Molecular and bioinformatic analysis of the Persea americana (Mill.) NPR1-dependent defence signalling pathway in response to Phytophthora cinnamomi infection

Abstract

The global avocado industry has experienced significant growth throughout the past two decades, with annual production doubling over that time. However, increased production is accompanied by an ever-increasing threat from a variety of pests and diseases. Phytophthora root rot (PRR) is currently considered the most devastating disease of avocado, causing significant economic losses annually. The causal agent, Phytophthora cinnamomi, is a hemibiotrophic oomycete; as such, it utilises both biotrophic and necrotrophic infection strategies to overwhelm its host. Plants use numerous phytohormone regulated defence response pathways, depending on the infection strategy employed by their pathogen. Typically, defence against biotrophic pathogens applies the salicylic acid (SA)-dependent defence response pathway; whereas defence against necrotrophic pathogens is associated with the jasmonic acid/ethylene (JA/ET)-dependent pathway. Notably, the nonexpressor of pathogenesis-related genes 1 (NPR1) co-transcription factor is crucial to most SA-dependent defence gene expression. Furthermore, it is essential to the establishment of systemic acquired resistance, a plant-wide state of heightened defence readiness. However, the mechanisms required to achieve SAR and effectively defend against different pathogens is exceedingly complex. Therefore, this dissertation aimed to identify and characterise NPR1-like proteins in Persea americana (avocado). Furthermore, this study attempted to understand the response of several NPR1 pathway-associated genes, both over-time and comparatively between PRR susceptible and partially resistant avocado rootstocks. A total of five NPR1-like coding genes were described in P. americana. Initial in silico analyses suggested that three PaNPR1-like proteins could be involved in defence; two of these, PaNPR1 and PaNPR2, were likely associated with positive SAR regulation while PaNPR4 probably served an opposing role. Meanwhile, the remaining two, PaNPR3 and PaNPR5, were most likely involved in tissue development. These suspicions were later confirmed by expression analysis following phytohormone application, P. cinnamomi inoculation and tissue-specific sampling. Interestingly, significant differences were observed when comparing the expression of the several PaNPR1-like genes in the PRR susceptible (R0.12) and partially resistant (Dusa®) rootstocks. Therefore, we identified and annotated 116 orthologs of Arabidopsis thaliana NPR1 pathway genes in the P. americana genome. Using dual RNA-sequencing data, we characterised the expression of all 116 genes over time in the PRR susceptible rootstock R0.12. Additionally, we compared the expression of the NPR1 pathway-associated genes between R0.12 and the partially PRR resistant rootstock, Dusa®. Our observations suggest that SAR was established in both avocado rootstocks; additionally, expression of the majority of NPR1 pathway-associated genes is regulated to some extent following P. cinnamomi challenge. However, significant differences were evident when comparing expression in R0.12 and Dusa®. Primarily, our observations suggest that the SA-defence response pathway is suppressed more effectively in Dusa® following the establishment of SAR. Thus, Dusa® likely responds more appropriately to the pathogen’s necrotrophic phase of infection. The work presented here represents the first step in fully characterising and understanding the NPR1 pathway in P. americana and its role in resistance against PRR. Furthermore, we believe that this study will form part of the foundation for further functional characterisation of disease resistance pathways in avocado.