Transfer of tomato immune receptor Ve1 confers Ave1-dependent Verticillium resistance in tobacco and cotton

Abstract

Verticillium wilts caused by soilborne fungal species of the Verticillium genus are economically important plant diseases that affect a wide range of host plants and are notoriously difficult to combat. Perception of pathogen(-induced) ligands by plant immune receptors is a key component of plant innate immunity. In tomato, race-specific resistance to Verticillium wilt is governed by the cell surface-localized immune receptor Ve1 through recognition of the effector protein Ave1 that is secreted by race 1 strains of Verticillium spp. It was previously demonstrated that transgenic expression of tomato Ve1 in the model plant Arabidopsis thaliana leads to Verticillium wilt resistance. Here, we investigated whether tomato Ve1 can confer Verticillium resistance when expressed in the crop species tobacco (Nicotiana tabcum) and cotton (Gossypium hirsutum). We show that transgenic tobacco and cotton plants constitutively expressing tomato Ve1 exhibit enhanced resistance against Verticillium wilt in an Ave1-dependent manner. Thus, we demonstrate that the functionality of tomato Ve1 in Verticillium wilt resistance through recognition of the Verticillium effector Ave1 is retained after transfer to tobacco and cotton, implying that the Ve1-mediated immune signalling pathway is evolutionary conserved across these plant species. Moreover, our results suggest that transfer of tomato Ve1 across sexually incompatible plant species can be exploited in breeding programmes to engineer Verticillium wilt resistance.

Keywords: cell surface receptor; pathogen resistance; receptor-like protein; vascular wilt; verticillium dahliae.

Figure 1

Generation and characterization of Ve1‐transgenic Nicotiana tabacum lines. (a) Schematic representation of the T‐DNA region of the binary vector pSol2095_Ve1 used for tobacco transformation. CaMV35Spro: CaMV35S promoter, eGFP: enhanced green fluorescent protein, CaMV35Ster: CaMV35S terminator; Kan ^r : kanamycin resistance gene, LB and RB: left and right T‐DNA borders, respectively. (b) Typical appearance of 6‐week‐old plants of four independent tobacco lines (Ve1 #0, #1, #2 and #3). (c) Accumulation of eGFP‐tagged Ve1 protein (~144 kDa) in leaves of the four independent Ve1 transgenic tobacco lines and wild‐type tobacco cv. Samsun transiently expressing the eGFP‐tagged Ve1 fusion protein (Ve1‐eGFP). Total protein extracts of transformed leaf tissue were subjected to immunoprecipitation (IP) using α‐GFP affinity beads. Proteins were subjected to sodium dodecyl sulphate–polyacrylamide electrophoresis (SDS/PAGE) and immunoblotted (IB) using α‐GFP antibody. Coomassie blue staining (CBS) of the blot containing total protein extracts is shown as a loading control based on the 50‐kDa RuBisCo (ribulose‐1,5‐bisphosphate carboxylase/oxygenase) band.

Figure 2

Tobacco plants expressing Ve1 show Ave1‐triggered resistance against Verticillium nonalfalfae. (a) Typical appearance of wild‐type tobacco cultivar Samsun plants (WT) and three independent Ve1 transgenic tobacco plants (#1, #2 and #3) that were engineered to express tomato Ve1 upon mock inoculation, inoculation with Ave1‐carrying V. nonalfalfae Vna5431 or an Ave1 deletion strain of V. nonalfalfae Vna5431 (ΔAve1) at 21 days postinoculation (dpi). Inoculation experiments were performed with at least 16 plants for each fungal strain and independently repeated three times. (b) Quantification of Verticillium‐induced plant stunting at 21 dpi. Bars represent averages with standard deviation. Different letter labels indicate statistically significant differences (Student's t‐test; P < 0.05). (c) Fungal biomass as determined with real‐time PCR at 21 dpi. Bars represent Verticillium ITS levels relative to tobacco actin levels (for equilibration) with standard deviation in a sample of three pooled plants. The fungal biomass in tobacco cv. Samsun plants upon inoculation with the wild‐type V. nonalfalfae strain Vna5431 is set to 100%. Asterisks indicate statistically significant differences when compared with tobacco cv. Samsun plants upon inoculation with the wild‐type V. nonalfalfae strain Vna5431 (Student's t‐test; P < 0.05). The data shown are representative of three independent experiments.

