Characterization of Vitis vinifera NPR1 homologs involved in the regulation of pathogenesis-related gene expression

Abstract

Background: Grapevine protection against diseases needs alternative strategies to the use of phytochemicals, implying a thorough knowledge of innate defense mechanisms. However, signalling pathways and regulatory elements leading to induction of defense responses have yet to be characterized in this species. In order to study defense response signalling to pathogens in Vitis vinifera, we took advantage of its recently completed genome sequence to characterize two putative orthologs of NPR1, a key player in salicylic acid (SA)-mediated resistance to biotrophic pathogens in Arabidopsis thaliana.

Results: Two cDNAs named VvNPR1.1 and VvNPR1.2 were isolated from Vitis vinifera cv chardonnay, encoding proteins showing 55% and 40% identity to Arabidopsis NPR1 respectively. Constitutive expression of VvNPR1.1 and VvNPR1.2 monitored in leaves of V. vinifera cv chardonnay was found to be enhanced by treatment with benzothiadiazole, a SA analog. In contrast, VvNPR1.1 and VvNPR1.2 transcript levels were not affected during infection of resistant Vitis riparia or susceptible V. vinifera with Plasmopara viticola, the causal agent of downy mildew, suggesting regulation of VvNPR1 activity at the protein level. VvNPR1.1-GFP and VvNPR1.2-GFP fusion proteins were transiently expressed by agroinfiltration in Nicotiana benthamiana leaves, where they localized predominantly to the nucleus. In this system, VvNPR1.1 and VvNPR1.2 expression was sufficient to trigger the accumulation of acidic SA-dependent pathogenesis-related proteins PR1 and PR2, but not of basic chitinases (PR3) in the absence of pathogen infection. Interestingly, when VvNPR1.1 or AtNPR1 were transiently overexpressed in Vitis vinifera leaves, the induction of grapevine PR1 was significantly enhanced in response to P. viticola.

Conclusion: In conclusion, our data identified grapevine homologs of NPR1, and their functional analysis showed that VvNPR1.1 and VvNPR1.2 likely control the expression of SA-dependent defense genes. Overexpression of VvNPR1 has thus the potential to enhance grapevine defensive capabilities upon fungal infection. As a consequence, manipulating VvNPR1 and other signalling elements could open ways to strengthen disease resistance mechanisms in this crop species.

Figure 1

Comparison of VvNPR1.1 and VvNPR1.2 with other NPR1 homologs and members of Arabidopsis thaliana NPR family. (A) Phylogenetic tree generated with the Phylo_win program using the neighbour-joining method [44]. Sequence alignment was previously realized using the ClustalW tool. Accession numbers are: AtNPR1 (At1g64280), AtNPR2 (At4g26120), AtNPR3 (At5g45110), AtNPR4 (At4g19660), AtBOP1 (At3g57130), AtBOP2 (At2g41370), Nicotiana tabacum (NtNPR1, AAM62410.1), Oryza sativa cv. japonica (OsNPR1, AAX18700.1), Lycopersicon esculentum (LeNPR1, AAT57637.1), Musa acuminata (MNPR1A, ABI93182.1; MNPR1B, ABL63913.1), Malus × domestica (MpNPR1-1, ACC77697.1) and Vitis vinifera (Genoscope accession numbers: VvNPR1.1, GSVIVP00016536001; VvNPR1.2, GSVIVP00031933001). Bootstrap values based on 500 replicates are indicated beside the branches. (B) Schematic representation comparing the structure of AtNPR1, VvNPR1.1 and VvNPR1.2, including the positions of the BTB/POZ domain, the ankyrin repeat domain (ARD) and the nuclear localization signals (NLS). (C) Multiple alignment of putative nuclear localization signals (NLS) at C-terminus of NPRs from different plant species. Basic amino acids are highlighted in grey and residues essential for AtNPR1 nuclear localization [30] are highlighted in black.

