Abstract
BackgroundAlamethicin is a membrane-active peptide isolated from the beneficial root-colonising fungus Trichoderma viride. This peptide can insert into membranes to form voltage-dependent pores. We have previously shown that alamethicin efficiently permeabilises the plasma membrane, mitochondria and plastids of cultured plant cells. In the present investigation, tobacco cells (Nicotiana tabacum L. cv Bright Yellow-2) were pre-treated with elicitors of defence responses to study whether this would affect permeabilisation.ResultsOxygen consumption experiments showed that added cellulase, already upon a limited cell wall digestion, induced a cellular resistance to alamethicin permeabilisation. This effect could not be elicited by xylanase or bacterial elicitors such as flg22 or elf18. The induction of alamethicin resistance was independent of novel protein synthesis. Also, the permeabilisation was unaffected by the membrane-depolarising agent FCCP. As judged by lipid analyses, isolated plasma membranes from cellulase-pretreated tobacco cells contained less negatively charged phospholipids (PS and PI), yet higher ratios of membrane lipid fatty acid to sterol and to protein, as compared to control membranes.ConclusionWe suggest that altered membrane lipid composition as induced by cellulase activity may render the cells resistant to alamethicin. This induced resistance could reflect a natural process where the plant cells alter their sensitivity to membrane pore-forming agents secreted by Trichoderma spp. to attack other microorganisms, and thus adding to the beneficial effect that Trichoderma has for plant root growth. Furthermore, our data extends previous reports on artificial membranes on the importance of lipid packing and charge for alamethicin permeabilisation to in vivo conditions.
Highlights
Alamethicin is a membrane-active peptide isolated from the beneficial root-colonising fungus Trichoderma viride
When the cells were pre-exposed to cell wall degrading enzymes for 4 h they retained 60% of the respiration after alamethicin addition compared to approximately 20% for cells incubated in Control medium (0.35 M mannitol, pH 5.0)
The concentrations of cellulase and macerozyme in the CM mixture (1% and 0.1%, respectively) are the ones commonly used in the isolation of protoplasts, but higher temperatures than used here are needed for a removal of the cell wall to occur within 4 h
Summary
Alamethicin is a membrane-active peptide isolated from the beneficial root-colonising fungus Trichoderma viride. This peptide can insert into membranes to form voltage-dependent pores. Besides being elicitors to defence responses, the channel-forming peptaibols secreted by Trichoderma kill pathogenic fungi and bacteria around the root [18,19]. Pores will only form through membranes that have a transmembrane potential, and only when the alamethicin is applied from the net positive compartment [21,25] Such a polarity of permeabilisation has been shown in vivo in tobacco cells, where the plasma membrane (negative transmembrane potential) but not the tonoplast (positive transmembrane potential) was permeabilised by alamethicin added to cells [26]. Varying the size of the headgroups in artificial phospholipid bilayers affected the concentration of alamethicin needed for permeabilisation [31]
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