MiAMP1 is a novel antimicrobial peptide, isolated from Macadamia integrifolia nut kernels. This peptide exerts broad spectrum antimicrobial actvity against a range of phytopathogens in vitro. This property provides MiAMPl with the potential to confer disease resistance in transgenic plants. To examine the effect of MiAMPl in planta, transgenic tobacco lines constitutively expressing native MiAMPl and either of two previously generated MiAMP1 variants, Mi46K/54V and Mi46K/54K, were produced. Four selected high expressing transgenic tobacco lines transformed with each of the two MiAMP1 variant constructs, two transgenic lines expressing native MiAMP1 and four untransformed control lines were inoculated with Cercospora nicotianae and Phytophthora parasitica in order to examine the effect of transgene expression on disease resistance. No significant increase in disease resistance was detected in the transgenic lines against either of the two tobacco pathogens.
In an attempt to increase the antimicrobial activity of MiAMP1, two new variants of MiAMP1 were produced by site-directed muatgenesis. These variants, termed Mi28K/46K/54V and Mil6A/28K/46K/54V, contained three and four amino acid substitutions, respectively. The amino acid substitutions were selected to increase net positive charge and hydrophobicity of the peptides. The antimicrobial activities of the two new MiAMP1 variants were tested by in vitro bioassays against several phytopathogens, including C. nicotianae, P parasitica and Verticillium dahliae. No significant difference in antimicrobial activity was found between either of the two MiAMP1 variants and native MiAMP1 for any of the tested pathogens. However, cation tolerance of the two variants appeared to be improved as both variants were significantly more active than native MiAMP1 in inhibiting the growth of C. nicotianae in the presence of elevated Ca2+ concentration after 48 hours incubation.
Mode of action studies of MiAMP1 were undertaken to gain insight into the underlying mechanism(s) behind the inhibitory activity of MiAMP1, and facilitate the design of more potent MiAMP1 variants for future studies. Yeast genes putatively involved in MiAMP1-yeast interaction were identified using a complementation approach, whereby DNA libraries made from MiAMP1-sensitive strains were screened for clones that restored MiAMP1-susceptibility when overexpressed in a mutant strain with increased MiAMP1-resistance. Five clones corresponding to five different genes were identified. These genes SKS1, YHR076W, YPL225W, RDN25 and RDN18 encode a Ser/Thr protein kinase, a Ser/Thr protein phosphatase, an unknown protein, and fragments of 25S rRNA and 18S rRNA, respectively. Overexpression of SKS1 and YHR076W were confirmed to be specific to the mutant genotype as no increase in MiAMP1 susceptibility was observed when either of the two clones were overexpressed in wild-type yeast. However, it is currently not clear how SKS1 and YHR076W contribute to MiAMP1 susceptibility in yeast. It is possible that the corresponding gene products are potentially involved in signal transduction pathways activated by MiAMP1.
Comparative studies of yeast mutants were performed to examine potential similarities between the mode of action of MiAMP1 and that of Williopsis mrakii yeast killer toxin, WmKT. No difference in MiAMP1-susceptibility was detected between wild-type and WmKT-resistant mutant yeast strain, demonstrating that MiAMP1 and WmKT inhibits yeast growth via different mechanisms, despite structural similarity between the two peptides. In accordance, no difference in WmKT-tolerance was detected between wildtype and MiAMP1-resistant mutant strains.
The effect of MiAMP1 on membrane permeabilisation was examined by analysing SYTOX Green influx. Yeast cells incubated with SYTOX Green and MiAMP1 (at a concentration confirmed to have a fungistatic effect) showed strong fluorescence in the cytosol, indicative of intracellular translocation of SYTOX Green and subsequent SYTOX Green-nucleic acid binding. Yeast cells incubated with SYTOX Green only, did not fluoresce. This result confirmed that MiAMP1 facilitates intracellular SYTOX Green uptake by increasing membrane permeabilisation.
Furthermore, the ability to bind to yeast cells was examined for MiAMP1. Immunological detection of MiAMP1 revealed high affinity binding of MiAMP1 to the yeast cell at inhibitory concentrations. This binding could not be prevented or reversed by monovalent or divalent cations, at concentrations shown to affect MiAMP1 activity, indicating that the binding is unlikely to be based on purely electrostatic interactions.
In conclusion, this PhD study has resulted in the generation of two MiAMP1 variants with reduced cation sensitivity. These may be useful for engineering of disease resistance in crop plants. In addition, identification and preliminary characterisation of yeast genes involved in MiAMP1 activity could open up future research areas that might eventually lead to the design of new and more effective agrochemicals.