Alfin-like transcription factor VqAL4 regulates a stilbene synthase to enhance powdery mildew resistance in grapevine

Abstract

Resveratrol is a phytoalexin that is synthesized by stilbene synthase (STS). Resveratrol in the human diet is known to have beneficial effects on health. We previously identified six novel STS (VqNSTS) transcripts from the transcriptome data of Vitis quinquangularis accession Danfeng-2. However, the functions of and defensive mechanisms triggered by these VqNSTS transcripts remain unknown. In the present study, we demonstrate that the expression of five of these six novel members, VqNSTS2-VqNSTS6, can be induced by the powdery mildew-causing fungus Uncinula necator. Additionally, overexpression of VqNSTS4 in the V. vinifera susceptible cultivar Thompson Seedless promoted accumulation of stilbenes and enhanced resistance to U. necator by activating salicylic acid (SA) signalling. Furthermore, our results indicate that the Alfin-like (AL) transcription factor VqAL4 can directly bind to the G-rich element (CACCTC) in the VqNSTS4 promoter and activate gene expression. Moreover, overexpression of VqAL4 in Thompson Seedless enhanced resistance to U. necator by promoting stilbene accumulation and activating SA signalling. Conversely, RNA interference-mediated silencing of VqNSTS4 and VqAL4 resulted in increased susceptibility to U. necator. Collectively, our results reveal that VqNSTS4, regulated by VqAL4, enhances grapevine resistance to powdery mildew by activating SA signalling. Our findings may be useful to improve disease resistance in perennial fruit trees.

Keywords: Chinese wild Vitis quinquangularis; VqAL4 transcription factor; VqNSTS; disease resistance; stilbene.

FIGURE 1

Identification and expression analysis of novel stilbene synthase transcripts in Vitis quinquangularis accession Danfeng‐2. (a) Expression analysis of novel transcripts in V. quinquangularis accession Danfeng‐2. Novel transcripts are grouped into 11 clusters. The numbers of transcripts from clusters 1 to 11 were 311, 322, 76, 53, 205, 380, 324, 460, 385, 328, and 6600, respectively. Expression of transcripts in cluster 11 was the highest at the ripe stage. (b) Analysis of the expression profiles of novel stilbene synthase transcripts in Danfeng‐2. Six stilbene synthase transcripts were isolated from cluster 11, with locus names c50176.graph_c0, c91313.graph_c0, c76190.graph_c0, c56362.graph_c0, c54055.graph_c0, and c11357.graph_c0. The four developmental stages of Danfeng‐2 berries were represented by DF_GH (green hard stage, 25 days after blooming), DF_BV (before véraison stage, 40 days after blooming), DF_V (véraison stage, 50 days after blooming), and DF_R (ripe stage, 80 days after blooming). (c) Multiple sequence alignment of VqNSTS1–6 amino acid sequences. The red rectangles represent the two conserved motifs. VqNSTS1 is a pseudogene, and VqNSTS5/6 contain mutations in the conserved motif. (d) The phylogenetic tree of VqNSTS1–6 with 41 reported VqSTSs and 48 reported VvSTSs. The 41 reported VqSTSs are from Danfeng‐2. All 48 reported VvSTSs are from V. vinifera ‘Pinot Noir’. They were divided into six subgroups: VqNSTS1, VqNSTS3, VqNSTS5, and VqNSTS6 belong to subgroup II; VqNSTS2 belongs to subgroup V; and VqNSTS4 belongs to subgroup I. (e–i) Reverse transcription‐quantitative PCR (RT‐qPCR) analysis was conducted to determine the relative transcript levels of VqNSTS2–6 after inoculation with Uncinula necator. VqNSTS2/4 showed high expression after U. necator inoculation, especially VqNSTS4. Asterisks indicate significant differences (*p < 0.05, **p < 0.01, Student's t test). (j–n) RT‐qPCR analysis was conducted to determine the relative transcript levels of VqNSTS2–6 after treatment with 100 μM abscisic acid (ABA), ethylene (Eth), salicylic acid (SA), or methyl jasmonate (MeJA). VqNSTS4 responded significantly to SA treatment. The standard deviation (SD) was calculated from three independent replicates. One‐way analysis of variance (Tukey's test) was carried out, with asterisks indicating significant differences at *p < 0.05, **p < 0.01.

