Genome-wide analysis of eukaryote thaumatin-like proteins (TLPs) with an emphasis on poplar

Abstract

Background: Plant inducible immunity includes the accumulation of a set of defense proteins during infection called pathogenesis-related (PR) proteins, which are grouped into families termed PR-1 to PR-17. The PR-5 family is composed of thaumatin-like proteins (TLPs), which are responsive to biotic and abiotic stress and are widely studied in plants. TLPs were also recently discovered in fungi and animals. In the poplar genome, TLPs are over-represented compared with annual species and their transcripts strongly accumulate during stress conditions.

Results: Our analysis of the poplar TLP family suggests that the expansion of this gene family was followed by diversification, as differences in expression patterns and predicted properties correlate with phylogeny. In particular, we identified a clade of poplar TLPs that cluster to a single 350 kb locus of chromosome I and that are up-regulated by poplar leaf rust infection. A wider phylogenetic analysis of eukaryote TLPs - including plant, animal and fungi sequences - shows that TLP gene content and diversity increased markedly during land plant evolution. Mapping the reported functions of characterized TLPs to the eukaryote phylogenetic tree showed that antifungal or glycan-lytic properties are widespread across eukaryote phylogeny, suggesting that these properties are shared by most TLPs and are likely associated with the presence of a conserved acidic cleft in their 3D structure. Also, we established an exhaustive catalog of TLPs with atypical architectures such as small-TLPs, TLP-kinases and small-TLP-kinases, which have potentially developed alternative functions (such as putative receptor kinases for pathogen sensing and signaling).

Conclusion: Our study, based on the most recent plant genome sequences, provides evidence for TLP gene family diversification during land plant evolution. We have shown that the diverse functions described for TLPs are not restricted to specific clades but seem to be universal among eukaryotes, with some exceptions likely attributable to atypical protein structures. In the perennial plant model Populus, we unravelled the TLPs likely involved in leaf rust resistance, which will provide the foundation for further functional investigations.

Figure 1

Thaumatin-like proteins (TLPs) from poplar. (A), Neighbour-joining tree of Populus trichocarpa TLPs. Branch lengths are proportional to phylogenetic distances. (B), Protein characteristics and natural selection of poplar TLPs. MW: mass weight in kDa; Ip: predicted iso-electric point; ns: neutral selection [39]. (C), Regulation of poplar TLPs during stress. Transcriptome analyses of 3 different studies on poplar leaves infected by Melampsora spp. are summarized [27,30,31]. Changes considered to be significant by the respective authors are in bold. I48: incompatible interaction at 48 hour post-inoculation (hpi); C48: compatible interaction at 48 hpi; Mmd: compatible interaction at 6 dpi; Mlp: compatible interaction at 6 dpi; Mxt: Mmd+Mlp; 1d, 3d, 7d, 9d: compatible interaction respectively at 1, 3, 7 and 9 dpi. Summarized data for expression during stress conditions were mined from the PopGenIE database [58] (non-underlined letters) or from the literature (underlined letters). 'up': up-regulated gene or increased protein accumulation; 'down': down-regulated gene; ns: no significant regulation; a letter alone indicates that the corresponding protein has been reported but no regulation information is available; a to d: ozone, UV, drought and cold stress respectively; e: wind exposed leaves; f: wounding; g: Populus/Melampsora compatible interaction; h: sap extract; i: sap extract after wounding; j: wood regeneration; k: copper stress. Corresponding references: [60,65-71] (D), Protein domain organisation and CDS structure. Light grey box: thaumatin domain; dark grey box: protein kinase domain; black box: exon. '-' in (A), (B) and (C) indicates missing information. ^aAccession number of the best Arabidopsis thaliana homolog.

Figure 2

Representation of genomic loci of TLP genes in the genome of Populus trichocarpa 'Nisqually-1'. (A), Position of TLP genes on scaffold 1. Transposable element coverage of the TLP cluster is presented below scaffold 1 (dark grey: LTR-retrotransposon; light grey: DNA transposon). (B), position of TLP genes on scaffolds 2 to 21. Black lines: scaffolds; triangles: TLP genes; triangles in rectangles: TLP-kinase genes. Grey and white triangles respectively correspond to regulated and non-regulated genes in rust-infected poplar leaves as shown in Figure 1.

