3.2 Identification and analysis of novel cell-wall derived DAMPs
3.2.6 Hydrolysis products of the barley β-1,3;1,4-glucan polymer induce defence responses in
The commercially available MLG tetramers and MLG trimers are derived from β-1,3;1,4-glucan polymers that were hydrolysed with enzymes that cleave β-1,3;1,4-glucan polymers (Megazyme).
Enzymes that act on β-1,3;1,4-glucan polymers exhibit different specificities regarding the position of the cleaved linkage. An endo-β-1,4-glucanase from Aspergillus japonicus hydrolyses β-1,4-linkages preceding a β-1,3-linkage resulting in MLG oligosaccharides with the structure G3G4nG with n describing the number of glucose units (Grishutin et al., 2006). However, the commercially available Bacillus subtilis endo-1,3:1,4-β-D-glucanase (lichenase) cleaves β-1,4-linkages adjacent to β-1,3-linkages and consequently, generates MLG oligosaccharides with the general structure
G4nG3G (Figure 18) (Planas, 2000). Full lichenase hydrolysis of β-1,3;1,4-polymers of grasses and cereals with the B. subtilis lichenase result mainly in the generation of the MLG tetramer G4G4G3G and the trimer G4G3G. However, intermediate products can be achieved upon a partial digest. These intermediate products can e.g. be a hexasaccharide with the structure G4G3G4G4G3G that eventually would by cleaved into two trimers (Figure 18) (Fry et al., 2008). Thus, the B. subtilis lichenase can be used to generate MLG oligosaccharides of varying length but the general structure G4nG3G.
Figure 18. Schematic representation of a β-1,3;1,4-glucan polymer and the obtained oligosaccharides upon treatment with B. subtilis lichenase. Abbreviations: G – glucose, n –number of residues linked together.
To further verify the ability of MLG oligosaccharides to elicit immune responses, MLG oligosaccharides of varying length were generated using the barley β-1,3;1,4-glucan polymer and the B. subtilis lichenase. In a next step, the obtained MLG oligosaccharides were analysed regarding their capacity to induce PTI responses including the calcium response, activation of MAPK and induction of defence gene expression.
First, optimal conditions for barley β-1,3;1,4-glucan polymer hydrolysis were identified by using different buffers with varying pHs and different enzyme concentrations. Samples were taken upon different incubation times and consequently, the lichenase was inactivated by incubating the hydrolysate for 15 min in boiling water. The hydrolysis was checked via Thin Layer Chromatography (TLC). The goal was to obtain hydrolysates in which the MLG oligosaccharide concentration increases over time. Optimal conditions were achieved by hydrolyzing the barley β-1,3;1,4-glucan polymer in 100 mM Sodium phosphate buffer (pH = 6.5) at 40°C with either 0.025 U ml-1 or 0.05 U ml-1 lichenase and taking samples upon incubation for 0, 5, 15, 30, 45, 60, 120 and 240 min (Figure S18). No MLG oligosaccharides can be found in the sample taken upon 0 min incubation time while the amount of MLG oligosaccharides increases over time. The MLG oligosaccharides obtained upon 45 min or longer incubation times co-migrated with the standards for G4G4G3G and G4G3G indicating that the tetramer as well as the trimer are abundant in the respective hydrolysates.
Additionally, MLG oligosaccharides were obtained that migrated more slowly than the MLG tetramer and the MLG trimer suggesting that these oligosaccharides are longer than the trimer and tetramer.
However, the exact length cannot be determined from the TLC since MLG standards with a length of
six or more glucose monomers are not commercially available. The β-1,3;1,4-glucan polymer can still be found in the hydrolysates, however, the hydrolysates were not further processed to obtain a polysaccharide-free MLG oligosaccharide mixture (Figure S18).
Again, to determine whether the enzymatically generated MLG oligosaccharides have the ability to induce the influx of Ca2+ ions, the calcium responses was monitored in Arabidopsis Col-0 seedlings expressing the Ca2+ sensor protein aequorin. Upon treatment with MLG oligosaccharides that were obtained upon incubation of the β-1,3;1,4-glucan polymer with either 0.05 U ml-1 or 0.025 U ml-1 active lichenase for 30, 60 or 240 min, a rapid increase in Ca2+ was observed. Comparing the intensities of the induced Ca2+ influx upon treatment with MLG oligosaccharides upon the three different incubation times, the MLG oligosaccharides obtained upon 60 min incubation induced the lowest Ca2+ influx while the strongest Ca2+ peak was observed with MLG oligosaccharides obtained upon incubation for 240 min. This suggests a positive correlation between the MLG oligosaccharide concentration and intensity of Ca2+ influx (Figure 19 A and B). In comparison to the Ca2+ peak upon chitin treatment, the Ca2+ spikes resulting from MLG oligosaccharide treatment occurred faster. No Ca2+ influx was induced upon control treatments with either Sodium phosphate buffer, the untreated β-1,3;1,4-glucan polymer or the β-1,3;1,4-glucan polymer incubated with heat-inactivated lichenase (0.05 U ml-1 or 0.025 U ml-1) indicating that only MLG oligosaccharides that were enzymatically produced from a MLG polymer can induce a Ca2+ influx (Figure 19 A and B).