FIGURE 2

VqNSTS4 transgenic Vitis vinifera lines exhibit disease resistance resulting from the accumulation of internal stilbene and the expression of resistance genes. (a) Photograph of VqNSTS4 transgenic lines and wild‐type Thompson Seedless before Uncinula necator inoculation. Bars = 1 cm. (b) Photograph of leaves of VqNSTS4 transgenic lines and wild‐type Thompson Seedless at 7 days postinoculation (dpi). Bars = 1 cm. (c) Trypan blue staining and 3,3′‐diaminobenzidine (DAB) staining show the hyphal growth of U. necator and H₂O₂ accumulation at 1, 3, 5, and 7 dpi. c, conidium; ap, appressorium; ph, primary hypha; sh, secondary hypha. Bars = 100 μm. (d) Scanning electron micrographs of the hyphae and appressoria (ap) of U. necator in VqNSTS4 transgenic lines and wild‐type Thompson Seedless. Upper figures, bars = 100 μm; lower figures, bars = 20 μm. (e) Aniline blue staining showing callose depositions in U. necator‐infected epidermal cells at 7 dpi. Bars = 50 μm. (f) Quantification of spores per mg fresh leaves from VqNSTS4 transgenic lines and wild‐type Thompson Seedless at 7 dpi. (g) Determination of five stilbene contents in the leaves of VqNSTS4 transgenic mutants and wild‐type Thompson Seedless at 7 dpi. (h) Free salicylic acid (SA) content in the leaves of VqNSTS4 transgenic lines and wild‐type Thompson Seedless at 0 dpi and 7 dpi. (i–k) Reverse transcription‐quantitative PCR analysis was conducted to determine the relative transcript levels of SA‐related genes in VqNSTS4 transgenic lines following U. necator inoculation. WT, wild‐type Thompson Seedless. The standard deviation (SD) was calculated from three independent replicates. One‐way analysis of variance (Tukey's test) was carried out. Asterisks indicate significant differences at *p < 0.05, **p < 0.01.

FIGURE 3

Transient silencing of VqNSTS4/STS1 in leaves of Vitis quinquangularis accession Danfeng‐2 reduces resistance to Uncinula necator. (a) Phenotypes of control EV and transiently silenced RNAi‐VqNSTS4/STS1‐GFP Danfeng‐2 leaves at 1 day postinoculation (dpi) and 7 dpi. EV, the empty vector pK7GWIWG2(II)‐35S‐GFP. (b) Trypan blue staining of leaves from EV and transiently silenced RNAi‐VqNSTS4/STS1‐GFP leaves at 1 dpi and 7 dpi. Bars = 100 μm. (c) Reverse transcription‐quantitative PCR (RT‐qPCR) analysis was conducted to determine the relative expression levels of VqNSTS4/STS1 in transiently transformed Danfeng‐2 leaves. (d) Quantification of spores per mg fresh leaves from EV and transiently silenced RNAi‐VqNSTS4/STS1‐GFP leaves at 7 dpi. (e, f) Stilbene contents in EV and transiently silenced RNAi‐VqNSTS4/STS1‐GFP leaves. (g) Free salicylic acid (SA) content in the leaves of EV and transiently silenced RNAi‐VqNSTS4/STS1‐GFP leaves at 7 dpi. (h–j) RT‐qPCR analysis was conducted to determine the relative transcript levels of SA‐related genes in EV and transiently silenced RNAi‐VqNSTS4/STS1‐GFP leaves following U. necator inoculation. The standard deviation (SD) was calculated from three independent replicates. Asterisks indicate significant differences (*p < 0.05, **p < 0.01, Student's t test).

