Unknown,Transcriptomics,Genomics,Proteomics

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The WOPR protein Ros1 is a master regulator of sporogenesis and effector gene expression in the maize pathogen Ustilago maydis [ChIP-Seq]

ABSTRACT: Goals: characterization of the transcription factor Ros1 in Ustilago maydis Methods: generation of deletion mutants, microscopic observations, ectopic expression of ros1, identification of Ros1 regulated genes by RNAseq and ChIP sequencing Results: Ros1 is not involved in plant colonization but is essential to trigger sporogenesis during late stages of infection. Premature expression of ros1 revealed that Ros1 counteracts the b-dependent filamentation program and induces morphological alterations resembling the early steps of sporogenesis. Transcriptional profiling and ChIP seq analyses revealed that Ros1 affects the expression of about 30 % of all U. maydis genes with 40% being direct targets. Cell wall remodeling and plasma membrane modifications are among the processes affected by Ros1 dependent regulation. Interestingly a large number of b-dependent genes including transcription factors and effector genes involved in biotrophy establishment were downregulated by Ros1 while a subset of novel â??late effectorsâ?? were upregulated. Taken together our results indicate that Ros1 is a master regulator of sporogenesis in U. maydis and that the switch to sporogenesis is accompanied by the differential regulation of 75% of the effector genes. 4 samples were analyzed in triplicate. The first two samples correspond to input and output DNA obtained from ChIP carried out on plant material infected with U. maydis strains expressing Ros1 with an HA tag. The other two other samples correspond to input and output DNA from ChIP carried out on plant material infected with U. maydis strains expressing Ros1 without a tag (control samples).

ORGANISM(S): Ustilago maydis

SUBMITTER: Marie Tollot

PROVIDER: E-GEOD-76230 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Glycosylation is an abundant post-translational modification of both intracellular and extracellular proteins [1]. The majority of glycans are classified as N-linked chains, where the carbohydrate moiety is attached to asparagine residues, or O-linked chains, most commonly linked to a serine or threonine. N-linked glycosylation is initiated by the oligosaccharyltransferase complex with only two paralogs of the catalytic subunit, whereas O-glycan initiation is more complex. There are several types of O-linked glycosylation, but among the most diverse is the mucin or GalNAc type (hereafter referred to as O-glycosylation). O-glycosylation is initiated by 20 evolutionarily conserved polypeptide GalNAc-transferases (GalNAc-Ts), which catalyze the first step in the O-glycosylation of proteins by adding GalNAc residues to threonine, serine, and tyrosine amino acids (Fig 1A). Each of the GalNAc-Ts are differentially expressed in various tissues and have both distinct and overlapping peptide substrate specificities [2-12]. Thus, the repertoire of GalNAc-Ts expressed in a given cell determines the subset and O-glycosite pattern of glycosylated proteins [13]. Substantial efforts have been made to characterize and predict the substrate specificities of GalNAc-Ts in vitro, but understanding of the in vivo specificities of the individual GalNAc-Ts or their biological functions is limited [13-15]. This lack of insight prevents an understanding of how site-specific O-linked glycosylation affects diseases, such as metabolic disorders, cardiovascular disease, and various malignancies, that have been associated with GalNAc-Ts through genome-wide association studies and other linkage studies [16-26]. Therefore, it is imperative that we establish how O-glycosylation at specific sites in proteins affects protein function. A major task in achieving this goal is to identify the non-redundant biological functions of site-specific O-glycosylation. We and others recently developed new strategies for identifying specific sites on proteins that undergo O-glycosylation in different cell types and tissues [27-31]. Characterization of the O-glycoproteomic landscape in isolated human cells and multiple human cell lines suggests that more than 80 % of all proteins that traffic through the secretory pathway are O-glycoproteins [28, 30]. Probing the non-redundant contributions of individual GalNAc-Ts in cells with and without specific GalNAc-Ts [32-34] has revealed broad substrate specificities for some of the individual isoforms, whereas others seem to have very restricted substrate specificities [33-35]. Assessing all of the mapped O-glycosylation sites to identify associations between O-glycosites and protein annotations, we recently found that O-glycans are over-represented close to tandem repeat regions, protease cleavage sites, within propeptides, and on a select group of protein domains [28, 30, 36]. Although such general associations between the location of O-glycans and protein functions may direct future investigations, the strategy does not define the function of site-specific glycosylation. Further progress in discovering and defining novel functions of site-specific glycosylation events requires direct quantitative analysis of potential biological responses induced by the loss of distinct GalNAc-T isoforms, and such biological responses are not easily observed in single cell culture systems. Instead, more complex model systems can be used to examine and dissect the molecular mechanisms underlying the important biological functions of site-specific glycosylation. We previously used an organotypic tissue model equipped with genetically engineered cells to decipher the function of elongated O-glycans [29]. In the present study, we use the model combined with quantitative O-glycoproteomics and phosphoproteomics to perform open-ended discovery of the biological functions of site-specific glycosylation governed by GalNAc-Ts (Fig 1B). With this combinatorial strategy, we demonstrate that loss of individual GalNAc-T isoforms has distinct phenotypic consequences through their effect on distinct biological pathways, suggesting specific roles during epithelial formation.

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The WOPR protein Ros1 is a master regulator of sporogenesis and effector gene expression in the maize pathogen Ustilago maydis [ChIP-Seq]

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