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. 2013 Sep 10;15(1):109–117. doi: 10.1111/mpp.12071

Chaperones of the endoplasmic reticulum are required for Ve1‐mediated resistance to Verticillium

Thomas W H Liebrand 1,2,[Link], Anja Kombrink 1,[Link], Zhao Zhang 1, Jan Sklenar 3, Alexandra M E Jones 3,4, Silke Robatzek 3, Bart P H J Thomma 1,2,[Link], Matthieu H A J Joosten 1,2,[Link],✉
PMCID: PMC6638731  PMID: 24015989

Summary

The tomato receptor‐like protein (RLP) Ve1 mediates resistance to the vascular fungal pathogen Verticillium dahliae. To identify the proteins required for Ve1 function, we transiently expressed and immunopurified functional Ve1‐enhanced green fluorescent protein (eGFP) from Nicotiana benthamiana leaves, followed by mass spectrometry. This resulted in the identification of peptides originating from the endoplasmic reticulum (ER)‐resident chaperones HSP70 binding proteins (BiPs) and a lectin‐type calreticulin (CRT). Knock‐down of the different BiPs and CRTs in tomato resulted in compromised Ve1‐mediated resistance to V. dahliae in most cases, showing that these chaperones play an important role in Ve1 functionality. Recently, it has been shown that one particular CRT is required for the biogenesis of the RLP‐type Cladosporium fulvum resistance protein Cf‐4 of tomato, as silencing of CRT3a resulted in a reduced pool of complex glycosylated Cf‐4 protein. In contrast, knock‐down of the various CRTs in N. benthamiana or N. tabacum did not result in reduced accumulation of mature complex glycosylated Ve1 protein. Together, this study shows that the BiP and CRT ER chaperones differentially contribute to Cf‐4‐ and Ve1‐mediated immunity.

Introduction

Recognition of microbial patterns or damage‐associated host molecules by immune receptors results in the activation of immune responses in plants (Boller and Felix, 2009; Jones and Dangl, 2006; Thomma et al., 2011). These immune receptors are localized either in the cytoplasm or at the cell surface (Beck et al., 2012; Jones and Dangl, 2006; Monaghan and Zipfel, 2012). Examples of well‐studied cell surface receptors involved in pathogen recognition include the Arabidopsis (Arabidopsis thaliana) receptor‐like kinases (RLKs) Flagellin Sensing‐2 (FLS2) and the elongation factor‐Tu (EF‐Tu) receptor (EFR), which are responsible for the recognition of bacterial flagellin and EF‐Tu, respectively (Gómez‐Gómez and Boller, 2000; Zipfel et al., 2006). Another important class of plant cell surface receptors is the receptor‐like proteins (RLPs). Similar to most RLKs, most RLPs carry extracellular leucine‐rich repeats (LRRs), but they lack a cytoplasmic kinase domain (Wang et al., 2010). Examples of RLPs that play a role in immunity are the tomato (Solanum lycopersicum) Cf proteins (Rivas and Thomas, 2005; Stergiopoulos and de Wit, 2009), Ve1 (Fradin et al., 2009), LeEix2 (Ron and Avni, 2004), Arabidopsis (Arabidopsis thaliana) AtRLP30 (Wang et al., 2008), apple (Malus domestica) HcrVf2 (Belfanti et al., 2004) and rapeseed (Brassica napus) LepR3 (Larkan et al., 2013).

The biogenesis of functional transmembrane receptors requires correct folding and glycosylation. In addition, the transport of non‐mature receptors to the plasma membrane should be prevented, as they may be incompetent in ligand binding and subsequent signalling. To ensure that all post‐translational modifications required for the maturation of transmembrane receptors take place correctly, endoplasmic reticulum (ER)‐resident chaperones regulate ER quality control (ER‐QC) (Anelli and Sitia, 2008; Eichmann and Schäfer, 2012; Saijo, 2010). For example, the ER‐resident HSP70 binding proteins (BiPs) interact with the HSP40‐like stromal‐derived factor‐2 (SDF2) and J‐domain‐containing proteins (ERdj3) (Jin et al., 2008; Nekrasov et al., 2009; Schott et al., 2010). The major function of these chaperones in ER‐QC is to prevent the accumulation of unfolded proteins by assisting in protein folding and initiating ER stress signalling when unfolded proteins start to accumulate (Eichmann and Schäfer, 2012; Liu and Howell, 2010). Another ER‐QC pathway involves N‐linked glycosylation and consists of lectin chaperone‐assisted folding by the lectin‐type calreticulin (CRT) and calnexin (CNX) chaperones. In the ER, a Glc3Man9GlcNAc2 oligosaccharide is first added to asparagine (Asn) (N) residues of nascent glycoproteins (Pattison and Amtmann, 2009). The subsequent action of glucosidases renders the glycoproteins accessible for CRT/CNX‐assisted folding (Anelli and Sitia, 2008). A third ER‐QC pathway is supported by protein disulphide isomerases and involves the formation of disulphide bridges between free thiol groups present in the client protein (Anelli and Sitia, 2008; Gruber et al., 2006). Eventually, only correctly folded transmembrane proteins are transported to the Golgi apparatus for further maturation and transport to the plasma membrane. Immature and incorrectly folded proteins either re‐enter the ER‐QC cycle or are degraded by the ER degradation machinery (Anelli and Sitia, 2008; Nakatsukasa and Brodsky, 2008). Consequently, a number of Arabidopsis mutants in ER chaperone components are immunocompromised for bacterial infections. sdf2 mutants, for example, show reduced immunity mediated by EFR (Nekrasov et al., 2009). Similarly, Arabidopsis mutants in the folding sensor UDP Glc glycoprotein glucosyltransferase (UGGT), the ERD2b HDEL receptor and CRT3 are compromised in EFR‐mediated defence responses (Christensen et al., 2010; Li et al., 2009; Nekrasov et al., 2009; Saijo et al., 2009).

