Cutinase activity in plant pathogen, Pseudomonas syringae

Abstract

Pseudomonas syringae pv. actinidiae (Psa) is a plant pathogen that causes kiwifruit blight in kiwifruit plants, resulting in inferior quality and economic losses. Symptoms usually include browning of the external vascular tissues and appearance of dark brown specks surrounded by yellow chlorotic haloes on the leaves. It is believed that Psa infects by first degrading the outer cutin layer of plants and then enters the internal tissues to cause infection. Degradation of the outer cutin layer can occur by the action of extracellular cutinase enzymes, believed to be secreted by the pathogen during the early phases of infection. Under the action of this enzyme, the ester bonds of cutin are hydrolysed resulting in the release of the cutin monomers, C16 and C18 hydroxy and epoxy fatty acids. Since at the time of the experiment, obtaining the Psa strain to study the cutinase enzyme was not possible, it was decided that the work be carried out using a similar Pseudomonas strain, Pseudomonas syringae pv. tomato (Pst). A study done on the actinomycete Thermobifida fusca reveals that cutinases are esterase-like enzymes with an α/β hydrolase fold structure and comprises of a Ser170- His248-Asp216 catalytic triad. BLAST searches of the Pst genome using the T. fusca cutinase as the query sequence indicated that Pst also possess a cutinase-like esterase gene, PSPTO_4843. The PSPTO_4843 gene was cloned and expressed as a 32kDa recombinant protein, which was tested for esterase and cutinase enzyme activity. Esterase activity was confirmed with synthetic substrates but convincing results for cutinase activity could not be demonstrated due to difficulties with the cutin preparations. In addition, as a comparative study the wild type strain has also been studied for the isolation and identification of the cutinase enzyme. Analysis of the protein extract revealed two possible enzymes. One was a 32kDa protein and the other was a 42kDa protein. Both these proteins were tested for esterase and cutinase activity along with the recombinant protein. Though not fully purified, these enzymes also displayed esterase activity. Understanding the molecular machinery behind the pathogenicity could lead to the development of effective control measures.