Figure 2

Expression patterns of VvNPR1.1 and VvNPR1.2 upon BTH treatment. Detached leaves of Vitis vinifera cv Chardonnay were sprayed with a solution of BTH (80 mg.L^-1) or water as control. Samples were collected at different time points. Hpt: hours post treatment; 0: untreated leaves at the beginning of the experiment. Actin (VvACT) was used as an internal control. Primer sequences are listed in table 1.

Figure 3

Expression patterns of VvNPR1.1 and VvNPR1.2 during a compatible or an incompatible interaction between grapevine and Plasmopara viticola. Leaves of plantlets of Vitis vinifera cv Chardonnay (grey bars) and Vitis riparia cv Gloire de Montpellier (dark bars) were inoculated with Plasmopara viticola (1.5 × 10⁵ spores mL^-1). Control leaves were sprayed with water. Leaves were collected at different time points as indicated. Hpi: Hours post inoculation. Transcript levels of each gene (Stilbene synthase VvSTS (A); VvNPR1.1 (B); VvNPR1.2 (C)) were normalized to actin transcript levels. The fold induction indicates normalized expression levels in inoculated leaves compared to normalized expression levels observed in water-treated leaves at the same time point. Expression ratio at the beginning of the experiment (0) is set to 1. Mean values and standard deviations were obtained from 2 duplicate experiments.

Figure 4

Subcellular localization of VvNPR1.1 and VvNPR1.2. N. benthamiana leaves were infiltrated with A. tumefaciens GV3101 containing empty vector (pK7FWG2) encoding free GFP (A, B), or AtNPR1 (C, D), VvNPR1.1 (E, G, H), and VvNPR1.2 (F) in pK7FWG2. Confocal images were captured 3 days after infiltration. GFP images (A, C, E, F, G) and differential contrast images (B, D, H) of N. benthamiana epidermal cells were compared to show the subcellular localization of GFP, AtNPR1-GFP, VvNPR1.1-GFP and VvNPR1.2-GFP. Bar = 10 μM.

Figure 5

Induction of PR1 and PR2 accumulation in N. benthamiana by transient expression of VvNPR1.1 and VvNPR1.2. N. benthamiana leaves were infiltrated with water (H₂O) or A. tumefaciens GV3101 containing VvNPR1.1, VvNPR1.2, or AtNPR1 in pK7FWG2 or empty vector. Leaves were harvested 3 days after agroinfiltration. Soluble proteins were extracted, submitted to SDS-PAGE and probed with sera against tobacco PR1, PR2 or basic chitinases (PR3).

Figure 6

Detection of AtNPR1 and VvNPR1.1 transgene expression in grapevine leaves. Leaves from in vitro grown V. vinifera cv Syrah were infiltrated with A. tumefaciens transformed with pBIN+ carrying AtNPR1 or VvNPR1.1. Control plants were infiltrated with water. Infiltrated leaves were challenged with P. viticola 3 days after agroinfiltration. Total RNAs were extracted 3 days after agro-infiltration (uninoculated) and 2 days after P. viticola inoculation. Full-lenght mRNA from each transgene was specifically amplified after reverse transcription with primers listed in table 1. VvACT was used as internal control.

Figure 7

Expression of VvPR1 and VvSTS after transient overexpression of AtNPR1 and VvNPR1.1 in grapevine leaves. (A, B) Expression levels of VvPR1 (A) and VvSTS (B), in uninoculated leaves, 3 days after agro-infiltration. (C, D) Expression levels of VvPR1 (C) and VvSTS (D) in uninoculated and inoculated leaves. Leaves were infiltrated with Agrobacterium carrying the different constructs and expression of VvPR1 and VvSTS was analyzed 3 days later (grey bars). Three days after agroinfiltration, leaves were inoculated with P. viticola and expression of genes of interest was analyzed 2 days after inoculation (black bars). Fold induction indicates expression levels in agroinfiltrated leaves compared to the expression in non-inoculated water-infiltrated leaves, which is set to 1. Mean values and standard deviations were obtained from 2 duplicate experiments.