MoRgs3 functions in intracellular reactive oxygen species perception-integrated cAMP signaling to promote appressorium formation in Magnaporthe oryzae.

Regulator of G-protein signaling (RGS) proteins exhibit GTPase-accelerating protein activities to govern G-protein function. In the rice blast fungus Magnaporthe oryzae, there is a family of at least eight RGS and RGS-like proteins (MoRgs1 to MoRgs8), each exhibiting distinct or shared functions in the growth, appressorium formation, and pathogenicity. MoRgs3 recently emerged as one of the crucial regulators that senses intracellular oxidation during appressorium formation. To explore this unique regulatory mechanism of MoRgs3, we identified the nucleoside diphosphate kinase MoNdk1 that interacts with MoRgs3. MoNdk1 phosphorylates MoRgs3 under induced intracellular reactive oxygen species levels, and MoRgs3 phosphorylation is required for appressorium formation and pathogenicity. In addition, we showed that MoRgs3 phosphorylation determines its interaction with MoCrn1, a coronin-like actin-binding protein homolog, which regulates MoRgs3 internalization. Finally, we provided evidence demonstrating that MoRgs3 functions in MoMagA-mediated cAMP signaling to regulate normal appressorium induction. By revealing a novel signal perception mechanism, our studies highlighted the complexity of regulation during the appressorium function and pathogenicity of the blast fungus. IMPORTANCE: We report that MoRgs3 becomes phosphorylated in an oxidative intracellular environment during the appressorium formation stage. We found that this phosphorylation is carried out by MoNdk1, a nucleoside diphosphate kinase. In addition, this phosphorylation leads to a higher binding affinity between MoRgs3 and MoCrn1, a coronin-like actin-binding protein that was implicated in the endocytic transport of several other RGS proteins of Magnaporthe oryzae. We further found that the internalization of MoRgs3 is indispensable for its GTPase-activating protein function toward the Gα subunit MoMagA. Importantly, we characterized how such cellular regulatory events coincide with cAMP signaling-regulated appressorium formation and pathogenicity in the blast fungus. Our studies uncovered a novel intracellular reactive oxygen species signal-transducing mechanism in a model pathogenic fungus with important basic and applied implications.

Csn5 inhibits autophagy by regulating the ubiquitination of Atg6 and Tor to mediate the pathogenicity of Magnaporthe oryzae.

Csn5 is subunit 5 of the COP9 signalosome (CSN), but the mechanism by which it strictly controls the pathogenicity of pathogenic fungi through autophagy remains unclear. Here, we found that Csn5 deficiency attenuated pathogenicity and enhanced autophagy in Magnaporthe oryzae. MoCSN5 knockout led to overubiquitination and overdegradation of MoTor (the core protein of the TORC1 complex [target of rapamycin]) thereby promoted autophagy. In addition, we identified MoCsn5 as a new interactor of MoAtg6. Atg6 was found to be ubiquitinated through linkage with lysine 48 (K48) in cells, which is necessary for infection-associated autophagy in pathogenic fungi. K48-ubiquitination of Atg6 enhanced its degradation and thereby inhibited autophagic activity. Our experimental results indicated that MoCsn5 promoted K48-ubiquitination of MoAtg6, which reduced the MoAtg6 protein content and thus inhibited autophagy. Aberrant ubiquitination and autophagy in ΔMocsn5 led to pleiotropic defects in the growth, development, stress resistance, and pathogenicity of M. oryzae. In summary, our study revealed a novel mechanism by which Csn5 regulates autophagy and pathogenicity in rice blast fungus through ubiquitination.

Phosphorylation of Mad1 at serine 18 by Mps1 is required for the full virulence of rice blast fungus, Magnaporthe oryzae.

