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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 positively regulate T-toxin biosynthesis genes.A. RT-PCR analysis of the genes known to be involved in T-toxin production in WT and mutant strains. Expression of ACT1 indicates relative RNA quantity in each sample. All Tox1 genes are up-regulated in the dark, relative to in the light in WT. Evidence of light regulation is erased in Chvel1 mutants. Most genes are weakly up-regulated in Chlae1 mutants, except for RED1, RED2 and RED3. B. Quantification of band intensity. Band intensity in Figure 2A was quantified by Image J. The band intensity ratio of each Tox1 gene and that of the corresponding control ACT1 gene was calculated and normalized to that of WT strain C4 in light. Note that band intensity of all genes is elevated in the dark in WT, while band intensity of all genes is minimal in the Chvel1 mutant grown in both the light and dark, and minimally elevated in the Chlae1 mutant grown in the dark. C. qPCR of PKS1 and PKS2. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Asterisks indicate p-value <0.001 in T-test analysis in which all the strains grown in the dark were compared to their corresponding strain grown in constant light. D. RT-PCR analysis of Tox1 genes in overexpression strains. Overexpression of ChLAE1 results in drastic up-regulation of all genes in the light and a moderate increase in the dark compared to WT. In contrast, ChVEL1 overexpression caused up-regulation of Tox1 gene expression in the dark but not the light. E. qPCR of ChLAE1 and ChVEL1. cDNA samples are the same as A. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Asterisks indicate p-value <0.01 in T-test analysis in which both overexpression strains were compared to WT grown in the same condition. F. qPCR of PKS1 and PKS2. Same as B. Asterisks indicate p-value <0.05 in T-test analysis in which both overexpression strains were compared to WT grown in the same condition.
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ppat-1002542-g002: ChLae1 and ChVel1 positively regulate T-toxin biosynthesis genes.A. RT-PCR analysis of the genes known to be involved in T-toxin production in WT and mutant strains. Expression of ACT1 indicates relative RNA quantity in each sample. All Tox1 genes are up-regulated in the dark, relative to in the light in WT. Evidence of light regulation is erased in Chvel1 mutants. Most genes are weakly up-regulated in Chlae1 mutants, except for RED1, RED2 and RED3. B. Quantification of band intensity. Band intensity in Figure 2A was quantified by Image J. The band intensity ratio of each Tox1 gene and that of the corresponding control ACT1 gene was calculated and normalized to that of WT strain C4 in light. Note that band intensity of all genes is elevated in the dark in WT, while band intensity of all genes is minimal in the Chvel1 mutant grown in both the light and dark, and minimally elevated in the Chlae1 mutant grown in the dark. C. qPCR of PKS1 and PKS2. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Asterisks indicate p-value <0.001 in T-test analysis in which all the strains grown in the dark were compared to their corresponding strain grown in constant light. D. RT-PCR analysis of Tox1 genes in overexpression strains. Overexpression of ChLAE1 results in drastic up-regulation of all genes in the light and a moderate increase in the dark compared to WT. In contrast, ChVEL1 overexpression caused up-regulation of Tox1 gene expression in the dark but not the light. E. qPCR of ChLAE1 and ChVEL1. cDNA samples are the same as A. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Asterisks indicate p-value <0.01 in T-test analysis in which both overexpression strains were compared to WT grown in the same condition. F. qPCR of PKS1 and PKS2. Same as B. Asterisks indicate p-value <0.05 in T-test analysis in which both overexpression strains were compared to WT grown in the same condition.

