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Recognition of a core fragment ofBeauveria bassiana hydrophobin gene promoter (P hyd1) and its special use in improving fungal biocontrol potential.

Wang ZL, Ying SH, Feng MG - Microb Biotechnol (2012)

Bottom Line: Further truncating P hyd1-t1 to 1179, 991 and 791 bp or mutating one of the binding domains of three transcription factors in P hyd1-t1 reduced significantly the expression of eGFP (enhanced green fluorescence protein).Therefore, P hyd1-t1 was used to integrate a bacterium-derived, insect midgut-specific toxin (vip3Aa1) gene into B. bassiana, yielding a transgenic strain (BbHV8) expressing 9.8-fold more toxin molecules in conidia than a counterpart strain (BbV28) expressing the toxin under the control of P gpdA, a promoter widely used for gene expression in fungi.Conclusively, P hyd1-t1 is a useful tool for enhancing beneficial protein expression, such as vip3Aa1, in fungal conidia, which are the active ingredients of mycoinsecticides.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China.

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Recognition of a core fragment in the hyd1 promoter region (Phyd1) of B. bassiana. a. Concatenation of expression elements in binary plasmids for insertion into wild-type strain (Bb2860) and comparison of eGFP expression levels (RFI) in the 4 day colonies of fungal transformants controlled by full-length, four truncated and three site-mutated fragments of Phyd1 and the widely used PgpdA respectively. Close and open symbols denote the normal and mutated binding domains of StuA (square), Mat-Mc (triangle) and NIT2 (circle) respectively. Error bars: SD of the mean of three transformants. b. Laser confocal fluorescence (left) and bright (right) images of samples of an eGFP-expressing colony (b1−b3: grown at 25°C for 2, 4 and 7 days respectively under Phyd1-t1 control; b4: the same images of a fungal mass from 4 day wild-type colony). Scale bars: 10 μm. c. RFI trend during the 7 day growth of three Phyd1-t1 controlled transformants on SDAY plates at 25°C.
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fig01: Recognition of a core fragment in the hyd1 promoter region (Phyd1) of B. bassiana. a. Concatenation of expression elements in binary plasmids for insertion into wild-type strain (Bb2860) and comparison of eGFP expression levels (RFI) in the 4 day colonies of fungal transformants controlled by full-length, four truncated and three site-mutated fragments of Phyd1 and the widely used PgpdA respectively. Close and open symbols denote the normal and mutated binding domains of StuA (square), Mat-Mc (triangle) and NIT2 (circle) respectively. Error bars: SD of the mean of three transformants. b. Laser confocal fluorescence (left) and bright (right) images of samples of an eGFP-expressing colony (b1−b3: grown at 25°C for 2, 4 and 7 days respectively under Phyd1-t1 control; b4: the same images of a fungal mass from 4 day wild-type colony). Scale bars: 10 μm. c. RFI trend during the 7 day growth of three Phyd1-t1 controlled transformants on SDAY plates at 25°C.

Mentions: The core fragment of Phyd1 was located using four truncated and three site-mutated Phyd1 fragments to drive eGFP expression in transgenic Bb2860. Relative fluorescence intensities (RFI) under their controls (Fig. 1a) differed significantly (F8,18 = 114, P < 0.0001 in one-way analysis of variance). The full-length Phyd1 was capable of driving eGFP expression but its efficiency was 1.6-fold lower than the Phyd1-t1 counterpart. This truncated fragment resulted in maximal RFI (1081 ± 104) in transgenic colonies. This RFI was 15.6-fold higher than that from the PgpdA-controlled transformants. However, further truncating Phyd1-t1 (1290 bp) to Phyd1-t2 (1179 bp), Phyd1-t3 (991 bp) and Phyd1-t4 (791 bp) reduced the eGFP expression by 16.7%, 71.3% and 98% respectively. All site mutations in Phyd1-t1 also led to significant RFI reductions in the transgenic colonies (Fisher's least significant difference, P < 0.05). The net RFI reductions were 17%, 52% and 81% for the mutated binding domains of Mat-Mc, NIT2 and StuA respectively. Apparently, Phyd1-t1 vectoring the essential binding domains of the three TFs was the core promoter fragment to maximize heterologous gene expression in B. bassiana.


