DGS1 improves rice disease resistance by elevating pathogen-associated molecular pattern-triggered immunity.

Rice yield and disease resistance are two crucial factors in determining the suitability of a gene for agricultural breeding. Decreased grain size1 (DGS1), encoding an RING-type E3 ligase, has been found to have a positive effect on rice yield by regulating rice grain number and 1000-grain weight. However, the role of DGS1 in rice blast resistance is still unknown. In this study, we report that DGS1 enhances disease resistance by improving PTI responses, including stronger ROS burst and MAPK activation, and also increased expression of defense-related genes. Furthermore, DGS1 works in conjunction with ubiquitin conjugating enzyme OsUBC45 as an E2-E3 pair to facilitate the ubiquitin-dependent degradation of OsGSK3 and OsPIP2;1, thereby influencing rice yield and immunity, respectively. Therefore, the DGS1-OsUBC45 module has the potential in facilitating rice agricultural breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00137-9.

Elucidation of Palmarumycin Spirobisnaphthalene Biosynthesis Reveals a Set of Previously Unrecognized Oxidases and Reductases.

Spirobisnaphthalenes (SBNs) are a class of highly oxygenated, fungal bisnaphthalenes containing an unique spiroketal bridge, that displayed diverse bioactivities. Among the reported SBNs, palmarumycins are the major type, which are precursors for the other type of SBNs structurally. However, the biosynthesis of SBNs is unclear. In this study, we elucidated the biosynthesis of palmarumycins, using gene disruption, heterologous expression, and substrate feeding experiments. The biosynthetic gene cluster for palmarumycins was identified to be distant from the polyketide synthase gene cluster, and included two cytochrome P450s (PalA and PalB), and one short chain dehydrogenase/reductase (PalC) encoding genes as key structural genes. PalA is an unusual, multifunctional P450 that catalyzes the oxidative dimerization of 1,8-dihydroxynaphthalene to generate the spiroketal linkage and 2,3-epoxy group. Chemical synthesis of key intermediate and in vitro biochemical assays proved that the oxidative dimerization proceeded via a binaphthyl ether. PalB installs the C-5 hydroxyl group, widely found in SBNs. PalC catalyzes 1-keto reduction, the reverse 1-dehydrogenation, and 2,3-epoxide reduction. Moreover, an FAD-dependent oxidoreductase, encoded by palD, which locates outside the cluster, functions as 1-dehydrogenase. These results provided the first genetic and biochemical evidence for the biosynthesis of palmarumycin SBNs.

First telomere-to-telomere gapless assembly of the rice blast fungus Pyricularia oryzae.

Rice blast caused by Pyricularia oryzae (syn., Magnaporthe oryzae) was one of the most destructive diseases of rice throughout the world. Genome assembly was fundamental to genetic variation identification and critically impacted the understanding of its ability to overcome host resistance. Here, we report a gapless genome assembly of rice blast fungus P. oryzae strain P131 using PacBio, Illumina and high throughput chromatin conformation capture (Hi-C) sequencing data. This assembly contained seven complete chromosomes (43,237,743 bp) and a circular mitochondrial genome (34,866 bp). Approximately 14.31% of this assembly carried repeat sequences, significantly greater than its previous assembled version. This assembly had a 99.9% complement in BUSCO evaluation. A total of 14,982 genes protein-coding genes were predicted. In summary, we assembled the first telomere-to-telomere gapless genome of P. oryzae, which would be a valuable genome resource for future research on the genome evolution and host adaptation.

The synthetic NLR RGA5HMA5 requires multiple interfaces within and outside the integrated domain for effector recognition.

Some plant sensor nucleotide-binding leucine-rich repeat (NLR) receptors detect pathogen effectors through their integrated domains (IDs). Rice RGA5 sensor NLR recognizes its corresponding effectors AVR-Pia and AVR1-CO39 from the blast fungus Magnaporthe oryzae through direct binding to its heavy metal-associated (HMA) ID to trigger the RGA4 helper NLR-dependent resistance in rice. Here, we report a mutant of RGA5 named RGA5HMA5 that confers complete resistance in transgenic rice plants to the M. oryzae strains expressing the noncorresponding effector AVR-PikD. RGA5HMA5 carries three engineered interfaces, two of which lie in the HMA ID and the other in the C-terminal Lys-rich stretch tailing the ID. However, RGA5 variants having one or two of the three interfaces, including replacing all the Lys residues with Glu residues in the Lys-rich stretch, failed to activate RGA4-dependent cell death of rice protoplasts. Altogether, this work demonstrates that sensor NLRs require a concerted action of multiple surfaces within and outside the IDs to both recognize effectors and activate helper NLR-mediated resistance, and has implications in structure-guided designing of sensor NLRs.

