the phenotypic examination, 29 (58%) and 22 (42%) entries were found to be highly resistant, 18 (36%) and 29 (57%) showed moderate resistance, and 05 (6%) and 01 (1%), respectively, were highly susceptible to leaf and neck blast. The genetic frequency of 25 major blast resistance genes ranged from 32 to 60%, with two genotypes having a maximum of 16 R-genes each. The 52 rice accessions were divided into two groups based on cluster and population structure analysis. The highly resistant and moderately resistant accessions are divided into different groups using the principal coordinate analysis. According to the analysis of molecular variance, the maximum diversity was found within the population, while the minimum diversity was found between the populations. Two markers (RM5647 and K39512), which correspond to the blast-resistant genes Pi36 and Pik, respectively, showed a significant association to the neck blast disease, whereas three markers (Pi2-i, Pita3, and k2167), which correspond to the blast-resistant genes Pi2, Pita/Pita2, and Pikm, respectively, showed a significant association to the leaf blast disease. The associated R-genes might be utilized in rice breeding programmes through marker-assisted breeding, and the identified resistant rice accessions could be used as prospective donors for the production of new resistant varieties in India and around the world. [ABSTRACT FROM AUTHOR]

Studies focused solely on single organisms can fail to identify the networks underlying host–pathogen gene-for-gene interactions. Here, we integrate genetic analyses of rice (Oryza sativa, host) and rice blast fungus (Magnaporthe oryzae, pathogen) and uncover a new pathogen recognition specificity of the rice nucleotide-binding domain and leucine-rich repeat protein (NLR) immune receptor Pik, which mediates resistance to M. oryzae expressing the avirulence effector gene AVR-Pik. Rice Piks-1, encoded by an allele of Pik-1, recognizes a previously unidentified effector encoded by the M. oryzae avirulence gene AVR-Mgk1, which is found on a mini-chromosome. AVR-Mgk1 has no sequence similarity to known AVR-Pik effectors and is prone to deletion from the mini-chromosome mediated by repeated Inago2 retrotransposon sequences. AVR-Mgk1 is detected by Piks-1 and by other Pik-1 alleles known to recognize AVR-Pik effectors; recognition is mediated by AVR-Mgk1 binding to the integrated heavy metal-associated (HMA) domain of Piks-1 and other Pik-1 alleles. Our findings highlight how complex gene-for-gene interaction networks can be disentangled by applying forward genetics approaches simultaneously to the host and pathogen. We demonstrate dynamic coevolution between an NLR integrated domain and multiple families of effector proteins. Studies focused solely on single organisms can fail to identify the networks underlying host–pathogen gene-for-gene interactions. Integrating genetic analyses of the pathogen rice blast fungus and its host plant helps to disentangle the complex interactions that determine the outcome of plant-pathogen interactions and reveals a previously overlooked pathogen effector. [ABSTRACT FROM AUTHOR]

Serine/arginine-rich (SR) proteins are well known as splicing factors in humans, model animals and plants. However, they are largely unknown in regulating pre-mRNA splicing of filamentous fungi. Here we report that the SR protein MoSrp1 enhances and suppresses alternative splicing in a model fungal plant pathogen Magnaporthe oryzae. Deletion of MoSRP1 caused multiple defects, including reduced virulence and thousands of aberrant alternative splicing events in mycelia, most of which were suppressed or enhanced intron splicing. A GUAG consensus bound by MoSrp1 was identified in more than 94% of the intron or/and proximate exons having the aberrant splicing. The dual functions of regulating alternative splicing of MoSrp1 were exemplified in enhancing and suppressing the consensus-mediated efficient splicing of the introns in MoATF1 and MoMTP1, respectively, which both were important for mycelial growth, conidiation, and virulence. Interestingly, MoSrp1 had a conserved sumoylation site that was essential to nuclear localization and enhancing GUAG binding. Further, we showed that MoSrp1 interacted with a splicing factor and two components of the exon-joining complex via its N-terminal RNA recognition domain, which was required to regulate mycelial growth, development and virulence. In contrast, the C-terminus was important only for virulence and stress responses but not for mycelial growth and development. In addition, only orthologues from Pezizomycotina species could completely rescue defects of the deletion mutants. This study reveals that the fungal conserved SR protein Srp1 regulates alternative splicing in a unique manner. Author summary: The primary roles of serine/arginine-rich (SR) proteins in pre-mRNA splicing have been well established in humans, model animals and plants. Some studies have suggested that fungal SR proteins are also involved in pre-mRNA splicing, but direct evidence is lacking to illustrate how they regulate pre-mRNA splicing in fungi. Here we describe that a fungal conserved SR protein MoSrp1 in the model plant fungal pathogen Magnaporthe oryzae regulates alternative pre-mRNA splicing with unique mechanisms. MoSrp1 enhances and suppresses alternative splicing by binding to the GUAG consensus in target pre-mRNAs. The GUAG consensus per se enhances and suppresses the intron splicing efficiency of MoATF1 and MoMTP1, both of which are important for mycelial growth, conidiation, and virulence. We also show that MoSrp1 is SUMOylatied for its nuclear localization and enhancing the GUAG binding capacity. Furthermore, we demonstrate that the N-terminal RNA recognition domain of MoSrp1 interacts with a splicing factor and two components of the exon-joining complex and is required for mycelial growth, development and virulence, and that the C-terminus was important only for virulence and stress responses but not for mycelial growth and development. [ABSTRACT FROM AUTHOR]

