Molecular cloning of BPL1, a pectate lyase-like gene in Brassica napus

Pectate lyase (EC 4.2.2.2) is an enzyme involved in the maceration and soft rotting of plant tissue via degradation of cell wall in organisms. Plants as well as bacteria and fungi are capable of producing pectate lyases. Here we report the cloning of a novel full-length cDNA of pectate lyase gene, designated BPL1, from Brassica napus by rapid amplification of cDNA ends. BPL1 cDNA is 1787 bp containing a 1503 bp ORF encoding a 500 amino acid protein precursor. The protein precursor has a potential signal peptide with 22 amino acids. Alignment of sequences shows that there are some extremely conserved amino acids among pectate lyase-like proteins from different plant species, and novel C-terminal domains are found in Arabidopsis and Brassica. Phylogenetic analysis of 50 pectate lyase-like proteins from various species demonstrates the obvious distinction among pectate lyase-like proteins from plants, bacteria and fungi, which are subsequently clustered into three groups. The cloning of BPL1 enables us to explore its diverse roles in higher plants and potential application in crop improvement.     

Cloning and characterization of pectate lyases expressed in the esophageal gland of the pine wood nematode Bursaphelenchus xylophilus

Two pectate lyase genes (Bx-pel-1 and Bx-pel-2) were cloned from the pine wood nematode, Bursaphelenchus xylophilus. The deduced amino acid sequences of these pectate lyases are most similar to polysaccharide lyase family 3 proteins. Recombinant BxPEL1 showed highest activity on polygalacturonic acid and lower activity on more highly methylated pectin. Recombinant BxPEL1 demonstrated full dependency on Ca2+ for activity and optimal activity at 55 degrees C and pH 8 to 10 like other pectate lyases of polysaccharide lyase family 3. The protein sequences have predicted signal peptides at their N-termini and the genes are expressed solely in the esophageal gland cells of the nematode, indicating that the pectate lyases could be secreted into plant tissues to help feeding and migration in the tree. This study suggests that pectate lyases are widely distributed in plant-parasitic nematodes and play an important role in plant-nematode interactions.     

Pectin methylesterases induce an abrupt increase of acidic pectin during strawberry fruit ripening

The decrease of strawberry (Fragariaxananassa Duch.) fruit firmness observed during ripening is partly attributed to pectolytic enzymes: polygalacturonases, pectate lyases and pectin methylesterases (PMEs). In this study, PME activity and pectin content and esterification degree were measured in cell walls from ripening fruits. Small green, large green, white, turning, red and over-ripe fruits from the Elsanta cultivar were analyzed. Using the 2F4 antibody directed against the calcium-induced egg box conformation of pectin, we show that calcium-bound acidic pectin was nearly absent from green and white fruits, but increased abruptly at the turning stage, while the total pectin content decreased only slightly as maturation proceeded. Isoelectrofocalisation performed on wall protein extracts revealed the expression of at least six different basic PME isoforms. Maximum PME activity was detected in green fruits and steadily decreased to reach a minimum in senescent fruits. The preliminary role of PMEs and subsequent pectin degradation by pectolytic enzymes is discussed.     

Functional implications of structure-based sequence alignment of proteins in the extracellular pectate lyase superfamily.

Pectate lyases are plant virulence factors that degrade the pectate component of the plant cell wall. The enzymes share considerable sequence homology with plant pollen and style proteins, suggesting a shared structural topology and possibly functional relationships as well. The three-dimensional structures of two Erwinia chrysanthemi pectate lyases, C and E, have been superimposed and the structurally conserved amino acids have been identified. There are 232 amino acids that superimpose with a root-mean-square deviation of 3 A or less. These amino acids have been used to correct the primary sequence alignment derived from evolution-based techniques. Subsequently, multiple alignment techniques have allowed the realignment of other extracellular pectate lyases as well as all sequence homologs, including pectin lyases and the plant pollen and style proteins. The new multiple sequence alignment reveals amino acids likely to participate in the parallel beta helix motif, those involved in binding Ca2+, and those invariant amino acids with potential catalytic properties. The latter amino acids cluster in two well-separated regions on the pectate lyase structures, suggesting two distinct enzymatic functions for extracellular pectate lyases and their sequence homologs.     

<Emphasis Type="Italic">In silico</Emphasis> analysis of pectin lyase and pectinase sequences

A total of 48 full-length protein sequences of pectin lyases from different source organisms available in NCBI were subjected to multiple sequence alignment, domain analysis, and phylogenetic tree construction. A phylogenetic tree constructed on the basis of the protein sequences revealed two distinct clusters representing pectin lyases from bacterial and fungal sources. Similarly, the multiple accessions of different source organisms representing bacterial and fungal pectin lyases also formed distinct clusters, showing sequence level homology. The sequence level similarities among different groups of pectinase enzymes, viz. pectin lyase, pectate lyase, polygalacturonase, and pectin esterase, were also analyzed by subjecting a single protein sequence from each group with common source organism to tree construction. Four distinct clusters representing different groups of pectinases with common source organisms were observed, indicating the existing sequence level similarity among them. Multiple sequence alignment of pectin lyase protein sequence of different source organisms along with pectinases with common source organisms revealed a conserved region, indicating homology at sequence level. A conserved domain Pec_Lyase_C was frequently observed in the protein sequences of pectin lyases and pectate lyases, while Glyco_hydro_28 domains and Pectate lyase-like β-helix clan domain are frequently observed in polygalacturonases and pectin esterases, respectively. The signature amino acid sequence of 41 amino acids, i.e. TYDNAGVLPITVN-SNKSLIGEGSKGVIKGKGLRIVSGAKNI, related with the Pec_Lyase_C is frequently observed in pectin lyase protein sequences and might be related with the structure and enzymatic function.     

