Colletotrichum gloeosporioides pelB is an important virulence factor in avocado fruit-fungus interaction

Colletotrichum gloeosporioides is an important pathogen of tropical and subtropical fruits. The C. gloeosporioides pelB gene was disrupted in the fungus via homologous recombination. Three independent isolates, GD-14, GD-23, and GD-29, did not produce or secrete pectate lyase B (PLB) and exhibited 25% lower pectate lyase (PL) and pectin lyase (PNL) activities and 15% higher polygalacturonase (PG) activity than the wild type. The PLB mutants exhibited no growth reduction on glucose, Na polypectate, or pectin as the sole carbon source at pH 3.8 or 6.0, except for a 15% reduction on pectin at pH 6.0. When pelB mutants were inoculated onto avocado fruits, however, a 36 to 45% reduction in estimated decay diameter was observed compared with the two controls, the wild type and undisrupted transformed isolate. In addition, these pelB mutants induced a significantly higher host phenylalanine ammonia lyase activity as well as the antifungal diene, which is indicative of higher host resistance. These results suggest that PLB is an important factor in the attack of C. gloeosporioides on avocado fruit, probably as a result of its virulence factor and role in the induction of host defense mechanisms.     

Local modulation of host pH by Colletotrichum species as a mechanism to increase virulence

The phytopathogenic fungus Colletotrichum gloeosporioides produces one pectate lyase (PL) that is a key virulence factor in disease development. During growth of C. gloeosporioides, Colletotrichum acutatum, and Colletotrichum coccodes in acidified yeast extract medium, the fungus secreted ammonia and increased the medium pH. Ammonia accumulation and the consequent pH change increased as a function of initial pH and buffer capacity of the medium. PL secretion by C. gloeosporioides correspondingly increased as the pH of the medium increased. The C. gloeosporioides pelB gene-disrupted mutant was able to increase ammonia accumulation and pH of the media similarly to the wild-type isolate. C. gloeosporioides in avocado, C. coccodes in tomato, and C. acutatum in apple showed ammonia accumulation in the infected area where pH increased to 7.5 to 8 and PL activity is optima. In nonhost interactions where C. gloeosporioides was inoculated in apples, the addition of ammonia-releasing compounds significantly enhanced pathogenicity to levels similar to those caused by the compatible C. acutatum-apple interaction. The results therefore suggest the importance of ammonia secretion as a virulence factor, enhancing environmental pH and pathogenicity of the Colletotrichum species.     

Role of ammonia secretion and pH modulation on pathogenicity of Colletotrichum coccodes on tomato fruit

Colletotrichum coccodes was found to alkalinize the decaying tissue of tomato fruit via accumulation and secretion of ammonia. Alkalinization dynamics caused by ammonia secretion from growing hyphae was examined microscopically using the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Values of pH of 7.9 observed in the host tissue close to the hyphal tips declined to pH 6.0 at 10 mm away from the hyphal tip, which was a value that was still higher than that detected in the healthy tissue, pH 4.2. Ammonia accumulation at the infection site depended on the initial environmental pH. Treatments with low (4.0) pH buffer at the infection site resulted in high levels of ammonia secretion and increased virulence of C. coccodes compared with similar treatments with buffer at pH 7.0. Significantly, mutants of C. coccodes defective in nitrogen utilization, nit-, and areA- were impaired in ammonia secretion and showed reduced decay development. The reduced infection rate of nit- mutants could be complemented by adding glutamine at the infection site. Thus, ammonia accumulation is a critical factor contributing to C. coccodes pathogenicity on tomato fruit. The results show that the initial acidic pH of the fruit is conducive to ammonia secretion and the subsequent alkalinization of the infection site, and facilitates fungal virulence and the transformation from the quiescent-biotrophic to active-necrotrophic state.     