Many species of fungal plant pathogens coexist as multiple lineages on the same host, but the factors underlying the origin and maintenance of population structure remain largely unknown. The rice blast fungus Pyricularia oryzae is a widespread model plant pathogen displaying population subdivision. However, most studies of natural variation in P. oryzae have been limited in genomic or geographic resolution, and host adaptation is the only factor that has been investigated extensively as a contributor to population subdivision. In an effort to complement previous studies, we analyzed genetic and phenotypic diversity in isolates of the rice blast fungus covering a broad geographical range. Using single-nucleotide polymorphism genotyping data for 886 isolates sampled from 152 sites in 51 countries, we showed that population subdivision of P. oryzae in one recombining and three clonal lineages with broad distributions persisted with deeper sampling. We also extended previous findings by showing further population subdivision of the recombining lineage into one international and three Asian clusters, and by providing evidence that the three clonal lineages of P. oryzae were found in areas with different prevailing environmental conditions, indicating niche separation. Pathogenicity tests and bioinformatic analyses using an extended set of isolates and rice varieties indicated that partial specialization to rice subgroups contributed to niche separation between lineages, and differences in repertoires of putative virulence effectors were consistent with differences in host range. Experimental crosses revealed that female sterility and early post-mating genetic incompatibilities acted as strong additional barriers to gene flow between clonal lineages. Our results demonstrate that the spread of a fungal pathogen across heterogeneous habitats and divergent populations of a crop species can lead to niche separation and reproductive isolation between distinct, widely distributed, lineages. Author summary: The spread of infectious plant diseases associated with introduced fungal pathogens can have devastating effects on agrosystems, ecosystems, and human livelihoods. In addition to the burden of emerging fungal infections, most major crops or domesticated trees are colonized by introduced pathogens that are widespread and have been present for extended periods of time. An understanding of the factors underlying the colonization success of fungal pathogens is essential to limit the risk of future pandemics. Here, we use genotyping, genome sequencing and phenotypic assays of a large collection of isolates of the rice blast fungus Pyricularia oryzae to investigate the factors underlying population differentiation and differences in life history strategies in this major plant pathogen. We found four lineages of P. oryzae that displayed significant differences in host range and repertoires of virulence genes, and thrived in areas with different types of environmental conditions. We also found that sexual compatibility between lineages is limited by the lack of fertile female structures and early, post-mating, reproductive barriers. Our study shows that niche separation, loss of sex and post-mating genetic incompatibilities can contribute to the maintenance of population structure in widely distributed fungal crop pathogens. [ABSTRACT FROM AUTHOR]

Fungi also share with animals several genetically encoded cell death functionalities for targeted cell removal. Because of these conditions, fungi are expected to share molecular underpinnings of their immune responses with animals, while being able to mount them in most vegetative cells of their bodies, like plants and in contrast to animals, which rely on specialized immune cells, phagocytes. Innate immunity is an ancient cell-autonomous property of eukaryotes that allows them to regulate interactions with antagonistic microbes, including bacteria. [Extracted from the article]

Rice blast, caused by the fungus Magnaporthe oryzae, is one of the three major diseases affecting rice production and quality; it reduces rice grain yield by nearly 30%. In the early stage of this study, a strain of Bacillus velezensis with strong inhibition of M. oryzae was isolated and named ZW10. In vitro assays indicated prolonged germination time of conidia of M. oryzae treated with the antifungal substances of ZW10, 78% of the conidia could not form appressorium, and the conidial tubes expanded to form vacuolar structure and then shrank. The results of FDA-PI composite dyes showed that the antifungal substances of ZW10 inhibited the normal activity of M. oryzae hyphae that were rarely able to infect the epidermal cells of rice leaf sheath in vivo tests. In addition, rice treated with the antifungal substances of ZW10 showed a variety of defense responses, including activation of defense-related enzymes, increased expression of the salicylic acid pathway genes, and accumulation of hydrogen peroxide (H2O2), which might function directly or indirectly in resistance to pathogen attack. The field experiment with rice blast infection in different periods showed that the antifungal substances of ZW10 had the same control effect as carbendazim. The significant biological control activity of ZW10 and its capacity to stimulate host defenses suggest that this B. velezensis strain has the potential to be developed into a biopesticide for the biocontrol of rice blast. [ABSTRACT FROM AUTHOR]

From appressorium to transpressorium - Defining the morphogenetic basis of host cell invasion by the rice blast fungus Graph: Fig 1 Infection structures of the rice blast fungus: The appressorium and transpressorium.A schematic figure describing the characteristics and developmental biology of appressoria and transpressoria - specialised infection structures formed by Magnaporthe oryzae to penetrate the host cuticle and traverse cell junctions, respectively. Only once the fungus has fully occupied the initial epidermal cell does it invade adjacent cells, normally in a highly synchronous manner, spreading from cell to cell and rapidly occupying host tissue (Fig 1). Cells in cells: morphogenetic and metabolic strategies conditioning rice infection by the blast fungus I Magnaporthe oryzae i . [Extracted from the article]

Transcriptional dynamic in response to environmental and developmental cues are fundamental to biology, yet many mechanistic aspects are poorly understood. One such example is fungal plant pathogens, which use secreted proteins and small molecules, termed effectors, to suppress host immunity and promote colonization. Effectors are highly expressed in planta but remain transcriptionally repressed ex planta, but our mechanistic understanding of these transcriptional dynamics remains limited. We tested the hypothesis that repressive histone modification at H3-Lys27 underlies transcriptional silencing ex planta, and that exchange for an active chemical modification contributes to transcription of in planta induced genes. Using genetics, chromatin immunoprecipitation and sequencing and RNA-sequencing, we determined that H3K27me3 provides significant local transcriptional repression. We detail how regions that lose H3K27me3 gain H3K27ac, and these changes are associated with increased transcription. Importantly, we observed that many in planta induced genes were marked by H3K27me3 during axenic growth, and detail how altered H3K27 modification influences transcription. ChIP-qPCR during in planta growth suggests that H3K27 modifications are generally stable, but can undergo dynamics at specific genomic locations. Our results support the hypothesis that dynamic histone modifications at H3K27 contributes to fungal genome regulation and specifically contributes to regulation of genes important during host infection. Author summary: Fungal pathogens of crops and humans pose annual threats to our food and health. There are many steps to the host infection process, during which fungal pathogens display unique growth, and use specific genes to cause disease. Despite this knowledge, many aspects of how pathogens regulate their genome to enact this process remain unknown. Here, we demonstrate how chemical modification of lysine residues on the histone H3, which helps organize and control DNA usage, play an important regulatory role in the model fungal pathogen causing rice blast disease. Our analysis shows a significant association between genes important for host infection and H3 lysine 27 methylation. We show that by experimentally changing histone modifications, many fungal genes normally used during plant infection are turned on outside of the host. Furthermore, we detail how histone modifications can change naturally in the fungus during plant infection. These findings help broaden our knowledge of genome regulation for these pathogens, and advances the goal of a more comprehensive understanding of the infection process. [ABSTRACT FROM AUTHOR]