Recognition of a core fragment ofBeauveria bassiana hydrophobin gene promoter (P hyd1) and its special use in improving fungal biocontrol potential.

Wang ZL, Ying SH, Feng MG - Microb Biotechnol (2012)

Recognition of a core fragment in the hyd1 promoter region (Phyd1) of B. bassiana. a. Concatenation of expression elements in binary plasmids for insertion into wild-type strain (Bb2860) and comparison of eGFP expression levels (RFI) in the 4 day colonies of fungal transformants controlled by full-length, four truncated and three site-mutated fragments of Phyd1 and the widely used PgpdA respectively. Close and open symbols denote the normal and mutated binding domains of StuA (square), Mat-Mc (triangle) and NIT2 (circle) respectively. Error bars: SD of the mean of three transformants. b. Laser confocal fluorescence (left) and bright (right) images of samples of an eGFP-expressing colony (b1−b3: grown at 25°C for 2, 4 and 7 days respectively under Phyd1-t1 control; b4: the same images of a fungal mass from 4 day wild-type colony). Scale bars: 10 μm. c. RFI trend during the 7 day growth of three Phyd1-t1 controlled transformants on SDAY plates at 25°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3815382&req=5

fig01: Recognition of a core fragment in the hyd1 promoter region (Phyd1) of B. bassiana. a. Concatenation of expression elements in binary plasmids for insertion into wild-type strain (Bb2860) and comparison of eGFP expression levels (RFI) in the 4 day colonies of fungal transformants controlled by full-length, four truncated and three site-mutated fragments of Phyd1 and the widely used PgpdA respectively. Close and open symbols denote the normal and mutated binding domains of StuA (square), Mat-Mc (triangle) and NIT2 (circle) respectively. Error bars: SD of the mean of three transformants. b. Laser confocal fluorescence (left) and bright (right) images of samples of an eGFP-expressing colony (b1−b3: grown at 25°C for 2, 4 and 7 days respectively under Phyd1-t1 control; b4: the same images of a fungal mass from 4 day wild-type colony). Scale bars: 10 μm. c. RFI trend during the 7 day growth of three Phyd1-t1 controlled transformants on SDAY plates at 25°C.
Mentions: The core fragment of Phyd1 was located using four truncated and three site-mutated Phyd1 fragments to drive eGFP expression in transgenic Bb2860. Relative fluorescence intensities (RFI) under their controls (Fig. 1a) differed significantly (F8,18 = 114, P < 0.0001 in one-way analysis of variance). The full-length Phyd1 was capable of driving eGFP expression but its efficiency was 1.6-fold lower than the Phyd1-t1 counterpart. This truncated fragment resulted in maximal RFI (1081 ± 104) in transgenic colonies. This RFI was 15.6-fold higher than that from the PgpdA-controlled transformants. However, further truncating Phyd1-t1 (1290 bp) to Phyd1-t2 (1179 bp), Phyd1-t3 (991 bp) and Phyd1-t4 (791 bp) reduced the eGFP expression by 16.7%, 71.3% and 98% respectively. All site mutations in Phyd1-t1 also led to significant RFI reductions in the transgenic colonies (Fisher's least significant difference, P < 0.05). The net RFI reductions were 17%, 52% and 81% for the mutated binding domains of Mat-Mc, NIT2 and StuA respectively. Apparently, Phyd1-t1 vectoring the essential binding domains of the three TFs was the core promoter fragment to maximize heterologous gene expression in B. bassiana.

Bottom Line: Further truncating P hyd1-t1 to 1179, 991 and 791 bp or mutating one of the binding domains of three transcription factors in P hyd1-t1 reduced significantly the expression of eGFP (enhanced green fluorescence protein).Therefore, P hyd1-t1 was used to integrate a bacterium-derived, insect midgut-specific toxin (vip3Aa1) gene into B. bassiana, yielding a transgenic strain (BbHV8) expressing 9.8-fold more toxin molecules in conidia than a counterpart strain (BbV28) expressing the toxin under the control of P gpdA, a promoter widely used for gene expression in fungi.Conclusively, P hyd1-t1 is a useful tool for enhancing beneficial protein expression, such as vip3Aa1, in fungal conidia, which are the active ingredients of mycoinsecticides.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China.

Show MeSH
Related in: MedlinePlus