Expression of pectate lyase from Colletotrichum gloesosporioides in C. magna promotes pathogenicity

To test the contribution of pectate lyase (PL) to promoting fungal pathogenicity, a pectate lyase gene (pel) from the avocado pathogen Colletotrichum gloeosporioides, isolate Cg-14, was expressed in C. magna isolate L-2.5, a pathogen of cucurbits that causes minor symptoms in watermelon seedlings and avocado fruits. Isolate L-2.5 was transformed with pPCPH-1 containing hph-B as a selectable marker and the 4.1-kb genomic pel clone. Southern hybridization, with the 4.1-kb genomic pel clone or 2.13-kb hph-B cassette as probes, detected integration of pel in transformed C. magna isolates Cm-PL-3 and Cm-PL-10. Western blot (immunoblot) analysis with antibodies against Cg-14 PL detected a single PL secreted by L-2.5 at a molecular mass of 41.5 kDa, whereas the PL of C. gloeosporioides had a molecular mass of 39 kDa. When PL activity was measured 4 days after inoculation in pectolytic enzyme-inducing media (PEIM), transformed isolates Cm-PL-3 and Cm-PL-10 showed additive PL activity relative to both Cg-14 and L-2.5. Transformed isolates also showed additive maceration capabilities on avocado pericarp relative to the wild-type C. magna alone, but did not reach the maceration ability of C. gloeosporioides. However, more severe maceration and damping off developed in watermelon seedlings inoculated with the transformed isolates compared with the two wild-type isolates, which showed no symptom development on these seedlings during the same period. Results clearly show the contribution of a single pel to the pathogenic abilities of C. magna and suggest that PL is a pathogenicity factor required for the penetration and colonization of Colletotrichum species.     

Characterization of the Erwinia carotovora pelB gene and its product pectate lyase.

The pelB gene encodes pectate lyase B, one of three pectate lyases identified in Erwinia carotovora EC. Pectate lyase B was purified from Escherichia coli containing the pelB gene on a recombinant plasmid. The activity of the protein was optimal at a pH of 8.3. The amino acid composition, N-terminal amino acid sequence, and C-terminal peptide sequence were determined and compared with the polypeptide sequence deduced from the DNA sequence of pelB. Purified pectate lyase B started at amino acid 23 of the predicted sequence, suggesting that a 22-amino-acid leader peptide had been removed. Pectate lyase B of E. carotovora EC and pectate lyase B of E. chrysanthemi EC16 contain 352 and 353 amino acids, respectively (N. T. Keen, S. Tanaki, W. Belser, D. Dahlbeck, and B. Staskawicz, J. Bacteriol. 168:595-606, 1986). The two proteins are 72% homologous on the basis of DNA sequence data, and 75% of the amino acids are identical.     

Erwinia chrysanthemi EC16 Produces a Second Set of Plant-Inducible Pectate Lyase Isozymes

The enterobacterium Erwinia chrysanthemi causes soft-rot diseases involving extensive tissue maceration in a wide variety of plants and secretes multiple pectic enzymes that degrade plant cell walls and middle lamellae. An E. chrysanthemi mutant with directed deletions or insertions in genes pehX, pelX, pelA, pelB, pelC, and pelE, which encode exo-poly-alpha-d-galacturonosidase, exopolygalacturonate lyase, and four isozymes of pectate lyase, respectively, was constructed by the marker exchange of a cloned pehX::TnphoA fragment into E. chrysanthemi CUCPB5010, a Delta(pelA pelE) Delta(pelB pelC)::28bp Delta(pelX)Delta4bp derivative of strain EC16. This mutant, E. chrysanthemi CUCPB5012, no longer caused pitting in a standard pectate semisolid agar medium used to detect pectolytic activity in bacteria. Nevertheless, the mutant still macerated leaves of chrysanthemum (Chrysanthemum morifolium), although with reduced virulence. The mutant was found to produce significant pectate lyase activity in rotting chrysanthemum tissue and in minimal media containing chrysanthemum extracts or cell walls as the sole carbon source. Activity-stained, ultra-thin-layer isoelectric focusing gels revealed the presence in these preparations of several pectate lyase isozymes with pIs ranging from highly acidic to highly alkaline. Sterile culture fluids containing these isozymes were able to macerate chrysanthemum leaf tissue. Unlike the products of the pelA, pelB, pelC, and pelE genes in E. chrysanthemi EC16, these plant-inducible pectate lyase isozymes were not produced in minimal medium containing pectate. The results suggest that E. chrysanthemi produces two sets of independently regulated pectate lyase isozymes that are capable of macerating plant tissues.     