Oligomycins are macrolide antibiotics, produced by Streptomyces spp. that show antagonistic effects against several microorganisms such as bacteria, fungi, nematodes and the oomycete Plasmopara viticola. Conidiogenesis, germination of conidia and formation of appressoria are determining factors pertaining to pathogenicity and successful diseases cycles of filamentous fungal phytopathogens. The goal of this research was to evaluate the in vitro suppressive effects of two oligomycins, oligomycin B and F along with a commercial fungicide Nativo® 75WG on hyphal growth, conidiogenesis, conidial germination, and appressorial formation of the wheat blast fungus, Magnaporthe oryzae Triticum (MoT) pathotype. We also determined the efficacy of these two oligomycins and the fungicide product in vivo in suppressing wheat blast with a detached leaf assay. Both oligomycins suppressed the growth of MoT mycelium in a dose dependent manner. Between the two natural products, oligomycin F provided higher inhibition of MoT hyphal growth compared to oligomycin B with a minimum inhibitory concentration of 0.005 and 0.05 μg/disk, respectively. The application of the compounds completely halted conidial formation of the MoT mycelium in agar medium. Further bioassays showed that these compounds significantly inhibited MoT conidia germination and induced lysis. The compounds also caused abnormal germ tube formation and suppressed appressorial formation of germinated spores. Interestingly, the application of these macrolides significantly inhibited wheat blast on detached leaves of wheat. This is the first report on the inhibition of mycelial growth, conidiogenesis, germination of conidia, deleterious morphological changes in germinated conidia, and suppression of blast disease of wheat by oligomycins from Streptomyces spp. Further study is needed to unravel the precise mode of action of these natural compounds and consider them as biopesticides for controlling wheat blast. [ABSTRACT FROM AUTHOR]

Mines and caves are unusual ecosystems containing unique fungi and are greatly understudied compared to other environments. The Soudan Mine in Tower, MN, an iron ore mine that closed in 1963 after operating for 80 years, was sampled to explore fungal diversity and to investigate taxa that tolerate heavy metals for potential bioprocessing technologies or as sources of bioactive molecules for drug discovery and possible biocontrol for white-nose syndrome (WNS) of bats. The mine is 714 m deep, has 18 levels and contains large quantities of wooden timbers, in contrast to many other oligotrophic subterranean environments. Fungi were cultured from samples and the ITS region was sequenced for identification and phylogenetic analysis. Results show Ascomycota are the dominant fungi followed by Basidiomycota and Mucoromycota. Out of 164 identified taxa, 108 belong to the Ascomycota and 26 and 31 to Basidiomycota and Mucoromycota, respectively. There are also 46 taxa that do not match (<97% BLAST GenBank identity) sequenced fungal species. Examples of the most commonly isolated Ascomycota include Scytalidium sp., Mariannaea comptospora, Hypocrea pachybasidioides, Oidiodendron griseum and Pochonia bulbillosa; Basidiomycota include Postia sp., Sistotrema brinkmannii, Calocera sp., Amylocorticiellum sp.; Mucoromycota include Mortierella parvispora, M. gamsii, M. hyaline, M. basiparvispora and Mortierella sp. Unusual growth forms were also found including large quantities of black rhizomorphs of Armillaria sinapina and white mycelial cords of Postia sp. mycelium, as well as Pseudogymnoascus species growing over large areas of mine walls and ceiling. The mine environment is a relatively extreme environment for fungi, with the presence of high levels of heavy metals, complete darkness and poor nutrient availability. Several genera are similar to those isolated in other extreme environments but phylogenetic analyses show differences in species between these environments. Results indicate this subterranean environment hosts a wide diversity of fungi, many of them not found in above ground environments. [ABSTRACT FROM AUTHOR]

Integrons play a major role in the evolution and spread of antimicrobial resistance in human pathogens, including Escherichia coli. This study describes the occurrence of class 1 integrons in human pathogenic E. coli, in three isolate collections involving three periods from the last 100 years (i) the Murray collection (n = 58 bacteria isolated from the 1910s to 1940s); (ii) the E. coli reference (ECOR) collection (n = 37 isolates mainly from the 1980s); and (iii) a recently assembled collection (n = 88 isolates obtained in 2016). High-quality whole genome sequences (WGSs) were available for all isolates. Integrons were detected in the WGSs with the program IntegronFinder and the results compared with three established methods: (i) polymerase chain reaction detection of the integrase gene; (ii) BLAST searching using draft genomes; and (iii) mapping of short reads. No integrons were found in any of the Murray Collection isolates; however, integrons were present in 3% of the isolates from ECOR collection, assembled in the 1980s, and 26% of the isolates from the 2010s. Similarly, antimicrobial resistance determinants were not present in the Murray Collection isolates, whereas they were present in 19% of the ECOR Collection isolates and in 55% of the isolates obtained in during the 2010s. [ABSTRACT FROM AUTHOR]

Secondary peat swamp forest (PSF) arise by degradation of primary PSF as a result of fire and human activities. Yeasts diversity of Kuan Kreng (KK) and Rayong Botanical Garden (RBG) PSF, which are two secondary PSF in southern and in eastern Thailand, respectively, were investigated. Yeasts were isolated from soil and peat soil by the dilution plate and enrichment techniques. From six samples collected from KK PSF, 35 strains were obtained, and they were identified based on the sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene 13 species in 12 genera, and one potential new species of the genus Galactomyces were detected. Thirty-two strains were obtained from six samples collected from RBG PSF and 26 strains were identified as 13 known yeast species in 11 genera, whereas six strains were found to represent two potential new species of the genera Papiliotrema and Moesziomyces. Among yeast strains isolated from KK PSF, the number of strains in the phylum Ascomycota and Basidiomycota were equal, whereas there were slightly fewer strains in Ascomycota than in Basidiomycota among the strains obtained from RBG PSF. The yeast strains were evaluated for their antagonistic activities against fungal pathogens which cause rice diseases (Fusarium moniliforme, Helminthosporium oryzae, Rhizoctonia solani, Curvularia lunata and Pyricularia grisea) and postharvest disease of fruits (Phytophthora palmivora, Lasiodiplodia theobromae and Colletotrichum gloeosporioides). Twelve strains of seven species were found to be antagonistic yeast strains. Starmerella kuoi DMKU-SPS13-6, Hanseniaspora lindneri DMKU ESS10-9 and Piskurozyma taiwanensis DMKU-SPS12-2 capable to inhibit R. solani by 70.1–76.2%, Wickerhamomyces anomalus DMKU SPS6-1 and three Rhodotorula taiwanensis strains (DMKU SPS8-1, DMKU ESS9-3, DMKU SPS9-2) inhibited C. lunata by 69.8–71.9%, Hanseniaspora lindneri DMKU ESS10-9 and Scheffersomyces spartinae DMKU SPS9-3 inhibited P. grisea by 81.9–84.4% and four Papiliotrema laurentii strains (DMKU-SPS15-1, DMKU-ESS11-2, DMKU-ESS8-2, DMKU-ESS6-4) inhibited P. palmivora by 53.2–59.5%. [ABSTRACT FROM AUTHOR]

