Fatty Acid Desaturases: Uncovering Their Involvement in Grapevine Defence against Downy Mildew

Abstract

Grapevine downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most severe and devastating diseases in viticulture. Unravelling the grapevine defence mechanisms is crucial to develop sustainable disease control measures. Here we provide new insights concerning fatty acid's (FA) desaturation, a fundamental process in lipid remodelling and signalling. Previously, we have provided evidence that lipid signalling is essential in the establishment of the incompatible interaction between grapevine and Plasmopara viticola. In the first hours after pathogen challenge, jasmonic acid (JA) accumulation, activation of its biosynthetic pathway and an accumulation of its precursor, the polyunsaturated α-linolenic acid (C18:3), were observed in the leaves of the tolerant genotype, Regent. This work was aimed at a better comprehension of the desaturation processes occurring after inoculation. We characterised, for the first time in Vitis vinifera, the gene family of the FA desaturases and evaluated their involvement in Regent response to Plasmopara viticola. Upon pathogen challenge, an up-regulation of the expression of plastidial FA desaturases genes was observed, resulting in a higher content of polyunsaturated fatty acids (PUFAs) of chloroplast lipids. This study highlights FA desaturases as key players in membrane remodelling and signalling in grapevine defence towards biotrophic pathogens.

Keywords: Plasmopara viticola; Vitis vinifera; biotrophy; fatty acid desaturases; fatty acid modulation; lipid signalling; plant defence.

Figure 1

Chromosomal location of Vitis vinifera FA desaturases genes. The proposed nomenclature of each V. vinifera FA desaturase is shown for each chromosome.

Figure 2

Maximum likelihood phylogenetic tree of the Vitis vinifera, Arabidopsis thaliana, Glycine max, and Oryza sativa FA desaturases superfamily. (A) Phylogenetic tree of the SAD proteins (B) Phylogenetic tree of the Δ3 desaturase proteins; (C) Phylogenetic tree of the Δ9 desaturase, Δ12/ω6 desaturase, Δ15/ω3 desaturase, sphingolipids Δ4 and the sphingolipid Δ8 desaturase. In (B), the root was truncated with a double dash totalling 0.49 changes per branch length. Scale bar represents the number of estimated changes per branch length.

Figure 3

Multiple alignments of the two grapevine FA desaturases groups representing the consensus and conserved motifs. Protein sequences were aligned for each FA desaturase group separately, applying the MAFFT tool. The consensus motifs are shown in shadow boxes according to percentage identity. (A) soluble fatty acid desaturases; (B) membrane-bound desaturases.

Figure 4

Protein domains and motifs of the grapevine FA desaturases.

Figure 5

Expression analysis of FA desaturases genes of V. vinifera cv. Regent leaves upon inoculation with P. viticola. The gene transcript’s fold-change relative to controls, at each time point (6, 24 and 48 hpi), are represented for: VviFAD2-1; VviFAD2-2; VviFAD3-1; VviFAD4; VviADS; VviFAD6; VviFAD7; VviFAD8; VviSAD-1; VviSAD-2. Fold-change values are relative to expression in mock-inoculated leaves. Asterisks indicate significant differences (p < 0.05).

Figure 6

Fatty acid-related parameters of major leaf lipid classes of V. vinifera cv. Regent mock control and inoculated leaves with P. viticola at 6 h. (A) Double bound index (DBI); (B) Ratio between C18:1 and C18:0; (C) Ratio between C18:2 and C18:1; (D) Ratio between C18:3 and C18:2. Values correspond to average values ± standard error, n = 5; Asterisks indicate significant differences (p < 0.05).

Figure 7

Lipid and FA metabolism, catalysed by the action of FA desaturases, in V. vinifera cv. Regent leaves upon inoculation with P. viticola. The gene expression regarding each grapevine desaturase encoding gene is represented by the respective heatmap as presented in (Figure 5). Abbreviations: acyl carrier protein (ACP); palmitic acid (C16:1); palmitoleic acid (C16:1); trans-9-hexadecenoic acid (C16:1 t); oleic acid (C18:1); linoleic acid (C18:2); α-linolenic acid (C18:3); Cytidine diphosphate diacylglycerol (CDP-DAG); coenzyme A (CoA); diacylglycerol (DAG); di–galactosyldiacylglycerol (DGDG); glyceraldehyde 3-phosphate (G3P); mono–galactosyldiacylglycerol (MGDG); phosphatidic acid (PA); phosphatidylcholine (PC); phosphatidylethanolamine (PE); phosphatidylglycerol (PG).