Indian Phytopathology

, Volume 71, Issue 2, pp 197–205 | Cite as

Characterization of four vital protein encoding genes of Candidatus Liberibacter asiaticus the causal agent of citrus greening disease

  • Manali Motghare
  • Pradeep Kumar Shukla
  • Pranav Kumar
  • A. K. Sharma
  • Dilip Kumar GhoshEmail author
Research Article


Candidatus Liberibacter asiaticus (Ca. L. asiaticus), a gram negative, phloem-limited, and unculturable bacteria is the causal agent of citrus greening disease. The disease is considered as one of the most serious threats to the citrus industry worldwide. It is responsible for heavy crop losses irrespective of citrus cultivar or rootstock used. During the infection process, the bacterium produces several pathogenesis-related proteins that are crucial for its survival and multiplication in plant phloem tissue and saliva of its insect vectors citrus psylla, inside which it multiplies efficiently. There is a tremendous potential of developing antimicrobial inhibitors against these proteins to reduce pathogen population in plant phloem tissue and in insect vectors as potential tool in disease management strategy. In the present study, we sequenced and characterized four crucial protein genes from 18 samples across India. The four protein genes includes two component response regulator protein, periplasmic solute binding protein, putative protease IV transmembrane protein and serine protease do-like protein of the causal bacterium. Secondary and tertiary structures of the proteins were also predicted using PSIPRED and TMHMM software, respectively. Phylogenetic analyses and secondary protein structure prediction reveals that these four proteins are unique, essential and conserved in Ca. L. asiaticus populations infecting different citrus cultivars prevalent in India. Further, these structures gave an idea about active sites which can act as perfect target sites for blocking. These bacterial encoded proteins are potential targets of antimicrobial inhibitors.