A gln-tRNA-based CRISPR/Cas9 knockout system enables the functional characterization of genes in the genetically recalcitrant brassica anthracnose fungus Colletotrichum higginsianum.

Colletotrichum higginsianum causes anthracnose disease in brassicas. The availability of the C. higginsianum genome has paved the way for the genome-wide exploration of genes associated with virulence/pathogenicity. However, delimiting the biological functions of these genes remains an arduous task due to the recalcitrance of C. higginsianum to genetic manipulations. Here, we report a CRISPR/Cas9-based system that can knock out the genes in C. higginsianum with a staggering 100% homologous recombination frequency (HRF). The system comprises two vectors: pCas9-Ch_tRp-sgRNA, in which a C. higginsianum glutaminyl-tRNA drives the expression of sgRNA, and pCE-Zero-HPT carrying a donor DNA cassette containing the marker gene HPT flanked by homology arms. Upon co-transformation of the C. higginsianum protoplasts, pCas9-Ch_tRp-sgRNA causes a DNA double-strand break in the targeted gene, followed by homology-directed replacement of the gene with HPT by pCE-Zero-HPT, thereby generating loss-of-function mutants. Using the system, we generated the knockout mutants of two effector candidates (ChBas3 and OBR06881) with a 100% HRF. Interestingly, the ΔChBas3 and ΔOBR06881 mutants did not seem to affect the C. higginsianum infection of Arabidopsis thaliana. Altogether, the CRISPR/Cas9 system developed in the study enables the targeted deletion of genes, including effectors, in C. higginsianum, thus determining their biological functions.

The GRAS protein OsDLA involves in brassinosteroid signalling and positively regulates blast resistance by forming a module with GSK2 and OsWRKY53 in rice.

Brassinosteroids (BRs) play a crucial role in shaping the architecture of rice (Oryza sativa) plants. However, the regulatory mechanism of BR signalling in rice immunity remains largely unexplored. Here we identify a rice mutant dla, which exhibits decreased leaf angles and is insensitive to 24-epiBL (a highly active synthetic BR), resembling the BR-deficient phenotype. The dla mutation caused by a T-DNA insertion in the OsDLA gene leads to downregulation of the causative gene. The OsDLA knockout plants display reduced leaf angles and less sensitivity to 24-epiBL. In addition, both dla mutant and OsDLA knockout plants are more susceptible to rice blast compared to the wild type. OsDLA is a GRAS transcription factor and interacts with the BR signalling core negative regulator, GSK2. GSK2 phosphorylates OsDLA for degradation via the 26S proteasome. The GSK2 RNAi line exhibits enhanced rice blast resistance, while the overexpression lines thereof show susceptibility to rice blast. Furthermore, we show that OsDLA interacts with and stabilizes the WRKY transcription factor OsWRKY53, which has been demonstrated to positively regulate BR signalling and blast resistance. OsWRKY53 directly binds the promoter of PBZ1 and activates its expression, and this activation can be enhanced by OsDLA. Together, our findings unravel a novel mechanism whereby the GSK2-OsDLA-OsWRKY53 module coordinates blast resistance and plant architecture via BR signalling in rice.

A study of typical plant growth changes in response to drainage water and salt in ditch wetland in arid area.

