A plant natriuretic peptide-like gene in the bacterial pathogen Xanthomonas axonopodis may induce hyper-hydration in the plant host: a hypothesis of molecular mimicry
© Nembaware et al; licensee BioMed Central Ltd. 2004
- Received: 08 January 2004
- Accepted: 24 March 2004
- Published: 24 March 2004
Plant natriuretic peptides (PNPs) are systemically mobile molecules that regulate homeostasis at nanomolar concentrations. PNPs are up-regulated under conditions of osmotic stress and PNP-dependent processes include changes in ion transport and increases of H2O uptake into protoplasts and whole tissue.
Presentation of the hypothesis
The bacterial citrus pathogen Xanthomonas axonopodis pv. Citri str. 306 contains a gene encoding a PNP-like protein. We hypothesise that this bacterial protein can alter plant cell homeostasis and thus is likely to represent an example of molecular mimicry that enables the pathogen to manipulate plant responses in order to bring about conditions favourable to the pathogen such as the induced plant tissue hyper-hydration seen in the wet edged lesions associated with Xanthomonas axonopodis infection.
Testing the hypothesis
We found a Xanthomonas axonopodis PNP-like protein that shares significant sequence similarity and identical domain organisation with PNPs. We also observed a significant excess of conserved residues between the two proteins within the domain previously identified as being sufficient to induce biological activity. Structural modelling predicts identical six stranded double-psi β barrel folds for both proteins thus supporting the hypothesis of similar modes of action. No significant similarity between the Xanthomonas axonopodis protein and other bacterial proteins from GenBank was found. Sequence similarity of the Xanthomonas axonopodis PNP-like protein with the Arabidopsis thaliana PNP (AtPNP-A), shared domain organisation and incongruent phylogeny suggest that the PNP-gene may have been acquired by the bacteria in an ancient lateral gene transfer event. Finally, activity of a recombinant Xanthomonas axonopodis protein in plant tissue and changes in symptoms induced by a Xanthomonas axonopodis mutant with a knocked-out PNP-like gene will be experimental proof of molecular mimicry.
Implication of the hypothesis
If the hypothesis is true, it could at least in part explain why the citrus pathogen Xanthomonas campestris that does not contain a PNP-like gene produces dry corky lesions while the closely related Xanthomonas axonopodis forms lesions with wet edges. It also suggests that genes typically found in the host, horizontally transferred or heterologous, can help to explain aspects of the physiology of the host-pathogen interactions.
- Horizontal Gene Transfer
- Horizontal Transfer
- Molecular Mimicry
- Significant Sequence Similarity
Plant natriuretic peptides (PNPs) are a novel class of plant molecules with biological activity at nanomolar concentrations. The PNP-dependent responses include concentration-dependent promotion of stomatal opening , rapid and transient increases in cellular cGMP levels  and modulation of K+, Na+ and H+ net fluxes  in Zea mays root tissue. PNPs also induce rapid increases in osmoticum-dependant H2O uptake into Solanum tuberosum and Arabidopsis thaliana protoplasts [4, 5]. We have also observed PNP-dependant increases in lateral H2O movement out of the conductive tissue (xylem) into the neighbouring parenchyma  and such a 'drawing' of water into cells and tissues together with an up-regulation under conditions of drought and NaCl stress are compatible with a role for these molecules in plant homeostasis. Incidentally, a PNP-like protein from Citrus jambhiri (CjBAp12) is expressed in root and stem tissue in response to a challenge from citrus blight  which proliferates in the conductive tissue of the host and severely affects host homeostasis eventually resulting in xylem plugging and consequent shoot wilting and host death. It is conceivable that the expression CjBAp12 is an early host response to counteract the pathogen induced limitation of water and nutrient availability.
Several lines of evidence suggest that PNPs can act systemically. Firstly, PNPs are associated with conductive tissues as demonstrated by in situ hybridisation and tissue printing . Secondly, biologically active PNP was isolated from xylem exudates , a tissue that is associated with transport and not protein synthesis. Amino acid sequence comparisons and structural modelling predict that PNPs do not contain the putative polysaccharide-binding C-terminal domain typical for the related expansins that act on the cell wall [9–11]. The absence of such a domain presumably results in increased extracellular mobility which in turn is a precondition for a systemic mode of action.