Regulation of the Aspergillus nidulans pectate lyase gene (pelA).

Aspergillus nidulans pectate lyase was purified from culture filtrates. The enzyme catalyzed a random eliminative cleavage reaction, had an apparent molecular weight of 40,000, and a pl of 4.2. Pectate lyase antisera were produced and used to identify pectate lyase clones in a cDNA expression library. Thirteen of 14 clones identified immunologically cross-hybridized. The identity of the single-copy pectate lyase gene, which we designated pelA, was confirmed in two ways. First, several cDNA clones expressed pectate lyase activity in Escherichia coli. Second, targeted mutation of the gene in A. nidulans resulted in complete loss of enzyme activity. pelA encodes a 1,300-nucleotide mRNA that was present in cells grown with polygalacturonic acid as carbon source but absent from cells grown with glucose or acetate as carbon source. Thus, pectate lyase expression is regulated at the level of mRNA accumulation.     

Pectate lyase gene regulatory mutants of Erwinia chrysanthemi.

The pelB gene, which encodes one of the five pectate lyase isoenzymes of Erwinia chrysanthemi 3937, was mutagenized with a mini-Mu transposable element that can form gene fusions to the neomycin phosphotransferase-encoding region. Secondary mutants resistant to kanamycin in the absence of polygalacturonate, an inducer of wild-type pectate lyase activities, were selected. Such mutants produced other pectate lyase isoenzymes in the absence of the inducer.     

Production of cell wall dissolving enzymes by Erwinia carotovora subsp. atroseptica in vitro at 27°C and 30°5C

Cultures of Erwinia carotovora subsp. atroseptica , grown at 27°C and 30·°C in different liquid media were assayed for activities of pectate lyase, polygalacturonase and cellulase. Total production of both pectate lyase and of polygalacturonase was 3–6 times less at 30·5°C than at 27°C; secretion of pectate lyase was similarly affected. Cellulase was cell bound and its production was not affected by the temperatures investigated. Growth, protein synthesis and protease activity were similar at the two temperatures and production of enzyme activity at 27°C and 30·5°C was independent of the growth medium.     

Enzymatic properties of the highly thermophilic and alkaline pectate lyase Pel-4B from alkaliphilic Bacillus sp. strain P-4-N and the entire nucleotide and amino acid sequences

We cloned two genes for alkaline pectate lyase, pel-4A and pel-4B, from alkaline pectinase-producing alkaliphilic Bacillus sp. strain P-4-N. The pel-4B gene product Pel-4B was purified to homogeneity and characterized. The purified enzyme had an isoelectric point of pH 9.6 and a molecular mass of 35 kDa, values close to those of the pel-4A gene product Pel-4A. The pH and temperature optima for activity were as high as 11.5 and 70 degrees C, respectively, which are the highest among the pectate lyases reported to date. The mature Pel-4B (304 amino acids; 33,868 Da) was structurally related to the enzymes in the polysaccharide lyase family 1 and showed 35.6% identity with Pel-4A on the amino acid level. It showed significant homology to other pectate lyases in the same family, such as the enzymes from alkaliphilic Bacillus sp. strains KSM-P7 and KSM-P103 and the fungi Aspergillus nidulans and Colletotrichum gloeosporioides f. sp. malvae.     

Thermodynamic characterization of a highly thermoactive extracellular pectate lyase from a new isolate Bacillus pumilus DKS1

An extracellular pectate lyase (EC 4.2.2.2) was purified from the culture filtrate of a newly isolated Bacillus pumilus DKS1 grown in pectin containing medium. Using ion-exchange and gel filtration chromatography, this enzyme was purified and found to have a molecular weight of around 35kDa. The purified enzyme exhibited maximal activity at a temperature of 75 degrees C and pH 8.5. The presence of 1mM calcium and manganese enhanced pectate lyase activity and was strongly inhibited by zinc, nickel and EDTA. The thermal inactivation studies revealed an entropy-enthalpy compensation pattern below a critical temperature. The alkaliphilicity and high thermostability of this pectate lyase may have potential implications in fibre degumming.     

