Investigating the role of downy mildew effectors in host resistance and susceptibility
Through specialised structures called haustoria, filamentous eukaryotic plant pathogens such as rusts and mildews can deliver “effector” proteins directly into plant cells in order to manipulate specific defence responses and processes. Hyaloperonospora arabidopsidis (Hpa) is a haustorium-producing biotrophic oomycete pathogen that causes downy mildew on Arabidopsis. Over 100 candidate effectors (called “HaRxLs”), carrying a characteristic RxLR protein motif, have been predicted from the Hpa genome sequence. This PhD work focused on the characterisation of some of these effectors and their interaction with plant defences. First, the Pseudomonas syringae pv. tomato (Pst) strain DC3000 type III secretion system was used to examine the effect of single oomycete effectors in planta. One of these, ATR13 from Hpa isolate Emco5 (ATR13Emco5) has been shown to be recognised in some Arabidopsis accessions by the RPP13 protein, preventing Hpa Emco5 from completing its life cycle on such resistant plants. Hpa Emco5 can however complete its life cycle on Ws-0 but paradoxically, ATR13Emco5 is also recognised in this accession. Here, I show that ATR13Emco5 is weakly recognised in Ws-0 by a single RPP13-independent, EDS1-independent dominant gene called RHA13, shown by rough mapping to position on Arabidopsis chromosome 4. Second, I contributed to two independent functional screens performed in our laboratory to experimentally characterise the Hpa effectorome. The first screen focused on HaRxLs effects on bacteria virulence using the EDV system. The second screen was based on HaRxLs subcellular localisation using Agrobacterium-mediated transient expression. As presented in this work, I showed that both screens identified putative interesting effectors which increased plant susceptibility to Pst and Hpa and localised to various plant subcellular compartments. One particular effector candidate, HaRxL79, localised to microtubules in planta and interacted with two microtubule-associated proteins in a yeast-two-hybrid assay. HaRxL79 was also found to interact with Arabidopsis histone chaperones (AtNAP1s) in the plant cytoplasm, which were observed to play a role in plant susceptibility to Hpa and partially to the necrotroph Botrytis cinerea, but not to the hemibiotrophs Pst and Phytophthora parasitica. AtNAP1s also interact with HaRxL67, a vacuole-associated Hpa effector. From this, I propose that AtNAP1s are susceptibility factors for Hpa, specifically targeted by Hpa effectors HaRxL67 and HaRxL79 in order to promote susceptibility and maintain biotrophy.
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