Phytophthora capsici causes serious diseases in numerous crop plants. Polygalacturonases (PGs) are cell wall-degrading enzymes that play an important role in pathogenesis in straminopilous pathogens. To understand PGs as they relate to the virulence of P. capsici, Pcipg2 was identified from a genomic library of a highly virulent P. capsici strain. Pcipg2 was strongly expressed during symptom development after the inoculation of pepper leaves with P. capsici. The wild protein (PCIPGII) was obtained from the expression of pcipg2 and found that increasing activity of PGs in PCIPGII-treated pepper leaves was consistent with increasing symptom development. Asp residues in active sites within pcipg2 affected PCIPGII activity or its virulence on pepper leaves. Results show that pcipg2 is an important gene among pcipg genes, and illustrate the benefit of analyzing mechanisms of pathogenicity during the period of host/parasite interaction.

Pectolytic enzymes are found mainly in fungi and bacteria. The most widely occurring enzymes are polygalacturonase (PGs), pectin methylesterase (PMEs) and pectate lyase (PLs) produced during the infection process and during culturing. The secretion of these enzymes results in the disorganization of the plant cell walls, which is responsible for the pathogenicity of the pathogens. These enzymes degrade the pectin of plants causing maceration of plant tissues and the enzyme activity increases under favourable environmental conditions. We have found that Phytophthora capsici, a pathogenic oomycete, produces levels of these three enzymes equal to those produced by soft-rotting Erwinia chrysanthemi. The activity of PGs, PLs and PMEs was investigated at the optimum temperature, pH and ionic strength in highly pathogenic P. capsici strains cultivated in two kinds of liquid medium containing either crude pepper extracts plus pectin or pectin as the carbon source. Virulence tests and enzymes activity showed that there was a high correlation between the enzyme activity and the pathogenicity of P. capsici. The effects of different carbon sources on the enzyme activity showed that pepper extract plus pectin was the best source for the carbon source.

ABSTRACT. To date, research on laccases has mostly been focused on plant and fungal laccases and their current use in biotechnological applications. In contrast, little is known about laccases from plant pathogens, although recent rapid progress in whole genome sequencing of an increasing number of organisms has facilitated their identification and ascertainment of their origins. In this study, a comparative analysis was performed to elucidate the distribution of laccases among bacteria, fungi, and oomycetes, and, through comparison of their amino acids, to determine the relationships between them. We retrieved the laccase genes for the 20 publicly available plant pathogen genomes. From these, 125 laccase genes were identified in total, including seven in bacterial genomes, 101 in fungal genomes, and 17 in oomycete genomes. Most of the predicted protein models of these genes shared typical fungal laccase characteristics, possessing four conserved domains with one cysteine and ten histidine residues at these domains. Phylogenetic analysis illustrated that laccases from bacteria and oomycetes were grouped into two distinct clades, whereas fungal laccases clustered in three main clades. These results 14019-14036 (2015) provide the theoretical groundwork regarding the role of laccases in plant pathogens and might be used to guide future research into these enzymes.

Laccases are blue copper oxidases (E.C. 1.10.3.2) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. A novel laccase gene pclac2 and its corresponding full-length cDNA were cloned and characterized from Phytophthora capsici for the first time. The 1683 bp full-length cDNA of pclac2 encoded a mature laccase protein containing 560 amino acids preceded by a signal peptide of 23 amino acids. The deduced protein sequence of PCLAC2 showed high similarity with other known fungal laccases and contained four copper-binding conserved domains of typical laccase protein. In order to achieve a high level secretion and full activity expression of PCLAC2, expression vector pPIC9K with the Pichia pastoris expression system was used. The recombinant PCLAC2 protein was purified and showed on SDS-PAGE as a single band with an apparent molecular weight ca. 68 kDa. The high activity of purified PCLAC2, 84 U/mL, at the seventh day induced with methanol, was observed with 2,2′-azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) as substrate. The optimum pH and temperature for ABTS were 4.0 and 30 °C, respectively. The reported data add a new piece to the knowledge about P. Capsici laccase multigene family and shed light on potential function about biotechnological and industrial applications of the individual laccase isoforms in oomycetes.

Phytophthora capsici inflicts damage on numerous crop plants by secreting a series of pectinase including pectate lyase (PEL). Here, we report a pectate lyase gene (Pcpel1) from a genomic library of a highly virulent P. capsici strain SD33. Pcpel1 was identified as an open reading frame of 1233 bp encoding a protein of 410 amino acids with a predicted amino-terminal signal sequence of 21 amino acids. The predicted protein of Pcpel1 has a calculated molecular mass of 43.8 kDa and a pI value of 6.8. Analysis of the amino acid sequence suggested that it was a member of the polysaccharide lyase family 1 that shows pectate lyase activity. Moreover, heterologous expression of Pcpel1 in Pichia pastoris produced proteins with molecular mass 66 kDa, very likely due to differential glycosylation by the yeast. By western blotting and northern blotting analysis, Pcpel1 was strongly expressed during interaction of P. capsici with the host plant, suggesting its involvement in the process of host infection. The role of Pcpel1 in cell wall disassembly and host/parasite interaction is discussed.