The spindle assembly checkpoint (SAC) proteins are conserved among eukaryotes safeguarding chromosome segregation fidelity during mitosis. However, their biological functions in plant-pathogenic fungi remain largely unknown. In this study, we found that the SAC protein MoMad1 in rice blast fungus (Magnaporthe oryzae) localizes on the nuclear envelope and is dispensable for M. oryzae vegetative growth and tolerance to microtubule depolymerizing agent treatment. MoMad1 plays an important role in M. oryzae infection-related development and pathogenicity. The monopolar spindle 1 homologue in M. oryzae (MoMps1) interacts with MoMad1 through its N-terminal domain and phosphorylates MoMad1 at Ser-18, which is conserved within the extended N termini of Mad1s from fungal plant pathogens. This phosphorylation is required for maintaining MoMad1 protein abundance and M. oryzae full virulence. Similar to the deletion of MoMad1, treatment with Mps1-IN-1 (an Mps1 inhibitor) caused compromised appressorium formation and decreased M. oryzae virulence, and these defects were dependent on its attenuating MoMad1 Ser-18 phosphorylation. Therefore, our study indicates the function of Mad1 in rice blast fungal pathogenicity and sheds light on the potential of blocking Mad1 phosphorylation by Mps1 to control crop fungal diseases.

The link between changing in host carbon allocation and resistance to Magnaporthe oryzae: a possible tactic for mitigating the rice blast fungus.

One of the most destructive diseases affecting rice is rice blast, which is brought on by the rice blast fungus Magnaporthe oryzae. The preventive measures, however, are not well established. To effectively reduce the negative effects of rice blasts on crop yields, it is imperative to comprehend the dynamic interactions between pathogen resistance and patterns of host carbon allocation. This review explores the relationship between variations in carbon allocation and rice plants' ability to withstand the damaging effects of M. oryzae. The review highlights potential strategies for altering host carbon allocation including transgenic, selective breeding, crop rotation, and nutrient management practices as a promising avenue for enhancing rice blast resistance. This study advances our knowledge of the interaction between plants' carbon allocation and M. oryzae resistance and provides stakeholders and farmers with practical guidance on mitigating the adverse effects of the rice blast globally. This information may be used in the future to create varieties that are resistant to M. oryzae.

Identification of Ossnrk1a-1 Regulated Genes Associated with Rice Immunity and Seed Set.

Sucrose non-fermenting-1-related protein kinase-1 (SnRK1) is a highly conserved serine-threonine kinase complex regulating plants' energy metabolisms and resistance to various types of stresses. However, the downstream genes regulated by SnRK1 in these plant physiological processes still need to be explored. In this study, we found that the knockout of OsSnRK1a resulted in no obvious defects in rice growth but notably decreased the seed setting rate. The ossnrk1a mutants were more sensitive to blast fungus (Magnaporthe oryzae) infection and showed compromised immune responses. Transcriptome analyses revealed that SnRK1a was an important intermediate in the energy metabolism and response to biotic stress. Further investigation confirmed that the transcription levels of OsNADH-GOGAT2, which positively controls rice yield, and the defense-related gene pathogenesis-related protein 1b (OsPR1b) were remarkably decreased in the ossnrk1a mutant. Moreover, we found that OsSnRK1a directly interacted with the regulatory subunits OsSnRK1ß1 and OsSnRK1ß3, which responded specifically to blast fungus infection and starvation stresses, respectively. Taken together, our findings provide an insight into the mechanism of OsSnRK1a, which forms a complex with specific ß subunits, contributing to rice seed set and resistance by regulating the transcription of related genes.

Enhanced ultraviolet-B radiation alleviates structural damages on rice leaf caused by Magnaporthe oryzae infection.

Enhanced ultraviolet-B (UV-B) radiation can change the interaction between crops and pathogens. The effects of single and compound stresses of enhanced UV-B radiation (5.0 kJ·m-2) and Magnaporthe oryzae on the morphology, anatomy, and ultrastructure of rice leaves were investigated. M. oryzae infection decreased the leaf area and thickness, reduced the stomatal area and density, and caused damages to the leaf ultrastructure, such as cytoplasm-cell wall separation, atrophy and sinking of fan-shaped bulliform cells, and chloroplast deformation. The enhanced UV-B radiation supplied before or during M. oryzae infection remarkably decreased the mycelia number of M. oryzae in leaf epidermis, increased the leaf area, leaf thickness, stomatal density, and mastoid number; and alleviated the ultrastructural damages induced by M. oryzae to keep an integral chloroplast. While the UV-B radiation was supplied after M. oryzae infection, its alleviation effects on the damages induced by M. oryzae infection on the morphology and structure of rice leaf were attenuated. Thus, the alleviation of enhanced UV-B radiation on damages induced by M. oryzae infection on rice leaves was related to its application period. The enhanced UV-B radiation supplied before or during M. oryzae infection allowed the rice leaf to resist M. oryzae infection.