Mentions: The effect of ChLAE1 and ChVEL1 deletions on T-toxin production in constant light and constant dark conditions was examined using the microbial assay [45]. T-toxin was produced by WT race T strain C4 under both light conditions tested, but much more so in the dark (Figure 1) and increasingly as the sample was taken from the center to the edge of 8 day old colonies (Figure 1A, lower left). No T-toxin was produced by control WT race O strain C5. In dark conditions, smaller halos were observed for Chlae1 and Chvel1 mutants compared to WT race T. In light conditions, this was still the case for Chlae1 mutants, while Chvel1 mutant plugs had bigger halos than in the dark and those taken from the edge of the colony were slightly larger than those of WT in the light (Figure 1A, top right). Overexpression of ChVEL1 caused a modest increase in T-toxin production in the dark (Figure 1B, top row), but not in the light when compared to WT strains. Overexpression of ChLAE1 lead to profuse over-production of T-toxin in both the dark and light compared to WT (Figure 1B, bottom row). These data suggest that both ChLae1 and ChVel1 positively regulate T-toxin production under dark conditions, however the observation that some toxin is produced by both mutants suggests that there must be an additional T-toxin biosynthesis regulatory mechanism(s) that is independent of ChLae1 and ChVel1. Copious production of toxin in the light by LAE1 overexpression strains suggests that regulatory mechanisms associated with light signals were disturbed. To further confirm that ChLae1 and ChVel1 act as positive regulators of T-toxin production, expression of all nine known Tox1 genes (PKS1, PKS2, DEC1, RED1, RED2, RED3, TOX9, OXI1, LAM1) [25]–[27] was examined for WT race T strain C4, Chlae1, and Chvel1 strains grown in constant light and constant dark at 19°C, the same conditions as culture plates used for the T-toxin assay (Figure 1). All Tox1 genes were up-regulated in the dark in WT, whereas dark-induced expression was not evident or reduced in Chvel1 and Chlae1 mutants, respectively (Figure 2A). Quantification of the RT PCR bands in Figure 2A showed that for Chvel1 mutants, most genes were not up-regulated, or were minimally up-regulated, in the dark. For Chlae1 mutants, expression levels of the nine genes were slightly more elevated than in Chvel1 mutants in the dark but in all cases less so than for WT in the dark (Figure 2B). We also carried out quantitative PCR (qPCR) analyses on the core PKS1 and PKS2 genes required for T-toxin production (Figure 2C). The two PKSs were minimally expressed in light and induced in dark in WT strain C4 (Figure 2C) and in laeA[LAE1] and vel1[VEL1]complemented strains (not shown). In contrast, expression of these genes in Chvel1 mutants was like that of WT in the light, regardless of the light conditions. Expression of these genes in Chlae1 mutants was slightly up in the dark compared to expression in the light, but significantly less than that of WT in the dark (Figure 2C). These results are consistent with the T-toxin microbial assay, in which T-toxin production was significantly decreased in Chlae1 and Chvel1 mutants grown under dark conditions, and demonstrate that ChLae1 and ChVel1 are required for positive regulation of T-toxin genes in the dark.


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 positively regulate T-toxin biosynthesis genes.A. RT-PCR analysis of the genes known to be involved in T-toxin production in WT and mutant strains. Expression of ACT1 indicates relative RNA quantity in each sample. All Tox1 genes are up-regulated in the dark, relative to in the light in WT. Evidence of light regulation is erased in Chvel1 mutants. Most genes are weakly up-regulated in Chlae1 mutants, except for RED1, RED2 and RED3. B. Quantification of band intensity. Band intensity in Figure 2A was quantified by Image J. The band intensity ratio of each Tox1 gene and that of the corresponding control ACT1 gene was calculated and normalized to that of WT strain C4 in light. Note that band intensity of all genes is elevated in the dark in WT, while band intensity of all genes is minimal in the Chvel1 mutant grown in both the light and dark, and minimally elevated in the Chlae1 mutant grown in the dark. C. qPCR of PKS1 and PKS2. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Asterisks indicate p-value <0.001 in T-test analysis in which all the strains grown in the dark were compared to their corresponding strain grown in constant light. D. RT-PCR analysis of Tox1 genes in overexpression strains. Overexpression of ChLAE1 results in drastic up-regulation of all genes in the light and a moderate increase in the dark compared to WT. In contrast, ChVEL1 overexpression caused up-regulation of Tox1 gene expression in the dark but not the light. E. qPCR of ChLAE1 and ChVEL1. cDNA samples are the same as A. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Asterisks indicate p-value <0.01 in T-test analysis in which both overexpression strains were compared to WT grown in the same condition. F. qPCR of PKS1 and PKS2. Same as B. Asterisks indicate p-value <0.05 in T-test analysis in which both overexpression strains were compared to WT grown in the same condition.
