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A novel antimicrobial protein for plant protection consisting of a Xanthomonas oryzae harpin and active domains of cecropin A and melittin.

Che YZ, Li YR, Zou HS, Zou LF, Zhang B, Chen GY - Microb Biotechnol (2011)

Bottom Line: The resulting chimeric protein maintained not only the HR-inducing property of the harpin, but also the antimicrobial activity of the cecropin A-melittin hybrid.Importantly, the protein acted as a potential pesticide by inducing disease resistance for viral, bacterial and fungal pathogens.This designed drug can be considered as a lead compound for use in plant protection, either for the development of new broad-spectrum pesticides or for expression in transgenic plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, Nanjing Agricultural University/Key Laboratory of Monitoring and Management for Plant Diseases and Insects, Ministry of Agriculture of China, Nanjing 210095, China.

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The coding sequence of Hcm1, the construction of the five chimeric proteins, Pep1, Pep2, Pep3, Pep4 and Hcm1, and results from their biological testing. A. The DNA and amino acid sequence of Hcm1, indicating the α‐helices of Hpa1, cecropin A and melittin (solid underline); the polylinker (dashed underline); and the flexible hinge (dashed‐dot underline). B. Construction of chimeric protein genes with corresponding primer pairs: pep1 (Hpa1‐F/P1); pep2 (Hpa1‐F/P2); pep3 (the fragment of Hpa1‐F/Hpa1‐R2 was fused with the DNA of P3/P2 at the BamHI site); pep4 (first amplified by Hpa1‐F/P4 and then by Hpa1‐F/P5); and hcm1 (the fragment of Hpa1‐F/Hpa1‐R2 was fused with the DNA of P3/P5 at the BamHI site). The arrows represent the location and orientation of the primers (Table 2). ‘+’ stands for HR induction in tobacco (N. tabacum cv. Xanthi nn) and antimicrobial activity against rice bacterial pathogen X. oryzae pv. oryzicola by gene products extracted as CFEPs from E. coli expression strains containing pep1, pep2, pep3, pep4 and hcm1 genes respectively. ‘−’ indicates no HR or no pathogen inhibition. Hpa1 was used as the control.
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f1: The coding sequence of Hcm1, the construction of the five chimeric proteins, Pep1, Pep2, Pep3, Pep4 and Hcm1, and results from their biological testing. A. The DNA and amino acid sequence of Hcm1, indicating the α‐helices of Hpa1, cecropin A and melittin (solid underline); the polylinker (dashed underline); and the flexible hinge (dashed‐dot underline). B. Construction of chimeric protein genes with corresponding primer pairs: pep1 (Hpa1‐F/P1); pep2 (Hpa1‐F/P2); pep3 (the fragment of Hpa1‐F/Hpa1‐R2 was fused with the DNA of P3/P2 at the BamHI site); pep4 (first amplified by Hpa1‐F/P4 and then by Hpa1‐F/P5); and hcm1 (the fragment of Hpa1‐F/Hpa1‐R2 was fused with the DNA of P3/P5 at the BamHI site). The arrows represent the location and orientation of the primers (Table 2). ‘+’ stands for HR induction in tobacco (N. tabacum cv. Xanthi nn) and antimicrobial activity against rice bacterial pathogen X. oryzae pv. oryzicola by gene products extracted as CFEPs from E. coli expression strains containing pep1, pep2, pep3, pep4 and hcm1 genes respectively. ‘−’ indicates no HR or no pathogen inhibition. Hpa1 was used as the control.

