Fig 1.
Isoprothiolane taming of M. oryzae.
(A) Proportion of stable IPT resistant mutants at different taming fungicide concentrations. (B) The IPT resistant mutants exhibited significant differences in their resistance to IPT. Data presented are the mean ± SD (n = 3). Bars followed by the same letter are not significantly different according to a LSD test at P = 0.01. (C) Detection of cross-resistance to IBP in IPT resistant mutants. Linear regression analysis was performed with EC50 values of IPT and IBP for IPT-resistant mutants.
Fig 2.
Assessment of fitness of different IPT resistant mutants.
(A) Growth rate of IPT resistant mutants on PDA and OTA. (B) Sporulation of different IPT resistant mutants. (C) Conidial germination of different IPT resistant mutants. (D) Detection of melanin accumulation in IPT resistant mutants. (E) Pathogenicity test of different IPT resistant mutants. (F) Mycelial inhibition rate heat map of IPT resistant mutants to different fungicides. H08-1a and IPT resistant mutants were inoculated on PDA or PDA amended with 0.15 μg/mL rapamycin (RAP), 5 μg/mL fludioxonil (FLU), 20 μg/mL iprodione (IPR), 10 μg/mL azoxystrobin (AZO), 20 μg/mL boscalid (BOS), 0.35 μg/mL carbendazim (CAR), 0.4 μg/mL tebuconazole (TEB). (G) Control efficacy of IPT resistant mutants. H08-1a and IPT resistant mutants were inoculated on PDA or PDA amended with 5 μg/mL IPT, 5 μg/mL FLU, combination of 5 μg/mL IPT and 5 μg/mL FLU. Data presented are the mean ± SD (n = 3). Bars followed by the same letter are not significantly different according to a LSD test at P = 0.01.
Fig 3.
Mutation of MoIRR was one of the main causes of IPT resistance in M. oryzae.
(A) Mutations of MoIRR gene in IPT resistant mutants. (B) Percentage of mutants with MoIRR mutations in all resistant mutants. (C) Mutation types in MR-2 mutants. (D) Mutation sites of MoIRR gene in MR-2 mutants. (E) Distribution of mutations in different domains in MR-2 mutants. Bars followed by the same letter are not significantly different according to a LSD test at P = 0.01.
Table 1.
Identification of candidate genes for low IPT resistance in M. oryzae
Fig 4.
MoVelB negatively regulated the low resistance to IPT in M. oryzae.
(A) Identification of IPT resistance loci in the LR mutant 3–15. (B) Phylogenetic tree of MoVelB homologous proteins. The amino acid sequences of proteins in phylogenetic tree were retrieved from the NCBI database. Domains were aligned with ClustalW, and the tree was constructed with the neighbor-joining method. The VelB velvet domain was indicated as green boxes. (C) Knockout transformant ΔVelB (ΔVelB-2) displayed low resistance to IPT. A 3-mm mycelial plug of each strain was inoculated on PDA or PDA amended with 4, 6, 8 μg/ mL IPT and then incubated at 27°C for 5 days (top panel), and the mycelial growth inhibition was calculated for each strain (below panel). (D) Expression of MoVelB in wild type isolate H08-1a at different concentrations of IPT. (E) Expression of MoIRR in different strains with or without IPT. (F) Expression of MoVelB in wild type isolate H08-1a, MoIRR knockout (ΔIRR-1) and overexpression (OEIRR-1) transformants with or without IPT. Ace: acetone, the solvent of IPT; IPT: isoprothiolane. The MoActin gene was used as the internal reference for normalization. Data presented are the mean ± SD (n = 3). Bars followed by the same letter are not significantly different according to a LSD test at P = 0.01.
Fig 5.
MoVelB regulated mycelial growth, sporulation, melanin synthesis, and oxidative stress.
(A) Comparisons in colony morphology among the parental isolate H08-1a, ΔVelB and the complemented transformant ΔVelB-C grown on CM, MM, PDA, or OTA, mycelial growth rate was calculated accordingly. (B) Sporulation of different types of strains. (C) Production of melanin of different strains cultured in PDB for 7 days. (D) The H08-1a, ΔVelB and ΔVelB-C strains were incubated on PDA amended with different stress agents at 27°C for 5 days and statistical analysis of the growth inhibition rate. (E) RT-qPCR analyses of the expression of Cat3 in ΔVelB, compared to H08-1a. The MoActin gene was used as the internal reference for normalization. (F) The H08-1a, ΔVelB and ΔVelB-C strains were incubated on PDA amended with different fungicides at 27°C for 5 days and statistical analysis of the growth inhibition rate. (G, H) RT-qPCR analyses of the expression of Gpd1, Gpp1 in ΔVelB, compared to H08-1a. The MoActin gene was used as the internal reference for normalization. Data presented are the mean ± SD (n = 3). Bars followed by the same letter are not significantly different according to a LSD test, lowercase letters indicate the p-value <0.05 and uppercase letters indicate the p-value <0.01.
Fig 6.
Involvement of velvet family proteins in resistance to IPT in M. oryzae.
(A) Velvet family proteins contain a velvet structural domain identified using the NBCI protein database (https://www.ncbi.nlm.nih.gov/cdd). (B) Knockout of velvet family proteins encoding genes led to reduced sensitivity to IPT. (C) Sensitivity of MoVeA and MoLaeA-related mutants to IPT. (D) Detection of MoVeA expression in OEVeA transformants by RT-qPCR. (E) Detection of MoLaeA expression in OELaeA transformants by RT-qPCR. (F) Expression of MoVeA at different concentrations of IPT. The MoActin gene was used as the internal reference for normalization. (G) The Co-IP assay revealed that MoVelB interacted directly with MoVeA. (H) MoVelB and MoVeA double knockout transformants reduced low resistance to IPT. Data presented are the mean ± SD (n = 3). Bars followed by the same letter are not significantly different according to a LSD test at P = 0.01.
Fig 7.
Involvement of MoVelB-MoVeA-MoLaeA complex in IPT resistance was possibly through regulation of secondary metabolism in M. oryzae.
(A) Volcano plot analysis of DEGs (log2(fold change ≥1 or ≤ -1 and P value of ≤ 0.05) in ΔVelB, compared to H08-1a. (B) KEGG enrichment analysis of DEGs in ΔVelB under IPT pressure. (C) Head map analysis of DEGs (log2(fold change ≥1.5 or ≤ -1.5)) in biosynthesis of secondary metabolites. (D) Regulation of DEGs in biosynthesis of secondary metabolites by MoVelB, MoVeA, MoLaeA determined by RT-qPCR. Data presented are the mean ± SD (n = 3). Statistical significance was determined using Student’s t test with a two-tailed distribution and two-sample unequal variance, *, P ≤ 0.05; **, P ≤ 0.01.
Fig 8.
Model of velvet family proteins in regulating low IPT resistance in M. oryzae.
VelB, VeA, LaeA, and KapA indicate the MoVelB, MoVeA, MoLaeA, and MoKapA, respectively.