Cloning and characterization of avrXa21 determinants from Xanthomonas oryzae pv. oryzae

A plasmid clone (plasmid 10.78) from a genomic library of Xanthomonas oryzae pv. oryzae (Xoo), PX099 strain (P6) was identified, that changed the phenotype of the Xoo Korean strain DY89031 (K1) from virulent to avirulent on Xa21 rice plants. Plasmid 10.78 contained a 9 kb fragment with eight putative open reading frames (ORFs). Three ORFs, xooST, xooA and xooB, are organized in an operon and comprised the minimum region conferring avirulence to K1. The predicted product of xooST showed similarity to Rhizobium sp. and human sulfotransferases, whereas those of xooA and xooB showed similarity to components of a type I secretion system. Because xooST, xooA and xooB do not seem to encode for the actual AvrXa21 elicitor, but most likely for modifier and transporter apparatus necessary for elicitor function, they were termed 'avirulence determinants'. Marker exchange mutagenesis revealed that these ORFs are required for full avirulence activity in P6. P6 strains with mutations in xooST or xooA were completely virulent on Xa21 plants, whereas xooB was partially virulent on Xa21 plants. These ORFs were cloned and sequenced from the Xoo strains, PX086 (P2) and DY89031 (K1), which are avirulent and virulent, respectively, on Xa21 rice plants. The DNA sequence of the xooST, xooA and xooB ORFs were almost identical between P2 and P6. The predicted proteins were 99.7%, 98.7% and 98.7% identical, respectively. In contrast, in K1, a 26-bp insertion was identified downstream of the putative start codon for the xooA ORF, causing a frame shift mutation. Additionally, the XooB protein from K1 has one amino acid change, an arginine in place of a methionine, in a highly conserved region among other ABC transporters, that might modify or eliminate transport or substrate recognition functions. xooST, xooA and xooB are conserved among most Xanthomonas species, but not in X. campestris pv. juglandis or other plant pathogenic bacteria as revealed by Southern blot hybridization analysis. The identification of avirulence determinants presented in this dissertation should facilitate the isolation of the AvrXa21 elicitor. Together with the isolation of this elicitor, this work will provide the first detailed evidence for the role of a type I secretion system in controlling the specificity of plant-bacteria interaction. Furthermore, because rice is a model system of other cereal crops, this work may provide improved ways for control of other bacterial diseases of cereals.