Lesion mimic mutants (LMMs) are valuable genetic resources for unraveling plant defense responses including programmed cell death. Here, we identified a rice (Oryza sativa) LMM, spotted leaf 38 (spl38), and demonstrated that spl38 is essential for the formation of hypersensitive response-like lesions and innate immunity. Map-based cloning revealed that SPL38 encodes MEDIATOR SUBUNIT 16 (OsMED16). The spl38 mutant showed enhanced resistance to rice pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo) and exhibited delayed flowering, while OsMED16-overexpressing plants showed increased rice susceptibility to M. oryzae. The OsMED16-edited rice lines were phenotypically similar to the spl38 mutant but were extremely weak, exhibited growth retardation, and eventually died. The C-terminus of OsMED16 showed interaction with the positive immune regulator PATHOGENESIS RELATED 3 (OsPR3), resulting in the competitive repression of its chitinase and chitin-binding activities. Furthermore, the ospr3 osmed16 double mutants did not exhibit the lesion mimic phenotype of the spl38 mutant. Strikingly, OsMED16 exhibited an opposite function in plant defense relative to that of Arabidopsis (Arabidopsis thaliana) AtMED16, most likely because of 2 amino acid substitutions between the monocot and dicot MED16s tested. Collectively, our findings suggest that OsMED16 negatively regulates cell death and immunity in rice, probably via the OsPR3-mediated chitin signaling pathway.

Rice yield is greatly reduced owing to rice blast, a polycyclic fungal disease caused by the ascomycete Magnaporthe oryzae. Previously, Bacillus cereus HS24, isolated from a rice farm, showed a strong antimicrobial effect toward M. oryzae. To better exploit it as a biocontrol agent, HS24 was studied for the mechanism that it uses to suppress rice blast. Conidium germination in M. oryzae was significantly inhibited by HS24, whereby inhibition reached 97.8% at the concentration of 10⁷CFU/ml. The transcription levels of Ca²⁺/calmodulin-dependent protein kinase II, PMC1, and CCH1, key genes involved in the M. oryzae Ca²⁺signaling pathway, were significantly decreased in HS24-treated conidia at high concentration. The treatment of M. oryzae with the corresponding Ca²⁺signaling pathway inhibitors KN-93, verapamil, and cyclopiazonic acid significantly reduced conidium germination. This inhibitory effect was found to be concentration dependent, similar to the HS24 treatment. We also found that HS24 was able to decrease the intracellular free Ca²⁺concentration in M. oryzae conidia significantly. The addition of exogenous Ca²⁺did not diminish the inhibitory effect of HS24 on the reduction of intracellular free Ca²⁺concentration and the level of conidium germination. In conclusion, B. cereus HS24 at high concentration prevents extracellular Ca²⁺from entering the conidia in M. oryzae, causes a significant reduction of intracellular free Ca²⁺concentration, and results in the inhibition of conidium germination.

Fungi secrete effector proteins, including extracellular redox enzymes, to inhibit host immunity. Redox enzymes have been hypothesized to inhibit host reactive oxygen species (ROS); however, how they suppress host immunity remains unknown. We characterized an extracellular ascorbate peroxidase (MoApx1) that is secreted into rice chloroplasts by the rice blast fungus Magnaporthe oryzae. MoApx1 displays multifunctional capabilities that significantly contribute to fungal virulence. Firstly, MoApx1 neutralizes host-derived H2O2 within the chloroplast through its peroxidase activity, thereby inhibiting chloroplast ROS (cROS)-mediated defense responses. Secondly, MoApx1 targets the photosystem I subunit OsPsaD, disrupting photosynthetic electron transport to further suppress cROS production. Most importantly, MoApx1 has evolved a fungal-specific starch-binding domain that binds host starch, inhibiting its degradation and disrupting the energy supply required for host resistance. Our findings underscore the importance of a novel multifaceted strategy, potentially widely employed by other fungal pathogens, in suppressing host immunity during host–microbe interactions.

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.