Development of a telomere vector-based approach to overcome limitations caused by lethal phenotypes in the study of essential genes in Magnaporthe oryzae.

Reverse genetic approaches are common tools in genomics for elucidating gene functions, involving techniques such as gene deletion followed by screening for aberrant phenotypes. If the generation of gene deletion mutants fails, the question arises whether the failure stems from technical issues or because the gene of interest (GOI) is essential, meaning that the deletion causes lethality. In this report, we introduce a novel method for assessing gene essentiality using the phytopathogenic ascomycete Magnaporthe oryzae. The method is based on the observation that telomere vectors are lost in transformants during cultivation without selection pressure. We tested the hypothesis that essential genes can be identified in deletion mutants co-transformed with a telomere vector. The M. oryzae gene MoPKC, described in literature as essential, was chosen as GOI. Using CRISPR/Cas9 technology transformants with deleted GOI were generated and backed up by a telomere vector carrying a copy of the GOI and conferring fenhexamid resistance. Transformants in which the GOI deletion in the genome was not successful lost the telomere vector on media without fenhexamid. In contrast, transformants with confirmed GOI deletion retained the telomere vector even in absence of fenhexamid selection. In the latter case, the maintenance of the telomere indicates that the GOI is essential for the surveillance of the fungi, as it would have been lost otherwise. The method presented here allows to test for essentiality of genes when no mutants can be obtained from gene deletion approaches, thereby expanding the toolbox for studying gene function in ascomycetes.

Transposable elements impact the population divergence of rice blast fungus Magnaporthe oryzae.

Dynamic transposition of transposable elements (TEs) in fungal pathogens has significant impact on genome stability, gene expression, and virulence to the host. In Magnaporthe oryzae, genome plasticity resulting from TE insertion is a major driving force leading to the rapid evolution and diversification of this fungus. Despite their importance in M. oryzae population evolution and divergence, our understanding of TEs in this context remains limited. Here, we conducted a genome-wide analysis of TE transposition dynamics in the 11 most abundant TE families in M. oryzae populations. Our results show that these TEs have specifically expanded in recently isolated M. oryzae rice populations, with the presence/absence polymorphism of TE insertions highly concordant with population divergence on Geng/Japonica and Xian/Indica rice cultivars. Notably, the genes targeted by clade-specific TEs showed clade-specific expression patterns and are involved in the pathogenic process, suggesting a transcriptional regulation of TEs on targeted genes. Our study provides a comprehensive analysis of TEs in M. oryzae populations and demonstrates a crucial role of recent TE bursts in adaptive evolution and diversification of the M. oryzae rice-infecting lineage. IMPORTANCE: Magnaporthe oryzae is the causal agent of the destructive blast disease, which caused massive loss of yield annually worldwide. The fungus diverged into distinct clades during adaptation toward the two rice subspecies, Xian/Indica and Geng/Japonica. Although the role of TEs in the adaptive evolution was well established, mechanisms underlying how TEs promote the population divergence of M. oryzae remain largely unknown. In this study, we reported that TEs shape the population divergence of M. oryzae by differentially regulating gene expression between Xian/Indica-infecting and Geng/Japonica-infecting populations. Our results revealed a TE insertion-mediated gene expression adaption that led to the divergence of M. oryzae population infecting different rice subspecies.

Rice-Magnaporthe oryzae interactions in resistant and susceptible rice cultivars under panicle blast infection based on defense-related enzyme activities and metabolomics.

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.

Distinct genomic contexts predict gene presence-absence variation in different pathotypes of Magnaporthe oryzae.

Fungi use the accessory gene content of their pangenomes to adapt to their environments. While gene presence-absence variation contributes to shaping accessory gene reservoirs, the genomic contexts that shape these events remain unclear. Since pangenome studies are typically species-wide and do not analyze different populations separately, it is yet to be uncovered whether presence-absence variation patterns and mechanisms are consistent across populations. Fungal plant pathogens are useful models for studying presence-absence variation because they rely on it to adapt to their hosts, and members of a species often infect distinct hosts. We analyzed gene presence-absence variation in the blast fungus, Magnaporthe oryzae (syn. Pyricularia oryzae), and found that presence-absence variation genes involved in host-pathogen and microbe-microbe interactions may drive the adaptation of the fungus to its environment. We then analyzed genomic and epigenomic features of presence-absence variation and observed that proximity to transposable elements, gene GC content, gene length, expression level in the host, and histone H3K27me3 marks were different between presence-absence variation genes and conserved genes. We used these features to construct a model that was able to predict whether a gene is likely to experience presence-absence variation with high precision (86.06%) and recall (92.88%) in M. oryzae. Finally, we found that presence-absence variation genes in the rice and wheat pathotypes of M. oryzae differed in their number and their genomic context. Our results suggest that genomic and epigenomic features of gene presence-absence variation can be used to better understand and predict fungal pangenome evolution. We also show that substantial intra-species variation can exist in these features.

