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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

Expression and subcellular localization of Cdc2A-GFP and Cdc2B-GFP fusion proteins.(A) Conidia and germlings (12 h) of the CDC2A-GFP (C2A-N4) and CDC2B-GFP (C2B-N2) transformants were examined by differential interference contrast (DIC) and epifluorescence (GFP) microscopy. The wild type PH-1 is shown as a negative control. Bar = 20 μm. (B) Western blots of Cdc2A-GFP (64.8 kDa) and Cdc2B-GFP (63.8 kDa) expression in C2A-N4 and C2B-N2 transformants, respectively, detected with the anti-GFP antibody. PH-1 is shown as a negative control. (C) Infectious hyphae formed by the CDC2A-GFP and CDC2B-GFP transformants inside wheat coleoptiles 48 hpi. GFP signals were observed in the nucleus in the CDC2A-GFP but not CDC2B-GFP transformant. Bar = 10 μm. (D) Ascospores of the CDC2A-GFP transformant. The young ascospore contains two nuclei had stronger GFP signals in the nucleus than the other mature ascospores with four nuclei. Bar = 20 μm.
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ppat.1004913.g006: Expression and subcellular localization of Cdc2A-GFP and Cdc2B-GFP fusion proteins.(A) Conidia and germlings (12 h) of the CDC2A-GFP (C2A-N4) and CDC2B-GFP (C2B-N2) transformants were examined by differential interference contrast (DIC) and epifluorescence (GFP) microscopy. The wild type PH-1 is shown as a negative control. Bar = 20 μm. (B) Western blots of Cdc2A-GFP (64.8 kDa) and Cdc2B-GFP (63.8 kDa) expression in C2A-N4 and C2B-N2 transformants, respectively, detected with the anti-GFP antibody. PH-1 is shown as a negative control. (C) Infectious hyphae formed by the CDC2A-GFP and CDC2B-GFP transformants inside wheat coleoptiles 48 hpi. GFP signals were observed in the nucleus in the CDC2A-GFP but not CDC2B-GFP transformant. Bar = 10 μm. (D) Ascospores of the CDC2A-GFP transformant. The young ascospore contains two nuclei had stronger GFP signals in the nucleus than the other mature ascospores with four nuclei. Bar = 20 μm.

Mentions: In both CDC2A- and CDC2B-GFP transformants, GFP signals were observed in conidia and vegetative hyphae, which is consistent with the fact that these two genes are constitutively expressed. In general, GFP signals appeared to be stronger in the CDC2A-GFP transformant than in the CDC2B-GFP transformant (Fig 6A). Western blot analysis confirmed the lower expression level of Cdc2B-GFP fusion compared to Cdc2A-GFP (Fig 6B). The Cdc2A-GFP fusion proteins were distributed throughout the cell but significantly enriched in the nucleus (Fig 6A and S6 Fig). In comparison with un-germinated conidia, young germ tubes had stronger GFP signals in the nucleus (Fig 6A), indicating that localization of Cdc2A to the nucleus was increased when nuclear division becomes active. In contrast, no significant changes in the subcellular localization of GFP signals were observed in the CDC2B-GFP transformant in conidia and germ tubes (Fig 6A and S6 Fig). GFP signals in the nucleus were faint in the cdc2B/CDC2B-GFP transformant.


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)

Expression and subcellular localization of Cdc2A-GFP and Cdc2B-GFP fusion proteins.(A) Conidia and germlings (12 h) of the CDC2A-GFP (C2A-N4) and CDC2B-GFP (C2B-N2) transformants were examined by differential interference contrast (DIC) and epifluorescence (GFP) microscopy. The wild type PH-1 is shown as a negative control. Bar = 20 μm. (B) Western blots of Cdc2A-GFP (64.8 kDa) and Cdc2B-GFP (63.8 kDa) expression in C2A-N4 and C2B-N2 transformants, respectively, detected with the anti-GFP antibody. PH-1 is shown as a negative control. (C) Infectious hyphae formed by the CDC2A-GFP and CDC2B-GFP transformants inside wheat coleoptiles 48 hpi. GFP signals were observed in the nucleus in the CDC2A-GFP but not CDC2B-GFP transformant. Bar = 10 μm. (D) Ascospores of the CDC2A-GFP transformant. The young ascospore contains two nuclei had stronger GFP signals in the nucleus than the other mature ascospores with four nuclei. Bar = 20 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4470668&req=5

ppat.1004913.g006: Expression and subcellular localization of Cdc2A-GFP and Cdc2B-GFP fusion proteins.(A) Conidia and germlings (12 h) of the CDC2A-GFP (C2A-N4) and CDC2B-GFP (C2B-N2) transformants were examined by differential interference contrast (DIC) and epifluorescence (GFP) microscopy. The wild type PH-1 is shown as a negative control. Bar = 20 μm. (B) Western blots of Cdc2A-GFP (64.8 kDa) and Cdc2B-GFP (63.8 kDa) expression in C2A-N4 and C2B-N2 transformants, respectively, detected with the anti-GFP antibody. PH-1 is shown as a negative control. (C) Infectious hyphae formed by the CDC2A-GFP and CDC2B-GFP transformants inside wheat coleoptiles 48 hpi. GFP signals were observed in the nucleus in the CDC2A-GFP but not CDC2B-GFP transformant. Bar = 10 μm. (D) Ascospores of the CDC2A-GFP transformant. The young ascospore contains two nuclei had stronger GFP signals in the nucleus than the other mature ascospores with four nuclei. Bar = 20 μm.
Mentions: In both CDC2A- and CDC2B-GFP transformants, GFP signals were observed in conidia and vegetative hyphae, which is consistent with the fact that these two genes are constitutively expressed. In general, GFP signals appeared to be stronger in the CDC2A-GFP transformant than in the CDC2B-GFP transformant (Fig 6A). Western blot analysis confirmed the lower expression level of Cdc2B-GFP fusion compared to Cdc2A-GFP (Fig 6B). The Cdc2A-GFP fusion proteins were distributed throughout the cell but significantly enriched in the nucleus (Fig 6A and S6 Fig). In comparison with un-germinated conidia, young germ tubes had stronger GFP signals in the nucleus (Fig 6A), indicating that localization of Cdc2A to the nucleus was increased when nuclear division becomes active. In contrast, no significant changes in the subcellular localization of GFP signals were observed in the CDC2B-GFP transformant in conidia and germ tubes (Fig 6A and S6 Fig). GFP signals in the nucleus were faint in the cdc2B/CDC2B-GFP transformant.

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