Molecular and genetic characterization of two pollen-expressed genes that have sequence similarity to pectate lyases of the plant pathogen Erwinia

A set of cDNAs that are expressed in tomato anthers were isolated [24]. We further characterized two of these cDNAs (LAT56 and LAT59) and their corresponding genomic clones. LAT56 and LAT59 show low levels of steady-state mRNA in immature anthers and maximal levels in mature anthers and pollen. The LAT56 and LAT59 genes are single-copy in the tomato genome, and are linked on chromosome 3, approximately 5 cM apart. Although these cDNAs did not cross-hybridize, their deduced protein sequences (P56 and P59) have 54% amino acid identity. The LAT56 and LAT59 genes each have two introns, but they are located in non-homologous positions. P56 and P59 show significant protein sequence similarity to pectate lyases of plant pathogenic bacteria. The similarity of P56 and P59 to the bacterial pectate lyases is equivalent to the homology described for different pectate lyase sequences of the genus Erwinia. We suggest that the pollen expression of LAT56 and LAT59 might relate to a requirement for pectin degradation during pollen tube growth.Abbreviations LAT, late anther tomato - bp, base pairs - MA, mature anther - PL, pectate lyase - kb, kilobase (pairs)     

Functional implications of the beta-helical protein fold: differences in chemical and thermal stabilities of Erwinia chrysanthemi EC16 pectate lyases B, C, and E

Colonization of plant tissue by the phytopathogen Erwinia chrysanthemi EC16 is aided by the activities of the pectate lyase isozymes (PLs), which depolymerize the polygalacturonic acid component (PGA) of plant cell walls. The bacterium secretes four pectate lyases (PLa, PLb, PLc, and PLe), two of which, PLc and PLe, have been shown to fold into a similar domain motif, the beta-helix. To understand the rationale behind the evolution and retention of these isoforms, the susceptibilities of pectate lyases B, C, and E to chemical and thermal denaturation and the resulting enzymatic inactivation were examined. With guanidine hydrochloride used as a denaturant, all three pectate lyases denatured with transition midpoint guanidine hydrochloride concentrations (Cm) of 1.3, 1.1, and 1.8 M for PLb, PLc, and PLe, respectively. Lyase activity decreased in direct response to loss of secondary structure in all enzymes. Pectate lyases B and C demonstrated increased enzymatic activity at temperatures above 30 degrees C, with maximal activity observed at 40 degrees C for PLb and 35 degrees C for PLc. Transition midpoints (Tm) as measured by circular dichroism were at 46.9 degrees C for PLb and 44.3 degrees C for PLc, indicating detectable conformational changes accompanying thermal inactivation. Decreased enzymatic activity of PLe was observed at all temperatures above 30 degrees C, and the enzyme was found to possess a Tm at 38.9 degrees C. The data demonstrate structural differences among these enzymes that may be the basis for different enzymatic efficiencies under the potential array of environmental conditions experienced by the bacterium. These differences, in turn, may play a part in the retention of these isozymes as virulence factors, allowing the successful colonization of susceptible plant hosts.     

Novel Pectate Lyase Genes of Heterodera glycines Play Key Roles in the Early Stage of Parasitism

Pectate lyases are known to play a key role in pectin degradation by catalyzing the random cleavage of internal polymer linkages (endo-pectinases). In this paper, four novel cDNAs, designated Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7, that encode pectate lyases were cloned and characterized from the soybean cyst nematode, Heterodera glycines. The predicted protein sequences of HG-PEL-3, HG-PEL-4 and HG-PEL-6 differed significantly in both their amino acid sequences and their genomic structures from other pectate lyases of H. glycines (HG-PEL-1, HG-PEL-2 and HG-PEL-7). A phylogenetic study revealed that the pectate lyase proteins of H. glycines are clustered into distinct clades and have distinct numbers and positioning of introns, which suggests that the pectate lyase genes of H. glycines may have evolved from at least two ancestral genes. A Southern blot analysis revealed that multiple Hg-pel-6-like genes were present in the H. glycines genome. In situ hybridization showed that four novel pectate lyases (Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7) were actively transcribed in the subventral esophageal gland cells. A semi-quantitative RT-PCR assay supported the finding that the expression of these genes was strong in the egg, pre-parasitic second-stage juvenile (J2) and early parasitic J2 stages and that it declined in further developmental stages of the nematode. This expression pattern suggests that these proteins play a role in the migratory phase of the nematode life cycle. Knocking down Hg-pel-6 using in vitro RNA interference resulted in a 46.9% reduction of the number of nematodes that invaded the plants and a 61.5% suppression of the development of H. glycines females within roots compared to the GFP-dsRNA control. Plant host-derived RNAi induced the silencing of the Hg-pel-6gene, which significantly reduced the nematode infection levels at 7 Days post inoculation (dpi). Similarly, this procedure reduced the number of female adults at 40 dpi, which suggests the important roles of this gene in the early stages of parasitism. Our combined data suggest that two types of pectate lyases are present in the H. glycines genome and may have different roles during infection.     

