MoAti1 mediates mitophagy by facilitating recruitment of MoAtg8 to promote invasive growth in Magnaporthe oryzae.

Mitophagy is a selective autophagy for the degradation of damaged or excessive mitochondria to maintain intracellular homeostasis. In Magnaporthe oryzae, a filamentous ascomycetous fungus that causes rice blast, the most devastating disease of rice, mitophagy occurs in the invasive hyphae to promote infection. To date, only a few proteins are known to participate in mitophagy and the mechanisms of mitophagy are largely unknown in pathogenic fungi. Here, by a yeast two-hybrid screen with the core autophagy-related protein MoAtg8 as a bait, we obtained a MoAtg8 interactor MoAti1 (MoAtg8-interacting protein 1). Fluorescent observations and protease digestion analyses revealed that MoAti1 is primarily localized to the peripheral mitochondrial outer membrane and is responsible for recruiting MoAtg8 to mitochondria under mitophagy induction conditions. MoAti1 is specifically required for mitophagy, but not for macroautophagy and pexophagy. Infection assays suggested that MoAti1 is required for mitophagy in invasive hyphae during pathogenesis. Notably, no homologues of MoAti1 were found in rice and human protein databases, indicating that MoAti1 may be used as a potential target to control rice blast. By the host-induced gene silencing (HIGS) strategy, transgenic rice plants targeted to silencing MoATI1 showed enhanced resistance against M. oryzae with unchanged agronomic traits. Our results suggest that MoATI1 is required for mitophagy and pathogenicity in M. oryzae and can be used as a target for reducing rice blast.

Draft genome sequence of Xylaria bambusicola isolate GMP-LS, the root and basal stem rot pathogen of sugarcane in Indonesia.

The first draft genome of X. bambusicola GMP-LS, the causal pathogen of the Root, and Basal Stem Rot disease in Sugarcane is presented based on single-molecule real-time PacBio sequencing. Xylaria genome (72.43 Mb) is predicted to encode 13,430 proteins and will contribute to molecular understanding of fungal pathogenesis.

The Ferroptosis landscape of biotic interactions in plants.

Ferroptosis is a cell death pathway that relies on iron- and reactive oxygen species-dependent lethal accumulation of lipid peroxides in the cytosol and/or plasma membrane. Interestingly, Ferroptosis is widely involved in modulating such regulated fatality in the host plant as well as the pathogen albeit with different outcome, dynamics, and interesting metabolic adaptations. Although the basic mechanism of Ferroptosis has been established recently in plants and associated microbes, the conservation, acclimatization, and application of such regulated cell death modality are now beginning to be explored further. Efforts towards this will certainly help better understand the origin, molecular mechanisms, and function of Ferroptosis-associated developmental regulation of biotic interactions in plants.

Plant growth promotion under phosphate deficiency and improved phosphate acquisition by new fungal strain, Penicillium olsonii TLL1.

Microbiomes in soil ecosystems play a significant role in solubilizing insoluble inorganic and organic phosphate sources with low availability and mobility in the soil. They transfer the phosphate ion to plants, thereby promoting plant growth. In this study, we isolated an unidentified fungal strain, POT1 (Penicillium olsonii TLL1) from indoor dust samples, and confirmed its ability to promote root growth, especially under phosphate deficiency, as well as solubilizing activity for insoluble phosphates such as AlPO4, FePO4·4H2O, Ca3(PO4)2, and hydroxyapatite. Indeed, in vermiculite containing low and insoluble phosphate, the shoot fresh weight of Arabidopsis and leafy vegetables increased by 2-fold and 3-fold, respectively, with POT1 inoculation. We also conducted tests on crops in Singapore's local soil, which contains highly insoluble phosphate. We confirmed that with POT1, Bok Choy showed a 2-fold increase in shoot fresh weight, and Rice displayed a 2-fold increase in grain yield. Furthermore, we demonstrated that plant growth promotion and phosphate solubilizing activity of POT1 were more effective than those of four different Penicillium strains such as Penicillium bilaiae, Penicillium chrysogenum, Penicillium janthinellum, and Penicillium simplicissimum under phosphate-limiting conditions. Our findings uncover a new fungal strain, provide a better understanding of symbiotic plant-fungal interactions, and suggest the potential use of POT1 as a biofertilizer to improve phosphate uptake and use efficiency in phosphate-limiting conditions.

Functional analysis of auxin derived from a symbiotic mycobiont.