Knock out of amino acid transporter gene OsLHT1 accelerates leaf senescence and enhances resistance to rice blast fungus.

Plant amino acid transporters regulate not only long-distance transport and reallocation of nitrogen (N) from source to sink organs, but also the amount of amino acids in leaves hijacked by invading pathogens. However, the function of amino acid transporters in plant defense responses to pathogen infection remains unknown. In this study, we found that the rice amino acid transporter gene OsLHT1 was expressed in leaves and up-regulated by maturation, N starvation, and inoculation of the blast fungus Magnaporthe oryzae. Knock out of OsLHT1 resulted in development stage- and N supply-dependent premature senescence of leaves at the vegetative growth stage. In comparison with the wild type, Oslht1 mutant lines showed sustained rusty red spots on fully mature leaf blades irrespective of N supply levels. Notably, no relationship between the severity of leaf rusty red spots and concentration of total N or amino acids was found in Oslht1 mutants at different developmental stages. Disruption of OsLHT1 altered transport and metabolism of amino acids and biosynthesis of flavones and flavonoids, enhanced expression of jasmonic acid- and salicylic acid-related defense genes, production of jasmonic acid and salicylic acid, and accumulation of reactive oxygen species. OsLHT1 inactivation dramatically prevented the leaf invasion by M. oryzae, a hemi-biotrophic ascomycete fungus. Overall, these results establish a link connecting the activity of an amino acid transporter with leaf metabolism and defense against rice blast fungus.

SH3P2, an SH3 domain-containing protein that interacts with both Pib and AvrPib, suppresses effector-triggered, Pib-mediated immunity in rice.

Plants usually keep resistance (R) proteins in a static state under normal conditions to avoid autoimmunity and save energy for growth, but R proteins can be rapidly activated upon perceiving pathogen invasion. Pib, the first cloned blast disease R gene in rice, encoding a nucleotide-binding leucine-rich repeat (NLR) protein, mediates resistance to the blast fungal (Magnaporthe oryzae) isolates carrying the avirulence gene AvrPib. However, the molecular mechanisms about how Pib recognizes AvrPib and how it is inactivated and activated remain largely unclear. In this study, through map-based cloning and CRISPR-Cas9 gene editing, we proved that Pib contributes to the blast disease resistance of rice cultivar Yunyin (YY). Furthermore, an SH3 domain-containing protein, SH3P2, was found to associate with Pib mainly at clathrin-coated vesicles in rice cells, via direct binding with the coiled-coil (CC) domain of Pib. Interestingly, overexpression of SH3P2 in YY compromised Pib-mediated resistance to M. oryzae isolates carrying AvrPib and Pib-AvrPib recognition-induced cell death. SH3P2 competitively inhibits the self-association of the Pib CC domain in vitro, suggesting that binding of SH3P2 with Pib undermines its homodimerization. Moreover, SH3P2 can also interact with AvrPib and displays higher affinity to AvrPib than to Pib, which leads to dissociation of SH3P2 from Pib in the presence of AvrPib. Taken together, our results suggest that SH3P2 functions as a "protector" to keep Pib in a static state by direct interaction during normal growth but could be triggered off by the invasion of AvrPib-carrying M. oryzae isolates. Our study reveals a new mechanism about how an NLR protein is inactivated under normal conditions but is activated upon pathogen infection.

A Subunit of the COP9 Signalosome, MoCsn6, Is Involved in Fungal Development, Pathogenicity, and Autophagy in Rice Blast Fungus.

