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

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.

Introduction

In order to activate immune responses to ward off invading microorganisms, plants deploy immune receptors that detect pathogen invasion through sensing pathogen(‐induced) ligands (Cook et al., 2015; Dodds and Rathjen, 2010; Thomma et al., 2011). The recognition of such ligands results in the activation of defence responses, which are sometimes accompanied by a hypersensitive response (HR) in which plant tissue surrounding the site of attempted penetration is sacrificed to restrict further pathogen invasion. Based on structure and subcellular location, immune receptors fall into two major classes: cell surface‐localized receptors that comprise receptor kinases (RKs) and receptor‐like proteins (RLPs) that monitor the extracellular space, and cytoplasm‐localized nucleotide‐binding domain leucine‐rich repeat receptors (NLRs) that survey the intracellular environment (Rodriguez‐Moreno et al., 2017).

Verticillium wilts are vascular wilt diseases caused by soilborne fungal pathogens that belong to the Verticillium genus (Fradin and Thomma, 2006; Klimes et al., 2015). Although Verticillium wilt symptoms may vary considerably between plant hosts, the most frequently observed symptoms of Verticillium wilt include stunting, wilting, chlorosis, necrosis, vascular discoloration and early senescence (Fradin and Thomma, 2006). Within the Verticillium genus, V. dahliae is the most notorious pathogenic species that can infect hundreds of dicotyledonous hosts, including ecologically important plants and many high‐value crops worldwide (Fradin and Thomma, 2006; Klosterman et al., 2009). V. albo‐atrum, V. alfalfae, V. nonalfalfae and V. longisporum are also economically important vascular pathogens, albeit with narrower host ranges (Agrios, 2005; Depotter et al., 2016; Fradin and Thomma, 2006; Inderbitzin et al., 2011; Klosterman et al., 2009; Pegg and Brady, 2002).

Tobacco (Nicotiana tabacum) is an agriculturally and economically important Solanaceae crop that is grown in many regions worldwide. Verticillium wilt has been considered as a limiting factor for tobacco production globally, and is a serious disease of tobacco in New Zealand, Chile and Canada (Pegg and Brady, 2002), although the impact of Verticillium wilt on tobacco leaf yield and quality can be slightly reduced by careful farm practices. Verticillium wilt significantly impacts production of the cash crop cotton (Gossypium spp.) in cotton‐growing areas worldwide (Cai et al., 2009). To date, only few sea‐island cotton (Gossypium barbadense) cultivars have been shown to have some degree of resistance against Verticillium wilt (Cai et al., 2009), but sea‐island cotton is only cultivated on a small scale with <2% of cotton production globally (Chen et al., 2007). The most common cultivated cotton species, Gossypium hirsutum (upland cotton), is used for more than 95% of the annual cotton production worldwide (Chen et al., 2007). However, upland cotton cultivars with stably inherited resistance to Verticillium wilt are not available, and complex genetics of resistance to Verticillium wilt in upland cotton cultivars has limited the cross‐breeding of upland cotton cultivars with Verticillium wilt‐resistant sea‐island cotton germplasm (Cai et al., 2009). Therefore, Verticillium wilt‐resistant upland cotton cultivars are lacking (Cai et al., 2009).

Verticillium wilt diseases are difficult to control due to the long viability of the resting structures, the wide host range of the pathogens and the inability of fungicides to affect the pathogen once in the plant vascular system. The most sustainable manner to control Verticillium wilt diseases is the use of resistant cultivars (Fradin and Thomma, 2006; Klosterman et al., 2009). In tomato (Solanum lycopersicum), a single dominant locus that confers Verticillium resistance has been identified as the Ve locus, which controls Verticillium isolates that are assigned to race 1 whereas race 2 strains of Verticillium escape recognition (Pegg, 1974; Schaible et al., 1951). The Ve locus contains two closely linked and inversely oriented genes, Ve1 and Ve2, both of which encode extracellular leucine‐rich repeat (eLRR) RLPs (Kawchuk et al., 2001; Wang et al., 2010). Of these, only Ve1 was found to confer resistance against race 1 isolates of Verticillium in tomato (Fradin et al., 2009). Interestingly, interfamily transfer of Ve1 from tomato to Arabidopsis thaliana leads to race‐specific Verticillium resistance in the latter species (Fradin et al., 2011, 2014; Zhang et al., 2014), implying that the underlying immune signalling cascade is evolutionary conserved (Fradin et al., 2011; Thomma et al., 2011). Moreover, homologs of tomato Ve1 that have the potential to recognize race 1 strains of V. dahliae have been characterized in other diverse plant species, suggesting an ancient origin of the tomato immune receptor Ve1 (Song et al., 2017a).