The tomato Ve1 receptor is an LRR‐RLP that mediates resistance to the fungal vascular wilt pathogens Verticillium dahliae and V. albo‐atrum (Fradin and Thomma, 2006; Fradin et al., 2009; Kawchuk et al., 2001). Ve1 provides resistance to race 1 strains of these pathogens on perception of the secreted Ave1 effector protein (de Jonge et al., 2012; Zhang et al., 2013a). Another group of defence‐associated LRR‐RLPs is the tomato Cf proteins, involved in resistance to the fungal biotrophic leaf pathogen Cladosporium fulvum (Rivas and Thomas, 2005). Recently, it has been discovered that Cf proteins interact with ER‐QC chaperones that are required for correct Cf function (Liebrand et al., 2012). Furthermore, it was found that both tomato and Nicotiana benthamiana contain four BiP homologues and three CRT‐like chaperones, and that Cf‐4 especially depends on CRT3a for its biogenesis (Liebrand et al., 2012). CRT3a is an isoform of the plant‐specific CRT3 class (Christensen et al., 2010), and silencing of CRT3a in N. benthamiana results in a reduced pool of mature complex glycosylated Cf‐4 protein, whereas the total Cf‐4 protein pool is not affected. Silencing of individual BiPs has no effect on Cf‐4 function, and silencing of multiple BiPs leads to lethality in N. benthamiana (Liebrand et al., 2012).

In this study, we investigated the role of the BiP and CRT chaperones in Ve1‐mediated immunity. We immunopurified a functional Ve1‐enhanced green fluorescent protein (eGFP) fusion protein from N. benthamiana and found that both the CRT‐ and BiP‐type ER chaperones interact with Ve1. Virus‐induced gene silencing (VIGS) of the genes encoding these ER‐QC chaperones in tomato resulted in reduced Ve1‐mediated resistance in most cases. Unexpectedly, we did not detect a reduction in complex glycosylation of Ve1 or a suppression of the Ve1‐mediated hypersensitive response (HR) on knock‐down of the different CRTs in N. tabacum or N. benthamiana. Together, our study shows that ER‐QC chaperones play an important role in Ve1‐mediated immunity. However, clear differences exist between the requirement and importance of the various ER‐QC chaperones in the maturation of the Cf‐4 and Ve1 proteins.

Results

Identification of ER‐resident chaperones as Ve1‐interacting proteins

Recently, a functional Ve1‐eGFP fusion protein has been generated (de Jonge et al., 2012; Zhang et al., 2013a), and we transiently expressed this fusion protein by Agrobacterium tumefaciens infiltration in N. benthamiana leaves. Ve1‐eGFP was subsequently immunopurified using GFP affinity beads, and a tryptic on‐bead digestion of the total immunoprecipitate was performed, after which the generated peptides were analysed by mass spectrometry to reveal co‐purifying proteins (Liebrand et al., 2012). Two peptides were found to match N. benthamiana (Nb) CRT2 (one of the three NbCRTs) and 32 peptides matched one or more of the four NbBiP homologues (Table S1, see Supporting Information). A list of additional co‐purifying proteins other than the BiPs and CRTs is presented in Table S2 (Supporting Information). In an alternative approach, Ve1‐eGFP was immunopurified from N. benthamiana after its transient expression, and the immunoprecipitate was run on sodium dodecylsulphate (SDS) gel and blotted. The blot was incubated with αBiP, detecting endogenous NbBiPs, revealing a clear band at the expected molecular weight of the four NbBiP proteins (Fig. 1A). Collectively, these results show that the NbCRTs and NbBiPs interact with Ve1 on transient expression in N. benthamiana.

