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. 1996 Aug;178(15):4661–4669. doi: 10.1128/jb.178.15.4661-4669.1996

Spontaneous and induced mutations in a single open reading frame alter both virulence and avirulence in Xanthomonas campestris pv. vesicatoria avrBs2.

K M Swords 1, D Dahlbeck 1, B Kearney 1, M Roy 1, B J Staskawicz 1
PMCID: PMC178237  PMID: 8755898

Abstract

Molecular characterization of the avrBs2 locus from Xanthomonas campestris pv. vesicatoria has revealed that expression of this gene triggers disease resistance in Bs2 pepper (Capsicum annuum) plants and contributes to virulence of the pathogen. Deletion analysis and site-directed mutagenesis established the avrBs2 gene as a 2,190-bp open reading frame encoding a putative 80.1-kDa protein. Two classes of Xanthomonas pathogens evading Bs2 host resistance and displaying reduced fitness were found to be specifically mutated in avrBs2. Members of one class contained a 5-bp insertion, while the second class was distinguished by a divergent 3' region of avrBs2; both mutant classes were complemented in trans by a plasmid-borne copy of avrBs2. A divergent avrBs2 homolog was cloned from the Brassica pathogen X. campestris pv. campestris. The predicted AvrBs2 proteins from the two Xanthomonas pathovars were strongly conserved and had predicted sequence similarity with both Agrobacterium tumefaciens agrocinopine synthase and Escherichia coli UgpQ, two enzymes involved in the synthesis or hydrolysis of phosphodiester linkages. On the basis of homology with agrocinopine synthase and UgpQ and the dual phenotype of avirulence and virulence, several models for the function of AvrBs2 are proposed.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Amemura A., Cabrera-Crespo J. Extracellular oligosaccharides and low-Mr polysaccharides containing (1----2)-beta-D-glucosidic linkages from strains of Xanthomonas, Escherichia coli and Klebsiella pneumoniae. J Gen Microbiol. 1986 Sep;132(9):2443–2452. doi: 10.1099/00221287-132-9-2443. [DOI] [PubMed] [Google Scholar]
  3. Bonas U., Stall R. E., Staskawicz B. Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria. Mol Gen Genet. 1989 Jul;218(1):127–136. doi: 10.1007/BF00330575. [DOI] [PubMed] [Google Scholar]
  4. Bonas U. hrp genes of phytopathogenic bacteria. Curr Top Microbiol Immunol. 1994;192:79–98. doi: 10.1007/978-3-642-78624-2_4. [DOI] [PubMed] [Google Scholar]
  5. Breedveld M. W., Miller K. J. Cyclic beta-glucans of members of the family Rhizobiaceae. Microbiol Rev. 1994 Jun;58(2):145–161. doi: 10.1128/mr.58.2.145-161.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brzoska P., Boos W. The ugp-encoded glycerophosphoryldiester phosphodiesterase, a transport-related enzyme of Escherichia coli. FEMS Microbiol Rev. 1989 Jun;5(1-2):115–124. doi: 10.1016/0168-6445(89)90015-6. [DOI] [PubMed] [Google Scholar]
  7. Cangelosi G. A., Martinetti G., Nester E. W. Osmosensitivity phenotypes of Agrobacterium tumefaciens mutants that lack periplasmic beta-1,2-glucan. J Bacteriol. 1990 Apr;172(4):2172–2174. doi: 10.1128/jb.172.4.2172-2174.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dangl J. L. The enigmatic avirulence genes of phytopathogenic bacteria. Curr Top Microbiol Immunol. 1994;192:99–118. doi: 10.1007/978-3-642-78624-2_5. [DOI] [PubMed] [Google Scholar]
  9. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dylan T., Helinski D. R., Ditta G. S. Hypoosmotic adaptation in Rhizobium meliloti requires beta-(1----2)-glucan. J Bacteriol. 1990 Mar;172(3):1400–1408. doi: 10.1128/jb.172.3.1400-1408.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fenselau S., Balbo I., Bonas U. Determinants of pathogenicity in Xanthomonas campestris pv. vesicatoria are related to proteins involved in secretion in bacterial pathogens of animals. Mol Plant Microbe Interact. 1992 Sep-Oct;5(5):390–396. doi: 10.1094/mpmi-5-390. [DOI] [PubMed] [Google Scholar]