Here we report the discovery of a gene in the completely sequenced genome of the plant pathogenic bacterium Xanthomonas axonopodis  that encodes a protein with significant sequence similarity to an Arabidopsis thaliana PNP (AtPNP-A) A. We propose that the presence of a PNP-like protein in Xanthomonas axonopodis has enabled the pathogen to affect plant homeostasis. Furthermore, we have investigated the origin of this PNP-like protein encoding gene in Xanthomonas axonopodis and have found evidence consistent with the possibility that it has been acquired by the bacterium through horizontal gene transfer. This has led us to search for other genes that show evidence of horizontal transfer with a view to establishing how many genes may have been acquired by Xanthomonas axonopodis through horizontal gene transfer from plants.
We found a PNP-like gene in the bacterial citrus pathogen Xanthomonas axonopodis pv. Citri str. 306 that has significant sequence similarity to the PNP encoding genes and hypothesise that the encoded protein can alter homeostasis of the host plant. Since PNP-like molecules are exported into the extracellular space, act systemically and promote significant ion and H2O uptake into cells it is very possible that the pathogen uses its PNP-like molecule to induce cell and tissue hyper-hydration in the host. Such hyper-hydration is typically seen in the wet rim of the lesions caused by Xanthomonas axonopodis and may suggest that PNP-like molecule benefits the pathogen by facilitating access to water and nutrients while severely disturbing the homeostasis of its host.
The observed similarity between the two proteins could be due to an ancient horizontal gene transfer event  from the plants to bacteria or to convergent evolution. However, we believe that lateral transfer is more likely because the bacterial and the plant genes also show some similarity outside of the region that we have shown to be essential and sufficient for the function of the protein (Figure 1). This similarity in domains not essential for osmotic function suggests that the overall similarity between the two molecules is not just a result of shared function but reflects common ancestry.
In AtPNP-A and the Xanthomonas axonopodis PNP-like protein the first psi loop connects strands β1 and β2, whereas the second psi loop connects strands β4 and β5 (Figure 3). The sequence conservation between AtPNP-A and the Xanthomonas axonopodis PNP-like molecule is greatest in the domain spanning the β2 and β3 strands which both flank the α helix (Figure 1 and 3). In AtPNP-A this structure (β2 – α helix – β3) has been demonstrated to be within the 33 amino acid long domain that is critical and sufficient for conferring biological activity . This domain also contains the first psi loop which is likely to be a part of the functional framework of AtPNP-A and the Xanthomonas axonopodis PNP-like molecule.
We also carried out a screen of all proteins from Xanthomonas axonopodis in order to discover whether other genes from this bacterial pathogen showed evidence of unexpected tree topology thus indicating horizontal gene transfer  from plants. All known proteins from the completely sequenced genomes of Xanthomonas axonopodis , Xanthomonas campestris , Pseudomonas putida , Escherichia coli  and Arabidopsis thaliana  were downloaded from GenBank (13/08/2003). The Xanthomonas axonopodis proteins were searched against the proteins from the remaining four organisms using BLASTp . CLUSTALW  was used to generate multiple sequence alignments and Neighbour-Joining phylogenetic trees from 4307 sets of five proteins consisting of one protein from each of the five organisms. Xanthomonas axonopodis proteins with greater than 25% identity to their Arabidopsis thaliana homologues that clustered with the Arabidopsis thaliana homologue on a phylogenetic tree were retained for further analysis. Each of these proteins was searched against the GenBank non-redundant protein database. Homologous sequences were downloaded and phylogenetic trees were constructed using the Neighbor-Joining method .
Analysis of putative laterally transferred genes obtained from the whole genome analyses.
amine oxidase related
Recently, another example of a pathogen mimicking an extracellular plant molecule has been reported . This protein (GrEXP1), a molecule with cell wall loosening (expansin) activity previously seen in plants  and other organisms with cell walls only , was found in the plant-parasitic roundworm Globodera rostochiensis. The infective juvenile nematodes express and secrete GrEXP1 in the subventral oesophageal glands  using this 'typical plant' protein to their advantage when invading the host root system.
