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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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Chlae1 and Chvel1 mutants are compromised in reproductive development.A.Chlae1 and Chvel1 mutants are female sterile. Top: a control cross between pigmented WT strain C4 and albino WT strain CB7. Both black and white pseudothecia (arrows) are produced indicating both strains are hermaphroditic. Middle and bottom rows: crosses between albino WT strain CB7 and pigmented Chlae1 or Chvel1, respectively. Only white pseudothecia (arrows) are produced, indicating the mutant strains are female sterile (color of pseudothecia reflects strain acting as female). Heavy black region (green arrow) on bottom image indicates profuse production of pigmented conidia of the Chvel1 mutant. B.ChVEL1 overexpression strains are more fertile than WT strains. Crosses with ChVEL1 OE strains produce both pigmented and white pseudothecia (bottom panel, arrows) at a ratio of 2∶1 while a WT cross produced nearly equal numbers of pigmented and white pseudothecia (top panel). ChLAE1 OE strains are female sterile (middle panel, white pseudothecia only), like Chlae1 mutants. C. Asexual sporulation is de-repressed in the Chvel1 mutant during mating. The average numbers of asexual spores formed on cross plates are shown. Error bars are standard deviation. Asterisks indicate p-value <0.05 in T-test analysis in which each strain was compared with WT C4. WT strain C4 and the Chlae1 mutant strain produce few vegetative spores during sexual reproduction, while asexual sporulation is de-repressed in the Chvel1 mutant (see also bottom panel in A, green arrow).
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ppat-1002542-g005: Chlae1 and Chvel1 mutants are compromised in reproductive development.A.Chlae1 and Chvel1 mutants are female sterile. Top: a control cross between pigmented WT strain C4 and albino WT strain CB7. Both black and white pseudothecia (arrows) are produced indicating both strains are hermaphroditic. Middle and bottom rows: crosses between albino WT strain CB7 and pigmented Chlae1 or Chvel1, respectively. Only white pseudothecia (arrows) are produced, indicating the mutant strains are female sterile (color of pseudothecia reflects strain acting as female). Heavy black region (green arrow) on bottom image indicates profuse production of pigmented conidia of the Chvel1 mutant. B.ChVEL1 overexpression strains are more fertile than WT strains. Crosses with ChVEL1 OE strains produce both pigmented and white pseudothecia (bottom panel, arrows) at a ratio of 2∶1 while a WT cross produced nearly equal numbers of pigmented and white pseudothecia (top panel). ChLAE1 OE strains are female sterile (middle panel, white pseudothecia only), like Chlae1 mutants. C. Asexual sporulation is de-repressed in the Chvel1 mutant during mating. The average numbers of asexual spores formed on cross plates are shown. Error bars are standard deviation. Asterisks indicate p-value <0.05 in T-test analysis in which each strain was compared with WT C4. WT strain C4 and the Chlae1 mutant strain produce few vegetative spores during sexual reproduction, while asexual sporulation is de-repressed in the Chvel1 mutant (see also bottom panel in A, green arrow).

Mentions: To determine if ChLae1 and ChVel1 control heterothallic reproductive development, as in homothallic A. nidulans, we tested ability of Chlae1 and Chvel1 mutants (both MAT1-2;ALB1) to undergo sexual development by crossing them to CB7, an albino tester strain of opposite mating type (MAT1-1;alb1). Control crosses of CB7 to WT strain C4 (MAT1-2;ALB1) produced both pigmented and albino pseudothecia, indicating that both parental strains were hermaphroditic. Crosses involving either the Chlae1 or Chvel1 pigmented mutants produced fertile albino pseudothecia, but failed to produce pigmented pseudothecia, indicating that both types of mutant are female sterile, since color of pseudothecia reflects which parent in a cross acted as the female (Figure 5A). Complementation of both Chlae1 and Chvel1 mutants restored WT crossing capability (data not shown). This result demonstrates that ChLae1 and ChVel1 have positive roles in sexual differentiation in C. heterostrophus, likely in the early stages of fruiting body formation, since no pigmented fruiting bodies were formed. Crosses of VEL1 OE strains to albino tester CB7 produced both black and white fertile pseudothecia, indicating that OE strains were fully mating competent (Figure 5B). Furthermore, the number of black pseudothecia was at least two times the number of white pseudothecia (data not shown). The ratio between black and albino pseudothecia is usually 1∶1 in crosses between pigmented WT and albino testers. This further confirmed that ChVel1 plays a major role in sexual reproduction. In contrast, when LAE1 OE strains were crossed to albino tester CB7, they were female sterile (Figure 5B). Thus, the excess of ChLae1, just like its absence, causes loss of ability to act as female, implying that ChLae1 might affect sexual development through influencing gene expression and/or protein levels of other members of the velvet complex, i.e. VelB, VeA, VosA [15].