  12. Fenselau S., Bonas U. Sequence and expression analysis of the hrpB pathogenicity operon of Xanthomonas campestris pv. vesicatoria which encodes eight proteins with similarity to components of the Hrp, Ysc, Spa, and Fli secretion systems. Mol Plant Microbe Interact. 1995 Nov-Dec;8(6):845–854. doi: 10.1094/mpmi-8-0845. [DOI] [PubMed] [Google Scholar]
  13. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Foster P. L., Trimarchi J. M. Adaptive reversion of a frameshift mutation in Escherichia coli by simple base deletions in homopolymeric runs. Science. 1994 Jul 15;265(5170):407–409. doi: 10.1126/science.8023164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Geiger O., Weissborn A. C., Kennedy E. P. Biosynthesis and excretion of cyclic glucans by Rhizobium meliloti 1021. J Bacteriol. 1991 May;173(9):3021–3024. doi: 10.1128/jb.173.9.3021-3024.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gough C. L., Genin S., Zischek C., Boucher C. A. hrp genes of Pseudomonas solanacearum are homologous to pathogenicity determinants of animal pathogenic bacteria and are conserved among plant pathogenic bacteria. Mol Plant Microbe Interact. 1992 Sep-Oct;5(5):384–389. doi: 10.1094/mpmi-5-384. [DOI] [PubMed] [Google Scholar]
  17. Huynh T. V., Dahlbeck D., Staskawicz B. J. Bacterial blight of soybean: regulation of a pathogen gene determining host cultivar specificity. Science. 1989 Sep 22;245(4924):1374–1377. doi: 10.1126/science.2781284. [DOI] [PubMed] [Google Scholar]
  18. Innes R. W., Bent A. F., Kunkel B. N., Bisgrove S. R., Staskawicz B. J. Molecular analysis of avirulence gene avrRpt2 and identification of a putative regulatory sequence common to all known Pseudomonas syringae avirulence genes. J Bacteriol. 1993 Aug;175(15):4859–4869. doi: 10.1128/jb.175.15.4859-4869.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Innes R. W., Hirose M. A., Kuempel P. L. Induction of nitrogen-fixing nodules on clover requires only 32 kilobase pairs of DNA from the Rhizobium trifolii symbiosis plasmid. J Bacteriol. 1988 Sep;170(9):3793–3802. doi: 10.1128/jb.170.9.3793-3802.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kearney B., Staskawicz B. J. Characterization of IS476 and its role in bacterial spot disease of tomato and pepper. J Bacteriol. 1990 Jan;172(1):143–148. doi: 10.1128/jb.172.1.143-148.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kearney B., Staskawicz B. J. Widespread distribution and fitness contribution of Xanthomonas campestris avirulence gene avrBs2. Nature. 1990 Jul 26;346(6282):385–386. doi: 10.1038/346385a0. [DOI] [PubMed] [Google Scholar]
  22. Keen N. T. Gene-for-gene complementarity in plant-pathogen interactions. Annu Rev Genet. 1990;24:447–463. doi: 10.1146/annurev.ge.24.120190.002311. [DOI] [PubMed] [Google Scholar]
  23. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  24. Levinson G., Gutman G. A. High frequencies of short frameshifts in poly-CA/TG tandem repeats borne by bacteriophage M13 in Escherichia coli K-12. Nucleic Acids Res. 1987 Jul 10;15(13):5323–5338. doi: 10.1093/nar/15.13.5323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Loubens I., Debarbieux L., Bohin A., Lacroix J. M., Bohin J. P. Homology between a genetic locus (mdoA) involved in the osmoregulated biosynthesis of periplasmic glucans in Escherichia coli and a genetic locus (hrpM) controlling pathogenicity of Pseudomonas syringae. Mol Microbiol. 1993 Oct;10(2):329–340. doi: 10.1111/j.1365-2958.1993.tb01959.x. [DOI] [PubMed] [Google Scholar]