Figure 3

Neighbour-joining tree of 598 thaumatin domains of TLP sequences from 100 eukaryote species. Branch lengths are proportional to phylogenetic distances. For clarity, protein names are not indicated but can be retrieved from individual phylogenetic trees of subgroups I, II and III respectively in Figure 5, Additional files 9 and 10. Red stars indicate sequences used for the alignment presented in Figure 4. Annotations of subgroups and clades are discussed in the text.

Figure 4

Alignment of thaumatin domains of selected eukaryote TLPs. Amino acid sequence comparison was carried out with ClustalW on MEGA 4 software with the parameters described by [6]. The alignment was then adjusted manually when necessary. ^aComplete protein reference: Glyma-Glyma11g14970.1.

Figure 5

Neighbour-joining tree of the 211 thaumatin domains of TLP subgroup I. Functionally characterized TLPs and corresponding functions are indicated. Poplar sequence names are in red. The 5 letter code before each protein ID corresponds to the 3 first letters of the genus name followed by the 2 first letters of the species name. The red arrow indicates PR-5d used for 3D structure alignment and black arrows indicate sequences used for alignment mapping on 3D Structure (see Figure 6). The red star indicates the Small-TLP-Kinase from Sorghum bicolor (Sb03g025670). The two columns successively indicate proteins with demonstrated antifungal activity and other functions. a: protection against abiotic stress; b: antifreeze activity; c: membrane permeabilization activity; d: xylanase inhibitor; e: α-amylase/trypsin inhibition; f: apoptosis-inducing in yeast; g: GPCR binding; h: CMV1-a binding; i: glycoprotein binding; j: endo-β-1,3-glucanase activity; k: solved 3D structures. References corresponding to these data are summarized in Additional file 8. Branch lengths are proportional to phylogenetic distances.

Figure 6

3D structure alignment (3D-SA) and alignment mapping on 3D structure (AM-3D) of eukaryote TLPs. Amino acids of the REDDD and FF motifs are represented with side-chains in balls and sticks. Color code of side-chains, red: negatively charged; blue: positively charged; yellow: hydrophobic. White dashed-lines indicate acidic cleft limits. (A), 3D-SA of tobacco PR-5d and cherry Pru av 2. Protein backbone color code, red: identical amino acids; blue: different amino acids; grey: unaligned residues, green: glycine/phenylalanine residues discussed in the text. Disulfide bonds are in orange. (B), AM-3D of 9 subgroup I TLPs using the PR-5d structure as template. The four-color code of the protein backbone (from red to blue) corresponds to a decrease in amino acid conservation. (C), AM-3D of 15 subgroup II TLPs using the Pru Av 2 structure as template. Color code and annotations are as in B. Amino acids under diversifying selection [39] are indicated by white asterisks. (D, E and F), Highlights of β-sheets forming the acidic cleft in A, B and C respectively. Color code is similar to that in A, B and C. In D, the residues forming the REDDD and FF motifs are numbered as in Figure 4. White arrows indicate motif differences discussed in the text. (G), AM-3D of the 9 small-TLPs indicated in Additional file 7 using the TLX1 structure as template. Color code is similar to that in B. A white dashed ellipse marks the missing acidic cleft.

Figure 7

Amino acid sequence comparison of plant TLP-kinases (TLP-Ks). (A), Neighbour-joining tree of the 14 TLP-Ks identified in plants. Branch lengths are proportional to phylogenetic distances. Black star: sTLP-K from Brachypodium distachyon; grey star: TLP-K from Arabidopsis thaliana described in [51]. (B), ClustalW amino acid alignment using the parameters described by [6] and manually adjusted. Thaumatin and protein kinase domains are respectively underlined in dark grey and black. Phobius [72] predicted transmembrane domain is underlined in light grey. Shaded boxes indicate highly conserved sequences. The arrow indicates the end of the predicted signal peptide. Vertical bars indicate cysteine residues in the thaumatin domain and aspartate residues forming the catalytic site of the kinase domain.