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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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Plant disease reduction by Hcm1. A. Hcm1 induces resistance to tobacco mosaic virus (TMV) infection. Three days after spraying N. tabacum cv. Xanthi nn plants with Hcm1 (1.5 µM), cotton swabs were used to gently and evenly apply freshly prepared TMV inoculum mixed with emery powder (see Experimental procedures) to the tobacco leaf surfaces. Necrotic spots formed by TMV were counted 3 days post inoculation (dpi). B. Application of Hcm1 to prevent rice blast caused by M. oryzae. Three days after the spraying of Hcm1 at 1.5 µM, a suspension of M. oryzae Guy11 spores (1 × 105 spore ml−1) was sprayed on leaves of 4‐week‐old O. sativa cv. CO‐39. The necrotic lesions of rice blast were counted 5 dpi. C. Prevention of tomato bacterial wilt disease by Hcm1. A fresh R. solanacearum suspension was adjusted to 1 × 108 cfu ml−1 and needle‐injected into stems of one‐month old seedling S. lycopersicum Suhong 2003 three days after spraying with Hcm1. The percentage of wilted leaves per plant was determined 8 and 16 dpi respectively. PBS buffer and Hpa1 at 1.5 µM were used as the negative and positive controls. The experiments shown were replicated three times and similar results were obtained. The different letters at the shoulders of PBS, Hpa1 and Hcm1 indicate significant differences in reducing disease severities at P = 0.01 by t‐test.
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f7: Plant disease reduction by Hcm1. A. Hcm1 induces resistance to tobacco mosaic virus (TMV) infection. Three days after spraying N. tabacum cv. Xanthi nn plants with Hcm1 (1.5 µM), cotton swabs were used to gently and evenly apply freshly prepared TMV inoculum mixed with emery powder (see Experimental procedures) to the tobacco leaf surfaces. Necrotic spots formed by TMV were counted 3 days post inoculation (dpi). B. Application of Hcm1 to prevent rice blast caused by M. oryzae. Three days after the spraying of Hcm1 at 1.5 µM, a suspension of M. oryzae Guy11 spores (1 × 105 spore ml−1) was sprayed on leaves of 4‐week‐old O. sativa cv. CO‐39. The necrotic lesions of rice blast were counted 5 dpi. C. Prevention of tomato bacterial wilt disease by Hcm1. A fresh R. solanacearum suspension was adjusted to 1 × 108 cfu ml−1 and needle‐injected into stems of one‐month old seedling S. lycopersicum Suhong 2003 three days after spraying with Hcm1. The percentage of wilted leaves per plant was determined 8 and 16 dpi respectively. PBS buffer and Hpa1 at 1.5 µM were used as the negative and positive controls. The experiments shown were replicated three times and similar results were obtained. The different letters at the shoulders of PBS, Hpa1 and Hcm1 indicate significant differences in reducing disease severities at P = 0.01 by t‐test.

