Ectopic Expression in Arabidopsis thaliana of an NB-ARC Encoding Putative Disease Resistance Gene from Wild Chinese Vitis pseudoreticulata Enhances Resistance to Phytopathogenic Fungi and Bacteria

Abstract

Plant resistance proteins mediate pathogen recognition and activate innate immune responses to restrict pathogen proliferation. One common feature of these proteins is an NB-ARC domain. In this study, we characterized a gene encoding a protein with an NB-ARC domain from wild Chinese grapevine Vitis pseudoreticulata accession "Baihe-35-1," which was identified in a transcriptome analysis of the leaves following inoculation with Erysiphe necator (Schw.), a causal agent of powdery mildew. Transcript levels of this gene, designated VpCN (GenBank accession number KT265084), increased strongly after challenge of grapevine leaves with E. necator. The deduced amino acid sequence was predicted to contain an NB-ARC domain in the C-terminus and an RxCC-like domain similar to CC domain of Rx protein in the N-terminus. Ectopic expression of VpCN in Arabidopsis thaliana resulted in either a wild-type phenotype or a dwarf phenotype. The phenotypically normal transgenic A. thaliana showed enhance resistance to A. thaliana powdery mildew Golovinomyces cichoracearum, as well as to a virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Moreover, promoter::GUS (β-glucuronidase) analysis revealed that powdery mildew infection induced the promoter activity of VpCN in grapevine leaves. Finally, a promoter deletion analysis showed that TC rich repeat elements likely play an important role in the response to E. necator infection. Taken together, our results suggest that VpCN contribute to powdery mildew disease resistant in grapevine.

Keywords: VpCN; disease resistance; powdery mildew; promoter analysis; wild Chinese Vitis.

Figure 1

Sequence analysis of VpCN and transcript level detection. (A) Schematic map of VpCN location and major motifs. (B) Multiple sequence alignment of the NB, ARC1 and ARC2 subdomains of NB-ARC in VpCN with closely related proteins. Domain borders are indicate as vertical green lines. Motifs are labeled by horizontal dark lines below the aligned sequences. gi30173240 (Bent et al., 1994), gi46395604 (Bevan et al., 1998), gi46395938 (Theologis et al., 2000), gi75318159 (Ori et al., 1997), gi325511400 (Theologis et al., 2000) (C) Structural model of the NB-ARC domain of VpCN. (D) Phylogenetic tree of VpCN and related proteins from other plant species. The tree was generated using the ClustalW function in the MegAlign program: Vitis vinifera (GenBank accession no. XP010661747), Nelumbo nucifera (GenBank accession no. XP0102588251), Glycine soja (GenBank accession no. KHN19144), Elaeis guineensis (GenBank accession no. XP010913221), Solanum lycopersicum (GenBank accession no. XP010319316), Beta vulgaris subsp. Vulgaris (GenBank accession no. XP010669409), Phoenix dactylifera (GenBank accession no. XP008791188), Camelina sativa (GenBank accession no. XP010426119), Citrus sinensis (GenBank accession no. XP006470644). The scale bar represents 0.05 substitutions per site. (D) Structure model of NB-ARC in VpCN. (E) Analysis of VpCN expression in response to E. necator inoculation. The third to fifth fully expanded young grapevine leaves beneath the apex were selected for samples. The experiment encompass three independent biological replicates, for each biological replicate three leaves haversted from three plant and three technical replicates were performed. Data represent means of three biological replicates ±SE, asterisksin indicate statistical significance in comparison with control (Student'st-test, significance levels of ^*P < 0.05, ^**P < 0.01 are indicated).