A key sphingolipid pathway gene, MoDES1, regulates conidiation, virulence and plasma membrane tension in Magnaporthe oryzae.

Rice blast, caused by the plant pathogenic fungus Magnaporthe oryzae, is a destructive disaster all over the earth that causes enormous losses in crop production. Sphingolipid, an important biological cell membrane lipid, is an essential structural component in the plasma membrane (PM) and has several biological functions, including cell mitosis, apoptosis, stress resistance, and signal transduction. Previous studies have suggested that sphingolipid and its derivatives play essential roles in the virulence of plant pathogenic fungi. However, the functions of sphingolipid biosynthesis-related proteins are not fully understood. In this article, we identified a key sphingolipid synthesis enzyme, MoDes1, and found that it is engaged in cell development and pathogenicity in M. oryzae. Deletion of MoDES1 gave rise to pleiotropic defects in vegetative growth, conidiation, plant penetration, and pathogenicity. MoDes1 is also required for lipid homeostasis and participates in the cell wall integrity (CWI) and Osm1-MAPK pathways. Notably, our results showed that there is negative feedback in the TORC2 signaling pathway to compensate for the decreased sphingolipid level due to the knockout of MoDES1 by regulating the phosphorylated Ypk1 level and PM tension. Furthermore, protein structure building has shown that MoDes1 is a potential drug target. These findings further refine the function of Des1 and deepen our understanding of the sphingolipid biosynthesis pathway in M. oryzae, laying a foundation for developing novel and specific drugs for rice blast control.

Rapid mini-chromosome divergence among fungal isolates causing wheat blast outbreaks in Bangladesh and Zambia.

The fungal pathogen, Magnaporthe oryzae Triticum pathotype, causing wheat blast disease was first identified in South America and recently spread across continents to South Asia and Africa. Here, we studied the genetic relationship among isolates found on the three continents. Magnaporthe oryzae strains closely related to a South American field isolate B71 were found to have caused the wheat blast outbreaks in South Asia and Africa. Genomic variation among isolates from the three continents was examined using an improved B71 reference genome and whole-genome sequences. We found strong evidence to support that the outbreaks in Bangladesh and Zambia were caused by the introductions of genetically separated isolates, although they were all close to B71 and, therefore, collectively referred to as the B71 branch. In addition, B71 branch strains carried at least one supernumerary mini-chromosome. Genome assembly of a Zambian strain revealed that its mini-chromosome was similar to the B71 mini-chromosome but with a high level of structural variation. Our findings show that while core genomes of the multiple introductions are highly similar, the mini-chromosomes have undergone marked diversification. The maintenance of the mini-chromosome and rapid genomic changes suggest the mini-chromosomes may serve important virulence or niche adaptation roles under diverse environmental conditions.

GERWR: Identifying the Key Pathogenicity- Associated sRNAs of Magnaporthe Oryzae Infection in Rice Based on Graph Embedding and Random Walk With Restart.

Rice blast, caused by Magnaporthe oryzae(M.oryzae), is a destructive rice disease that reduces rice yield by 10% to 30% annually. It also affects other cereal crops such as barley, wheat, rye, millet, sorghum, and maize. Small RNAs (sRNAs) play an essential regulatory role in fungus-plant interaction during the fungal invasion, but studies on pathogenic sRNAs during the fungal invasion of plants based on multi-omics data integration are rare. This paper proposes a novel approach called Graph Embedding combined with Random Walk with Restart (GERWR) to identify pathogenic sRNAs based on multi-omics data integration during M.oryzae invasion. By constructing a multi-omics network (MRMO), we identified 29 pathogenic sRNAs of rice blast fungus. Further analysis revealed that these sRNAs regulate rice genes in a many-to-many relationship, playing a significant regulatory role in the pathogenesis of rice blast disease. This paper explores the pathogenic factors of rice blast disease from the perspective of multi-omics data analysis, revealing the inherent connection between pathogenic factors of different omics. It has essential scientific significance for studying the pathogenic mechanism of rice blast fungus, the rice blast fungus-rice model system, and the pathogen-host interaction in related fields.

