Jessie Fernandez

Rice blast, caused by the hemibiotrophic fungus Magnaporthe oryzae , is one of the most destructive diseases affecting rice production worldwide. During infection, M. oryzae secretes effector proteins that suppress host immunity and facilitate colonization. Here, we characterized MoALE1, a predicted effector that is structurally similar to members of the heat-labile enterotoxin/cholera toxin family. Using a yeast secretion trap assay, we demonstrated that MoALE1 contains a functional signal peptide. A MoALE1-GFP fusion localized to the biotrophic interfacial complex, indicating that MoALE1 is a cytoplasmic effector. Expression analyses revealed that MoALE1 is expressed during biotrophy, from 16 to 48 h post-inoculation. Transient expression in Nicotiana benthamiana showed that MoALE1 suppresses BAX-mediated cell death, suggesting a role in host immune suppression. To investigate its contribution to pathogenicity, a Δ moale1 mutant was generated and evaluated in rice infection assays. Deletion of MoALE1 significantly reduced disease severity but did not affect fungal growth, stress tolerance, sporulation, appressorium development, host penetration, or early invasive growth. In contrast, infection by the Δ moale1 mutant resulted in enhanced host H₂O₂ accumulation and increased expression of ROS-associated defense genes. Together, these findings identify MoALE1 as a biotrophy-associated cytoplasmic effector that promotes rice blast pathogenicity by suppressing host immune responses and provide new insight into the function of ADP-ribosyltransferase (ART)-like effector candidates in M. oryzae .

Rice blast disease caused by the hemibiotrophic fungal pathogen Magnaporthe oryzae poses a major threat to global rice production, creating an urgent need for sustainable disease management strategies. Because plant-associated microbiomes can contribute to pathogen suppression and host defense, their role in rice blast resistance is of considerable interest. However, interactions among M. oryzae , rice, and the rice microbiome remain poorly understood. Using a gnotobiotic rice pathosystem, we investigated how microbiome colonization influences host responses to infection and how bacterial community structure changes following pathogen challenge. Microbiome colonization significantly reduced blast disease severity, while differential abundance analyses identified infection-associated shifts in bacterial community composition, including enrichment of Brevibacillus -associated taxa. Functional characterization of cultured endophytes revealed plant growth-promoting traits and antagonistic activity against M. oryzae . Two synthetic communities assembled from healthy- and infected-derived isolates significantly reduced disease severity, demonstrating that disease suppression can be recapitulated using simplified microbial communities. Interestingly, axenically grown plants exhibited elevated basal expression of multiple defense-associated genes but greater disease severity and reduced chitin-induced ROS production compared with holoxenic plants. In contrast, microbiome-colonized plants displayed enhanced ROS responsiveness and reduced disease, suggesting that the rice microbiome promotes a more effective and regulated immune state rather than simply increasing defense activation. Together, these findings advance our understanding of rice-microbiome- M. oryzae interactions and establish a tractable experimental platform for mechanistic studies of microbiome-mediated disease suppression. Highlights: Gnotobiotic rice pathosystem enables controlled microbiome manipulation. Microbiome colonization significantly suppresses rice blast disease. Axenic plants show elevated defense-gene expression but greater disease. Rice microbiome colonization enhances chitin-induced ROS responses. Synthetic communities recapitulate microbiome-mediated disease suppression.

Poly(ADP-ribose) polymerases (PARPs) are important regulators of DNA repair and cellular stress responses in eukaryotes. Although mammalian PARPs have been extensively characterized, comparatively little is known about PARPs in pathogenic filamentous fungi. Here, we define biochemical features of MoPARP1 from the plant-pathogenic fungus Magnaporthe oryzae . Mutation of the conserved catalytic glutamate E714 abolished detectable PARylation activity, whereas PAR generated by MoPARP1 was hydrolyzed by human PARG, suggesting synthesis of polymeric ADP-ribose. Several mammalian PARP inhibitors were effective against MoPARP1 in vitro, consistent with conservation of the catalytic inhibitor-binding pocket. DNA substrates containing 5′ phosphate enhanced MoPARP1 catalytic activity, while both wild-type MoPARP1 and the catalytically inactive MoPARP1 E714A bound diverse DNA substrates. Quantitative analyses revealed that structurally distinct DNA substrates exhibit similar apparent binding affinities yet yield markedly different catalytic outputs, suggesting that DNA engagement alone does not determine productive PAR synthesis. MoPARP1 lacks the canonical N-terminal zinc-finger domains of human PARP1 and instead depends on its WGR-containing region for DNA association. Domain truncation analyses showed the BRCT-WGR region supports high-affinity DNA binding, whereas the WGR-PARP region retains catalytic competence despite weaker DNA affinity. Together, these findings establish a mechanistic framework for DNA-dependent PARylation in a filamentous fungal plant pathogen.

