Characterisation of the WAK / WAKL gene family, with a focus on members in Persea americana implicated in defense against Phytophthora cinnamomi

Abstract

Phytophthora cinnamomi is a devastating oomycete pathogen, which has a significant negative impact on global avocado (Persea americana) production. The wall associated kinases (WAKs) and wall associated kinase-likes (WAKLs) are pattern recognition receptors which detect fragmented pectin (oligogalacturonides formed during pathogen infection) and activate downstream plant defence responses. The identification and charaterisation of this gene family shows many inconsistencies which needed to be addressed before delving into the avocado WAK/WAKL gene family and describing which member are implicated in the defence response of P. cinnamomi. Thus, this dissertation aimed to critically evaluate the in silico characterization of the WAK and WAKL gene family as a whole and then in avocado, with a particular focus on the genes involved in defence against P. cinnamomi. The study begins with a critical evaluation of current methodologies for identifying and classifying WAK/WAKL genes across various plant species, revealing inconsistencies in identification, classification and characterisation protocols that hinder broader gene family conclusions. The review provides a streamlined approach to help increase the standardisation in this gene family so that all genes defined as WAKs or WAKLs contain the same properties, such as a standardised set of predicted protein domains in the downstream proteins. The review also provides recommendations for characterising the family to allow a broader description to be concatenated for large-scale analyses in the future. The core of the research is the comprehensive in silico characterization of the avocado WAK/WAKL (PaWAK/WAKL) gene family. The study describes the PaWAK/WAKL family composition, gene placement and structure, the protein properties and cellular localisation, phylogenetic relationships, their expression patterns during pathogen infection, and the potential impact of cis-acting elements in targeted regulation during defence. Protein 3D structures and the binding affinities of WAK/WAKLs and the damage-associated molecular pattern, oligogalacturonide, were predicted for defence-implicated proteins. This dissertation examines the PaWAK/WAKL on a genomic, transcriptomic, and proteomic level to identify differences between the partially-resistant Dusa® and susceptible R0.12 rootstocks (while also assessing the partially-resistant LeolaTM) to predict how these factors, in combination, contribute to the increased defence efficiency against P. cinnamomi. These findings highlight the significance of specific PaWAK/WAKL genes in avocado's defence against P. cinnamomi on a multi-omic level. This research not only enhances our understanding of this disease interaction but also serves as a foundation for developing molecular tools to screen for resistant rootstocks for commercial use.