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Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software
Published on: July 24, 2021
Phosphoproteomic and transcriptomic analyses reveal multiple functions for Aspergillus nidulans MpkA independent of
Cynthia L Chelius1, Liliane F C Ribeiro1, Walker Huso1
1Department of Chemical, Biochemical, and Environmental Engineering, University of Maryland Baltimore County, Baltimore, MD 21250, United States.
Abstract:
The protein kinase MpkA plays a prominent role in the cell wall integrity signaling (CWIS) pathway, acting as the terminal MAPK activating expression of genes which encode cell wall biosynthetic enzymes and other repair functions. Numerous studies focus on MpkA function during cell wall perturbation. Here, we focus on the role MpkA plays outside of cell wall stress, during steady state growth. In an effort to seek other, as yet unknown, connections to this pathway, an mpkA deletion mutant (ΔmpkA) was subjected to phosphoproteomic and transcriptomic analysis. When compared to the control (isogenic parent of ΔmpkA), there is strong evidence suggesting MpkA is involved with maintaining cell wall strength, branching regulation, and the iron starvation pathway, among others. Particle-size analysis during shake flask growth revealed ΔmpkA mycelia were about 4 times smaller than the control strain and more than 90 cell wall related genes show significantly altered expression levels. The deletion mutant had a significantly higher branching rate than the control and phosphoproteomic results show putative branching-regulation proteins, such as CotA, LagA, and Cdc24, have a significantly different level of phosphorylation. When grown in iron limited conditions, ΔmpkA had no difference in growth rate or production of siderophores, whereas the control strain showed decreased growth rate and increased siderophore production. Transcriptomic data revealed over 25 iron related genes with altered transcript levels. Results suggest MpkA is involved with regulation of broad cellular functions in the absence of stress.
Insights
The protein kinase MpkA regulates cell wall strength and branching during normal growth. This study reveals MpkA
Area of Science:
- Cellular biology
- Molecular biology
- Biochemistry
Background:
- The protein kinase MpkA is a key component of the cell wall integrity signaling (CWIS) pathway.
- MpkA activates genes involved in cell wall biosynthesis and repair.
- Previous research primarily focused on MpkA's role during cell wall stress.
Purpose of the Study:
- To investigate the function of MpkA during steady-state growth, outside of cell wall stress conditions.
- To identify novel pathways and cellular processes regulated by MpkA.
Main Methods:
- Comparative phosphoproteomic and transcriptomic analysis of an mpkA deletion mutant (ΔmpkA) and its isogenic parent control.
- Particle-size analysis to assess mycelial morphology.
- Growth rate and siderophore production assays under iron-limited conditions.
Main Results:
- ΔmpkA showed significantly smaller mycelia (4x reduction) and altered expression of over 90 cell wall-related genes.
- MpkA deletion led to a higher branching rate, with altered phosphorylation of key branching regulators (CotA, LagA, Cdc24).
- Under iron limitation, ΔmpkA exhibited normal growth and siderophore production, unlike the control strain, with altered expression of over 25 iron-related genes.
Conclusions:
- MpkA plays a significant role in maintaining cell wall strength and regulating hyphal branching during steady-state growth.
- MpkA is involved in the iron starvation response pathway, independent of direct cell wall stress.
- MpkA regulates a broader range of cellular functions than previously recognized, even in the absence of external stress.
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