Three hrfA (hypersensitive response-functioning faction A) homologues (hrf1, hrf2 and hrf3) are cloned from 12 strains of Xanthomonas oryzae using PCR based techniques. Hrf1, hrf2 and hrf3 are derived from strains bel...Three hrfA (hypersensitive response-functioning faction A) homologues (hrf1, hrf2 and hrf3) are cloned from 12 strains of Xanthomonas oryzae using PCR based techniques. Hrf1, hrf2 and hrf3 are derived from strains belonging to X. o. pv. oryzae, X. o. pv. oryzicola and X. o. pv. oryzae respectively. Sequence analysis shows that all three genes encode glycine-rich pro-teins with various numbers of GGG-GG motifs. They all share a conserved cysteine residue at position 45 or 47. Hrf1 and hrf3 encode HarpinXoo while hrf2 encodes Harpinxooc. Hrf1 and hrf3 encodes two different types of HarpinXoo proteins. Hrf1 from X. o. pv. oryzae strains ( JxoIII, JxoIV, Jxov, Pxo61, Pxo76, Pxo79, Pxo99, Pxo99 and Pxo124) encodes a 15.6 kD HarpinXoo with 3 GGG-GG motifs while Hrf3 from strain Pxo86 and Pxo112 encodes a 15.9 kD Harpinxoo with 4 GGG-GG motifs. Harpinxooc encoded by hrf2 from X. o. pv. oryzicola (strain RS105) has the mo-lecular weight of 15.3 kD and contains 2 GGG-GG motifs. Cluster analysis is performed using deduced sequences of hrf1, hrf2 and hrf3 as well as previously reported Hpa1 and XopI protein sequence. The results indicated that Harpinxoo and Harpinxooc belong to two closely related sub-groups. Hrf, hrf2 and hrf3 are expressed in E. coli strain BL21 successfully. Under the same condition, hrf1, hrf2 and hrf3 are expressed at the level of 0.389, 0.530 and 0.083 mg/mL re-spectively. All expressed hrf1, hrf2 and hrf3 proteins (Harpins) are shown to be able to induce hypersensitive reaction and TMV resistance on tobacco. Among the three proteins, Hrf2 has the highest activity while Hrf3 has the lowest activity.展开更多
Using harpin protein gene from apple fire blight pathogen Erwinia amylavora and potato prp1-1 promoter as main DNA elements, the feasibility of using pathogen infection-induced hypersensitive response was explored as ...Using harpin protein gene from apple fire blight pathogen Erwinia amylavora and potato prp1-1 promoter as main DNA elements, the feasibility of using pathogen infection-induced hypersensitive response was explored as a new strategy of engineering fungal disease resistance. Three plant transformation vectors were constructed and 68 transgenic potato plants were produced through Agrobacterium mediated transformation method. Southern, Northern and Western blot analysis demonstrated the insertion, transcription and protein expression of harpin protein gene in transgenic plants. Disease resistance test using a complex race of Phytophthora infestans as challenging pathogen showed that both constitutive and pathogen infection-induced expression of harpin protein gene in transgenic potato reduced the lesion growth rate of fungus. Among plants where harpin protein gene expression was induced only by fungus infection, two plants were found to be highly resistant to P. infestans infection. Fungal hyphae were not produced on total of 30 inoculated leaves from the two resistant plants and necrotic lesion was limited to inoculation area. The results highlighted that engineered hypersensitive response in plants was a very promising approach to produce fungal disease resistant genotype.展开更多
基金This work was supported the National Key Basic Research Plan of China(973-G20000016201,2003CB 114204)National Natural Science Foundation of China(Grant No.30230240)Science and Technology Plan of Nanjing(2002ZB0114).
文摘Three hrfA (hypersensitive response-functioning faction A) homologues (hrf1, hrf2 and hrf3) are cloned from 12 strains of Xanthomonas oryzae using PCR based techniques. Hrf1, hrf2 and hrf3 are derived from strains belonging to X. o. pv. oryzae, X. o. pv. oryzicola and X. o. pv. oryzae respectively. Sequence analysis shows that all three genes encode glycine-rich pro-teins with various numbers of GGG-GG motifs. They all share a conserved cysteine residue at position 45 or 47. Hrf1 and hrf3 encode HarpinXoo while hrf2 encodes Harpinxooc. Hrf1 and hrf3 encodes two different types of HarpinXoo proteins. Hrf1 from X. o. pv. oryzae strains ( JxoIII, JxoIV, Jxov, Pxo61, Pxo76, Pxo79, Pxo99, Pxo99 and Pxo124) encodes a 15.6 kD HarpinXoo with 3 GGG-GG motifs while Hrf3 from strain Pxo86 and Pxo112 encodes a 15.9 kD Harpinxoo with 4 GGG-GG motifs. Harpinxooc encoded by hrf2 from X. o. pv. oryzicola (strain RS105) has the mo-lecular weight of 15.3 kD and contains 2 GGG-GG motifs. Cluster analysis is performed using deduced sequences of hrf1, hrf2 and hrf3 as well as previously reported Hpa1 and XopI protein sequence. The results indicated that Harpinxoo and Harpinxooc belong to two closely related sub-groups. Hrf, hrf2 and hrf3 are expressed in E. coli strain BL21 successfully. Under the same condition, hrf1, hrf2 and hrf3 are expressed at the level of 0.389, 0.530 and 0.083 mg/mL re-spectively. All expressed hrf1, hrf2 and hrf3 proteins (Harpins) are shown to be able to induce hypersensitive reaction and TMV resistance on tobacco. Among the three proteins, Hrf2 has the highest activity while Hrf3 has the lowest activity.
基金Project partly supported by the Molecular Biology Laboratory of CPRO-DLO in Netherlands.
文摘Using harpin protein gene from apple fire blight pathogen Erwinia amylavora and potato prp1-1 promoter as main DNA elements, the feasibility of using pathogen infection-induced hypersensitive response was explored as a new strategy of engineering fungal disease resistance. Three plant transformation vectors were constructed and 68 transgenic potato plants were produced through Agrobacterium mediated transformation method. Southern, Northern and Western blot analysis demonstrated the insertion, transcription and protein expression of harpin protein gene in transgenic plants. Disease resistance test using a complex race of Phytophthora infestans as challenging pathogen showed that both constitutive and pathogen infection-induced expression of harpin protein gene in transgenic potato reduced the lesion growth rate of fungus. Among plants where harpin protein gene expression was induced only by fungus infection, two plants were found to be highly resistant to P. infestans infection. Fungal hyphae were not produced on total of 30 inoculated leaves from the two resistant plants and necrotic lesion was limited to inoculation area. The results highlighted that engineered hypersensitive response in plants was a very promising approach to produce fungal disease resistant genotype.
