The PpPep2-Triggered PTI-like Response in Peach Trees Is Mediated by miRNAs

Abstract

Plant diseases diminish crop yields and put the world's food supply at risk. Plant elicitor peptides (Peps) are innate danger signals inducing defense responses both naturally and after external application onto plants. Pep-triggered defense networks are compatible with pattern-triggered immunity (PTI). Nevertheless, in complex regulatory pathways, there is crosstalk among different signaling pathways, involving noncoding RNAs in the natural response to pathogen attack. Here, we used Prunus persica, PpPep2 and a miRNA-Seq approach to show for the first time that Peps regulate, in parallel with a set of protein-coding genes, a set of plant miRNAs (~15%). Some PpPep2-regulated miRNAs have been described to participate in the response to pathogens in various plant-pathogen systems. In addition, numerous predicted target mRNAs of PpPep2-regulated miRNAs are themselves regulated by PpPep2 in peach trees. As an example, peach miRNA156 and miRNA390 probably have a role in plant development regulation under stress conditions, while others, such as miRNA482 and miRNA395, would be involved in the regulation of resistance (R) genes and sulfate-mediated protection against oxygen free radicals, respectively. This adds to the established role of Peps in triggering plant defense systems by incorporating the miRNA regulatory network and to the possible use of Peps as sustainable phytosanitary products.

Keywords: Prunus; differential network analysis; gene set enrichment analysis; miRNA sequencing; plant defense; plant elicitor peptide (Pep).

Figure 1

Principal component analysis (PCA) of log2 normalized expression data from the top 50 most variable miRNAs (DESeq2 1.20.0 package, [38]). Two principal components, PC1 and PC2, are represented. They have Eigenvalues above 1 and explain 48.7% and 23.91% of the variability, respectively. Colors represent the different peptide treatments: orange, time zero control (t0); green, 1 h PpPep2 (t1); and blue, 24 h PpPep2 (t24). Three biological replicates per sample are shown.

Figure 2

Volcano plots showing miRNA expression (expressed as log2FoldChange) 1 h after PpPep2 application in comparison to untreated control (A) and 24 h after PpPep2 application in comparison to untreated control (B); and the adj. p-value of the same comparisons (expressed as log10[adj. p]). The dashed line delimits differentially expressed miRNAs (DEMs), shown in red (upregulated DEMs, log2FC > 1) and blue (downregulated DEMs, log2FC < 1), from unregulated miRNAs (gray) using adj. p < 0.05. DEMs regulated at both comparisons are named and represented in triangles.

Figure 3

Enriched GO terms associated with DEM-Ts 1 h (A) and 24 h (B) after PpPep2 treatment of peach leaves. The x-axis indicates the rich factor (ratio of genes in the input list that are annotated to the function); and the y-axis shows the GO terms grouped into upper families shown in rainbow colors. GO terms are also classified into molecular function (MF), biological process (BP), or cellular component (CC) categories. Dot colors indicate p-values, shown in blue (0.05) to red (0.00). Dot radiuses are proportional to gene number (number of genes contributing to each GO term enrichment).

Figure 4

Enriched GO terms associated with DEM-Ts that were differentially expressed after 1 µM of PpPep2 treatment of peach leaves for 1 h, 24 h or 48 h. The x-axis indicates the rich factor (ratio of genes in the input list that are annotated to the function), and the y-axis shows the GO terms. GO terms are also classified into molecular function (MF) or biological process (BP) categories. Dot color indicate p-values, shown in blue (0.05) to red (0.00). Dot radiuses are proportional to the gene number (number of genes contributing to each GO term enrichment).