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Mitogen-activated protein kinases are associated with the regulation of physiological traits and virulence in Fusarium oxysporum f. sp. cubense.

Ding Z, Li M, Sun F, Xi P, Sun L, Zhang L, Jiang Z - PLoS ONE (2015)

Bottom Line: Transcriptional analysis revealed that the MAP kinase signaling pathway plays a key role in regulation of the genes encoding production of chitin, peroxidase, beauvericin and fusaric acid.Moreover, disruption of the MAP kinase genes resulted in abnormal hypha and increased sensitivity to Congo Red, Calcofluor White and H2O2.Taken together, these results depict the critical roles of MAP kinases in regulation of FOC physiology and virulence.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, South China Agricultural University, Guangzhou 510642, China; Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, China.

ABSTRACT
Fusarium oxysporum f. sp. cubense (FOC) is an important soil-borne fungal pathogen causing devastating vascular wilt disease of banana plants and has become a great concern threatening banana production worldwide. However, little information is known about the molecular mechanisms that govern the expression of virulence determinants of this important fungal pathogen. In this study, we showed that mutation of three mitogen-activated protein (MAP) kinase genes, designated as FoSlt2, FoMkk2 and FoBck1, respectively, led to substantial attenuation in fungal virulence on banana plants. Transcriptional analysis revealed that the MAP kinase signaling pathway plays a key role in regulation of the genes encoding production of chitin, peroxidase, beauvericin and fusaric acid. Biochemical analysis further confirmed the essential role of MAP kinases in modulating the production of fusaric acid, which was a crucial phytotoxin in accelerating development of Fusarium wilt symptoms in banana plants. Additionally, we found that the MAP kinase FoSlt2 was required for siderophore biosynthesis under iron-depletion conditions. Moreover, disruption of the MAP kinase genes resulted in abnormal hypha and increased sensitivity to Congo Red, Calcofluor White and H2O2. Taken together, these results depict the critical roles of MAP kinases in regulation of FOC physiology and virulence.

No MeSH data available.


Related in: MedlinePlus

Hypha morphology of WT, mutants ΔFoSlt2, ΔFoMkk2 and ΔFoBck1, and complemented strains ΔFoSlt2-c and ΔFoMkk2-c.Strains were incubated at 28°C for 4 days on PDA plates. A: ΔFoSlt2, B: ΔFoMkk2, C: ΔFoBck1, D: WT, E: ΔFoSlt2-c, F: ΔFoMkk2-c. Bars: 10μm.
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pone.0122634.g001: Hypha morphology of WT, mutants ΔFoSlt2, ΔFoMkk2 and ΔFoBck1, and complemented strains ΔFoSlt2-c and ΔFoMkk2-c.Strains were incubated at 28°C for 4 days on PDA plates. A: ΔFoSlt2, B: ΔFoMkk2, C: ΔFoBck1, D: WT, E: ΔFoSlt2-c, F: ΔFoMkk2-c. Bars: 10μm.

Mentions: The high similarity of MAP kinase genes among Fusarium species facilitated generation of corresponding mutants in FOC race 4 strain XJZ2. To generate the knockout mutants ΔFoSlt2, ΔFoMkk2 and ΔFoBck1, the upstream and downstream sequences of the MAP kinase genes FoSlt2 (Slt2), FoMkk2 (Mkk2), and FoBck1 (Bck1) were amplified by PCR (S3 Fig, S1 Table), and fused with the hph gene encoding hygromycin resistance, respectively (S3 Fig). Thus these three MAP kinase genes were replaced separately by hph through homologous recombination. The complemented strains ΔFoSlt2-c and ΔFoMkk2-c were generated by cloning of the wild type FoSlt2 and FoMkk2 under the control of native promoter in the vector pMD18-T before inserting a zeocin resistance cassette (S3 Fig). The knockout mutants and complemented strains were selected in the medium containing appropriate antibiotics, and validated by PCR, Southern blot analysis and quantitative real-time PCR (S3–S6 Figs, S1 Table). Bright field microscopy revealed that the three mutants had flexuous hyphal structures and contained more branches compared with WT (Fig 1A–1D), which were restored in the complemented strains ΔFoSlt2-c and ΔFoMkk2-c (Fig 1E and 1F). On PDA or MM plates, the mutants ΔFoSlt2, ΔFoMkk2 and ΔFoBck1 showed similar morphologies with their colonies being smaller and more compact than that of WT (Fig 2A). Additionally, while WT produced abundant aerial hyphae on PDA plates, the three mutants produced fewer and shorter aerial hyphae (Fig 2A). The hyphal growth rate of the three mutants was assayed on MM plates with WT as a control. The results showed that the hyphal growth rate of the three mutants was lower than that of WT (Table 1). Moreover, the colony morphology of the mutants could be restored to the WT levels on MM plates supplemented with 1.2 M sorbitol (Fig 2A), indicating that the integrity of the cell wall is to some extent complemented by osmotic stabilizer.


