Back
 AiM  Vol.7 No.1 , January 2017
Identification of New Genes Related to Virulence of Xanthomonas axonopodis Pv. Citri during Citrus Host Interactions
Abstract: A mutant library of the bacterium Xanthomonas citri subsp. citri strain 306 pathotype A (Xac), the causative agent of most aggressive Asiatic type A citrus canker, was screened regarding altered canker symptoms after inoculations into Citrus sinensis and Citrus limonia host leaves. Twenty-six mutants have shown phenotypic virulence changes and have respectively knocked out gene identified by sequencing. In vivo growth curves were obtained for nine mutants to quantify how the mutations could affect pathogen’s adaptability to growth inside and attack host plant infected tissue. Among identified genes in mutated strains, we could find those that until now had not been reported as being involved in Xac adaptation and/or virulence, such as predicted to encode for xylose repressor-like protein (XACΔxylR), Fe-S oxidoredutase (XACΔaslB), helicase IV (XACΔhelD), ubiquinol cytochrome c oxidoreductase iron-sulfur subunit (XACΔpetA), chromosome partitioning protein (XACΔparB) and cell division protein FtsB (XACΔftsB), in addition to genes predicted to encode for hypothetical proteins. The new genes found in this study as being relevant to adaptation and virulence, improve the understanding of Xac fitness during citrus plant attack and canker symptoms development.
Cite this paper: Ferreira, C. , Moreira, L. , Brigati, J. , Lima, L. , Ferro, J. , Ferro, M. and Oliveira, J. (2017) Identification of New Genes Related to Virulence of Xanthomonas axonopodis Pv. Citri during Citrus Host Interactions. Advances in Microbiology, 7, 22-46. doi: 10.4236/aim.2017.71003.
References

[1]   Schaad, N.W., Postnikova, E., Lacy, G.H., Sechler, A., Agarkova, I., Stromberg, P.E., Stromberg, V.K. and Vidaver, A.K. (2005) Reclassification of Xanthomonas campestris pv. citri (ex Hasse 1915) Dye 1978 Forms A, B/C/D, and E as X. smithii subsp. citri (ex Hasse) sp. nov. nom. rev. comb. nov., X. fuscans subsp. aurantifolii (ex Gabriel 1989) sp. nov. nom. rev. comb. nov., and X. alfalfae subsp. citrumelo (ex Riker and Jones) Gabriel et al., 1989 sp. nov. nom. rev. comb. nov.; X. campestris pv. malvacearum (ex Smith 1901) Dye 1978 as X. smithii subsp. smithii nov. comb. nov. nom. nov.; X. campestris pv. alfalfae (ex Riker and Jones, 1935) Dye 1978 as X. alfalfae subsp. alfalfae (ex Riker et al., 1935) sp. nov. nom. rev.; and “var. fuscans” of X. campestris pv. phaseoli (ex Smith, 1987) Dye 1978 as X. fuscans subsp. fuscans sp. nov. Systematic and Applied Microbiology, 28, 494-518.
https://doi.org/10.1016/j.syapm.2005.03.017

[2]   Brunings, A.M. and Gabriel, D.W. (2003) Xanthomonas citri: Breaking the Surface. Molecular Plant Pathology, 4, 141-157. https://doi.org/10.1046/j.1364-3703.2003.00163.x

[3]   da Silva, A.C., Ferro, J.A., Reinach, F.C., Farah, C.S., Furlan, L.R., Quaggio, R.B., Monteiro-Vitorello, C.B., Van Sluys, M.A., Almeida, N.F., Alves, L.M., et al. (2002) Comparison of the Genomes of Two Xanthomonas Pathogens with Differing Host Specificities. Nature, 417, 459-463. https://doi.org/10.1038/417459a

[4]   Moreira, L.M., Almeida Jr., N.F., Potnis, N., Digiampietri, L.A., Adi, S.S., Bortolossi, J.C., da Silva, A.C., da Silva, A.M., de Moraes, F.E., de Oliveira, J.C., et al. (2010) Novel Insights into the Genomic Basis of Citrus Canker Based on the Genome Sequences of Two Strains of Xanthomonas fuscans subsp. aurantifolii. BMC Genomics, 11, 238.
https://doi.org/10.1186/1471-2164-11-238