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)

Chlae1 and Chvel1 mutants are compromised in reproductive development.A.Chlae1 and Chvel1 mutants are female sterile. Top: a control cross between pigmented WT strain C4 and albino WT strain CB7. Both black and white pseudothecia (arrows) are produced indicating both strains are hermaphroditic. Middle and bottom rows: crosses between albino WT strain CB7 and pigmented Chlae1 or Chvel1, respectively. Only white pseudothecia (arrows) are produced, indicating the mutant strains are female sterile (color of pseudothecia reflects strain acting as female). Heavy black region (green arrow) on bottom image indicates profuse production of pigmented conidia of the Chvel1 mutant. B.ChVEL1 overexpression strains are more fertile than WT strains. Crosses with ChVEL1 OE strains produce both pigmented and white pseudothecia (bottom panel, arrows) at a ratio of 2∶1 while a WT cross produced nearly equal numbers of pigmented and white pseudothecia (top panel). ChLAE1 OE strains are female sterile (middle panel, white pseudothecia only), like Chlae1 mutants. C. Asexual sporulation is de-repressed in the Chvel1 mutant during mating. The average numbers of asexual spores formed on cross plates are shown. Error bars are standard deviation. Asterisks indicate p-value <0.05 in T-test analysis in which each strain was compared with WT C4. WT strain C4 and the Chlae1 mutant strain produce few vegetative spores during sexual reproduction, while asexual sporulation is de-repressed in the Chvel1 mutant (see also bottom panel in A, green arrow).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285592&req=5

ppat-1002542-g005: Chlae1 and Chvel1 mutants are compromised in reproductive development.A.Chlae1 and Chvel1 mutants are female sterile. Top: a control cross between pigmented WT strain C4 and albino WT strain CB7. Both black and white pseudothecia (arrows) are produced indicating both strains are hermaphroditic. Middle and bottom rows: crosses between albino WT strain CB7 and pigmented Chlae1 or Chvel1, respectively. Only white pseudothecia (arrows) are produced, indicating the mutant strains are female sterile (color of pseudothecia reflects strain acting as female). Heavy black region (green arrow) on bottom image indicates profuse production of pigmented conidia of the Chvel1 mutant. B.ChVEL1 overexpression strains are more fertile than WT strains. Crosses with ChVEL1 OE strains produce both pigmented and white pseudothecia (bottom panel, arrows) at a ratio of 2∶1 while a WT cross produced nearly equal numbers of pigmented and white pseudothecia (top panel). ChLAE1 OE strains are female sterile (middle panel, white pseudothecia only), like Chlae1 mutants. C. Asexual sporulation is de-repressed in the Chvel1 mutant during mating. The average numbers of asexual spores formed on cross plates are shown. Error bars are standard deviation. Asterisks indicate p-value <0.05 in T-test analysis in which each strain was compared with WT C4. WT strain C4 and the Chlae1 mutant strain produce few vegetative spores during sexual reproduction, while asexual sporulation is de-repressed in the Chvel1 mutant (see also bottom panel in A, green arrow).
Mentions: To determine if ChLae1 and ChVel1 control heterothallic reproductive development, as in homothallic A. nidulans, we tested ability of Chlae1 and Chvel1 mutants (both MAT1-2;ALB1) to undergo sexual development by crossing them to CB7, an albino tester strain of opposite mating type (MAT1-1;alb1). Control crosses of CB7 to WT strain C4 (MAT1-2;ALB1) produced both pigmented and albino pseudothecia, indicating that both parental strains were hermaphroditic. Crosses involving either the Chlae1 or Chvel1 pigmented mutants produced fertile albino pseudothecia, but failed to produce pigmented pseudothecia, indicating that both types of mutant are female sterile, since color of pseudothecia reflects which parent in a cross acted as the female (Figure 5A). Complementation of both Chlae1 and Chvel1 mutants restored WT crossing capability (data not shown). This result demonstrates that ChLae1 and ChVel1 have positive roles in sexual differentiation in C. heterostrophus, likely in the early stages of fruiting body formation, since no pigmented fruiting bodies were formed. Crosses of VEL1 OE strains to albino tester CB7 produced both black and white fertile pseudothecia, indicating that OE strains were fully mating competent (Figure 5B). Furthermore, the number of black pseudothecia was at least two times the number of white pseudothecia (data not shown). The ratio between black and albino pseudothecia is usually 1∶1 in crosses between pigmented WT and albino testers. This further confirmed that ChVel1 plays a major role in sexual reproduction. In contrast, when LAE1 OE strains were crossed to albino tester CB7, they were female sterile (Figure 5B). Thus, the excess of ChLae1, just like its absence, causes loss of ability to act as female, implying that ChLae1 might affect sexual development through influencing gene expression and/or protein levels of other members of the velvet complex, i.e. VelB, VeA, VosA [15].

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