PMR6, a pectate lyase-like gene required for powdery mildew susceptibility in Arabidopsis

The plant genes required for the growth and reproduction of plant pathogens are largely unknown. In an effort to identify these genes, we isolated Arabidopsis mutants that do not support the normal growth of the powdery mildew pathogen Erysiphe cichoracearum. Here, we report on the cloning and characterization of one of these genes, PMR6. PMR6 encodes a pectate lyase-like protein with a novel C-terminal domain. Consistent with its predicted gene function, mutations in PMR6 alter the composition of the plant cell wall, as shown by Fourier transform infrared spectroscopy. pmr6-mediated resistance requires neither salicylic acid nor the ability to perceive jasmonic acid or ethylene, indicating that the resistance mechanism does not require the activation of well-described defense pathways. Thus, pmr6 resistance represents a novel form of disease resistance based on the loss of a gene required during a compatible interaction rather than the activation of known host defense pathways.     

Cloning and characterization of an esophageal-gland-specific pectate lyase from the root-knot nematode Meloidogyne javanica

Root-knot nematodes (Meloidogynejavanica) are obligate sedentary endoparasites that must penetrate the host root to initiate their life cycle. Many enzymes are secreted by the nematode to facilitate host penetration; required enzymes may include pectate lyases and cellulases. Using differential screening, a class III pectate lyase, Mj-pel-1 (M. javanica pectate lyase 1), was cloned from a library enriched for esophageal gland genes. DNA gel blotting confirmed that the Mj-pel-1 gene was of nematode origin and a member of a small multigene family. In situ hybridization localized the expression of Mj-pel-1 to the basal cells of the esophageal glands, while immunolocalization detected the protein in the esophageal glands as well as on the exterior of the nematode, confirming that the protein is secreted. When MJ-PEL-1 was expressed in Pichia pastoris, the resulting protein was active. The pH optimum of MJ-PEL-1 was 10.0, and the enzyme was five times more active on pectate than on pectin. Like other class III pectate lyases, MJ-PEL-1 also displayed an absolute requirement for Ca2+. The root-knot nematode migrates through the middle lamella of the plant root; therefore, MJ-PEL-1 may be an important enzyme early in the infection process.     

Carbon catabolite repression in pectin digestion by the phytopathogen Dickeya dadantii

The catabolism of pectin from plant cell walls plays a crucial role in the virulence of the phytopathogen Dickeya dadantii. In particular, the timely expression of pel genes encoding major pectate lyases is essential to circumvent the plant defense systems and induce massive pectinolytic activity during the maceration phase. Previous studies identified the role of a positive feedback loop specific to the pectin-degradation pathway, whereas the precise signals controlling the dynamics of pectate lyase expression were unclear. Here, we show that the latter is controlled by a metabolic switch involving both glucose and pectin. We measured the HPLC concentration profiles of the key metabolites related to these two sources of carbon, cAMP and 2-keto-3-deoxygluconate, and developed a dynamic and quantitative model of the process integrating the associated regulators, cAMP receptor protein and KdgR. The model describes the regulatory events occurring at the promoters of two major pel genes, pelE and pelD. It highlights that their activity is controlled by a mechanism of carbon catabolite repression, which directly controls the virulence of D. dadantii. The model also shows that quantitative differences in the binding properties of common regulators at these two promoters resulted in a qualitatively different role of pelD and pelE in the metabolic switch, and also likely in conditions of infection, justifying their evolutionary conservation as separate genes in this species.     

Analysis of promoter activity of members of the <Emphasis Type="Italic">PECTATE LYASE-LIKE (PLL)</Emphasis> gene family in cell separation in Arabidopsis

Background  

Pectate lyases depolymerize pectins by catalyzing the eliminative cleavage of α-1,4-linked galacturonic acid. Pectate lyase-like (PLL) genes make up among the largest and most complex families in plants, but their cellular and organismal roles have not been well characterized, and the activity of these genes has been assessed only at the level of entire organs or plant parts, potentially obscuring important sub-organ or cell-type-specific activities. As a first step to understand the potential functional diversity of PLL genes in plants and specificity of individual genes, we utilized a reporter gene approach to document the spatial and temporal promoter activity for 23 of the 26 members of the Arabidopsis thaliana (Arabidopsis) PLL gene family throughout development, focusing on processes involving cell separation.