Figure 1.

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HSP70 binding proteins (BiPs) of Nicotiana benthamiana co‐purify with Ve1 and Ve1 co‐purifies with tomato lectin‐type calreticulins (CRTs). Ve1‐enhanced green fluorescent protein (eGFP) was expressed in N. benthamiana (A) and Ve1‐haemagglutinin (HA) was co‐expressed with SlCRT2‐eGFP, SlCRT3a‐eGFP or SlCRT3b‐eGFP in N. benthamiana (B). Total protein extracts of the agroinfiltrated leaf tissue were subjected to immunopurification using GFP affinity beads. Total proteins (Input) and immunopurified proteins (IP) were separated by sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and blotted. Blots were incubated with αGFP antibody to detect immunopurified Ve1‐eGFP (A) or SlCRT‐eGFP (B) fusion proteins. Duplicate blots were incubated with αBiP to detect co‐immunopurifying BiPs (A) or with αHA antibody to detect co‐immunopurifying Ve1‐HA. The Coomassie‐stained blot shows the 50‐kDa Rubisco band present in the input to confirm equal loading. The experiment was performed twice with similar results and a representative example is shown.

Previously, C‐terminally‐tagged eGFP fusion proteins of the S. lycopersicum (Sl) CRTs have been generated (Liebrand et al., 2012), and we investigated here whether Ve1 interacts with the three individual SlCRTs. Therefore, SlCRT2, SlCRT3a and SlCRT3b, all fused to eGFP, were transiently co‐expressed with the fusion protein Ve1‐haemagglutinin (Ve1‐HA) by Agrobacterium infiltration in N. benthamiana. CRT immunoprecipitation using GFP affinity beads, followed by the detection of Ve1‐HA using αHA antibody, revealed that Ve1 interacts with all three SlCRTs, as Ve1‐HA is co‐immunopurified in all cases (Fig. 1B).

Targeting of tomato BiPs by VIGS compromises Ve1‐mediated immunity

To study whether the different BiPs play a role in Ve1‐mediated immunity, Tobacco rattle virus (TRV)‐based VIGS was employed (Fradin et al., 2009). Tomato cultivar Motelle plants, carrying the Ve1 resistance gene, were Agrobacterium inoculated with TRV constructs targeting the expression of the individual BiPs (TRV:Sl‐BiP1, TRV:Sl‐BiP2, TRV:Sl‐BiP3 and TRV:Sl‐BiP4) (Liebrand et al., 2012). As a control, plants were inoculated with TRV:GFP. Two weeks after inoculation with the recombinant viruses, plants were either inoculated with a V. dahliae strain expressing Ave1 or mock inoculated. Subsequently, plants were monitored for a period of 2 weeks for stunted growth, which is a typical disease symptom caused by V. dahliae infection. Targeting of SlBiP1–SlBiP3 resulted in stunted growth of Motelle plants on infection with V. dahliae, whereas targeting of SlBiP4 did not compromise Ve1 resistance (Fig. 2). As expected, TRV:GFP inoculation did not result in compromised growth of the plants on inoculation with V. dahliae, showing that these plants remained fully resistant to the fungus (Fig. 2). We determined the efficiency of BiP silencing by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) with specific primers for the individual BiPs (Fig. S1A, see Supporting Information). We detected the knock‐down of BiP1 and BiP2 in lines inoculated with TRV:Sl‐BiP1 and TRV:Sl‐BiP2, respectively (Fig. S1A). However, no reduced expression of SlBiP3 and SlBiP4 was detected on inoculation with TRV constructs targeting these genes. This is most likely because expression levels of SlBiP3 and SlBiP4 are much lower than those of SlBiP1 and SlBiP2 (not shown).

Figure 2.

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Targeting of individual HSP70 binding protein genes (BiPs) by virus‐induced gene silencing (VIGS) results in compromised Ve1‐mediated resistance to Verticillium dahliae. Tomato cultivar Motelle, carrying the Ve1 resistance gene, was subjected to VIGS by inoculation with the indicated TRV:Sl‐BiP constructs. TRV:GFP served as a control. GFP, green fluorescent protein; TRV, Tobacco rattle virus. Two weeks after inoculation with the recombinant viruses, plants were either mock inoculated (left plant in each panel) or inoculated with a race 1 strain of V. dahliae expressing Ave1 (right plant in each panel). Photographs were taken at 10 days after V. dahliae inoculation. Compromised resistance is reflected by the stunted appearance of the V. dahliae‐inoculated plants when compared with the mock‐inoculated plants. Three independent experiments were performed and a representative example is shown.