The COP9 signalosome (CSN) is a highly conserved protein complex in eukaryotes, affecting various development and signaling processes. To date, the biological functions of the COP9 signalosome and its subunits have not been determined in Magnaporthe oryzae. In this study, we characterized the CSN in M. oryzae (which we named MoCsn6) and analyzed its biological functions. MoCsn6 is involved in fungal development, autophagy, and plant pathogenicity. Compared with the wild-type strain 70-15, ΔMocsn6 mutants showed a significantly reduced growth rate, sporulation rate, and germ tube germination rate. Pathogenicity assays showed that the ΔMocsn6 mutants did not cause or significantly reduced the number of disease spots on isolated barley leaves. After the MoCSN6 gene was complemented into the ΔMocsn6 mutant, vegetative growth, sporulation, and pathogenicity were restored. The Osm1 and Pmk1 phosphorylation pathways were also disrupted in the ΔMocsn6 mutants. Furthermore, we found that MoCsn6 participates in the autophagy pathway by interacting with the autophagy core protein MoAtg6 and regulating its ubiquitination level. Deletion of MoCSN6 resulted in rapid lipidation of MoAtg8 and degradation of the autophagic marker protein green fluorescent protein-tagged MoAtg8 under nutrient and starvation conditions, suggesting that MoCsn6 negatively regulates autophagic activity. Taken together, our results demonstrate that MoCsn6 plays a crucial role in regulating fungal development, pathogenicity, and autophagy in M. oryzae. IMPORTANCE Magnaporthe oryzae, a filamentous fungus, is the cause of many cereal diseases. Autophagy is involved in fungal development and pathogenicity. The COP9 signalosome (CSN) has been extensively studied in ubiquitin pathways, but its regulation of autophagy has rarely been reported in plant-pathogenic fungi. Investigations on the relationship between CSN and autophagy will deepen our understanding of the pathogenic mechanism of M. oryzae and provide new insights into the development of new drug targets to control fungal diseases. In this study, the important function of Csn6 in the autophagy regulation pathway and its impact on the pathogenicity of M. oryzae were determined. We showed that Csn6 manages autophagy by interacting with the autophagy core protein Atg6 and regulating its ubiquitination level. Furthermore, future investigations that explore the function of CSN will deepen our understanding of autophagy mechanisms in rice blast fungus.

The Plant Homeodomain Protein Clp1 Regulates Fungal Development, Virulence, and Autophagy Homeostasis in Magnaporthe oryzae.

Rice blast disease caused by Magnaporthe oryzae is a serious threat to global grain yield and food security. Cti6 is a nuclear protein containing a plant homeodomain (PHD) that is involved in transcriptional regulation in Saccharomyces cerevisiae. The biological function of its homologous protein in M. oryzae has been elusive. Here, we report Clp1 with a PHD domain in M. oryzae, a homologous protein of the yeast Cti6. Clp1 was mainly located in the nucleus and partly in the vesicles. Clp1 colocalized and interacted with the autophagy-related proteins Atg5, Atg7, Atg16, Atg24, and Atg28 at preautophagosomal structures (PAS) and autophagosomes, and the loss of Clp1 increased the fungal background autophagy level. Δclp1 displayed reduced hyphal growth and hyperbranching, abnormal fungal morphology (including colony, spore, and appressorium), hindered appressorial glycogen metabolism and turgor production, weakened plant infection, and decreased virulence. The PHD is indispensable for the function of Clp1. Therefore, this study revealed that Clp1 regulates development and pathogenicity by maintaining autophagy homeostasis and affecting gene transcription in M. oryzae. IMPORTANCE The fungal pathogen Magnaporthe oryzae causes serious diseases of grasses such as rice and wheat. Autophagy plays an indispensable role in the pathogenic process of M. oryzae. Here, we report a Cti6-like protein, Clp1, that is involved in fungal development and infection of plants through controlling autophagy homeostasis in the cytoplasm and gene transcription in the nucleus in M. oryzae. This study will help us to understand an elaborated molecular mechanism of autophagy, gene transcription, and virulence in the rice blast fungus.

Homeostasis of cell wall integrity pathway phosphorylation is required for the growth and pathogenicity of Magnaporthe oryzae.

The cell wall provides a crucial barrier to stress imposed by the external environment. In the rice blast fungus Magnaporthe oryzae, this stress response is mediated by the cell wall integrity (CWI) pathway, consisting of a well-characterized protein phosphorylation cascade. However, other regulators that maintain CWI phosphorylation homeostasis, such as protein phosphatases (PPases), remain unclear. Here, we identified two PPases, MoPtc1 and MoPtc2, that function as negative regulators of the CWI pathway. MoPtc1 and MoPtc2 interact with MoMkk1, one of the key components of the CWI pathway, and are crucial for the vegetative growth, conidial formation, and virulence of M. oryzae. We also demonstrate that both MoPtc1 and MoPtc2 dephosphorylate MoMkk1 in vivo and in vitro, and that CWI stress leads to enhanced interaction between MoPtc1 and MoMkk1. CWI stress abolishes the interaction between MoPtc2 and MoMkk1, providing a means of deactivation for CWI signalling. Our studies reveal that CWI signalling in M. oryzae is a highly coordinated regulatory mechanism vital for stress response and pathogenicity.