Through comparative population genomics of race 1 and race 2 strains of V. dahliae, the effector protein that activates Ve1‐mediated immunity was identified as Ave1 (for Avirulence on Ve1 tomato) (de Jonge et al., 2012). No allelic variation was found among the identified Ave1 alleles from V. dahliae as well as from V. alfalfae and V. nonalfalfae (de Jonge et al., 2012; Song et al., 2017b). Interestingly, homologues of Ave1 were found in the bacterial plant pathogen Xanthomonas axonopodis pv. citri (XacPNP) and in the plant‐pathogenic fungi Colletotrichum higginsianum (ChAve1), Cercospora beticola (CbAve1), Fusarium oxysporum f. sp. lycopersici (FoAve1), as well as in plants (de Jonge et al., 2012). A few of these homologues are differentially recognized by tomato Ve1 in Nicotiana tabacum (de Jonge et al., 2012; Song et al., 2017b). Although the intrinsic function of Ave1 remains unknown, it is clear that Ave1 contributes to fungal virulence on susceptible plant genotypes (de Jonge et al., 2012).

Plant immune receptors are pivotal elements of the plant immune system that act as sentinels against pathogens. Engineering plants via transfer of immune components, such as plant immune receptors, have the potential to improve disease resistance in crops (Rodriguez‐Moreno et al., 2017). Previous reports showed that the transfer of individual cell surface immune receptors into crops confers enhanced disease resistance against diverse pathogens, including bacteria, fungi and oomycetes. For example, transfer of the Arabidopsis cell surface immune receptor EFR results in responsiveness to bacterial elongation factor Tu and bacterial resistance in tomato (Lacombe et al., 2010), rice (Lu et al., 2015; Schwessinger et al., 2015) and wheat (Schoonbeek et al., 2015). Similarly, introduction of the rice Xa21 confers bacterial resistance in sweet orange (Mendes et al., 2010), tomato (Afroz et al., 2011) and banana (Tripathi et al., 2014). Heterologous expression of the Nicotiana benthamiana FLS2 in citrus leads to increased disease resistance to citrus canker (Hao et al., 2016). Moreover, the Arabidopsis DORN1/LecRK‐I.9 enhances resistance to Phytophthora infestans in potato (Bouwmeester et al., 2014). Finally, ectopic expression of the Arabidopsis RLP23 in potato plants enhances immunity to the oomycete and fungal plant pathogens P. infestans and Sclerotinia sclerotiorum (Albert et al., 2015). In this study, we investigated whether the immune receptor gene Ve1 can confer Verticillium wilt resistance when transferred from tomato to the closely related crop species tobacco (N. tabacum cv. Samsun) and the distantly related crop species cotton (G. hirsutum).

Results

Generation of Ve1‐expressing Nicotiana tabacum plants

Previously, it was shown that co‐expression of Ve1 and Ave1 by agroinfiltration induces an HR in N. tabacum (Zhang et al., 2013a), suggesting that required signalling components acting downstream of tomato Ve1 are functionally conserved in tobacco. To further test whether tomato Ve1 can confer resistance to race 1 Verticillium spp., transgenic tobacco lines expressing tomato Ve1 were generated. The binary plasmid pSol2095_Ve1 encoding C‐terminally eGFP‐tagged Ve1 (Zhang et al., 2013a; Figure 1a) was transferred to N. tabacum cv. Samsun via Agrobacterium‐mediated transformation. Primary transformants were selected in tissue culture by their ability to regenerate in the presence of kanamycin, and eight independent T0 transformation events expressing Ve1 were obtained (Figure S1a).

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 : 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.

Intriguingly, 45 of 56 progeny (T1 plants) derived from the eight T0 transformation events were significantly smaller in size when compared to the parental line, while 11 plants displayed a normal stature (Figure S1b,c). We assumed these 45 are Ve1‐transgenic plants while the 11 correspond to segregating wild‐type plants. To assay whether Ve1 protein accumulated in these plants, we isolated proteins from a line (Ve1 #0) that exhibited normal growth and three lines (Ve1 #1, #2 and #3) that showed a dwarfed phenotype (Figure 1b) and performed immunoblotting analysis using anti‐GFP‐HRP antibody. This analysis showed that eGFP‐tagged Ve1 protein was clearly detected in the three dwarfed lines, but not in the line that displays a normal stature (Figure 1c), suggesting that dwarfing of these tobacco lines is due to Ve1 expression. Nevertheless, the progeny of these three dwarfed lines was used for further assays to assess the contribution of Ve1 expression to Verticillium wilt resistance.

