Transcriptomic analysis of gene expression of Verticillium dahliae upon treatment of the cotton root exudates

et al. 2020

Preprint

Background. Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains a number of pseudoenzymes predicted to lack enzyme activity and with poorly characterized molecular function. The genome of the yeast S. cerevisiae encodes one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. In order to better understand the function of such pseudoenzymes, we studied the structure and function of Ecm14 in S. cerevisiae. Results. A phylogenetic analysis of Ecm14 in fungi found it to be conserved throughout the ascomycete phylum, with a group of related pseudoenzymes found in basidiomycetes. To investigate the structure and function of this conserved protein, his6-tagged Ecm14 was overexpressed in Sf9 cells and purified. The prodomain of Ecm14 was cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism; however, no activity was detectable using standard carboxypeptidase substrates. In order to determine the function of Ecm14 using an unbiased screen, we undertook a synthetic lethal assay. Upon screening approximately 27,000 yeast colonies, twenty-two putative synthetic lethal clones were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker genes and requiring multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14. Conclusions. We show in this study that Ecm14, although lacking detectable enzyme activity, is a conserved carboxypeptidase-like protein that is secreted from cells and is processed to a mature form by the action of an endopeptidase. Our study and datasets from other recent large-scale screens suggest a role for Ecm14 in processes such as vesicle-mediated transport and aggregate invasion, a fungal process that has been selected against in modern laboratory strains of S. cerevisiae.

Section: Discussionmentioning

confidence: 99%

Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase

et al. 2020

Preprint

“…This hints at a role in aggregate invasion, although the equivalent experiment performed in S. cerevisiae of the Σ1278b background, which can undergo aggregate invasive growth, did not result in a phenotype [44], suggesting either a false positive in the rst screen or the use of inappropriate conditions for this assay with S. cerevisiae. A transcriptomics study of the plant pathogen Verticillium dahliae showed that the expression of Ecm14, along with a variety of cell wall degrading hydrolases, was upregulated in response to exudates from V. dahliaesusceptible cotton roots [45]. This again points to a possible role for Ecm14 in invasion.…”

Section: Discussionmentioning

confidence: 99%

Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase

et al. 2020

Preprint

Background. Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains a number of pseudoenzymes predicted to lack enzyme activity and with poorly characterized molecular function. The genome of the yeast Saccharomyces cerevisiae encodes one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. In order to better understand the function of such pseudoenzymes, we studied the structure and function of Ecm14 in S. cerevisiae. Results. A phylogenetic analysis of Ecm14 in fungi found it to be conserved throughout the ascomycete phylum, with a group of related pseudoenzymes found in basidiomycetes. To investigate the structure and function of this conserved protein, His6-tagged Ecm14 was overexpressed in Sf9 cells and purified. The prodomain of Ecm14 was cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism; however, no activity was detectable using standard carboxypeptidase substrates. In order to determine the function of Ecm14 using an unbiased screen, we undertook a synthetic lethal assay. Upon screening approximately 27,000 yeast colonies, twenty-two putative synthetic lethal clones were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker genes and requiring multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14Δ. Conclusions. We show in this study that Ecm14, although lacking detectable enzyme activity, is a conserved carboxypeptidase-like protein that is secreted from cells and is processed to a mature form by the action of an endopeptidase. Our study and datasets from other recent large-scale screens suggest a role for Ecm14 in processes such as vesicle-mediated transport and aggregate invasion, a fungal process that has been selected against in modern laboratory strains of S. cerevisiae.

“…This hints at a role in aggregate invasion, although the equivalent experiment performed in S. cerevisiae of the Σ1278b background, which can undergo aggregate invasive growth, did not result in a phenotype [ 44 ], suggesting either a false positive in the first screen or the use of inappropriate conditions for this assay with S. cerevisiae . A transcriptomics study of the plant pathogen Verticillium dahliae showed that the expression of Ecm14, along with a variety of cell wall degrading hydrolases, was upregulated in response to exudates from V. dahliae -susceptible cotton roots [ 45 ]. This again points to a possible role for Ecm14 in invasion.…”

Section: Discussionmentioning

confidence: 99%

Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase

et al. 2020

BMC Mol and Cell Biol

Background Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains a number of pseudoenzymes predicted to lack enzyme activity and with poorly characterized molecular function. The genome of the yeast Saccharomyces cerevisiae encodes one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. In order to better understand the function of such pseudoenzymes, we studied the structure and function of Ecm14 in S. cerevisiae. Results A phylogenetic analysis of Ecm14 in fungi found it to be conserved throughout the ascomycete phylum, with a group of related pseudoenzymes found in basidiomycetes. To investigate the structure and function of this conserved protein, His6-tagged Ecm14 was overexpressed in Sf9 cells and purified. The prodomain of Ecm14 was cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism; however, no activity was detectable using standard carboxypeptidase substrates. In order to determine the function of Ecm14 using an unbiased screen, we undertook a synthetic lethal assay. Upon screening approximately 27,000 yeast colonies, twenty-two putative synthetic lethal clones were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker genes and requiring multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14Δ. Conclusions We show in this study that Ecm14, although lacking detectable enzyme activity, is a conserved carboxypeptidase-like protein that is secreted from cells and is processed to a mature form by the action of an endopeptidase. Our study and datasets from other recent large-scale screens suggest a role for Ecm14 in processes such as vesicle-mediated transport and aggregate invasion, a fungal process that has been selected against in modern laboratory strains of S. cerevisiae.