JXB Advance Access originally published online on January 12, 2006
Journal of Experimental Botany 2006 57(3):599-608; doi:10.1093/jxb/erj044
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RESEARCH PAPER |
Affinity-tags are removed from Cladosporium fulvum effector proteins expressed in the tomato leaf apoplast
Laboratory of Phytopathology, Centre for Biosystems Genomics (CBSG), Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands
To whom correspondence should be addressed. E-mail: bart.thomma{at}wur.nl
Received 5 July 2005; Accepted 1 November 2005
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Abstract |
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Cladosporium fulvum (syn. Passalora fulva) is a biotrophic fungal pathogen that causes leaf mould on tomato (Solanum esculentum). The fungus grows exclusively in the tomato leaf apoplast where it secretes several small (<15 kDa) cysteine-rich proteins that are thought to play a role in disease establishment. To investigate the role of these proteins, and to identify their in planta targets, a targeted proteomics approach was undertaken. C. fulvum proteins were expressed as recombinant fusion proteins carrying various affinity-tags at either their C- or N-terminus. Although these fusion proteins were correctly expressed and secreted into the leaf apoplast, detection of affinity-tagged C. fulvum proteins failed, and affinity purification did not result in the recovery of these proteins. However, when using C. fulvum effector protein-specific antibodies, specific signals were obtained for the different proteins. It is concluded that the stability of the in planta expressed recombinant fusion proteins is insufficient, which results in removal of the affinity-tag from the fusion proteins, irrespective of the C- or N-terminal fusion or the nature of the affinity-tag. Similar phenomena were observed when the fusion proteins were expressed in other Solanaceous species, but not when expressed in Arabidopsis thaliana.
Key words: Avirulence, extracellular, c-myc, FLAG, His, StrepII, TAP, targeted proteomics
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cladosporium fulvum (syn. Passalora fulva) is a biotrophic fungal pathogen that causes leaf mould on tomato (Solanum esculentum) (Thomma et al., 2005
). The fungus grows exclusively in the tomato leaf apoplast without forming any known feeding structures like haustoria. This implies that during the infection process, molecular components that play a role in the interaction between pathogen and host need to pass the host apoplast. Eight C. fulvum effector proteins, secreted by the fungus during infection, have been characterized in detail and their corresponding genes have been cloned (Thomma et al., 2005). These comprise the race-specific avirulence proteins Avr2, Avr4, Avr4E, and Avr9, and the extracellular proteins Ecp1, Ecp2, Ecp4, and Ecp5, of which the corresponding genes are thus far found in all C. fulvum strains (Thomma et al., 2005). All effector proteins are relatively small (ranging between 3–15 kDa) and contain a high number of cysteine residues that are involved in disulphide bridge formation (Kooman-Gersmann et al., 1997; van den Burg et al., 2003). These bridges provide a compact tertiary structure for the C. fulvum effector proteins in the tomato apoplast which is reported to be rich in proteases (Tornero et al., 1997; Jorda et al., 1999; Krüger et al., 2002).
Tomato resistance against C. fulvum is genetically determined by the presence of Cf resistance genes in a so-called ‘gene-for-gene’ relationship (Kruijt et al., 2005). For all four cloned C. fulvum Avr genes, the corresponding Cf resistance genes have also been cloned (Jones et al., 1994; Dixon et al., 1996; Thomas et al., 1997; Takken et al., 1998). Furthermore, Cf resistance genes that confer recognition of Ecp genes have been described (Laugé et al., 1998; de Kock et al., 2005; Kruijt et al., 2005). Since Avr and Ecp genes have been maintained within the C. fulvum population they are likely to provide a specific fitness benefit to the fungus, either in planta during the infection, or in the absence of the natural host (Thomma et al., 2005). The observation that they are highly expressed in planta at the onset of the infection suggests they play a role in disease establishment (van Kan et al., 1991; van den Ackerveken et al., 1993a; Joosten et al., 1997). This hypothesis is supported by the observation that the Avr4 and Avr2 proteins display biological activities that suggests they are true virulence proteins (Krüger et al., 2002; van den Burg et al., 2003; Rooney et al., 2005).