Identification of Scytalone Dehydratase Inhibitors Effective against Melanin Biosynthesis Dehydratase Inhibitor-Resistant Pyricularia oryzae.

Melanin is a secondary metabolite required for the infection of the rice blast fungus Pyricularia oryzae. Melanin biosynthesis enzymes are targets for controlling rice blast disease, and three types of commercial melanin biosynthesis inhibitors (MBIs) including MBI-R, MBI-D, and MBI-P have been developed. However, the occurrence of MBI-D-resistant strains containing scytalone dehydratase (SDH1/RSY1) with V75M mutations has been recently reported. In this study, we aimed to identify inhibitors of SDH1-V75M. We screened the RIKEN Natural Products Depository chemical library using chemical array technology and evaluated the inhibition of SDH1-V75M by candidate compounds. NPD13731 strongly inhibited the activity of wild-type and mutant SDH1. The structure-activity relationship data were used to create a more potent inhibitor 16, which controlled rice blast disease in rice plants infected with MBI-D-resistant P. oryzae. Compound 16, which we named melabiostin, may be used to develop fungicides for controlling rice blast infections.

Gene deletion and constitutive expression of the pectate lyase gene 1 (MoPL1) lead to diminished virulence of Magnaporthe oryzae.

Phytopathogenic fungi are known to secrete specific proteins which act as virulence factors and promote host colonization. Some of them are enzymes with plant cell wall degradation capability, like pectate lyases (Pls). In this work, we examined the involvement of Pls in the infection process of Magnaporthe oryzae, the causal agent of rice blast disease. From three Plgenes annotated in the M. oryzae genome, only transcripts of MoPL1 considerably accumulated during the infection process with a peak at 72 h post inoculation. Both, gene deletion and a constitutive expression of MoPL1 in M. oryzae led to a significant reduction in virulence. By contrast, mutants that constitutively expressed an enzymatic inactive version of MoPl1 did not differ in virulence compared to the wild type isolate. This indicates that the enzymatic activity of MoPl1 is responsible for diminished virulence, which is presumably due to degradation products recognized as danger associated molecular patterns (DAMPs), which strengthen the plant immune response. Microscopic analysis of infection sites pointed to an increased plant defense response. Additionally, MoPl1 tagged with mRFP, and not the enzymatic inactive version, focally accumulated in attacked plant cells beneath appressoria and at sites where fungal hyphae transverse from one to another cell. These findings shed new light on the role of pectate lyases during tissue colonization in the necrotrophic stage of M. oryzae's life cycle.

Type 2C Protein Phosphatases MoPtc5 and MoPtc7 Are Crucial for Multiple Stress Tolerance, Conidiogenesis and Pathogenesis of Magnaporthe oryzae.

Protein kinases and phosphatases catalyze the phosphorylation and dephosphorylation of their protein substrates, respectively, and these are important mechanisms in cellular signal transduction. The rice blast fungus Magnaporthe oryzae possesses 6 protein phosphatases of type 2C class, including MoPtc1, 2, 5, 6, 7 and 8. However, only very little is known about the roles of these phosphatases in filamentous fungi. Here in, we deployed genetics and molecular biology techniques to identify, characterize and establish the roles of MoPtc5 and MoPtc7 in M. oryzae development and pathogenicity. We found that during pathogen-host interaction, MoPTC7 is differentially expressed. Double deletion of MoPTC7 and MoPTC5 suppressed the fungal vegetative growth, altered its cell wall integrity and reduced its virulence. The two genes were found indispensable for stress tolerance in the phytopathogen. We also demonstrated that disruption of any of the two genes highly affected appressorium turgor generation and Mps1 and Osm1 phosphorylation levels. Lastly, we demonstrated that both MoPtc5 and MoPtc7 are localized to mitochondria of different cellular compartments in the blast fungus. Taken together, our study revealed synergistic coordination of M. oryzae development and pathogenesis by the type 2C protein phosphatases.

MoSnf5 Regulates Fungal Virulence, Growth, and Conidiation in Magnaporthe oryzae.

