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Two Cdc2 Kinase Genes with Distinct Functions in Vegetative and Infectious Hyphae in Fusarium graminearum.

Liu H, Zhang S, Ma J, Dai Y, Li C, Lyu X, Wang C, Xu JR - PLoS Pathog. (2015)

Bottom Line: However, whereas the cdc2B mutant was normal, the cdc2A mutant was significantly reduced in virulence and rarely produced ascospores.Although deletion of CDC2A had no obvious effect on the formation of penetration branches or hyphopodia, the cdc2A mutant was limited in the differentiation and growth of infectious growth in wheat tissues.Overall, our data indicate that cell cycle regulation is different between vegetative and infectious hyphae in F. graminearum and Cdc2A, possibly by interacting with a stage-specific cyclin, plays a more important role than Cdc2B during ascosporogenesis and plant infection.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest Agriculture and Forestry University, Yangling, Shaanxi, China; Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana, United States of America.

ABSTRACT
Eukaryotic cell cycle involves a number of protein kinases important for the onset and progression through mitosis, most of which are well characterized in the budding and fission yeasts and conserved in other fungi. However, unlike the model yeast and filamentous fungi that have a single Cdc2 essential for cell cycle progression, the wheat scab fungus Fusarium graminearum contains two CDC2 orthologs. The cdc2A and cdc2B mutants had no obvious defects in growth rate and conidiation but deletion of both of them is lethal, indicating that these two CDC2 orthologs have redundant functions during vegetative growth and asexual reproduction. However, whereas the cdc2B mutant was normal, the cdc2A mutant was significantly reduced in virulence and rarely produced ascospores. Although deletion of CDC2A had no obvious effect on the formation of penetration branches or hyphopodia, the cdc2A mutant was limited in the differentiation and growth of infectious growth in wheat tissues. Therefore, CDC2A plays stage-specific roles in cell cycle regulation during infectious growth and sexual reproduction. Both CDC2A and CDC2B are constitutively expressed but only CDC2A was up-regulated during plant infection and ascosporogenesis. Localization of Cdc2A- GFP to the nucleus but not Cdc2B-GFP was observed in vegetative hyphae, ascospores, and infectious hyphae. Complementation assays with chimeric fusion constructs showed that both the N- and C-terminal regions of Cdc2A are important for its functions in pathogenesis and ascosporogenesis but only the N-terminal region is important for its subcellular localization. Among the Sordariomycetes, only three Fusarium species closely related to F. graminearum have two CDC2 genes. Furthermore, F. graminearum uniquely has two Aurora kinase genes and one additional putative cyclin gene, and its orthologs of CAK1 and other four essential mitotic kinases in the budding yeast are dispensable for viability. Overall, our data indicate that cell cycle regulation is different between vegetative and infectious hyphae in F. graminearum and Cdc2A, possibly by interacting with a stage-specific cyclin, plays a more important role than Cdc2B during ascosporogenesis and plant infection.

No MeSH data available.


Related in: MedlinePlus

Yeast two-hybrid and co-immunoprecipitation (co-IP) assays for the interaction of FgCak1 with Cdc2A and Cdc2B.(A) Different concentrations of yeast cells (cells/ml) of the transformants expressing the marked bait and prey constructs (labelled on the left) were assayed for growth on SD-His plates. The positive and negative controls are shown. The auto-activation controls AD-empty+Cdc2A and AD-empty+Cdc2B are also shown. The same set of yeast transformants was assayed for β-galactosidase (LacZ) activities. (B) Co-IP assays. Immunoblots of total proteins (Total) and proteins eluted from the anti-FLAG M2 beads (Elution) from F. graminearum transformants expressing the GFP fusion construct alone or co-expressing GFP and 3xFLAG fusion constructs as labeled on the top. Western blots were detected with anti-FLAG, anti-GFP or anti-Histone H3 antibody. Total proteins isolated from the wild-type (WT) PH-1 were included as the control.
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ppat.1004913.g009: Yeast two-hybrid and co-immunoprecipitation (co-IP) assays for the interaction of FgCak1 with Cdc2A and Cdc2B.(A) Different concentrations of yeast cells (cells/ml) of the transformants expressing the marked bait and prey constructs (labelled on the left) were assayed for growth on SD-His plates. The positive and negative controls are shown. The auto-activation controls AD-empty+Cdc2A and AD-empty+Cdc2B are also shown. The same set of yeast transformants was assayed for β-galactosidase (LacZ) activities. (B) Co-IP assays. Immunoblots of total proteins (Total) and proteins eluted from the anti-FLAG M2 beads (Elution) from F. graminearum transformants expressing the GFP fusion construct alone or co-expressing GFP and 3xFLAG fusion constructs as labeled on the top. Western blots were detected with anti-FLAG, anti-GFP or anti-Histone H3 antibody. Total proteins isolated from the wild-type (WT) PH-1 were included as the control.

Mentions: In addition to binding with cyclins, the complete activation of Cdc28 requires phosphorylation by the Cak1 kinase at the conserved T residue in the T-loop in S. cerevisiae [3, 4]. A single CAK1 ortholog (FGSG_04947) was identified in F. graminearum. In yeast two-hybrid assays, FgCak1 interacted with both Cdc2A and Cdc2B (Fig 9A). To confirm their interactions by co-IP assays, the FgCAK1-3xFLAG construct was co-transformed with the CDC2A- or CDC2B-GFP fusion construct into PH-1. In the resulting transformants (Table 1), the Cdc2A-GFP or Cdc2B-GFP band could be detected with an anti-GFP antibody in total proteins and proteins eluted from anti-FLAG beads (Fig 9B). These results indicate that both Cdc2A and Cdc2B interacted with FgCak1.