Mitogen-activated protein kinases are associated with the regulation of physiological traits and virulence in Fusarium oxysporum f. sp. cubense.

Ding Z, Li M, Sun F, Xi P, Sun L, Zhang L, Jiang Z - PLoS ONE (2015)

Hypha morphology of WT, mutants ΔFoSlt2, ΔFoMkk2 and ΔFoBck1, and complemented strains ΔFoSlt2-c and ΔFoMkk2-c.Strains were incubated at 28°C for 4 days on PDA plates. A: ΔFoSlt2, B: ΔFoMkk2, C: ΔFoBck1, D: WT, E: ΔFoSlt2-c, F: ΔFoMkk2-c. Bars: 10μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4388850&req=5

pone.0122634.g001: Hypha morphology of WT, mutants ΔFoSlt2, ΔFoMkk2 and ΔFoBck1, and complemented strains ΔFoSlt2-c and ΔFoMkk2-c.Strains were incubated at 28°C for 4 days on PDA plates. A: ΔFoSlt2, B: ΔFoMkk2, C: ΔFoBck1, D: WT, E: ΔFoSlt2-c, F: ΔFoMkk2-c. Bars: 10μm.
Mentions: The high similarity of MAP kinase genes among Fusarium species facilitated generation of corresponding mutants in FOC race 4 strain XJZ2. To generate the knockout mutants ΔFoSlt2, ΔFoMkk2 and ΔFoBck1, the upstream and downstream sequences of the MAP kinase genes FoSlt2 (Slt2), FoMkk2 (Mkk2), and FoBck1 (Bck1) were amplified by PCR (S3 Fig, S1 Table), and fused with the hph gene encoding hygromycin resistance, respectively (S3 Fig). Thus these three MAP kinase genes were replaced separately by hph through homologous recombination. The complemented strains ΔFoSlt2-c and ΔFoMkk2-c were generated by cloning of the wild type FoSlt2 and FoMkk2 under the control of native promoter in the vector pMD18-T before inserting a zeocin resistance cassette (S3 Fig). The knockout mutants and complemented strains were selected in the medium containing appropriate antibiotics, and validated by PCR, Southern blot analysis and quantitative real-time PCR (S3–S6 Figs, S1 Table). Bright field microscopy revealed that the three mutants had flexuous hyphal structures and contained more branches compared with WT (Fig 1A–1D), which were restored in the complemented strains ΔFoSlt2-c and ΔFoMkk2-c (Fig 1E and 1F). On PDA or MM plates, the mutants ΔFoSlt2, ΔFoMkk2 and ΔFoBck1 showed similar morphologies with their colonies being smaller and more compact than that of WT (Fig 2A). Additionally, while WT produced abundant aerial hyphae on PDA plates, the three mutants produced fewer and shorter aerial hyphae (Fig 2A). The hyphal growth rate of the three mutants was assayed on MM plates with WT as a control. The results showed that the hyphal growth rate of the three mutants was lower than that of WT (Table 1). Moreover, the colony morphology of the mutants could be restored to the WT levels on MM plates supplemented with 1.2 M sorbitol (Fig 2A), indicating that the integrity of the cell wall is to some extent complemented by osmotic stabilizer.

Bottom Line: Transcriptional analysis revealed that the MAP kinase signaling pathway plays a key role in regulation of the genes encoding production of chitin, peroxidase, beauvericin and fusaric acid.Moreover, disruption of the MAP kinase genes resulted in abnormal hypha and increased sensitivity to Congo Red, Calcofluor White and H2O2.Taken together, these results depict the critical roles of MAP kinases in regulation of FOC physiology and virulence.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, South China Agricultural University, Guangzhou 510642, China; Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, China.

ABSTRACT
Fusarium oxysporum f. sp. cubense (FOC) is an important soil-borne fungal pathogen causing devastating vascular wilt disease of banana plants and has become a great concern threatening banana production worldwide. However, little information is known about the molecular mechanisms that govern the expression of virulence determinants of this important fungal pathogen. In this study, we showed that mutation of three mitogen-activated protein (MAP) kinase genes, designated as FoSlt2, FoMkk2 and FoBck1, respectively, led to substantial attenuation in fungal virulence on banana plants. Transcriptional analysis revealed that the MAP kinase signaling pathway plays a key role in regulation of the genes encoding production of chitin, peroxidase, beauvericin and fusaric acid. Biochemical analysis further confirmed the essential role of MAP kinases in modulating the production of fusaric acid, which was a crucial phytotoxin in accelerating development of Fusarium wilt symptoms in banana plants. Additionally, we found that the MAP kinase FoSlt2 was required for siderophore biosynthesis under iron-depletion conditions. Moreover, disruption of the MAP kinase genes resulted in abnormal hypha and increased sensitivity to Congo Red, Calcofluor White and H2O2. Taken together, these results depict the critical roles of MAP kinases in regulation of FOC physiology and virulence.

No MeSH data available.


Related in: MedlinePlus