Magnaporthe oryzae causes rice blast disease, but little is known about the dynamic restructuring of the actin cytoskeleton during its polarized tip growth and pathogenesis. Here, we used super-resolution live-cell imaging to investigate the dynamic organization of the actin cytoskeleton in M. oryzae during hyphal tip growth and pathogenesis. We observed a dense actin network at the apical region of the hyphae and actin filaments originating from the Spitzenkörper (Spk, the organizing center for hyphal growth and development) that formed branched actin bundles radiating to the cell membrane. The actin cross-linking protein Fimbrin (MoFim1) helps organize this actin distribution. MoFim1 localizes to the actin at the subapical collar, the actin bundles, and actin at the Spk. Knockout of MoFim1 resulted in impaired Spk maintenance and reduced actin bundle formation, preventing polar growth, vesicle transport, and the expansion of hyphae in plant cells. Finally, transgenic rice (Oryza sativa) expressing RNA hairpins targeting MoFim1 exhibited improved resistance to M. oryzae infection, indicating that MoFim1 represents an excellent candidate for M. oryzae control. These results reveal the dynamics of actin assembly in M. oryzae during hyphal tip development and pathogenesis, and they suggest a mechanism in which MoFim1 organizes such actin networks. Author summary: The actin cytoskeleton plays crucial roles in M. oryzae growth and pathogenesis. However, the organizing principles and dynamic behavior of the cytoskeleton in M. oryzae growth and expansion in plant cells remain enigmatic. In this study, through super-resolution observation, we found a dense network of actin cables dynamically gathered near the Spitzenkörper, the organizing center for hyphal growth and development. The actin cytoskeleton also accumulated at the narrow penetration site where the hypha invaded the plant cell. Furthermore, the actin cross-linking protein MoFim1 contributes to organizing these actin cables, thus facilitating hyphal polarity, vesicle transport, and invasive expansion of hyphae in the host plant cell. In all, our results uncovered a mechanism by which M. oryzae organizes and relies on a unique actin distribution pattern in hyphae during cell growth and pathogenesis. [ABSTRACT FROM AUTHOR]

Rice blast disease caused by Magnaporthe oryzae (M. oryzae) is one of the most serious diseases. Although previous research using two-dimensional gel-based proteomics to assess the proteins related to the rice blast resistance had been done, few proteins were identified. Here, we used the iTRAQ method to detect the differentially expressed proteins (DEPs) in the durable resistant rice variety Gangyuan8 (GY8) and the susceptible rice variety Lijiangxintuanheigu (LTH) in response to M. oryzae invasion, and then transcriptome sequencing was used to assist analysis A total of 193 and 672 DEPs were specifically identified in GY8 and LTH, respectively, with only 46 similarly expressed DEPs being shared by GY8 and LTH.39 DEPs involved in plant-pathogen interaction, plant hormone signal transduction, fatty acid metabolism and peroxisome biosynthesis were significantly different between compatible interaction (LTH) and incompatible interaction (GY8). Some proteins participated in peroxide signal transduction and biosynthesis was down-regulated in GY8 but up-regulated in LTH. A lot of genes encoding pathogenesis-related gene (PR), such as chitinase and glucanase, were significantly up-regulated at both the transcriptome and proteome levels at 24 hours post-inoculation in GY8, but up-regulated at the transcriptome level and down-regulated at the proteome level in LTH. Our study reveals that the pathogen-associated molecular pattern (PAMP)-triggered immunity defense system may be activated at the transcriptome level but was inhibited at the protein level in susceptible rice varieties after inoculation. The results may facilitate future studies of the molecular mechanisms of rice blast resistance. [ABSTRACT FROM AUTHOR]

Newly emerged wheat blast disease is a serious threat to global wheat production. Wheat blast is caused by a distinct, exceptionally diverse lineage of the fungus causing rice blast disease. Through sequencing a recent field isolate, we report a reference genome that includes seven core chromosomes and mini-chromosome sequences that harbor effector genes normally found on ends of core chromosomes in other strains. No mini-chromosomes were observed in an early field strain, and at least two from another isolate each contain different effector genes and core chromosome end sequences. The mini-chromosome is enriched in transposons occurring most frequently at core chromosome ends. Additionally, transposons in mini-chromosomes lack the characteristic signature for inactivation by repeat-induced point (RIP) mutation genome defenses. Our results, collectively, indicate that dispensable mini-chromosomes and core chromosomes undergo divergent evolutionary trajectories, and mini-chromosomes and core chromosome ends are coupled as a mobile, fast-evolving effector compartment in the wheat pathogen genome. [ABSTRACT FROM AUTHOR]

The accumulation of the reactive oxygen species (ROS) in rice is important in its interaction with the rice blast fungus Magnaporthe oryzae during which the pathogen scavenges ROS through the production of extracellular enzymes that promote blast. We previously characterized the MoYvh1 protein phosphatase from M. oryzae that plays a role in scavenging of ROS. To understand the underlying mechanism, we found that MoYvh1 is translocated into the nucleus following oxidative stress and that this translocation is dependent on MoSsb1 and MoSsz1 that are homologous to heat-shock protein 70 (Hsp70) proteins. In addition, we established a link between MoYvh1 and MoMrt4, a ribosome maturation factor homolog whose function also involves shuttling between the cytoplasm and the nucleus. Moreover, we found that MoYvh1 regulates the production of extracellular proteins that modulate rice-immunity. Taking together, our evidence suggests that functions of MoYvh1 in regulating ROS scavenging require its nucleocytoplasmic shuttling and the partner proteins MoSsb1 and MoSsz1, as well as MoMrt4. Our findings provide novel insights into the mechanism by which M. oryzae responds to and subverts host immunity through the regulation of ribosome biogenesis and protein biosynthesis. [ABSTRACT FROM AUTHOR]

