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ChLae1 and ChVel1 regulate T-toxin production, virulence, oxidative stress response, and development of the maize pathogen Cochliobolus heterostrophus.

Wu D, Oide S, Zhang N, Choi MY, Turgeon BG - PLoS Pathog. (2012)

Bottom Line: T-toxin production is significantly increased in the dark in wild type (WT), whereas Chvel1 and Chlae1 mutant toxin levels are much reduced in the dark compared to WT.Deletion of ChLAE1 or ChVEL1 reduces tolerance to H(2)O(2).ChLae1 and ChVel1 repress expression of 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis genes, and, accordingly, melanization is enhanced in Chlae1 and Chvel1 mutants, and reduced in OE strains.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Plant Pathology & Plant-Microbe Biology, Cornell University, Ithaca, New York, USA.

ABSTRACT
LaeA and VeA coordinate secondary metabolism and differentiation in response to light signals in Aspergillus spp. Their orthologs, ChLae1 and ChVel1, were identified in the maize pathogen Cochliobolus heterostrophus, known to produce a wealth of secondary metabolites, including the host selective toxin, T-toxin. Produced by race T, T-toxin promotes high virulence to maize carrying Texas male sterile cytoplasm (T-cms). T-toxin production is significantly increased in the dark in wild type (WT), whereas Chvel1 and Chlae1 mutant toxin levels are much reduced in the dark compared to WT. Correspondingly, expression of T-toxin biosynthetic genes (Tox1) is up-regulated in the dark in WT, while dark-induced expression is much reduced/minimal in Chvel1 and Chlae1 mutants. Toxin production and Tox1 gene expression are increased in ChVEL1 overexpression (OE) strains grown in the dark and in ChLAE1 strains grown in either light or dark, compared to WT. These observations establish ChLae1 and ChVel1 as the first factors known to regulate host selective toxin production. Virulence of Chlae1 and Chvel1 mutants and OE strains is altered on both T-cms and normal cytoplasm maize, indicating that both T-toxin mediated super virulence and basic pathogenic ability are affected. Deletion of ChLAE1 or ChVEL1 reduces tolerance to H(2)O(2). Expression of CAT3, one of the three catalase genes, is reduced in the Chvel1 mutant. Chlae1 and Chvel1 mutants also show decreased aerial hyphal growth, increased asexual sporulation and female sterility. ChLAE1 OE strains are female sterile, while ChVEL1 OE strains are more fertile than WT. ChLae1 and ChVel1 repress expression of 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis genes, and, accordingly, melanization is enhanced in Chlae1 and Chvel1 mutants, and reduced in OE strains. Thus, ChLae1 and ChVel1 positively regulate T-toxin biosynthesis, pathogenicity and super virulence, oxidative stress responses, sexual development, and aerial hyphal growth, and negatively control melanin biosynthesis and asexual differentiation.

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Lae1 and Vel1 proteins negatively regulate mycelial pigmentation of C. heterostrophus.A. Bottom of culture plates incubated in constant light or dark for 9 days. Pictures were taken after removal of conidia. Note heavy pigmentation of mycelia of Chlae1 and Chvel1 mutants in both light and dark compared to WT. B. Mycelial pellet of WT strain C4, Chvel1, Chlae1 mutants, and complemented strains (vel1[VEL1], lae1[LAE1]) at different time points indicated. Supernatant is shown only for the 64 hour samples. Chlae1 strain is melanized by 40 hrs, clearly ahead of other strains. Melanization starts by 48 hrs in Chvel1, while pigmentation is not evident in WT at this time. The supernatant of the Chlae1 strain is pigmented indicating secretion of pigment(s) into the medium (no conidia are present). C. qPCR analyses of PKS18. Expression was examined for the samples from B at 40 and 48 hours. Expression level relative to the WT strain C4 sample at 40 hours is shown. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Single asterisks indicate p-value <0.05, double asterisks indicate p-value <0.001 in T-test analysis in which each strain was compared with corresponding WT C4 at the same time point. Matching the enhanced pigmentation (B), PKS18 is induced in Chlae1 by 40 hrs and in Chvel1 by 48 hrs. D. qPCR analyses of CMR1. Expression was examined for the samples from B at 40 and 48 hours. Expression level relative to the WT, C4 sample at 40 hours is shown. Error bars and T-test analysis are same as C. Like the enhanced pigmentation in B, CMR1 expression increased 1.7 and 3.8 fold in Chvel1 and Chlae1 mutants, respectively, compared to WT by 48 hrs. Note scale is different from C.
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ppat-1002542-g007: Lae1 and Vel1 proteins negatively regulate mycelial pigmentation of C. heterostrophus.A. Bottom of culture plates incubated in constant light or dark for 9 days. Pictures were taken after removal of conidia. Note heavy pigmentation of mycelia of Chlae1 and Chvel1 mutants in both light and dark compared to WT. B. Mycelial pellet of WT strain C4, Chvel1, Chlae1 mutants, and complemented strains (vel1[VEL1], lae1[LAE1]) at different time points indicated. Supernatant is shown only for the 64 hour samples. Chlae1 strain is melanized by 40 hrs, clearly ahead of other strains. Melanization starts by 48 hrs in Chvel1, while pigmentation is not evident in WT at this time. The supernatant of the Chlae1 strain is pigmented indicating secretion of pigment(s) into the medium (no conidia are present). C. qPCR analyses of PKS18. Expression was examined for the samples from B at 40 and 48 hours. Expression level relative to the WT strain C4 sample at 40 hours is shown. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Single asterisks indicate p-value <0.05, double asterisks indicate p-value <0.001 in T-test analysis in which each strain was compared with corresponding WT C4 at the same time point. Matching the enhanced pigmentation (B), PKS18 is induced in Chlae1 by 40 hrs and in Chvel1 by 48 hrs. D. qPCR analyses of CMR1. Expression was examined for the samples from B at 40 and 48 hours. Expression level relative to the WT, C4 sample at 40 hours is shown. Error bars and T-test analysis are same as C. Like the enhanced pigmentation in B, CMR1 expression increased 1.7 and 3.8 fold in Chvel1 and Chlae1 mutants, respectively, compared to WT by 48 hrs. Note scale is different from C.