Figure 2

Generation of CaMV 35S promoter-VpCN constructs used for transformation of Arabidopsis thaliana, morphology of wild type and transgenic Arabidopsis thaliana plants, with transgenic plants showing enhanced disease resistance to G. cichoracearum after ectopic expression of VpCN. (A) Structure of the CaMV 35S promoter-VpCN ectopic expression construct. LB, left border; RB, right border; 35S, CaMV 35S promoter; NOS, terminator; NPT II, aminoglycoside-3′- phosphotransferase. (B) Indicate T2 transgenic plants displayed either normal phenotypes or dwarfism. Blue arrows indicate the dwarf phenotype in 4 week old plants. (C) Transgenic A. thaliana leaves developed fungal spores 8 dpi with G. cichoracearum. (D) Disease symptoms developed on the leaves of transgenic lines and wild type plants 8 dpi with G. cichoracearum. (E) A. thaliana PR1 transcript levels in T3 lines and wild-type after inoculation with G. cichoracearum. Total RNA was extracted from A. thaliana leaves 0, 12, 24, 36, and 48 h post-inoculation (hpi) with G. Cichoracearum. The experiment encompass three independent biological replicates, for each biological replicate six rosette leaves were harvested from three plant and three technical replicates were performed. Data represent means of three biological replicates ±SE, asterisksin indicate statistical significance in comparison with WT (Student's t-test, significance levels of ^*P < 0.05, ^**P < 0.01 are indicated).

Figure 3

Ectopic expression of VpCNin Arabidopsis thaliana enhanced disease resistance to Pseudomonas syringae pv. tomato DC3000. (A) P. st DC3000 was diluted to OD₆₀₀ 0.02 and injected into the middle of a leaf with needleless syringes. The injected leaves were marked with white pipette tips, and pictures taken 3 dpi. (B) Disease symptoms developed on the leaves of transgenic lines and wild type plants 3 dpi with P.st DC3000. (C) Transgenic plants and wild type leaves were stained with trypan blue 12 hpi with P.st DC3000. (D) Transgenic plants and wild type leaves were stained with nitro blue terazolium (NBT). (E) Microscopic observation of callose deposition after 3 dpi. Bars = 50 μm. (F) The numbers of bacterial cells in the leaves were determined at 3 and 5 dpi. (G) Detection of H₂O₂ concentration in Arabidopsis leaf samples harvested at 24 hpi. (H) Quantification of dead cells at 12 hpi. (I) Quantification of callose from A. thaliana leaves at 3 dpi. The experiment encompass three independent biological replicates, for each biological replicate six rosette leaves were harvested from three plant and three technical replicates were performed. Data represent means of three biological replicates ±SE, asterisksin indicate statistical significance in comparison with WT (Student's t-test, significance levels of ^*P < 0.05, ^**P < 0.01 are indicated).

Figure 4

The main predicted cis-acting elements in the pVpCN promoter sequence, structure of the VpCN promoter fused to the GUS reporter gene and GUS staining of the transient constructs in transformed grapevine leaves. (A) Schematic diagram of the main predicted cis-acting elements in the VpCN promoter sequence of Chinese wild V. pseudoreticulata. (B) The pVpCN promoter was fused to the GUS gene. The plasmid pCaMV35S:GUS was used as a positive control and pC0380:GUS was used as a negative control. (C) The fully expanded grapevine leaves of V. vinifera “Red globe” were collect from a grape germplasm resources orchard and used for agroinfiltration.

Figure 5

Schematic map of the pVpCN promoter-GUS gene fusion deletion constructs, histochemical analysis of GUS expression in transiently transformed V. vinifera “Red globe” leaves after inoculation with E.necator, and fluorometric analysis of GUS activity in the transiently transformed grapevine leaves. (A) The GUS gene was driven by the VpCN promoter deletions, the exact locations of the promoter fragments are shown in Supplement Figure 2. The deletion size is indicated at the far right. (B) GUS staining was carried out 2 days after treatment with sterile water (upper panel) or E. necator (lower panel). (C) The various deletion fragments of the VpCN promoter fused to GUS and relative GUS activity driven in the transiently transformed grapevine leaves. The dark bars indicate the average GUS activity for deletion constructs in transiently transformed grapevine leaves treated with E. necator, the gray bars indicate the mock treatment (sterile water). Numbers adjacent to the bars indicate the fold difference in GUS activity leaves harboring the various constructs challenged with E. necator relative to the mock samples. The mean GUS activity (±SD) is averaged from three independent experiments (n = 3), the errors bars indicate the stand deviation. Significant difference between treatment and mock conditions was analyzed using one sided paired t-test (^** and ^* meaning P < 0.0.1 or P < 0.05, respectively).