Project description:RNA-directed DNA methylation (RdDM) plays an essential role in transposable element (TE) silencing in plants. In the Arabidopsis RdDM pathway, the DDR complex containing DRD1, DMS3, and RDM1, is necessary for recruiting Pol V to transcribe scaffold RNA. Although the role of DDR is known, the mechanism by which the DDR complex is regulated remains unexplored. Here, we demonstrate that the Anaphase Promoting Complex/Cyclosome (APC/C) monitors the assembly of the DDR complex by targeting DMS3 for degradation. We show that a subset of Pol V-dependent RdDM loci are de-repressed in apc/c mutants, accompanied by defective recruitment and transcription of Pol V. APC/C targets DMS3 for ubiquitination and degradation in a D box-dependent manner, and the D-box-mutated DMS3 fails to complement the dms3 mutant. Competitive binding assays shows that the dosage of DMS3 is critical for the assembly of the DDR complex, and in vivo gel filtration analysis shows that the assembly of both DDR and Pol V is compromised in the apc8 mutant. These findings uncover a safeguard role of APC/C-mediated DMS3 degradation in the assembly of the DDR complex, and provide a direct link between selective protein degradation and RdDM.
Project description:RNA-directed DNA methylation (RdDM) plays an essential role in transposable element (TE) silencing in plants. In the Arabidopsis RdDM pathway, the DDR complex containing DRD1, DMS3, and RDM1, is necessary for recruiting Pol V to transcribe scaffold RNA. Although the role of DDR is known, the mechanism by which the DDR complex is regulated remains unexplored. Here, we demonstrate that the Anaphase Promoting Complex/Cyclosome (APC/C) monitors the assembly of the DDR complex by targeting DMS3 for degradation. We show that a subset of Pol V-dependent RdDM loci are de-repressed in apc/c mutants, accompanied by defective recruitment and transcription of Pol V. APC/C targets DMS3 for ubiquitination and degradation in a D box-dependent manner, and the D-box-mutated DMS3 fails to complement the dms3 mutant. Competitive binding assays shows that the dosage of DMS3 is critical for the assembly of the DDR complex, and in vivo gel filtration analysis shows that the assembly of both DDR and Pol V is compromised in the apc8 mutant. These findings uncover a safeguard role of APC/C-mediated DMS3 degradation in the assembly of the DDR complex, and provide a direct link between selective protein degradation and RdDM.
Project description:This series represents the gene expression study of phages DT1 and 2972 during the whole process of infection. Gene expression was measured at nine time intervals (0, 2, 7, 12, 17, 22, 27, 32, 37 minutes) during phage infection.
Project description:Some phages encode anti-CRISPR (acr) genes, which antagonize bacterial CRISPR-Cas immune systems by binding components of its machinery, but it is less clear how deployment of these acr genes impacts phage replication and epidemiology. Here, we demonstrate that bacteria with CRISPR-Cas resistance are still partially immune to Acr-encoding phage. As a consequence, Acr-phages often need to cooperate in order to overcome CRISPR resistance, with a first phage blocking the host CRISPR-Cas immune system to allow a second Acr-phage to successfully replicate. This cooperation leads to epidemiological tipping points in which the initial density of Acr-phage tips the balance from phage extinction to a phage epidemic. Furthermore, both higher levels of CRISPR-Cas immunity and weaker Acr activities shift the tipping points toward higher initial phage densities. Collectively, these data help elucidate how interactions between phage-encoded immune suppressors and the CRISPR systems they target shape bacteria-phage population dynamics.