MLG oligosaccharides from a second company can induce immune responses in barley

To verify the ability of MLGs to induce immune responses in barley and Arabidopsis, one MLG tetramer (G4G3G4G) as well as two MLG trimers (G3G4G and G4G3G) were purchased from a second supplier for carbohydrates, Carbosynth (Compton, UK) and tested for their ability to induce immune responses. The two MLG trimers as well as the tetramer G4G4G3G are structurally the same as the MLGs obtained from Megazyme. However, Carbosynth offered the MLG tetramer G4G3G4G

as a single substance while it was only available as a mixture (G4G3G4G + G3G4G4G) at Megazyme.

Again, the generation of ROS as well as the activation of MAPK upon MLG treatment were analysed in barley. The MLG tetramer G3G4G4G but not the MLG trimers G3G4G and G4G3G did induce the generation of ROS (Figure 13 A). Furthermore, the activation of MAPK was clearly induced upon treatment with the tetramer G4G3G4G and comparable to the intensity of MAPK phosphorylation upon flg22. Also, the MLG trimers G3G4G and G4G3G did induce the MAPK activation in barley, however, the level of MAPK activation was lower in comparison to the induction upon MLG tetramer treatment (Figure 13 B).

Figure 13. ROS burst generation and activation of MAPK in barley by MLG oligomers from Carbosynth.

(A) Generation of ROS in barley upon treatment with a MLG tetramer and two MLG trimers. Leaf discs of second leaves of 10-day old barley plants were treated with water, 100 µg ml^-1 chitin, 100 nM flg22 or 100 µg ml^-1 MLG oligomers. Data show mean of eight leaf discs and error bars represent SEM. The experiment was performed three times with similar results. (B) MAPK activation upon MLG treatment. Leaf discs of second leaves of barley plants were treated for 12 min with water, 10 µg ml^-1 chitin, 50 nM flg22 or 10 µg ml^-1 MLG oligomers. Activation of MAPK was analysed via Western Blot using the p44/42-antibody. Lower panel shows Coomassie Brilliant Blue (CBB) staining as loading control. The experiment was repeated two times with similar results.

To determine if the three MLGs from Carbosynth are able to induce immune responses in Arabidopsis, Arabidopsis Col-0 seedlings expressing the Ca²⁺ sensor protein aequorin were used to monitor the calcium response. The tetramer G4G3G4G as well as the two trimers G3G4G and G4G3G induced the influx of Ca²⁺ ions. The Ca²⁺ peak induced by the MLG tetramer was stronger in comparison to the Ca²⁺ influx induced by the two MLG trimers. Also, the Ca²⁺ influx upon MLG treatment was faster than upon flg22 treatment (Figure 14 A). Next, a luminol-based assay was performed to determine whether MLGs induce the generation of ROS. The ROS burst generation was clearly observed upon treatment with chitin and flg22, however, could not be observed upon treatment with MLG oligomers in Col-0 (Figure 14 B). ROS generation was also not observed in Ws-0 and Ws-4 upon MLG elicitation (Figure S17 A and B). Western blot analysis using the p44/42-antibody revealed that MAPK6 and MAPK3 were phosphorylated upon treatment with the MLG tetramer G4G3G4G and very slightly upon treatment with the MLG trimers G3G4G and G4G3G in Col-0, Ws-0 and Ws-4 (Figure 14 C, Figure S17 C).

Figure 14. Activation of immune responses in Arabidopsis Col-0 upon treatment with MLG oligomers from Carbosynth. (A) Influx of Ca²⁺ upon MLG oligomer treatment. 8-10-day old Arabidopsis Col-0 seedlings expressing the Ca²⁺ sensor protein aequorin were treated with water, 100 µg ml^-1 chitin, 100 nM flg22 or 100 µg ml^-1 MLG oligomers. Elevation in Ca²⁺ was measured in 6 sec intervals for 1200 sec. Upon treatment, the total remaining luminescence (Lmax) was obtained by adding CaCl2 to the wells and luminescence was recorded for 3 min in 6 sec intervals. For normalization, luminescence upon elicitor treatment per 6 sec (L) was divided by Lmax.Data shown represent mean of 12 seedlings with SEM. The experiment was repeated three times with similar results. (B) ROS burst generation upon MLG treatment. Leaf discs of 5-7 week old Arabidopsis Col-0 plants were treated with water, 100 µg ml^-1 chitin, 100 nM flg22 or 100 µg ml^-1 MLG oligomers and relative light units (RLU) were recorded every minute for 60 min. The shown data represent mean of 8 leaf discs with SEM.

The experiment was repeated two times with similar results. (C) Activation of MAPK upon MLG treatment. In-vitro grown 14-day old Col-0 seedlings were treated with water, 10 µg ml^-1 chitin, 50 nM flg22 or 10 µg ml^-1 MLG oligomersfor 12 min. Activation of MAPK6, MAPK3 and MAPK4 was analysed via Western Blot using the p-44/42 antibody. The lower panel shows Coomassie Brilliant Blue (CBB) staining as loading control. The experiment was repeated three times with similar results.

Additionally, it was tested whether the three MLGs from Carbosynth are able to induce defence gene expression. Thus, the expression of WRKY33 and WRKY53 upon MLG elicitation was analysed via qRT-PCR. The tetramer G4G3G4G induced the transcriptional upregulation of WRKY33 and WRKY53 10-fold and 12-fold, respectively, compared to water (Figure 15). The trimer G3G4G did significantly induce the transcription of WRKY53 (Figure 15). As observed before, the induction of defence gene expression was more pronounced upon treatment with the MLG tetramer than with the MLG trimers.

All ordered MLGs from the second company Carbosynth did induce immune responses in barley as well as in Arabidopsis indicating that MLGs display elicitor activities.

Figure 15. Expression of defence genes upon treatment with MLG oligomers from Carbosynth. In-vitro grown 14-day old Arabidopsis Col-0 seedlings were treated with water, 10 µg ml^-1 chitin, 50 nM flg22 or 10 µg ml^-1 MLG oligomers for 30 min. Expression of the genes WRKY33 (A) and WRKY53 (B) were tested using qRT-PCR. UBIQUITIN5 served as reference gene. The bars show the mean of three biological replicates consisting of three technical replicates each and error bar represents standard deviation. Statistical significance of the elicitor treatments compared to water treatment is indicated by 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.