Identification of fungal proteases responsible for proteolytic cleavage of tomato chitinases

CS-5.4 - Plants defend themselves against fungal pathogens by secreting enzymes with antifungal activities such as chitinases that degrade the fungal cell wall. In response, fungal pathogens secrete chitin-binding proteins such as Avr4 which can protect them against plant chitinases. This protection might be incomplete, so released chitin oligomers may still be detected by plant recognition receptors and induce defence. Fungi like C. fulvum secrete Ecp6 which can sequester chitin oligomers preventing them to be detected. Previously, degradation of PR proteins has been proposed as a component of virulence of some plant pathogens. Here, we employed a combined biochemical, proteomics, bioinformatics and mass spectrometry approach to identify the chitinase modifying protein(s). We have produced four chitin binding tomato chitinases (CBD-tomato chitinases) in Pichia pastoris and analysed eight tomato fungal pathogens for their ability to degrade their CBD-. We showed that enzymes present in culture filtrate of some of these tomato pathogens can degrade two of the four CBD-tomato chitinases. Culture filtrates with proteolytic activity were fractionated on Superdex 75 and analysed. Mass spectrometry confirmed the presence of at least two novel secreted serine proteases in the active fractions. We also performed expression profiling for the fungal proteases and CBD-tomato chitinases during infection of susceptible and resistant tomato cultivars. In addition, we are testing whether the two serine proteases play a role in virulence.