Snf5 (sucrose nonfermenting) is a core component of the SWI/SNF complexes and regulates diverse cellular processes in model eukaryotes. In plant pathogenic fungi, its biological function and underlying mechanisms remain unexplored. In this study, we investigated the biological roles of MoSnf5 in plant infection and fungal development in the rice blast pathogen Magnaporthe oryzae. The gene deletion mutants of MoSNF5 exhibited slower vegetative hyphal growth, severe defects in conidiogenesis, and impaired virulence and galactose utilization capacities. Domain dissection assays showed that the Snf5 domain and the N- and C-termini of MoSnf5 were all required for its full functions. Co-immunoprecipitation and yeast two-hybrid assays showed that MoSnf5 physically interacts with four proteins, including a transcription initiation factor MoTaf14. Interestingly, the ∆MoTaf14 mutants showed similar phenotypes as the ∆Mosnf5 mutants on fungal virulence and development. Moreover, assays on GFP-MoAtg8 expression and localization showed that both the ∆Mosnf5 and ∆MoTaf14 mutants were defective in autophagy. Taken together, MoSnf5 regulates fungal virulence, growth, and conidiation, possibly through regulating galactose utilization and autophagy in M. oryzae.

Characterization of the MYB Genes Reveals Insights Into Their Evolutionary Conservation, Structural Diversity, and Functional Roles in Magnaporthe oryzae.

The myeloblastosis (MYB) transcription factor family is evolutionarily conserved among plants, animals, and fungi, and contributes to their growth and development. We identified and analyzed 10 putative MYB genes in Magnaporthe oryzae (MoMYB) and determined their phylogenetic relationships, revealing high divergence and variability. Although MYB domains are generally defined by three tandem repeats, MoMYBs contain one or two weakly conserved repeats embedded in extensive disordered regions. We characterized the secondary domain organization, disordered segments, and functional contributions of each MoMYB. During infection, MoMYBs are distinctively expressed and can be subdivided into two clades of being either up- or down-regulated. Among these, MoMYB1 and MoMYB8 are up-regulated during infection and vegetative growth, respectively. We found MoMYB1 localized predominantly to the cytosol during the formation of infection structures. ΔMomyb1 exhibited reduced virulence on intact rice leaves corresponding to the diminished ability to form hypha-driven appressorium (HDA). We discovered that MoMYB1 regulates HDA formation on hard, hydrophobic surfaces, whereas host surfaces partially restored HDA formation in ΔMomyb1. Lipid droplet accumulation in hyphal tips and expression of HDA-associated genes were strongly perturbed in ΔMomyb1 indicating genetic interaction of MoMYB1 with downstream components critical to HDA formation. We also found that MoMYB8 is necessary for fungal growth, dark-induced melanization of hyphae, and involved in higher abiotic stress tolerance. Taken together, we revealed a multifaceted picture of the MoMYB family, wherein a low degree of conservation has led to the development of distinct structures and functions, ranging from fungal growth to virulence.

Transcriptional Landscapes of Long Non-coding RNAs and Alternative Splicing in Pyricularia oryzae Revealed by RNA-Seq.

Pyricularia oryzae causes the rice blast, which is one of the most devastating crop diseases worldwide, and is a model fungal pathogen widely used for dissecting the molecular mechanisms underlying fungal virulence/pathogenicity. Although the whole genome sequence of P. oryzae is publicly available, its current transcriptomes remain incomplete, lacking the information on non-protein coding genes and alternative splicing. Here, we performed and analyzed RNA-Seq of conidia and hyphae, resulting in the identification of 3,374 novel genes. Interestingly, the vast majority of these novel genes likely transcribed long non-coding RNAs (lncRNAs), and most of them were localized in the intergenic regions. Notably, their expressions were concomitant with the transcription of neighboring genes thereof in conidia and hyphae. In addition, 2,358 genes were found to undergo alternative splicing events. Furthermore, we exemplified that a lncRNA was important for hyphal growth likely by regulating the neighboring protein-coding gene and that alternative splicing of the transcription factor gene CON7 was required for appressorium formation. In summary, results from this study indicate that lncRNA transcripts and alternative splicing events are two important mechanisms for regulating the expression of genes important for conidiation, hyphal growth, and pathogenesis, and provide new insights into transcriptomes and gene regulation in the rice blast fungus.

Predicting Protein-Protein Interactions Between Rice and Blast Fungus Using Structure-Based Approaches.