Figure 19. Calcium influx in Arabidopsis Col-0 upon treatment with MLG oligosaccharides. To obtain MLG oligosaccharides of varying length, 10 mg ml-1 barley β-1,3;1,4-glucan polymer dissolved in 100 mM Sodium Phosphate buffer (pH = 6.5) was hydrolysed with 0.05 (A) or 0.025 (B) U ml-1 lichenase and the reaction was stopped upon 0, 5, 15, 30, 45, 60, 120 or 240 min incubation. 8-10 day old Arabidopsis Col-0 seedlings expressing the Ca2+ sensor protein aequorin were treated with 10 µg ml-1 chitin, 10 mM Sodium Phosphate buffer, 1 mg ml-1 β-1,3;1,4-glucan, 0.05 or 0.025 U ml-1 heat inactivated lichenase, 1 mg ml-1 β-1,3;1,4-glucan plus either 0.05 or 0.025 U ml-1 heat-treated lichenase or a 1:10 dilution of MLG oligosaccharides (β-1,3;1,4-glucan + 0.05 or 0.025 U ml-1 active lichenase incubated for 0, 30, 60 or 240 min).
The Ca2+ elevation upon treatment (L) was recorded in 6 sec intervals for 1200 sec. To obtain the total remaining luminescence (Lmax), CaCl2 was added to the wells and luminescence was recorded for 3 min in 6 sec intervals.
For normalization, luminescence counts per 6 sec upon treatment (L) were divided by Lmax. Data shown represent mean of 12 seedlings with SEM. The experiment was performed twice with similar results.
Furthermore, the MAPK activation upon MLG oligosaccharide elicitation was tested via immunoblot analysis using the p44/42-antibody. Treatment with active lichenase did not induce MAPK activation in Arabidopsis Col-0 suggesting that Col-0 does not contain MLGs itself which could be cleaved upon pathogen attack and consequently, lead to the activation of immune responses. The activity of the lichenase was verified by testing the hydrolysis of barley β-1,3;1,4-glucan polymer in ½ MS plus sucrose medium and the hydrolysis was analysed via TLC (Figure S19). Additionally, the control treatments as well as the β-1,3;1,4-glucan polymer incubated with either 0.05 U ml-1 or 0.025 U ml-1
lichenase for 0 min did not induce MAPK activation. MAPK6 and MAPK3 phosphorylation was only induced upon treatment with MLG oligosaccharides obtained from β-1,3;1,4-glucan polymer incubated with either 0.05 U ml-1 or 0.025 U ml-1 lichenase for at least 15 min (Figure 20). The intensity of the MAPK activation increased over time, thus correlating with increasing amounts of MLG oligosaccharides. This result demonstrates that oligomeric MLGs act as elicitor of immune responses (Figure 20).
Figure 20. Activation of MAPK in Arabidopsis Col-0 upon treatment with MLG oligosaccharides. To obtain MLG oligosaccharides of varying length, 10 mg ml-1 barley β-1,3;1,4-glucan polymer dissolved in 100 mM Sodium Phosphate buffer was hydrolysed with 0.05 or 0.025 U ml-1 lichenase and the reaction was stopped upon 0, 5, 15, 30, 45, 60, 120 or 240 min incubation. In-vitro grown 14-day old Arabidopsis Col-0 seedlings were treated for 12 min with 10 µg mL-1 chitin, 50 nM flg22, 10 mM Sodium Phosphate buffer, 1 mg ml-1 β-1,3;1,4-glucan, 0.005 or 0.0025 U ml-1 heat inactivated lichenase, 1 mg ml-1 β-1,3;1,4-glucan with 0.0025 or 0.005 U ml-1 heat-treated lichenase or a 1:10 dilution of MLG oligosaccharides (β-1,3;1,4-glucan + active lichenase upon different incubation times). Activation of MAPK6, MAPK3 and MAPK4 was analysed via Western Blot with the p44/42-antibody. Lower panel shows Coomassie Brilliant Blue (CBB) staining as loading control.
The experiment was performed two times with similar results.
To further investigate whether the enzymatically generated MLG oligosaccharides trigger immune responses in Arabidopsis, the expression of the defence genes WRKY33 and WRKY53 was tested via qRT-PCR. The expression of the defence gene WRKY33 is significantly induced upon treatment with MLG oligosaccharides upon hydrolysis of β-1,3;1,4-glucan polymer for all different incubation times (Figure 21 A and C). Similarly, WRKY53 expression was induced significantly upon treatment with MLG oligosaccharides obtained from β-1,3;1,4-glucan polymer incubated with 0.05 U ml-1 lichenase for 15, 30, 120 and 240 min or MLG oligosaccharides obtained from β-1,3;1,4-glucan polymer incubated with 0.025 U ml-1 lichenase for 15, 30, 45, 60 and 240 min (Figure 21 B and D). As the expression of the two tested defence genes was only significantly induced upon treatment with MLG oligosaccharides but not upon control treatments including Sodium phosphate buffer, this result
confirms that oligomeric MLGs are perceived by Arabidopsis which subsequently leads to the initiation of defence responses.
