Unknown,Transcriptomics,Genomics,Proteomics

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RNA-sequencing of transgenic Arabidopsis seedlings constitutively expressing a Colletotrichum higginsianum effector

ABSTRACT: The fungal pathogen Colletotrichum higginsianum secretes effector proteins during infection of Arabidopsis. To explore the host processes perturbed by the effector candidate ChEC108 in host plants, transgenic Arabidopsis lines were generated to express AtUBQ10pro:ChEC108-eGFP or AtUBQ10pro:ChEC108^D164R-eGFP in the Col-0 background. D164R refers to a mutation that prevents binding of ChEC108 to the Arabidopsis protein, HIPP6. Three replicate samples (A, B, C) were collected per genotype (Col-0, AtUBQ10pro:ChEC108-eGFP or AtUBQ10pro:ChEC108^D164R-eGFP). Total RNA was extracted from 19-day-old whole seedlings grown on sterile media.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Christine Faulkner

PROVIDER: E-MTAB-16996 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The cholera disease bacterium V. cholerae, can adopt planktonic or biofilm lifestyles depending on the intracellular concentration of the second messenger cyclic diguanylic acid (c-di-GMP). Biofilm formation protects Vibrios from stressful conditions and facilitates disease transmission by enhancing infectivity. The histone-like nucleoid structuring protein (H-NS) is a global regulator of genes associated with pathogenicity and responses to environmental stresses. H-NS represses the transcription of genes vpsT, vpsA and vpsL, which are required for the biosynthesis of the biofilm exopolysacchide matrix. Here we demonstrate that the c-di-GMP-binding protein VpsT disrupts H-NS nucleoprotein complexes at the vpsA and vpsL promoters and that this effect is enhanced by c-di-GMP. We used ChIP coupled with Next Generation Sequencing (ChIP-Seq) and transcriptome analysis (RNA-Seq) to identify additional loci repressed by H-NS affecting biofilm formation. This study showed that H-NS directly represses the transcription of genes encoding proteins present in the biofilm matrix such as the rbmA-F cluster, hemolysin and chitinase. Similar to vpsA and vpsL, the promoter region of vpsU, rbmA and rbmF exhibited overlapping H-NS and VpsT binding motifs. Deletion of vpsT increased H-NS occupancy at the vpsU, vpsA, vpsL, rbmA and rbmF promoters. Conversely, artificially increasing the c-di-GMP pool diminished H-NS occupancy at the above promoters. Deletion of vpsT did not affect H-NS occupancy at its own promoter. However, deletion of genes encoding the regulators AphA and VpsR significantly increased H-NS occupancy at the vpsT promoter. In sum, our study shows that c-di-GMP enhances biofilm formation by acting through VpsT to activate an H-NS anti-repression cascade. The Binding profile of V. cholerae H-NS to the genome was determined by ChIP followed by Next Generation Sequencing (ChIP-Seq) using the Illumina HiSeq2000 platform. V. cholerae C7258 cells expressing H-NS-FLAG fusion protein from the hns transcription and translation signals were collected from LB cultures grown to mid-exponential phase (OD600 0.5). An anti-FLAG Immunoprecipitation (IP) and an Input samples were used for the analysis.

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RNA-sequencing of transgenic Arabidopsis seedlings constitutively expressing a Colletotrichum higginsianum effector

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