Mentions: Since Hcm1 activates plant defence genes in planta (Fig. 2) and shows antimicrobial activity in vitro (Figs 2, 4–6), we investigated whether Hcm1 reduces plant disease severity by spraying it on plants prior to inoculation with plant pathogens. To test different types of pathogens on different plants, we chose tobacco mosaic virus (TMV), the bacterium R. solanacearum and the fungus M. oryzae, which cause the diseases tobacco mosaic, bacterial wilt of tomato, and rice blast respectively. We investigated whether prior treatment of plants with Hcm1 can: (i) increase resistance to TMV in Nicotiana tabacum cv. Xanthi nn, which does not contain the N gene and produces HR‐like necrotic‐like lesions when TMV is rubbed on leaves (Enyedi et al., 1992; Ehrenfeld et al., 2008); (ii) reduce the number of brown necrotic spots caused when M. oryzae Guy11 infects seedlings of Oryza sativa cv. CO‐39 (Zhang et al., 2009a); and (iii) reduce the bacterial wilt seen in Solanum lycopersicum cv. Suhong 2003 when infected by R. solanacearum ZJ3721 (Li et al., 2010). Ten plants each of 2‐month‐old tobacco, 1‐month‐old rice and 1‐month‐old tomato were sprayed fully with Hcm1 or Hpa1 (1.5 µM), and then sprayed again 3 days later. Three days after the second spraying, the plants were inoculated – the tobacco by softly rubbing the leaf surfaces with cotton tips containing a TMV‐emery powder mixture, the rice by spraying fresh M. oryzae spores (1 × 105 spore ml−1) on the leaf surfaces, and the tomato by injecting R. solanacearum (1 × 108 cfu ml−1) into the stems with needled syringes. Disease symptoms were assessed at different times following inoculation as indicated in the legend to Fig. 7. The number of necrotic spots and the necrotic area per leaf were measured for the tobacco plants inoculated with TMV and the rice plants inoculated with M. oryzae (which produced brown dark spindle spots), and the severity of plant wilting was assessed for tomato bacterial wilt. The data in Fig. 7 and Table 4 show clearly the effectiveness of Hcm1‐induced resistance against TMV, M. oryzae and R. solanacearum infections. The control plants initially sprayed with PBS showed significantly more infection by all three pathogens than the Hcm1‐treated plants (Table 4). Prior application of Hpa1, which had been shown previously to induce systemic resistance against plant pathogen infections (Fontanilla et al., 2005; Zhao et al., 2006; Shao et al., 2008), also significantly reduced the number of necrotic spots produced by TMV or M. oryzae in our experiments, but it was not as effective as Hcm1 in reducing the incidence of bacterial wilt in tomato (Fig. 7, Table 4). The significantly better (P = 0.01, t‐test) protection seen with prior treatment with Hcm1 versus Hpa1 against both rice blast and bacterial wilt in tomato (Fig. 7D, Table 4) may be due to the addition in Hcm1 of the cecropin A‐melittin hybrid peptide at its C‐terminus. This suggests that application of the rationally designed protein Hcm1 to crop plants may be a new and effective way to control plant diseases.


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)

Plant disease reduction by Hcm1. A. Hcm1 induces resistance to tobacco mosaic virus (TMV) infection. Three days after spraying N. tabacum cv. Xanthi nn plants with Hcm1 (1.5 µM), cotton swabs were used to gently and evenly apply freshly prepared TMV inoculum mixed with emery powder (see Experimental procedures) to the tobacco leaf surfaces. Necrotic spots formed by TMV were counted 3 days post inoculation (dpi). B. Application of Hcm1 to prevent rice blast caused by M. oryzae. Three days after the spraying of Hcm1 at 1.5 µM, a suspension of M. oryzae Guy11 spores (1 × 105 spore ml−1) was sprayed on leaves of 4‐week‐old O. sativa cv. CO‐39. The necrotic lesions of rice blast were counted 5 dpi. C. Prevention of tomato bacterial wilt disease by Hcm1. A fresh R. solanacearum suspension was adjusted to 1 × 108 cfu ml−1 and needle‐injected into stems of one‐month old seedling S. lycopersicum Suhong 2003 three days after spraying with Hcm1. The percentage of wilted leaves per plant was determined 8 and 16 dpi respectively. PBS buffer and Hpa1 at 1.5 µM were used as the negative and positive controls. The experiments shown were replicated three times and similar results were obtained. The different letters at the shoulders of PBS, Hpa1 and Hcm1 indicate significant differences in reducing disease severities at P = 0.01 by t‐test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815413&req=5