The cell-end protein Tea4 spatially regulates hyphal branch initiation and appressorium remodeling in the blast fungus Magnaporthe oryzae.

The differentiation of specialized infection cells, called appressoria, from polarized germ tubes of the blast fungus Magnaporthe oryzae, requires remarkable remodeling of cell polarity and architecture, yet our understanding of this process remains incomplete. Here we investigate the behavior and role of cell-end marker proteins in appressorium remodeling and hyphal branch emergence. We show that the SH3 domain-containing protein Tea4 is required for the normal formation of an F-actin ring at Tea1-GFP-labeled polarity nodes, which contributes to the remodeling of septin structures and repolarization of the appressorium. Further, we show that Tea1 localizes to a cortical structure during hyphal septation which, unlike contractile septin rings, persists after septum formation, and, in combination with other polarity determinants, likely spatially regulates branch emergence. Genetic loss of Tea4 leads to mislocalization of Tea1 at the hyphal apex and with it, impaired growth directionality. In contrast, Tea1 is largely depleted from septation events in Δtea4 mutants and branching and septation are significantly reduced. Together, our data provide new insight into polarity remodeling during infection-related and vegetative growth by the blast fungus.

Hydroxamate siderophores secreted by plant endophytic Pseudomonas putida elicit defense against blast disease in rice incited by Magnaporthe oryzae.

Our study focuses on hydroxamate-type siderophores from Pseudomonas putida BP25, known for chelating ferric iron and aiding microbial growth in iron-deficient environments. Confirmed through CAS-agar and tetrazolium tests, a purified siderophore extract was obtained via ion-exchange chromatography. Applying varying concentrations of this siderophore to rice seedlings demonstrated concentration-dependent effects on shoot and root phenotypes. Prophylactic application on rice leaves significantly reduced blast severity (68.7%-97.0%), surpassing curative application (47.5%-86.87%). Additionally, the siderophore treatment elevated peroxidase, polyphenol oxidase, and total phenols in rice plants. Defense-related genes linked to salicylic acid (OsPR1.1, OsNPR1, and OsPDF2.2), and other pathways (Oshox24, OsCLE, and OsGLP3-3, OsEIN2.4, and OsCSE) promoting blast suppression showed upregulation. However, the OsACS6 gene associated with ethylene-induced internodal elongation was significantly downregulated. Overall, our findings propose that the siderophore from P. putida BP25 induces defense gene transcription, offering potential for sustainable rice production via bio-formulation.

Magnaporthe oryzae effector MoSPAB1 directly activates rice Bsr-d1 expression to facilitate pathogenesis.

Fungal pathogens typically use secreted effector proteins to suppress host immune activators to facilitate invasion. However, there is rarely evidence supporting the idea that fungal secretory proteins contribute to pathogenesis by transactivating host genes that suppress defense. We previously found that pathogen Magnaporthe oryzae induces rice Bsr-d1 to facilitate infection and hypothesized that a fungal effector mediates this induction. Here, we report that MoSPAB1 secreted by M. oryzae directly binds to the Bsr-d1 promoter to induce its expression, facilitating pathogenesis. Amino acids 103-123 of MoSPAB1 are required for its binding to the Bsr-d1 promoter. Both MoSPAB1 and rice MYBS1 compete for binding to the Bsr-d1 promoter to regulate Bsr-d1 expression. Furthermore, MoSPAB1 homologues are highly conserved among fungi. In particular, Colletotrichum fructicola CfSPAB1 and Colletotrichum sublineola CsSPAB1 activate kiwifruit AcBsr-d1 and sorghum SbBsr-d1 respectively, to facilitate pathogenesis. Taken together, our findings reveal a conserved module that may be widely utilized by fungi to enhance pathogenesis.