Finally, if the PNP-like gene was indeed horizontally transferred from a plant to Xanthomonas axonopodis it is also consistent with the complexity theory of gene transfer  which postulates that a major factor in the more frequent horizontal transfer of operational genes such as expansins and PNPs as compared to informational genes is that they are structurally and functionally less complex. This bias is explained by the increased chance of transfer of a functional unit advantageous to the recipient. It would also appear that particularly in the case of a pathogen, extracellular signals, transporters or surface components perceived by the host can cause systemic host responses that give the pathogen a significant advantage. A point in case are eukaryotic genes found in Mycobacterium tuberculosis many of which directly modulate host responses and have a role in the specific pathogenesis induced by the bacterium .
The experimental test of the hypothesis of molecular mimicry of the Xanthomonas axonopodis PNP-like molecule will require two types of investigations. In the first, a recombinant Xanthomonas axonopodis protein must be obtained and tested for effects on (host) plant tissue. Molecular mimicry would require that net H2O uptake is increased and ion transport is affected in the host tissue in response to the recombinant peptide. In a second experiment, a Xanthomonas axonopodis mutant with a knocked-out PNP-like gene must be obtained. If the mutant induces altered host symptoms and in particular an absence of watery edges of the lesions, then the hypothesis can be considered proven.
If the hypothesis is true, then the bacterial PNP-like protein plays a role in manipulating the homeostatic balance of the host. Such mimicry could at least in part explain why the citrus pathogen Xanthomonas campestris that does not contain a PNP-like gene produces dry corky lesions while the closely related Xanthomonas axonopodis forms wet lesions . Furthermore, the hypothesis suggests that the presence of "typical" and functional host genes in pathogens can explain key aspects of host-pathogen interactions in general and can help elucidate the specific molecular and cellular interactions between hosts and pathogens.
The project was supported by the South African National Research Foundation and the Royal Society (UK).
- Billington T, Pharmawati M, Gehring CA: Isolation and immunoaffinity purification of biologically active plant natriuretic peptide. Biochem Biophys Res Commun. 1997, 235: 722-725. 10.1006/bbrc.1997.6883.View ArticlePubMedGoogle Scholar
- Pharmawati M, Gehring CA, Irving HR: An immunoaffinity purified plant natriuretic peptide analogue modulates cGMP level in the Zea mays root stele. Plant Sci. 1998, 137: 107-115. 10.1016/S0168-9452(98)00135-6.View ArticleGoogle Scholar
- Pharmawati M, Shabala SN, Newman IA, Gehring CA: Natriuretic peptides and cGMP modulate K+, Na+ and H+ fluxes in Zea mays roots. Mol Cell Biol Res Commun. 1999, 2: 53-57. 10.1006/mcbr.1999.0151.View ArticlePubMedGoogle Scholar
- Maryani MM, Bradley G, Cahill DM, Gehring CA: Natriuretic peptides and immunoreactants modify osmoticum-dependent volume changes in Solanum tuberosum L. mesophyll cell protoplasts. Plant Sci. 2001, 161: 443-452. 10.1016/S0168-9452(01)00423-X.View ArticleGoogle Scholar
- Morse M, Proncheva G, Gehring C: AtPNP-A is a systemically mobile natriuretic peptide immunoanalogue with a role in Arabidopsis thaliana cell volume regulation. FEBS Lett. 2004, 556: 99-103. 10.1016/S0014-5793(03)01384-X.View ArticlePubMedGoogle Scholar
