Elicitation of Roots and AC-DC with PEP-13 Peptide Shows Differential Defense Responses in Multi-Omics

Abstract

The root extracellular trap (RET) has emerged as a specialized compartment consisting of root AC-DC and mucilage. However, the RET's contribution to plant defense is still poorly understood. While the roles of polysaccharides and glycoproteins secreted by root AC-DC have started to be elucidated, how the low-molecular-weight exudates of the RET contribute to root defense is poorly known. In order to better understand the RET and its defense response, the transcriptomes, proteomes and metabolomes of roots, root AC-DC and mucilage of soybean (Glycine max (L.) Merr, var. Castetis) upon elicitation with the peptide PEP-13 were investigated. This peptide is derived from the pathogenic oomycete Phytophthora sojae. In this study, the root and the RET responses to elicitation were dissected and sequenced using transcriptional, proteomic and metabolomic approaches. The major finding is increased synthesis and secretion of specialized metabolites upon induced defense activation following PEP-13 peptide elicitation. This study provides novel findings related to the pivotal role of the root extracellular trap in root defense.

Keywords: Glycine max; PEP-13 elicitor; omics; root-associated cap-derived cells (root AC-DC).

Figure 1

Differential expression analysis of genes (A) and proteins (B) in root AC-DC and roots. These volcano plots were obtained from transcriptomic (A) and proteomic (B) data. Blue points correspond to genes (A) and proteins (B) under-expressed in root AC-DC compared to roots. Red points correspond to genes (A) and proteins (B) over-expressed in root AC-DC compared to roots. The number of under- and over-expressed genes and proteins for each method appears in blue (under-expressed) and red (over-expressed) squares. DEG = differentially expressed genes; DEP = differentially expressed proteins (fold change ≤ 0.66 or fold change ≥ 1.5; p-value ≤ 0.05).

Figure 2

Cell wall over-expressed genes and proteins in root AC-DC compared to roots in constitutive conditions. Each square corresponds to a gene or protein (squares with *), dark green squares correspond to highly overexpressed genes or proteins compared to roots, light green squares correspond to lowly overexpressed genes or proteins in root AC-DC compared to roots (fold change ≤ 0.66 or fold change ≥ 1.5; p-value ≤ 0.05).

Figure 3

Differential expression analysis of genes (A,B) and proteins (C,D) upon PEP-13 elicitation in roots (B,D) and root AC-DC (A,C). These volcano plots were obtained from transcriptomic (A,B) and proteomic (C,D) data. Blue points correspond to genes and proteins that are under-expressed upon PEP-13 elicitation. Red points correspond to genes and proteins that are over-expressed upon PEP-13 elicitation. The number of under- and over-expressed genes and proteins for each method appears in blue (under-expressed) and red (over-expressed) squares. DEG = differentially expressed genes; DEP = differentially expressed proteins (fold change ≤ 0.66 or fold change ≥ 1.5; p-value ≤ 0.05).

Figure 4

Heatmap of most-discriminative metabolites upon PEP-13 elicitation. Metabolite abundances were analyzed using UPLC–QTOF–MS–MS. Multivariate supervised classification OPLS-DA was established for each compartment (root AC-DC, MUC and roots) to discriminate between control (NE) and PEP-13 elicited (E) samples. The heatmap shows the peak abundance of all metabolites with a VIP > 2 in root AC-DC and Muc. The maximum abundance in each row is shown in red and the minimum in blue. MUC = mucilage; AC-DC = root-associated cap-derived cells; NE = non-elicited (control); PEP = PEP-13 elicited.

Figure 5

Pathway analysis of root, root AC-DC and mucilage upon induced defense response with PEP-13. In root AC-DC (A), mucilage (B) and roots (C), pathways are represented according to their impact value (pathway impact) and their p-value (−log10(p)). Pathways with impact value > 0.1 and −log10(p) > 1.3 are considered to be impacted.

Figure 6

Malate and phenylalanine use in root AC-DC and mucilage is modified upon PEP-13 elicitation. The tricarboxylic acid (TCA) cycle in root AC-DC seems to be modified in control conditions (NE: non-elicited) in order to transfer malic acid in the mucilage. Upon elicitation with PEP-13 (E), a malate deshydrogenase is over-expressed, enabling the conversion of malate in oxaloacetate during the TCA cycle. In control conditions, phenylalanine is found in root AC-DC along with an over-expressed phenylamonialyase. These two actors might induce the synthesis of phenylpropanoids in root AC-DC. Upon elicitation, phenylalanine is found in mucilage and might be used as a defense signal for other cells and roots by activating their phenylpropanoid synthesis. Yellow circles and stars represent beneficial soil bacteria.

Figure 7

Overview of root AC-DC basal and induced defense responses. (A) In root AC-DC, constitutive differences with roots mainly consist of highly expressed cell wall genes and proteins and metabolite storage. (B): Upon PEP-13 elicitation, a transfer of metabolites was observed from root AC-DC to the mucilage.