Transcriptional regulation of stilbene synthases in grapevine germplasm differentially susceptible to downy mildew

Abstract

Background: To limit the impact of the downy mildew disease of grapevine and reduce the need to recur to chemical treatments, an effective strategy might be recovering adaptive resistance traits in both cultivated and wild V. vinifera germplasm. Considering that stilbenes represent the most important class of phytoalexins in the Vitaceae, the constitutive expression and transcriptional activation of all the functional members of the stilbene synthase gene family were analysed in a group of nine grapevine genotypes following artificial infection with the oomycete Plasmopara viticola, the causal agent of the disease. In addition, in the same genotypes we analyzed the expression of genes encoding for two transcription factors involved in the transcriptional regulation of the stilbene synthase genes, namely VvMYB14 and VvMYB15, and of genes encoding for chalcone synthases.

Results: Downy mildew incidence and severity ranged from nihil to high in the grapevine genotypes considered, being low to moderate in a subgroup of V. vinifera genotypes. The constitutive expression of the stilbene synthase genes as well as the extent of their transcriptional activation following P. viticola inoculation appeared to be inversely related to the proneness to develop disease symptoms upon infection. In a specular manner, following P. viticola inoculation all the chalcone synthase genes were up-regulated in the susceptible grapevine genotypes and down-regulated in the resistant ones. The infection brought by P. viticola appeared to elicit a co-ordinated and sequential transcriptional activation of distinct stilbene synthase genes subsets, each of which may be regulated by a distinct and specific MYB transcription factor.

Conclusions: The present results suggest that the induction of stilbene biosynthesis may contribute to the basal immunity against the downy mildew of grapevine, thus representing an adaptive resistance trait to recover, in both cultivated and wild V. vinifera germplasm. During the early stages of P. viticola infection, an antagonistic interaction between flavonol and stilbene biosynthesis might occur, whose outcome might determine the subsequent extent of disease symptoms. Further studies are needed to decipher the possible regulatory mechanisms involved in the antagonistic crosstalk between these two metabolic pathways in resistant and susceptible genotypes in response to P. viticola.

Keywords: Chalcone synthase; Downy mildew susceptibility; Grapevine germplasm; MYB14 and MYB15; Plant-pathogen interactions; Plasmopara viticola; Stilbene synthase; Vitis vinifera L.

Fig. 1

Mean values of disease incidence (plain bars, left axis, small letters) and severity (pattern bars, right axis, capital letters) evaluated in nine different Vitis vinifera genotypes 7 days after inoculation with Plasmopara viticola. Bars represent standard error of the mean. Different letters denote significant differences according to the Tukey’s test (p ≤ 0.05). Grapevine genotypes: CHA = Chasselas, ALE = Aleatico, CAN=Canaiolo nero, TRE = Trebbiano toscano, ROS = Rossetto, ROM = Romanesco, SYL = accession of V. vinifera subsp. sylvestris, ISA = Isabella, SOL = Solaris

Fig. 2

Phylogenetic tree of the deduced amino acid sequences of the Vitis vinifera stilbene synthase genes (VvSTS) identified in the NCBI database. The multiple alignment of 34 protein sequences (31 VvSTS and three V. vinifera chalcone synthases) was performed by ClustalX 1.83 software and the phylogenetic tree was constructed by the neighbor-joining (NJ) method and evaluated by bootstrap analysis (MEGA 7). The numbers on the main branches indicate bootstrap percentages for 1000 replicates. The three phylogenetic groups (A, B and C) identified in the grapevine STS family are highlighted with square brackets

Fig. 3

Relative expression levels of the group A Vitis vinifera stilbene synthase gene family members (VvSTS1/2) and of selected members of groups C and B in the leaves of the nine grapevine genotypes of Fig. 1 collected before inoculation with Plasmopara viticola. The expression data of each gene were normalized using the geometric average of the two reference genes VvEF1α and VvSAND. Relative expression levels of the different VvSTS genes were referred to a calibrator, set to the value 1, which was represented by the gene in the nine genotypes with the lowest expression (VvSTS7/8 in the cv. Aleatico, ALE). Each reported value is the mean ± SD of three biological replicates, each of which was analyzed in triplicate. Grapevine genotypes: CHA = Chasselas, ALE = Aleatico, CAN=Canaiolo nero, TRE = Trebbiano toscano, ROS = Rossetto, ROM = Romanesco, SYL = accession of V. vinifera subsp. sylvestris, ISA = Isabella, SOL = Solaris

