Genetic diversity of Harpins from Xanthomonas oryzae and their activity to induce hypersensitive response and disease resistance in tobacco

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.