MoWhi2 Participates in Mitophagy and Pathogenesis by Modulating MoAti1 Protein Level in Magnaporthe Oryzae

doi:10.1016/j.rsci.2026.03.009

Abstract

Abstract:

Rice blast caused by the fungus Magnaporthe oryzae is one of the most devastating diseases affecting rice production. It was previously demonstrated that MoWHI2 is indispensable for mitophagy and full virulence in M. oryzae, yet the molecular mechanisms underlying its control of mitophagy remained unresolved. In this study, we found that MoWhi2 interacts with MoAti1, a SUN family protein that mediates mitophagy by recruiting the autophagy-associated protein MoAtg8. Phenotypic analysis showed that the defects of the ΔMowhi2/ΔMoati1 mutant in growth, conidiation, and pathogenicity were comparable to those of ΔMoati1, but more severe than those of ΔMowhi2. Furthermore, compared with the wild type, the ΔMowhi2/ΔMoati1 mutant exhibited a lower mitophagy level similar to that of the single deletion mutants ΔMowhi2 or ΔMoati1. In addition, deletion of MoWHI2 resulted in a decreased protein level of MoAti1, suggesting that MoWhi2 might regulate mitophagy by modulating the protein levels of MoAti1. Due to the absence of MoWhi2 orthologues in both rice and humans, we employed host-induced gene silencing (HIGS) targeting MoWHI2 in rice plants; the resultant transgenic rice lines displayed resistance to blast without agronomic penalties. Collectively, our data established that MoWhi2 regulates mitophagy and pathogenicity of M. oryzae by interacting with MoAti1, and can serve as a target for rice blast control strategies.

Key words: rice blast, pathogenesis, fungal disease, mitophagy, Whi2, host-induced gene silencing

Zhang Ying, Shi Huanbin, Meng Shuai, Wen Hui, Chen Ya, Liu Li, Kou Yanjun. MoWhi2 Participates in Mitophagy and Pathogenesis by Modulating MoAti1 Protein Level in Magnaporthe Oryzae[J]. Rice Science, 2026, 33(4): 545-558.

Figures/Tables 5

Fig. 1. Interaction and co-localization of MoWhi2 and MoAti1. A, DUAL membrane yeast two-hybrid system assay confirming the MoWhi2-MoAti1 interaction. The paired plasmids of pTSUC2-APP and NubG-Fe65 were used as the positive control, while pTSUC2-APP and pPR3-N served as the negative control. SD-WLHA, SD-Leu/-Trp/-His/-Ade; SD-WL, SD-Leu/-Trp; dilu, Dilution. BD-E, Empty pGBKT7; AD-E, Empty pGADT7. B, Pull-down assay detecting the interaction between MoWhi2 and MoAti1. The GST-MoWhi2 and His-MoAti1 co-incubated mixture served as the experimental sample, while the GST and His-MoAti1 co-incubated mixture served as the negative control. C, Validation of MoWhi2-MoAti1 interaction via a Co-IP (Co-immunoprecipitation) assay. The protein MoWhi2-mCherry (59 kDa) was co-precipitated with MoAti1-GFP (71 kDa). The MoWhi2-mCherry/GFP strain served as a negative control, and the MoWhi2-mCherry/MoAti1-GFP strain served as the experimental sample. D, Subcellular localization of MoWhi2 and MoAti1 at different developmental stages of MoWhi2-GFP/MoAti1-mCherry co-expression strain. Scale bars, 5 μm; line-scan fluorescence intensity profile is shown. Arrows indicate the analyzed regions. The intensity is given in arbitrary units (a.u.).

