MAMP-induced generation of reactive oxygen species in CLR1 mutants

Activation of PRRs results in rapid production of reactive oxygen species (ROS) by NADPH oxidases at the cell surface, which is a commonly analysed early MAMP-induced defence response (Torres et al., 2006). The ability of the different clr1 T-DNA mutant lines to generate ROS upon treatment with the fungal elicitor chitin was investigated in a luminol-based assay with L-012 (Figure 7).

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Figure 7. clr1 mutants show reduced ROS-generation upon chitin treatment. Leaf discs of five-week-old Arabidopsis plants were either treated with 100 µg/ml polymeric chitin (A) or mock treated with buffer (B).

Relative luminescence units (RLU) were measured for 35 min after the respective treatment. The data are shown as mean of 12 leaf discs per genotype ± SE. The experiment was repeated three times with similar results.

In addition to the three clr1 alleles, wild type Col-0, cerk1-2 and pbl27-1 were included as controls. Although PBL27 is involved in regulating chitin-induced defence responses the generation of ROS is not impaired in pbl27 mutants (Shinya et al., 2014). As described previously (Miya et al., 2007; Petutschnig et al., 2010) chitin did not induce a ROS burst in cerk1-2 (Figure 7 A). All three clr1 mutants showed reduced chitin-induced ROS generation compared to Col-0 in the three independent experiments, suggesting that CLR1 is required for full activation of ROS generating enzymes (Figure 7 A; Suppl. Figure 1). In pbl27-1 the ROS burst was not decreased in comparison to Col-0, which is in agreement with previous reports (Shinya et al., 2014). pbl27-1 even appeared to have a slightly higher chitin-induced ROS production, but more sensitive methods would be required to investigate the significance of this. None of the tested lines showed induction of ROS burst without elicitor treatment in the mock-treated assay (Figure 7 B).

3.2.4 clr1 T-DNA mutants show reduced expression of MAMP-induced genes after chitin polymer and chitin heptamer treatment

After the very early MAMP-triggered signalling events such as receptor phosphorylation, ROS burst and MAPK activation, defence-related genes are induced. Among many others, the expression of several genes from the WRKY-transcription factor family was shown to be upregulated upon treatment with chitin and other MAMPs (Wan et al., 2004; 2008). To test whether lack of functional CLR1 leads to alterations in chitin-induced expression of defence

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genes, qRT-PCR was performed. For this, the expression of WRKY30, WRKY33 and WRKY53, was monitored in response to different polymeric chitin concentrations in the clr1 mutants, Col-0 and cerk1-2. In all of the tested lines, apart from cerk1-2, a clear dose-dependent induction in expression of the three WRKYs was visible (Figure 8). The expression of WRKY30 and WRKY53 in the clr1-3 and clr1-4 mutants was significantly lower than in Col-0 at all tested chitin concentrations (Figure 8 A and C). clr1-1 showed an intermediate phenotype between Col-0 and the other two clr1 mutant alleles. The expression of WRKY33 was reduced in clr1-3 and clr1-4 only at lower chitin concentrations (Figure 8 B). The obtained results lead to the assumption that CLR1 contributes to chitin-induced expression of defence-related genes and that different genes might be affected to different degrees.

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Figure 8. Chitin-induced expression of WRKY30, WRKY33 and WRKY53 is reduced in the clr1 mutants. Two-week-old in vitro grown seedlings of the depicted clr1 Arabidopsis mutants were treated with the chitin concentrations indicated, ranging from 0 µg/ml to 100 µg/ml, for 30 min. Col-0 and cerk1-2 were included as positive and negative control, respectively. qRT-PCR of the following genes was performed: (A) WRKY30, (B) WRKY33, (C) WRKY53. ACTIN8 served as a reference gene. The bars represent the mean ± STDEV of three biological replicates consisting of 4 technical repetitions each. Asterisks indicate statistical significance of the mutants compared to Col-0 (**** = p ≤ 0.0001, *** = p ≤ 0.001, ** = p ≤0.01, * = p ≤ 0.05, ns = p > 0.05). P-values were calculated using the unpaired student’s t-test.

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Besides polymeric chitin, also fully acetylated chitin oligomers are able to induce CERK1-mediated immune responses in Arabidopsis (Miya et al., 2007; Wan et al., 2008; Petutschnig et al., 2010; Liu et al., 2012). Chitin oligomers with a polymerization degree ≥ 5 are efficiently able to induce CERK1-dependent immune responses as for example CERK1 band shift, ROS burst and MAPK activation (Petutschnig et al., 2010).

To analyse whether chitin oligomers have the same effect on WRKY gene expression as observed for seedlings treated with polymeric chitin (Figure 8), the previous experiments were repeated with chitin heptamer (7mer; Figure 9). Similar to the results obtained after treatment with polymeric chitin, the 7mer treatment resulted in a significant induction of WRKY30, WRKY33 and WRKY53 expression compared to mock treatment in all tested genotypes apart from cerk1-2. Also, the 7mer-induced expression of the WRKYs was significantly reduced in clr1-3 and clr1-4 compared to Col-0 (Figure 9). The reduction of WRKY expression levels in clr1-1 however was not significant.

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Figure 9. Expression levels of WRKY30, WRKY33 and WRKY53 are significantly reduced in clr1-3 and clr1-4 mutants after treatment with chitin heptamer (7mer). Two-week-old in vitro grown Arabidopsis seedlings of the indicated clr1 mutants were treated with 1 µM 7mer for 30 min. Col-0 and cerk1-2 were included as positive and negative control, respectively. qRT-PCR of the following genes was performed: (A) WRKY30, (B) WRKY33, (C) WRKY53. ACTIN8 served as a reference gene. The bars represent the mean ± STDEV of three biological replicates consisting of 4 technical repetitions. Asterisks indicate statistical significance of the mutants compared to Col-0 (**** = p ≤ 0.0001, *** = p ≤ 0.001, ** = p ≤0.01, * = p ≤ 0.05, ns = p > 0.05). P-values were calculated using the unpaired student’s t-test.

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3.2.5 Identification of specifically chitin-induced genes and their analysis in clr1 mutants