Bioinformatics Advance Access originally published online on December 14, 2004
Bioinformatics 2005 21(8):1304-1307; doi:10.1093/bioinformatics/bti221
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The WHy domain mediates the response to desiccation in plants and bacteria
European Molecular Biology Laboratory 69012 Heidelberg, Meyerhofstrasse 1, Germany
*To whom the correspondence should be addressed.
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Abstract |
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 TOP Abstract INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Motivation: The hypersensitive response (HR) is a process activated by plants after microbial infection. Its main phenotypic effects are both a programmed death of the plant cells near the infection site and a reduction of the microbial proliferation. Although many resistance genes (R genes) associated to HR have been identified, very little is known about the molecular mechanisms activated after their expression.
Results: The analysis of the product of one of the R genes, the Hin1 protein, led to the identification of a novel domain, which we named WHy because it is detectable in proteins involved in Water stress and Hypersensitive response. The expression of this domain during both biotic infection and response to desiccation points to a molecular machinery common to these two stress conditions. Moreover, its presence in a restricted number of bacteria suggests a possible use for marking plant pathogenicity.
Contact: francesca.ciccarelli{at}embl.de
Supplementary information: Supplementary data (Figures S1 and S2 and Table S1) and the alignment in clustal format are available at http://www.bork.embl.de/~ciccarel/WHy_add_data.html
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INTRODUCTION |
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TOPAbstractINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Plants react to microbial infections through a process known as hypersensitive response (HR). The main effects of its activation are a rapid programmed cell death (PCD) of the cells near the infection site, and a reduction of bacterial growth and spread. The molecular machinery through which the HR acts is still largely unknown. However, it has been shown that this defense response is initiated by the secretion of bacterial molecules which are able to induce the expression of plant resistance genes (R genes) in a process called ‘gene-for-gene response’ (Hueck 1998; Cornelis and Van Gijsegem, 2000). According to the currently accepted view, bacteria start their aggression translocating specific proteins into the plant cell via the Hrp pathway (reviewed in Cornelis and Van Gijsegem, 2000). The presence of these proteins in the plant cytosol stimulates the expression of the plant R genes, which in turn initiate the HR. Several families of R gene products have been isolated, some of which share common sequence features (Hammond-Kosack and Jones, 1997). The largest group bears a nucleotide binding motif (NBS) and a leucine-rich repeat (LRR), and its members are consequently known as NBS-LRR proteins (Ellis and Jones, 1998).
Other components of HR are poorly characterized, among them the Hin1 genes, which have been isolated during a screen of genes induced after bacterial infection (Gopalan et al., 1996). Hin1 seems to take part in a non-specific pathway activated as a general response of plants to pathogenic infection (Gopalan et al., 1996). Differently from other R genes, Hin1 does not share significant sequence similarity to any functionally characterized eukaryotic protein family and its function is unknown.
Here we report the identification of a novel domain that links the Hin1 proteins to the plant family LEA-14, which is expressed under water stress conditions and during late embryogenesis and to a variety of uncharacterized bacterial and archaeal proteins. We named this module WHy for Water stress and Hypersensitive response domain.
The detection of a common domain in two families of proteins both expressed during the plant response to external stresses indicates a common molecular mechanism, involving the activation of similar proteins. Interestingly, the domain is also found in bacteria and archaea, which suggests a possible similar pathway for the stress response, maybe acquired by horizontal transfer.
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METHODS |
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TOPAbstractINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The protein sequence of Nicotiana tabacum Hin1 (P93353) was used as a query for standalone PSI-Blast (Altschul et al., 1997) against the protein nr-database. After three iterations, which retrieved only homologs of the Hin1 family in several plants, the first protein from the bacterium Ralstonia metallidurans was detected (ZP_00273651, E = 0.0003). This is an uncharacterized protein whose homology with the plant Hin1 almost covers the entire sequence. The search converged at the 6th iteration, although the grey zone just after the E-value threshold of 0.001 was clearly enriched in false negatives, such as some plant sequences (Q9C573, Q7X8V0, E = 0.006) and one protein from the bacterium Burkholderia pseudomallei (CAH37099 [GenBank] E = 0.009). Indeed, using these sequences as queries for PSI-Blast, both the Hin1 family and the bacterial sequences were retrieved. The three sequences were then manually added to the alignment and the resulting profile was used to continue the PSI-Blast search. More bacterial sequences were retrieved until the search converged again at the 9th iteration. A hidden Markov model (HMM) profile was built using non-redundant representatives of all the sequences detected with PSI-Blast (seq.id. <80%). The borders of the shared region were defined according to the PSI-Blast pairwise alignments. With the resulting profile a HMM search (Eddy, 1998) against the nr-database was run. This search added two new families to the alignment: the family of plant LEA-14 (Late Embryogenesis Abundant) proteins (E = 1.2x 10–13 at the 2nd iteration) and some archaeal sequences with a duplication of the domain (E = 3.2 x 10–5 at the 3rd iteration). The HMM search converged at the 4th iteration. Two distinct Pfam family domains are detectable in the Hin1 and the LEA-14 proteins (Hin1 and LEA_2, respectively). In both cases the domains span the whole protein length and extend, as their names suggest, only to members of the two families.
The secondary structure predictions were assigned using PHD (Rost, 1996), PsiPred (McGuffin et al., 2000) and SAM-T99 (Karplus et al., 1998).