Targeting the tomato CRTs by VIGS compromises Ve1‐mediated immunity

To determine whether the CRTs are involved in Ve1‐mediated immunity, Motelle plants were Agrobacterium inoculated with the constructs TRV:Sl‐CRT2, TRV:Sl‐CRT3a and TRV:Sl‐CRT3b (Liebrand et al., 2012), targeting the expression of the different SlCRTs, and TRV:GFP as a control. Three weeks after TRV inoculation, the plants were inoculated with V. dahliae expressing Ave1 and monitored for stunted growth. Interestingly, targeting of the three individual CRTs in each case resulted in stunting of the plants on inoculation with V. dahliae (Fig. 3). This suggests that all three CRTs play a role in Ve1‐mediated immunity to V. dahliae. Again, the TRV:GFP‐inoculated plants remained fully resistant to the fungus (Fig. 3). Successful knock‐down of the expression of the different SlCRTs in the TRV:Sl‐CRT‐inoculated plants was shown by qRT‐PCR (Fig. S1B).

Figure 3.

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Targeting of individual lectin‐type calreticulin genes (CRTs) by virus‐induced gene silencing (VIGS) results in compromised Ve1‐mediated resistance to Verticillium dahliae. Tomato cultivar Motelle, carrying the Ve1 resistance gene, was subjected to VIGS by inoculation with the indicated Tobacco rattle virus (TRV) constructs. TRV:GFP (GFP, green fluorescent protein) served as a control. Two weeks after inoculation with the recombinant viruses, plants were either mock inoculated (left plant in each panel) or inoculated with a race 1 strain of V. dahliae expressing Ave1 (right plant in each panel). Photographs were taken at 10 days after V. dahliae inoculation. Compromised resistance is reflected by the stunted appearance of the V. dahliae‐inoculated plants when compared with the mock‐inoculated plants. Three independent experiments were performed and a representative example is shown.

Targeting of the CRTs in N. benthamiana or N. tabacum does not lead to reduced Ve1 accumulation or compromised complex glycosylation of Ve1

Knock‐down of NbCRT3a by VIGS in N. benthamiana compromises the Cf‐4‐mediated HR and is associated with a reduced accumulation of mature, complex N‐linked glycosylated Cf‐4 protein (Liebrand et al., 2012). To reveal whether the N‐linked complex glycosylation status of Ve1 changes on targeting of the expression of the different CRTs, we inoculated N. benthamiana and N. tabacum cultivar Samsun with the TRV constructs TRV:Sl‐CRT2, TRV:Nb‐CRT3a and TRV:Sl‐CRT3b which target the different N. benthamiana CRTs (Liebrand et al., 2012). In addition, TRV:GUS was used as a control. Nicotiana tabacum cultivar Samsun was included because Ve1‐mediated recognition of the Ave1 effector results in an HR in this species, whereas this does not occur in N. benthamiana (de Jonge et al., 2012; Zhang et al., 2013a). Next, Ve1‐eGFP was transiently expressed by Agrobacterium infiltration into the leaves of the TRV‐inoculated plants. As a control, we also transiently expressed Cf‐4‐eGFP in the TRV‐inoculated N. benthamiana plants. Two days later, eGFP‐tagged proteins were immunoprecipitated using GFP affinity beads. The total amount of purified Ve1‐eGFP and Cf‐4‐eGFP proteins was subsequently determined by immunoblotting and detection of the fusion proteins with αGFP antibody, whereas the amounts of complex N‐linked glycosylated Ve1‐eGFP and Cf‐4‐eGFP were determined using an anti‐horseradish peroxidase (HRP) antibody. This polyclonal antibody recognizes the HRP protein by binding to β(1,2)‐xylose and α(1,3)‐fucose residues that are added to the precursor glycan in the Golgi apparatus (Henquet et al., 2008). Consequently, this antibody cross‐reacts with other proteins that are also complex N‐linked glycosylated (Henquet et al., 2008; Liebrand et al., 2012). Because complex N‐linked glycosylation takes place in the Golgi, this antibody can be used as a tool to determine the amount of mature, complex glycosylated Ve1‐eGFP that has reached the Golgi apparatus.