Agricultural drainage significantly affected the changes of soil moisture and salinity in ditch wetlands. These changes can profoundly impact the spatial distribution and evolution of ditch wetland vegetation, thereby affecting the ecological environmental effects of these wetlands. Consequently, it is imperative to investigate the response of typical plant growth to drainage and soil salt in ditch wetlands in arid regions. Based on the classical metapopulation conceptual framework model (Levins model), this study established a new model of plant growth change in ditch wetlands, incorporating the key variables (water level and soil salinity) of arid area ditch wetlands. The application of the Gaussian model facilitates the resolution of species growth rates and mortality rates within this model. The study focused on the main drainage ditch (ditch M) and the drainage bucket ditch (ditch N) in the Lubotan saline-alkali land in Fuping, Shaanxi Province. The results revealed the following key findings: 1) the model effectively simulates the response of plant growth changes to water level and soil salinity in ditch wetlands in arid regions, particularly plants in the reed area and transition area disturbed by single factors such as water level and soil salinity; 2) the germination period of Phragmites australis in the reed area thrives in a shallow moisture environment, and adjusting the water level of the drainage ditch can maintain optimal growth conditions for Phragmites australis; 3) during the germination period of Suaeda salsa in the transition area, soil salinity should not be excessively high, though a moderate increase in soil salinity can promote the germination and growth of Suaeda salsa; and 4) Suaeda salsa in the symbiotic area has a higher adaptability to the soil salinity, with change in biomass consistent with plants in the transition area. The model provides an explanation and prediction for the growth changes of plant communities in ditch wetlands under drainage conditions. By integrating this model with the impact of farmland drainage on water level and soil salinity in drainage ditches, effective drainage management measures can be formulated, offering scientific support for the construction of ecological irrigation areas.

Dual-Specificity Inhibitor Targets Enzymes of the Trehalose Biosynthesis Pathway.

To reduce the risk of resistance development, a novel fungicide with dual specificity is demanded. Trehalose is absent in animals, and its synthases, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), are safe fungicide targets. Here, we report the discovery of a dual-specificity inhibitor of MoTps1 (Magnaporthe oryzae Tps1, TPS) and MoTps2 (M. oryzae Tps2, TPP). The inhibitor, named A1-4, was obtained from a virtual screening and subsequent surface plasmon resonance screening. In in vitro assays, A1-4 interacts with MoTps1 and MoTps2-TPP (MoTps2 TPP domain) and inhibits their enzyme activities. In biological activity assays, A1-4 not only inhibits the virulence of M. oryzae on host but also causes aggregation of conidia cytosol, which is a characteristic phenotype of MoTps2. Furthermore, hydrogen/deuterium exchange mass spectrometry assays support the notion that A1-4 binds to the substrate pockets of TPS and TPP. Collectively, A1-4 is a promising hit compound for the development of safe fungicide with dual-target specificity.

The plant immune receptor SNC1 monitors helper NLRs targeted by a bacterial effector.

Plants deploy intracellular receptors to counteract pathogen effectors that suppress cell-surface-receptor-mediated immunity. To what extent pathogens manipulate intracellular receptor-mediated immunity, and how plants tackle such manipulation, remains unknown. Arabidopsis thaliana encodes three similar ADR1 class helper nucleotide-binding domain leucine-rich repeat receptors (ADR1, ADR1-L1, and ADR1-L2), which are crucial in plant immunity initiated by intracellular receptors. Here, we report that Pseudomonas syringae effector AvrPtoB suppresses ADR1-L1- and ADR1-L2-mediated cell death. ADR1, however, evades such suppression by diversifying into two ubiquitination sites targeted by AvrPtoB. The intracellular sensor SNC1 interacts with and guards the CCR domains of ADR1-L1/L2. Removal of ADR1-L1/L2 or delivery of AvrPtoB activates SNC1, which then signals through ADR1 to trigger immunity. Our work elucidates the long-sought-after function of SNC1 in defense, and also how plants can use dual strategies, sequence diversification, and a multi-layered guard-guardee system, to counteract pathogen's attack on core immunity functions.

Plant immunity suppression by an exo-ß-1,3-glucanase and an elongation factor 1α of the rice blast fungus.

Fungal cell walls undergo continual remodeling that generates ß-1,3-glucan fragments as products of endo-glycosyl hydrolases (GHs), which can be recognized as pathogen-associated molecular patterns (PAMPs) and trigger plant immune responses. How fungal pathogens suppress those responses is often poorly understood. Here, we study mechanisms underlying the suppression of ß-1,3-glucan-triggered plant immunity by the blast fungus Magnaporthe oryzae. We show that an exo-ß-1,3-glucanase of the GH17 family, named Ebg1, is important for fungal cell wall integrity and virulence of M. oryzae. Ebg1 can hydrolyze ß-1,3-glucan and laminarin into glucose, thus suppressing ß-1,3-glucan-triggered plant immunity. However, in addition, Ebg1 seems to act as a PAMP, independent of its hydrolase activity. This Ebg1-induced immunity appears to be dampened by the secretion of an elongation factor 1 alpha protein (EF1α), which interacts and co-localizes with Ebg1 in the apoplast. Future work is needed to understand the mechanisms behind Ebg1-induced immunity and its suppression by EF1α.