The biosynthesis of auxin or indole-3-acetic acid by microorganisms has a major impact on plant-microbe interactions. Several beneficial microbiota are known to produce auxin, which largely influences root development and growth in the host plants. Akin to findings in rhizobacteria, recent studies have confirmed the production of auxin by plant growth-promoting fungi too. Here, we show that Penicillium citrinum isolate B9 produces auxin as deduced by liquid chromatography tandem-mass spectrometry analysis. Such fungal auxin is secreted and contributes directly to enhanced root and shoot development and overall plant growth in Arabidopsis thaliana. Furthermore, auxin production by P. citrinum likely involves more than one tryptophan-dependent pathway. Using auxin biosynthesis inhibitor L-Kynurenine, we show that the indole-3-pyruvate pathway might be one of the key biosynthetic routes involved in such auxin production. Confocal microscopy of the DR5rev:GFP Arabidopsis reporter line helped demonstrate that P. citrunum B9-derived auxin is biologically active and is able to significantly enhance auxin signaling in roots during such improved root growth and plant development. Furthermore, the phenotypic growth defects arising from impaired auxin signaling in Arabidopsis taa1 mutant or upon L-Kynurenine treatment of wild-type Arabidopsis seedlings could be significantly alleviated by fungus B9-derived auxin, thus suggesting its positive role in plant growth promotion. Collectively, our results provide clear evidence that the production of auxin is one of the main mechanisms involved in induction of the beneficial plant growth by P. citrinum.

Rab7/Retromer-based endolysosomal trafficking is essential for proper host invasion in rice blast.

Secretion is a fundamental process that plant pathogens utilize to deliver effectors into the host to downregulate immunity and promote infection. Here, we uncover a fascinating membrane trafficking and delivery route that originates from vacuolar membranes in Magnaporthe oryzae and conduits to the host interface and plasma membrane. To perform such secretory/trafficking function, MoRab7 first recruits the retromer complex to the vacuolar membrane, enabling recognition of a family of SNARE proteins, including MoSnc1. Live-cell imaging confirmed a highly dynamic vesicular trafficking of the retromer complex component(s) and MoSnc1 toward and across the host interface or plasma membrane, and subsequent fusion with target membranes. Interestingly, disruption of the MoRab7/Retromer/MoSnc1-based endolysosomal cascade affects effector secretion and fungal pathogenicity. Taken together, we discovered an unconventional protein and membrane trafficking route starting from the fungal endolysosomes to the M. oryzae-rice interaction interface and dissect the role of MoRab7/Retromer/MoSnc1 sorting machinery in effector secretion during biotrophy and invasive growth in rice blast fungus.

Penicillium citrinum Provides Transkingdom Growth Benefits in Choy Sum (Brassica rapa var. parachinensis).

Soil-borne beneficial microbes establish symbioses with plant hosts and play key roles during growth and development therein. In this study, two fungal strains, FLP7 and B9, were isolated from the rhizosphere microbiome associated with Choy Sum (Brassica rapa var. parachinensis) and barley (Hordeum vulgare), respectively. Sequence analyses of the internal transcribed spacer and 18S ribosomal RNA genes combined with colony and conidial morphology identified FLP7 and B9 to be Penicillium citrinum strains/isolates. Plant-fungus interaction assays revealed that isolate B9 showed significant growth promotion effects in Choy Sum plants cultivated in normal soil, as well as under phosphate-limiting conditions. In comparison to the mock control, B9-inoculated plants showed a 34% increase in growth in aerial parts and an 85% upsurge in the fresh weight of roots when cultivated in sterilized soil. The dry biomass of such fungus-inoculated Choy Sum increased by 39% and 74% for the shoots and roots, respectively. Root colonization assays showed that P. citrinum associates directly with the root surface but does not enter or invade the root cortex of the inoculated Choy Sum plants. Preliminary results also indicated that P. citrinum can promote growth in Choy Sum via volatile metabolites too. Interestingly, we detected relatively higher amounts of gibberellins and cytokinins in axenic P. citrinum culture filtrates through liquid chromatography-mass spectrometry analyses. This could plausibly explain the overall growth induction in P. citrinum-inoculated Choy Sum plants. Furthermore, the phenotypic growth defects associated with the Arabidopsis ga1 mutant could be chemically complemented by the exogenous application of P. citrinum culture filtrate, which also showed accumulation of fungus-derived active gibberellins. Our study underscores the importance of transkingdom beneficial effects of such mycobiome-assisted nutrient assimilation and beneficial fungus-derived phytohormone-like metabolites in the induction of robust growth in urban farmed crops.

Warm temperature compromises JA-regulated basal resistance to enhance Magnaporthe oryzae infection in rice.