Fig. 4

Relative expression levels of the whole set of the Vitis vinifera stilbene synthase genes (VvSTS) in the leaves of the nine grapevine genotypes of Fig. 1 collected before inoculation with Plasmopara viticola. The letter denoting the phylogenetic group to which each gene belongs is given in parentheses. For each gene, relative expression levels were calculated by setting a value of 1 for the lowest value among the nine genotypes. Normalization and sample replication as in Fig. 3. Different letters denote significant differences according to the Tukey’s test (p ≤ 0.01). Grapevine genotypes: CHA = Chasselas, ALE = Aleatico, CAN=Canaiolo nero, TRE = Trebbiano toscano, ROS = Rossetto, ROM = Romanesco, SYL = accession of V. vinifera subsp. sylvestris, ISA = Isabella, SOL = Solaris

Fig. 5

Relative expression levels of the whole set of the Vitis vinifera stilbene synthase genes (VvSTS) in the leaves of the nine grapevine genotypes of Fig. 1 collected up to 72 h after inoculation with P. viticola. The letter denoting the phylogenetic group to which each gene belongs is given in parentheses. For each gene, relative expression levels were calculated by setting a value of 1 for the lowest value among the nine genotypes in each of the five time points considered. Normalization and sample replication as in Fig. 3. For each gene, statistical evaluation of the differences among the nine grapevine genotypes and among the five sampling times is reported in Additional file 2: Table S6. Grapevine genotypes: CHA = Chasselas, ALE = Aleatico, CAN=Canaiolo nero, TRE = Trebbiano toscano, ROS = Rossetto, ROM = Romanesco, SYL = accession of V. vinifera subsp. sylvestris, ISA = Isabella, SOL = Solaris

Fig. 6

Relative gene expression levels of the genes coding for the two transcription factors VvMYB14 and VvMYB15 in the leaves of the nine grapevine genotypes of Fig. 1 collected either before inoculation with Plasmopara viticola (left panels) or up to 72 h after inoculation (right panels). For each gene, relative expression levels were calculated by setting a value of 1 for the lowest value among the nine genotypes in each of the five time points considered. Normalization and sample replication as in Fig. 3. For the left panels, different letters denote significant differences according to the Tukey’s test (p ≤ 0.01). In the right panels, statistical evaluation of the differences among the nine grapevine genotypes and among the five sampling times is reported in Additional file 3: Table S7. Grapevine genotypes: CHA = Chasselas, ALE = Aleatico, CAN=Canaiolo nero, TRE = Trebbiano toscano, ROS = Rossetto, ROM = Romanesco, SYL = accession of V. vinifera subsp. sylvestris, ISA = Isabella, SOL = Solaris

Fig. 7

Relative expression levels of the Vitis vinifera chalcone synthase genes (VvCHS) in the leaves of the nine grapevine genotypes of Fig. 1 collected either before inoculation with Plasmopara viticola (left panels) or up to 72 h after inoculation (right panels). For each gene, relative expression levels were calculated by setting a value of 1 for the lowest value among the nine genotypes in each of the five time points considered. Normalization and sample replication as in Fig. 3. For the left panels, different letters denote significant differences according to the Tukey’s test (p ≤ 0.01). In the right panels, statistical evaluation of the differences among the nine grapevine genotypes and among the five sampling times is reported in Additional file 3: Table S8. Grapevine genotypes: CHA = Chasselas, ALE = Aleatico, CAN=Canaiolo nero, TRE = Trebbiano toscano, ROS = Rossetto, ROM = Romanesco, SYL = accession of V. vinifera subsp. sylvestris, ISA = Isabella, SOL = Solaris