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most important bacterial disease in rice (Oryza sativa L.). Our previous studies have revealed that the bacterial blight resistance gene Xa23 from wild rice O. rufipogon Griff. confers the broadest-spectrum resistance against all the naturally occurring Xoo races. As a novel executor R gene, Xa23 is transcriptionally activated by the bacterial avirulence (Avr) protein AvrXa23 via binding to a 28-bp DNA element (EBEAvrXa23) in the promoter region. So far, the evolutionary mechanism of Xa23 remains to be illustrated. Here, a rice germplasm collection of 97 accessions, including 29 rice cultivars (indica and japonica) and 68 wild relatives, was used to analyze the evolution, phylogeographic relationship and association of Xa23 alleles with bacterial blight resistance. All the ~ 473 bp DNA fragments consisting of promoter and coding regions of Xa23 alleles in the germplasm accessions were PCR-amplified and sequenced, and nine single nucleotide polymorphisms (SNPs) were detected in the promoter regions (~131 bp sequence upstream from the start codon ATG) of Xa23/xa23 alleles while only two SNPs were found in the coding regions. The SNPs in the promoter regions formed 5 haplotypes (Pro-A, B, C, D, E) which showed no significant difference in geographic distribution among these 97 rice accessions. However, haplotype association analysis indicated that Pro-A is the most favored haplotype for bacterial blight resistance. Moreover, SNP changes among the 5 haplotypes mostly located in the EBE/ebe regions (EBEAvrXa23 and corresponding ebes located in promoters of xa23 alleles), confirming that the EBE region is the key factor to confer bacterial blight resistance by altering gene expression. Polymorphism analysis and neutral test implied that Xa23 had undergone a bottleneck effect, and selection process of Xa23 was not detected in cultivated rice. In addition, the Xa23 coding region was found highly conserved in the Oryza genus but absent in other plant species by searching the plant database, suggesting that Xa23 originated along with the diversification of the Oryza genus from the grass family during evolution. This research offers a potential for flexible use of novel Xa23 alleles in rice breeding programs and provide a model for evolution analysis of other executor R genes. [ABSTRACT FROM AUTHOR]

The lysin motif (LysM) containing proteins can bind chitin and are ubiquitous in various organisms including fungi. In plant pathogenic fungi, a few LysM proteins have been characterized as effectors to suppress chitin-induced immunity in plant hosts and therefore contribute to fungal virulence. The effector mechanism is still questioned in fungus-animal interactions. In this study, we found that LysM proteins are also present in animal pathogenic fungi and have evolved divergently. The genome of the insect pathogen Beauveria bassiana encodes 12 LysM proteins, and the genes were differentially transcribed by the fungus when grown in different conditions. Deletion of six genes that were expressed by the fungus growing in insects revealed that two, Blys2 and Blys5, were required for full fungal virulence. Both proteins could bind chitin and Blys5 (containing two LysM domains) could additionally bind chitosan and cellulose. Truncation analysis of Blys2 (containing five LysM domains) indicated that the combination of LysM domains could determine protein-binding affinity and specificity for different carbohydrates. Relative to the wild-type strain, loss of Blys2 or Blys5 could impair fungal propagation in insect hemocoels and lead to the upregulation of antifungal gene in insects. Interestingly, the virulence defects of ΔBlys2 and ΔBlys5 could be fully restored by complementation with the Slp1 effector from the rice blast fungus Magnaporthe oryzae. In contrast to Slp1 and Blys2, Blys5 could potentially protect fungal hyphae against chitinase hydrolysis. The results of this study not only advance the understanding of LysM protein evolution but also establish the effector mechanism of fungus-animal interactions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The establishment of polarity is a critical process in pathogenic fungi, mediating infection-related morphogenesis and host tissue invasion. Here, we report the identification of TPC1 (Transcription factor for Polarity Control 1), which regulates invasive polarized growth in the rice blast fungus Magnaporthe oryzae. TPC1 encodes a putative transcription factor of the fungal Zn(II)2Cys6 family, exclusive to filamentous fungi. Tpc1-deficient mutants show severe defects in conidiogenesis, infection-associated autophagy, glycogen and lipid metabolism, and plant tissue colonisation. By tracking actin-binding proteins, septin-5 and autophagosome components, we show that Tpc1 regulates cytoskeletal dynamics and infection-associated autophagy during appressorium-mediated plant penetration. We found that Tpc1 interacts with Mst12 and modulates its DNA-binding activity, while Tpc1 nuclear localisation also depends on the MAP kinase Pmk1, consistent with the involvement of Tpc1 in this signalling pathway, which is critical for appressorium development. Importantly, Tpc1 directly regulates NOXD expression, the p22phox subunit of the fungal NADPH oxidase complex via an interaction with Mst12. Tpc1 therefore controls spatial and temporal regulation of cortical F-actin through regulation of the NADPH oxidase complex during appressorium re-polarisation. Consequently, Tpc1 is a core developmental regulator in filamentous fungi, linking the regulated synthesis of reactive oxygen species and the Pmk1 pathway, with polarity control during host invasion. [ABSTRACT FROM AUTHOR]

Rice blast disease caused by Magnaporthe oryzae is one of the most destructive disease causing huge losses to rice yield in different parts of the world. Therefore, an attempt has been made to find out the resistance by screening and studying the genetic diversity of eighty released rice varieties by National Rice Research Institute, Cuttack (NRVs) using molecular markers linked to twelve major blast resistance (R) genes viz Pib, Piz, Piz-t, Pik, Pik-p, Pikm Pik-h, Pita/Pita-2, Pi2, Pi9, Pi1 and Pi5. Out of which, nineteen varieties (23.75%) showed resistance, twenty one were moderately resistant (26.25%) while remaining forty varieties (50%) showed susceptible in uniform blast nursery. Rice varieties possessing blast resistance genes varied from four to twelve and the frequencies of the resistance genes ranged from 0 to 100%. The cluster analysis grouped the eighty NRVs into two major clusters at 63% level of genetic similarity coefficient. The PIC value for seventeen markers varied from 0 to 0.37 at an average of 0.20. Out of seventeen markers, only five markers, 195R-1, Pi9-i, Pita3, YL155/YL87 and 40N23r corresponded to three broad spectrum R genes viz. Pi9, Pita/Pita2 and Pi5 were found to be significantly associated with the blast disease with explaining phenotypic variance from 3.5% to 7.7%. The population structure analysis and PCoA divided the entire 80 NRVs into two sub-groups. The outcome of this study would help to formulate strategies for improving rice blast resistance through genetic studies, plant-pathogen interaction, identification of novel R genes, development of new resistant varieties through marker-assisted breeding for improving rice blast resistance in India and worldwide. [ABSTRACT FROM AUTHOR]