The blots incubated with αGFP revealed that similar amounts of Ve1‐eGFP were purified from N. benthamiana and N. tabacum inoculated with the different TRV constructs that target the CRTs, or with TRV:GUS (Fig. 4). Replicate blots incubated with αHRP revealed that the pool of complex N‐linked glycosylated Ve1‐eGFP is equal, regardless of which TRV construct is used for the inoculation of the plants, both for N. benthamiana and N. tabacum (Fig. 4A,B). As expected, N. benthamiana plants inoculated with TRV:Nb‐CRT3a showed a strongly reduced complex glycosylation on Cf‐4‐eGFP (Fig. 4B) (Liebrand et al., 2012). To a lesser extent, this was also observed for plants inoculated with TRV: Sl‐CRT3b (Fig. 4B). These results show that the Ve1 protein is indeed complex N‐linked glycosylated, but targeting of the individual CRTs does not reduce the amount of complex glycosylated Ve1 protein (Fig. 4A,B). This is in contrast with Cf‐4, for which the amount of complex glycosylated protein is strongly reduced when SlCRT3a is targeted. Hence, the compromised resistance to V. dahliae on targeting of the different CRTs is probably not a result of reduced accumulation of complex mature N‐linked glycosylated Ve1 protein.

Figure 4.

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Ve1‐enhanced green fluorescent protein (eGFP) contains complex N‐linked glycans and virus‐induced gene silencing (VIGS) of the different lectin‐type calreticulin genes (CRTs) does not affect Ve1 glycosylation. Ve1‐eGFP was transiently expressed in Nicotiana tabacum (cultivar Samsun) (A) or N. benthamiana (B), silenced for the different CRTs. As a control in (B), Cf‐4‐eGFP was also transiently expressed in N. benthamiana silenced for the different CRTs. The fusion proteins were subsequently immunopurified using GFP affinity beads, run on sodium dodecylsulphate (SDS) gel and blotted. Blots were incubated with αGFP antibody to reveal the total amount of immunopurified Ve1 and Cf‐4 proteins, and duplicate blots were incubated with a horseradish peroxidase antibody (αHRP) to reveal the pool of mature complex glycosylated purified protein. The experiment was performed twice with similar results and a representative example is shown. TRV, Tobacco rattle virus.

Targeting the BiPs and CRTs in N. tabacum does not compromise the Ve1‐mediated HR

To determine whether the knock‐down of CRTs and BiPs compromises the Ve1‐triggered HR, N. tabacum (cv. Samsun) (Nt) was inoculated with TRV:Sl‐BiP1, TRV:Sl‐BiP2, TRV:Sl‐BiP3 and TRV:Sl‐BiP4 targeting the different N. benthamiana BiPs (Liebrand et al., 2012), as well as with TRV:Sl‐CRT2, TRV:Nb‐CRT3a and TRV:Sl‐CRT3b targeting the CRTs. Although its genome has not yet been sequenced, we anticipated that, based on the very high genome sequence homology between N. benthamiana and N. tabacum, these constructs would also target the corresponding close homologues of N. tabacum. Three weeks post‐viral inoculation, expanded leaf sections were Agrobacterium infiltrated to transiently co‐express Ve1 and the matching effector Ave1. As controls, we included TRV:GUS and TRV:Nb‐SOBIR1/Nb‐SOBIR1‐like. TRV:GUS does not have a plant target, whereas TRV:Nb‐SOBIR1/Nb‐SOBIR1‐like targets the homologues of the RLK SOBIR1, required for the Ve1‐mediated HR in N. tabacum (Liebrand et al., 2013).

Remarkably, targeting of the different BiP and CRT homologues in N. tabacum did not significantly compromise the Ve1‐mediated HR (Fig. 5). As expected, inoculation with the TRV construct targeting NtSOBIR1 resulted in a compromised Ve1‐mediated HR (Fig. 5). These results are in contrast with the reduced Ve1‐mediated resistance in tomato (Figs 2 and 3), but in agreement with the presence of N‐linked glycosylated Ve1 receptor protein in the plants inoculated with the TRV:CRT constructs (Fig. 4).

Figure 5.

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Targeting of the individual HSP70 binding protein (BiP) and lectin‐type calreticulin (CRT) genes in Nicotiana tabacum does not lead to a compromised Ve1‐mediated hypersensitive response (HR). Nicotiana tabacum cv. Samsun was subjected to virus‐induced gene silencing (VIGS) by inoculation with the indicated constructs. Three weeks later, Ve1 was transiently co‐expressed with the matching ligand Ave1. Photographs of infiltrated spots were taken another 4 days later. TRV:GUS and TRV:Nb‐SOBIR1/Nb‐SOBIR1 ‐like were included as controls. Three independent experiments were performed and representative examples are shown. TRV, Tobacco rattle virus.

