Final Report Summary - POETIC (Functional analysis of plant-pathogenic oomycete effectors and their targets inside plant cell) Although filamentous phytopathogens such as fungal rusts and powdery mildews and oomycete downy mildews and white rusts are more damaging to agriculture than bacteria, their effector functions are more poorly understood. To understand the mode of action of the secreted molecules during pathogen attack, I developed an in vivo cell biology approach to analyse both sides of the compatible interaction between the downy mildew oomycete Hpa and its host Arabidopsis. We first analysed the plant side of the interaction, by monitoring at a subcellular level the restructuring of the plant mesophyll cell during Hpa haustoria ontogenesis. We then analysed the pathogen side of the interaction by screened for the subcellular localisation of over 50 Hpa RxLR effector candidates in the plant cell (Caillaud et al., TPJ 2012). This screening led to the selection of Hpa effectors that localise to the host nucleus or vesicle trafficking machinery. By generating transgenic lines expressing HaRxLs candidates, I then selected Hpa effectors that confer alterations in PTI response for further investigation (Fabro et al., PLoS Patho 2011; Caillaud et al., TPJ 2012). I then analysed the subcellular localisation of three Hpa effectors (HaRxL17, HaRxL77 and HaRxL495) during oomycete infection. This work led to the identification of tonoplast-localised effector HaRxL17 that increases plant susceptibility to Hpa during both compatible and incompatible interactions and localises at the membrane around oomycete haustoria during infection (Caillaud et al., TPJ 2012; Caillaud et al., PSB 2012). In addition, we found that 66 % of the HaRxLs accumulate in the plant cell nucleus and identified 14 effectors (including ATR13 and HaRxL44) that strictly localise to the plant cell nucleus. We then generated transgenic lines that express tagged versions of Hpa nuclear effectors (35S::GFP-HaRxLs). Using this approach, nuclear-HaRxLs, including ATR13 and HaRxL44 were shown to suppress plant defence and increase plant susceptibility to pathogens. Independent transgenic lines expressing some nuclear-localised HaRxLs show developmental phenotypes, suggesting that nuclear effectors might interfere with signalling networks involved in diverse plant processes (Caillaud et al., submitted). Thus, detailed functional analysis of nuclear-HaRxLs and the identification of their plant targets may reveal new aspects of fundamental plant mechanisms, and unknown processes involved in host defence and essential for Hpa virulence. For follow-up studies, I selected HaRxL44 because: (i) it is expressed during Hpa infection in Arabidopsis; (ii) it is present in all Hpa races; (iii) it enhances Pst virulence in at least 6/12 Arabidopsis ecotypes when delivered via the EDV system; (iv) it enhances plant susceptibility to both Hpa and Pst when stably expressed in planta; (v) it suppresses PTI when delivered via EDV or stably expressed in planta; (vi) it localised to the plant cell nucleus; (vii) interacts with regulators of the transcriptional machinery. Arabidopsis transgenic lines expressing the nuclear Hpa effector HaRxL44 show growth inhibition and a curvy leaf that resembles HYL1, Serrate mutants. In yeast two hybrid and BiFC, HaRxL44 interacts with AtMed19a. AtMed19a is a component of Mediator, a conserved multi-subunit complex that acts as a molecular bridge between transcriptional regulators at promoter enhancer sequences, and the activation of transcription by ribonucleic acid (RNA) polymerase II at the transcription start site (Backstrom et al., 2007; Conaway and Conaway, 2011). In Arabidopsis, Mediator likely plays a role in plant defence because Med16, Med21, Med25 are important for plant resistance against hemibiotroph or necrotroph pathogens (Dhawan et al., 2009; Kidd et al., 2009; Wathugala et al., 2012). However, the role of AtMed19a in plant immunity remains unknown. Transfer deoxyribonucleic acid (T-DNA) insertion mutants and over-expression lines of AtMed19a show altered susceptibility to Hpa, demonstrating that AtMed19a contributes to Hpa resistance. In addition, we found that Arabidopsis transgenic lines expressing the pathogen effector HaRxL44 are more susceptible to Hpa. Expression profiling of 35S::HaRXL44 Arabidopsis lines in comparison with wild type shows a positive correlation between messenger RNA (Mrna) profiles of Arabidopsis genes after Jasmonic acid (JA) treatment with genes differentially expressed in 35S::HaRXL44 Arabidopsis lines. We hypothesise HaRxL44 effector activates jasmonate (JA) signalling at a transcriptional level via its interaction with the Mediator complex, in order to promote Hpa virulence. Perspective; how might knowledge of effector mechanisms enable elevated disease resistance in crops? Optimising disease resistance in crops can in part be achieved by breeding in classical R genes. However, pathogen evolution can overcome such resistances. Alternatively, one can aim to elevate basal resistance. If an effector is well defined, and its host target is known, we can define the physical basis of such interactions and use this knowledge to generate alleles of the effector target that still carry out their host function, but are refractory to the action of the effector. We aim to do this for HaRxL44 targets. Since this approach is novel, it is reasonable to test it in Arabidopsis since many more 'effector-refractory' constructs can be assayed in whole plants than is feasible in a crop.