Rice blast is a destructive disease caused by Magnaporthe oryzae, and it has a large impact on rice production worldwide. Compared with leaf blast resistance, our understanding of panicle blast resistance is limited. The japonica landrace Jiangnanwan from Taihu Lake region in China shows highly resistance to panicle and leaf blast. In this study, three generations (F2:5, F2:6, F2:7) consisting of 221 RILs (recombination inbreeding lines), developed from the cross of Jiangnanwan and Suyunuo, a susceptible-blast japonica variety, were evaluated for panicle blast resistance in the fields and leaf blast resistance in greenhouse in Nanjing in 2013, 2014 and 2015. A blast resistance gene Pi-jnw1 referring to panicle blast resistance and leaf blast resistance was identified in the three generations and located in the region of RM27273 and RM27381 in chromosome 11. The RIL18 line harboring Pi-jnw1 was selected to be backcrossed with Suyunuo to develop BC2F2 populations. According to the genotyping of 1,150 BC2F2 individuals and panicle blast and leaf blast resistance evaluation of 47 recombinants between RM27150 and RM27381, Pi-jnw1 was finally mapped to the 282 kb region between markers W28 and BS39. This study revealed that Jiangnanwan harboring a panicle blast and leaf blast resistance gene Pi-jnw1 could be a genetic source for breeding new rice cultivars with panicle blast resistance. [ABSTRACT FROM AUTHOR]

In a previous transcriptome analysis of early response genes in rice during Magnaporthe oryzae infection, we identified a CONSTANS-like (COL) gene OsCOL9. In the present study, we investigated the functional roles of OsCOL9 in blast resistance. OsCOL9 belonged to group II of the COL protein family, and it contained a BB-box and a C-terminal CCT (CONSTANS, COL and TOC1) domain. OsCOL9 was found in the nucleus of rice cells, and it exerted transcriptional activation activities through its middle region (MR). Magnaporthe oryzae infection induced OsCOL9 expression, and transgenic OsCOL9 knock-out rice plants showed increased pathogen susceptibility. OsCOL9 was a critical regulator of pathogen-related genes, especially PR1b, which were also activated by exogenous salicylic acid (SA) and 1-aminocyclopropane-1-carboxylicacid (ACC), the precursor of ethylene (ET). Further analysis indicated that OsCOL9 over-expression increased the expressions of phytohormone biosynthetic genes, NPR1, WRKY45, OsACO1 and OsACS1, which were related to SA and ET biosynthesis. Interestingly, we found that OsCOL9 physically interacted with the scaffold protein OsRACK1 through its CCT domain, and the OsRACK1 expression was induced in response to exogenous SA and ACC as well as M. oryzae infection. Taken together, these results indicated that the COL protein OsCOL9 interacted with OsRACK1, and it enhanced the rice blast resistance through SA and ET signaling pathways. [ABSTRACT FROM AUTHOR]

S3 Table lists short read sequence data of six isolates as provided on request. (XLSX) Reference 1 Thierry M, Charriat F, Milazzo J, Adreit H, Ravel S, Cros-Arteil S, et al. (2022) Maintenance of divergent lineages of the Rice Blast Fungus Pyricularia oryzae through niche separation, loss of sex and post-mating genetic incompatibilities. [Extracted from the article]

Rice blast disease caused by Magnaporthe oryzae is one of the most devastating fungal diseases of rice and results in a huge loss of rice productivity worldwide. During the infection process, M. oryzae secretes a large number of glycosyl hydrolase proteins into the host apoplast to digest the cell wall and facilitate fungal ingression into host tissues. In this study, we identified a novel arabinofuranosidase-B (MoAbfB) protein that is secreted by M. oryzae during fungal infection. Deletion of MoAbfB from M. oryzae resulted in reduced disease severity in rice. Biochemical assays revealed that the MoAbfB protein exhibited arabinofuranosidase activity and caused degradation of rice cell wall components. Interestingly, pre-treatment of rice with the MoAbfB protein inhibited fungal infection by priming defence gene expression. Our findings suggest that MoAbfB secretion affects M. oryzae pathogenicity by breaking down the host cell wall, releasing oligosaccharides that may be recognized by the host to trigger innate immune responses. [ABSTRACT FROM AUTHOR]

Blast disease caused by the Magnaporthe species is a major factor affecting the productivity of rice, wheat and millets. This study was aimed at generating genomic information for rice and non-rice Magnaporthe isolates to understand the extent of genetic variation. We have sequenced the whole genome of the Magnaporthe isolates, infecting rice (leaf and neck), finger millet (leaf and neck), foxtail millet (leaf) and buffel grass (leaf). Rice and finger millet isolates infecting both leaf and neck tissues were sequenced, since the damage and yield loss caused due to neck blast is much higher as compared to leaf blast. The genome-wide comparison was carried out to study the variability in gene content, candidate effectors, repeat element distribution, genes involved in carbohydrate metabolism and SNPs. The analysis of repeat element footprints revealed some genes such as naringenin, 2-oxoglutarate 3-dioxygenase being targeted by Pot2 and Occan, in isolates from different host species. Some repeat insertions were host-specific while other insertions were randomly shared between isolates. The distributions of repeat elements, secretory proteins, CAZymes and SNPs showed significant variation across host-specific lineages of Magnaporthe indicating an independent genome evolution orchestrated by multiple genomic factors. [ABSTRACT FROM AUTHOR]

N-linked glycosylation is a way of glycosylation for newly synthesized protein, which plays a key role in the maturation and transport of proteins. Glycoside hydrolases (GHs) are essential in this process, and are involved in processing of N-linked glycoproteins or degradation of carbohydrate structures. Here, we identified and characterized MoGls2 in Magnaporthe oryzae, which is a yeast glucosidase II homolog Gls2 and is required for trimming the final glucose in N-linked glycans and normal cell wall synthesis. Target deletion of MoGLS2 in M. oryzae resulted in a reduced mycelial growth, an increased conidial production, delayed conidial germination and loss the ability of sexual reproduction. Pathogenicity assays revealed that the ΔMogls2 mutant showed significantly decreased in virulence and infectious growth. Further studies showed that the mutant was less sensitive to salt and osmotic stress, and increased sensitivity to cell wall stresses. Additionally, the ΔMogls2 mutant showed a defect in cell wall integrity. Our results indicate that MoGls2 is a key protein for the growth and development of M. oryzae, involving in the regulation of asexual/sexual development, stress response, cell wall integrity and infectious growth. [ABSTRACT FROM AUTHOR]