- Suwastika IN, Gehring CA: Natriuretic peptide hormones promote radial water movements from the xylem of Tradescantia shoots. Cell Mol Life Sci. 1998, 54: 1161-1167. 10.1007/s000180050244.View ArticleGoogle Scholar
- Ceccardi TL, Barthe GA, Derrick KS: A novel protein associated with citrus blight has sequence similarities to expansin. Plant Mol Biol. 1998, 38: 775-783. 10.1023/A:1006039016393.View ArticlePubMedGoogle Scholar
- Maryani MM, Morse MV, Bradley G, Irving HR, Cahill DM, Gehring C: In situ localisation associates biologically active natriuretic peptide immuno-analogues with conductive tissue and stomata. J Exp Bot. 2003, 54: 1553-1564. 10.1093/jxb/erg174.View ArticlePubMedGoogle Scholar
- Barre A, Rouge P: Homology modeling of the cellulose-binding domain of a pollen allergen from rye grass: Structural basis for the cellulose recognition and associated allergenic properties. Biochem Biophys Res Commun. 2002, 296: 1346-1351. 10.1016/S0006-291X(02)02091-0.View ArticlePubMedGoogle Scholar
- Linder M, Teeri TT: The roles and function of cellulose-binding domains. J Biotech. 1997, 57: 15-28. 10.1016/S0168-1656(97)00087-4.View ArticleGoogle Scholar
- Ludidi NN, Heazlewood JL, Seoighe CJ, Irving HR, Gehring CA: Expansin-like molecules: Novel functions derived from common domains. J Mol Evol. 2002, 54: 587-594. 10.1007/s00239-001-0055-4.View ArticlePubMedGoogle Scholar
- da Silva ACR, Ferro JA, Reinach FC, Farah CS, Furlan LR, Quaggio RB, Monteiro-Vitorello CB, Van Sluys MA, Almeida NF, Alves LM, do Amaral AM, Bertolini MC, Camargo LE, Camarotte G, Cannavan F, Cardozo J, Chambergo F, Ciapina LP, Cicarelli RM, Coutinho LL, Cursino-Santos JR, El-Dorry H, Faria JB, Ferreira AJ, Ferreira RC, Ferro MI, Formighieri EF, Franco MC, Greggio CC, Gruber A, Katsuyama AM, Kishi LT, Leite RP, Lemos EG, Lemos MV, Locali EC, Machado MA, Madeira AM, Martinez-Rossi NM, Martins EC, Meidanis J, Menck CF, Miyaki CY, Moon DH, Moreira LM, Novo MT, Okura VK, Oliveira MC, Oliveira VR, Pereira HA, Rossi A, Sena JA, Silva C, de Souza RF, Spinola LA, Takita MA, Tamura RE, Teixeira EC, Tezza RI, Trindade dos Santos M, Truffi D, Tsai SM, White FF, Setubal JC, Kitajima JP: Comparison of the genomes of two Xanthomonas pathogens with different host specificities. Nature. 2002, 417: 459-463. 10.1038/417459a.View ArticlePubMedGoogle Scholar
- Gehring C: Natriuretic peptides – a new class of plant hormone?. Ann Bot. 1999, 83: 329-334. 10.1006/anbo.1998.0834.View ArticleGoogle Scholar
- Koonin E, Makarova K, Aravind L: Horizontal gene transfer in prokaryotes: Quantification and classification. Ann Rev Microbiol. 2001, 55: 709-742. 10.1146/annurev.micro.55.1.709.View ArticleGoogle Scholar
- Williams M, Shirai H, Shi J, Nagendra H, Mueller J, Mizuguchi K, Miguel RN, Lovell SC, Innis CA, Deane CM, Chen L, Campillo N, Burke DF, Blundell TL, de Bakker PI: Sequence-structure homology recognition by iterative alignment refinement and comparative modeling. Protein. 2001, 5 (suppl): 92-97. 10.1002/prot.1169.View ArticleGoogle Scholar
- Bujnicki J, Elofsson A, Fischer D, Rychlewski L: Structure prediction meta server. Bioinfo. 2001, 17: 750-751. 10.1093/bioinformatics/17.8.750.View ArticleGoogle Scholar
- Shi J, Blundell T, Mizuguchi K: FUGUE: Sequence-structure homology recognition using environment-specific substitution tables and structure- dependent gap penalties. J Mol Biol. 2001, 310: 243-257. 10.1006/jmbi.2001.4762.View ArticlePubMedGoogle Scholar
- Sali A, Blundell T: Comparative protein modeling by satisfaction of spatial restraints. J Mol Biol. 1993, 234: 779-781. 10.1006/jmbi.1993.1626.View ArticlePubMedGoogle Scholar