Insertion of Transposable Elements in AVR-Pib of Magnaporthe oryzae Leading to LOSS of the Avirulent Function.

Rice blast is a very serious disease caused by Magnaporthe oryzae, which threatens rice production and food supply throughout the world. The avirulence (AVR) genes of rice blast are perceived by the corresponding rice blast resistance (R) genes and prompt specific resistance. A mutation in AVR is a major force for new virulence. Exploring mutations in AVR among M. oryzae isolates from rice production fields could aid assessment of the efficacy and durability of R genes. We studied the probable molecular-evolutionary patterns of AVR-Pib alleles by assaying their DNA-sequence diversification and examining their avirulence to the corresponding Pib resistance gene under natural conditions in the extremely genetically diverse of rice resources of Yunnan, China. PCRs detected results from M. oryzae genomic DNA and revealed that 162 out of 366 isolates collected from Yunnan Province contained AVR-Pib alleles. Among them, 36.1-73.3% isolates from six different rice production areas of Yunnan contained AVR-Pib alleles. Furthermore, 36 (28.6%) out of 126 isolates had a transposable element (TE) insertion in AVR-Pib, which resulted in altered virulence. The TE insertion was identified in isolates from rice rather than from Musa nana Lour. Twelve AVR-Pib haplotypes encoding three novel AVR-Pib variants were identified among the remaining 90 isolates. AVR-Pib alleles evolved to virulent forms from avirulent forms by base substitution and TE insertion of Pot2 and Pot3 in the 5' untranslated region of AVR-Pib. These findings support the hypothesis that functional AVR-Pib possesses varied sequence structures and can escape surveillance by hosts via multiple variation manners.

Intraspecific Variation of Transposable Elements Reveals Differences in the Evolutionary History of Fungal Phytopathogen Pathotypes.

Transposable elements (TEs) contribute to intraspecific variation and play important roles in the evolution of fungal genomes. However, our understanding of the processes that shape TE landscapes is limited, as is our understanding of the relationship between TE content, population structure, and evolutionary history of fungal species. Fungal plant pathogens, which often have host-specific populations, are useful systems in which to study intraspecific TE content diversity. Here, we describe TE dynamics in five lineages of Magnaporthe oryzae, the fungus that causes blast disease of rice, wheat, and many other grasses. We identified differences in TE content across these lineages and showed that recent lineage-specific expansions of certain TEs have contributed to overall greater TE content in rice-infecting and Setaria-infecting lineages. We reconstructed the evolutionary histories of long terminal repeat-retrotransposon expansions and found that in some cases they were caused by complex proliferation dynamics of one element and in others by multiple elements from an older population of TEs multiplying in parallel. Additionally, we found evidence suggesting the recent transfer of a DNA transposon between rice- and wheat-infecting M. oryzae lineages and a region showing evidence of homologous recombination between those lineages, which could have facilitated such a transfer. By investigating intraspecific TE content variation, we uncovered key differences in the proliferation dynamics of TEs in various pathotypes of a fungal plant pathogen, giving us a better understanding of the evolutionary history of the pathogen itself.

Recent co-evolution of two pandemic plant diseases in a multi-hybrid swarm.

Most plant pathogens exhibit host specificity but when former barriers to infection break down, new diseases can rapidly emerge. For a number of fungal diseases, there is increasing evidence that hybridization plays a major role in driving host jumps. However, the relative contributions of existing variation versus new mutations in adapting to new host(s) is unclear. Here we reconstruct the evolutionary history of two recently emerged populations of the fungus Pyricularia oryzae that are responsible for two new plant diseases: wheat blast and grey leaf spot of ryegrasses. We provide evidence that wheat blast/grey leaf spot evolved through two distinct mating episodes: the first occurred ~60 years ago, when a fungal individual adapted to Eleusine mated with another individual from Urochloa. Then, about 10 years later, a single progeny from this cross underwent a series of matings with a small number of individuals from three additional host-specialized populations. These matings introduced non-functional alleles of two key host-specificity factors, whose recombination in a multi-hybrid swarm probably facilitated the host jump. We show that very few mutations have arisen since the founding event and a majority are private to individual isolates. Thus, adaptation to the wheat or Lolium hosts appears to have been instantaneous, and driven entirely by selection on repartitioned standing variation, with no obvious role for newly formed mutations.

