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The mRNA stability factor Khd4 defines a specific RNA regulon for membrane trafficking in the pathogen Ustilago maydis [hyperTRIBE]

ABSTRACT: Fungal pathogens depend on sophisticated gene expression programs for successful infection. A crucial component is RNA regulation mediated by RNA-binding proteins. In the biotrophic fungal plant pathogen Ustilago maydis, loss of the multi KH domain containing RBP, Khd4, causes aberrant cell morphology, disturbed hyphal growth and impaired virulence. To investigate the role of Khd4 in the polar growth of infectious hyphae, we established the RNA-editing based hyperTRIBE method to detect in vivo mRNA targets.This technique is especially suited to investigate proteins of high molecular weight or low expression that are difficult to purify (McMahon et al., 2016, Xu et al., 2018, Rahman et al., 2018). HyperTRIBE exploits the RNA editing activity of the catalytic domain of ADAR (Adenosine Deaminase Acting on RNA) from D. melanogaster that additionally carries the mutation E488Q to increase editing (Kuttan and Bass, 2012; designated Ada). We fused the codon-optimized catalytic Ada domain with the green fluorescent protein (see Materials and methods; enhanced version, designated Gfp, Clontech) to the C terminus of Khd4 (Khd4-Ada-Gfp). As a control for background editing, we used the strain expressing Ada-Gfp protein with N-terminal Kat fusion in the wildtype background (control-Ada).

ORGANISM(S): Mycosarcoma maydis

PROVIDER: GSE224485 | GEO | 2023/07/26

REPOSITORIES: GEO

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The mRNA stability factor Khd4 defines a specific RNA regulon for membrane trafficking in the pathogen Ustilago maydis [RNA-Seq]

Project description:Fungal pathogens depend on sophisticated gene expression programs for successful infection. A crucial component is RNA regulation mediated by RNA-binding proteins. In the biotrophic fungal plant pathogen Ustilago maydis, loss of the multi KH domain containing RBP, Khd4, causes aberrant cell morphology, disturbed hyphal growth and impaired virulence. To investigate the role of Khd4 in the polar growth of infectious hyphae, we established the RNA-editing based hyperTRIBE method to detect in vivo mRNA targets.This technique is especially suited to investigate proteins of high molecular weight or low expression that are difficult to purify (McMahon et al., 2016, Xu et al., 2018, Rahman et al., 2018). HyperTRIBE exploits the RNA editing activity of the catalytic domain of ADAR (Adenosine Deaminase Acting on RNA) from D. melanogaster that additionally carries the mutation E488Q to increase editing (Kuttan and Bass, 2012; designated Ada). We fused the codon-optimized catalytic Ada domain with the green fluorescent protein (see Materials and methods; enhanced version, designated Gfp, Clontech) to the C terminus of Khd4 (Khd4-Ada-Gfp). As a control for background editing, we used the strain expressing Ada-Gfp protein with N-terminal Kat fusion in the wildtype background (control-Ada). We then performed differential gene expression analysis to determine the pathological consequence of dysregulated Khd4-mediated mRNA regulation.

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