Discussion

Ve1 is a transmembrane RLP carrying extracellular LRRs which mediates resistance to race 1 strains of the vascular wilt fungi V. dahliae and V. albo‐atrum (Fradin and Thomma, 2006; Fradin et al., 2009; Kawchuk et al., 2001). Hence, Ve1 requires post‐translational modifications and ER‐QC‐assisted folding before being guided to the plasma membrane (Anelli and Sitia, 2008). Indeed, we observed that Ve1 is associated with complex N‐linked glycans (Fig. 4). Furthermore, the protein interacts with BiP and CRT ER‐QC chaperones (Fig. 1). Here, we have shown that targeting of BiP1, BiP2 and BiP3 genes by VIGS in tomato compromises Ve1‐mediated immunity to V. dahliae, whereas targeting of BiP4 does not affect resistance to the pathogen (Fig. 2). Targeting of the CRT chaperones by VIGS in tomato also compromises Ve1‐mediated resistance to V. dahliae (Fig. 3), revealing that multiple BiPs and CRTs play a role in Ve1‐mediated immunity. Remarkably, targeting of the CRTs in N. tabacum does not affect the Ve1‐mediated HR (Fig. 5). This suggests that the Ve1‐activated pathway triggering the HR is still intact, and that the HR may thus not be required for Ve1‐mediated resistance against Verticillium. In support of this, it has been shown recently that Ve1‐mediated resistance in Arabidopsis does not require the HR (Zhang et al., 2013b).

Recently, it has been shown that tomato Cf proteins carry complex N‐linked glycans and interact with various ER‐QC chaperones (Liebrand et al., 2012). For example, Cf‐4 has been found to interact with BiP and CRTs in co‐immunopurification assays. In addition, silencing of the N. benthamiana and tomato CRT3a homologues strongly compromises Cf‐4‐mediated immune responses (Liebrand et al., 2012). In another study on Cf‐4‐mediated responses, it was found that BiPs are differentially regulated during mounting of the Cf‐4/Avr4‐triggered HR (Xu et al., 2012).

Ve1 function does not seem to depend on one particular CRT. Indeed, targeting the three SlCRTs individually by VIGS resulted in a compromised Ve1‐mediated resistance. This is in contrast with the findings for Cf‐4, which specifically requires CRT3a for its functionality. This chaperone was found to be particularly required for the accumulation of complex, N‐linked glycosylated Cf‐4, but not for the stability of the total Cf‐4 protein pool in N. benthamiana (Fig. 4) (Liebrand et al., 2012). Interestingly, Ve1 is still complex glycosylated on targeting CRT3a and the Ve1 protein accumulates to similar levels as in control plants (Fig. 4). In addition, the Ve1‐triggered HR in TRV:Nb‐CRT3a‐inoculated N. tabacum plants was still intact. Thus, a reduced accumulation of complex glycosylated Ve1 probably does not explain the compromised Ve1‐mediated resistance.

Therefore, the question remains on how CRT silencing affects Ve1 function. One explanation could be that CRTs play a role in Ve1‐mediated immunity of tomato, which is independent of their role in N‐linked glycosylation. Arabidopsis CRT2 has been shown to be involved in the salicylic acid‐dependent expression of defence genes (Qiu et al., 2012). Silencing of different CRTs may thus hamper an adequate response after ligand perception by Ve1. Alternatively, other downstream components involved in Ve1‐mediated immunity may rely on CRT‐mediated ER‐QC. Indeed, several proteins required for Ve1 signalling have been identified, such as the RLKs SOBIR1, SERK1 and SERK3/BAK1 (Fradin et al., 2009, 2011; Liebrand et al., 2013), and targeting the CRTs may affect the folding and glycosylation of these RLKs. Thus, Ve1‐mediated immunity to V. dahliae may be affected indirectly when the CRTs are targeted. These results reveal a differential requirement of CRTs in Ve1‐ and Cf‐4‐mediated immunity. Although Cf‐4 relies strongly on SlCRT3a but not on SlCRT2 or SlCRT3b (Liebrand et al., 2012), Ve1 requires all three CRTs for the RLP to mediate immunity.