FIGURE 4

VqNSTS4 has unconventional transcriptional regulation activity and carries specific VqAL4 binding sites. (a) cis‐Regulatory element analysis in the promoters of VqNSTS genes. MYB and WRKY binding elements were found in the ProVqNSTS2, ProVqNSTS3, and ProVqNSTS6 promoters. The ProVqNSTS4 promoter included a G‐rich element in addition to the MYB and WRKY binding elements. (b) Yeast one‐hybrid assays were carried out to determine whether VqWRKYs, VqMYBs, and VqAL4 could bind directly to the promoters of VqNSTS genes. VqMYB154 can bind to ProVqNSTS2, VqWRKY3 and VqWRKY53 can bind to ProVqNSTS3, VqMYB14 can bind to ProVqNSTS6, and VqAL4 can bind to ProVqNSTS4. (c) Yeast one‐hybrid assays were conducted to demonstrate that VqAL4 cannot bind to ProVqNSTS4 ^m2 , ProVqNSTS4 ^m3 , ProVqNSTS4 ^d , ProVqNSTS4 ^m5 , or ProVqNSTS4 ^m6 . (d) Luminescence intensity. (e) Ratio of firefly luciferase (LUC) to Renilla luciferase (REN) activity. The standard deviation (SD) was calculated from three independent replicates. One‐way analysis of variance (Tukey's test) was carried out. Asterisks indicate significant differences at *p < 0.05, **p < 0.01. (f) Chromatin immunoprecipitation–quantitative PCR assays were carried out to demonstrate that VqAL4 binds to the promoter of VqNSTS4 via the CACCTC/GAGGTG element. The SD was calculated from three independent replicates. Asterisks indicate significant differences (*p < 0.05, **p < 0.01, Student's t test).

FIGURE 5

Transient expression of transcription factor VqAL4 in Vitis quinquangularis accession Danfeng‐2 promoted transcription of VqNSTS4 and antifungal stilbene accumulation. (a) Schematic diagram of overexpression and RNAi vectors. (b, c) Transient transformation of 35S‐VqAL4‐GFP and RNAi‐VqAL4‐GFP in Danfeng‐2 leaves using the Agrobacterium vacuum infiltration method. (d) Western blot assays were carried out to determine the expression of 35S‐VqAL4‐GFP in transiently transformed Danfeng‐2 leaves. EV, the empty vector pCAMBIA2300‐35S‐GFP was used as the control. (e) Western blot assays were carried out to determine the expression of RNAi‐VqAL4‐GFP in transiently transformed Danfeng‐2 leaves. EV, the empty vector pK7GWIWG2(II)‐35S‐GFP was used as the control. (f, i) Reverse transcription‐quantitative PCR analysis was conducted to determine the relative expression levels of VqAL4 and VqNSTS4 in transiently transformed Danfeng‐2 leaves. (g, j) Determination of five stilbene contents in transiently transformed Danfeng‐2 leaves. (h, k) Free salicylic acid (SA) content in transiently transformed Danfeng‐2 leaves. The standard deviation (SD) was calculated from three independent replicates. One‐way analysis of variance (Tukey's test) was carried out. Asterisks indicate significant differences at *p < 0.05, **p < 0.01.

FIGURE 6

Location and structural analysis of VqAL4 isolated from Vitis quinquangularis accession Danfeng‐2. (a) Chromosomal localization of VqAL4. (b) Phylogenetic analysis of VqAL4 and the Alfin‐like proteins in Vitis vinifera, Brassica rapa, and Arabidopsis thaliana. (c) Multiple sequence alignment of amino acid sequences of VqAL4 and other group IV members, including VvAL4, VvAL1, VvAL3, AtAL4, BrAL3, BrAL7, and BrAL10. (d) Subcellular localization of VqAL4 in A. thaliana protoplasts. (e) Transcriptional activation of VqAL4 in yeast. pGADT7‐T/pGBKT7‐53 was used as a positive control, and pGADT7‐T/pGBKT7‐Lam was used as a negative control.

