Fig. 1. Characterization of Ustilaginoidea virens mitovirus 1 (UvMV1). A, RT-PCR was conducted to identify potential mycoviruses in U. virens strain Uv488. M, DNA marker (DL2000 bp). B, RT-PCR was conducted on the viral genome using mycovirus- specific primers both before and after treatments with S1 nuclease for the digestion of ssDNA or ssRNA. M, DNA marker (DL2000 bp).C, Schematic illustrates the genome organization of UvMV1, with boxes representing putative open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp), and gray lines indicate untranslated regions. D, Predicted secondary structure of the 5ʹ and 3ʹ terminal sequences of UvMV1.

Fig. 2. Effect of UvMV1 on biological characteristics of Ustilaginoidea virens. A, Colony morphology of the UvMV1-free strain HWD2-27 compared with UvMV1-infected strain UvMV1-H1 grown on potato sucrose agar (PSA) plates at 28 ºC for 14 d. B, Comparison of colony diameter of strains HWD2-27 and UvMV1-H1 grown on PSA plates for 14 d. C, Comparison of the number of conidia produced by strains HWD2-27 and UvMV1-H1 when cultured on potato sucrose (PS) liquid medium under shaking conditions for 7 d. D, Conidial germination rates of strains HWD2-27 and UvMV1-H1 at 9 h after incubation.E, Spores of strains HWD2-27 and UvMV1-H1 were examined using microscopy at both 0 and 9 h after incubation. Scale bars, 20 μm. F, Toxicity assessment of culture filtrates from 7-day-old strains HWD2-27 and UvMV1-H1 to evaluate their effects on rice seed germination. Scale bars, 1 cm. G, Shoot lengths of rice seeds following a 5-day treatment with culture filtrates derived from strains HWD2-27 and UvMV1-H1. H and I, Colony morphologies (H) and growth inhibition rates (I) of UvMV1-free strain HWD2-27 and UvMV1-infected strains UvMV1-H1 and UvMV1-H3 after culturing on PSA medium plates containing various environmental stress factors [120 μg/mL Congo red, 120 μg/mL Calcofluor white (CFW), 0.5 mol/L sorbitol, or 0.3 mol/L NaCl] for 14 d. J, qRT-PCR analysis of the transcript levels of UvBI-1, UvPmk1, UvPRO1, UvCom1, UvCDC2, UvHog1, Uvt3277, and UvMK1 in mock- and UvMV1-infected U. virens. The mock- and UvMV1-infected strains were cultured in PS liquid medium for 5 d with shaking in the dark, after which total RNA was extracted for qRT-PCR analyses. The housekeeping gene β-tubulin serves as the internal control. Data are Mean ± SD (n = 3) in B‒D, G, I, and J. Student’s t-test was used for analyses: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Lowercase letters above the bars indicate significant differences among the treatments.

Fig. 3. UvMV1 infection induces autophagy in Ustilaginoide virens to increase viral titers. A, qRT-PCR analysis of the transcript levels of UvAtg2, UvAtg4, UvAtg6, UvAtg8, UvAtg9, and UvAtg14 in mock- and UvMV1-infected U. virens. The mock- and UvMV1-infected strains were cultured in potato sucrose (PS) liquid medium for 5 d with shaking in the dark, after which total RNA was extracted for qRT-PCR analyses. B, qRT-PCR analysis shows the impact of 3-methyladenine (3-MA) and rapamycin on the transcript levels of the UvMV1 RNA-dependent RNA polymerase genes in U. virens. The UvMV1-infected strains were initially cultured in PS medium for 2 d, and then 3-MA or rapamycin was added to PS liquid medium for 24 h, respectively. The UvMV1-infected strains were treated by washing them with ddH2O and then incubated on regular PS medium for 3 d. Dimethylsulfoxide (DMSO)-treated U. virens were used as a control. The levels of UvMV1 RNA transcripts in various samples were individually detected using qRT-PCR. Values represent the mean relative to the mock-treated strains, with the housekeeping gene β-tubulin serving as the internal control. Student’s t-test was used for analyses: *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, Not significant.

Fig. 4. Overall transcriptomic results and analysis of differentially expressed genes (DEGs) in UvMV1-infected and UvMV1-free strains of Ustilaginoidea virens. A, Sample correlation heatmap. HWD2-27-1, -2, and -3 as well as UvMV1-H1, -3, and -4 are three biological replicates, respectively. B, Volcano plots illustrating differential gene expression levels between UvMV1-infected strains and UvMV1-free strains. FDR, False discovery rate.C, Heatmap illustrating DEGs in UvMV1-infected strains compared with UvMV1-free strains.

Fig. 5. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enhancement analyses of differentially expressed genes (DEGs). A and B, GO terms that are enriched in up-regulated (A) and down-regulated genes (B), as identified through GO enrichment analysis. The bubble size represents the number of members detected in the GO enrichment and the color of the bubble represents the P-value. BP, Biological process; CC, Cellular component; MF, Molecular function.C, KEGG pathway enrichment analysis of DEGs. The rich factor is the ratio of DEG numbers annotated in this pathway term to all gene numbers annotated in this pathway term. The bubble size represents the number of members detected in the KEGG enrichment and the color of the bubble represents the P-value.

Fig. 6. Differentially expressed genes involved in MAPK signaling pathway. The pathways representing cell wall stress and high osmolarity are illustrated from left to right, respectively. Expression values were presented as log2(Fold change), with red indicating up-regulation and blue indicating down-regulation. In the heatmap, each column represents the expression of HWD2-27-1, HWD2-27-2, HWD2-27-3, UvMV1-H1, UvMV1-H3, and UvMV1-H4 from left to right, and each row represents one gene. MAPK, Mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPK kinase kinase; TF, Transcription factor.

Fig. 7. Accuracy of RNA-seq was validated through qRT-PCR analysis. The expression levels of 12 DEGs identified from the RNA-seq analysis, comprising 6 up-regulated (UV8b_06886, UV8b_02392, UV8b_02631, UV8b_06468, UV8b_05743, and UV8b_00346) and 6 down-regulated genes (UV8b_05392, UV8b_07837, UV8b_03106, UV8b_08214, UV8b_05560, and UV8b_05223), were quantified using qRT-PCR. Histograms were generated from the data obtained via qRT-PCR, while corresponding line charts were constructed from the fragments per kilobase of transcript per million mapped reads (FPKM) values derived from the RNA-seq analysis, with distinct colors representing different samples. Each bar in the histograms denotes the mean and standard error of three independent experiments.