In this study, an attempt was made to find virulence targets for C. fulvum effector proteins. As it was desired to address the virulence function of C. fulvum effector proteins, these studies were carried out in the absence of Cf resistance genes (Cf-0) to mimic the situation in a compatible interaction. In this targeted proteomics approach, affinity-tagged C. fulvum effector proteins were used as bait to fish for their in planta targets. However, affinity purification of the affinity-tagged effector protein fusions failed, and subsequent experiments demonstrated that no, or only very low amounts of affinity-tagged proteins could be detected in the apoplast of tomato leaves. These data suggest that, irrespective of the sequence of the affinity-tag or the C- or N-terminal fusion, the affinity-tags are removed when the fusion proteins are deposited into the apoplast of Solanaceous species, but not in the apoplast of Arabidopsis thaliana. This phenomenon will have great implications for the in planta use of affinity-tagged apoplastic proteins in Solanaceous species.
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Materials and methods |
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Heterologous production of His6-FLAG-tagged C. fulvum effector proteins in Pichia pastoris
Plasmids for the expression of affinity-tagged C. fulvum proteins in the yeast Pichia pastoris were generated as described (Rooney et al., 2005
). Briefly, vector pPIC-9 (Invitrogen, Carlsbad, CA, USA) was modified by inserting an adaptor, encoding the His6-tag and SmaI, ApaI, and SacII restriction sites, resulting in vector pPIC-9His. To create His6-FLAG-tagged effector proteins, cDNA for each effector was amplified (see primers for Pichia pastoris expression; Table 1) and cloned into pPIC-9His using the SmaI (blunt) and EcoRI restriction sites. Subsequently, Pichia pastoris strain GS115 (Invitrogen, Carlsbad, CA, USA) was transformed.
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Fermentation was performed as previously described (van den Burg et al., 2001
; Rooney et al., 2005). Proteins in the culture supernatant were separated on a Tricine SDS–PAGE gel, and stained with Coomassie Brilliant Blue or analysed on western blots. After removal of cells and concentration of the supernatant, the His6-FLAG-tagged proteins were purified using a Ni2+-NTA Superflow column (Qiagen, Leusden, the Netherlands), according to the manufacturer's protocol. The eluted protein fractions were pooled and dialysed using Milli-Q water. Protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). In addition, proteins were tested for their hypersensitive response (HR-) inducing activity by injection into leaves of tomato plants carrying the corresponding Cf resistance genes.
PVX-mediated expression of affinity-tagged C. fulvum effector proteins in planta
The binary PVX vector pGr106 (Jones et al., 1999) was used as a backbone for all PVX expression constructs used in this study. From left border to right border the T-DNA of this vector consists of a CaMV 35S promoter-driven PVX sequence containing the replicase gene, the triple gene block, the duplicated coat protein promoter, and the coat protein gene (Fig. 1A). The multiple cloning site is located directly downstream of the duplicated coat protein promoter. The P. pastoris expression vectors described above were used to amplify the His6-FLAG-tagged effector proteins. The cloning strategy for the various constructs is described below (see Table 1 for primer sequences).
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Constructs for N-terminally His6-FLAG-tagged effector proteins:
First, the N. benthamiana PR1a signal sequence was amplified introducing a 5' ClaI restriction site (primers PR1a-ClaI and PR1a-His1). In addition, all His6-FLAG-tagged effector proteins were amplified from the P. pastoris expression vectors using a forward primer annealing to the His6-tag (primer PR1a-His2) and a reverse primer annealing to the pPIC-9 backbone (primer 3' AOX1). Subsequently, utilizing overlap extension PCR, the coding region for the PR1a signal sequence was fused to that of the His6-FLAG-tagged effector proteins (primers PR1a-ClaI and 3' AOX1).
Constructs for N-terminally His6-FLAG-StrepII-tagged effector proteins:
The coding sequence for the effector proteins was amplified from the P. pastoris expression vectors using gene-specific forward primers including a 5' StrepII coding sequence overhang and a reverse primer annealing to the pPIC-9 backbone (primer 3' AOX1). In addition, constructs encoding the N-terminally His6-FLAG-tagged effector proteins were used to amplify the PR1a signal sequence fused to the His6-FLAG-tag, including a 3' StrepII coding sequence overhang using the primers PR1a-ClaI and FLAG-Strep. For each construct, both PCR products were fused by overlap extension PCR with the primers PR1a-ClaI and 3' AOX1.
Constructs for C-terminally StrepII- or c-myc-tagged effector proteins:
First, the coding sequence for the effector proteins was amplified from the P. pastoris expression vectors using gene-specific forward primers including a 5' overhang for the PR1a coding sequence, and gene-specific reverse primers with an overhang to include the coding sequence for the StrepII or c-myc affinity-tag followed by a stop codon and a NotI restriction site. In addition, the PR1a signal sequence was amplified using the primer PR1a-ClaI and a set of reverse primers containing effector gene-specific overhangs. Subsequently, PCR products were fused by overlap extension PCR with the PR1a-ClaI forward primer and the gene-specific reverse primers that were used to add the StrepII or c-myc affinity-tag.