An ERAD-related ubiquitin-conjugating enzyme boosts broad-spectrum disease resistance and yield in rice.

Rice is a staple crop for over half of the global population. However, blast disease caused by Magnaporthe orzae can result in more than a 30% loss in rice yield in epidemic years. Although some major resistance genes bolstering blast resistance have been identified in rice, their stacking in elite cultivars usually leads to yield penalties. Here we report that OsUBC45, a ubiquitin-conjugating enzyme functioning in the endoplasmic reticulum-associated protein degradation system, promotes broad-spectrum disease resistance and yield in rice. OsUBC45 is induced upon infection by M. oryzae, and its overexpression enhances resistance to blast disease and bacterial leaf blight by elevating pathogen-associated molecular pattern-triggered immunity (PTI) while nullifying the gene-attenuated PTI. The OsUBC45 overexpression also increases grain yield by over 10%. Further, OsUBC45 enhances the degradation of glycogen synthase kinase 3 OsGSK3 and aquaporin OsPIP2;1, which negatively regulate the grain size and PTI, respectively. The OsUBC45 reported in our study has the potential for improving yield and disease resistance for sustainable rice production.

Structural mechanism of heavy metal-associated integrated domain engineering of paired nucleotide-binding and leucine-rich repeat proteins in rice.

Plant nucleotide-binding and leucine-rich repeat (NLR) proteins are immune sensors that detect pathogen effectors and initiate a strong immune response. In many cases, single NLR proteins are sufficient for both effector recognition and signaling activation. These proteins possess a conserved architecture, including a C-terminal leucine-rich repeat (LRR) domain, a central nucleotide-binding (NB) domain, and a variable N-terminal domain. Nevertheless, many paired NLRs linked in a head-to-head configuration have now been identified. The ones carrying integrated domains (IDs) can recognize pathogen effector proteins by various modes; these are known as sensor NLR (sNLR) proteins. Structural and biochemical studies have provided insights into the molecular basis of heavy metal-associated IDs (HMA IDs) from paired NLRs in rice and revealed the co-evolution between pathogens and hosts by combining naturally occurring favorable interactions across diverse interfaces. Focusing on structural and molecular models, here we highlight advances in structure-guided engineering to expand and enhance the response profile of paired NLR-HMA IDs in rice to variants of the rice blast pathogen MAX-effectors (Magnaporthe oryzae AVRs and ToxB-like). These results demonstrate that the HMA IDs-based design of rice materials with broad and enhanced resistance profiles possesses great application potential but also face considerable challenges.

CRISPR/Cas9-mediated deletion of large chromosomal segments identifies a minichromosome modulating the Colletotrichum graminicola virulence on maize.

Colletotrichum graminicola causes anthracnose on maize, an economically significant disease worldwide. To decipher how the pathogen controls its virulence/pathogenicity on maize at the minichromosomal level, we sequenced the genome and transcriptome of the C. graminicola strain T1-3-3. The 61.91 Mb genome contains three transcriptionally repressed, full-length strain-specific minichromosomes (<1 Mb; Chr11 through Chr13). A CRISPR/Cas9-based system was developed to knock out large chromosomal segments; it involved the generation of multiple simultaneous DNA double-strand breaks across a targeted genomic region, followed by homology-directed replacement thereof with a donor DNA template carrying the selectable marker hygromycin phosphotransferase gene flanked by homologous sequence arms of the targeted region. Using this system, we obtained distinct mutants functionally nullisomic for individual minichromosomes. Only the ΔChr12 mutant lacking the 498.44 Kb genomic region carrying all of the 31 genes of Chr12 exhibited attenuated virulence on maize and was indistinguishable from T1-3-3 in fungal growth and conidiation, indicating that Chr12 is a conditionally dispensable minichromosome and imparts full virulence to C. graminicola on maize. The CRISPR/Cas9-mediated genome editing system developed in this study will enable the determination of the biological functions of minichromosomes or large chromosomal segments in fungal plant pathogens.