ER‐QC chaperones play a role in the biogenesis and functionality of a number of additional plant transmembrane receptors. In N. tabacum, CRT2 and CRT3a have been shown to be required for the accumulation of the plasma membrane‐localized induced receptor kinase (IRK), which is involved in N‐mediated resistance to Tobacco mosaic virus (Caplan et al., 2009). In rice (Oryza sativa), OsBiP3 interacts with the plasma membrane‐localized RLK Xa21 involved in resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae. In this case, overexpression of OsBiP3 results in enhanced resistance to the bacterium (Park et al., 2010). In Arabidopsis, mutant forms of the brassinosteroid receptor BRI1 are retained in the ER by CRT3 (Hong et al., 2008; Jin et al., 2007, 2009). Furthermore, mutations in a number of ER‐QC chaperones strongly compromise the functionality of the immune receptor EFR in Arabidopsis (Li et al., 2009; Lu et al., 2009; Nekrasov et al., 2009; Saijo et al., 2009). In particular, crt3 mutants accumulate EFR to reduced amounts. In contrast, FLS2 accumulation and downstream responses are hardly affected in these ER‐QC chaperone mutants. Perturbation of the N‐linked glycosylation status of EFR and FLS2 reveals that under‐glycosylation strongly affects EFR function, whereas this is not the case for FLS2 (Häweker et al., 2010; Saijo, 2010; Sun et al., 2012). Interestingly, weakly defective crt3 Arabidopsis mutants accumulate EFR to wild‐type levels, but display compromised EFR‐triggered responses, showing that abundance control and QC of the EFR receptor can be uncoupled (Saijo et al., 2009). This may also explain why Ve1‐mediated defence is reduced on silencing of the individual CRTs, whereas Ve1 protein accumulation remains unaltered. Together, our results show that Ve1‐mediated immunity to V. dahliae depends on ER‐QC‐assisted folding mediated by the BiP and CRT families.

Experimental Procedures

Plant material and growth conditions

Nicotiana benthamiana, N. tabacum cultivar Samsun and tomato (Solanum lycopersicum) cultivar Motelle plants, carrying the Ve1 gene, were grown in the glasshouse under 16 h of light at 21 °C and 8 h of darkness at 19 °C. Supplemental light of 100 W/m2 was applied when the light intensity dropped below 150 W/m2. The relative humidity was approximately 75%.

Binary vectors for Agrobacterium infiltrations and VIGS

The constructs pBIN‐KS‐Ve1‐eGFP (pSOL2095Ve1::GFP) and pBIN‐KS‐Cf‐4‐eGFP have been described previously (Liebrand et al., 2012; Zhang et al., 2013a). pB7K40‐Ve1‐HA, p35S:Ve1 and pSOLl292‐Ave1 have been described elsewhere (Fradin et al., 2009; Fradin et al., unpublished data; Zhang et al., 2013a). Plasmids directing the expression of the SlCRT‐eGFP fusions and all TRV constructs for VIGS experiments have been described previously (Liebrand et al., 2012; Zhang et al., 2013a). All binary plasmids were transformed to A. tumefaciens strain C58C1, carrying helper plasmid pCH32, for Agrobacterium infiltration assays. Agrobacterium infiltrations were performed as described previously (van der Hoorn et al., 2000; Zhang et al., 2013a).

VIGS experiments and V. dahliae disease assays

VIGS in N. benthamiana, N. tabacum cultivar Samsun and tomato cultivar Motelle was essentially performed as described previously (Fradin et al., 2009; Liebrand et al., 2012; Zhang et al., 2013a). We used Agrobacterium inoculation to express both pTRV:RNA1 and pTRV:RNA2 (Liu et al., 2002). Verticillium dahliae race 1 strain JR2 was used for the inoculation of tomato. Fungal inoculations and disease scoring were performed as described previously (Fradin et al., 2009, 2011). Agrobacterium‐mediated co‐expression of p35S:Ve1 and pSOLl292‐Ave1 in TRV‐inoculated N. tabacum cv. Samsun was performed at a final optical density at 600 nm (OD600) = 1 for each construct (Zhang et al., 2013a).

RNA extraction and qRT‐PCR

For qRT‐PCR, leaves of four plants inoculated with a TRV‐silencing construct were ground and subjected to RNA extraction plus first‐strand cDNA synthesis, as described previously (Liebrand et al., 2012). Primers matching the individual SlBiPs, SlCRTs and SlActin are given in Table S3 (see Supporting Information). qRT‐PCR was performed as described previously (Liebrand et al., 2012).

Protein identification by mass spectrometry

Immunopurification of the Ve1‐eGFP fusion protein and GFP‐HA control from transiently transformed N. benthamiana leaves was performed as described previously for Cf‐4‐eGFP (Liebrand et al., 2012). In brief, proteins were extracted using extraction buffer containing 150 mm NaCl, 1% IGEPAL CA‐630 (NP40), 50 mm Tris, pH 8, and one tablet of protease inhibitor cocktail (Roche, Almere, the Netherlands) per 50 mL. One gram of leaf material (fresh weight) per 2 mL of extraction buffer was used and 10 mL of total extract was subjected to immunopurification using 60 μL (50% slurry) of GFP_TrapA affinity beads (Chromotek, Planegg, Germany). The beads were incubated with the protein extract for 1 h, after which they were washed five times with extraction buffer. Subsequently, a trypsin on‐bead digestion was performed and the peptide mixture was analysed by mass spectrometry using an Orbitrap XL mass spectrometer (Thermo Scientific, Etten‐Leur, the Netherlands), as described previously (Liebrand et al., 2012).