Generation of isogenic Verticillium genotypes lacking or expressing Ave1

To identify Verticillium strains that can be used for inoculation assays on tobacco, six strains, comprising V. dahliae strains JR2 and St14.01, V. nonalfalfae strains Vna5431, CBS385.91 and Vna1, and V. alfalfae strain Va2 (Table S1) were inoculated onto wild‐type tobacco cv. Samsun plants, and Verticillium wilt symptoms were scored up to 14 days postinoculation (dpi). As anticipated, the various Verticillium strains caused different degrees of Verticillium wilt symptoms on these plants (Figure S2a). Among these six Verticillium genotypes, V. alfalfae strain Va2 and V. nonalfalfae strain Vna5431 caused the most severe disease symptoms (Figure S2a,b) and were selected for Verticillium wilt disease assays on tobacco. As V. nonalfalfae Vna5431 carries the Ave1 gene, it belongs to race 1, while V. alfalfae Va2 belongs to race 2 as it lacks Ave1 (Figure S2c).

To thoroughly investigate Ve1‐mediated Verticillium wilt resistance in tobacco, isogenic Verticillium genotypes lacking or expressing Ave1 are required. To this end, we deleted Ave1 from the genome of V. nonalfalfae Vna5431 and simultaneously introduced Ave1 into V. alfalfae Va2 genome. Subsequently, the wild‐type V. nonalfalfae strain Vna5431 and two independent Ave1 deletion mutants were used to inoculate Ve1 tomato plants and tomato plants that lack Ve1 (Figure S3a). As expected, targeted deletion of Ave1 resulted in gain of virulence on Ve1 tomato plants (Figure S3b,c). Moreover, these Ave1 deletion mutants displayed reduced virulence on tomato plants lacking Ve1 when compared to the corresponding wild‐type V. nonalfalfae strain Vna5431 (Figure S3b,d). These results show that, also for V. nonalfalfae strain, Vna5431 Ave1 acts as a virulence factor on tomato, and confirm that deletion of Ave1 leads to escape of Ve1‐mediated resistance.

Simultaneously, the wild‐type V. alfalfae strain Va2 and two independent Ave1 expression strains were inoculated onto tomato plants that express or lack Ve1 (Figure S4a). However, the wild‐type V. alfalfae strain Va2, as well as the two Ave1‐expressing V. alfalfae strains, failed to cause visible disease symptoms on tomato plants (Figure S4b,d,e), suggesting that the V. alfalfae strain Va2 does not have the capacity to infect tomato. Subsequently, we inoculated these strains on N. glutinosa, which is resistant to race 1 strains of Verticillium due to the occurrence of an endogenous Ve1 allele (Song et al., 2017a; Zhang et al., 2013a). As expected, V. alfalfae strain Va2 was able to infect N. glutinosa plants, while the Ave1 expression strains failed to cause infection (Figure S4c,f). These results reveal that ectopic expression of Ave1 in V. alfalfae strain Va2 can activate Ve1‐mediated resistance against Verticillium wilt.

Ave1 acts as a virulence factor on tobacco

It was previously determined that Ave1 acts as a virulence factor of V. dahliae on tomato and A. thaliana (de Jonge et al., 2012). To investigate the contribution of Ave1 to Verticillium virulence on tobacco, isogenic Ave1 mutants and the corresponding wild‐type Verticillium strain were inoculated onto tobacco cv. Samsun plants. Interestingly, Ave1 deletion strains of V. nonalfalfae Vna5431 displayed significantly reduced virulence on tobacco plants when compared with the corresponding wild‐type V. nonalfalfae strain Vna5431 at 21 dpi (Figure S5a), as inoculation with the Ave1 deletion strains resulted in reduced stunting (Figure S5b) and compromised fungal colonization (Figure S5c). Conversely, the two Ave1 expression strains in V. alfalfae Va2 showed clearly increased aggressiveness on tobacco plants when compared with the corresponding wild‐type strain (Figure S5d–f). These experiments show that Ave1 acts as a virulence factor on tobacco plants.