[5]   Verniere, C., Hartung, J.S., Pruvost, O.P., Civerolo, E.L., Alvarez, A.M., Maestri, P. and Luisetti, J. (1998) Characterization of Phenotypically Distinct Strains of Xanthomonas axonopodis pv. citri from Southwest Asia. European Journal of Plant Pathology, 104, 477-487.
https://doi.org/10.1023/A:1008676508688

[6]   Sun, X., Stall, R.E., Jones, J.B., Cubero, J., Gottwald, T.R., Graham, J.H., Dixon, W.H., Schubert, T.S., Chaloux, P.H., Stromberg, V.K., et al. (2004) Detection and Characterization of a New Strain of Citrus Canker Bacteria from Key/Mexican Lime and Alemow in South Florida. Plant Disease, 88, 1179-1188. https://doi.org/10.1094/PDIS.2004.88.11.1179

[7]   Chen, G., Zou, H., Ye, G., Song, X., Ji, Z. and Ma, W. (2013) Completed Genome of Xanthomonas axonopodis Sp. Xac29-1. In EMBL/GenBank/DDBJ Databases.

[8]   Jalan, N., Kumar, D., Yu, F., Jones, J.B., Graham, J.H. and Wang, N. (2013) Complete Genome Sequence of Xanthomonas citri subsp. citri Strain Aw12879, a Restricted-Host-Range Citrus Canker-Causing Bacterium. Genome Announcements, 1, e00235-13.
https://doi.org/10.1128/genomeA.00235-13

[9]   Gottig, N., Garavaglia, B.S., Daurelio, L.D., Valentine, A., Gehring, C., Orellano, E.G. and Ottado, J. (2008) Xanthomonas axonopodis pv. citri Uses a Plant Natriuretic Peptide-Like Protein to Modify Host Homeostasis. Proceedings of the National Academy of Sciences of the United States of America, 105, 18631-18636. https://doi.org/10.1073/pnas.0810107105

[10]   Gottig, N., Garavaglia, B.S., Garofalo, C.G., Orellano, E.G. and Ottado, J. (2009) A Filamentous Hemagglutinin-Like Protein of Xanthomonas axonopodis pv. citri, the Phytopathogen Responsible for Citrus Canker, Is Involved in Bacterial Virulence. PLoS ONE, 4, e4358.
https://doi.org/10.1371/journal.pone.0004358

[11]   Baptista, J.C., Machado, M.A., Homem, R.A., Torres, P.S., Vojnov, A.A. and do Amaral, A.M. (2010) Mutation in the XpsD Gene of Xanthomonas axonopodis pv. citri Affects Cellulose Degradation and Virulence. Genetics and Molecular Biology, 33, 146-153.
https://doi.org/10.1590/S1415-47572009005000110

[12]   Cappelletti, P.A., dos Santos, R.F., do Amaral, A.M., Homem, R.A., Souza, T.S., Machado, M.A. and Farah, C.S. (2011) Structure-Function Analysis of the HrpB2-HrcU Interaction in the Xanthomonas citri Type III Secretion System. PLoS ONE, 6, e17614.
https://doi.org/10.1371/journal.pone.0017614

[13]   Facincani, A.P., Moreira, L.M., Soares, M.R., Ferreira, C.B., Ferreira, R.M., Ferro, M.I.T., Ferro, J.A., Gozzo, F.C. and Oliveira, J.C.F. (2013) Comparative Proteomic Analysis Reveals that T3SS, Tfp, and Xanthan Gum Are Key Factors in Initial Stages of Citrus sinensis Infection by Xanthomonas citri subsp. citri. Functional & Integrative Genomics, 14, 205-217.
https://doi.org/10.1007/s10142-013-0340-5