The phylogeny was reconstructed using the non-redundant alignment of 44 protein sequences and both minimum evolution (ME) and Bayesian inferences implemented in MEGA (Kumar et al., 2001) and MrBayes (Huelsenbeck and Ronquist, 2001), respectively. For the ME estimation, the bootstrap test of phylogeny was set to 1000 replicates, and the initial tree was calculated using the Neighbor-Joining algorithm. In the Bayesian analysis, the TTJ model for amino acid substitution (Jones et al., 1992) was used and each run was set for 105 generations, with three heated and one cold chains. Convergence was assessed by repeating the analysis for four runs. Comparable tree topologies were obtained using both methods.
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RESULTS AND DISCUSSION |
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TOPAbstractINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The WHy domain is an
100 amino acid long module, with an alternation of hydrophilic and hydrophobic residues and an almost invariable NPN motif at its N-terminus (Fig. 1). The predicted secondary structure is mostly composed of beta strands with a C-terminal 
-helix (Fig. 1). No similarity with any of the known structural folds could be assigned using the metaserver 3D-Jury (Ginalski et al., 2003). The modular nature of the WHy domain is supported by the fact that it is present in several protein families with different domain architectures and by its duplication in archaea (Fig. 2A).
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For two of the families bearing the WHy domain, namely the Hin1 and the LEA-14 proteins, some functional information is available. The Hin1 proteins are expressed during the response to bacterial infection and play an uncharacterized role during HR. The LEA-14 proteins are expressed in late embryogenesis but also as part of the plant response to desiccation (Galau et al., 1993). These proteins do not share any sequence similarity with other members of the large LEA family (Maitra and Cushman, 1994). The identification of a common domain between proteins expressed in HR and during desiccation suggests a shared mechanism in plant response to these two stress conditions. Many different observations support this hypothesis. First, genes involved in the response to both biotic and abiotic stresses partly share the same signal transduction pathway (Xing et al., 2001). Second, a direct link between HR and water stress has been recently postulated (Wright and Beattie, 2004). It has been observed that the water potential in the bacterial cell reduces during HR, becoming sufficiently low to prevent cell division in many cells of the bacterial colony (Wright and Beattie, 2004). Although experimental proofs to this hypothesis are required, such a link suggests a role of the Hin1 family in the desiccation-like process taking place during the HR and leading to the microbial death.
The WHy domain is widespread among plants, but it is absent in fungi and animals. Interestingly, it is also detectable in prokaryotes such as some proteobacteria, Deinococcus radiodurans and few euryarchaeota (Fig. 2B and Supplementary Table S1). This patchy distribution can be explained either by a selective retention of the domain, and the function it accomplishes during evolution or by its lateral transfer from plant to prokaryotes. This latter case would be an example of capture of plant defense genes by bacteria either to integrate them in their own defense system or to interfere with the host signal pathway. The tree topology obtained most frequently seems to support this interpretation, pointing to an early appearance of the WHy module in plants followed by horizontal transfers from plant to bacteria and archaea (Fig. 2C). A first lateral transfer probably took place between plants and proteobacteria and a second one between plants and archaea after the duplication of the plant domain and the divergence between Hin1 and LEA (Fig. 2C). Several observations support this scenario. First, the HR process had an early origin in plants (Meyers et al., 1999). Indeed, we could find a hit for Hin1 in the ancient green alga Chlamydomonas reinhardtii (AV395132 [GenBank] ), but no hits for LEA. The fact that the C.reinhardtii genome is not complete does not allow for a final statement, but this result at least confirms the early origin of Hin1 in the plant kingdom. Second, the domain distribution among bacteria seems to be restricted to plant pathogens or at least symbionts (Supplementary Table S1). The only bacterial species not belonging to proteobacteria is D.radiodurans, which is known to be prone to horizontal transfers (Makarova et al., 2001). Indeed, the position of the D.radiodurans sequence in the WHy domain tree does not reflect the usual phylogenetic relationships within bacteria (Supplementary Figure S2). Finally, horizontal transfer among domains of life as a mechanism for the acquisition of novel functions has been reported in several cases (Koonin et al., 2001; Ponting et al., 1999; Schultz, 2004). While the horizontal transfer of WHy from plants to symbiotic or pathogenic bacteria can be seen as the acquisition of a function putatively related to the cell defense, the lateral acquisition by euryarchaeota is more difficult to explain. In general, horizontal transfer of domains from eukaryotes to archaea does not seem to be a frequent event (Ponting et al., 1999). In addition to that, the lifestyle of hyperthermophilic euryarchaeota does not allow an easy interpretation of the domain acquisition, except in the context of a general response to stress conditions. Indeed, these organisms live in extreme environments which make the contact with eukaryotes difficult. Nevertheless, examples have been reported of horizontal transfer from eukaryotes to hyperthermophilic microbes, as in the case of the TrpRS gene (Wolf et al., 1999).
Although horizontal transfer seems the most suitable explanation, we cannot exclude an alternative interpretation for the evolution of the WHy domain. It implies its appearance in the bacterial kingdom and a selective retention only in plants, some proteobacteria and few euryarchaeota. This scenario requires several gene losses in bacteria and in the common ancestor between animal and fungi (Fig. 2B), and one horizontal gene transfer from archaea to plants (Fig. 2C). In this case, the patchy distribution of the WHy domain would be the result of a selective retention of defense mechanisms during evolution.
In either scenario, the actual presence of the domain in a restricted number of bacteria makes it suitable as a marker for detecting plant pathogenicity and to eventually develop new bactericidal strategies.
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Acknowledgments |
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The authors are grateful to David Torrents and Christian von Mering for their critical reading of the manuscript.
Received on November 4, 2004; revised on November 28, 2004; accepted on December 12, 2004
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