Changes in global temperatures profoundly affect the occurrence of plant diseases. It is well known that rice blast can easily become epidemic in relatively warm weather. However, the molecular mechanism remains unclear. In this study, we show that enhanced blast development at a warm temperature (22°C) compared with the normal growth temperature (28°C) is rice plant-determined. Comparative transcriptome analysis revealed that jasmonic acid (JA) biosynthesis and signaling genes in rice could be effectively induced by Magnaporthe oryzae at 28°C but not at 22°C. Phenotypic analyses of the osaoc1 and osmyc2 mutants, OsCOI1 RNAi lines, and OsMYC2-OE plants further demonstrated that compromised M. oryzae-induced JA biosynthesis and signaling lead to enhanced blast susceptibility at the warm temperature. Consistent with these results, we found that exogenous application of methyl jasmonate served as an effective strategy for improving blast resistance under the warm environmental conditions. Furthermore, decreased activation of JA signaling resulted in the downregulated expression of some key basal resistance genes at 22°C when compared with 28°C. Among these affected genes, OsCEBiP (chitin elicitor-binding protein precursor) was found to be directly regulated by OsMYB22 and its interacting protein OsMYC2, a key component of JA signaling, and this contributed to temperature-modulated blast resistance. Taken together, these results suggest that warm temperature compromises basal resistance in rice and enhances M. oryzae infection by reducing JA biosynthesis and signaling, providing potential new strategies for managing rice blast disease under warm climate conditions.

Identification and Characterization of Auxin/IAA Biosynthesis Pathway in the Rice Blast Fungus Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae has been known to produce the phytohormone auxin/IAA from its hyphae and conidia, but the detailed biological function and biosynthesis pathway is largely unknown. By sequence homology, we identified a complete indole-3-pyruvic acid (IPA)-based IAA biosynthesis pathway in M. oryzae, consisting of the tryptophan aminotransferase (MoTam1) and the indole-3-pyruvate decarboxylase (MoIpd1). In comparison to the wild type, IAA production was significantly reduced in the motam1Δ mutant, and further reduced in the moipd1Δ mutant. Correspondingly, mycelial growth, conidiation, and pathogenicity were defective in the motam1Δ and the moipd1Δ mutants to various degrees. Targeted metabolomics analysis further confirmed the presence of a functional IPA pathway, catalyzed by MoIpd1, which contributes to IAA/auxin production in M. oryzae. Furthermore, the well-established IAA biosynthesis inhibitor, yucasin, suppressed mycelial growth, conidiation, and pathogenicity in M. oryzae. Overall, this study identified an IPA-dependent IAA synthesis pathway crucial for M. oryzae mycelial growth and pathogenic development.

Tangeretin inhibits fungal ferroptosis to suppress rice blast.

Flavonoids are polyphenolic secondary metabolites that function as signaling molecules, allopathic compounds, phytoalexins, detoxifying agents and antimicrobial defensive compounds in plants. Blast caused by the fungus Magnaporthe oryzae is a serious disease affecting rice cultivation. In this study, we revealed that a natural flavonoid, tangeretin, substantially delays the formation of M. oryzae appressoria and blocks the development of blast lesions on rice plants. Our data suggest that tangeretin has antioxidant activity that interferes with conidial cell death/ferroptosis, which is critical for M. oryzae pathogenicity. Tangeretin showed a ferroptosis inhibition efficacy comparable to the well-established liproxstatin-1. Furthermore, overexpression of the NADPH oxidases NOX1 or NOX2 significantly decreased sensitivity toward tangeretin treatment, suggesting Nox-mediated lipid peroxidation as a possible target for tangeretin in regulating redox signaling and ferroptosis in M. oryzae. Our nursery and field tests showed that application of tangeretin can effectively mitigate overall disease symptoms and prevent leaf blast. Our study reveals the plant-derived fungal ferroptosis inhibitor tangeretin as a potential and novel antifungal agrochemical for the sustainable prevention of the devastating blast disease in important cereal crops.

Fungal Jasmonate as a Novel Morphogenetic Signal for Pathogenesis.