- Cosgrove DJ, Li L, Cho HT, Hoffmann-Benning S, Moore RC, Blecker D: The growing world of expansins. Plant Cell Physiol. 2002, 43: 1436-1444. 10.1093/pcp/pcf180.View ArticlePubMedGoogle Scholar
- Li Y, Darley CP, Ongaro V, Fleming A, Schipper O, Baldauf SL, McQueen-Mason SJ: Plant expansins are a complex multigene family with an ancient evolutionary origin. Plant Physiol. 2002, 128: 854-864. 10.1104/pp.010658.PubMed CentralView ArticlePubMedGoogle Scholar
- Castillo RM, Mizuguchi K, Dhanaraj V, Albert A, Blundell TA, Murzin A: A six-stranded double-psi beta barrel is shared by several protein superfamilies. Structure. 1999, 7: 227-236. 10.1016/S0969-2126(99)80028-8.View ArticlePubMedGoogle Scholar
- Nelson K, Weinel C, Paulsen I, Dodson R, Hilbert H, Martins dos Santos VA, Fouts DE, Gill SR, Pop M, Holmes M, Brinkac L, Beanan M, DeBoy RT, Daugherty S, Kolonay J, Madupu R, Nelson W, White O, Peterson J, Khouri H, Hance I, Chris Lee P, Holtzapple E, Scanlan D, Tran K, Moazzez A, Utterback T, Rizzo M, Lee K, Kosack D, Moestl D, Wedler H, Lauber J, Stjepandic D, Hoheisel J, Straetz M, Heim S, Kiewitz C, Eisen JA, Timmis KN, Dusterhoft A, Tummler B, Fraser CM: Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol. 2002, 12: 799-808. 10.1046/j.1462-2920.2002.00366.x.View ArticleGoogle Scholar
- Blattner F, Plunkett G, Bloch C, Perna N, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y: The complete genome sequence of Escherichia coli K-12. Science. 1997, 277: 1453-1474. 10.1126/science.277.5331.1453.View ArticlePubMedGoogle Scholar
- The Arabidopsis Genome Initiative: Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature. 2000, 408: 796-815. 10.1038/35048692.Google Scholar
- Altschul S, Madden T, Schäffer A, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucl Acids Res. 1997, 25: 3389-3402. 10.1093/nar/25.17.3389.PubMed CentralView ArticlePubMedGoogle Scholar
- Thompson JD, Gibson TJ, Plewiak F, Jeanmougin F, Higgins DG: The ClustalX windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res. 1997, 24: 4876-4882. 10.1093/nar/25.24.4876.View ArticleGoogle Scholar
- Qin L, Kudla U, Roze E, Goverse A, Popeijus H, Nieuwland J, Overmars H, Jones JT, Schots A, Smant G, Bakker J, Helder J: Plant degradation: A nematode expansin acting on plants. Nature. 2004, 427: 30-10.1038/427030a.View ArticlePubMedGoogle Scholar
- Cosgrove DJ: Loosening of plant cell walls by expansins. Nature. 2000, 407: 321-326. 10.1038/35030000.View ArticlePubMedGoogle Scholar
- Darley C, Li Y, Schaap P, McQueen-Mason S: Expression of a family of expansin-like proteins during the development of Dictyostelium discoideum. FEBS Lett. 2003, 546: 416-418. 10.1016/S0014-5793(03)00598-2.View ArticlePubMedGoogle Scholar
- Jain R, Rivera M, Lake J: Horizontal gene transfer among genomes: The complexity hypothesis. Proc Natl Acad Sci USA. 1999, 96: 3801-3806. 10.1073/pnas.96.7.3801.PubMed CentralView ArticlePubMedGoogle Scholar
- Gamieldien J, Ptitsyn A, Hide W: Eukaryotic genes in Mycobacterium tuberculosis could have a role in pathogenesis and immunomodulation. TRENDS Gen. 2002, 18: 5-8. 10.1016/S0168-9525(01)02529-X.View ArticleGoogle Scholar
- Civerolo E: Bacterial canker disease of citrus. J Rio Gde Val Hort Soc. 1984, 37: 127-145.Google Scholar
- Kumar S, Tamura K, Jakobsen I, Nei M: Molecular evolutionary genetics analysis software. Bioinfo. 2001, 17: 1244-1245. 10.1093/bioinformatics/17.12.1244.View ArticleGoogle Scholar
- Kraulis P: MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures. J Appl Cryst. 1991, 24: 946-950. 10.1107/S0021889891004399.View ArticleGoogle Scholar
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