| Sclerotinia rot of oilseed caused by Sclerotinia sclerotiorum is widespread disease. Coniothyrium minitans is a mycoparasite fungus of S. sclerotiorum, and plays an important role in controlling sclerotinia disease caused by S. sclerotiorum. Previous studies indicated that S. sclerotiorum usually creates an acidic pH environment by production and secretion of oxalic acid(OA). C. minitans produces antifungal substances(AFS) inhibitory to hyphal growth and sclerotial germination of S. sclerotiorum in an acidic pH environment. On the other hand, degradation of OA by C. minitans can increase ambient pH, thus creating an environment with weakly acidic and neutral pH, which significantly enhances the production and activity of cell wall degrading enzymes related with mycoparasitism by C. minitans. These results indicate that ambient pH play an important role in interaction between C. minitans and S. sclerotiorum. Previous study indicates that the pacC/Rim101 homologue(CmpacC) has been cloned from the wild type strain Chy-1 of C. minitans. However, the function of CmpacC and other homologue Pal-like genes in the pH sensing/response system remains unknown. Aiming at these problems, the function of CmpacC and CmpalA, CmpalB, CmpalC, CmpalF, CmpalH, CmpalI by the disruption and complementary of these genes using the PEG mediated protoplast transformation has been analyzed, and we also studied the effects of these Pal-like genes on mycoparasitism, oxalate degradation, and antifungal activity of C. minitans. Results achieved are summarized below:Firstly, the effect of the transcriptional factor CmpacC on the mycelial growth and stress tolerance of C. minitans was investigated. Three CmpacC-disruption mutants designated as ?CmpacC-21, ?CmpacC-29, ?CmpacC-46, and the corresponding complementary mutant ?CmpacC-29 C were obtained by the protoplast PEG transformation. Compaired to the wild type strain Chy-1, the mycelial growth and spore production of the deletion mutant ?CmpacC-29 were significantly suppressed under neutral or alkaline pH. Moreover, the deletion of CmpacC increased the sensitivity to salt/hypertonic stress, but the mutant ?CmpacC-29 showed similar sensitivity to oxidative or metal stress in compairision to the wild type strain Chy-1. These results indicated that CmpacC may play an important role in the regulation of the resistance to salt/hypertonic stress in C. minitans.Secondly, the regulation of the transcription factor CmpacC on mycoparasitism, oxalate degradation, antifungal activity was investigated. The results indicate that compaired to the wild type strain Chy-1 and the mutant ?CmpacC-29 C, the disruption mutant ?CmpacC-29 were compromised in infecting mycelia or sclerotia of S. sclerotiorum, but increased oxalate degradation and antifungal activity. To elucidate the regulation mechanism by CmpacC responsible for mycoparasitism, oxalate degradation and antifungal activity, qRT-PCR and enzymae activity assay were conducted. The results showed that the expression and activity of β-1, 3-glucanase, chitinase, and proteases were significantly reduced in the mutant ?CmpacC-29, compaired to the wild type strain Chy-1, however ?CmpacC-29 showed higher transcript levels of Cmoxdc1 and higher activity of total oxalate decarboxylase. Transcripttional activation test and EMSA further confirmed that CmpacC is a transcription factor, which could bind to the promoter consensus binding site(GCCARG) of Cmg1, Cmch1 and Cmoxdc1. These results suggested that CmpacC was involved in the positive regulation on mycoparasitism, but negative control of oxalate degradation and antifungal activity.Thirdly, five Pal-like homologue genes were cloned from the wild type strain Chy-1, which were designated as CmpalA(GenBank Acc. No. KP747602), CmpalB(GenBank Acc. No. KP747603), CmpalC(GenBank Acc. No. KP747604), CmpalF(GenBank Acc. No. KP747605), CmpalH(GenBank Acc. No. KP747606), CmpalI(GenBank Acc. No. KP747607). Phylogenetic analysis showed CmpalA, CmpalC, CmpalF and CmpalH were closely related to the homologue genes in Leptosphaeria maculans, CmpalI was closely related to the homologue gene Pal1 in Phaeosphaeria nodorum.Fourthly, five Pal-like genes(CmpalA, CmpalB, CmpalC, CmpalF, CmpalH) disruption mutants designated as ?CmpalA-33, ?CmpalB-13, ?CmpalC-5, ?CmpalF-50 and ?CmpalH-26 respectively were obtained using the PEG mediated protoplast transformation. However, we still do not obtain the CmpalI-disruption mutant. Compaired to the wild type strain Chy-1, these Pal-like genes-disruption mutants ?CmpalA-33, ?CmpalB-13, ?CmpalC-5, ?CmpalF-50 and ?CmpalH-26 showed poor mycelia growth at pH 6 or higher pH. These results indicated that the disruption of these Pal-like genes increases sensitivity to neutral or alkaline pH. qRT-PCR results indicated that the transcript levels of CmpacC in these Pal-like genes-disruption mutants were gradually decreased. We speculated that five Pal-like genes(CmpalA, CmpalB, CmpalC, CmpalF and CmpalH) may be located at the upstream of CmpacC in the Pal pathway, the deletion of these genes may block a signal transduction which was able to trigger the conversion of the transcription factor encoded by CmpacC to an active form able to enter the nucleus and activate the CmpacC self-expression.Finally, effects of CmpalA, CmpalB, CmpalC, CmpalF and CmpalH on mycoparasitism and antifungal activity of C. minitans were investigated. The results showed that compaired to the wild type strain Chy-1, the mutants ?CmpalA-33, ?CmpalB-13, ?CmpalC-5 were compromised in infecting mycelium of the OA-producing S. sclerotiorum strain A5, and the mutants ?CmpalF-50, ?CmpalH-26 showed no differences in colonizing colonies of A5. However, all mutants(?CmpalA-33, ?CmpalB-13, ?CmpalC-5, ?CmpalF-50 and ?CmpalH-26) were significantly compromised in invading the scleratia of S. sclerotiorum. The antifungal activity of ?CmpalA-33, ?CmpalB-13, ?CmpalC-5, ?CmpalF-50 and ?CmpalH-26 in MCD culture at pH 4 and 8 was determined. The results indicate that at pH 8, all mutants showed higher antifungal activity against S. sclerotiorum compaired to strain Chy-1. These results could be concluded that the lower transcript levels of CmpacC which can repress the expression of the antifungal-related genes in these mutants may contribute to higher antifungal activity.In conclusion, the above results revealed that CmpacC and the Pal signal pathway may play an important role in the interaction between C. minitans and S. sclerotiorum. At the early interaction stage, the acidic pH created by oxalic acid(OA) from S. sclerotiorum triggers expression of Cmoxdc1 in C. minitans. The resulting oxalate decarboxylase then mediates OA degradation, thus elevating the ambient pH. At the later stage, the Pal genes in C. minitans(CmpalA, CmpalB, CmpalC, CmpalF, CmpalI, CmpalH, CmpalH) sense the high-pH environment, express and mediate proteolytic processing of the Cmpac C protein encoded by CmpacC. The active form of Cmpac C then activates transcription of Cmch1 and Cmg1 and meanwhile represses transcription of Cmoxdc1 and the genes for AFS biosynthesis. |
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