[14]   Laia, M.L., Moreira, L.M., Dezajacomo, J., Brigati, J.B., Ferreira, C.B., Ferro, M.I., Silva, A.C., Ferro, J.A. and Oliveira, J.C. (2009) New Genes of Xanthomonas citri subsp. citri Involved in Pathogenesis and Adaptation Revealed by a Transposon-Based Mutant Library. BMC Microbiology, 9, 12. https://doi.org/10.1186/1471-2180-9-12

[15]   Li, J. and Wang, N. (2011) Genome-Wide Mutagenesis of Xanthomonas axonopodis pv. citri Reveals Novel Genetic Determinants and Regulation Mechanisms of Biofilm Formation. PLoS ONE, 6, e21804. https://doi.org/10.1371/journal.pone.0021804

[16]   Song, X., Guo, J., Ma, W.X., Ji, Z.Y., Zou, L.F., Chen, G.Y. and Zou, H.S. (2015) Identification of Seven Novel Virulence Genes from Xanthomonas citri subsp. citri by Tn5-Based Random Mutagenesis. Journal of Microbiology, 53, 330-336.
https://doi.org/10.1007/s12275-015-4589-3

[17]   Goryshin, I.Y., Jendrisak, J., Hoffman, L.M., Meis, R. and Reznikoff, W.S. (2000) Insertional Transposon Mutagenesis by Electroporation of Released Tn5 Transposition Complexes. Nature Biotechnology, 18, 97-100. https://doi.org/10.1038/72017

[18]   Schaad, N.W., Jones, J.B. and Chun, W. (2001) Laboratory Guide for Identification of Plant Pathogenic Bacteria. APS Press, St. Paul.

[19]   Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic Local Alignment Search Tool. Journal of Molecular Biology, 215, 403-410.
https://doi.org/10.1016/S0022-2836(05)80360-2

[20]   Kanehisa, M. and Goto, S. (2000) KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Research, 28, 27-30. https://doi.org/10.1093/nar/28.1.27

[21]   Parkinson, N., Cowie, C., Heeney, J. and Stead, D. (2009) Phylogenetic Structure of Xanthomonas Determined by Comparison of GyrB Sequences. International Journal of Systematic and Evolutionary Microbiology, 59, 264-274. https://doi.org/10.1099/ijs.0.65825-0

[22]   Rodriguez, R.L., Grajales, A., Arrieta-Ortiz, M.L., Salazar, C., Restrepo, S. and Bernal, A. (2012) Genomes-Based Phylogeny of the Genus Xanthomonas. BMC Microbiology, 12, 43.
https://doi.org/10.1186/1471-2180-12-43

[23]   Song, W.Y., Kim, H.M., Hwang, C.Y. and Schaad, N.W. (2004) Detection of Acidovorax avenae Ssp. avenae in Rice Seeds Using BIO-PCR. Journal of Phytopathology, 152, 667-676.
https://doi.org/10.1111/j.1439-0434.2004.00914.x

[24]   Brock, T.D., Madigan, M.T., Martinko, J.M. and Parker, J.E. (2000) Biology of Microorganisms. 9th Edition, Prentice Hall, Upper Saddle River.

[25]   Reslewic, S., Zhou, S., Place, M., Zhang, Y., Briska, A., Goldstein, S., Churas, C., Runnheim, R., Forrest, D., Lim, A., et al. (2005) Whole-Genome Shotgun Optical Mapping of Rhodospirillum rubrum. Applied and Environmental Microbiology, 71, 5511-5522.
https://doi.org/10.1128/AEM.71.9.5511-5522.2005

[26]   Marchler-Bauer, A., Zheng, C., Chitsaz, F., Derbyshire, M.K., Geer, L.Y., Geer, R.C., Gonzales, N.R., Gwadz, M., Hurwitz, D.I., Lanczycki, C.J., et al. (2013) CDD: Conserved Domains and Protein Three-Dimensional Structure. Nucleic Acids Research, 41, D348-D352.
https://doi.org/10.1093/nar/gks1243

