Interfamily transfer of tomato Ve1 mediates Verticillium resistance in Arabidopsis

Abstract

Vascular wilts caused by soil-borne fungal species of the Verticillium genus are devastating plant diseases. The most common species, Verticillium dahliae and Verticillium albo-atrum, have broad host ranges and are notoriously difficult to control. Therefore, genetic resistance is the preferred method for disease control. Only from tomato (Solanum lycopersicum) has a Verticillium resistance locus been cloned, comprising the Ve1 gene that encodes a receptor-like protein-type cell surface receptor. Due to lack of a suitable model for receptor-like protein (RLP)-mediated resistance signaling in Arabidopsis (Arabidopsis thaliana), so far relatively little is known about RLP signaling in pathogen resistance. Here, we show that Ve1 remains fully functional after interfamily transfer to Arabidopsis and that Ve1-transgenic Arabidopsis is resistant to race 1 but not to race 2 strains of V. dahliae and V. albo-atrum, nor to the Brassicaceae-specific pathogen Verticillium longisporum. Furthermore, we show that signaling components utilized by Ve1 in Arabidopsis to establish Verticillium resistance overlap with those required in tomato and include SERK3/BAK1, EDS1, and NDR1, which strongly suggests that critical components for resistance signaling are conserved. We subsequently investigated the requirement of SERK family members for Ve1 resistance in Arabidopsis, revealing that SERK1 is required in addition to SERK3/BAK1. Using virus-induced gene silencing, the requirement of SERK1 for Ve1-mediated resistance was confirmed in tomato. Moreover, we show the requirement of SERK1 for resistance against the foliar fungal pathogen Cladosporium fulvum mediated by the RLP Cf-4. Our results demonstrate that Arabidopsis can be used as model to unravel the genetics of Ve1-mediated resistance.

Figure 1.

Transgenic expression of Ve1, but not of Ve2, mediates Verticillium resistance in Arabidopsis. Arabidopsis engineered to express tomato CaMV 35S-driven Ve1 or Ve2 (P35S:Ve1 and P35S:Ve2). A, Typical appearance of nontransgenic control and transgenic lines upon mock inoculation or inoculation with race 1 or race 2 strains of V. dahliae and V. albo-atrum (V. a-a) at 21 d after inoculation. B, Typical appearance of nontransgenic Ws-0 control and P35S:Ve1 at 21 d after inoculation with four V. longisporum strains (1–4). [See online article for color version of this figure.]

Figure 2.

Transgenic expression of Ve1, but not of Ve2, reduces Verticillium wilt symptoms and fungal biomass upon inoculation with V. dahliae race 1. Quantification of Verticillium wilt symptoms (Sympt.) in Arabidopsis Col-0 engineered to express CaMV 35S-driven tomato Ve1 (A) or Ve2 (B) at 21 d after inoculation. Bars represent quantification of symptom development as percentage of diseased rosette leaves with sd. Col-0 (control) is set to 100%. Fungal biomass determined by quantitative real-time PCR (R.Q.) in Arabidopsis Col-0 engineered to express CaMV 35S-driven Ve1 (C) or Ve2 (D). Bars represent Verticillium ITS transcript levels relative to Arabidopsis Rubisco transcript levels (for equilibration) with sd in a sample of four pooled plants. Col-0 (control) is set to 100%. A to D, Two transgenic lines per construct are shown (1 and 2). Asterisks indicate significant differences when compared with Col-0 (P < 0.05).

Figure 3.

Expression of Ve1, but not of Ve2, driven by their respective tomato native promoters reduces Verticillium wilt disease in Arabidopsis. Arabidopsis Col-0 engineered to express tomato Ve1 or Ve2 driven by their respective native promoters (PVe1:Ve1 and PVe2:Ve2, respectively). A, Typical appearance of nontransgenic Col-0 (control) and transgenic lines upon mock inoculation or inoculation with V. dahliae race 1 at 21 d after inoculation. Quantification of Verticillium wilt symptoms (Sympt.) in Arabidopsis Col-0 engineered to express tomato Ve1 (B) or Ve2 (C). Bars represent quantification of symptom development shown as percentage of diseased rosette leaves with sd. Col-0 (control) is set to 100%. Fungal biomass determined by quantitative real-time PCR (R.Q.) in Arabidopsis Col-0 engineered to express tomato Ve1 (D) or Ve2 (E). Bars represent Verticillium ITS transcript levels relative to Arabidopsis Rubisco transcript levels (for equilibration) with sd in a sample of four pooled plants. Col-0 (control) is set to 100%. B to E, Two transgenic lines are shown per construct (1 and 2). Asterisks indicate significant differences when compared with Col-0 (P < 0.05). [See online article for color version of this figure.]

