Abstract
Kauri trees have become a threatened species, due to kauri dieback disease, which is caused by the oomycete pathogen Phytophthora agathidicida. Infection of kauri trees is initiated through motile zoospores, with chemotaxis occurring through G protein-coupled receptors (GPCRs). Phytophthora species have novel GPCR chimeric proteins which consist of the seven transmembrane helices of a GPCR fused with a catalytic C-terminal domain. These “GPCR-bigrams” have been proposed as potential novel drug targets for the chemical control of pathogens in the Phytophthora genus.
This thesis aimed to characterize GPCR-bigrams from P. agathidicida to better understand their cellular signalling and function, in relation to kauri dieback disease. With the absence of specific ligands for P. agathidicida GPCR-bigrams, this was achieved with chimeric GPCR-bigram fusions, fusing the catalytic domain of P. agathidicida GPCR- bigrams to either a beta2-adrenergic receptor (β2AR) or muscarinic acetylcholine receptor M1 (M1R). This enabled the use of known ligands to activate the GPCR to investigate whether the bigram was still capable of directly activating G protein signalling, and whether agonist stimulation of the GPCR domain drove C-terminal domain activity.
While G protein signalling was observed in β2AR-bigram fusions, indicating that GPCR-bigrams can signal through the G protein machinery of the receptor; this was not observed for the M1R-bigram fusion. The absence of signalling from the M1R-bigram fusion protein may be the result of poor transfection efficiency. Agonist stimulation of the GPCR domains in β2AR-bigram and M1R-bigram fusions did not appear to drive C-terminal domain activity. A negative result in this study is challenging to interpret, as it may suggest a lack of direct activation of the C-terminal protein, or could be due to the designed biosensors or experimental protocols used. Overall, this study highlighted many of the challenges in GPCR deorphanization, but has begun to lead to a better functional understanding of GPCR-bigrams from P. agathidicida and their signalling outputs.