Investigation of Apoplastic Effectors from Arabidopsis Downy Mildew Pathogen Hyaloperonospora arabidopsidis

Downy mildew is a plant disease, caused by a group of obligate oomycete pathogens, which results in significant economic loss by affecting plants such as, tomato, potato, grapevine, lettuce, onion and brassicas. The downy mildew pathogens secrete molecules called effectors, which modulate plant innate immunity and enable parasitic colonisation. Despite the increasing attention, the knowledge of these effectors is highly limited. Especially, the apoplastic effectors are understudied with respect to cytoplasmic effectors. Therefore, in this study, we focused on identifying novel apoplastic effectors, taking advantage of an established model pathosystem of the Arabidopsis thaliana and its adapted downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). With this purpose, two different approaches were followed: Firstly, five candidate genes were determined from the Expressed Sequence Tag library of Hpa-Emoy2 isolate using bioinformatic tools. The candidates were Hpa 804480, Hpa 806249, Hpa 814231, Hpa 814014 and Hpa 813915, all of which contained signal peptides and lacked known motifs of cytoplasmic effector and intracellular functions. The candidate genes were validated for their expression by challenging the susceptible Ws-eds1 plants with the pathogen. It was observed that the candidates were expressed at varying times and levels during the infection suggesting that some encoded proteins by the candidate genes can be put forward by the pathogen right away or as a second layer of attack to suppress the PTI later during the infection. Additional assays also showed that the candidate genes were not triggering hypersensitive response in Nicotiana plants. Moreover, the candidate genes were examined for their variations across other Hpa isolates, and the results suggested they are under less pressure to evolve with respect to cytoplasmic effectors; thus it is possible that they may be avoiding recognition. Secondly, the intercellular washing fluid (IWF) of the Hpa infected Arabidopsis plants were scrutinized for the capability of triggering defence responses in wild type and receptor-impaired plants. GUS reporter assays and cumulative quantification of Reactive Oxygen Species assays suggested that the infected IWF, indeed, was inducing immune activation on both wild-type and mutant plants. The complex IWF was then simplified to identify the active fraction. One fraction, the flow-through, was found to be responsible for triggering defence responses with the same assays. In addition, the IWF sample was analysed via MALDI-TOF mass spectrometry, and results showed that there were cysteine-rich proteins from Hpa with putative apoplastic effector characteristics. Two genes from the cysteine-rich proteins, Hpa 806256 and Hpa 813024 were tried in in vitro expression assays. Additionally, MALDI-TOF revealed A. thaliana that were annotated as LRR family proteins with signal peptides; which carried high potential of recognizing apoplastic effectors. T-DNA insertional A. thaliana mutant lines for those LRR protein encoding genes, AT1G33610.1, AT3G20820.1 and AT1G49750.1 were use in assessment of interaction phenotypes and recognition of the molecules within the active fraction. The assays suggested that these candidate receptors may not be the corresponding receptors, or they may need co-receptors to function properly. Overall, we believe, the findings of this research will indeed contribute to future studies on apoplastic effectors and their recognition.