[27]   Darrasse, A., Carrere, S., Barbe, V., Boureau, T., Arrieta-Ortiz, M.L., Bonneau, S., Briand, M., Brin, C., Cociancich, S., Durand, K., et al. (2013) Genome Sequence of Xanthomonas fuscans subsp. fuscans Strain 4834-R Reveals That Flagellar Motility Is Not a General Feature of Xanthomonads. BMC Genomics, 14, 761. https://doi.org/10.1186/1471-2164-14-761

[28]   Jalan, N., Aritua, V., Kumar, D., Yu, F., Jones, J.B., Graham, J.H., Setubal, J.C. and Wang, N. (2011) Comparative Genomic Analysis of Xanthomonas axonopodis pv. citrumelo F1, Which Causes Citrus Bacterial Spot Disease, and Related Strains Provides Insights into Virulence and Host Specificity. Journal of Bacteriology, 193, 6342-6357. https://doi.org/10.1128/JB.05777-11

[29]   Thieme, F., Koebnik, R., Bekel, T., Berger, C., Boch, J., Buttner, D., Caldana, C., Gaigalat, L., Goesmann, A., Kay, S., et al. (2005) Insights into Genome Plasticity and Pathogenicity of the Plant Pathogenic Bacterium Xanthomonas campestris pv. vesicatoria Revealed by the Complete Genome Sequence. Journal of Bacteriology, 187, 7254-7266.
https://doi.org/10.1128/JB.187.21.7254-7266.2005

[30]   Mendonca, V.M., Klepin, H.D. and Matson, S.W. (1995) DNA Helicases in Recombination and Repair: Construction of a Delta UvrD Delta HelD Delta RecQ Mutant Deficient in Recombination and Repair. Journal of Bacteriology, 177, 1326-1335.
https://doi.org/10.1128/jb.177.5.1326-1335.1995

[31]   Wiedermannova, J., Sudzinova, P., Koval, T., Rabatinova, A., Sanderova, H., Ramaniuk, O., Rittich, S., Dohnalek, J., Fu, Z., Halada, P., et al. (2004) Characterization of HelD, an Interacting Partner of RNA Polymerase from Bacillus subtilis. Nucleic Acids Research, 42, 5151-5163.
https://doi.org/10.1093/nar/gku113

[32]   Zygmunt, M.S., Hagius, S.D., Walker, J.V. and Elzer, P.H. (2006) Identification of Brucella melitensis 16M Genes Required for Bacterial Survival in the Caprine Host. Microbes and Infection, 8, 2849-2854. https://doi.org/10.1016/j.micinf.2006.09.002

[33]   Gomelsky, M. and Klug, G. (2002) BLUF: A Novel FAD-Binding Domain Involved in Sensory Transduction in Microorganisms. Trends in Biochemical Sciences, 27, 497-500.
https://doi.org/10.1016/S0968-0004(02)02181-3

[34]   Van der Spek, P.J., Kobayashi, K., Bootsma, D., Takao, M., Eker, A.P. and Yasui, A. (1996) Cloning, Tissue Expression, and Mapping of a Human Photolyase Homolog with Similarity to Plant Blue-Light Receptors. Genomics, 37, 177-182. https://doi.org/10.1006/geno.1996.0539

[35]   Kraiselburd, I., Alet, A.I., Tondo, M.L., Petrocelli, S., Daurelio, L.D., Monzon, J., Ruiz, O.A., Losi, A. and Orellano, E.G. (2012) A LOV Protein Modulates the Physiological Attributes of Xanthomonas axonopodis pv. citri Relevant for Host Plant Colonization. PLoS ONE, 7, e38226.
https://doi.org/10.1371/journal.pone.0038226

[36]   Feil, H., Feil, W.S., Chain, P., Larimer, F., DiBartolo, G., Copeland, A., Lykidis, A., Trong, S., Nolan, M., Goltsman, E., et al. (2005) Comparison of the Complete Genome Sequences of Pseudomonas syringae pv. syringae B728a and pv. tomato DC3000. Proceedings of the National Academy of Sciences of the United States of America, 102, 11064-11069.
https://doi.org/10.1073/pnas.0504930102

[37]   Plumbridge, J. (2001) Regulation of PTS Gene Expression by the Homologous Transcriptional Regulators, Mlc and NagC, in Escherichia coli (or How Two Similar Repressors Can Behave Differently). Journal of Molecular Microbiology and Biotechnology, 3, 371-380.

