Laminarin-triggered defence responses are geographically dependent in natural populations of Solanum chilense

Abstract

Natural plant populations are polymorphic and show intraspecific variation in resistance properties against pathogens. The activation of the underlying defence responses can depend on variation in perception of pathogen-associated molecular patterns or elicitors. To dissect such variation, we evaluated the responses induced by laminarin (a glucan, representing an elicitor from oomycetes) in the wild tomato species Solanum chilense and correlated this to observed infection frequencies of Phytophthora infestans. We measured reactive oxygen species burst and levels of diverse phytohormones upon elicitation in 83 plants originating from nine populations. We found high diversity in basal and elicitor-induced levels of each component. Further we generated linear models to explain the observed infection frequency of P. infestans. The effect of individual components differed dependent on the geographical origin of the plants. We found that the resistance in the southern coastal region, but not in the other regions, was directly correlated to ethylene responses and confirmed this positive correlation using ethylene inhibition assays. Our findings reveal high diversity in the strength of defence responses within a species and the involvement of different components with a quantitatively different contribution of individual components to resistance in geographically separated populations of a wild plant species.

Keywords: Phytopthora infestans; Solanum chilense; Diversity; early immune response; ethylene; laminarin; phytohormones; reactive oxygen species resistance; tomato.

Fig. 1.

RNAseq analysis of the central population LA3111 (nine plants pooled per treatment) of S. chilense 3 h after P. infestans (3000 sporongia ml⁻¹), laminarin (1 mg ml⁻¹), and water treatment. (A–C) Differentially expressed genes (DEGs) overlap in different treatments: overall (A), up-regulated (B), and down-regulated (C). (D, E) Gene Ontology (GO) analysis of RNA-seq data showing percentage genes with signals for gene ontology terms for biological process (D) and molecular function (E) for laminarin versus P. infestans treatment.

Fig. 2.

ROS accumulation in the leaf discs from Solanum chilense measured from 0 to 180 min after elicitation with laminarin (1 mg ml⁻¹). (A) Overview for each of the populations. Each boxplot represents a populations, and each black dot is the mean measured value for one plant, obtained from three or four individual repetitions. Statistically significant pairwise comparisons between populations are shown in Supplementary Table S6. (B) Examples highlighting the within-population diversity for three of nine populations. Each boxplot represents an individual plant per population with data from one leaf disc represented as one data point accounting for up to 10–12 leaf discs per plant. Individual measurements were performed on three different dates (n=3–4 each date; 3 × 3 (4)=10 (12) leaf discs per plant). Each individual data point in (A) corresponds to the median of one of the boxplots shown in (B). Within-population pairwise comparison statistics are shown in Supplementary Table S7. (C) Differences in ROS kinetics for the three highlighted populations from (B). x-axis is minutes post-treatment; y-axis shows relative luminescence unit (RLU). Colours of the boxplots or header bars represent the geographical location of the population. Extended panels like (B) and (C) for all populations can be found in Supplementary Figs S3, S4. Each data point is a median similar to (B) and the error bars represent standard error to give a clear visualization of the different plants.

Fig. 3.

Ethylene (ET) accumulation in the leaf discs from S. chilense 3 h after elicitation with laminarin (1 mg ml⁻¹) and mock (milliQ-H₂O). (A) Each bar pair (light grey and coloured) represents an population. Each bar shows the mean of the population, and each dot represents the mean of one plant from three individual repetitions (as in B). (B) Each bar pair (light grey and dark grey) represents an individual from the population. Each bar shows mean of seven to nine data points that represent seven to nine sample measurements performed on three different dates (n=2–3 samples for each date), each sample containing three leaf discs. Significantly different ET accumulation in laminarin-treated samples from the mock-treated samples in an individual is represented by the star on the bar pair (ANOVA with post-hoc Tukey test on complete dataset). y-axis shows ET accumulation in pmol ml⁻¹ headspace of the samples. Each panel in (B) shows a different population and colours represent the geographical location of the population. Panels for all additional populations can be found in Supplementary Fig. S6A. Statistically significant ET accumulation upon laminarin treatment is denoted with stars and other pairwise comparisons of within and between populations can be found in Supplementary Tables S9–S11.

Fig. 4.

Phytohormone measures in the leaf discs from S. chilense 3 h after elicitation with laminarin (1 mg ml⁻¹) and mock (milliQ-H₂O). Each bar pair (light grey and coloured) represents a population. Each dot represents the mean of a single individual measured with at least three independent repetitions. Results for each individual plant can be found in Supplementary Fig. S6. y-axis shows phytohormone accumulation in ng g⁻¹ of the samples. Colours represent the geographical location of the population. Statistically significant phytohormone accumulation upon laminarin treatment is denoted with stars and other pairwise comparisons of within and between populations can be found in Supplementary Tables S13–S17.

Fig. 5.

Correlations between the P. infestans infection frequency (IF, x-axis) and the measured Ethylene (ET) or phaseic acid (PA) phytohormone accumulation in ng g⁻¹ sample or pmol ml⁻¹, respectively (y-axis). Each dot represents the mean value of an individual plant from three individual repetitions. Infection frequencies were obtained from an independent experiment from the same plants as presented in Kahlon et al. (2021a). IF of zero indicates plants that are fully resistant and IF at 1 indicates 100% infection rate upon inoculation. Pearson’s correlation was calculated per geographic group. The star indicates significant correlations for the central and southern coastal populations for PA and ET, respectively.

Fig. 6.

Ethylene (ET) inhibition assay on LA4107 plant 05 and plant 10. (A) ET accumulation in the leaf discs from S. chilense LA4107 plant 05 and 10, 3 h after elicitation with laminarin (1 mg ml⁻¹), AVG (5µM), laminarin (1 mg ml⁻¹) + AVG (5µM), and mock (milliQ-H₂O). Light and dark grey pairs of bars represents plants with and without treatment with laminarin (1 mg ml⁻¹), respectively. Each bar is the mean of three samples measured. y-axis shows ET accumulation in pmol ml⁻¹ air of the samples. (B) Detached leaf infection assay of LA4107 plant 05 and 10 upon drop inoculation with Phytopathora infestans Pi100 (3000 sporangia ml⁻¹) with and without AVG (5µM) treatment. y-axis represents infection frequency which is the ratio of infected leaflets divided by inoculated leaflets. Each dot represents the ratio from one leaf. Statistical significance is denoted by stars.