Fig. 2. MoWhi2 and MoAti1 regulate growth, conidiation, and virulence in Magnaporthe oryzae. A-C, Colony morphology of wild-type strain B157, mutant strains (ΔMowhi2, ΔMoati1, and ΔMowhi2/ΔMoati1), and complemented strains (ΔMowhi2-C and ΔMoati1-C) grown on complete medium (CM) for 7 d. The lesion diameter (B) and conidia production (C) of the mutants were lower than those of the wild-type and complemented strains. D and E, Relative lesion area (D) and fungal biomass (E) measurements of wild-type strain B157, mutant strains (ΔMowhi2, ΔMoati1, and ΔMowhi2/ΔMoati1), and complemented strains (ΔMowhi2-C and ΔMoati1-C) after 7 d of inoculation. The statistical analysis was conducted with Photoshop 2021 and Prism 7.0. Lesion number, area, and fungal biomass were lower in the mutants than in the wild-type and complemented strains. F, Rice seedling infection assay of the wild-type strain B157, mutant strains (ΔMowhi2, ΔMoati1, and ΔMowhi2/ΔMoati1), and complemented strains (ΔMowhi2-C and ΔMoati1-C). Rice seedlings were spraying inoculated with conidial suspensions and photographed at 5 dpi. Scale bars, 1cm. Data are presented as mean ± SD (n = 3). Statistically significant differences between wild-type strain B157, ΔMowhi2, ΔMoati1, ΔMowhi2/ΔMoati1, and complemented strains (ΔMowhi2-C and ΔMoati1-C) were determined by one-way ANOVA tests (P < 0.05). Post-hoc pairwise comparisons were conducted using Tukey’s Honestly Significant Difference (HSD) test (P < 0.05). In the figures, different lowercase letters above bars indicate significant differences among groups.

Fig. 3. MoWhi2 and MoAti1 regulate mitophagy in Magnaporthe oryzae. A, Impaired Mito-GFP degradation in ΔMoatg8/Mito-GFP, ΔMowhi2/Mito-GFP, ΔMoati1/Mito-GFP, and ΔMowhi2/ΔMoati1/Mito-GFP strains. Hyphae were cultured in liquid complete medium (CM) for 24 h, transferred to glycerol-supplemented basal medium (BM)-G for another 30 h, then to nitrogen-starvation (SD-N) medium for 6-8 h, and stained with 7-amino-4-chloromethylcoumarin (CMAC) before imaging. Arrows indicate the analyzed regions. Scale bars, 5 μm. B, Linescan analysis of the arrow-indicated region in A. The intensity is given in arbitrary units (a.u.).

Fig. 4. MoWhi2 may be essential for maintaining MoAti1 protein levels. A, Fluorescence microscopy showing reduced fluorescence intensity of MoAti1-mCherry in the ΔMowhi2 mutant compared with the MoAti1-mCherry/GFP-MoAtg8 strain. Hyphae were stained with 7-amino-4-chloromethylcoumarin (CMAC). Arrows indicate the analyzed regions. Scale bars, 5 μm. CM, Complete medium; SD-N, Nitrogen-starvation medium. B, Line-scan analysis of fluorescence intensity across the region marked by the arrow in A. The intensity is given in arbitrary units (a.u.). C, Knockout of MoWHI2 reduced MoAti1 protein level. Western blot analysis was performed to examine the protein level of MoAti1-mCherry in response to nitrogen starvation. Protein extracts from the indicated strains were western-blotted with anti-mCherry and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (loading control) antibodies.

Fig. 5. Growth phenotype and agronomic traits of MoWHI2-RNAi transgenic plants. A, Disease symptoms on rice leaves of wild type TP309 and MoWHI2-RNAi plants at 7 d post-inoculation with Magnaporthe oryzae (strain RB22). Scale bar, 1 cm. B and C, Statistical analysis of lesion area on infected rice leaves (B) and quantification of relative fungal biomass in infected rice leaves (C), as determined by qPCR. Data are presented as mean ± SD (n = 3). D, Phenotypic comparison between wild type TP309 and MoWHI2-RNAi transgenic plants at the maturity stage. Scale bars, 15 cm. E and F, Plant height (E) and the number of panicles (F) of RNAi plants show no significant difference compared with wild type TP309. Data are presented as mean ± SD (n = 8). G and H, Grain length (G) and grain width (H) of TP309 and MoWHI2-RNAi plants. Scale bars, 5 mm. I-K, Statistical analysis of 1000-grain weight (I), grain length (J), and grain width (K) of wild-type TP309 and MoWHI2-RNAi plants. Data are presented as mean ± SD (n = 15). Statistically significant differences between TP309 and MoWHI2-RNAi plants were determined by one-way ANOVA tests (P < 0.05).

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