Co‐immunopurifications and immunoblotting

Co‐immunopurifications were performed as described previously (Liebrand et al., 2012). We used 15 μL (50% slurry) of GFP_TrapA affinity beads for the immunopurifications. The beads were washed five times with the extraction buffer before eluting with SDS loading buffer. After blotting to poly(vinylidene difluoride) (PVDF) membrane (Bio‐Rad, Veenendaal, the Netherlands), the membranes were incubated with αGFP (anti‐GFP‐HRP, 130‐091‐833, MACS antibodies, Leiden, the Netherlands), αHA (anti‐HA‐peroxidase clone 3F10, 12013819001, Roche) or αHRP (rabbit anti‐HRP, 323‐005‐021, Jackson ImmunoResearch, Suffolk, UK) as a primary antibody, and goat anti‐rabbit‐HRP (anti‐rabbit IgG‐HRP, A9169, Sigma, Zwijndrecht, the Netherlands) as a secondary antibody. All antibodies were used at the dilutions recommended by the manufacturer.

Supporting information

Fig. S1 Determination of the expression levels of Solanum lycopersicum HSP70 binding protein (SlBiP) (A) and lectin‐type calreticulin (SlCRT) (B) genes in Tobacco rattle virus (TRV)‐inoculated tomato plants. Tomato plants were inoculated with the indicated TRV‐silencing contructs and relative transcript levels of SlBiP1 and SlBiP2 (A) as well as of SlCRT2, SlCRT3a and SlCRT3b (B) were determined by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Expression levels were standardized to those in the TRV:GUS‐inoculated plants and normalized to endogenous SlActin. RNA used as a template for qRT‐PCR was extracted from a mixture of four individually silenced tomato plants. The standard deviation shows the variation between three technical repeats.

Click here for additional data file. (12.6MB, tif)

Table S1 Sequences and Mascot ion scores of peptides matching co‐purifying endoplasmic reticulum (ER)‐resident chaperones, identified by mass spectrometry of a tryptic digest of immunopurified Ve1‐enhanced green fluorescent protein (eGFP), transiently expressed in Nicotiana benthamiana.

Click here for additional data file. (50KB, doc)

Table S2 Additional Ve1‐enhanced green fluorescent protein (eGFP) co‐purifying proteins identified by mass spectrometry. Shown are the number of unique mass spectra divided by the number of unique peptides identified for each protein in the Ve1‐eGFP and GFP‐haemagglutinin (HA) control immunopurification.

Click here for additional data file. (70KB, doc)

Table S3 List of oligonucleotide primers used.

Click here for additional data file. (35KB, doc)

Acknowledgements

Bert Essenstam and Henk Smid are acknowledged for excellent plant care. TWHL, BPHJT and MHAJJ are supported by the Centre for BioSystems Genomics (part of the Netherlands Genomics Initiative and the Netherlands Organization for Scientific Research). BPHJT is supported by a Vidi grant of the Netherlands Organization for Scientific Research. JS, AMEJ and SR are supported by the Gatsby Charitable Foundation. The authors declare no conflict of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1 Determination of the expression levels of Solanum lycopersicum HSP70 binding protein (SlBiP) (A) and lectin‐type calreticulin (SlCRT) (B) genes in Tobacco rattle virus (TRV)‐inoculated tomato plants. Tomato plants were inoculated with the indicated TRV‐silencing contructs and relative transcript levels of SlBiP1 and SlBiP2 (A) as well as of SlCRT2, SlCRT3a and SlCRT3b (B) were determined by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Expression levels were standardized to those in the TRV:GUS‐inoculated plants and normalized to endogenous SlActin. RNA used as a template for qRT‐PCR was extracted from a mixture of four individually silenced tomato plants. The standard deviation shows the variation between three technical repeats.

Click here for additional data file. (12.6MB, tif)

Table S1 Sequences and Mascot ion scores of peptides matching co‐purifying endoplasmic reticulum (ER)‐resident chaperones, identified by mass spectrometry of a tryptic digest of immunopurified Ve1‐enhanced green fluorescent protein (eGFP), transiently expressed in Nicotiana benthamiana.

Click here for additional data file. (50KB, doc)

Table S2 Additional Ve1‐enhanced green fluorescent protein (eGFP) co‐purifying proteins identified by mass spectrometry. Shown are the number of unique mass spectra divided by the number of unique peptides identified for each protein in the Ve1‐eGFP and GFP‐haemagglutinin (HA) control immunopurification.

Click here for additional data file. (70KB, doc)

Table S3 List of oligonucleotide primers used.

Click here for additional data file. (35KB, doc)

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