A novel and ubiquitous miRNA-involved regulatory module ensures precise phosphorylation of RNA polymerase II and proper transcription
Fig 5
PcCDK7 directly phosphorylates RNPII and modulates sporangia formation in P. capsici.
(A and B) PcCDK7 was localized in the nucleus. The subcellular localization of PcCDK7 was examined by confocal observation (A) and western blotting based on nuclear-/cytoplasmic-fraction isolation (B). Histone H3 and GAPDH were used as markers of nucleus and cytoplasm, respectively. (C) CDK7 inhibitor THZ1 had no effect on hypha growth in wild-type P. capsici LT1534 (WT) and the PcCDK7 overexpression isolate oePcCDK7. CK represents the DMSO treatment. (D and I) THZ1 could efficiently inhibit sporangia formation in WT but not in oePcCDK7. Sporangia development was impaired in WT grown on V8 medium with THZ1 added, while oePcCDK7 was not influenced by THZ1. Measurement and imaging were performed 4 days after the isolate was inoculated onto the medium. Data are presented as the mean ± SD of 10 biological replicates. Statistical significance compared to water treatment was determined using Student’s t-test (* P < 0.05). Scale bar: 100 μm. (E and F) RNPII Ser5 phosphorylation was elevated in oePcCDK7 than in WT and empty vector isolate (EV). The relative intensity of Ser5-phosphorylated RNPII and unphosphorylated RNPII in the sporangia-generated hypha stage of WT, EV, and oePcCDK7 was quantified with ImageJ. The WT band detected by each antibody was given a value of 1.00. Data presented in F are as the mean ± SD of three replicates. Statistical significance compared to the WT was determined using Student’s t-test (* P < 0.01). β-tubulin was used as the loading control and its intensity in each sample was used to normalize the data between samples. (G and H) THZ1 could repress RNPII Ser5 phosphorylation in P. capsici. LT1534 was grown on V8 medium with or without THZ1 for 5 days, the sporulated hypha was collected and used for protein extraction, and the relative intensity of Ser5-phosphorylated RNPII and unphosphorylated RNPII was quantified by western blotting. The WT-CK band detected by each antibody was given a value of 1.00. Data presented in H are as the mean ± SD of three replicates. Statistical significance compared to the WT-CK was determined using Student’s t-test (* P < 0.01). The protein loading control is shown by Ponceau staining.