Genetic and molecular analysis of leaf blast resistance in Tetep derived line RIL4 and its relationship to genes at Pita/Pita2 locus.

The Vietnamese indica landrace 'Tetep' is known worldwide for its durable and broad spectrum-resistance to blast. We performed genetic and molecular analyses of leaf blast resistance in a Tetep derived recombinant inbred line 'RIL4' which is resistant to both leaf and neck blast. Phenotypic analysis of segregating F2 progenies suggested that leaf blast resistance in RIL4 was controlled by a dominant gene tentatively designated as Pi-l(t). The gene was mapped to a 2.4 cm region close to the centromere of chromosome 12. The search for the gene content in the equivalent genomic region of reference cv. Nipponbare revealed the presence of five NBS-LRR genes, two of which corresponded to the alleles of Pita and Pi67 genes previously identified from Tetep. The two other genes, LOC_Os12g17090, and LOC_Os12g17490 represented the homologs of stripe rust resistance gene Yr10. The allelic tests with Pita2 and Pi67 lines suggested that the leaf blast resistance gene in RIL4 is either allelic or tightly linked to these genes. The genomic position of the leaf blast resistance gene in RIL4 perfectly coincided with the genomic position of a neck blast resistance gene Pb2 previously identified from this line suggesting that the same gene confers resistance to leaf and neck blast. The present results were discussed in juxtaposition with past studies on the genes of Pita/Pita2 resistance gene complex.

Pijx confers broad-spectrum seedling and panicle blast resistance by promoting the degradation of ATP ß subunit and OsRbohC-mediated ROS burst in rice.

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is one of the most important diseases of rice. Utilization of blast-resistance genes is the most economical, effective, and environmentally friendly way to control the disease. However, genetic resources with broad-spectrum resistance (BSR) that is effective throughout the rice growth period are rare. In this work, using a genome-wide association study, we identify a new blast-resistance gene, Pijx, which encodes a typical CC-NBS-LRR protein. Pijx is derived from a wild rice species and confers BSR to M. oryzae at both the seedling and panicle stages. The functions of the resistant haplotypes of Pijx are confirmed by gene knockout and overexpression experiments. Mechanistically, the LRR domain in Pijx interacts with and promotes the degradation of the ATP synthase ß subunit (ATPb) via the 26S proteasome pathway. ATPb acts as a negative regulator of Pijx-mediated panicle blast resistance, and interacts with OsRbohC to promote its degradation. Consistently, loss of ATPb function causes an increase in NAPDH content and ROS burst. Remarkably, when Pijx is introgressed into two japonica rice varieties, the introgression lines show BSR and increased yields that are approximately 51.59% and 79.31% higher compared with those of their parents in a natural blast disease nursery. In addition, we generate PPLPijx Pigm and PPLPijx Piz-t pyramided lines and these lines also have higher BSR to panicle blast compared with Pigm- or Piz-t-containing rice plants. Collectively, this study demonstrates that Pijx not only confers BSR to M. oryzae but also maintains high and stable rice yield, providing new genetic resources and molecular targets for breeding rice varieties with broad-spectrum blast resistance.

Loss of PWT7, Located on a Supernumerary Chromosome, Is Associated with Parasitic Specialization of Pyricularia oryzae on Wheat.

Pyricularia oryzae, a blast fungus of gramineous plants, is composed of various host genus-specific pathotypes. The avirulence of an Avena isolate on wheat is conditioned by PWT3 and PWT4. We isolated the third avirulence gene from the Avena isolate and designated it as PWT7. PWT7 was effective as an avirulence gene only at the seedling stage or on leaves. PWT7 homologs were widely distributed in a subpopulation of the Eleusine pathotype and the Lolium pathotype but completely absent in the Triticum pathotype (the wheat blast fungus). The PWT7 homolog found in the Eleusine pathotype was one of the five genes involved in its avirulence on wheat. A comparative analysis of distribution of PWT7 and the other two genes previously identified in the Eleusine pathotype suggested that, in the course of parasitic specialization toward the wheat blast fungus, a common ancestor of the Eleusine, Lolium, Avena, and Triticum pathotypes first lost PWT6, secondly PWT7, and, finally, the function of PWT3. PWT7 or its homologs were located on core chromosomes in Setaria and Eleusine isolates but on supernumerary chromosomes in Lolium and Avena isolates. This is an example of interchromosomal translocations of effector genes between core and supernumerary chromosomes. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Melatonin targets MoIcl1 and works synergistically with fungicide isoprothiolane in rice blast control.

