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The transcriptomic signature of RacA activation and inactivation provides new insights into the morphogenetic network of Aspergillus niger

ABSTRACT: RacA is the main Rho GTPase in Aspergillus niger regulating polarity maintenance via controlling actin dynamics. We have previously shown that both deletion and dominant activation of RacA (RacG18V) provoke an actin localization defect and thereby loss of polarized tip extension. This loss of apical dominance results in frequent dichotomous branching in the racA deletion strain and an apolar growing phenotype for RacG18V. In the current study we investigated the transcriptomics and physiological consequences of these morphological changes and compared the data with our previously established morphogenetic network model for the dichotomous branching mutant ramosa-1. This integrated approach revealed that polar tip growth is most likely orchestrated by the concerted activities of phospholipid signaling, sphingolipid signaling, TORC2 signaling, calcium signaling and CWI signaling pathways. The transcriptomic signatures and the reconstructed network model for all three morphology mutants imply that these pathways become integrated to bring about different physiological adaptations including changes in sterol, zinc and amino acid metabolism and changes in ion transport and protein trafficking. We furthermore followed that fate of exocytotic (SncA) and endocytotic (AbpA, SlaB) markers in the dichotomous branching racA deletion mutant, and provide data demonstrating that hyperbranching does not per se result in increased protein secretion.

ORGANISM(S): Aspergillus niger Aspergillus niger CBS 513.88

PROVIDER: GSE42258 | GEO | 2013/08/25

SECONDARY ACCESSION(S): PRJNA179484

REPOSITORIES: GEO

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Project description:RacA is the main Rho GTPase in Aspergillus niger regulating polarity maintenance via controlling actin dynamics. We have previously shown that both deletion and dominant activation of RacA (RacG18V) provoke an actin localization defect and thereby loss of polarized tip extension. This loss of apical dominance results in frequent dichotomous branching in the racA deletion strain and an apolar growing phenotype for RacG18V. In the current study we investigated the transcriptomics and physiological consequences of these morphological changes and compared the data with our previously established morphogenetic network model for the dichotomous branching mutant ramosa-1. This integrated approach revealed that polar tip growth is most likely orchestrated by the concerted activities of phospholipid signaling, sphingolipid signaling, TORC2 signaling, calcium signaling and CWI signaling pathways. The transcriptomic signatures and the reconstructed network model for all three morphology mutants imply that these pathways become integrated to bring about different physiological adaptations including changes in sterol, zinc and amino acid metabolism and changes in ion transport and protein trafficking. We furthermore followed that fate of exocytotic (SncA) and endocytotic (AbpA, SlaB) markers in the dichotomous branching racA deletion mutant, and provide data demonstrating that hyperbranching does not per se result in increased protein secretion. This data set consists of 14 samples in total and covers the genome-wide transcriptional responses of Aspergillus niger towards deletion of racA and expression of a dominant active racA allele (G18V). The genome-wide transcriptional effect of deleting racA was monitored by comparison with the A. niger wild type. For both, the racA deletion mutant and the wild type, samples consist of biological triplicates (6 in total). The genome-wide transcriptional effect of expressing the dominant active racA mutant allele was monitored by sampling 2 and 4 hours after induction with maltose. For comparison, the racA wild type allele was induced and expressed similarly and samples were taken at corresponding time points. The experimental setup for the dominant active racA expression consisted of biological triplicates (8 in total).

Project description:In this study we used the maize (Zea mays) inflorescence to investigate gene networks that modulate determinacy, specifically the decision to allow branch growth. We characterized developmental transitions by associating spatiotemporal expression profiles with morphological changes resulting from genetic perturbations that disrupt steps in a pathway controlling branching. These are the RNA-seq datasets used in this study. We profiled changes in gene expression during normal maize ear and tassel development and in developing maize ear primordia upon genetic perturbation of the RAMOSA branching pathway. For the wild-type ear and tassel developmental series, greenhouse-grown B73 inbred plants were used. 10mm ears were collected and sectioned as follows from tip to base along the developmental gradient: tip 1mm sampled (tip; Inflorescence Meristem/Spikelet Pair Meristem), next 1mm discarded, next 1mm sampled (mid; Spikelet Meristem), next 2mm discarded, next 2 mm sampled (base; Floral Meristem), and immediately frozen in liquid nitrogen. Sections from ~30 sampled ears were pooled for each of 2 biological replicates to represent tip, mid, and base stages. Tassels were hand-dissected, measured, separated by stage: 1-2mm (stg1), 3-4mm (stg2), and 5-7mm (stg3), and immediately frozen in liquid N. For each stage, ~20-30 tassels were pooled for each of 2 biological replicates. For ramosa mutant series, segregating families (1:1) of ra1-R, ra2-R, and ra3-fea1 mutant alleles, all introgressed at least 6 times into the B73 inbred background, were grown at CSHL Uplands Farm. Field-grown plants were genotyped and collected 6-7 weeks after germination (V7-V8 stage). First and second ear primordia were immediately hand-dissected, measured, and frozen in liquid nitrogen. For ra1, ra2 and ra3 mutants and wild-type controls, ears were pooled into two size classes: 1) 1mm class included a range of 0.7-1.5mm sized ears and nine ears were pooled for each of 2 biological replicates; 2) 2mm class included a range of 1.8-2.5mm sized ears and six ears were pooled for each of three biological replicates. Wild-type samples were proportional mixtures of heterozygote siblings segregating in ra1, ra2, and ra3 populations. Variability factors (e.g. ear size within class, ear rank on the plant, and time of collection) were distributed evenly across pooled samples.

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The transcriptomic signature of RacA activation and inactivation provides new insights into the morphogenetic network of Aspergillus niger

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