Tomato Ve1 confers Ave1‐dependent Verticillium wilt resistance in tobacco

To test whether constitutive Ve1 expression in tobacco confers resistance against Verticillium wilt in an Ave1‐dependent manner, three independent Ve1‐transgenic lines (Ve1 #1, #2 and #3) as well as nontransgenic control plants were challenged with either the wild‐type race 1 V. nonalfalfae strain Vna5431, or an Ave1 deletion mutant (V. nonalfalfae Vna5431ΔAve1) and inspected for Verticillium wilt symptoms up to 21 dpi. Interestingly, Ve1‐transgenic tobacco plants were clearly more resistant to the race 1 V. nonalfalfae strain Vna5431, as significantly fewer Verticillium wilt symptoms developed when compared with nontransgenic controls (Figure 2a,b). Importantly, despite the fact that the Ave1 deletion mutant of V. nonalfalfae Vna5431 displays compromised virulence on wild‐type tobacco plants, Ve1‐transgenic tobacco plants were clearly susceptible to this Ave1 deletion mutant (Figure 2a,b). The phenotypes correlated with the degree of fungal colonization as determined by real‐time PCR (Figure 2c). Additionally, the three independent Ve1‐transgenic tobacco lines and nontransgenic controls were inoculated with either the wild‐type V. alfalfae strain Va2, or the two independent Ave1 expression strains, and monitored for the development of Verticillium wilt symptoms at 14 dpi. Intriguingly, upon inoculation with the Ave1 expression strains, no symptoms of Verticillium wilt were observed on the Ve1‐expressing tobacco plants, whereas the nontransgenic controls displayed clear symptoms of Verticillium wilt (Figure 3). Importantly, all Ve1‐transgenic lines were susceptible to the wild‐type V. alfalfae strain Va2. Collectively, these data show that tobacco plants expressing Ve1 display enhanced Verticillium wilt resistance in an Ave1‐dependent manner.

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 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 (p 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 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.

Generation of isogenic V. dahliae strains lacking Ave1

Verticillium wilt of cotton is mostly caused by V. dahliae, and thus far effective tools to control Verticillium wilt in cotton are lacking (Cai et al., 2009). In a previous attempt to investigate whether tomato Ve1 can confer resistance against Verticillium wilt in cotton, transgenic cotton (G. hirsutum cv. YZ‐1) lines that express tomato Ve1 were generated, but no increased Verticillium wilt resistance was observed (Liu et al., 2014). However, it was realized later on that the V. dahliae strains used in this study did not contain Ave1 (Liu et al., 2014).

To re‐address the potential value of Ve1 to engineer Verticillium wilt resistance in cotton, we pursued Ave1‐carrying V. dahliae that can cause clear Verticillium wilt symptoms on G. hirsutum cv. YZ‐1 plants. To this end, we tested V. dahliae strains JR2, V4, V991 and V117 (Table S1) on cotton cultivar YZ‐1, and the development of Verticillium wilt symptoms was monitored at 21 dpi. As expected, differential degrees of Verticillium wilt symptoms were observed on these cotton plants (Figure S6a). Whereas V. dahliae strain JR2 that carries Ave1 only induced mild symptoms on cotton, V. dahliae strain V4 that similarly carries Ave1, and V. dahliae strains V991 and V117 that both lack Ave1 induced considerably stronger Verticillium wilt symptoms (Figure S6). Thus, race 1 V. dahliae strain V4 was selected for further assays on cotton.

To obtain an isogenic line that lacks Ave1, targeted replacement of Ave1 in V. dahliae strain V4 through homologous recombination was pursed (Figure S7a). To test whether the Ave1 deletion strains of V. dahliae V4 indeed overcome recognition by Ve1, two independent Ave1 deletion strains were inoculated onto tomato plants that express or lack Ve1 (Figure S7a). As expected, Ve1 tomato plants that were inoculated with two independent Ave1 deletion strains of V. dahliae V4 showed a similar disease phenotype as Ve1 tomato plants inoculated with the V. dahliae JR2ΔAve1 strain (Figure S7b,c; de Jonge et al., 2012), whereas Ve1 tomato plants inoculated with wild‐type V. dahliae strains V4 and JR2 resembled mock‐inoculated Ve1 tomato plants (Figure S7b,c). Moreover, the Ave1 deletion strains of V. dahliae strain V4 displayed significantly reduced virulence on susceptible tomato plants when compared with the corresponding wild‐type race 1 V. dahliae strain V4 (Figure S7b,d). These results are in line with previous results that show that Ave1 acts as a virulence factor on tomato, and confirm that deletion of Ave1 leads to escape of Ve1‐mediated Verticillium wilt resistance (de Jonge et al., 2012).