In all cases, PCR fragments were purified from agarose gel (QIAquick, Qiagen, Leusden, the Netherlands) and cloned into the pGEM®-T easy vector (Promega, Mannheim, Germany). After DNA sequencing, inserts were obtained using the ClaI and NotI restriction enzymes and ligated into pGr106. The resulting plasmids were transformed into Agrobacterium tumefaciens (GV3101) by electroporation.
A. tumefaciens strains containing PVX constructs for the expression of C. fulvum effector proteins were cultured on plates containing modified LB medium (10 g l–1 bacto-peptone; 5 g l–1 yeast extract; 2.5 g l–1 NaCl; 10 g l–1 mannitol) for 48 h at 28 °C. Subsequently, colonies were selected and inoculated on 2-week-old tomato plants by toothpick inoculation.
Western blot analyses
Apoplastic fluid (AF) was isolated from leaf material, using demineralized water for vacuum infiltration (de Wit and Spikman, 1982). Total leaf extracts were prepared by homogenizing leaf material in demineralized water. Both total extracts and AF were denatured by boiling for 5 min in an equal volume of denaturing solution (0.0625 M TRIS–HCl, pH 6.8, 2% (w/v) SDS, 10% (w/v) glycerol, 5% (v/v) ß-mercaptoethanol, and 0.001% (w/v) bromophenol blue). Proteins separated on Tricine gels were transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA) and blocked overnight at 4 °C with phosphate buffered saline, pH 7.3, 3% (w/v), and BSA, 0.1% (v/v) Tween 20). Subsequently, blots were incubated for 2 h at room temperature with primary (rabbit) antibody (10 µl in 10 ml antibody buffer: PBS, pH 7.3, 0.3% (w/v) BSA, and 0.1% (v/v) Tween 20). After washing in antibody buffer, blots were incubated (2 h at room temperature) with secondary horseradish peroxidase (HRP)-conjugated antibody. After washing, the HRP-conjugate was activated (SuperSignal, Pierce, Rockford, IL, USA) and detected on film. Western blot analysis with StrepTactin-HRP (IBA, Göttingen, Germany), monoclonal anti-FLAG bioM2 (Sigma, St Louis, MO, USA) and anti-myc-HRP antibody (Invitrogen, Carlsbad, CA, USA) was performed according to the manufacturer's specifications. Polyclonal antibodies raised against the individual C. fulvum effector proteins were produced upon immunization of rabbits according to standard procedures (Eurogentec, Seraing, Belgium). For immunization, enterokinase-treated (for affinity-tag removal), P. pastoris-produced C. fulvum effectors were used.
Affinity purification using the His6-tag
For His6-based affinity purification, AF was isolated from leaf material by vacuum infiltration (de Wit and Spikman, 1982) using a buffer (50 mM NaH2PO4, pH 7.0, 300 mM NaCl, 10 mM imidazole, and 0.005% (v/v) Tween 20) compatible with subsequent purification steps using Ni2+-NTA magnetic agarose beads (Qiagen, Leusden, the Netherlands). The same buffer was used for the preparation of total protein extracts. Total protein extracts were prepared by homogenizing 2 g of leaf material frozen in liquid nitrogen after which 2 ml of buffer was added. Subsequently, 50 µl of Ni2+-NTA magnetic agarose beads suspension was added to 2 ml of AF or total protein extract and incubated for 60 min at 21 °C. The beads were recovered by using a magnetic separator and washed four times in 2 ml washing buffer (50 mM NaH2PO4, pH 7.0, 300 mM NaCl, 20 mM imidazole, and 0.005% (v/v) Tween 20). Elution was performed in 50 µl of elution buffer (50 mM NaH2PO4, pH 7.0, 300 mM NaCl, 250 mM imidazole, and 0.005% (v/v) Tween 20). Eluates were separated on a 16% Tricine gel and visualized by silver-staining (Blum et al., 1987).