Mentions: Unlike A. nidulans and A. fumigatus LaeA, deletion of which leads to loss of mycelial pigmentation [12], lack of either ChLae1 or ChVel1 caused increased pigmentation of mycelia grown either on solid (Figure 7A) or in liquid (Figure 7B) medium, in both dark and light, indicating that ChLae1 and ChVel1 negatively regulate mycelial pigmentation in C. heterostrophus. Pigment was also evident in the supernatant of the Chlae1 mutant at 64 hrs (Figure 7B) suggesting that it is secreted into the medium. Hyphal pigmentation develops more slowly in the Chvel1 mutant than in the Chlae1 mutant, but faster than in WT. By 64 hrs, all strains were darkly pigmented. Reintroduction of ChVEL1 or ChLAE1 restored the WT phenotype (Figure 7B).


ChLae1 and ChVel1 regulate T-toxin production, virulence, oxidative stress response, and development of the maize pathogen Cochliobolus heterostrophus.

Wu D, Oide S, Zhang N, Choi MY, Turgeon BG - PLoS Pathog. (2012)

Lae1 and Vel1 proteins negatively regulate mycelial pigmentation of C. heterostrophus.A. Bottom of culture plates incubated in constant light or dark for 9 days. Pictures were taken after removal of conidia. Note heavy pigmentation of mycelia of Chlae1 and Chvel1 mutants in both light and dark compared to WT. B. Mycelial pellet of WT strain C4, Chvel1, Chlae1 mutants, and complemented strains (vel1[VEL1], lae1[LAE1]) at different time points indicated. Supernatant is shown only for the 64 hour samples. Chlae1 strain is melanized by 40 hrs, clearly ahead of other strains. Melanization starts by 48 hrs in Chvel1, while pigmentation is not evident in WT at this time. The supernatant of the Chlae1 strain is pigmented indicating secretion of pigment(s) into the medium (no conidia are present). C. qPCR analyses of PKS18. Expression was examined for the samples from B at 40 and 48 hours. Expression level relative to the WT strain C4 sample at 40 hours is shown. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Single asterisks indicate p-value <0.05, double asterisks indicate p-value <0.001 in T-test analysis in which each strain was compared with corresponding WT C4 at the same time point. Matching the enhanced pigmentation (B), PKS18 is induced in Chlae1 by 40 hrs and in Chvel1 by 48 hrs. D. qPCR analyses of CMR1. Expression was examined for the samples from B at 40 and 48 hours. Expression level relative to the WT, C4 sample at 40 hours is shown. Error bars and T-test analysis are same as C. Like the enhanced pigmentation in B, CMR1 expression increased 1.7 and 3.8 fold in Chvel1 and Chlae1 mutants, respectively, compared to WT by 48 hrs. Note scale is different from C.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285592&req=5