Figure 3

Tobacco plants expressing Ve1 exhibit Ave1‐triggered resistance against Verticillium alfalfae. (a) Typical appearance of wild‐type tobacco cultivar Samsun plants (WT) and three independent Ve1 transgenic tobacco plants (#1, #2 and #3) that were engineered to express tomato Ve1 upon mock inoculation, inoculation with V. alfalfae Va2 or two Ave1‐expressing strains of V. alfalfae Va2 (pA ve1::Ave1 #1 and #2) at 14 dpi. Inoculation experiments were performed with at least 16 plants for each fungal strain and independently repeated three times. (b) Quantification of the canopy area of tobacco plants at 14 dpi. Bars represent averages with standard deviation. Different letter labels indicate statistically significant differences (Student's t‐test; P < 0.05). (c) Fungal biomass as determined with real‐time PCR at 14 dpi. Bars represent Verticillium ITS levels relative to tobacco actin levels (for equilibration) with standard deviation in a sample of three pooled plants. The fungal biomass in tobacco cv. Samsun plants upon inoculation with the wild‐type V. alfalfae strain Va2 is set to 100%. Asterisks indicate statistically significant differences when compared with tobacco cv. Samsun plants upon inoculation with the wild‐type V. alfalfae strain Va2 (Student's t‐test; P < 0.05). The data shown are representative of three independent experiments.

Figure 4

Cotton plants expressing Ve1 display Ave1‐triggered resistance against Verticillium dahliae. (a) Typical appearance of wild‐type cotton cultivar YZ‐1 plants (WT) and two independent Ve1 transgenic cotton plants (Ve1‐4 and Ve1‐6) upon mock inoculation, inoculation with Ave1‐carrying V. dahliae V4 or an Ave1 deletion strain of V. dahliae V4 (ΔAve1) at 28 dpi. Inoculation experiments were performed with at least 20 plants for each fungal strain and independently repeated three times. (b) Quantification of Verticillium‐induced plant stunting at 28 dpi. Bars represent averages with standard deviation. Different letter labels indicate statistically significant differences (Student's t‐test; P < 0.05). (c) Fungal biomass as determined with real‐time PCR at 28 dpi. Bars represent Verticillium ITS levels relative to cotton ubiquitin levels (for equilibration) with standard deviation in a sample of three pooled plants. The fungal biomass in cotton cv. YZ‐1 plants upon inoculation with the wild‐type V. dahliae strain V4 is set to 100%. Asterisks indicate statistically significant differences when compared with cotton cv. YZ‐1 plants upon inoculation with the wild‐type V. dahliae strain V4 (Student's t‐test; P < 0.05). (d) Expression of tomato Ve1 in individual transgenic cotton plants and nontransgenic controls of cotton cv. YZ‐1 (WT) as detected with reverse transcription‐PCR (RT‐PCR). As an endogenous control, a fragment of the cotton ubiquitin gene (GhUB ) was amplified. The data shown are representative of three independent experiments.

Figure 5

Ve1‐transgenic and nontransgenic cotton plants are equally susceptible to Verticillium dahliae lacking Ave1. (a) Typical appearance of wild‐type cotton cultivar YZ‐1 plants (WT) and transgenic cotton plants expressing tomato Ve1 upon mock inoculation or inoculation with V. dahliae strain V991 at 28 dpi. Inoculation experiments were performed with at least 20 plants for V. dahliae strain V991 and independently repeated three times. (b) Expression of tomato Ve1 in individual cotton plants from wild‐type controls and transgenic lines as detected with reverse transcription‐PCR (RT‐PCR). As an endogenous control, a fragment of the cotton ubiquitin gene (GhUB ) was amplified. (c) Quantification of Verticillium‐induced plant stunting at 28 dpi. Bars represent averages with standard deviation. Different letter labels indicate statistically significant differences (Student's t‐test; P < 0.05). (d) Fungal biomass as determined with real‐time PCR at 28 dpi. Bars represent Verticillium ITS levels relative to cotton ubiquitin levels (for equilibration) with standard deviation in a sample of three pooled plants. The fungal biomass in cotton cv. YZ‐1 plants upon inoculation with the V. dahliae strain V991 is set to 100%. Same letter labels indicate no statistically significant differences (Student's t‐test; P > 0.05). The data shown are representative of three independent experiments.