Plant cells can directly or indirectly detect bacterial effectors, triggering a hypersensitive response to defend against pathogen infection. One extensively studied effector, AvrB, is a Fido (Fic, Doc, AvrB) domain-containing protein that acts as a glycosyltransferase. AvrC is an elusive Pseudomonas syringae avirulence effector protein with significant sequence and structural similarity to AvrB. Combining biochemistry, mass spectrometry, and AlphaFold prediction tools, we show that AvrC is a glycosyltransferase with auto-rhamnosylation activity. Like AvrB, AvrC can rhamnosylate a threonine residue (T166) on the A. thaliana guardee protein RIN4. In vitro assays revealed rhamnosylation substrates for AvrC also include plant coatomer subunits COPE1 and COPZ1. Collectively, our findings indicate that AvrC is a rhamnosyltransferase with broad substrate specificity. Our experimental strategies and findings provide valuable insights into future studies on the characterization of other Fido proteins.

Biological control strategies are increasingly being explored as sustainable alternatives for managing rice blast disease caused by Magnaporthe oryzae. In this study, we characterized three Bacillus pumilus isolates (DC01, DC09, and DC13) and evaluated their antifungal and plant-beneficial properties against M. oryzae. Whole-genome sequencing revealed multiple biosynthetic gene clusters associated with the production of antimicrobial metabolites. All three isolates inhibited fungal growth in dual-culture assays, whereas heat-stable diffusible antifungal activity was primarily associated with the cell-free supernatants of DC09 and DC13. Exposure to bacterial supernatants disrupted fungal development, inducing abnormal hyphal morphology in M. oryzae characterized by bulbous swelling, altered polarity, and increased branching. Volatile organic compound assays further revealed that the DC isolates suppress fungal growth in the absence of physical contact. The isolates additionally inhibited the growth of other phytopathogenic fungi and selected human bacterial pathogens. All strains exhibited plant growth-promoting traits, including indole-3-acetic acid production and osmotic stress tolerance, whereas DC09 also displayed phosphate-solubilizing activity. Importantly, soil inoculation with the DC isolates significantly reduced rice blast disease severity and induced expression of defense-associated genes involved in jasmonic acid/ethylene signaling and immune priming. Collectively, these findings identify the DC isolates, particularly DC09 and DC13, as promising multi-mechanistic biological control agents for sustainable rice blast management.

Biological control strategies are increasingly being explored as sustainable alternatives for managing rice blast disease caused by Magnaporthe oryzae . In this study, we characterized three Bacillus pumilus isolates (DC01, DC09, and DC13) and evaluated their antifungal and plant-beneficial properties against M. oryzae . Whole genome sequencing revealed multiple biosynthetic gene clusters associated with the production of antimicrobial metabolites. All three isolates inhibited fungal growth in dual-culture assays, whereas heat-stable diffusible antifungal activity was primarily associated with the cell-free supernatants of DC09 and DC13. Exposure to bacterial supernatants disrupted fungal development, inducing abnormal hyphal morphology characterized by bulbous swelling, altered polarity, and increased branching in M. oryzae . Volatile organic compound assays further revealed that the DC isolates suppress fungal growth in the absence of physical contact. The isolates additionally inhibited the growth of other phytopathogenic fungi and selected human bacterial pathogens. All strains exhibited plant growth-promoting traits, including indole-3-acetic acid production and osmotic stress tolerance, whereas DC09 also displayed phosphate-solubilizing activity. Importantly, root inoculation with the DC isolates significantly reduced rice blast disease severity and induced expression of defense-associated genes involved in jasmonic acid/ethylene signaling and immune priming. Collectively, these findings identify the DC isolates, particularly DC09 and DC13, as promising multi-mechanistic biological control agents for sustainable rice blast management.