Identification and Characterization of Novel Candidate Effector Proteins from Magnaporthe oryzae.

The fungal pathogen Magnaporthe oryzae secretes a large number of effector proteins to facilitate infection, most of which are not functionally characterized. We selected potential candidate effector genes from the genome of M. oryzae, field isolate P131, and cloned 69 putative effector genes for functional screening. Utilizing a rice protoplast transient expression system, we identified that four candidate effector genes, GAS1, BAS2, MoCEP1 and MoCEP2 induced cell death in rice. In particular, MoCEP2 also induced cell death in Nicotiana benthamiana leaves through Agrobacteria-mediated transient gene expression. We further identified that six candidate effector genes, MoCEP3 to MoCEP8, suppress flg22-induced ROS burst in N. benthamiana leaves upon transient expression. These effector genes were highly expressed at a different stage after M. oryzae infection. We successfully knocked out five genes in M. oryzae, MoCEP1, MoCEP2, MoCEP3, MoCEP5 and MoCEP7. The virulence tests suggested that the deletion mutants of MoCEP2, MoCEP3 and MoCEP5 showed reduced virulence on rice and barley plants. Therefore, those genes play an important role in pathogenicity.

An Agrobacterium-Mediated Transient Expression Method for Functional Assay of Genes Promoting Disease in Monocots.

Agrobacterium-mediated transient expression (AMTE) has been widely used for high-throughput assays of gene function in diverse plant species. However, its application in monocots is still limited due to low expression efficiency. Here, by using histochemical staining and a quantitative fluorescence assay of ß-glucuronidase (GUS) gene expression, we investigated factors affecting the efficiency of AMTE on intact barley plants. We found prominent variation in GUS expression levels across diverse vectors commonly used for stable transformation and that the vector pCBEP produced the highest expression. Additionally, concurrent treatments of plants with one day of high humidity and two days of darkness following agro-infiltration also significantly increased GUS expression efficiency. We thus established an optimized method for efficient AMTE on barley and further demonstrated its efficiency on wheat and rice plants. We showed that this approach could produce enough proteins suitable for split-luciferase assays of protein-protein interactions on barley leaves. Moreover, we incorporated the AMTE protocol into the functional dissection of a complex biological process such as plant disease. Based on our previous research, we used the pCBEP vector to construct a full-length cDNA library of genes upregulated during the early stage of rice blast disease. A subsequent screen of the library by AMTE identified 15 candidate genes (out of ~2000 clones) promoting blast disease on barley plants. Four identified genes encode chloroplast-related proteins: OsNYC3, OsNUDX21, OsMRS2-9, and OsAk2. These genes were induced during rice blast disease; however, constitutive overexpression of these genes conferred enhanced disease susceptibility to Colletotrichum higginsianum in Arabidopsis. These observations highlight the power of the optimized AMTE approach on monocots as an effective tool for facilitating functional assays of genes mediating complex processes such as plant-microbe interactions.

Structure-Aided Identification of an Inhibitor Targets Mps1 for the Management of Plant-Pathogenic Fungi.

