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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

Assays for inhibitory phosphorylation of Cdc2A and Cdc2B and defects of the Fgwee1 deletion mutants in plant infection.(A) Total proteins were isolated from the wild-type (PH-1), CDC2A-GFP (C2A-N4) and CDC2B-GFP (C2B-N1 and C2B-N2) transformants, and the Fgcak1 (AK2) and Fgwee1 (WE6) deletion mutants. The Cdc2A, Cdc2B, Cdc2A-GFP and Cdc2B-GFP proteins were detected with the Cdc2 p34 (PSTAIRE) antibody. The phosphorylation of them at tyrosine 15 was detected with the Phospho-Cdc2 (Tyr15) antibody. The band of Cdc2A (36.8 kDa), Cdc2B (35.8 kDa), Cdc2A-GFP (64.8 kDa) and Cdc2B-GFP (63.8 kDa) is indicated. (B) Flowering wheat heads inoculated with the wild type (PH-1) and Fgwee1 mutant (WE6). Disease symptoms were examined 14 dpi.
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ppat.1004913.g008: Assays for inhibitory phosphorylation of Cdc2A and Cdc2B and defects of the Fgwee1 deletion mutants in plant infection.(A) Total proteins were isolated from the wild-type (PH-1), CDC2A-GFP (C2A-N4) and CDC2B-GFP (C2B-N1 and C2B-N2) transformants, and the Fgcak1 (AK2) and Fgwee1 (WE6) deletion mutants. The Cdc2A, Cdc2B, Cdc2A-GFP and Cdc2B-GFP proteins were detected with the Cdc2 p34 (PSTAIRE) antibody. The phosphorylation of them at tyrosine 15 was detected with the Phospho-Cdc2 (Tyr15) antibody. The band of Cdc2A (36.8 kDa), Cdc2B (35.8 kDa), Cdc2A-GFP (64.8 kDa) and Cdc2B-GFP (63.8 kDa) is indicated. (B) Flowering wheat heads inoculated with the wild type (PH-1) and Fgwee1 mutant (WE6). Disease symptoms were examined 14 dpi.

Mentions: Inhibitory phosphorylation at the well-conserved Tyr15 is known to affect CDK activation and nuclear localization [27]. In Ustilago maydis inhibitory phosphorylation of Cdk1 is required for conjugation tube formation and plant penetration [28]. Therefore, we used the anti-Tyr15 antibody [29, 30] to assay inhibitory phosphorylation levels of Cdc2A and Cdc2B in F. graminearum. The expression level of Cdc2A in the two cdc2B/CDC2B-GFP transformants (C2B-N1 and C2B-N2) was comparable to that of Cdc2B in the cdc2A/CDC2A-GFP transformant (C2A-N4). However, the inhibitory phosphorylation level of Cdc2A was obviously higher than that of Cdc2B (Fig 8A). This result was confirmed by at least three independent phosphorylation assays.


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)

Assays for inhibitory phosphorylation of Cdc2A and Cdc2B and defects of the Fgwee1 deletion mutants in plant infection.(A) Total proteins were isolated from the wild-type (PH-1), CDC2A-GFP (C2A-N4) and CDC2B-GFP (C2B-N1 and C2B-N2) transformants, and the Fgcak1 (AK2) and Fgwee1 (WE6) deletion mutants. The Cdc2A, Cdc2B, Cdc2A-GFP and Cdc2B-GFP proteins were detected with the Cdc2 p34 (PSTAIRE) antibody. The phosphorylation of them at tyrosine 15 was detected with the Phospho-Cdc2 (Tyr15) antibody. The band of Cdc2A (36.8 kDa), Cdc2B (35.8 kDa), Cdc2A-GFP (64.8 kDa) and Cdc2B-GFP (63.8 kDa) is indicated. (B) Flowering wheat heads inoculated with the wild type (PH-1) and Fgwee1 mutant (WE6). Disease symptoms were examined 14 dpi.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4470668&req=5

ppat.1004913.g008: Assays for inhibitory phosphorylation of Cdc2A and Cdc2B and defects of the Fgwee1 deletion mutants in plant infection.(A) Total proteins were isolated from the wild-type (PH-1), CDC2A-GFP (C2A-N4) and CDC2B-GFP (C2B-N1 and C2B-N2) transformants, and the Fgcak1 (AK2) and Fgwee1 (WE6) deletion mutants. The Cdc2A, Cdc2B, Cdc2A-GFP and Cdc2B-GFP proteins were detected with the Cdc2 p34 (PSTAIRE) antibody. The phosphorylation of them at tyrosine 15 was detected with the Phospho-Cdc2 (Tyr15) antibody. The band of Cdc2A (36.8 kDa), Cdc2B (35.8 kDa), Cdc2A-GFP (64.8 kDa) and Cdc2B-GFP (63.8 kDa) is indicated. (B) Flowering wheat heads inoculated with the wild type (PH-1) and Fgwee1 mutant (WE6). Disease symptoms were examined 14 dpi.
Mentions: Inhibitory phosphorylation at the well-conserved Tyr15 is known to affect CDK activation and nuclear localization [27]. In Ustilago maydis inhibitory phosphorylation of Cdk1 is required for conjugation tube formation and plant penetration [28]. Therefore, we used the anti-Tyr15 antibody [29, 30] to assay inhibitory phosphorylation levels of Cdc2A and Cdc2B in F. graminearum. The expression level of Cdc2A in the two cdc2B/CDC2B-GFP transformants (C2B-N1 and C2B-N2) was comparable to that of Cdc2B in the cdc2A/CDC2A-GFP transformant (C2A-N4). However, the inhibitory phosphorylation level of Cdc2A was obviously higher than that of Cdc2B (Fig 8A). This result was confirmed by at least three independent phosphorylation assays.

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