Trichoderma viride cellulase induces resistance to the antibiotic pore-forming peptide alamethicin associated with changes in the plasma membrane lipid composition of tobacco BY-2 cells

Abstract

Background: 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. 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.

Results: Oxygen 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.

Conclusion: We 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.

Figure 1

The effect of alamethicin on oxygen consumption of tobacco cells pretreated with cellulase and macerozyme (CM). (A) Respiration in Control cells (upper trace) and cells treated for 4 h with CM (lower trace). Alam, addition of alamethicin. (B) Alamethicin resistance after different incubation times in Control medium and CM, respectively. Resistance was measured as per cent of respiration rate remaining after 10 min incubation with 20 µg ml^-1 alamethicin compared to the initial rate. Squares are control samples, open circles are CM-treated samples, and filled circles are samples treated with boiled CM. Values represent the mean of three biological replicates and the error bars denote SE.

Figure 2

Propidium iodide staining of alamethicin-treated tobacco cells. Bright field (A) and (C) and fluorescent (B) and (D) images are shown for cells after incubation with 20 µg ml^-1 alamethicin for 10 min. Before addition of alamethicin, cells were pretreated with either Control medium for 4 h (A, B) or CM medium for 20 min followed by 220 min with Control medium (C, D). The bar is valid for all images.

Figure 3

Remaining respiration in control and CM-treated tobacco cells after adding different concentrations of alamethicin. Open circles, control cells; filled circles CM-treated cells. Resistance was measured as per cent of respiration rate remaining after 10 min incubation with 20 µg ml^-1 alamethicin compared to the initial rate. Each data point represents the mean of four biological replicates and the error bars represent SE. Significant differences (Student's t-test) between CM cells and control are denoted with * for p < 0.05 and *** for p < 0.001.

Figure 4

Effect of inhibition of cellulase activity on the induction of alamethicin resistance of tobacco cells. Resistance was measured as per cent of respiration rate remaining after 10 min incubation with 20 µg ml^-1 alamethicin compared to the initial rate. Samples were pre-incubated with combinations of 1% cellulase, 0.1% macerozyme, 0.1 M glucose, and 0.1 M cellobiose in 0.35 M mannitol for 20 min followed by 220 min with control medium only. Where glucose or cellobiose was included, the concentration of mannitol in the control medium was reduced to give a similar molarity. M, control cells, CM, CM-treated cells, G, glucose, C, cellobiose. Data shown are averages of two biological replicates and error bars represent SD. Student's t-test was performed relative to the CM sample with * denoting p< 0.05 and *** denoting p < 0.001.

Figure 5

Resistance to alamethicin after preincubation of tobacco cells with known plant defence elicitors. Resistance was measured as per cent of respiration rate remaining after 10 min incubation with 20 µg ml^-1 alamethicin compared to the initial rate. Data points are averages of three to five measurements and error bars represents SE.

Figure 6

The effect of the uncoupler FCCP (A) and protein synthesis inhibitor cycloheximide (B) on the CM-induced alamethicin resistance of tobacco cells. Resistance was measured as per cent of respiration rate remaining after 10 min incubation with 20 µg ml^-1 alamethicin compared to the initial rate. Average of two independent experiments are shown with error bars representing SD. FCCP was added just before alamethicin addition, whereas cycloheximide was added before CM treatment (as described in Methods). The respiration increased 1.6 ± 0.1 and 1.7 ± 0.4 times in control and CM-treated cells, respectively, by the addition of FCCP, showing that respiration in the cell cultures became equally uncoupled from ATP synthesis.

Figure 7

Protein, fatty acid and sterol ratios in plasma membranes isolated from control and CM-treated cells. Dark grey bars, control cells; light grey bars, CM-treated cells. Values used are averages of two plasma membrane preparations and error bars denote SD.

Figure 8

Phospholipid analysis of tobacco cell plasma membranes. (A) Percents of different phospholipids of plasma membranes from CM-treated cells relative to control cells. The CM/Control ratio for PS+PI was significantly different from that for PE+PG (p < 0.05). (B) Fatty acid composition of plasma membranes isolated from control and CM-treated cells. Dark grey bars, control cells; light grey bars, CM-treated cells. Values used are averages of two plasma membrane preparations and error bars denote SD.