Figure 4.

Quantification of V. dahliae biomass in Arabidopsis defense signaling mutants. Fungal biomass was determined by quantitative real-time PCR (R.Q.) in Col-0 and defense signaling mutants at 21 d after inoculation. Mutants that show enhanced (A) or reduced (B) susceptibility towards V. dahliae. C, Mutants for which fungal biomass is comparable to Col-0. A to C, Bars represent Verticillium ITS transcript levels relative to Arabidopsis Rubisco transcript levels (for equilibration) with sd in a sample of four pooled plants. Col-0 is set to 100%. Asterisks indicate significant differences when compared with Col-0 (P < 0.05).

Figure 5.

Ve1-mediated reduction of V. dahliae biomass in defense signaling mutants. Mutants for which Ve1-mediated resistance is compromised (A) or not compromised (B). Fungal biomass was determined by quantitative real-time PCR and represents Verticillium ITS transcript levels relative to Arabidopsis Rubisco transcript levels (for equilibration). Bars represent the percentage of Ve1-mediated fungal biomass reduction (B.R.) in Ve1-expressing lines when compared to the fungal biomass accumulated in the respective nontransformed progenitors, with sd in a sample of four pooled plants. Ve1-mediated fungal biomass reduction in Col-0 is set to 100%. Two independent transgenic lines expressing Ve1 are shown per construct (1 and 2). Asterisks indicate significant differences when compared with Col-0 (P < 0.05).

Figure 6.

Overview of Ve1-transgenic Serk mutants challenged with V. dahliae race 1. A, Typical appearance of nontransgenic (top row) and Ve1-transgenic (bottom row; P35S:Ve1) Col-0 and Serk mutant plants at 21 d after Verticillium inoculation. B, Quantification of V. dahliae biomass in nontransgenic Serk mutants when compared with Col-0. Bars represent Verticillium quantification (R.Q.) with sd in a sample of four pooled plants. Col-0 is set to 100%. C, Ve1-mediated reduction of V. dahliae biomass in Serk mutants when compared with Col-0. Bars represent the percentage of Ve1-mediated fungal biomass reduction (B.R.) in Ve1-expressing lines when compared to the fungal biomass accumulated in the respective nontransformed progenitors, with sd in a sample of four pooled plants. B and C, Fungal biomass was determined by quantitative real-time PCR and represents Verticillium ITS transcript levels relative to Arabidopsis Rubisco transcript levels (for equilibration). Ve1-mediated fungal biomass reduction in Col-0 is set to 100%. Two independent transgenic lines expressing Ve1 are shown per construct (1 and 2). Asterisks indicate significant differences when compared with Col-0 (P < 0.05). [See online article for color version of this figure.]

Figure 7.

VIGS of SlSerk1 impairs Ve1-mediated Verticillium resistance and Cf-4-mediated Cladosporium resistance in tomato. A, Motelle (Ve1/Ve1; resistant) plants were treated with an empty recombinant TRV vector (TRV:00), a TRV vector targeting Ve1 (TRV:Ve1), the 3′-UTR of SlSerk1 (TRV:SlSerk1-UTR), or the CDS of SlSerk1 (TRV:SlSerk1-CDS). Two weeks after treatment, the plants were mock inoculated (control) or inoculated with a race 1 strain of V. dahliae. Photographs were taken 14 d after V. dahliae inoculation, and compromised resistance is shown by a stunted appearance of the V. dahliae-inoculated plants when compared with mock-inoculated control plants. B, Two weeks after V. dahliae inoculation, stem sections were plated, allowing fungal outgrowth as a measure for fungal colonization. Photographs were taken at 14 d after plating. C, Cf4 tomato plants were treated with a TRV vector targeting GUS (TRV:GUS) as a control or a TRV vector targeting the 3′-UTR of SlSerk1 (TRV:SlSerk1-UTR) and challenged with transgenic C. fulvum expressing GUS. Representative leaflets after destaining are shown, revealing that full C. fulvum resistance is compromised when Serk1 is targeted. D, Quantitation of fungal growth in TRV:GUS and TRV:SlSerk1-UTR treated plants. Bars represent the degree of fungal colonization, expressed as the ratio between blue and total leaf area, in leaves from four independent experiments with se. The asterisk indicates a statistically significant difference (P < 0.05). [See online article for color version of this figure.]