The interplay between membrane lipids and phospholipase A family members in grapevine resistance against Plasmopara viticola

Abstract

Grapevine downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most important diseases in modern viticulture. The search for sustainable disease control measure is of extreme importance, thus becoming imperative to fully characterize the mechanisms leading to an incompatible interaction. We have previously shown that lipid signalling events play an important role in grapevine's response to this pathogen, namely through changes in linolenic acid content, lipid peroxidation and jasmonic acid synthesis. Here, we have characterized the modulation of lipid metabolism in leaves from two V. vinifera cultivars (resistant and susceptible to P. viticola) in the first hours after pathogen inoculation. Prior to pathogen inoculation both genotypes present an inherently different fatty acid composition that is highly modulated in the resistant genotype after pathogen challenge. Such changes involve modulation of phospholipase A activity suggesting that the source of lipids mobilized upon pathogen infection are the chloroplast membranes. This work thus provides original evidence on the involvement of lipid signalling and phospholipases in grapevine immune responses to pathogen infection. The results are discussed considering the implications on the plant's physiological status and the use of discriminating lipid/fatty acids pattern in future selection procedures of cultivars.

Figure 1

Canonical Analysis of Principal (CAP) coordinates plot based in the Euclidean distances between samples considering the complete leaves fatty acid profile of the mock inoculated groups of V. vinifera cv. Trincadeira and Regent (A), mock inoculated (hm) and inoculated (hpi) samples of Trincadeira (B) and Regent (C) varieties along the time course.

Figure 2

Fatty acid composition of V. vinifera cv. Regent mock inoculated (light grey) and inoculated (dark grey) leaves with P. viticola at 6 (A), 12 (B) and 24 (C) hours; (D) Double bound index (DBI); (E) Ratio between unsaturated and saturated FA. Values correspond to average relative percentage ± standard error, n = 4; Asterisks indicate significant differences (p < 0.05).

Figure 3

Lipid composition of V. vinifera cv. Regent mock inoculated (hm) and inoculated (hpi) leaves with P. viticola at 6 hours. (A) Total of lipids content; (B) Percentage of total FA present in MGDG; (C) Percentage of total FA in DGDG; (D) Percentage of total FA in PG. Values correspond to average relative percentage ± standard error, n = 3; Asterisks indicate significant differences (p < 0.05). Abbreviations: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PI), phosphatidic acid (PA), free fatty acids (FA) and triacylglycerol (TAG).

Figure 4

Locations of Vitis vinifera PLA genes in chromosomes. Proposed V. vinifera PLA nomenclature is shown in each chromosome.

Figure 5

Maximum likelihood phylogenetic tree of the grapevine PLA superfamily. The numbers above branches show bootstrap values. Scale bar represents the number of estimated changes per branch length. Root was truncated with double dash totalling 0.3 changes per branch length.

Figure 6

Multiple alignments of four grapevine PLA families representing the consensus and conserved motifs. Protein sequences were aligned for each PLA family, separately, applying MAFFT tool. The consensus motifs have been shown in shadow boxes according BLOSUM62. (A) VviPLA₁; (B) VviPA-PLA₁; (C) VvisPLA₂; (D) VvipPLA.

Figure 7

Gene expression profiles in Regent inoculated leaves. For each time point (6, 12 and 24 hpi) gene transcripts fold-change relative to controls are represented for VviPLA₁-Iβ1; VviPLA₁-Iγ1; VviPLA₁-IIδ; VvisPLA₂; VvipPLA-I; VvipPLA-IIβ; VvipPLA-IIδ2; VvipPLA-IIIβ. Fold-change values are relative to expression in mock inoculated leaves. Asterisks indicate significant differences (p < 0.05).

Figure 8

Lipid and FA modulation in Vitis vinifera cv. Regent at first hours upon infection with P. viticola. Fatty acids role in lipid signalling pathway, by phospholipases action, in plant defence mechanisms, upon its release from lipids, serving as signalling molecules or as substrate for oxylipins biosynthesis. Abbreviations: (9S,13S)-12-oxo-cis-10,15-phytodienoic acid (9S,13S/cis(+)-OPDA), 12,13-epoxy-9-Z,11,15-Z-octadecatrienoic acid (12,13-EOT), 13S-hydroperoxy-(9Z,11E,15)-octadecatrienoic acid (13-HPOT), (+)-7-iso-jasmonic acid ((+)-7-iso-JA), (−)-jasmonic acid ((−)-JA), abscisic acid (ABA), allene oxide cyclase (AOC), allene oxide synthase (AOS), azelaic acid (AzA), oleic acid (C18:1), linoleic acid (C18:2), α-linolenic acid (C18:3), calcium (Ca2⁺), diacylglycerol (DAG), di–galactosyldiacylglycerol (DGDG), diacylglycerol kinase (DGK), fatty acids desaturases 6/7/8 (FAD 6/7/8), inositol triphosphate (IP3), jasmonates-amide synthetase (JAR1), lipoxygenase 2 (LOX2), mono–galactosyldiacylglycerol (MGDG), nitric oxide (NO), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidilglycerol (PG), phosphatidylinositol (PI), phosphatidylinositol 4,5-bisphosphate (PIP₂), phospholipase C (PLC), phospholipase D (PLD), monocarboxylic acid 9-oxononanoic acid (ONA), oxophytodienoate reductase 3 (OPR3), reactive oxygen species (ROS), salicylic acid (SA), systemic acquired resistance (SAR).