Candidatus Liberibacter asiaticus Citrus greening Protein gene loci 


  1. Akula N, Zheng H, Han FQ, Wang N (2011) Discovery of novel SecA inhibitors of Candidatus Liberibacter asiaticus by structure based design. Bioorg Med Chem Lett 21:4183–4188CrossRefPubMedGoogle Scholar
  2. Bhose S, Misra P, Ramteke PW, Ghosh DK (2015a) Sequence analysis of ribosomal protein gene of ‘Candidatus Liberibacter asiaticus’ infecting major citrus cultivars in western Maharashtra of India. Indian Phytopathol 68(3):334–341Google Scholar
  3. Bhose S, Misra P, Shukla PK, Ghosh DK (2015b) Comparative analysis of 16S/23S intergenic region of Candidatus Liberibacter asiaticus associated with citrus greening disease in different citrus cultivars of Assam. Indian Phytopathol 68(4):432–437Google Scholar
  4. Bové JM (2006) Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. J Plant Pathol 88:7–37Google Scholar
  5. Cong Q, Kinch LN, Kim B-H, Grishin NV (2012) Predictive sequence analysis of the Candidatus Liberibacter asiaticus proteome. PLoS One 7(7):e41071. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Davidson AL, Dassa E, Orelle C, Chen J (2008) Structure, function, and evolution of bacterial ATP-binding cassette systems. Microbiol Mol Biol Rev 72:317–364CrossRefPubMedPubMedCentralGoogle Scholar
  7. Deng X, Chen J, Feng Z, Shan Z, Guo H, Zhu J, Li H, Civerolo E (2008) Identification and characterization of the Huanglongbing bacterium in pummel from multiple locations in Guangdong, P. R. China. Plant Dis 92:513–518CrossRefGoogle Scholar
  8. Doige CA, Ames GF (1993) ATP-dependent transport systems in bacteria and humans: relevance to cystic fibrosis and multidrug resistance. Ann Rev Microbiol 47:291–319CrossRefGoogle Scholar
  9. Duan Y, Zhou L, Hall DG, Li W, Doddapaneni H, Lin H, Liu L, Vahling CM, Gabriel DW, Williams KP, Dickerman A, Sun Y, Gottwald T (2009) Complete genome sequence of citrus Huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’ obtained through metagenomics. Mol Plant Microb Interact 22:1011–1020CrossRefGoogle Scholar
  10. Ekici OD, Paetzel M, Dalbey RE (2008) Unconventional serine proteases: variations on the catalytic Ser/His/Asp triad configuration. Protein Sci 17(12):2023–2037CrossRefPubMedPubMedCentralGoogle Scholar
  11. Gao R, Stock AM (2009) Biological insights from structures of two-component proteins. Ann Rev Microbiol 63:133–154CrossRefGoogle Scholar
  12. Gardy JL, Laird MR, Chen F, Rey S, Walsh CJ, Ester M, Brinkman FS (2005) PSORTb v. 2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis. Bioinformatics 21:617–623CrossRefPubMedGoogle Scholar
  13. Garnier M, Bove JM (1992) Citrus greening disease and the greening bacterium. In: Proceeding 12th Conference on IOCV. IOCV, Riverside, pp. 212–219Google Scholar
  14. Ghosh DK, Bhose S, Mukherjee K, Baranwal VK (2013) Sequence and evolutionary analysis of ribosomal DNA from Huanglongbing (HLB) isolates of western India. Phytopara 41:295–305CrossRefGoogle Scholar
  15. Ghosh DK, Bhose S, Motghare M, Warghane AJ, Mukherjee K, Ghosh DK, Sharma AK, Ladaniya MS, Gowda S (2015) Genetic diversity of the Indian populations of ‘Candidatus Liberibacter asiaticus’ based on the tandem repeat variability in a genomic locus. Phytopathology 105:1043–1049CrossRefPubMedGoogle Scholar
  16. Harris BZ, Lim WA (2001) Mechanism and role of PDZ domains in signaling complex assembly. J Cell Sci 114:3219–3231PubMedGoogle Scholar
  17. Jagoueix S, Bove JM, Garnier M (1994) The phloem-limited bacterium of greening disease of citrus is a member of the alpha-subdivision of proteobacteria. Int J Syst Bacteriol 44:379–386CrossRefPubMedGoogle Scholar
  18. Kim AC, Oliver DC, Paetzel M (2008) Crystal structure of a bacterial signal Peptide peptidase. J Mol Biol 15:376–382Google Scholar
  19. Krogh A, Larsson B, Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580CrossRefPubMedPubMedCentralGoogle Scholar
  20. Li W, Hartung JS, Levy L (2006) Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. J Microbiol Methods 66:104–115CrossRefPubMedGoogle Scholar
  21. Linton KJ, Higgins CF (1998) The Escherichia coli ATP-binding cassette (ABC) proteins. Mol Microbiol 28:5–13CrossRefPubMedGoogle Scholar
  22. Ng SYM, Chaban B, VanDyke DJ, Jarrell KF (2007) Archaeal signal peptidases. Microbiology 153(2):305–314CrossRefPubMedGoogle Scholar
  23. Rao M, Miyata S, Ghosh D, Irey M, Garnsey S, Gowda S (2013) Development of rapid, sensitive and non-radioactive tissue-blot diagnostic method for the detection of citrus greening. Mol Cell Probes 27:176–183CrossRefGoogle Scholar
  24. Schneider E, Hunke S (1998) ATP-binding-cassette (ABC) transport systems: functional and structural aspects of the ATP-hydrolyzing subunits/domains. FEMS Microbiol Rev 22:1–20CrossRefPubMedGoogle Scholar
  25. Sharma N, Selvakumar P, Bhose S, Ghosh DK, Kumar P, Sharma AK (2015) Crystal structure of a periplasmic solute binding protein in metal-free, intermediate and metal-bound states from Candidatus Liberibacter asiaticus. J Struct Biol 189:184–194CrossRefPubMedGoogle Scholar
  26. Sharma N, Selvakumar P, Saini G, Warghane A, Ghosh DK, Sharma AK (2016) Crystal structure analysis in Zn2 + -bound state and biophysical characterization of CLas-ZnuA2. Biochim Biophys Acta 1864:1649–1657CrossRefPubMedGoogle Scholar
  27. Shen W, Cevallos-Cevallos JM, da Rocha UN, Arevalo HA, Stansly PA (2013) Relation between plant nutrition, hormones, insecticide applications, bacterial endophytes, and Candidatus Liberibacter Ct values in citrus trees infected with huanglongbing. Eur J Plant Pathol 137(4):727–742CrossRefGoogle Scholar
  28. Vahling CM, Duan Y, Lin H (2010) Characterization of an ATP translocase identified in the destructive plant pathogen Candidatus Liberibacter asiaticus. J Bacteriol 192:834–840CrossRefPubMedGoogle Scholar
  29. Wang P, Shim E, Cravatt B, Jacobsen R, Schoeniger J, Kim AC, Paetzel M, Dalbey RE (2008) Escherichia coli signal peptide peptidase A is a serine-lysine protease with a lysine recruited to the nonconserved amino-terminal domain in the S49 protease family. Biochemistry 47(24):6361–6369CrossRefPubMedPubMedCentralGoogle Scholar
  30. Wolanin PM, Webre DJ, Stock JB (2003) Mechanism of phosphatase activity in the chemotaxis response regulator CheY. Biochemistry 42(47):14075–14082CrossRefPubMedGoogle Scholar
  31. Zhang M, Powell CA, Zhou L, He Z, Stover E, Duan Y (2011) Chemical compounds effective against the citrus huanglongbing bacterium Candidatus Liberibacter asiaticus in planta. Phytopathology 101:1097–1103CrossRefPubMedGoogle Scholar

Copyright information

© Indian Phytopathological Society 2018

Authors and Affiliations

  • Manali Motghare
    • 1
    • 2
  • Pradeep Kumar Shukla
    • 2
  • Pranav Kumar
    • 3
  • A. K. Sharma
    • 3
  • Dilip Kumar Ghosh
    • 1
    Email author
  1. 1.Plant Virology LaboratoryICAR-Central Citrus Research InstituteNagpurIndia
  2. 2.Department of Biological SciencesSam Higginbottom University of Agriculture, Technology and SciencesAllahabadIndia
  3. 3.Department of BiotechnologyIndian Institute of TechnologyRoorkeeIndia

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