Mentions: The HR induction in tobacco by Hpa1Xoo from X. oryzae pv. oryzae, the causal agent of rice bacterial blight, is attributed to two α‐helices involved in coiled‐coil protein interactions, SEKQLDQLLCQLISALLQ and PFTQMLMHIVGEILQAQ, at the N‐ and C‐termini of the protein respectively (Ji et al., 2010). The use of SMART software (http://smart.embl‐heidelberg.de) to analyse Hpa1 of X. oryzae pv. oryzicola, a homologue of Hpa1Xoo (Zou et al., 2006), predicted two α‐helices, ISEKQLDQLLCQLIQALL and ASPLTQMLNIVGEILQAQ, also at the N‐ and C‐termini (Fig. 1). Since mutations in the N‐terminal α‐helices of Hpa1Xoo and HpaG of X. axonopodis pv. glycines led to the loss of HR induction in tobacco (Oh et al., 2007; Wang et al., 2007; Ji et al., 2010), a rational design approach for developing a new antimicrobial protein prompts us to maintain the α‐helix structures in Hpa1 while adding other AMPs to its C‐terminus. For one of these, cecropin A, the antibacterial activity is due to the α‐helix at its N‐terminus (Andreu et al., 1983; Hancock, 2001; Shai, 2002; Ferre et al., 2006; Makovitzki et al., 2007); for the second, melittin, the cytolytic and toxic activity towards microbes depends on an α‐helix at its C‐terminus (Hristova et al., 2001; Ferre et al., 2006; Glättli et al., 2006; Raghuraman and Chattopadhyay, 2007). A hybrid molecule that includes both of these α‐helices linked by a flexible hinge, GQGIG, shows high antibacterial activity (Ferre et al., 2006; Saugar et al., 2006; Xu et al., 2007). To investigate whether linkers between Hpa1 and the α‐helix of cecropin A, and between the α‐helix of cecropin A and the α‐helix of melittin, are required for the chimeric proteins to induce HR in plants and to show antimicrobial activity in vitro, we constructed five chimeric genes (Fig. 1): pep1, where the hpa1 gene without a stop codon at its 3′ terminus was fused directly to the sequence encoding the α‐helix, KLFKKIEKV, of cecropin A, plus a stop codon; pep2, where the pep1 gene was linked directly at its 3′ terminus to the sequence encoding the α‐helix, AVLKVLTTGL, of melittin, plus a stop codon; pep3, where a polylinker, DPGGFGGKW (Wriggers et al., 2005), that maintained the reading frame was used to join the 3′ end of the hpa1 gene, without a stop codon, to the sequence encoding the α‐helices of both cecropin A and melittin (without a flexible hinge between them); pep4, where the 3′ terminus of the hpa1 gene without a stop codon was fused directly (without the polylinker) to the sequence for the hybrid peptide, where cecropin A and melittin were linked via the flexible hinge between them; and hcm1, where the polylinker mentioned above linked hpa1 and the α‐helix sequence of cecropin A, and the flexible hinge linked the α‐helix sequences of cecropin A and melittin. The unmodified hpa1 gene was used as the control. The above chimeric genes were cloned with a His‐tag sequence into plasmid pET30a(+), producing the recombinant constructs pPep1, pPep2, pPep3, pPep4 and pHcm1 respectively (Table 1, Fig. 1).


A novel antimicrobial protein for plant protection consisting of a Xanthomonas oryzae harpin and active domains of cecropin A and melittin.

Che YZ, Li YR, Zou HS, Zou LF, Zhang B, Chen GY - Microb Biotechnol (2011)

The coding sequence of Hcm1, the construction of the five chimeric proteins, Pep1, Pep2, Pep3, Pep4 and Hcm1, and results from their biological testing. A. The DNA and amino acid sequence of Hcm1, indicating the α‐helices of Hpa1, cecropin A and melittin (solid underline); the polylinker (dashed underline); and the flexible hinge (dashed‐dot underline). B. Construction of chimeric protein genes with corresponding primer pairs: pep1 (Hpa1‐F/P1); pep2 (Hpa1‐F/P2); pep3 (the fragment of Hpa1‐F/Hpa1‐R2 was fused with the DNA of P3/P2 at the BamHI site); pep4 (first amplified by Hpa1‐F/P4 and then by Hpa1‐F/P5); and hcm1 (the fragment of Hpa1‐F/Hpa1‐R2 was fused with the DNA of P3/P5 at the BamHI site). The arrows represent the location and orientation of the primers (Table 2). ‘+’ stands for HR induction in tobacco (N. tabacum cv. Xanthi nn) and antimicrobial activity against rice bacterial pathogen X. oryzae pv. oryzicola by gene products extracted as CFEPs from E. coli expression strains containing pep1, pep2, pep3, pep4 and hcm1 genes respectively. ‘−’ indicates no HR or no pathogen inhibition. Hpa1 was used as the control.
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Related In: Results  -  Collection