Ave1 acts as a virulence factor on cotton

To investigate whether Ave1 acts as a virulence factor on cotton, two independent Ave1 deletion strains and the corresponding wild‐type strain V4 were used to inoculate cotton cv. YZ‐1 plants. Interestingly, the Ave1 deletion strains of V. dahliae V4 displayed clearly reduced virulence on wild‐type cotton plants when compared with the corresponding wild‐type strain up to 28 dpi (Figure S8a), as inoculation with Ave1 deletion mutants resulted in significantly reduced stunting (Figure S8b) and compromised fungal colonization (Figure S8c). This assay demonstrates that Ave1 acts as a virulence factor also on cotton.

Cotton plants expressing Ve1 exhibit enhanced Verticillium wilt resistance in an Ave1‐dependent manner

To investigate whether cotton plants constitutively expressing tomato Ve1 display enhanced resistance against race 1 V. dahliae, two Ve1‐trangenic lines (Ve1‐4 and Ve1‐6) as well as nontransgenic control plants were inoculated with either the race 1 V. dahliae strain V4 or an Ave1 deletion mutant (V. dahliae V4ΔAve1), and monitored for Verticillium wilt symptoms up to 28 dpi. As expected, clear Verticillium wilt symptoms were observed on nontransgenic plants upon inoculation with V. dahliae strain V4 and with the corresponding Ave1 deletion mutant (Figure 4a–c), despite the observation that Ave1 deletion compromises virulence on cotton. Interestingly, Ve1‐expressing cotton plants exhibited significantly enhanced resistance against V. dahliae strain V4, as less Verticillium wilt symptoms were observed when compared with nontransgenic controls (Figure 4). When the two Ve1‐transgenic lines and nontransgenic controls were challenged with V. dahliae strain V991 that does not carry Ave1, Ve1‐expressing cotton lines were as susceptible as nontransgenic controls (Figure 5), confirming that the enhanced Verticillium wilt resistance upon Ve1 expression is Ave1‐dependent. Taken together, these data demonstrate that transfer of tomato immune receptor Ve1 into cotton confers Ave1‐dependent Verticillium wilt resistance.

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 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 (Gh) 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 (Gh) 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 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.

Discussion

Major management strategies for Verticillium wilt diseases in crops include chemical and biological control and cultivation practices, and for the use of disease‐resistant cultivars (Fradin and Thomma, 2006; Klosterman et al., 2009). Although chemical control has been proven to be successful for many diseases and pests, no truly effective fungicides are commercially available to control Verticillium wilt diseases once plants have been infected (Fradin and Thomma, 2006; Klosterman et al., 2009). Biocontrol measures and cultivation practices for controlling Verticillium wilts are time‐consuming and laborious, and control effectiveness largely depends on the field conditions. Therefore, breeding for disease‐resistant cultivars has been considered as the most sustainable approach to control Verticillium wilt diseases in crops (Fradin and Thomma, 2006; Klosterman et al., 2009). Presently, genetic resistance against Verticillium spp. has been described in several plant species, including tomato, potato, hop, alfalfa, cotton, strawberry, sunflower and lettuce (Antanaviciute et al., 2015; Barrow, 1970; Bolek et al., 2005; Christopoulou et al., 2015; Hayes et al., 2011; Jakse et al., 2013; Lynch et al., 1997; Mert et al., 2005; Putt, 1964; Schaible et al., 1951; Simko et al., 2004; Wang et al., 2008; Yang et al., 2008). However, only tomato Ve1 has been cloned and characterized as a dominant gene responsible for race 1 Verticillium wilt resistance (Fradin et al., 2009; Kawchuk et al., 2001). We previously reported that homologues of tomato Ve1 occur widespread in phylogenetically distant plant species (Song et al., 2017a). However, despite being widespread, Ve1 homologues occur in a scattered fashion throughout plant phylogeny. For instance, a functional Ve1 allele was identified in N. glutinosa, but not in other species within the genus Nicotiana (Song et al., 2017a; Zhang et al., 2013a). This finding implies that, most likely, many plant species lost their functional Ve1 homologues, but underlying immune signalling cascade may have been retained. Indeed, we have previously shown that heterologous expression of tomato Ve1 in the model plant A. thaliana that does not normally respond to Ave1 results in resistance against race 1 Verticillium spp. (Fradin et al., 2011; de Jonge et al., 2012; Zhang et al., 2013b). In this study, we investigated whether tomato Ve1 can confer Verticillium wilt resistance when expressed in the crop species tobacco and cotton. We show that transgenic tobacco and cotton plants constitutively expressing tomato Ve1 display enhanced resistance against Verticillium wilt in an Ave1‐dependent manner. Thus, our results reveal that the functionality of tomato Ve1 in resistance against Verticillium wilt through recognition of the Verticillium effector Ave1 is retained after transfer to these plant species, and further support the view that the underlying immune signalling cascade mediated by Ve1 is retained in these plant species.

