Molecular Profiling of Pierce's Disease Outlines the Response Circuitry of Vitis vinifera to Xylella fastidiosa Infection

Abstract

Pierce's disease is a major threat to grapevines caused by the bacterium Xylella fastidiosa. Although devoid of a type 3 secretion system commonly employed by bacterial pathogens to deliver effectors inside host cells, this pathogen is able to influence host parenchymal cells from the xylem lumen by secreting a battery of hydrolytic enzymes. Defining the cellular and biochemical changes induced during disease can foster the development of novel therapeutic strategies aimed at reducing the pathogen fitness and increasing plant health. To this end, we investigated the transcriptional, proteomic, and metabolomic responses of diseased Vitis vinifera compared to healthy plants. We found that several antioxidant strategies were induced, including the accumulation of gamma-aminobutyric acid (GABA) and polyamine metabolism, as well as iron and copper chelation, but these were insufficient to protect the plant from chronic oxidative stress and disease symptom development. Notable upregulation of phytoalexins, pathogenesis-related proteins, and various aromatic acid metabolites was part of the host responses observed. Moreover, upregulation of various cell wall modification enzymes followed the proliferation of the pathogen within xylem vessels, consistent with the intensive thickening of vessels' secondary walls observed by magnetic resonance imaging. By interpreting the molecular profile changes taking place in symptomatic tissues, we report a set of molecular markers that can be further explored to aid in disease detection, breeding for resistance, and developing therapeutics.

Keywords: Xanthomonadaceae; defense response; metabolome; plant–bacteria interaction; proteome; transcriptome; vascular pathogen.

FIGURE 1

Grapevines developing Pierce’s disease in controlled inoculations. (A) Twelve weeks after inoculation of X. fastidiosa, grapevines already displayed Pierce’s disease symptoms as leaf scorching, match stick petioles from fallen leaves, and darkened patches on stems. (B) Detail of leaf showing scorching symptoms. (C) Nuclear magnetic resonance imaging of representative transversal cuts of stems exhibiting intensive secondary wall deposition in infected vines. Yellow arrows point to electron-denser material more abundant in infected samples. Scale bar is 6 mm for both images.

FIGURE 2

Multivariate analysis of datasets used in this work. (A) Circos plot of correlation matrix of all 32 samples, including infected (I) and non-infected (N), transcriptome (T), proteome (P), and metabolome (M) samples. Samples are represented by segments on the circumference and ribbons connecting samples indicate the relative contribution of each interaction to the total of that sample. Interactions only among the 10 transcriptome samples (B), 6 proteome samples (C), and 16 metabolome samples (D) are also shown. Correlation matrix is shown in Supplementary Table S1.

FIGURE 3

Evaluation of expression levels of selected CDS and proteins. (A) RT-qPCR analysis of CDS selected based on RNA-seq data encompassing various functions and modulation intensities in samples from diseased leaves. Fold changes compared to healthy control plants were calculated from three biological replicas assayed in triplicate each. One and two asterisks indicate, respectively, p-values < 0.05 and 0.01, according to two-tailed t-test for two paired samples with alpha = 0.05. (B) Immunodetection of ferritin 3 in leaf samples (P1, P2: two plants per treatment) infected with X. fastidiosa (Xf).

FIGURE 4

Molecular functions affected by Pierce’s disease. Comparative transcriptome data grouped by functional categories are available in MapMan. Color scale represents log2 ratios of diseased vs. healthy grapevine leaves, in which each protein coding sequence is represented by a square within a selected functional category. Leaf diagram represents the transition of from healthy to diseased tissues analyzed in this comparison. Pale rods on leaf represent X. fastidiosa cells occurring within xylem vessels, at higher density near the petiole, and gradually decreasing toward margin, where outer membrane vesicles and secreted proteins are still abundant (n = 5 diseased vs. 5 healthy plants).

FIGURE 5

Selected functional categories responsive to Pierce’s disease. Representation showing expression ratio and level of transcripts with altered abundance in grapevines leaves showing Pierce’s disease symptoms mentioned in this work. Log₂ expression ratios were calculated from averages of five symptomatic samples compared to five healthy samples, with padj < 0.05. Normalized expression levels are represented in reads per kilobase of transcript per million mapped fragments (FPKM), from the sum of average values of symptomatic and healthy samples. ¹Al. deh. = alcohol and aldehyde dehydrogenases and ²RFO = raffinose family of oligosaccharides. A complete list of genes and values shown in this figure is given in Supplementary Table S11.

FIGURE 6

Selection of paralogs responsive to Pierce’s disease. Four examples are shown, in which the members of paralogous groups that are considered responsive to disease can be selected based on confidence levels (variability among replicas). In each panel, the green line indicates the p-value of 0.05 selection threshold used by us. (A) A total of 291 leucine-rich repeats receptor-like kinases were detected as expressed, and while only 19 were considered responsive to disease, 14 showed increased transcript abundance, and 5 reduced. (B) Seventeen thaumatin-like genes were detected as expressed, being three with increased transcript abundance. (C) From the 122 2-oxoglutarate oxygenase paralogs detected as expressed, seven were responsive to disease, with five showing increased transcript abundance. (D) From the 74 cellulose synthases detected as expressed, 12 were responsive to disease and of these only one shows reduced transcript abundance.

FIGURE 7

Major metabolites with increased abundance in V. vinifera affected by Pierce’s disease. PubChem structures of metabolites with known antimicrobial and signaling functions accumulated in symptomatic grapevines (n = 8 diseased vs. 8 healthy plants).

FIGURE 8

Model of molecular events occurring in V. vinifera with Pierce’s disease. Depiction based on transcriptomic, proteomic, and metabolomic analyses, highlighting perception of X. fastidiosa PAMPs and DAMPs, signaling cascades and stress response. The lower box lists prominent members in each functional category based on their expression ratios between diseased and healthy grapevines. Gene identifiers are based on the Ensembl Gramene release 51 V. vinifera annotation.