ppat-1002542-g007: Lae1 and Vel1 proteins negatively regulate mycelial pigmentation of C. heterostrophus.A. Bottom of culture plates incubated in constant light or dark for 9 days. Pictures were taken after removal of conidia. Note heavy pigmentation of mycelia of Chlae1 and Chvel1 mutants in both light and dark compared to WT. B. Mycelial pellet of WT strain C4, Chvel1, Chlae1 mutants, and complemented strains (vel1[VEL1], lae1[LAE1]) at different time points indicated. Supernatant is shown only for the 64 hour samples. Chlae1 strain is melanized by 40 hrs, clearly ahead of other strains. Melanization starts by 48 hrs in Chvel1, while pigmentation is not evident in WT at this time. The supernatant of the Chlae1 strain is pigmented indicating secretion of pigment(s) into the medium (no conidia are present). C. qPCR analyses of PKS18. Expression was examined for the samples from B at 40 and 48 hours. Expression level relative to the WT strain C4 sample at 40 hours is shown. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Single asterisks indicate p-value <0.05, double asterisks indicate p-value <0.001 in T-test analysis in which each strain was compared with corresponding WT C4 at the same time point. Matching the enhanced pigmentation (B), PKS18 is induced in Chlae1 by 40 hrs and in Chvel1 by 48 hrs. D. qPCR analyses of CMR1. Expression was examined for the samples from B at 40 and 48 hours. Expression level relative to the WT, C4 sample at 40 hours is shown. Error bars and T-test analysis are same as C. Like the enhanced pigmentation in B, CMR1 expression increased 1.7 and 3.8 fold in Chvel1 and Chlae1 mutants, respectively, compared to WT by 48 hrs. Note scale is different from C.
Mentions: Unlike A. nidulans and A. fumigatus LaeA, deletion of which leads to loss of mycelial pigmentation [12], lack of either ChLae1 or ChVel1 caused increased pigmentation of mycelia grown either on solid (Figure 7A) or in liquid (Figure 7B) medium, in both dark and light, indicating that ChLae1 and ChVel1 negatively regulate mycelial pigmentation in C. heterostrophus. Pigment was also evident in the supernatant of the Chlae1 mutant at 64 hrs (Figure 7B) suggesting that it is secreted into the medium. Hyphal pigmentation develops more slowly in the Chvel1 mutant than in the Chlae1 mutant, but faster than in WT. By 64 hrs, all strains were darkly pigmented. Reintroduction of ChVEL1 or ChLAE1 restored the WT phenotype (Figure 7B).

Bottom Line: T-toxin production is significantly increased in the dark in wild type (WT), whereas Chvel1 and Chlae1 mutant toxin levels are much reduced in the dark compared to WT.Deletion of ChLAE1 or ChVEL1 reduces tolerance to H(2)O(2).ChLae1 and ChVel1 repress expression of 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis genes, and, accordingly, melanization is enhanced in Chlae1 and Chvel1 mutants, and reduced in OE strains.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Plant Pathology & Plant-Microbe Biology, Cornell University, Ithaca, New York, USA.

ABSTRACT
LaeA and VeA coordinate secondary metabolism and differentiation in response to light signals in Aspergillus spp. Their orthologs, ChLae1 and ChVel1, were identified in the maize pathogen Cochliobolus heterostrophus, known to produce a wealth of secondary metabolites, including the host selective toxin, T-toxin. Produced by race T, T-toxin promotes high virulence to maize carrying Texas male sterile cytoplasm (T-cms). T-toxin production is significantly increased in the dark in wild type (WT), whereas Chvel1 and Chlae1 mutant toxin levels are much reduced in the dark compared to WT. Correspondingly, expression of T-toxin biosynthetic genes (Tox1) is up-regulated in the dark in WT, while dark-induced expression is much reduced/minimal in Chvel1 and Chlae1 mutants. Toxin production and Tox1 gene expression are increased in ChVEL1 overexpression (OE) strains grown in the dark and in ChLAE1 strains grown in either light or dark, compared to WT. These observations establish ChLae1 and ChVel1 as the first factors known to regulate host selective toxin production. Virulence of Chlae1 and Chvel1 mutants and OE strains is altered on both T-cms and normal cytoplasm maize, indicating that both T-toxin mediated super virulence and basic pathogenic ability are affected. Deletion of ChLAE1 or ChVEL1 reduces tolerance to H(2)O(2). Expression of CAT3, one of the three catalase genes, is reduced in the Chvel1 mutant. Chlae1 and Chvel1 mutants also show decreased aerial hyphal growth, increased asexual sporulation and female sterility. ChLAE1 OE strains are female sterile, while ChVEL1 OE strains are more fertile than WT. ChLae1 and ChVel1 repress expression of 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis genes, and, accordingly, melanization is enhanced in Chlae1 and Chvel1 mutants, and reduced in OE strains. Thus, ChLae1 and ChVel1 positively regulate T-toxin biosynthesis, pathogenicity and super virulence, oxidative stress responses, sexual development, and aerial hyphal growth, and negatively control melanin biosynthesis and asexual differentiation.

Show MeSH
Related in: MedlinePlus