Magnaporthe oryzae, the causative organism of rice blast, infects cereal crops and grasses at various stages of plant development. A comprehensive understanding of its metabolism and the implications on pathogenesis is necessary for countering this devastating crop disease. We present the role of the CorA magnesium transporters, MoAlr2 and MoMnr2, in development and pathogenicity of M. oryzae. The MoALR2 and MoMNR2 genes individually complement the Mg2+ uptake defects of a S. cerevisiae CorA transporter double mutant. MoALR2 and MoMNR2 respond to extracellular Mg2+ and Ca2+ levels and their expression is elevated under Mg2+ scarce conditions. RNA silencing mediated knockdown of MoALR2 (WT+siALR2, Δmnr2+siALR2 and ALR2+MNR2 simultaneous silencing) drastically alters intracellular cation concentrations and sensitivity to metal ions. MoALR2 silencing is detrimental to vegetative growth and surface hydrophobicity of mycelia, and the transformants display loss of cell wall integrity. MoALR2 is required for conidiogenesis and appressorium development, and is essential for infection. Investigation of knockdown transformants reveal low cAMP levels and altered expression of genes encoding proteins involved in MoMps1 cell wall integrity and cAMP MoPmk1 driven MAP Kinase signaling pathways. In contrast to MoALR2 knockdowns, the MoMNR2 deletion (Δmnr2) shows increased sensitivity to CorA inhibitors as well as altered cation sensitivity, but has limited effect on surface hydrophobicity and severity of plant infection. Interestingly, MoALR2 expression is elevated in Δmnr2. Impairment of development and infectivity of knockdown transformants and altered intracellular cation composition suggest that CorA transporters are essential for Mg2+ homeostasis within the cell, and are crucial to maintaining normal gene expression associated with cell structure, signal transduction and surface hydrophobicity in M. oryzae. We suggest that CorA transporters, and especially MoALR2, constitute an attractive target for the development of antifungal agents against this pathogen. [ABSTRACT FROM AUTHOR]

MoHrip2 Magnaporthe oryzae hypersensitive protein 2 is an elicitor protein of rice blast fungus M. oryzae. Rice seedlings treated with MoHrip2 have shown an induced resistance to rice blast. To elucidate the mechanism underlying this MoHrip2 elicitation in rice, we used differential-display 2-D gel electrophoresis and qRT-PCR to assess the differential expression among the total proteins extracted from rice leaves at 24 h after treatment with MoHrip2 and buffer as a control. Among ~1000 protein spots detected on each gel, 10 proteins were newly induced, 4 were up-regulated, and 3 were down-regulated in MoHrip2-treated samples compared with the buffer control. Seventeen differentially expressed proteins were detected using MS/MS analysis and categorized into six groups according to their putative function: defense-related transcriptional factors, signal transduction-related proteins, reactive oxygen species (ROS) production, programmed cell death (PCD), defense-related proteins, and photosynthesis and energy-related proteins. The qPCR results (relative expression level of genes) further supported the differential expression of proteins in MoHrip2-treated rice leaves identified with 2D-gel, suggesting that MoHrip2 triggers an early defense response in rice leaves via stress-related pathways, and the results provide evidence for elicitor-induced resistance at the protein level. [ABSTRACT FROM AUTHOR]

Rice blast is one of the most destructive diseases affecting rice worldwide. The adoption of host resistance has proven to be the most economical and effective approach to control rice blast. In recent years, sequence-specific nucleases (SSNs) have been demonstrated to be powerful tools for the improvement of crops via gene-specific genome editing, and CRISPR/Cas9 is thought to be the most effective SSN. Here, we report the improvement of rice blast resistance by engineering a CRISPR/Cas9 SSN (C-ERF922) targeting the OsERF922 gene in rice. Twenty-one C-ERF922-induced mutant plants (42.0%) were identified from 50 T0 transgenic plants. Sanger sequencing revealed that these plants harbored various insertion or deletion (InDel) mutations at the target site. We showed that all of the C-ERF922-induced allele mutations were transmitted to subsequent generations. Mutant plants harboring the desired gene modification but not containing the transferred DNA were obtained by segregation in the T1 and T2 generations. Six T2 homozygous mutant lines were further examined for a blast resistance phenotype and agronomic traits, such as plant height, flag leaf length and width, number of productive panicles, panicle length, number of grains per panicle, seed setting percentage and thousand seed weight. The results revealed that the number of blast lesions formed following pathogen infection was significantly decreased in all 6 mutant lines compared with wild-type plants at both the seedling and tillering stages. Furthermore, there were no significant differences between any of the 6 T2 mutant lines and the wild-type plants with regard to the agronomic traits tested. We also simultaneously targeted multiple sites within OsERF922 by using Cas9/Multi-target-sgRNAs (C-ERF922S1S2 and C-ERF922S1S2S3) to obtain plants harboring mutations at two or three sites. Our results indicate that gene modification via CRISPR/Cas9 is a useful approach for enhancing blast resistance in rice. [ABSTRACT FROM AUTHOR]

Oryza minuta (Poaceae family) is a tetraploid wild relative of cultivated rice with a BBCC genome. O. minuta has the potential to resist against various pathogenic diseases such as bacterial blight (BB), white backed planthopper (WBPH) and brown plant hopper (BPH). Here, we sequenced and annotated the complete mitochondrial genome of O. minuta. The mtDNA genome is 515,022 bp, containing 60 protein coding genes, 31 tRNA genes and two rRNA genes. The mitochondrial genome organization and the gene content at the nucleotide level are highly similar (89%) to that of O. rufipogon. Comparison with other related species revealed that most of the genes with known function are conserved among the Poaceae members. Similarly, O. minuta mt genome shared 24 protein-coding genes, 15 tRNA genes and 1 ribosomal RNA gene with other rice species (indica and japonica). The evolutionary relationship and phylogenetic analysis revealed that O. minuta is more closely related to O. rufipogon than to any other related species. Such studies are essential to understand the evolutionary divergence among species and analyze common gene pools to combat risks in the current scenario of a changing environment. [ABSTRACT FROM AUTHOR]