Affinity purification using the StrepII-tag
AF was isolated from leaf material by vacuum infiltration (de Wit and Spikman, 1982) using demineralized water containing a protease inhibitor cocktail (complete Protease Inhibitor Cocktail Tablets, Roche, Basel, Switzerland). AF was concentrated 8x by using a 5 kDa cut-off filter (Vivaspin 4, Vivascience GA, Germany). Purification of the protein was carried out essentially as described by Witte et al. (2004). To 200 µl of concentrated AF, 60 µl of a 5x buffer [500 mM TRIS, pH 8.0, 25 mM EGTA, 25 mM EDTA, 750 mM NaCl, 50 mM DTT, 2.5 mM AEBSF (4-(2-aminoethyl)benzenesulphonyl fluoride hydrochloride), and 2.5% (v/v) Triton X-100] was added plus 40 µl of avidin (1 mg ml–1) (Witte et al., 2004). After 15 min of incubation at 4 °C, 50 µl StrepTactin sepharose (IBA, Göttingen, Germany) was added. After 30 min of incubation at 4 °C the slurry was transferred to a glass wool column (200 µl void volume) and washed twice with 1 ml and four times with 0.5 ml wash buffer (50 mM TRIS, pH 8.0, 2.5 mM EDTA, 150 mM NaCl, 2 mM DTT, and 0.05% (v/v) Triton X-100). Elution was performed by filling the void volume of the column with elution buffer (10 mM TRIS, pH 8.0, 10 mM desthiobiotin, 2 mM DTT, and 0.05% (v/v) Triton X-100). Subsequently, seven fractions of 50 µl were collected by stepwise adding 50 µl of elution buffer. As a final elution step, the glass-wool was removed from the column, taking care that the sepharose was also transferred, and boiled in 200 µl 2x denaturing buffer (0.0625 M TRIS–HCl, pH 6.8, 2% (w/v) SDS, 10% (w/v) glycerol, 5% (v/v) ß-mercaptoethanol, and 0.001% (w/v) bromophenol blue). Washing and eluted fractions were separated on a 16% Tricine gels and were visualized by silver-staining (Blum et al., 1987).
Plant cultivation
All tomato plants were grown under standard greenhouse conditions: at 21/19 °C over the 16/8 h day/night period, 70% relative humidity and 100 W m–2 supplemental light when the sunlight influx intensity was below 150 W m–2.
Similarly, Arabidopsis plants were also grown under greenhouse conditions of 21/18 °C during the 16/8 h day/night, 60% relative humidity and 100 W m–2 supplemental light when the sunlight influx intensity was below 150 W m–2.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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To investigate the intrinsic function of C. fulvum proteins secreted during infection, a search for their plant interactors that may act as virulence targets was initiated. To this end, fusion proteins consisting of the mature Avr or Ecp proteins and an affinity-tag were expressed (Table 2; Fig. 1), in principle allowing affinity purification of the Avr or Ecp protein together with possible interactors. To allow systemic expression throughout the whole plant, and thus boost the total amount of protein produced, a binary potato virus X (PVX)-based expression system was used (Fig. 1A), permitting Agrobacterium tumefaciens-mediated inoculation of the virus (Luderer et al., 2002
; Takken et al., 2000; Westerink et al., 2004).
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Initially, two sets of binary PVX constructs were produced; the first encoding N-terminally His6-FLAG-tagged Avr and Ecp effector proteins and the second set encoding N-terminally His6-FLAG-StrepII-tagged effector proteins (Fig. 1B). In all cases the sequence encoding the tobacco PR1a signal peptide was used for targeting of the fusion proteins towards the apoplast, where the effector proteins are also secreted by the fungus during infection. To test whether expression of the constructs in planta results in biologically active proteins, A. tumefaciens strains carrying the various constructs were inoculated on 2-week-old MoneyMaker tomato plants carrying the corresponding Cf resistance genes. About 2–3 weeks post-inoculation, a systemically spreading HR appeared (Fig. 2A). This response was not observed when the constructs were expressed in tomato plants without any functional resistance gene (Cf-0 plants) (Fig. 2B), nor on tomato plants carrying non-corresponding Cf resistance genes (data not shown). This indicated that biologically active proteins were produced. To determine whether the PVX-expressed recombinant fusion proteins were correctly targeted towards the leaf apoplast, Cf-0 plants were inoculated with individual A. tumefaciens strains harbouring the different constructs. After 15 d, AF was isolated, and subsequently injected into leaves of tomato plants carrying the corresponding Cf resistance genes. This resulted in triggering of effector-specific, HR-like symptoms in the injected sectors (Fig. 2C, D). It was therefore concluded that upon expression of affinity-tagged Avr and Ecp proteins by PVX, biologically active effector proteins are produced and secreted into the leaf apoplast.
