Identification and expression analysis of putative NLR genes in avocado, Persea americana, during infection by the oomycete, Phytophthora cinnamomi

Abstract

Avocado production in South Africa is significantly affected by the pathogen Phytophthora cinnamomi. This pathogen causes root necrosis in infected rootstocks, which may ultimately lead to plant death and thus economic losses. The complex molecular interactions between avocado and P. cinnamomi are not well understood and might be the key to breeding P. cinnamomi resistant rootstocks. Understanding the molecular mechanisms behind successful immune responses against P. cinnamomi is thus of utmost importance. Nucleotide binding-Leucine rich repeat (NLR) proteins play an important role in activating the Effector triggered immune (ETI) response after pathogen effector recognition. This immune response leads to a Hypersensitive response and cell death, which restricts P. cinnamomi growth during the pathogen’s biotrophic stage. Furthermore, previous studies have shown that differential expression patterns of NLR genes influences the amplitude of the ETI response. The importance of NLR proteins during P. cinnamomi infection and whether they play a role in rootstock resistance levels remain unclear. Chapter 1 of this thesis provides a detailed review of NLR proteins. The structure of these proteins is described, together with the molecular mechanisms which regulate the expression of NLR genes. Various examples are given regarding how NLR gene expression changes after pathogen infection in other plant species, and how NLR gene sequence variations within a plant species influence resistance levels towards various pathogens. Furthermore, methods for NLR identification and characterization using a genomic approach are discussed. Lastly, a summary is given on NLR sequences found in woody tree species and how this knowledge can be applied to avocado trees. Chapter 2 reports on the PaNLRs identified in avocado using a bioinformatic approach and the VC75 avocado genome. Gene clusters were identified, and phylogenetic analysis was performed to investigate possible PaNLR gene duplication events. PaNLR expression analysis was done using dual RNA-seq data obtained from a partially resistant and susceptible rootstock inoculated with P. cinnamomi. The expression data was used to indicate which PaNLRs are potentially important for the detection of P. cinnamomi infection, and to assess whether a difference in PaNLR expression was evident between the two rootstocks.