f7: Plant disease reduction by Hcm1. A. Hcm1 induces resistance to tobacco mosaic virus (TMV) infection. Three days after spraying N. tabacum cv. Xanthi nn plants with Hcm1 (1.5 µM), cotton swabs were used to gently and evenly apply freshly prepared TMV inoculum mixed with emery powder (see Experimental procedures) to the tobacco leaf surfaces. Necrotic spots formed by TMV were counted 3 days post inoculation (dpi). B. Application of Hcm1 to prevent rice blast caused by M. oryzae. Three days after the spraying of Hcm1 at 1.5 µM, a suspension of M. oryzae Guy11 spores (1 × 105 spore ml−1) was sprayed on leaves of 4‐week‐old O. sativa cv. CO‐39. The necrotic lesions of rice blast were counted 5 dpi. C. Prevention of tomato bacterial wilt disease by Hcm1. A fresh R. solanacearum suspension was adjusted to 1 × 108 cfu ml−1 and needle‐injected into stems of one‐month old seedling S. lycopersicum Suhong 2003 three days after spraying with Hcm1. The percentage of wilted leaves per plant was determined 8 and 16 dpi respectively. PBS buffer and Hpa1 at 1.5 µM were used as the negative and positive controls. The experiments shown were replicated three times and similar results were obtained. The different letters at the shoulders of PBS, Hpa1 and Hcm1 indicate significant differences in reducing disease severities at P = 0.01 by t‐test.
Mentions: Since Hcm1 activates plant defence genes in planta (Fig. 2) and shows antimicrobial activity in vitro (Figs 2, 4–6), we investigated whether Hcm1 reduces plant disease severity by spraying it on plants prior to inoculation with plant pathogens. To test different types of pathogens on different plants, we chose tobacco mosaic virus (TMV), the bacterium R. solanacearum and the fungus M. oryzae, which cause the diseases tobacco mosaic, bacterial wilt of tomato, and rice blast respectively. We investigated whether prior treatment of plants with Hcm1 can: (i) increase resistance to TMV in Nicotiana tabacum cv. Xanthi nn, which does not contain the N gene and produces HR‐like necrotic‐like lesions when TMV is rubbed on leaves (Enyedi et al., 1992; Ehrenfeld et al., 2008); (ii) reduce the number of brown necrotic spots caused when M. oryzae Guy11 infects seedlings of Oryza sativa cv. CO‐39 (Zhang et al., 2009a); and (iii) reduce the bacterial wilt seen in Solanum lycopersicum cv. Suhong 2003 when infected by R. solanacearum ZJ3721 (Li et al., 2010). Ten plants each of 2‐month‐old tobacco, 1‐month‐old rice and 1‐month‐old tomato were sprayed fully with Hcm1 or Hpa1 (1.5 µM), and then sprayed again 3 days later. Three days after the second spraying, the plants were inoculated – the tobacco by softly rubbing the leaf surfaces with cotton tips containing a TMV‐emery powder mixture, the rice by spraying fresh M. oryzae spores (1 × 105 spore ml−1) on the leaf surfaces, and the tomato by injecting R. solanacearum (1 × 108 cfu ml−1) into the stems with needled syringes. Disease symptoms were assessed at different times following inoculation as indicated in the legend to Fig. 7. The number of necrotic spots and the necrotic area per leaf were measured for the tobacco plants inoculated with TMV and the rice plants inoculated with M. oryzae (which produced brown dark spindle spots), and the severity of plant wilting was assessed for tomato bacterial wilt. The data in Fig. 7 and Table 4 show clearly the effectiveness of Hcm1‐induced resistance against TMV, M. oryzae and R. solanacearum infections. The control plants initially sprayed with PBS showed significantly more infection by all three pathogens than the Hcm1‐treated plants (Table 4). Prior application of Hpa1, which had been shown previously to induce systemic resistance against plant pathogen infections (Fontanilla et al., 2005; Zhao et al., 2006; Shao et al., 2008), also significantly reduced the number of necrotic spots produced by TMV or M. oryzae in our experiments, but it was not as effective as Hcm1 in reducing the incidence of bacterial wilt in tomato (Fig. 7, Table 4). The significantly better (P = 0.01, t‐test) protection seen with prior treatment with Hcm1 versus Hpa1 against both rice blast and bacterial wilt in tomato (Fig. 7D, Table 4) may be due to the addition in Hcm1 of the cecropin A‐melittin hybrid peptide at its C‐terminus. This suggests that application of the rationally designed protein Hcm1 to crop plants may be a new and effective way to control plant diseases.

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