Culture conditions for the production of pectinolytic enzymes by the white-rot fungus Trametes trogii on a laboratory scale

Different cultural parameters that regulate pectinolytic enzyme production in vitro by Trametes trogii were studied. When grown in a medium containing pectin, T. trogii produced extracellular polymethylgalacturonase, polygalacturonase and pectin lyase but no pectate lyase activity. No significant differences in the maximum enzyme activities measured were observed with the addition of xylan, carboxymethylcellulose or both to the medium containing pectin. The addition of glucose to that medium considerably decreases all the activities studied, and in a medium with glucose as the sole carbon source no galacturonase activity could be measured, and pectin lyase activity was at its minimum. The low synthesis of pectin lyase in cultures containing glucose suggests that this enzyme is constitutive in contrast to the polygalacturonases that were not detected. The increase in pectin concentration stimulated growth and enzyme production. The highest specific activities were attained with the greatest concentration tested (15 g/l). Casamino acids were the best nitrogen source for enzyme production. Maximum growth was measured at pH 3.3; pH values of around 4.5 stimulated enzyme production, but high pectinase activities were also detected in media with more alkaline initial pH values (6.2 for galacturonases and 6.6 for lyases), probably owing to the specific induction of particular isoforms. In the range of 23 to 28°C, good results were obtained in growth as well as in enzyme production. The addition of Tween 80 promoted growth and gave the highest yield of polymethylgalacturonase and pectin lyase (0.37 and 36.2 E.U./ml, respectively). The highest polygalacturonase activity (1.1 E.U/ml) was achieved with polyethylene glycol. Tween 20 and Triton X-100 inhibited growth and pectinase production.     

Production of lyases byPhoma exigua associated with seed-rot ofVigna radiata

Phoma exigua associated with seed-rot ofVigna radiata produced lyases which varied with the media tested. The production of lyases was higher in pectin-supplemented media.Vigna seed meal medium was not suitable for induction of lyase production. The pectin lyase and pectate lyase was maximum after 11 d of incubation by which time the pH was shifted to alkaline side. Temperature of 25 °C and pH 9 was found to be optimum for the activity of pectin lyase and pectate lyase. Fungicides (antracol and panoctine), phenols (pyrocatechol and gallic acid) and growth substances (gibberellic acid and yeast extract) adversely affected the enzyme secretion.     

Characterization of a family 3 polysaccharide lyase with broad temperature adaptability, thermo-alkali stability, and ethanol tolerance

The 774-bp pectate lyase gene plyAI4 from Bacillus sp. I4 was cloned and expressed in E. coli. The gene encodes a 257-residue polypeptide (PlyAI4, 28.3 kDa) with the highest identities of 97.3% with a putative pectate lyase from Bacillus subtilis BSn5 (ADV94306) and 60.3% with an identified pectate lyase of the polysaccharide lyase family (PL) 3 from Paenibacillus amylolyticus 27C64 (ADB78774). The purified recombinant PlyAI4 (rPlyAI4) exhibited apparently optimal activity at pH 10.5 ?? 11.0 and 50°C. Compared with the majority of reported alkaline pectate lyases, rPlyAI4 exhibited more residual enzyme activity at 20°C (??45%) or at 70°C (??50%) and better thermostability at 70°C (??60 min half-life at 70°C). In the presence of 20% (v/v) ethanol, pectate lyase activity was enhanced by 0.2 fold. After incubation in 40% (v/v) ethanol at 37°C and pH 8.5 for 1 h, the purified rPelAI4 retained more than 75% of the initial activity. Sequence analysis proposed a new signature block, A-D-G-[V/I]-H, for PL 3 pectate lyases. These properties may prove to be important with regards to PlyAI4 for basic research and industrial application.