[38]   Plumbridge, J. (2001) DNA Binding Sites for the Mlc and NagC Proteins: Regulation of nagE, Encoding the N-Acetylglucosamine-Specific Transporter in Escherichia coli. Nucleic Acids Research, 29, 506-514. https://doi.org/10.1093/nar/29.2.506

[39]   Titgemeyer, F., Reizer, J., Reizer, A. and Saier, M.H.J. (1994) Evolutionary Relationships between Sugar Kinases and Transcriptional Repressors in Bacteria. Microbiology, 140, 2349-2354. https://doi.org/10.1099/13500872-140-9-2349

[40]   Carvalho, F.M., Oliveira, J.C., Laia, M.L., Jacob, T.R., Ferreira, R.M., Ferro, M.I., Tezza, R.I., Zingaretti, S.M., Silva, C.F. and Ferro, J.A. (2016) Mapping and Validation of Xanthomonas citri Subsp citri Genes Regulated by Putative Plant-Inducible Promoter Box (PIP-Box). Genetics and Molecular Research, 15.

[41]   Fenselau, S. and Bonas, U. (1995) 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. Molecular Plant-Microbe Interactions, 8, 845-854. https://doi.org/10.1094/MPMI-8-0845

[42]   Dejean, G., Blanvillain-Baufume, S., Boulanger, A., Darrasse, A., de Duge, B.T., Girard, A.L., Carrere, S., Jamet, S., Zischek, C., Lautier, M., et al. (2013) The Xylan Utilization System of the Plant Pathogen Xanthomonas campestris Pv campestris Controls Epiphytic Life and Reveals Common Features with Oligotrophic Bacteria and Animal Gut Symbionts. New Phytologist, 198, 899-915. https://doi.org/10.1111/nph.12187

[43]   Mohl, D.A., Easter, J.J. and Gober, J.W. (2001) The Chromosome Partitioning Protein, ParB, Is Required for Cytokinesis in Caulobacter crescentus. Molecular Microbiology, 42, 741-755.
https://doi.org/10.1046/j.1365-2958.2001.02643.x

[44]   Sengupta, M. and Austin, S. (2011) Prevalence and Significance of Plasmid Maintenance Functions in the Virulence Plasmids of Pathogenic Bacteria. Infection and Immunity, 79, 2502-2509. https://doi.org/10.1128/IAI.00127-11

[45]   Villanelo, F., Ordenes, A., Brunet, J., Lagos, R. and Monasterio, O. (2011) A Model for the Escherichia coli FtsB/FtsL/FtsQ Cell Division Complex. BMC Structural Biology, 11, 28. https://doi.org/10.1186/1472-6807-11-28

[46]   Bateman, A., Coin, L., Durbin, R., Finn, R.D., Hollich, V., Griffiths-Jones, S., Khanna, A., Marshall, M., Moxon, S., Sonnhammer, E.L., et al. (2004) The Pfam Protein Families Database. Nucleic Acids Research, 32, D138-D141. https://doi.org/10.1093/nar/gkh121

[47]   Ciurli, S. and Musiani, F. (2005) High Potential Iron-Sulfur Proteins and Their Role as Soluble Electron Carriers in Bacterial Photosynthesis: Tale of a Discovery. Photosynthesis Research, 85, 115-131. https://doi.org/10.1007/s11120-004-6556-4

[48]   Bruschi, M. and Guerlesquin, F. (1988) Structure, Function and Evolution of Bacterial Ferredoxins. FEMS Microbiology Reviews, 54, 155-175.
https://doi.org/10.1111/j.1574-6968.1988.tb02741.x

