Identification and expression analysis of phytophthora cinnamomi CRN effector genes during infection of persea americana (Mill.)

Abstract

Phytophthora cinnamomi is a hemi-biotrophic plant pathogen impacting the South African avocado industry. Like other Phytophthora spp., this pathogen employs a repertoire of effectors to suppress host defence mechanisms and facilitate successful infection in host plants. Because P. cinnamomi is a hemi-biotroph, the pathogen would benefit from the suppression of cell death during the biotrophic phase early in infection and the subsequent induction of cell death later in infection when the pathogen switches to a necrotrophic phase. P. cinnamomi achieves this beneficial pattern of cell death through the differential expression and delivery of effectors at different stages of infection. Multiple effectors have been shown to play a role in host cell death manipulation in Phytophthora, but CRNs are of ongoing interest due to their ability to both supress and induce cell death during infection. The research in this thesis is a continuation of honours research conducted by me in 2020, where a pipeline was created to identify and validate P. cinnamomi CRN (PcinCRN) effector protein sequences from the P. cinnamomi GKB4 transcriptome. In the work conducted for this MSc degree, P. cinnamomi CRN expression profiles were analysed using dual RNA-seq data from P. cinnamomi infected avocado rootstocks. Candidate CRNs were selected based on their expression profile and their coding sequences were confirmed via cloning and Sanger sequencing. Amino acid sequences of CRN candidates and their variants were extracted and compared to other Phytophthora CRN sequences that have been functionally characterised as cell death inducers or suppressors during infection of host plant species. Tertiary protein structures for each CRN were predicted and their domains were assessed. Putative functions in cell death manipulation during infection of avocado were assigned to P. cinnamomi CRNs based on expression profiles, relatedness to other Phytophthora CRNs, domain analyses and protein folding predictions. From a list of 25 full-length P. cinnamomi CRNs, 13 CRN genes demonstrated expression profiles corresponding to a potential role in cell death manipulation during infection of avocado. The coding sequences of 10 candidate CRNs were confirmed, of which six were found to have two alleles. Additionally, one was found to have the potential to undergo alternative splicing. PcinCRN52 was molecular characterised as a cell death inducer during late infection and PcinCRN30, PcinCRN77, PcinCRN81 and PcinCRN86 were molecularly characterised as cell death suppressors during early infection. PcinCRN11, PcinCRN53, PcinCRN73, PcinCRN75 and PcinCRN95 were found to have variants that potentially play contradicting roles in cell death or where one variant may function in a different host plant species. The findings from this study suggest that P. cinnamomi CRNs may function by regulating each other, to either suppress or induce cell death at appropriate stages to promote successful colonisation of avocado. This study highlights the complex and dynamic interactions between CRN effectors and their potential impact on the outcome of P. cinnamomi infection of the host plant.