A key question that has remained unanswered is how pathogenic fungi switch from vegetative growth to infection-related morphogenesis during a disease cycle. Here, we identify a fungal oxylipin analogous to the phytohormone jasmonic acid (JA), as the principal regulator of such a developmental switch to isotropic growth and pathogenicity in the rice-blast fungus Magnaporthe oryzae. Using specific inhibitors and mutant analyses, we determined the molecular function of intrinsic jasmonates during M. oryzae pathogenesis. Loss of 12-Oxo-phytodienoic Acid (OPDA) Reductase and/or consequent reduction of jasmonate biosynthesis, prolonged germ tube growth and caused delayed initiation and improper development of infection structures in M. oryzae, reminiscent of phenotypic defects upon impaired cyclic AMP (cAMP) signaling. Chemical- or genetic-complementation completely restored proper vegetative growth and appressoria in opr1Δ. Mass spectrometry-based quantification revealed increased OPDA accumulation and significantly decreased jasmonate levels in opr1Δ. Most interestingly, exogenous jasmonate restored proper appressorium formation in pth11Δ that lacks G protein/cAMP signaling; but failed to do so in the Mitogen-activated protein (MAP) kinase mutants. Epistasis analysis placed jasmonate upstream of the cAMP pathway in rice blast. Mechanistically, intrinsic jasmonate orchestrates timely cessation of the vegetative phase and induces pathogenic development via a complex regulatory interaction with the cAMP-PKA cascade and redox signaling in rice blast.

The Histone Deacetylases MoRpd3 and MoHst4 Regulate Growth, Conidiation, and Pathogenicity in the Rice Blast Fungus Magnaporthe oryzae.

As the causal agent of the blast disease, Magnaporthe oryzae is one of the most destructive fungal pathogens of rice. Histone acetylation/deacetylation is important for remodeling of chromatin superstructure and thus altering gene expression. In this study, two genes encoding histone deacetylases, namely, MoRPD3 and MoHST4, were identified and functionally characterized in M. oryzae. MoHst4 was required for proper mycelial growth and pathogenicity, whereas overproduction of MoRpd3 led to loss of pathogenicity, likely due to a block in conidial cell death and restricted invasive growth within the host plants. Green fluorescent protein (GFP)-MoRpd3 localized to the nucleus and cytoplasm in vegetative hyphae and developing conidia. By comparative transcriptomics analysis, we identified potential target genes epigenetically regulated by histone deacetylases (HDACs) containing MoRpd3 or MoHst4, which may contribute to conidia formation and/or conidial cell death, which is a prerequisite for successful appressorium-mediated host invasion. Taken together, our results suggest that histone deacetylases MoRpd3 and MoHst4 differentially regulate mycelial growth, asexual development, and pathogenesis in M. oryzae. IMPORTANCE HDACs (histone deacetylases) regulate various aspects of growth, development, and pathogenesis in plant-pathogenic fungi. Most members of HDAC classes I to III have been functionally characterized, except for orthologous Rpd3 and Hst4, in the rice blast fungus Magnaporthe oryzae. In this study, we assessed the function of MoRpd3 and MoHst4 by reverse genetics and found that they differentially regulate M. oryzae vegetative growth, asexual development, and infection. Particularly, MoRpd3 negatively regulates M. oryzae pathogenicity, likely through suppression of conidial cell death, which we recently reported as being critical for appressorium maturation and functioning. Overall, this study broadens our understanding of fungal pathobiology and its critical regulation by histone modification(s) during cell death and in planta differentiation.

The retromer CSC subcomplex is recruited by MoYpt7 and sequentially sorted by MoVps17 for effective conidiation and pathogenicity of the rice blast fungus.

In eukaryotic cells, Rab GTPases and the retromer complex are important regulators of intracellular protein transport. However, the mechanistic relationship between Rab GTPases and the retromer complex in relation to filamentous fungal development and pathogenesis is unknown. In this study, we used Magnaporthe oryzae, an important pathogen of rice and other cereals, as a model filamentous fungus to dissect this knowledge gap. Our data demonstrate that the core retromer subunit MoVps35 interacts with the Rab GTPase MoYpt7 and they colocalize to the endosome. Without MoYpt7, MoVps35 is mislocalized in the cytoplasm, indicating that MoYpt7 plays an important role in the recruitment of MoVps35. We further demonstrate that the expression of an inactive MoYpt7-DN (GDP-bound form) mutant in M. oryzae mimicks the phenotype defects of retromer cargo-sorting complex (CSC) null mutants and blocks the proper localization of MoVps35. In addition, our data establish that MoVps17, a member of the sorting nexin family, is situated at the endosome independent of retromer CSC but regulates the sorting function of MoVps35 after its recruitment to the endosomal membrane by MoYpt7. Taken together, these results provide insight into the precise mechanism of retromer CSC recruitment to the endosome by MoYpt7 and subsequent sorting by MoVps17 for efficient conidiation and pathogenicity of M. oryzae.

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.

Species-independent analytical tools for next-generation agriculture.

