Cloning and characterization of a pectin lyase gene from Colletotrichum lindemuthianum and comparative phylogenetic/structural analyses with genes from phytopathogenic and saprophytic/opportunistic microorganisms

Abstract

Background: Microorganisms produce cell-wall-degrading enzymes as part of their strategies for plant invasion/nutrition. Among these, pectin lyases (PNLs) catalyze the depolymerization of esterified pectin by a β-elimination mechanism. PNLs are grouped together with pectate lyases (PL) in Family 1 of the polysaccharide lyases, as they share a conserved structure in a parallel β-helix. The best-characterized fungal pectin lyases are obtained from saprophytic/opportunistic fungi in the genera Aspergillus and Penicillium and from some pathogens such as Colletotrichum gloeosporioides.The organism used in the present study, Colletotrichum lindemuthianum, is a phytopathogenic fungus that can be subdivided into different physiological races with different capacities to infect its host, Phaseolus vulgaris. These include the non-pathogenic and pathogenic strains known as races 0 and 1472, respectively.

Results: Here we report the isolation and sequence analysis of the Clpnl2 gene, which encodes the pectin lyase 2 of C. lindemuthianum, and its expression in pathogenic and non-pathogenic races of C. lindemuthianum grown on different carbon sources. In addition, we performed a phylogenetic analysis of the deduced amino acid sequence of Clpnl2 based on reported sequences of PNLs from other sources and compared the three-dimensional structure of Clpnl2, as predicted by homology modeling, with those of other organisms. Both analyses revealed an early separation of bacterial pectin lyases from those found in fungi and oomycetes. Furthermore, two groups could be distinguished among the enzymes from fungi and oomycetes: one comprising enzymes from mostly saprophytic/opportunistic fungi and the other formed mainly by enzymes from pathogenic fungi and oomycetes. Clpnl2 was found in the latter group and was grouped together with the pectin lyase from C. gloeosporioides.

Conclusions: The Clpnl2 gene of C. lindemuthianum shares the characteristic elements of genes coding for pectin lyases. A time-course analysis revealed significant differences between the two fungal races in terms of the expression of Clpnl2 encoding for pectin lyase 2. According to the results, pectin lyases from bacteria and fungi separated early during evolution. Likewise, the enzymes from fungi and oomycetes diverged in accordance with their differing lifestyles. It is possible that the diversity and nature of the assimilatory carbon substrates processed by these organisms played a determinant role in this phenomenon.

Figure 1

Nucleotide and deduced amino acid sequence of the Clpnl2 gene. Intron and exon sequences are in lowercase and uppercase, respectively. The signal peptide sequence is boxed. The possible binding sequences of RAP1 and AbaA are underlined with a dotted line. The putative transcription start point is underlined, and the putative Kozak sequence is shaded. The sequences of the 3'-terminal region are underlined. An asterisk (*) marks the translation stop codon. The potential N-glycosylation site is circled. This sequence has been deposited in the GenBank nucleotide sequence database under accession number JN034038.

Figure 2

Southern blot analysis of total DNA from C. lindemuthianum. Total DNA was digested with BamHI (1), EcoRI (2), HindIII (3), XhoI (4), EcoRI/BamHI (5), or HindIII/XhoI (6), analyzed on a 0.8% agarose gel, transferred to nylon membrane and hybridized with a ³²P-radiolabeled Clpnl2 fragment.

Figure 3

Three-dimensional structure of Clpnl2 from C. lindemuthianum showing highly conserved residues involved in catalysis.

Figure 4

Alignment of the amino acid sequences of pectin lyases of bacteria, fungi and oomycetes used in phylogenetic analyses. Identical residues are marked with an asterisk (*). Dashes represent gaps introduced to preserve alignment. Conserved catalytic residues are indicated in boxes.

Figure 5

Phylogenetic tree of pectin lyases. The phylogeny shown is the Bayesian topology and branch lengths inferred using MrBayes vs. 3.1.2, with the Blosum 62 + G model. Numbers above the diagonal indicate posterior probability values from Bayesian analysis. Numbers below the diagonal indicate bootstrap percentage values from a bootstrap analysis inferred using the same alignment with PAUP*4.0 and Neighbor-J, respectively. A. thaliana pectate lyase was used as an outgroup. The asterisks represent branches that were not supported in 50% or more of the bootstraps. The scale bar represents the number of substitutions per site. The phylogenetic tree was edited using Dendroscope software [77].

Figure 6

Clustering the three-dimensional structures of pectin lyases. The pectin lyase dataset was clustered by the un-weighted pair group method using the arithmetic mean (UPGMA) [53] with a similarity matrix obtained by the Voronoi contact method [51] using the ProCKSI-Server [52]. The tree image was generated using Dendroscope software [77]. A. Three-dimensional structure of PEL B from A. niger [PDB:1QCX]. B-C. Three-dimensional structures of the PNLs from C. lindemuthianum [GenBank: JN034039] and P. carotovorum [GenBank: AAA24856] respectively, predicted by homology modeling using the Swiss-Model Server [48].

Figure 7

Analysis of the relative gene expression of Clpnl2 in races 0 and 1472 of C. lindemuthianum. A-B. Gel-like images showing the expression of Clpnl2 in races 0 and 1472, respectively, on the different carbon sources tested. C. Semi-quantitative data for the expression of Clpnl2 in both races on the carbon sources. Total RNA was isolated from induced mycelia and amplified by RT-PCR with specific primers to yield the cDNA of Clpnl2. Amplification products were checked and quantified on a Bioanalyzer (2100 Agilent Bioanalyzer). The data were normalized using 18S rRNA as a control, and the results are expressed in μg/μl of amplified product.