Low level of polymorphism in two putative NPR1 homologs in the Vitaceae family

Abstract

Background: Grapevine is subjected to numerous pests and diseases resulting in the use of phytochemicals in large quantities. The will to decrease the use of phytochemicals leads to attempts to find alternative strategies, implying knowledge of defence mechanisms. Numerous studies have led to the identification of signalling pathways and regulatory elements involved in defence in various plant species. Nonexpressor of Pathogenesis Related 1 (NPR1) is an important regulatory component of systemic acquired resistance (SAR) in Arabidopsis thaliana.

Results: Two putative homologs of NPR1 gene were found in the two sequenced grapevine genomes available in the Genoscope database for line 40024 and in the IASMA database for Pinot noir ENTAV 115. We named these two NPR1 genes of Vitis vinifera : VvNPR1.1 and VvNPR1.2. A PCR-based strategy with primers designed on exons was used to successfully amplify NPR1 gene fragments from different Vitaceae accessions. Sequence analyses show that NPR1.1 and NPR1.2 are highly conserved among the different accessions not only V. vinifera cultivars but also other species. We report nucleotide polymorphisms in NPR1.1 and NPR1.2 from fifteen accessions belonging to the Vitaceae family. The ratio of nonsynonymous to synonymous nucleotide substitutions determines the evolutionary pressures acting on the Vitaceae NPR1 genes. These genes appear to be experiencing purifying selection. In some of the species we have analysed one of the two alleles of NPR1.1 contains a premature stop codon. The deduced amino acid sequences share structural features with known NPR1-like proteins: ankyrin repeats, BTB/POZ domains, nuclear localization signature and cysteines. Phylogenetic analyses of deduced amino acid sequences show that VvNPR1.1 belongs to a first group of NPR1 proteins known as positive regulators of SAR and VvNPR1.2 belongs to a second group of NPR1 proteins whose principal members are AtNPR3 and AtNPR4 defined as negative regulators of SAR.

Conclusion: Our study shows that NPR1.1 and NPR1.2 are highly conserved among different accessions in the Vitaceae family. VvNPR1.1 and VvNPR1.2 are phylogenetically closer to the group of positive or negative SAR regulators respectively.

Figure 1

Schematic representation of VvNPR1.1 (A) and VvNPR1.2 (B). The positions of the BTB/POZ, ankyrin repeat domains, nuclear localization signature and conserved cysteine residues represented by stars are shown. Numbers represent amino acid positions.

Figure 2

Comparison of VvNPR1.1, VvNPR1.2 and putative Blade-on-petiole (VvBOP) with NPR proteins from Arabidopsis thaliana and other plant species. Deduced sequences of VvNPR1.1 and VvNPR1.2 were compared to sequences of proteins annotated as being homologs of AtNPR1 from different plant species. Accession numbers used in the alignments are listed in table 1. All sequences annotated as NPR1 or closely related to VvNPR1 genes after blast were recovered in the NCBI database. The numbers beside the branches represent bootstrap values based on 5,000 replicates. The scale at the top indicates genetic distance proportional to the number of substitutions per site.

Figure 3

Partial classification of Vitaceae [according to Gallet (1967) [41]].

Figure 4

Distribution of SNPs in NPR1.1 and NPR1.2 along exons 1 to 4. From Ampelopsis japonica, Muscadinia rotundifolia Carlos, Parthenocissus quinquefolia and five American Vitis species. The horizontal scale indicates the nucleotide number. The vertical scale indicates the number of SNPs counted at each position among the eight species in comparison to line 40024. Numbers represent missense substitutions.

Figure 5

Distribution of SNPs in NPR1.1 and NPR1.2 along exons 1 to 4. From six Vitis vinifera cultivars (Gouais blanc, Muscat reine des vignes, Cabernet Sauvignon, Riesling, Pinot noir, Gewurztraminer). The horizontal scale indicates the nucleotide number. The vertical scale indicates the number of SNPs counted at each position among the eight species in comparison to line 40024. Numbers represent missense substitutions.

Figure 6

Multiple alignment (clustal W) of VvNPR1.1 with AtNPR1, AtNPR2 and other NPR1 homologs. Vertical rectangles and stars represent conserved cysteine residues. The BTB/POZ domain is underlined. Dashes indicate gaps introduced to maximize alignments.

Figure 7

Multiple alignment (Clustal W) of VvNPR1.2 with AtNPR1, AtNPR3, AtNPR4 and other NPR1 homologs. Vertical rectangles and stars represent conserved cysteine residues. The BTB/POZ domain is underlined. Dashes indicate gaps introduced to maximize alignments.