Innovative approaches are urgently required to alleviate the growing pressure on agriculture to meet the rising demand for food. A key challenge for plant biology is to bridge the notable knowledge gap between our detailed understanding of model plants grown under laboratory conditions and the agriculturally important crops cultivated in fields or production facilities. This Perspective highlights the recent development of new analytical tools that are rapid and non-destructive and provide tissue-, cell- or organelle-specific information on living plants in real time, with the potential to extend across multiple species in field applications. We evaluate the utility of engineered plant nanosensors and portable Raman spectroscopy to detect biotic and abiotic stresses, monitor plant hormonal signalling as well as characterize the soil, phytobiome and crop health in a non- or minimally invasive manner. We propose leveraging these tools to bridge the aforementioned fundamental gap with new synthesis and integration of expertise from plant biology, engineering and data science. Lastly, we assess the economic potential and discuss implementation strategies that will ensure the acceptance and successful integration of these modern tools in future farming practices in traditional as well as urban agriculture.

Ferroptosis contributes to developmental cell death in rice blast.

Ferroptosis, an iron-dependent cell death process, was found to occur in Magnaporthe oryzae, and plays a key role in infection-related development therein. Ferroptosis in the rice-blast fungus was confirmed based on five basic criteria. We confirmed the dependence of ferroptosis on ferric ions, and optimized ratio-fluorescence imaging of C11-BODIPY581/591 as a precise sensor for lipid peroxides that mediate ferroptosis in M. oryzae. We uncovered an important regulatory function for reduced glutathione and NADPH oxidases in modulating the superoxide moieties required for ferroptotic cell death. We found ferroptosis to be necessary for the developmental cell death of conidia during appressorium maturation in rice blast. Such ferroptotic cell death initiated first in the terminal cell and progressed sequentially to the entire conidium. Iron chelation or chemical inhibition of ferroptosis caused conidial cells to remain viable, and led to strong defects in host invasion by M. oryzae. Ferroptosis induction exclusively in the host severely constrained the invasive growth of M. oryzae. We found inter-reliant and independent roles for ferroptosis and autophagy in controlling such precise cell death in M. oryzae during pathogenic differentiation. Our study provides significant molecular insights into the role of developmental cell death and iron homeostasis in fungal pathogenesis.

Detection of Fungal Jasmonates by Liquid Chromatography Paired with Mass Spectrometry.

Liquid chromatography-mass spectrometry (LC-MS) is one of the most important analytical chemistry techniques for the detection and characterization of biologically active compounds of low abundance-for example, hormones. Gas chromatography (GC) coupled with mass spectrometry has been a method of choice to detect jasmonic acid, the well-known defense hormone in plants. Recently, we identified structural and functional analogs of phytohormone jasmonic acid (JA) and its derivatives, in the rice-blast fungus Magnaporthe oryzae. Here, we describe protocols of LC-MS/MS-based identification and quantification of fungal jasmonates, especially during pathogenic development in the rice blast fungus.

Metabolomics Analysis Identifies Sphingolipids as Key Signaling Moieties in Appressorium Morphogenesis and Function in Magnaporthe oryzae.

The blast fungus initiates infection using a heavily melanized, dome-shaped infection structure known as the appressorium, which forcibly ruptures the cuticle to enter the rice leaf tissue. How this process takes place remains not fully understood. Here, we used untargeted metabolomics analyses to profile the metabolome of developing appressoria and identified significant changes in six key metabolic pathways, including early sphingolipid biosynthesis. Analyses employing small molecule inhibitors, gene disruption, or genetic and chemical complementation demonstrated that ceramide compounds of the sphingolipid biosynthesis pathway are essential for normal appressorial development controlled by mitosis. In addition, ceramide was found to act upstream from the protein kinase C-mediated cell wall integrity pathway during appressorium repolarization and pathogenicity in rice blast. Further discovery of the sphingolipid biosynthesis pathway revealed that glucosylceramide (GlcCer) synthesized by ceramide is the key substance affecting the pathogenicity of Magnaporthe oryzae Our results provide new insights into the chemical moieties involved in the infection-related signaling networks, thereby revealing a potential target for the development of novel control agents against the major disease of rice and other cereals.IMPORTANCE Our untargeted analysis of metabolomics throughout the course of pathogenic development gave us an unprecedented high-resolution view of major shifts in metabolism that occur in the topmost fungal pathogen that infects rice, wheat, barley, and millet. Guided by these metabolic insights, we demonstrated their practical application by using two different small-molecule inhibitors of sphingolipid biosynthesis enzymes to successfully block the pathogenicity of M. oryzae Our study thus defines the sphingolipid biosynthesis pathway as a key step and potential target that can be exploited for the development of antifungal agents. Furthermore, future investigations that exploit such important metabolic intermediates will further deepen our basic understanding of the molecular mechanisms underlying the establishment of fungal blast disease in important cereal crops.