Two Cdc2 Kinase Genes with Distinct Functions in Vegetative and Infectious Hyphae in Fusarium graminearum.

Liu H, Zhang S, Ma J, Dai Y, Li C, Lyu X, Wang C, Xu JR - PLoS Pathog. (2015)

Yeast two-hybrid and co-immunoprecipitation (co-IP) assays for the interaction of FgCak1 with Cdc2A and Cdc2B.(A) Different concentrations of yeast cells (cells/ml) of the transformants expressing the marked bait and prey constructs (labelled on the left) were assayed for growth on SD-His plates. The positive and negative controls are shown. The auto-activation controls AD-empty+Cdc2A and AD-empty+Cdc2B are also shown. The same set of yeast transformants was assayed for β-galactosidase (LacZ) activities. (B) Co-IP assays. Immunoblots of total proteins (Total) and proteins eluted from the anti-FLAG M2 beads (Elution) from F. graminearum transformants expressing the GFP fusion construct alone or co-expressing GFP and 3xFLAG fusion constructs as labeled on the top. Western blots were detected with anti-FLAG, anti-GFP or anti-Histone H3 antibody. Total proteins isolated from the wild-type (WT) PH-1 were included as the control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4470668&req=5

ppat.1004913.g009: Yeast two-hybrid and co-immunoprecipitation (co-IP) assays for the interaction of FgCak1 with Cdc2A and Cdc2B.(A) Different concentrations of yeast cells (cells/ml) of the transformants expressing the marked bait and prey constructs (labelled on the left) were assayed for growth on SD-His plates. The positive and negative controls are shown. The auto-activation controls AD-empty+Cdc2A and AD-empty+Cdc2B are also shown. The same set of yeast transformants was assayed for β-galactosidase (LacZ) activities. (B) Co-IP assays. Immunoblots of total proteins (Total) and proteins eluted from the anti-FLAG M2 beads (Elution) from F. graminearum transformants expressing the GFP fusion construct alone or co-expressing GFP and 3xFLAG fusion constructs as labeled on the top. Western blots were detected with anti-FLAG, anti-GFP or anti-Histone H3 antibody. Total proteins isolated from the wild-type (WT) PH-1 were included as the control.
Mentions: In addition to binding with cyclins, the complete activation of Cdc28 requires phosphorylation by the Cak1 kinase at the conserved T residue in the T-loop in S. cerevisiae [3, 4]. A single CAK1 ortholog (FGSG_04947) was identified in F. graminearum. In yeast two-hybrid assays, FgCak1 interacted with both Cdc2A and Cdc2B (Fig 9A). To confirm their interactions by co-IP assays, the FgCAK1-3xFLAG construct was co-transformed with the CDC2A- or CDC2B-GFP fusion construct into PH-1. In the resulting transformants (Table 1), the Cdc2A-GFP or Cdc2B-GFP band could be detected with an anti-GFP antibody in total proteins and proteins eluted from anti-FLAG beads (Fig 9B). These results indicate that both Cdc2A and Cdc2B interacted with FgCak1.

Bottom Line: However, whereas the cdc2B mutant was normal, the cdc2A mutant was significantly reduced in virulence and rarely produced ascospores.Although deletion of CDC2A had no obvious effect on the formation of penetration branches or hyphopodia, the cdc2A mutant was limited in the differentiation and growth of infectious growth in wheat tissues.Overall, our data indicate that cell cycle regulation is different between vegetative and infectious hyphae in F. graminearum and Cdc2A, possibly by interacting with a stage-specific cyclin, plays a more important role than Cdc2B during ascosporogenesis and plant infection.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest Agriculture and Forestry University, Yangling, Shaanxi, China; Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana, United States of America.

ABSTRACT
Eukaryotic cell cycle involves a number of protein kinases important for the onset and progression through mitosis, most of which are well characterized in the budding and fission yeasts and conserved in other fungi. However, unlike the model yeast and filamentous fungi that have a single Cdc2 essential for cell cycle progression, the wheat scab fungus Fusarium graminearum contains two CDC2 orthologs. The cdc2A and cdc2B mutants had no obvious defects in growth rate and conidiation but deletion of both of them is lethal, indicating that these two CDC2 orthologs have redundant functions during vegetative growth and asexual reproduction. However, whereas the cdc2B mutant was normal, the cdc2A mutant was significantly reduced in virulence and rarely produced ascospores. Although deletion of CDC2A had no obvious effect on the formation of penetration branches or hyphopodia, the cdc2A mutant was limited in the differentiation and growth of infectious growth in wheat tissues. Therefore, CDC2A plays stage-specific roles in cell cycle regulation during infectious growth and sexual reproduction. Both CDC2A and CDC2B are constitutively expressed but only CDC2A was up-regulated during plant infection and ascosporogenesis. Localization of Cdc2A- GFP to the nucleus but not Cdc2B-GFP was observed in vegetative hyphae, ascospores, and infectious hyphae. Complementation assays with chimeric fusion constructs showed that both the N- and C-terminal regions of Cdc2A are important for its functions in pathogenesis and ascosporogenesis but only the N-terminal region is important for its subcellular localization. Among the Sordariomycetes, only three Fusarium species closely related to F. graminearum have two CDC2 genes. Furthermore, F. graminearum uniquely has two Aurora kinase genes and one additional putative cyclin gene, and its orthologs of CAK1 and other four essential mitotic kinases in the budding yeast are dispensable for viability. Overall, our data indicate that cell cycle regulation is different between vegetative and infectious hyphae in F. graminearum and Cdc2A, possibly by interacting with a stage-specific cyclin, plays a more important role than Cdc2B during ascosporogenesis and plant infection.

No MeSH data available.


Related in: MedlinePlus