Blast disease caused by Magnaporthe oryzae threatens rice production worldwide, and chemical control is one of the main methods of its management. The high mutation rate of the M. oryzae genome results in drug resistance, which calls for novel fungicide targets. Fungal proteins that function during the infection process might be potential candidates, and Mps1 (M. oryzae mitogen-activated protein kinase 1) is such a protein that plays a critical role in appressorium penetration of the plant cell wall. Here, we report the structure-aided identification of a small-molecule inhibitor of Mps1. High-throughput screening was performed with Mps1 against a DNA-encoded compound library, and one compound, named A378-0, with the best performance was selected for further verification. A378-0 exhibits a higher binding affinity than the kinase cosubstrate ATP and can inhibit the enzyme activity of Mps1. Cocrystallization of A378-0 with Mps1 revealed that A378-0 binds to the catalytic pocket of Mps1, while the three ring-type substructures of A378-0 constitute a triangle that squeezes into the pocket. In planta assays showed that A378-0 could inhibit both the appressorium penetration and invasive growth but not the appressorium development of M. oryzae, which is consistent with the biological function of Mps1. Furthermore, A378-0 exhibits binding and activity inhibition abilities against Mpk1, the Mps1 ortholog of the soilborne fungal pathogen Fusarium oxysporum. Collectively, these results show that Mps1 as well as its orthologs can be regarded as fungicide targets, and A378-0 might be used as a hit compound for the development of a broad-spectrum fungicide. IMPORTANCE M. oryzae is the causal agent of rice blast, one of the most devastating diseases of cultivated rice. Chemical control is still the main strategy for its management, and the identification of novel fungicide targets is indispensable for overcoming existing problems such as drug resistance and food safety. With a combination of structural, biochemical, and in planta assays, our research shows that Mps1 may serve as a fungicide target and confirms that compound A378-0 binds to Mps1 and possesses bioactivity in inhibiting M. oryzae virulence. As fungal orthologs of Mps1 are conserved, A378-0 may serve as a hit for broad-spectrum fungicide development, as evidenced with Mpk1, the Mps1 ortholog of F. oxysporum. Additionally, A378-0 contains a novel chemical scaffold that has not been reported in approved kinase inhibitors, suggesting its potential to be considered the basis for the development of other kinase inhibitors.

Magnaporthe oryzae endoplasmic reticulum membrane complex regulates the biogenesis of membrane proteins for pathogenicity.

In eukaryotes, the majority of newly synthesized integral membrane proteins are inserted into the endoplasmic reticulum (ER) membrane before transferred to their functional sites. The conserved ER membrane complex (EMC) takes part in the insertion process for tail-anchored membrane proteins. However, the function of EMC in phytopathogenic fungi has not been characterized. Here, we report the identification and functional characterization of two EMC subunits MoEmc5 and MoEmc2 in Magnaporthe oryzae. The knockout mutants ΔMoemc5 and ΔMoemc2 exhibit substantial defect in autophagy, pathogenicity, cell wall integrity, and magnesium ion sensitivity. We demonstrate that the autophagy process was severely impaired in the ΔMoemc5 and ΔMoemc2 mutants because of the low-protein steady-state level of Atg9, the sole membrane-associated autophagy protein. Furthermore, the protein level of membrane proteins Chs4, Fks1, and MoMnr2 is also significantly reduced in the ΔMoemc5 and ΔMoemc2 strains, leading to their supersensitivity to Calcofluor white, Congo red, and magnesium. In addition, MoEmc5, but not MoEmc2, acts as a magnesium transporter independent of its EMC function. Magnaporthe oryzae EMC regulates the biogenesis of membrane proteins for autophagy and virulence; therefore, EMC subunits could be potential targets for fungicide design in the future.

The Hidden Truths of Fungal Virulence and Adaptation on Hosts: Unraveling the Conditional Dispensability of Minichromosomes in the Hemibiotrophic Colletotrichum Pathogens.

Colletotrichum spp. are ascomycete fungi and cause anthracnose disease in numerous crops of economic significance. The genomes of these fungi are distributed among ten core chromosomes and two to three minichromosomes. While the core chromosomes regulate fungal growth, development and virulence, the extent to which the minichromosomes are involved in these processes is still uncertain. Here, we discuss the minichromosomes of three hemibiotrophic Colletotrichum pathogens, i.e., C. graminicola, C. higginsianum and C. lentis. These minichromosomes are typically less than one megabase in length, characterized by containing higher repetitive DNA elements, lower GC content, higher frequency of repeat-induced point mutations (RIPMs) and sparse gene distribution. Molecular genetics and functional analyses have revealed that these pathogens harbor one conditionally dispensable minichromosome, which is dispensable for fungal growth and development but indispensable for fungal virulence on hosts. They appear to be strain-specific innovations and are highly compartmentalized into AT-rich and GC-rich blocks, resulting from RIPMs, which may help protect the conditionally dispensable minichromosomes from erosion of already scarce genes, thereby helping the Colletotrichum pathogens maintain adaptability on hosts. Overall, understanding the mechanisms underlying the conditional dispensability of these minichromosomes could lead to new strategies for controlling anthracnose disease in crops.

The rice blast fungus SR protein 1 regulates alternative splicing with unique mechanisms.

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.