In roots of Arabidopsis thaliana, the damage-associated molecular pattern AtPep1 is a stronger elicitor of immune signalling than flg22 or the chitin heptamer

Abstract

Plants interpret their immediate environment through perception of small molecules. Microbe-associated molecular patterns (MAMPs) such as flagellin and chitin are likely to be more abundant in the rhizosphere than plant-derived damage-associated molecular patterns (DAMPs). We investigated how the Arabidopsis thaliana root interprets MAMPs and DAMPs as danger signals. We monitored root development during exposure to increasing concentrations of the MAMPs flg22 and the chitin heptamer as well as of the DAMP AtPep1. The tissue-specific expression of defence-related genes in roots was analysed using a toolkit of promoter::YFPN lines reporting jasmonic acid (JA)-, salicylic acid (SA)-, ethylene (ET)- and reactive oxygen species (ROS)- dependent signalling. Finally, marker responses were analysed during invasion by the root pathogen Fusarium oxysporum. The DAMP AtPep1 triggered a stronger activation of the defence markers compared to flg22 and the chitin heptamer. In contrast to the tested MAMPs, AtPep1 induced SA- and JA-signalling markers in the root and caused a severe inhibition of root growth. Fungal attack resulted in a strong activation of defence genes in tissues close to the invading fungal hyphae. The results collectively suggest that AtPep1 presents a stronger danger signal to the Arabidopsis root than the MAMPs flg22 and chitin heptamer.

Fig 1. Responses of PAMP-triggered immunity in roots.

(a) Luminol-based detection of ROS production in isolated roots of Col-0 seedlings treated with 1 μM flg22, chi7 or AtPep1 or without elicitor. Data represent the mean of 12 replicates ± SE. RLU—relative luminescence units (b) Activation of MAP kinases detected by western blot (MPK). Isolated roots from 2-week old seedlings of Col-0 and transgenic lines were treated with 1 μM flg22, chi7 or AtPep1 or without elicitor for 10 min before sampling. Ponceau S staining was used as loading control (PS). The experiment was performed three times with similar results.

Fig 2. Root growth inhibition by AtPep1.

(a) Col-0 wild-type and transgenic lines were germinated and grown vertically for 12 days on 0.5x MS containing 10 nM, 100 nM or 1 μM flg22, chi7 or AtPep1 or without elicitor. (b) Analysis of primary root length was performed using Fiji on images recorded under (a) and is shown as box plots representing ≥ 20 roots per sample. The experiment was performed three times for Col-0 and two times for pepr1 pepr2. Statistical analysis was performed using a Student’s t-test comparing with roots grown on control medium: * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 3. Elicitor-triggered responses of pHEL::YFP_N.

(a) Microscopic analysis of root developmental zones of 7-days old pHEL::YFP_N seedlings following 24 h treatment with 100 nM flg22, chi7, AtPep1 or 0.5x MS as control. RC—root cap / meristem, TZ—transition zone, DZ—differentiation zone, MZ—mature zone. Bar 100 μM. (b) Quantification of fluorescent signals from microscopic analysis of different developmental zones of pHEL::YFP_N as described under (a) using Fiji. Bars represent the mean of ≥ 3 images ± SD.

Fig 4. Elicitor-dependent activation of defence-related promoters in the TZ and DZ of the root.

(a) Quantification of microscopic analysis of Promoter::YFP_N constructs in the differentiation zone of 7-days old seedlings following treatment with 100 nM flg22, chi7, AtPep1 or 0.5x MS as control. Images were analysed using Fiji. Bars represent the mean of ≥ 3 images ± SD. (b) Overview of inductive and suppressive responses of Promoter::YFP_N constructs in different developmental root zones analysed as described under (a). Numbers in the upper row indicate the sum of significant marker responses in one of the four analysed root zones for a given elicitor, while numbers in the lower row indicate the total sum of significant responses from all markers and all root zones for a given elicitor. Black colour indicates induction, white colour suppression and grey colour non-significant changes.

Fig 5. Verification of elicitor-triggered responses of promoter::YFP_N markers by qPCR.

(a) Signal quantification using micrographs from roots expressing pMYB51::YFP_N and pACS6::YFP_N. 7-day old seedlings were treated with 100 nM flg22, chi7 or AtPep1 or 0.5x MS without elicitor and analysed in four developmental zones. Bars represent the sum of average signals obtained for each of the four developmental zones from 2–3 independent transformation lines per construct. (b) Expression of pMYB51::YFP_N and pACS6::YFP_N in the differentiation zone of 7-day old seedlings treated with 100 nM flg22, chi7 or AtPep1 or 0.5x MS without elicitor. Bar 100 μm. (c) Expression of MYB51 and ACS6 analysed by quantitative RT-PCR following 2 h treatment of 7-day old seedlings with 100 nM flg22, chi7 or AtPep1 or 0.5x MS without elicitor. Transcript levels were normalized to the reference gene UBIQUITIN5 before calculation of expression relative to the control. The experiment was repeated three times with similar results.

Fig 6. Expression of promoter::YFP_N constructs in roots infected with F. oxysporum.

(a) Microscopic analysis of the infection site of 12-day old roots 2 days after inoculation with spores from F. oxysporum. Fluorescence derived from promoter::YFP_N constructs is localized in the root nuclei and shown in green, while propidium iodide was used as a counterstain of cell walls and dead cells and is shown in red. Bar 100 μm. (b) Quantification of microscopic analysis of promoter::YFP_N constructs as depicted in (a) using Fiji. Bars represent the mean of ≥ 6 images ± SE. Statistical analysis was performed using a Student’s t-test: * p < 0.05, ** p < 0.01, *** p < 0.001.