Melatonina natural harmless molecule-displays versatile roles in human health and crop disease control such as for rice blast. Rice blast, caused by the filamentous fungus Magnaporthe oryzae, is one devastating disease of rice. Application of fungicides is one of the major measures in the control of various crop diseases. However, fungicide resistance in the pathogen and relevant environmental pollution are becoming serious problems. By screening for possible synergistic combinations, here, we discovered an eco-friendly combination for rice blast control, melatonin, and the fungicide isoprothiolane. These compounds together exhibited significant synergistic inhibitory effects on vegetative growth, conidial germination, appressorium formation, penetration, and plant infection by M. oryzae. The combination of melatonin and isoprothiolane reduced the effective concentration of isoprothiolane by over 10-fold as well as residual levels of isoprothiolane. Transcriptomics and lipidomics revealed that melatonin and isoprothiolane synergistically interfered with lipid metabolism by regulating many common targets, including the predicted isocitrate lyase-encoding gene MoICL1. Furthermore, using different techniques, we show that melatonin and isoprothiolane interact with MoIcl1. This study demonstrates that melatonin and isoprothiolane function synergistically and can be used to reduce the dosage and residual level of isoprothiolane, potentially contributing to the environment-friendly and sustainable control of crop diseases.

A molecular mechanosensor for real-time visualization of appressorium membrane tension in Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae uses a pressurized infection cell called an appressorium to drive a rigid penetration peg through the leaf cuticle. The vast internal pressure of an appressorium is very challenging to investigate, leaving our understanding of the cellular mechanics of plant infection incomplete. Here, using fluorescence lifetime imaging of a membrane-targeting molecular mechanoprobe, we quantify changes in membrane tension in M. oryzae. We show that extreme pressure in the appressorium leads to large-scale spatial heterogeneities in membrane mechanics, much greater than those observed in any cell type previously. By contrast, non-pathogenic melanin-deficient mutants, exhibit low spatially homogeneous membrane tension. The sensor kinase ∆sln1 mutant displays significantly higher membrane tension during inflation of the appressorium, providing evidence that Sln1 controls turgor throughout plant infection. This non-invasive, live cell imaging technique therefore provides new insight into the enormous invasive forces deployed by pathogenic fungi to invade their hosts, offering the potential for new disease intervention strategies.

The RasGEF MoCdc25 regulates vegetative growth, conidiation and appressorium-mediated infection in the rice blast fungus Magnaporthe oryzae.

Ras guanine nucleotide exchange factors (RasGEFs) can trigger Ras GTPase activities and play important roles in controlling various cellular processes in eukaryotes. Recently, it has been exhibited that RasGEF Cdc25 regulates morphological differentiation and pathogenicity in several plant pathogenic fungi. However, the role of RasGEFs in Magnaporthe oryzae is largely unknown. In this study, we identified and functionally characterized a RasGEF gene MoCDC25 in M. oryzae, which is orthologous to Saccharomyces cerevisiae CDC25. Targeted gene deletion mutants (ΔMocdc25) were completely nonpathogenic and were severely impaired in hyphal growth, conidiation and appressorium formation. The mutants exhibited highly sensitive response to osmotic, cell wall integrity or oxidative stresses. MoCdc25 physically interacts with the MAPK scaffold Mst50 and the putative Cdc42GEF MoScd1 in yeast two-hybrid assays. Moreover, we found that MoCdc25 was involved in regulating the phosphorylation of the MAP kinases (Pmk1, Mps1, and Osm1). In addition, the intracellular cAMP content in hyphae of the ΔMocdc25 mutants was significantly reduced compared to the parent strain Ku80 and the defect of appressorium formation of the mutants could be partially restored by the supplement of exogenous cAMP. Taken together, we conclude that the RasGEF MoCdc25 regulates vegetative growth, conidiation, appressorium formation and pathogenicity via MAPK and cAMP response pathways in M. oryzae.