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getmorefigures.php?uid=PMC3285592&req=5

ppat-1002542-g002: ChLae1 and ChVel1 positively regulate T-toxin biosynthesis genes.A. RT-PCR analysis of the genes known to be involved in T-toxin production in WT and mutant strains. Expression of ACT1 indicates relative RNA quantity in each sample. All Tox1 genes are up-regulated in the dark, relative to in the light in WT. Evidence of light regulation is erased in Chvel1 mutants. Most genes are weakly up-regulated in Chlae1 mutants, except for RED1, RED2 and RED3. B. Quantification of band intensity. Band intensity in Figure 2A was quantified by Image J. The band intensity ratio of each Tox1 gene and that of the corresponding control ACT1 gene was calculated and normalized to that of WT strain C4 in light. Note that band intensity of all genes is elevated in the dark in WT, while band intensity of all genes is minimal in the Chvel1 mutant grown in both the light and dark, and minimally elevated in the Chlae1 mutant grown in the dark. C. qPCR of PKS1 and PKS2. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Asterisks indicate p-value <0.001 in T-test analysis in which all the strains grown in the dark were compared to their corresponding strain grown in constant light. D. RT-PCR analysis of Tox1 genes in overexpression strains. Overexpression of ChLAE1 results in drastic up-regulation of all genes in the light and a moderate increase in the dark compared to WT. In contrast, ChVEL1 overexpression caused up-regulation of Tox1 gene expression in the dark but not the light. E. qPCR of ChLAE1 and ChVEL1. cDNA samples are the same as A. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Asterisks indicate p-value <0.01 in T-test analysis in which both overexpression strains were compared to WT grown in the same condition. F. qPCR of PKS1 and PKS2. Same as B. Asterisks indicate p-value <0.05 in T-test analysis in which both overexpression strains were compared to WT grown in the same condition.
Mentions: The effect of ChLAE1 and ChVEL1 deletions on T-toxin production in constant light and constant dark conditions was examined using the microbial assay [45]. T-toxin was produced by WT race T strain C4 under both light conditions tested, but much more so in the dark (Figure 1) and increasingly as the sample was taken from the center to the edge of 8 day old colonies (Figure 1A, lower left). No T-toxin was produced by control WT race O strain C5. In dark conditions, smaller halos were observed for Chlae1 and Chvel1 mutants compared to WT race T. In light conditions, this was still the case for Chlae1 mutants, while Chvel1 mutant plugs had bigger halos than in the dark and those taken from the edge of the colony were slightly larger than those of WT in the light (Figure 1A, top right). Overexpression of ChVEL1 caused a modest increase in T-toxin production in the dark (Figure 1B, top row), but not in the light when compared to WT strains. Overexpression of ChLAE1 lead to profuse over-production of T-toxin in both the dark and light compared to WT (Figure 1B, bottom row). These data suggest that both ChLae1 and ChVel1 positively regulate T-toxin production under dark conditions, however the observation that some toxin is produced by both mutants suggests that there must be an additional T-toxin biosynthesis regulatory mechanism(s) that is independent of ChLae1 and ChVel1. Copious production of toxin in the light by LAE1 overexpression strains suggests that regulatory mechanisms associated with light signals were disturbed. To further confirm that ChLae1 and ChVel1 act as positive regulators of T-toxin production, expression of all nine known Tox1 genes (PKS1, PKS2, DEC1, RED1, RED2, RED3, TOX9, OXI1, LAM1) [25]–[27] was examined for WT race T strain C4, Chlae1, and Chvel1 strains grown in constant light and constant dark at 19°C, the same conditions as culture plates used for the T-toxin assay (Figure 1). All Tox1 genes were up-regulated in the dark in WT, whereas dark-induced expression was not evident or reduced in Chvel1 and Chlae1 mutants, respectively (Figure 2A). Quantification of the RT PCR bands in Figure 2A showed that for Chvel1 mutants, most genes were not up-regulated, or were minimally up-regulated, in the dark. For Chlae1 mutants, expression levels of the nine genes were slightly more elevated than in Chvel1 mutants in the dark but in all cases less so than for WT in the dark (Figure 2B). We also carried out quantitative PCR (qPCR) analyses on the core PKS1 and PKS2 genes required for T-toxin production (Figure 2C). The two PKSs were minimally expressed in light and induced in dark in WT strain C4 (Figure 2C) and in laeA[LAE1] and vel1[VEL1]complemented strains (not shown). In contrast, expression of these genes in Chvel1 mutants was like that of WT in the light, regardless of the light conditions. Expression of these genes in Chlae1 mutants was slightly up in the dark compared to expression in the light, but significantly less than that of WT in the dark (Figure 2C). These results are consistent with the T-toxin microbial assay, in which T-toxin production was significantly decreased in Chlae1 and Chvel1 mutants grown under dark conditions, and demonstrate that ChLae1 and ChVel1 are required for positive regulation of T-toxin genes in the dark.

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