FIGURE 7

Overexpression of VqAL4 in grapevine enhances resistance to Uncinula necator by promoting stilbene accumulation and activating expression of resistance genes. (a) Photograph of VqAL4 transgenic lines and wild‐type Thompson Seedless before U. necator inoculation. Bars = 1 cm. (b) Photograph of leaves of VqAL4 transgenic lines and wild‐type Thompson Seedless at 7 days postinoculation (dpi). Bars = 1 cm. (c) Trypan blue staining and 3,3′‐diaminobenzidine (DAB) staining show the hyphal growth of U. necator and H₂O₂ accumulation at 1, 3, 5, and 7 dpi. c, conidium; ap, appressorium; ph, primary hypha; sh, secondary hypha. Bars = 100 μm. (d) Scanning electron micrographs of the hyphae and appressoria (ap) of U. necator in VqAL4 transgenic lines and wild‐type Thompson Seedless. Upper figures, bars = 100 μm; lower figures, bars = 20 μm. (e) Aniline blue staining showing callose depositions in U. necator‐infected epidermal cells at 7 dpi. Bars = 50 μm. (f) Quantification of spores per mg fresh leaves from VqAL4 transgenic lines and wild‐type Thompson Seedless at 7 dpi. (g) H₂O₂ content of VqAL4 transgenic lines and wild‐type Thompson Seedless leaves at 7 dpi. (h) Free salicylic acid (SA) content in the leaves of VqAL4 transgenic lines and wild‐type Thompson Seedless at 0 dpi and 7 dpi. (i) Determination of five stilbene contents in the leaves of VqAL4 transgenic lines and wild‐type Thompson Seedless at 7 dpi. (j, k) Reverse transcription‐quantitative PCR (RT‐qPCR) analysis was conducted to determine the relative transcript levels of H₂O₂‐related genes in VqAL4 transgenic lines after U. necator inoculation. WT, wild‐type Thompson Seedless. (l–n) RT‐qPCR analysis was conducted to determine the relative transcript levels of SA‐related genes in VqAL4 transgenic mutants after U. necator inoculation. The standard deviation (SD) was calculated from three independent replicates. One‐way analysis of variance (Tukey's test) was carried out. Asterisks indicate significant differences at *p < 0.05, **p < 0.01.

FIGURE 8

Transient silencing of VqAL4 in leaves of Danfeng‐2 reduces resistance to Uncinula necator. (a) Phenotypes of control EV and transiently silenced RNAi‐VqAL4‐GFP Danfeng‐2 leaves at 1 day postinoculation (dpi) and 7 dpi. EV, the empty vector pK7GWIWG2(II)‐35S‐GFP. (b) Trypan blue staining of leaves from EV and transiently silenced RNAi‐VqAL4‐GFP leaves at 1 dpi and 7 dpi. Bars = 100 μm. (c) Quantification of spores per mg fresh leaves from EV and transiently silenced RNAi‐VqAL4‐GFP leaves at 7 dpi. (d) Free salicylic acid (SA) content in the leaves of EV and transiently silenced RNAi‐VqAL4‐GFP leaves at 7 dpi. (e–g) Reverse transcription‐quantitative PCR analysis was conducted to determine the relative transcript levels of SA‐related genes in EV and transiently silenced RNAi‐VqAL4‐GFP leaves following U. necator inoculation. The standard deviation (SD) was calculated from three independent replicates. One‐way analysis of variance (Tukey's test) was carried out. Asterisks indicate significant differences at *p < 0.05, **p < 0.01.

FIGURE 9

Proposed model of VqNSTS4‐mediated disease resistance to Uncinula necator and its regulatory mechanism. U. necator infection induces expression of VqNSTS4 and VqAL4. VqAL4 positively regulates the transcription of VqNSTS4. VqNSTS4 or VqAL4 transgenic grapevines show enhanced resistance to U. necator by producing more phytoalexin and activating SA signalling. ROS, reactive oxygen species. SA, salicylic acid.