Trichoderma viride alamethicin and plant cells - Action, response and resistance

Sammanfattning: Alamethicin is a membrane active peptide secreted from the soil-living fungus Trichoderma viride. When coexisting with T. Viride, plant roots are exposed to alamethicin, which has the ability to elicit formation of volatile compounds involved in pathogen defence. Alamethicin can make pores across membranes with a negative membrane potential, such as the plant plasma membrane. The alamethicin pores allow passage of metabolites and ions and alamethicin can thus be used as a molecular tool for in situ enzyme measurements. Its main advantage is the small size of the pores created as this keeps cytosolic proteins confined to the cell interior and gives minimal effect on membranes. In addition alamethicin will not permeabilise the tonoplast, which would otherwise dilute and change the composition of the cytosol. In this thesis alamethicin was used for in situ investigations on Nicotiana tabacum BY-2 and Arabidopsis thaliana cell suspension cells. I have successfully used alamethicin to study the regulation of the membrane protein callose synthase and its interactions with the fragile cytoskeleton. Callose is regulated by the presence of microtubules, divalent ions and ethanol. I could also induce resistance against the pore-forming properties of alamethicin in BY-2 cells by incubating cells with the cell wall degrading enzyme cellulase. The amount of phyosphatidylserine in the plasma membrane was reduced in resistant cells, accompanied by an increased amount of fatty acids compared to proteins as well as a reduction of the sterol to fatty acid ratio. In addition, visualising the plasma membrane using the styryl dye FM4-64 showed a change from an evenly stained plasma membrane to a patchy pattern. The changes may help to explain the cause of alamethicin resistance in cellulase treated cells. The molecular conditions that affects alamethicin membrane association and pore formation, in plasma membranes in vivo, remains to be characterised. Also, the cellulase-induced signalling leading to alamethicin resistance needs to be investigated. With this knowledge the possible advantages for the plant of being able to develop a fungus-induced resistance to a membrane active peptide such as alamethicin could also be clarified.