To date, several examples of transgenic expression of cell surface immune receptor genes resulting in enhanced disease resistance have been reported (Rodriguez‐Moreno et al., 2017). Although transgenic expression of such receptors enhanced disease resistance, in some cases it also has adverse effects on plant fitness, such as growth retardation or leaf necrosis (Bouwmeester et al., 2011, 2014; Wang et al., 2016). In this study, we observed that expression of tomato Ve1 in tobacco caused stunted growth (Figures 1 and S1). A similar growth defect has previously been observed in N. benthamiana plants that constitutively express Ve1 (Fradin, 2011). In contrast, potato (Kawchuk et al., 2001), tomato (Fradin et al., 2009), A. thaliana (Fradin et al., 2011) and cotton (Figures 4 and 5) plants expressing Ve1 do not suffer from such growth defects. Based on these findings, we speculate that expression of tomato Ve1 in the genus Nicotiana may lead to a constitutive activation of downstream signalling cascade of tomato Ve1 that causes growth retardation. Alternatively, a ligand that is endogenous to these tobacco genotypes is recognized, leading to immune signalling activation.

Previously, we have shown that Ave1 acts as a virulence factor on tomato as well as on A. thaliana (de Jonge et al., 2012). In the present study, we observed that targeted Ave1 deletion results in significantly compromised virulence of Verticillium spp. on tobacco (Figure S5) and cotton (Figure S8), demonstrating that Ave1 acts as a virulence factor also on these plants. Previously, the bacterial homolog XacPNP from X. axonopodis pv. citri was characterized as a virulence factor on citrus trees (Gottig et al., 2008; Nembaware et al., 2004). More recently, Ave1 homologs from the fungal tomato wilt pathogen F. oxysporum f. sp. lycopersici (FoAve1), and the fungal sugar beet leaf spot pathogen C. beticola (CbAve1) were characterized as virulence factors too. Although the intrinsic function of the fungal Ave1 homologs remains enigmatic, XacPNP is thought to manipulate the physiology of the host through plant natriuretic peptide (PNP; immunological analogues of mammalian atrial natriuretic peptides) activity that affects water homoeostasis, stomatal opening and photosynthesis to promote bacterial proliferation (Garavaglia et al., 2010; Gottig et al., 2008). It is tempting to speculate that the fungal homologs promote virulence through a similar activity, but this remains to be demonstrated. In any case, the finding that Ave1 promotes virulence on tomato, tobacco, cotton as well as A. thaliana suggests that the molecular target of the effector is widely conserved in the plant kingdom.

In summary, our data demonstrate that transfer of tomato Ve1 into the closely related crop species tobacco and the distantly related crop species leads to enhanced resistance against Verticillium wilt in an Ave1‐dependent manner. Given that Ave1 homologues were found in a number of pathogenic microbes (Gan et al., 2013; de Jonge et al., 2012; Nembaware et al., 2004), and these homologs were differentially recognized by tomato Ve1 (de Jonge et al., 2012; Song et al., 2017b), our findings may further broaden biotechnological avenues to exploit tomato Ve1 for engineering disease resistance in an Ave1(homolog)‐dependent manner, for instance through transfer or artificial evolution of tomato Ve1.

Experimental procedures

Plant materials and growth conditions

Nicotiana tabacum cv. Samsun, N. glutinosa, cotton (Gossypium hirsutum cv. YZ‐1) and tomato (Solanum lycopersicum cv. Moneymaker (ve1) or 35S::Ve1 tomato (Ve1); Fradin et al., 2009) plants were used in this study and grown in commercial potting soil (Horticoop, Bleiswijk, the Netherlands) under controlled greenhouse conditions (Unifarm, Wageningen, the Netherlands).

Nicotiana tabacum cv. Samsun seeds were surface‐sterilized by 70% ethanol and 1% commercial bleach, and grown on Murashige‐Skoog (MS) medium (4.4 g MS salt, 20 g sucrose and 8 g agar in 1 L) or MS medium supplemented with antibiotics in a conditioned growth chamber at 21°C/19°C during 16‐h/8‐h light/dark photoperiods, respectively, and a relative humidity of ~75%.