  26. Miller K. J., Gore R. S., Benesi A. J. Phosphoglycerol substituents present on the cyclic beta-1,2-glucans of Rhizobium meliloti 1021 are derived from phosphatidylglycerol. J Bacteriol. 1988 Oct;170(10):4569–4575. doi: 10.1128/jb.170.10.4569-4575.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Miller K. J., Hadley J. A., Gustine D. L. Cyclic [beta]-1,6-1,3-Glucans of Bradyrhizobium japonicum USDA 110 Elicit Isoflavonoid Production in the Soybean (Glycine max) Host. Plant Physiol. 1994 Mar;104(3):917–923. doi: 10.1104/pp.104.3.917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Miller K. J., Kennedy E. P., Reinhold V. N. Osmotic adaptation by gram-negative bacteria: possible role for periplasmic oligosaccharides. Science. 1986 Jan 3;231(4733):48–51. doi: 10.1126/science.3941890. [DOI] [PubMed] [Google Scholar]
  29. Murillo J., Shen H., Gerhold D., Sharma A., Cooksey D. A., Keen N. T. Characterization of pPT23B, the plasmid involved in syringolide production by Pseudomonas syringae pv. tomato PT23. Plasmid. 1994 May;31(3):275–287. doi: 10.1006/plas.1994.1029. [DOI] [PubMed] [Google Scholar]
  30. Paulus F., Otten L. Functional and mutated agrocinopine synthase genes on octopine T-DNAs. Mol Plant Microbe Interact. 1993 May-Jun;6(3):393–402. doi: 10.1094/mpmi-6-393. [DOI] [PubMed] [Google Scholar]
  31. Ritter C., Dangl J. L. The avrRpm1 gene of Pseudomonas syringae pv. maculicola is required for virulence on Arabidopsis. Mol Plant Microbe Interact. 1995 May-Jun;8(3):444–453. doi: 10.1094/mpmi-8-0444. [DOI] [PubMed] [Google Scholar]
  32. Ryder M. H., Tate M. E., Jones G. P. Agrocinopine A, a tumor-inducing plasmid-coded enzyme product, is a phosphodiester of sucrose and L-arabinose. J Biol Chem. 1984 Aug 10;259(15):9704–9710. [PubMed] [Google Scholar]
  33. Salmeron J. M., Staskawicz B. J. Molecular characterization and hrp dependence of the avirulence gene avrPto from Pseudomonas syringae pv. tomato [corrected]. Mol Gen Genet. 1993 May;239(1-2):6–16. doi: 10.1007/BF00281595. [DOI] [PubMed] [Google Scholar]
  34. Shen H., Keen N. T. Characterization of the promoter of avirulence gene D from Pseudomonas syringae pv. tomato. J Bacteriol. 1993 Sep;175(18):5916–5924. doi: 10.1128/jb.175.18.5916-5924.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Staskawicz B. J., Ausubel F. M., Baker B. J., Ellis J. G., Jones J. D. Molecular genetics of plant disease resistance. Science. 1995 May 5;268(5211):661–667. doi: 10.1126/science.7732374. [DOI] [PubMed] [Google Scholar]
  37. Staskawicz B., Dahlbeck D., Keen N., Napoli C. Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol. 1987 Dec;169(12):5789–5794. doi: 10.1128/jb.169.12.5789-5794.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Strand M., Prolla T. A., Liskay R. M., Petes T. D. Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature. 1993 Sep 16;365(6443):274–276. doi: 10.1038/365274a0. [DOI] [PubMed] [Google Scholar]
  39. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tommassen J., Eiglmeier K., Cole S. T., Overduin P., Larson T. J., Boos W. Characterization of two genes, glpQ and ugpQ, encoding glycerophosphoryl diester phosphodiesterases of Escherichia coli. Mol Gen Genet. 1991 Apr;226(1-2):321–327. doi: 10.1007/BF00273621. [DOI] [PubMed] [Google Scholar]
  41. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  42. Whalen M. C., Wang J. F., Carland F. M., Heiskell M. E., Dahlbeck D., Minsavage G. V., Jones J. B., Scott J. W., Stall R. E., Staskawicz B. J. Avirulence gene avrRxv from Xanthomonas campestris pv. vesicatoria specifies resistance on tomato line Hawaii 7998. Mol Plant Microbe Interact. 1993 Sep-Oct;6(5):616–627. doi: 10.1094/mpmi-6-616. [DOI] [PubMed] [Google Scholar]
  43. Xiao Y., Lu Y., Heu S., Hutcheson S. W. Organization and environmental regulation of the Pseudomonas syringae pv. syringae 61 hrp cluster. J Bacteriol. 1992 Mar;174(6):1734–1741. doi: 10.1128/jb.174.6.1734-1741.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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