Identification of a Vitis vinifera endo-β-1,3-glucanase with antimicrobial activity against Plasmopara viticola

Abstract

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis-related (PR) proteins. Endo-β-1,3-glucanases (EGases) belong to the PR-2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti-P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR-2 family from Vitis.

Keywords: PR-2; Plasmopara viticola; Vitis vinifera; endo-β-1,3-glucanase; grapevine.

Figure 1

Analysis of grapevine endo‐β‐1,3‐glucanase (EGase) sequences. (a) Phylogenetic tree containing grapevine (black and green), Arabidopsis (magenta), tobacco (blue) and soybean (orange) endoglucanases. Grapevine sequences in green indicate proteins with a predicted signal peptide. Asterisks indicate grapevine genes for which evidence of expression is available. Numbers on the nodes indicate bootstrap values. Sequences selected as EGase1, EGase2 and EGase3 are specified. Grapevine and Arabidopsis endoglucanases are named according to the Grapevine Genome Database (v2) at CRIBI and The Arabidopsis Information Resource (TAIR), respectively (see Experimental procedures). The accession numbers in the tree correspond to the two original sequences used in the blast search. Accession numbers for tobacco and soybean glucanases are as follows: Tobacco1, CAA38324; Tobacco2, AAA34079; Tobacco3, AAD33880; Tobacco4, AAA34078; Tobacco5, AAA34103.1; SoybeanA, AAA33946; SoybeanB, AAR26001. (b) Alignment of the three selected grapevine endoglucanases and a tobacco endoglucanase possessing a vacuolar targeting signal. The black background shows identical amino acid residues. The grey background represents similar amino acids. Asterisks indicate predicted catalytic glutamic acid (Glu) residues. Blue bars show the divergent region in the vicinity of a catalytic Glu. Red bars show the glycosyl hydrolase family 17 signature.

Figure 2

Expression of grapevine endoglucanases on Plasmopara viticola infection. (a) Semi‐quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) showing the expression of grapevine endo‐β‐1,3‐glucanases (EGases) at 0, 6, 24, 48, 72, 96 and 168 h post‐inoculation (hpi). Non‐inoculated leaves were used as control (C). PvACT, Plasmopara viticola actin; VvACT, Vitis vinifera actin. The results are representative of two independent experiments. (b) Expression of EGases on grapevine leaves based on RNA‐Seq data from Vannozzi et al. (2012). Pv 24hpi, P. viticola‐infected leaves at 24 hpi; Pv 48hpi, P. viticola‐infected leaves at 48 hpi; FPKM, fragments per kilobase of exon per million fragments mapped. (c) Expression of EGases on grapevine leaves based on RNA‐Seq data from Perazzolli et al. (2012). Pv 24h, P. viticola‐infected leaves at 24 hpi; T39, leaves treated with Trichoderma harzianum T39, which induces resistance to downy mildew; T39 Pv 24h, P. viticola‐infected leaves following treatment with T39 at 24 hpi; FPKM, fragments per kilobase of exon per million fragments mapped. Data shown are the means and standard errors of three biological replicates.

Figure 3

Sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of purified grapevine endoglucanases. H, soluble lysate containing recombinant endo‐β‐1,3‐glucanase (EGase); FT, flow‐through of affinity Ni‐TED column; EL, purified recombinant EGase, indicated by an asterisk. Two micrograms of each enzyme were run on a 12% acrylamide gel and stained with colloidal Coomassie blue. MW, molecular weight.

Figure 4

Effect of pH and temperature on grapevine glucanase enzymatic activity. Effect of pH and temperature on the activity of EGase1 (a), EGase2 (b) and EGase3 (c). Effect of pH (left panels) was measured at a constant temperature of 37°C. Effect of temperature (right panels) was measured at a constant pH of 5.4. The pH of the buffer was not affected by temperature. In both cases, the activity is expressed relative to the maximum activity of 100%. Data are the means of three independent experiments with no technical repetitions, and the error bars represent the standard deviation.

Figure 5

Activity of grapevine endoglucanases produced in planta. Glucanase activity in crude extracts of Nicotiana benthamiana leaves transiently expressing grapevine endoglucanases. Plants agroinfiltrated with an empty vector (Ev) were used as controls. Mean value and standard error were obtained from three biological repetitions, each including two technical repetitions. Asterisks indicate samples significantly different from Ev in a Dunett's test at *P = 0.1 and ***P = 0.01.

Figure 6

Effect of endoglucanases on germinated Plasmopara viticola spores. (a) Glucanase assay on P. viticola germinated spores. Each line represents a different treatment, where purified endo‐β‐1,3‐glucanases (EGases) or buffer as control were added to the spore suspension. The results of each treatment are illustrated by a photograph of the same microscopic field taken at different times (t = 0 min and t = 12 min) under two different light sources. Fluorescent light reveals fluorescein diacetate (FDA) fluorescence and indicates live zoospores, whereas visible light allows the counting of structurally intact zoospores. The effect on spore viability can be observed for all three EGases (fluorescence panels). The effect of treatment on spore integrity is visible for EGase3. Bar represents 15 µm. (b) Percentage of spores showing structural integrity after 12 min of treatment. Percentages were calculated by counting spores at t = 0 min and t = 12 min in visible light. (c) Percentage of spore viability after 12 min of treatment. Percentages were calculated by counting spores at t = 0 min and t = 12 min in fluorescent light. In (b) and (c), boxplots show the results of three independent experiments without technical repetitions. Boxes with the same letter are not significantly different in a Tukey's honestly significant difference (HSD) test at P = 0.05. Raw data for spore counting are detailed in Table S2.