Plants produce cytokinin (CK) hormones for controlling key developmental processes like source/sink distribution, cell division or programmed cell-death. Some plant pathogens have been shown to produce CKs but the function of this mimicry production by non-tumor inducing pathogens, has yet to be established. Here we identify a gene required for CK biosynthesis, CKS1, in the rice blast fungus Magnaporthe oryzae. The fungal-secreted CKs are likely perceived by the plant during infection since the transcriptional regulation of rice CK-responsive genes is altered in plants infected by the mutants in which CKS1 gene was deleted. Although cks1 mutants showed normal in vitro growth and development, they were severely affected for in planta growth and virulence. Moreover, we showed that the cks1 mutant triggered enhanced induction of plant defenses as manifested by an elevated oxidative burst and expression of defense-related markers. In addition, the contents of sugars and key amino acids for fungal growth were altered in and around the infection site by the cks1 mutant in a different manner than by the control strain. These results suggest that fungal-derived CKs are key effectors required for dampening host defenses and affecting sugar and amino acid distribution in and around the infection site. [ABSTRACT FROM AUTHOR]

The retromer mediates protein trafficking through recycling cargo from endosomes to the trans-Golgi network in eukaryotes. However, the role of such trafficking events during pathogen-host interaction remains unclear. Here, we report that the cargo-recognition complex (MoVps35, MoVps26 and MoVps29) of the retromer is essential for appressorium-mediated host penetration by Magnaporthe oryzae, the causal pathogen of the blast disease in rice. Loss of retromer function blocked glycogen distribution and turnover of lipid bodies, delayed nuclear degeneration and reduced turgor during appressorial development. Cytological observation revealed dynamic MoVps35-GFP foci co-localized with autophagy-related protein RFP-MoAtg8 at the periphery of autolysosomes. Furthermore, RFP-MoAtg8 interacted with MoVps35-GFP in vivo, RFP-MoAtg8 was mislocalized to the vacuole and failed to recycle from the autolysosome in the absence of the retromer function, leading to impaired biogenesis of autophagosomes. We therefore conclude that retromer is essential for autophagy-dependent plant infection by the rice blast fungus. [ABSTRACT FROM AUTHOR]

Rice blast fungus, Magnaporthe oryzae, is the most destructive pathogen in the rice-growing area. This fungus has a biotrophic phase early in infection and later switches to a necrotrophic lifestyle. During the biotrophic phase, the fungus competes with its host for nutrients and oxygen. Continuous uptake of oxygen is essential for successful establishment of blast disease of this pathogen. Here, we report transcriptional responses of the fungus to oxygen limitation. Transcriptome analysis using RNA-Seq identified that 1,047 genes were up-regulated in response to hypoxia. Those genes are involved in mycelial development, sterol biosynthesis, and metal ion transport based on hierarchical GO terms, and are well-conserved among three fungal species. In addition, null mutants of two hypoxia-responsive genes were generated and their roles in fungal development and pathogenicity tested. The mutant for the sterol regulatory element-binding protein gene, MoSRE1, exhibited increased sensitivity to a hypoxia-mimicking agent, increased conidiation, and delayed invasive growth within host cells, which is suggestive of important roles in fungal development. However, such defects did not cause any significant decrease in disease severity. The other null mutant, for the alcohol dehydrogenase gene MoADH1, showed no defect in the hypoxia-mimicking condition (using cobalt chloride) and fungal development. Taken together, this comprehensive transcriptional profiling in response to a hypoxic condition with experimental validations would provide new insights into fungal development and pathogenicity in plant pathogenic fungi. [ABSTRACT FROM AUTHOR]

Peroxisomes play important roles in metabolisms of eukaryotes and infection of plant fungal pathogens. These organelles proliferate by de novo formation or division in response to environmental stimulation. Although the assembly of peroxisomes was documented in fungal pathogens, their division and its relationship to pathogenicity remain obscure. In present work, we analyzed the roles of three Pex11 family members in peroxisomal division and pathogenicity of the rice blast fungus Magnaporthe oryzae. Deletion of MoPEX11A led to fewer but enlarged peroxisomes, and impaired the separation of Woronin bodies from peroxisomes, while deletion of MoPEX11B or MoPEX11C put no evident impacts to peroxisomal profiles. MoPEX11A mutant exhibited typical peroxisome related defects, delayed conidial germination and appressoria formation, and decreased appressorial turgor and host penetration. As a result, the virulence of MoPEX11A mutant was greatly reduced. Deletion of MoPEX11B and MoPEX11C did not alter the virulence of the fungus. Further, double or triple deletions of the three genes were unable to enhance the virulence decrease in MoPEX11A mutant. Our data indicated that MoPEX11A is the main factor modulating peroxisomal division and is required for full virulence of the fungus. [ABSTRACT FROM AUTHOR]

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

Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection. [ABSTRACT FROM AUTHOR]

Magnaporthe oryzae is a hemibiotrophic fungal pathogen that causes rice blast disease. A compatible interaction requires overcoming plant defense responses to initiate colonization during the early infection process. Nitric oxide (NO) plays important roles in defense responses during host-pathogen interactions. Microbes generally protect themselves against NO-induced damage by using enzymes. Here, we characterized an S-(hydroxymethyl)- glutathione dehydrogenase gene in M. oryzae, MoSFA1, the homologs of which are involved in NO metabolism by specifically catalyzing the reduction of S-nitrosoglutathione (GSNO) in yeasts and plants. As expected from the activities of S-(hydroxymethyl)glutathione dehydrogenase in formaldehyde detoxification and GSNO reduction, MoSFA1 deletion mutants were lethal in formaldehyde containing medium, sensitive to exogenous NO and exhibited a higher level of S-nitrosothiols (SNOs) than that of the wild type. Notably, the mutants showed severe reduction of conidiation and appressoria turgor pressure, as well as significantly attenuated the virulence on rice cultivar CO-39. However, the virulence of MoSFA1 deletion mutants on wounded rice leaf was not affected. An infection assay on barley leaf further revealed that MoSFA1 deletion mutants exhibited a lower infection rate, and growth of infectious hyphae of the mutants was retarded not only in primary infected cells but also in expansion from cell to cell. Furthermore, barley leaf cell infected by MoSFA1 deletion mutants exhibited a stronger accumulation of H2O2 at 24 and 36 hpi. MoSFA1 deletion mutants displayed hypersensitivity to different oxidants, reduced activities of superoxide dismutases and peroxidases, and lower glutathione content in cells, compared with the wild type. These results imply that MoSFA1-mediated NO metabolism is important in redox homeostasis in response to development and host infection of M. oryzae. Taken together, this work identifies that MoSFA1 is required for conidiation and contributes to virulence in the penetration and biotrophic phases in M. oryzae. [ABSTRACT FROM AUTHOR]