Generation of Ve1‐transgenic plants

Agrobacterium tumefaciens GV3101 (pMP90) carrying the binary vector pSol2095_Ve1 to encode C‐terminally eGFP‐tagged Ve1 (Figure 1a; Zhang et al., 2013a) was used for transformation of tobacco N. tabacum cv. Samsun. Transformation was performed by the leaf disc method as previously described (Wang et al., 2016). The generated plantlets were transferred to half‐strength MS medium containing 200 mg/L kanamycin to allow root development. Upon root generation, plantlets were transferred into soil and grown in the greenhouse for seed production. Independent tobacco transformation lines were confirmed by PCR and reverse transcription‐PCR (RT‐PCR).

Two independent T0 generation cotton lines expressing tomato Ve1 gene driven by the cauliflower mosaic virus 35S promoter (Ve1‐4 and Ve1‐6; Liu et al., 2014) were self‐pollinated to generate seeds. After two generations of selfing, T2 seeds were used for further experiments.

Protein extraction, immunoprecipitation and immunoblotting

To test whether eGFP‐tagged Ve1 protein accumulated in transgenic tobacco lines, leaves of six‐week‐old transgenic tobacco lines were harvested and ground into a fine powder in liquid nitrogen. As a positive control, A. tumefaciens carrying the binary vector pSol2095_Ve1 was infiltrated into mature N. tabacum cv. Samsun leaves as described previously (Zhang et al., 2013a). Total proteins were extracted using extraction buffer (150 mm NaCl, 50 mm Tris‐HCl pH 8.0, 1.0% IGEPAL® CA‐630 [NP‐40] (Sigma‐Aldrich Chemie BV, Zwijndrecht, the Netherlands) and one protease inhibitor cocktail tablet (Roche, Basel, Switzerland) per 50 mL extract buffer). Samples were centrifuged at 21 191  for 20 min at 4°C, and then, 2 mL of supernatant was incubated with 10 μL (50% slurry) of GFP‐trap®_A beads (ChromoTek, Munich, Germany) at 4°C for 1 h. After incubation, GFP‐trap®_A beads with proteins were spun down by 110  centrifugation and subsequently washed for six times in 1 mL extraction buffer. After each wash step, the GFP‐trap®_A beads were collected by 1000 rpm centrifugation. Proteins were released from GFP‐trap®_A beads by boiling for 5 min, separated on a 10% SDS‐PAGE gel and wet‐electroblotted onto PVDF membrane (Bio‐Rad, Hercules, CA). Accumulation of eGFP‐tagged Ve1 was detected by immunoblotting using anti‐GFP‐HRP antibody (Miltenyi Biotec, Bergisch Gladbach, Germany). SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA) was used for signal development. Coomassie blue staining was used as loading control.

Generation of Ave1 mutant strains

Verticillium strains (Table S1) were grown on potato dextrose agar (PDA; Oxoid, Basingstoke, UK) at 22°C. The Ave1 knockout construct pRF‐HU2_Ave1 that was described previously (de Jonge et al., 2012) was used to generate Ave1 deletion mutants in V. nonalfalfae strain Vna 5431 and V. dahliae strain V4 (Table S1). The Ave1 complementation construct pFBT 005_pAve1::Ave1 that was described earlier (Song et al., 2017b) was used to generate Ave1 expression strains in V. alfalfae strain Va2 (Table S1).

Agrobacterium tumefaciens‐mediated Verticillium transformation was performed as described previously (Santhanam, 2012), and Verticillium deletion transformants were selected on PDA (Oxoid, Basingstoke, UK) containing 200 μg/mL cefotaxime and 50 μg/mL hygromycin (Duchefa, Haarlem, the Netherlands). Ave1 expression transformants were selected on PDA supplemented with 200 μg/mL cefotaxime (Duchefa, Haarlem, the Netherlands) and 50 μg/mL nourseothricin sulphate (Sigma‐Aldrich Chemie BV, Zwijndrecht, the Netherlands). Putative Verticillium transformants were tested by PCR, and subsequent inoculation on Ve1 tomato plants (Ve1) and tomato cultivar Moneymaker plants (ve1) (Fradin et al., 2009), or N. glutionsa plants carrying a functional Ve1 homolog (Song et al., 2017a).