Figure 21. Defence gene expression in Arabidopsis Col-0 upon treatment with MLG oligosaccharides. To obtain MLG oligosaccharides of varying length, 10 mg ml-1 barley β-1,3;1,4-glucan polymer dissolved in 100 mM Sodium Phosphate buffer was hydrolysed with 0.05 U ml-1 lichenase (A and B) or 0.025 U ml-1 lichenase (C and D) and the reaction was stopped upon 0, 15, 30, 45, 60, 120 or 240 min incubation. 14-day old in-vitro grown Arabidopsis Col-0 seedlings were treated for 30 min with 10 µg mL-1 chitin, 50 nM flg22, 10 mM Sodium phosphate buffer, 1 mg ml-1 β-1,3;1,4-glucan, 0.005 U ml-1 or 0.025 U ml-1 heat inactivated lichenase, 1 mg ml-1 β-1,3;1,4-glucan with 0.005 U ml-1 or 0.025 U ml-1 heat-treated lichenase or a 1:10 dilution of MLG oligosaccharides (β-1,3;1,4-glucan + active lichenase upon different incubation times). The expression of the defence genes WRKY33 (A and C) and WRKY53 (B and D) was analysed via qRT PCR. UBIQUITIN5 served as reference gene. The bars represent means of two biological replicates with each three technical replicates. Error bars represent STDEV. Statistical significance is indicated with asterisks with not significant (ns) = p > 0.5,
* = p ≤ 0.5, ** = p ≤ 0.001 and *** = p ≤ 0.001. The unpaired student’s t-teat was used to calculate p-values.
3.2.7 Hydrolysis products of the barley β-1,3;1,4-glucan polymer do not inhibit seedling growth
Typically, seedlings show an inhibition of growth when they are exposed to high concentrations of elicitors e.g. flg22 or elf18 (Gómez-Gómez and Boller, 2000; Zipfel et al., 2006). The receptor for elf18 was identified in a reverse genetic screen in which several T-DNA insertion lines were tested for their sensitivity towards elf18 in seedling growth experiments (Zipfel et al., 2006). Similarly, if MLGs negatively influence seedling growth, receptors or co-receptors required for MLG perception could be identified with a forward genetic screen using the seedling growth assay.
To test whether MLG oligosaccharides can inhibit seedling growth, 5-day old in-vitro grown Col-0 seedlings were transferred to ½ MS medium plus sucrose containing either no elicitor, Sodium phosphate buffer, flg22 or MLG oligosaccharides that were generated upon β-1,3;1,4-glucan hydrolysis (Figure S20). The seedlings were grown for eight further days and then, the size of the seedlings as well as the dry weight of the 13-day old seedlings was analysed. A dramatic reduction in growth was only directly observed for seedlings that were grown in the presence of the bacterial MAMP flg22 but not for seedlings grown in medium containing MLG oligosaccharides or Sodium phosphate buffer (Figure 22 A). Furthermore, the dry weight of seedlings growing in medium containing MLG oligosaccharides was not significantly reduced in comparison to seedlings growing in
½ MS plus sucrose medium without elicitor indicating that MLG oligosaccharides do not influence seedling growth (Figure 22 B). Consequently, testing the sensitivity of several mutants or ecotypes to MLGs in a seedling growth assay cannot be used to identify molecular components required for MLG perception.
Figure 22. Effect of MLG oligosaccharides on seedling growth. To obtain MLG oligosaccharides of varying length, 10 mg ml-1 barley β-1,3;1,4-polymer dissolved in 100 mM Sodium phosphate buffer was hydrolysed with 1 U ml-1 lichenase and the reaction was stopped upon 1h. 5-day old in-vitro grown Arabidopsis Col-0 seedlings were transferred to liquid ½ MS plus sucrose medium containing no elicitor, 10 mM Sodium Phosphate buffer (pH = 6.5), 1 µM flg22 or a 1:10 dilution of MLG oligosaccharides and grown for 8 further days. (A) Pictures were taken of 13-day old seedlings. Scale bar represents 1 cm. (B) 13-day old seedlings were dried and the weight of 7 to 8 seedlings per treatment was measured. The weight of one seedling was calculated by dividing the weight of all seedlings of one treatment by the total number of seedlings. Bars represent the average weight of one seedling of two biological replicated consisting of 7 to 8 seedlings each. Error bars represent STDEV. Statistical significance is indicated by asterisk with not significant (ns) = p > 0.5, * = p ≤ 0.5, ** = p ≤ 0.01 and
*** = p ≤ 0.001. To calculate p values, the unpaired student’s t-teat was used.