Trichoderma harzianum transformant has high extracellular alkaline proteinase expression during specific mycoparasitic interactions

The mycoparasite Trichoderma harzianum produces an alkaline proteinase that may be specifically involved in mycoparasitism. We have constructed transformant strains of this fungus that overexpress this alkaline proteinase. Some of the transformants were assessed for alkaline proteinase activity, and those with higher activity than the wild type were selected for further studies. One of these transformant strains produced an elevated and constitutive pbr1 mRNA level during mycoparasitic interactions with Rhizoctonia solani.

Authorship

Maria Helena S. Goldman

Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de BiologiaFaculdade de Filosofia, Ciências e Letras de Ribeirão PretoFaculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia

Gustavo H. Goldman

USP, Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão PretoUSPUSP, Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto

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Figures (4)

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Figure 1
- Northern blot of the mRNA obtained during specific mycoparasitic interactions between Trichoderma harzianum and Rhizoctonia solani. A: Different stages of the interaction. B: Northern analysis of the pbr1 mRNA obtained during dual interactions. The left panel shows hybridization with pbr1 gene, whereas the right panel shows hybridization with the tub1 gene. C: Northern analysis of the pgk mRNA obtained during dual interactions. The left panel shows hybridization with pgk gene, whereas the right panel shows hybridization with the tub1 gene. In panel AII, T. harzianum has grown over R. solani mycelia. R: R. solani; T: T. harzianum; G: T. harzianum grown in MM plates with 0.5% glucose; CW: T. harzianum grown in MM broth with 0.1% cell walls of R. solani.

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Figure 2
- Southern blot analysis of transformants of Trichoderma harzianum DNA. Genomic DNA (10 µg) from five transformants (pbr1-2A, 16A, 36A, 55A, and 57A) and from the wild type (WT) was digested with PstI (A) or HindIII (B), separated by agarose gel electrophoresis, and analyzed by the Southern blot technique. Blots were probed with a ³²P-labelled 5.0 kb EcoRI fragment containing the pbr1 gene from T. harzianum.

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Figure 3
- Alkaline protease activity of Trichodema harzianum transformants. T. harzianum wild type and transformants were grown for 72 h in MM + glucose 2% at 28^oC. Afterwards, mycelia were transferred to MM + colloidal chitin 0.1% (open bars) or colloidal chitin 0.1% combined with glucose 2% (grid bars). These cultures were grown for 48 h at 28^oC, and supernatants were assayed for alkaline protease activity. Activity was expressed as a percentage of wild type strain activity. These results are the means of three experiments.

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Figure 4
- Northern blot was performed on the mRNA obtained during specific mycoparasitic interactions using Trichoderma harzianum wild type and the transformant strain pbr1-2A against Rhizoctonia solani. A: Different stages of the interaction. B: Northern analysis of the pbr1 mRNA obtained during dual interactions. The left panel shows dual interactions between the wild type and R. solani, whereas the right panel shows dual interactions between pbr1-2A and R. solani. C: Northern analysis of the tub1 mRNA obtained during dual interactions. The left panel shows dual interactions between the wild type and R. solani, whereas the right panel shows dual interactions between pbr1-2A and R. solani. In panel AIII, T. harzianum has grown over R. solani mycelia.

Figure 1 - Northern blot of the mRNA obtained during specific mycoparasitic interactions between Trichoderma harzianum and Rhizoctonia solani. A: Different stages of the interaction. B: Northern analysis of the pbr1 mRNA obtained during dual interactions. The left panel shows hybridization with pbr1 gene, whereas the right panel shows hybridization with the tub1 gene. C: Northern analysis of the pgk mRNA obtained during dual interactions. The left panel shows hybridization with pgk gene, whereas the right panel shows hybridization with the tub1 gene. In panel AII, T. harzianum has grown over R. solani mycelia. R: R. solani; T: T. harzianum; G: T. harzianum grown in MM plates with 0.5% glucose; CW: T. harzianum grown in MM broth with 0.1% cell walls of R. solani.

Figure 2 - Southern blot analysis of transformants of Trichoderma harzianum DNA. Genomic DNA (10 µg) from five transformants (pbr1-2A, 16A, 36A, 55A, and 57A) and from the wild type (WT) was digested with PstI (A) or HindIII (B), separated by agarose gel electrophoresis, and analyzed by the Southern blot technique. Blots were probed with a ³²P-labelled 5.0 kb EcoRI fragment containing the pbr1 gene from T. harzianum.

Figure 3 - Alkaline protease activity of Trichodema harzianum transformants. T. harzianum wild type and transformants were grown for 72 h in MM + glucose 2% at 28^oC. Afterwards, mycelia were transferred to MM + colloidal chitin 0.1% (open bars) or colloidal chitin 0.1% combined with glucose 2% (grid bars). These cultures were grown for 48 h at 28^oC, and supernatants were assayed for alkaline protease activity. Activity was expressed as a percentage of wild type strain activity. These results are the means of three experiments.

Figure 4 - Northern blot was performed on the mRNA obtained during specific mycoparasitic interactions using Trichoderma harzianum wild type and the transformant strain pbr1-2A against Rhizoctonia solani. A: Different stages of the interaction. B: Northern analysis of the pbr1 mRNA obtained during dual interactions. The left panel shows dual interactions between the wild type and R. solani, whereas the right panel shows dual interactions between pbr1-2A and R. solani. C: Northern analysis of the tub1 mRNA obtained during dual interactions. The left panel shows dual interactions between the wild type and R. solani, whereas the right panel shows dual interactions between pbr1-2A and R. solani. In panel AIII, T. harzianum has grown over R. solani mycelia.

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