Dragon fruit canker (DFC), caused by Neoscytalidium dimidiatum , is a major disease affecting dragon fruit production. However, the pathogen’s nutrient utilization strategies and enzymatic activities remain poorly characterized. Using calcofluor white-based fluorescence microscopy, we show that nutrient limitation restricts fungal growth and development, impairing melanization and arthrosporulation. Carbon utilization assays revealed a preference for maltose, suggesting adaptation to starch-derived sugars. Nitrogen utilization assays indicated efficient assimilation of complex organic nitrogen sources. In addition, we experimentally validated extracellular enzymatic activities involved in host macromolecule degradation, including cellulase, amylase, pectinase and protease activities. Together, these findings provide insight into the physiological traits of N. dimidiatum associated with growth and development under varying nutrient conditions.

Poly(ADP-ribose) polymerases (PARPs) regulate genome maintenance through NAD⁺-dependent ADP-ribosylation, yet PARP function in fungi remains poorly defined. Here, we reconstituted the activity of the Magnaporthe oryzae PARP1 homolog (MoPARP1) in Saccharomyces cerevisiae, a genetically tractable organism that lacks endogenous PARP enzymes. Upon galactose induction, expression of MoPARP1 reduced yeast growth, whereas a catalytically inactive mutant showed no defect, indicating that the growth phenotype depends on PARP catalytic activity. Consistent with this requirement, PARylation was detected in MoPARP1-expressing yeast cells but not in the catalytic mutant. In a multidrug transporter-deficient background, the PARP inhibitor 3-aminobenzamide and the clinically used PARP inhibitor olaparib rescued the growth of MoPARP1-expressing strains, establishing a framework for inhibitor testing in vivo. Finally, MoPARP1-GFP localized to the nucleus independent of catalytic activity, supporting correct targeting in this heterologous system. Together, these findings establish yeast as a platform to dissect fungal PARP biology and evaluate chemical inhibition.

Poly(ADP-ribose) polymerases (PARPs) regulate genome maintenance through NAD⁺-dependent ADP-ribosylation, yet PARP function in fungi remains poorly defined. Here, we reconstituted the activity of the Magnaporthe oryzae PARP1 homolog (MoPARP1) in Saccharomyces cerevisiae, a genetically tractable organism that lacks endogenous PARP enzymes. Galactose-induced expression of MoPARP1 reduced yeast growth, whereas a catalytically inactive mutant showed no defect, indicating that the growth phenotype depends on PARP catalytic activity. In a multidrug transporter-deficient background, the PARP inhibitor 3-aminobenzamide and the clinically used PARP inhibitor olaparib rescued the growth of MoPARP1-expressing strains, establishing a framework for inhibitor testing in vivo. Finally, MoPARP1-GFP localized to the nucleus independent of catalytic activity, supporting correct targeting in this heterologous system. Together, these findings establish yeast as a platform to dissect fungal PARP biology and evaluate chemical inhibition.

Dragon fruit canker (DFC), caused by the pathogenic fungus Neoscytalidium dimidiatum , is a severe disease that threatens dragon fruit production worldwide. Current management efforts largely rely on fungicide applications and sanitation measures; however, the pathogen's molecular genetic characterization, nutrient utilization preferences, and extracellular enzyme activities remain poorly understood. Using calcofluor white-based fluorescence microscopy, we demonstrate that nutrient limitation significantly restricts the growth and maturation of N. dimidiatum , impairing melanization and sporulation. Carbon utilization assays revealed a preference for maltose, suggesting reliance on starch-derived sugars. In addition, nitrogen utilization assays indicated efficient assimilation of complex organic nitrogen sources rich in peptides and amino acids. Finally, we experimentally validated extracellular enzymatic activities involved in host macromolecule degradation, including cellulase, amylase, pectinase, and protease activities. Collectively, these findings provide new insights into the physiology and pathogenic potential of N. dimidiatum and establish a foundation for developing improved strategies to mitigate DFC.