Abstract
Penicillium digitatum is the major postharvest pathogen of citrus fruit under Mediterranean climate conditions. Previous results have shown that proteases is the largest enzyme family induced by P. digitatum during fruit infection. In the present work, we addressed the study of the role of P. digitatum’s proteases in virulence following two complementary approaches. In the first approach, we undertook the functional characterization of the P. digitatum prtT gene, which codes for a putative transcription factor previously shown to regulate extracellular proteases in other filamentous fungi. Deletion of prtT caused a significant loss in secreted protease activity during in vitro growth assays. However, there was no effect on virulence. Gene expression of the two major secreted acid proteases was barely affected in the ΔprtT deletant during infection of citrus fruit. Hence, no conclusion could be drawn on the role of these secreted acidic proteases on the virulence of P. digitatum. In the second approach, we studied the effect of different protease inhibitors and chelators on virulence. Co-inoculation of citrus fruit with P. digitatum conidia and a cocktail of protease inhibitors resulted in almost a complete absence of disease development. Analysis of individual inhibitors revealed that the metalloprotease inhibitor, 1,10-phenanthroline, was responsible for the observed effect. The application of metal ions reverted the protective effect caused by the metallopeptidase inhibitor. These results may set the basis for the development of new alternative treatments to combat this important postharvest pathogen.
Highlights
Proteases, denoted as peptidases, proteinases, or proteolytic enzymes, can be classified according to the nature of the functional group at the active site
Our results showed that 1,10-phenanthroline, a metalloprotease inhibitor, is able to control the development of P. digitatum in citrus fruit
Our results showed that only the mixtures containing 1,10-phenanthroline were able to control the infection of Pd1 in Moicrroaonrggaensis,mcso2n0fi19r,m7,ixng that this compound was responsible for the reduction of the P. digitatum in11feocft1i9on in citrus fruit
Summary
Denoted as peptidases, proteinases, or proteolytic enzymes, can be classified according to the nature of the functional group at the active site. Most proteases belong to one of the four major families: Aspartic, cysteine, metallo, and serine peptidases. They are widely used in biotechnology, mainly in the food, leather, and detergent industries, in ecological bioremediation processes, and to produce therapeutic peptides [1]. Species in the genera, Aspergillus or Penicillium, contain more than 200 and 100 annotated genes encoding for putative proteases in the MEROPS database (https://www.ebi.ac.uk/merops/), respectively. These enzymes play a major role in the physiology, morphogenesis, and metabolism of fungi. Single or double deletant mutants in five genes encoding aspartic proteases did not result in any defect in virulence [12]
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