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f1: The coding sequence of Hcm1, the construction of the five chimeric proteins, Pep1, Pep2, Pep3, Pep4 and Hcm1, and results from their biological testing. A. The DNA and amino acid sequence of Hcm1, indicating the α‐helices of Hpa1, cecropin A and melittin (solid underline); the polylinker (dashed underline); and the flexible hinge (dashed‐dot underline). B. Construction of chimeric protein genes with corresponding primer pairs: pep1 (Hpa1‐F/P1); pep2 (Hpa1‐F/P2); pep3 (the fragment of Hpa1‐F/Hpa1‐R2 was fused with the DNA of P3/P2 at the BamHI site); pep4 (first amplified by Hpa1‐F/P4 and then by Hpa1‐F/P5); and hcm1 (the fragment of Hpa1‐F/Hpa1‐R2 was fused with the DNA of P3/P5 at the BamHI site). The arrows represent the location and orientation of the primers (Table 2). ‘+’ stands for HR induction in tobacco (N. tabacum cv. Xanthi nn) and antimicrobial activity against rice bacterial pathogen X. oryzae pv. oryzicola by gene products extracted as CFEPs from E. coli expression strains containing pep1, pep2, pep3, pep4 and hcm1 genes respectively. ‘−’ indicates no HR or no pathogen inhibition. Hpa1 was used as the control.
Mentions: The HR induction in tobacco by Hpa1Xoo from X. oryzae pv. oryzae, the causal agent of rice bacterial blight, is attributed to two α‐helices involved in coiled‐coil protein interactions, SEKQLDQLLCQLISALLQ and PFTQMLMHIVGEILQAQ, at the N‐ and C‐termini of the protein respectively (Ji et al., 2010). The use of SMART software (http://smart.embl‐heidelberg.de) to analyse Hpa1 of X. oryzae pv. oryzicola, a homologue of Hpa1Xoo (Zou et al., 2006), predicted two α‐helices, ISEKQLDQLLCQLIQALL and ASPLTQMLNIVGEILQAQ, also at the N‐ and C‐termini (Fig. 1). Since mutations in the N‐terminal α‐helices of Hpa1Xoo and HpaG of X. axonopodis pv. glycines led to the loss of HR induction in tobacco (Oh et al., 2007; Wang et al., 2007; Ji et al., 2010), a rational design approach for developing a new antimicrobial protein prompts us to maintain the α‐helix structures in Hpa1 while adding other AMPs to its C‐terminus. For one of these, cecropin A, the antibacterial activity is due to the α‐helix at its N‐terminus (Andreu et al., 1983; Hancock, 2001; Shai, 2002; Ferre et al., 2006; Makovitzki et al., 2007); for the second, melittin, the cytolytic and toxic activity towards microbes depends on an α‐helix at its C‐terminus (Hristova et al., 2001; Ferre et al., 2006; Glättli et al., 2006; Raghuraman and Chattopadhyay, 2007). A hybrid molecule that includes both of these α‐helices linked by a flexible hinge, GQGIG, shows high antibacterial activity (Ferre et al., 2006; Saugar et al., 2006; Xu et al., 2007). To investigate whether linkers between Hpa1 and the α‐helix of cecropin A, and between the α‐helix of cecropin A and the α‐helix of melittin, are required for the chimeric proteins to induce HR in plants and to show antimicrobial activity in vitro, we constructed five chimeric genes (Fig. 1): pep1, where the hpa1 gene without a stop codon at its 3′ terminus was fused directly to the sequence encoding the α‐helix, KLFKKIEKV, of cecropin A, plus a stop codon; pep2, where the pep1 gene was linked directly at its 3′ terminus to the sequence encoding the α‐helix, AVLKVLTTGL, of melittin, plus a stop codon; pep3, where a polylinker, DPGGFGGKW (Wriggers et al., 2005), that maintained the reading frame was used to join the 3′ end of the hpa1 gene, without a stop codon, to the sequence encoding the α‐helices of both cecropin A and melittin (without a flexible hinge between them); pep4, where the 3′ terminus of the hpa1 gene without a stop codon was fused directly (without the polylinker) to the sequence for the hybrid peptide, where cecropin A and melittin were linked via the flexible hinge between them; and hcm1, where the polylinker mentioned above linked hpa1 and the α‐helix sequence of cecropin A, and the flexible hinge linked the α‐helix sequences of cecropin A and melittin. The unmodified hpa1 gene was used as the control. The above chimeric genes were cloned with a His‐tag sequence into plasmid pET30a(+), producing the recombinant constructs pPep1, pPep2, pPep3, pPep4 and pHcm1 respectively (Table 1, Fig. 1).

Bottom Line: The resulting chimeric protein maintained not only the HR-inducing property of the harpin, but also the antimicrobial activity of the cecropin A-melittin hybrid.Importantly, the protein acted as a potential pesticide by inducing disease resistance for viral, bacterial and fungal pathogens.This designed drug can be considered as a lead compound for use in plant protection, either for the development of new broad-spectrum pesticides or for expression in transgenic plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, Nanjing Agricultural University/Key Laboratory of Monitoring and Management for Plant Diseases and Insects, Ministry of Agriculture of China, Nanjing 210095, China.

Show MeSH
Related in: MedlinePlus