Rice blast, caused by the fungus Magnaporthe oryzae, is the most devastating disease affecting rice production. Identification of protein-protein interactions (PPIs) is a critical step toward understanding the molecular mechanisms underlying resistance to blast fungus in rice. In this study, we presented a computational framework for predicting plant-pathogen PPIs based on structural information. Compared with the sequence-based methods, the structure-based approach showed to be more powerful in discovering new PPIs between plants and pathogens. Using the structure-based method, we generated a global PPI network consisted of 2,018 interacting protein pairs involving 1,344 rice proteins and 418 blast fungus proteins. The network analysis showed that blast resistance genes were enriched in the PPI network. The network-based prediction enabled systematic discovery of new blast resistance genes in rice. The network provided a global map to help accelerate the identification of blast resistance genes and advance our understanding of plant-pathogen interactions.

Biosynthesis and biological function of secondary metabolites of the rice blast fungus Pyricularia oryzae.

Filamentous fungi have many secondary metabolism genes and produce a wide variety of secondary metabolites with complex and unique structures. However, the role of most secondary metabolites remains unclear. Moreover, most fungal secondary metabolism genes are silent or poorly expressed under laboratory conditions and are difficult to utilize. Pyricularia oryzae, the causal pathogen of rice blast disease, is a well-characterized plant pathogenic fungus. P. oryzae also has a large number of secondary metabolism genes and appears to be a suitable organism for analyzing secondary metabolites. However, in case of this fungus, biosynthetic genes for only four groups of secondary metabolites have been well characterized. Among two of the four groups of secondary metabolites, biosynthetic genes were identified by activating secondary metabolism. These secondary metabolites include melanin, a polyketide compound required for rice infection; tenuazonic acid, a well-known mycotoxin produced by various plant pathogenic fungi and biosynthesized by a unique nonribosomal peptide synthetase-polyketide synthase hybrid enzyme; nectriapyrones, antibacterial polyketide compounds produced mainly by symbiotic fungi, including plant pathogens and endophytes, and pyriculols, phytotoxic polyketide compounds. This review mainly focuses on the biosynthesis and biological functions of the four groups of P. oryzae secondary metabolites.

A VASt-domain protein regulates autophagy, membrane tension, and sterol homeostasis in rice blast fungus.

Sterols are a class of lipids critical for fundamental biological processes and membrane dynamics. These molecules are synthesized in the endoplasmic reticulum (ER) and are transported bi-directionally between the ER and plasma membrane (PM). However, the trafficking mechanism of sterols and their relationship with macroautophagy/autophagy are still poorly understood in the rice blast fungus Magnaporthe oryzae. Here, we identified the VAD1 Analog of StAR-related lipid transfer (VASt) domain-containing protein MoVast1 via co-immunoprecipitation in M. oryzae. Loss of MoVAST1 resulted in conidial defects, impaired appressorium development, and reduced pathogenicity. The MoTor (target of rapamycin in M. oryzae) activity is inhibited because MoVast1 deletion leads to high levels of sterol accumulation in the PM. Site-directed mutagenesis showed that the 902 T site is essential for localization and function of MoVast1. Through filipin or Flipper-TR staining, autophagic flux detection, MoAtg8 lipidation, and drug sensitivity assays, we uncovered that MoVast1 acts as a novel autophagy inhibition factor that monitors tension in the PM by regulating the sterol content, which in turn modulates the activity of MoTor. Lipidomics and transcriptomics analyses further confirmed that MoVast1 is an important regulator of lipid metabolism and the autophagy pathway. Our results revealed and characterized a novel sterol transfer protein important for M. oryzae pathogenicity.Abbreviations: AmB: amphotericin B; ATMT: Agrobacterium tumefaciens-mediated transformation; CM: complete medium; dpi: days post-inoculation; ER: endoplasmic reticulum; Flipper-TR: fluorescent lipid tension reporter; GO: Gene ontology; hpi: hours post-inoculation; IH: invasive hyphae; KEGG: kyoto encyclopedia of genes and genomes; MoTor: target of rapamycin in Magnaporthe oryzae; PalmC: palmitoylcarnitine; PM: plasma membrane; SD-N: synthetic defined medium without amino acids and ammonium sulfate; TOR: target of rapamycin; VASt: VAD1 Analog of StAR-related lipid transfer; YFP, yellow fluorescent protein.