Abstract
CEP peptide was synthesized and tested for induction of disease susceptibility using Arabidopsis Col-0. When Colletotrichum tropicale was used as a non-adapted fungal pathogen, the conidia germinated to form hyphal-like structures, which successfully penetrated epidermis, eventually causing disease symptoms. In such case, PEN2-, but not PEN3-dependent resistance was likely suppressed by CEP peptide. Similarly, the CEP peptide-mediated disease susceptibility was also effective to a non-adapted bacterial pathogen. Notably, such induced susceptibility was also evident on Arabidopsis mutants lacking the previously identified receptors, suggesting that the CEP peptide modulates Arabidopsis immunity through an unidentified receptor(s).
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Acknowledgements
ANF thanks the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) for financial support during the doctoral program. We thank Prof. Dr. Yoshitaka Takano (Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan) for providing Colletotrichum tropicale. The cepr1-1 (psi_00082), cepr2-1 (psi00083) and cepr1-1 cepr2-1 (psi_00084) mutants was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. This research was supported in part by the Grants-in-Aid for Scientific Research (21K05597) from the Japan Society for Promotion of Science (JSPS).
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10327_2022_1077_MOESM1_ESM.pptx
Supplementary file1 Fig. S1. CEP5phyp does not change Colletotrichum tropicale (Ctro) infection behavior when inoculated on ethanol-killed onion epidermal strips. Inner part of onion epidermis was cut into 1 cm2 strips, immersed overnight in absolute ethanol, then washed extensively with distilled water before use. Conidial suspension of Ctro (5 x 105 conidia/ml) were spotted on the hydrophobic side of epidermal strips along with CEP5phyp at 1 mM (final conc.) or with distilled water as a control (10 μl in total per strips). Onion epidermal strips were observed using an Olympus BX60 light microscope at 8 and 12 hpi. Microscopic observation showed that the conidia (c) of Ctro germinated to differentiate appressoria (ap), which directly penetrated epidermis to form infection hyphae (ih) in the presence of CEP5phyp. Scale bar = 25 μm. Photos were taken at 12 hpi. Rate of appressoria and infection hyphae formation were calculated as percentages of the number conidia forming appressoria or the number of appressoria forming infection hyphae per total of germinated conidia. The means and SDs were calculated from six independent onion strips with three images taken per strip. Asterisks indicate significant difference among treatments using two-way ANOVA followed by Tukey’s multiple comparison test (ns p > 0.12). The experiment was repeated twice with similar results and representative results were shown. Fig. S2. CEP5phyp induces susceptibility in Arabidopsis thaliana to non-adapted Mycosphaerella pinodes. a Detached leaves of Col-0 and jar1-1 were drop-inoculated with spore suspension (5 x 105 spores/ml) of M. pinodes (Mp) strain OMP-1 as described previously (Suzuki at al. 2017) with the presence or absence of synthetic CEP5phyp (1 mM). Distilled water was used as the control. Mp caused more severe symptoms in jar1-1 mutant plant in the presence of CEP5phyp. Arrows indicate lesions developed on the Arabidosis leaves. Photos were taken at 3 dpi. Scale bar = 1 cm. b Quantitative analysis of lesion development on Col-0 and jar1-1 leaves challenged with Mp spore suspension in the absence or presence of CEP5phyp. The means and SDs were calculated from three independent experiments using Fiji/ImageJ NIH (n=3). Asterisks indicate significant difference among treatments using two-way ANOVA followed by Tukey’s multiple comparison test (ns p > 0.12; * p < 0.033). Fig. S3. CEP5phyp induces susceptibility in Arabidopsis cepr mutant plants to non-adapted fungal pathogen, Mycosphaerella pinodes (Mp). a Detached leaves of 5-week-old Arabidopsis No-0, cepr1-1, cepr2-1 and cepr1-1; cepr2-1 were drop-inoculated with spore suspension of Mp (5 x 105 spore/ml) in the presence of CEP5phyp or water as control treatment. Arrows indicate lesions developed on the Arabidosis leaves. Photos were taken at 3 dpi. Scale bar = 1 cm. b Detached leaves of Col-0 and cepr1-3 were inoculated with Mp spore suspension in the presence of CEP5phyp or distilled water. Arrows indicate lesions developed on the leaves. Photos were taken at 3 dpi. Scale bar = 1 cm. Fig. S4. CEP5phyp dose not affect the growth of Pseudomonas syringae pv. tabaci 6605 (Pta 6605) in liquid KB media. CEP5phyp was added to the media at 10 or 100 μM (final conc.) and the OD600 values of Pta 6605 cultures were monitored at different time points using Biochrom WPA Colorimeter CO7500 (Biochrom Ltd., Cambridge, UK). The means with SDs were calculated from three replicates in each experiment. Two-way ANOVA followed by Tukey’s multiple comparison test was conducted (p > 0.05). (PPTX 2733 KB)
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Fitrianti, A.N., Mai, T.L., Phuong, L.T. et al. CEP peptide induces susceptibility of Arabidopsis thaliana to non-adapted pathogens. J Gen Plant Pathol 88, 287–292 (2022). https://doi.org/10.1007/s10327-022-01077-2
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DOI: https://doi.org/10.1007/s10327-022-01077-2