[49]   Rudolph, K. (1993) Infection of the Plant by Xanthomonas. In: Swings, J.G. and Civerolo, E.L., Eds., Xanthomonas, Chapman and Hall, New York, 193-254.
https://doi.org/10.1007/978-94-011-1526-1_4

[50]   Chou, F.L., Chou, H.C., Lin, Y.S., Yang, B.Y., Lin, N.T., Weng, S.F. and Tseng, Y.H. (1997) The Xanthomonas campestris gumD Gene Required for Synthesis of Xanthan Gum Is Involved in Normal Pigmentation and Virulence in Causing Black Rot. Biochemical and Biophysical Research Communications, 233, 265-269. https://doi.org/10.1006/bbrc.1997.6365

[51]   Katzen, F., Ferreiro, D.U., Oddo, C.G., Ielmini, M.V., Becker, A., Puhler, A. and Ielpi, L. (1998) Xanthomonas campestris pv. campestris Gum Mutants: Effects on Xanthan Biosynthesis and Plant Virulence. Journal of Bacteriology, 180, 1607-1617.

[52]   Kemp, B.P. (2004) Xanthomonas axonopodis pv. manihotis gumD Gene Is Essential for EPS Production and Pathogenicity and Enhances Epiphytic Survival on Cassava (Manihot esculenta). Physiology Molecular Plant Pathology, 64, 209-218.
https://doi.org/10.1016/j.pmpp.2004.08.007

[53]   Chakrabarty, A.M. (1998) Nucleoside Diphosphate Kinase: Role in Bacterial Growth, Virulence, Cell Signalling and Polysaccharide Synthesis. Molecular Microbiology, 28, 875-882.
https://doi.org/10.1046/j.1365-2958.1998.00846.x

[54]   Yang, W., Zhou, Y., Dai, H., Bi, L., Zhang, Z., Zhang, X., Leng, Y. and Zhang, X. (2008) Application of Methyl Parathion Hydrolase (MPH) as a Labeling Enzyme. Analytical and Bioanalytical Chemistry, 390, 2133-2140. https://doi.org/10.1007/s00216-008-1987-y

[55]   Marger, M.D. and Saier, M.H. (1993) A Major Superfamily of Transmembrane Facilitators that Catalyze Uniport, Symport and Antiport. Trends in Biochemical Sciences, 18, 13-20.
https://doi.org/10.1016/0968-0004(93)90081-W

[56]   Fry, W.E. (1982) Principles of Plant Disease Management. Elsevier Academic Press, San Diego.

[57]   Agrios, G.N. (2004) Plant Pathology. Elsevier Academic Press, San Diego.

[58]   Intile, P.J., Balzer, G.J., Wolfgang, M.C. and Yahr, T.L. (2015) The RNA Helicase DeaD Stimulates ExsA Translation to Promote Expression of the Pseudomonas aeruginosa Type III Secretion System. Journal of Bacteriology, 197, 2664-2674.
https://doi.org/10.1128/JB.00231-15

[59]   Ito, T., Tyler, J.K., Bulger, M., Kobayashi, R. and Kadonaga, J.T. (1996) ATP-Facilitated Chromatin Assembly with a Nucleoplasmin-Like Protein from Drosophila melanogaster. The Journal of Biological Chemistry, 271, 25041-25048. https://doi.org/10.1074/jbc.271.40.25041

[60]   Gu, Y.Q. and Walling, L.L. (2002) Identification of Residues Critical for Activity of the Wound-Induced Leucine Aminopeptidase (LAP-A) of Tomato. European Journal of Biochemistry/FEBS, 269, 1630-1640.

[61]   Plener, L., Boistard, P., Gonzalez, A., Boucher, C. and Genin, S. (2012) Metabolic Adaptation of Ralstonia solanacearum during Plant Infection: A Methionine Biosynthesis Case Study. PLoS ONE, 7, e36877. https://doi.org/10.1371/journal.pone.0036877

[62]   Moran, N.A. (2002) Microbial Minimalism: Genome Reduction in Bacterial Pathogens. Cell, 108, 583-586. https://doi.org/10.1016/S0092-8674(02)00665-7

 
 
Top