Figure Legends
Figure 1. Characterisation of chitosan-induced resistance in tomato and Arabidopsis. (a) Disease lesions in tomato and (b) in Arabidopsis at 3 days post inoculation. Values represent means ± SEM (n=4-10). (c) Callose deposition triggered by chitosan treatment in tomato and (d) in Arabidopsis 1 day post treatment. Values represent means ± SEM (n=8-10) of % of callose per leaf area. Different letters indicate statistically significant differences among treatments (Least Significant Differences for graph a and Dunnett T3 Post-Hoc test for graphs b, c and d, α=0.05).
Figure 2. Chitosan-induced resistance is based on priming .(a) Disease lesions in tomato at 3 days post inoculation (dpi) 2 weeks after treatment with water (Control) or 1% chitosan. Values represent means ± SEM (n=8). Asterisk indicates statistically significant differences among treatments (Student’s T. test, α=0.05).(b) Percentage of callose deposited at the infection site in water (Control) and chitosan (0.01%)-treated plants compared to the fungal lesion diameter at 1 day after infection with B. cinerea . Values represent means ± SEM (n=4). Asterisk indicates statistically significant differences among treatments (Student’s T. test, α=0.05).(c) Representative pictures of chitosan-induced priming of callose at the infection site. Blue colours correspond to fungal growth whereas yellow colours correspond with the callose deposition at the infection site. Scale bars= 0.5 mm. (d) Mass-spectrometry quantification (ng/mL) of Salicylic acid (SA), Jasmonic acid (JA) and Abscisic acid (ABA) at 24h post infection. Values represent means ± SEM (n=4). letters indicates statistically significant differences among treatments (Least Significant Differences, α=0.05, n.s. = not significant).
Figure 3. Transcriptome analysis of chitosan-induced resistance against Botrytis cinerea. (a) Principal component analysis (PCA) of whole transcriptional microarray at 6, 9 and 12 hour post infection (hpi). (b) Heatmap of differentially expressed genes (2-way ANOVA p < 0.01, Bonferoni), clustered by expression. Profiles are shown +/- treatment with chitosan and infection with B. cinerea at 6, 9, 12 hpi logarithmic scale fold induced (red) or repressed (blue) compared to Water + Mock. Hierarchical clusters on expression profile are broadly classed as i, ii, iii or iv.(c) Venn diagram of statistically significant data set (2-way ANOVA p < 0.01, Benjamin–Hochberg) of differentially expressed genes. Pairwise Student’s T-test comparisons were performed (Volcano plots: p < 0.05, 2-fold cut-off) for the three test treatments (Chitosan + Mock, Water + B. cinerea and Chitosan + B. cinerea ) compared to control treatment (Water + Mock) at 6, 9 and 12 hpi.
Figure 4. Functional characterisation of ACRE genes .(a) Chitosan-induced resistance in Nicotiana benthamiana . Disease lesions at 2 days post inoculation (dpi). Values represent means ± SEM (n=18). Asterisk indicates statistically significant differences between treatments (Student’s T. test, α=0.05).(b) Transient expression of constitutively active SlACRE75, SlACRE180, NbACRE75 and NbACRE180 in N. benthamiana againstB. cinerea . Lesion size measurements were performed at 4 days post-infection (dpi). Values presented are means ± SEM (n=6). Different letters indicate statistically significant differences (ANOVA p < 0.05 followed by Tukey‘s Post-hoc at 4 dpi). (c)A. thaliana transformed overexpression stable SlACRE75, SlACRE180, NbACRE75 and NbACRE180 infected with B. cinerea . Lesion sizes were measured at 6 days after inoculation (dpi). Values presented are means ± SEM (n=8-16). Asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01; *** p < 0.001).
Figure S1. Chitosan-induced resistance in Solanaceae melongena (aubergine) . Disease lesions at 3 dpi. Values represent means ± SEM (n=10). Different letters indicate statistically significant differences among treatments (Least Significant Differences, α=0.05).
Figure S2. Chitosan and Switch fungicide antifungal activity against Botrytis cinerea . Bars represent means of fungal growth diameter (± SEM, n=5) at 4 days after inoculating PDA-containing Petri dishes with 5 mm agar plugs of actively growing B. cinereamycelia. Different letters indicate statistically significant differences among treatments (Least Significant Differences, α=0.05)
Figure S3. Chitosan-induced resistance is based on priming .(a) Relative Growth Rate (RGR) per week of tomato plants 1 and 2 weeks after treatment with 0.01% chitosan. Values represent means ± SEM (n=10). Asterisk indicates statistically significant differences among treatments (Student’s T. test, α=0.05) (b)Mass-spectrometry quantification (% of peak area) of Jasmonic acid-isoleucine (JA-ile) at 24h post inoculation. Values represent means ± SEM (n=4). Different letters indicate statistically significant differences among treatments (Least Significant Differences, α=0.05)
Figure S4. Validation of microarray expression results . Expression profile obtained in the microarray (a) and in the analysis by RT-q-PCR (b) of a subset of 9 genes at 9 hpi with Botrytis cinerea
Figure S5. Western Blot analysis . Expression of proteins by immunoblot analysis of GFP-SlACRE75, GFP-SlACRE180, GFP-NbACRE75 and GFP-NbACRE180 fusion proteins in N. benthamiana leaves at 48 h after agroinfiltration. Expected protein sizes were (i) SlACRE75= 14.79 + 26 KDa GFP= 40.8 KDa; (ii) SlACRE180= 10.86 +26= 36.8 KDa; (iii) NbACRE180= 11.74+26= 37.7 KDa; and (iv) NbACRE75= 14.6+26= 40.7 Kda. Proteins were separated by SDS–PAGE and analysed by immunoblotting. A GFP-specific antibody was used for detection of GFP-fusion protein. Equal loading of total proteins was examined by Ponceau staining (PS). Three lanes represent 3 replicates per construct GFP-SlACRE75, GFP-SlACRE180, GFP-NbACRE75, GFP-NbACRE180, and a GFP-non-protein/ empty vector (control).
Figure S6. Subcellular location of ACRE proteins. Confocal microscopy observation of (a) pFlub vector as a RFP-peroxisome tagged marker, (b) nucleus mRFP marker, (c) ER mRFP marker, (d) free GFP in cytoplasm and the nucleus, (e)GFP-SlACRE75 and (f) GFP-NbACRE75 fusions in the nucleus and nucleolus, (g) GFP-SlACRE180 fusion in the ER and (h)GFP-NbACRE180 fusion in the peroxisomes.
Figure S7. Growth analysis. Perimeter in cm of rosettes from Arabidopsis lines overexpressing GFP-Empty vector (EV), GFP-SlACRE75, GFP-SlACRE180, GFP-NbACRE75 and GFP-NbACRE180 constructs. Values represent means ± SEM (n=8-16). n.s = not significant differences between treatments (One-way ANOVA, α=0.05)