Disease assays

Verticillium conidiospores were collected from 7‐ to 10‐day‐old cultures on PDA plates and washed with tap water. Disease assays were performed on tomato, tobacco and cotton plants using the root‐dipping inoculation method as previously described (Fradin et al., 2009). Briefly, 10‐day‐old Ve1 or ve1 tomato seedlings (for inoculation with Verticillium Ave1 deletion strains), or four‐week‐old tobacco (N. tabacum cv. Samsun or N. glutinosa) plants or 10‐day‐old cotton seedlings were uprooted. Next, the roots were rinsed in water, dipped for 5 min in a suspension of 106 conidiospores/mL water while the roots of mock plants (control) were dipped in tap water without conidiospores, and subsequently transplanted to fresh commercial potting soil (Horticoop, Bleiswijk, the Netherlands). Disease symptoms were scored up to 14 days postinoculation (dpi) (tomato, N. glutinosa and N. tabacum cv. Samsun), or 21 dpi (N. tabacum cv. Samsun), or 28 dpi (cotton). To this end, plants were photographed, and Image J was used to determine the canopy area (for quantification of stunting) while the rectilinear scale was used to measure the plant height (for quantification of growth). For fungal biomass quantification in planta, stems of three inoculated plants were harvested at 14 dpi (for N. tabacum cv. Samsun upon V. alfalfae inoculation), 21 dpi (for N. tabacum cv. Samsun upon V. nonalfalfae inoculation) or 28 dpi (cotton upon V. dahliae inoculation). The samples were ground into a fine powder in liquid nitrogen, and genomic DNA was isolated. Real‐time PCR was conducted using the fungus‐specific primers ITS‐F and ITS‐R (Table S2) with primers for tobacco actin (GenBank accession number: X69885; for Verticillium‐infected tobacco) or cotton ubiquitin (GenBank accession number: DQ116441; for Verticillium‐infected cotton) (Table S2) as an endogenous plant control, employing an ABI 7300 PCR system (Applied Biosystems, Foster City, CA) with the qPCR Core kit for SYBR Green I (Eurogentec Nederland BV, Maastricht, the Netherlands).

Gene expression analysis

For the expression of Ve1 in transgenic tobacco plants, leaves of six‐week‐old tobacco plants were harvested and ground into a fine powder in liquid nitrogen. Tobacco total RNA isolation and cDNA synthesis were performed as previously described (Song et al., 2017a). RT‐PCR was conducted using the primers Ve1‐F(PCR) and Ve1‐R(PCR) (Table S2), and N. tabacum actin gene (NtACT) (Table S2) was used as the endogenous control.

To check whether the Ve1 gene is expressed in individual T2 cotton plants, leaves of five‐week‐old cotton plants were collected separately, flash frozen in liquid nitrogen and stored at −80°C for total RNA isolation. Cotton total RNA was isolated using Spectrum™ Plant Total RNA Kit (Sigma‐Aldrich Chemie BV, Zwijndrecht, the Netherlands) following the manufacturer's instructions. First‐strand cDNA synthesis was performed using M‐MLV reverse transcriptase system (Promega, Madison, WI). RT‐PCR was conducted with primers Ve1‐F(RT) and Ve1‐R(RT) (Table S2) in a total volume of 25 μL with 17.9 sterilized water, 5 μL 5× PCR buffer, 0.5 μL dNTPs, 0.5 μL of each primer, 0.1 μL GoTag DNA polymerase (Promega) and 1.0 μL of first‐strand cDNA. Primers GhUb‐F and GhUb‐R (Table S2) were used to amplify the cotton ubiquitin gene as endogenous loading control. PCR amplification consisted of an initial denaturation step of 95°C for 5 min, followed by denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 40 s with 35 cycles. The resulting PCR products were subjected to agarose gel electrophoresis.

Figure S1 Characterisation of Ve1‐transgenic Nicotiana tabacum cv. Samsun plants.

Figure S2 Verticillium strains induce differential degrees of Verticillium wilt symptoms on N. tabacum cv. Samsun plants.

Figure S3 Analysis of Ave1 deletion strains of V. nonalfalfae Vna5431.

Figure S4 Analysis of ectopic expression Ave1 strains in V. alfalfae Va2.

Figure S5 Ave1 acts as a virulence factor on tobacco cv. Samsun plants.

Figure S6 V. dahliae strains induce differential degrees of Verticillium wilt on cotton (Gossypium hirsutum) cv. YZ‐1 plants.

Figure S7 Analysis of Ave1 deletion strains of V. dahliae V4.

Figure S8 Ave1 acts as a virulence factor on cotton plants.

Table S1 Verticillium strains used in this study.

Table S2 Primers used in this study.

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