DOI: 10.25403/upresearchdata.28296932.v1
title: Nucleotide-binding leucine-rich repeat (NLR) proteins in Dusa®, which play a critical role in effector-triggered immunity (ETI) and hypersensitive response (HR)

description: <i>Persea americana</i> Mill., commonly known as avocado, has gained considerable popularity in recent years as a fruit associated with global health food trends. One of the primary threats affecting avocado production in South Africa is <i>Phytophthora cinnamomi</i>, a hemibiotrophic, oomycete pathogen that attacks avocado feeder root systems to cause Phytophthora root rot (PRR). Most control measures gravitate towards phosphite-based fungicide applications and the use of a partially resistant rootstock, Dusa®. It is unclear what kind of immune repertoire Dusa® possesses, which gives it a competitive edge against <i>P. cinnamomi</i> compared to susceptible rootstocks such as R0.12. We directed our focus towards a group of resistance proteins known as the nucleotide-binding leucine-rich repeat (NLR) proteins. NLRs recognise pathogen effector proteins during effector-triggered immunity (ETI) to cause a type of immunogenic cell death known as the hypersensitive response (HR). The HR arrests pathogen infection to a localised site and prevents systemic spread to the rest of the plant. Differences in NLR protein repertoires have been observed between and within plant hosts which has implications for corresponding levels of resistance. Previous studies have ascertained variations in NLR repertoires between the partially resistant Dusa® and the susceptible R0.12 rootstocks. This study sought to functionally characterise a subset of <i>P. americana</i> NLR (PaNLR) proteins present in the Dusa® rootstock. We first established a model system involving <i>Nicotiana benthamiana-P. cinnamomi</i> to understand which NLR networks within <i>N. benthamiana</i> were activated in response to <i>P. cinnamomi</i> infection and to understand the timing of the biotrophic and necrotrophic stages of pathogen infection. The <i>NLR-required for cell death (NRC)</i> network indicated a higher level of expression compared to the <i>N requirement gene 1 and activated disease resistance 1 (NRG1/ADR1)</i> network in response to <i>P. cinnamomi</i> infection. We also identified that 3-12 hours post infection (hpi) is approximately the timing of <i>P. cinnamomi</i> biotrophy within <i>N. benthamiana</i> while 72-120 hpi is likely the necrotrophic phase. The juncture at which the switch between biotrophy and necrotrophy occurs is around 24-48 hpi. We then applied this knowledge to the second chapter to time the introduction of PaNLRs against the biotrophic phase to maximise conditions for the occurrence of an HR. PaNLR candidates from Dusa® were selected based on ability to recognise <i>P. cinnamomi </i>effectors, presence of defence motifs and high, significant (p&lt;0.05) expression during early infection. The candidates were analysed using transient expression assays using <i>Agrobacterium tumefaciens</i> and confocal microscopy. When expressed on their own, five PaNLR candidates did not cause HR or any form of cell death, while one PaNLR candidate caused a cell death phenotype. In the absence of infection, five of the PaNLRs localised to the nucleus and one PaNLR localised to the cytoplasm. Upon introduction of <i>P. cinnamom</i>i, all PaNLR candidates caused a strong HR which limits uncontrolled necrosis by the pathogen, suggesting that the candidates play a very active role in recognising cognate effectors to defend the avocado host during infection. The results of the study suggest that at the resting stage, some NLRs conserve their cellular resources whilst others are poised for cell death induction. We also demonstrated that the expression of PaNLR proteins during the biotrophic phase of infection allows it to successfully cordon off infection through induction of the HR. Diverse cellular localisations further suggest that PaNLRs have adapted complex ways to interact with various plant apoptotic pathways to induce immunogenic cell death during defence. This study provides the first molecular characterisation of PaNLRs and provides a foundation to study intricate avocado immunity interactions.