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Having verified the correct expression and targeting of the recombinant fusion proteins, it was attempted to recover the expressed affinity-tagged Avr and Ecp proteins from PVX-inoculated tomato Cf-0 plants. To this end, AF, as well as total leaf extract, was isolated. Subsequent affinity purification using Ni2+-NTA magnetic agarose beads (with high affinity for the His6-tag) or StrepTactin sepharose (with high affinity for the StrepII-tag), however, did not result in recovery of detectable amounts of affinity-tagged effector proteins (data not shown).
Two additional sets of binary PVX constructs were generated to investigate whether the N-terminal fusion, or the biochemical nature of the affinity-tags, interfered with the stability or the level of production of the recombinant fusion proteins. These sets consisted of C-terminally StrepII-tagged and C-terminally c-myc-tagged fusions of Avr2, Avr4E and Ecp2 (Fig. 1B). Expression, biological activity, and localization were confirmed in a similar fashion as for the N-terminal fusion proteins. However, the C-terminal c-myc or StrepII fusions could also not be detected using an anti-c-myc antibody or StrepTactin-HRP, neither in AF, nor in total leaf extract from tomato Cf-0 plants expressing the different fusion constructs (data not shown).
As all attempts to purify or detect affinity-tagged C. fulvum effector proteins from tomato AF failed, an attempt was made to determine whether (part of) the affinity-tags were still attached to the secreted effector proteins. To this end, AF isolated from Cf-0 tomato plants expressing the various constructs was denatured, separated on Tricine gels, and blotted to PVDF membranes. Subsequent western blot analysis using anti-FLAG antibodies, anti-c-myc antibodies, or StrepTactin-HRP did not result in detection of PVX-expressed effector proteins (see Fig. 3 for detection using anti-FLAG as an example). However, when using polyclonal antibodies raised against the individual C. fulvum effector proteins themselves (no antibodies were available for Ecp1 and Avr9), specific signals were detected for all effector proteins, except for Avr4, showing that the presence of most effector proteins could be detected in the AF from inoculated Cf-0 tomato plants (Fig. 3). The inability to detect Avr4 can be attributed to the characteristics of the polyclonal antibodies, or to the level of production of stable Avr4 protein in tomato AF.
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-FLAG) or against the individual C. fulvum effector proteins (It cannot be excluded that the ability to detect the proteins with effector-specific polyclonal antibodies, but not with affinity-tag-specific antibodies, is caused by superior sensitivity of the effector-specific polyclonal antibodies. To test this hypothesis, a 5-fold dilution series of P. pastoris-produced His6-FLAG-tagged Avrs and Ecps was dot-blotted and used in a western analysis to compare the sensitivity of the anti-FLAG antibody to the effector-specific polyclonal antibodies. This demonstrated that the anti-FLAG antibody generally displays a higher sensitivity compared with the antibodies raised against the individual C. fulvum effector proteins (Fig. 4). These data altogether suggest that the affinity-tags, irrespective of whether they are fused to the N- or the C-terminus of the effector proteins, are removed from the recombinant fusion proteins in the tomato apoplast.
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The tomato leaf apoplast is known to contain many proteases. Furthermore, it has been shown that upon C. fulvum infection the protease activity in the apoplast increases (Solomon and Oliver, 2001
; Krüger et al., 2002; Rooney et al., 2005). To test the stability of affinity-tagged C. fulvum proteins in tomato AF, an in vitro assay was performed using N-terminally His6-FLAG-tagged P. pastoris-produced Avr2 protein. Twelve µg of Avr2 protein was incubated in 300 µl of AF isolated from leaves of 4-week-old MoneyMaker Cf-0 tomato plants. At regular intervals, subsamples were taken that were separated on a Tricine gel, and stained. The appearance of a band of a lower molecular weight than the His6-FLAG-tagged Avr2 indicated that degradation of the tagged Avr2 protein already occurs 1 h after incubation in AF (Fig. 5). N-terminal sequencing of the smaller protein band demonstrated that the affinity-tag was cleaved from the intact Avr2 protein. Although the major part of the FLAG-tag is still attached to the Avr2 protein, the removal of the N-terminal aspartic acid destroys the core epitope for the FLAG-specific antibody (Hopp et al., 1988; Miceli et al., 1994).
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To determine whether removal of the affinity-tags from PVX-expressed C. fulvum proteins also occurs in other Solanaceous species, His6-FLAG-tagged Avr2 was expressed in several Nicotiana species (N. benthamiana, N. clevelandi, N. glutinosa, N. tabacum). After 14 d, AF was isolated, denatured, and analysed on western blots. The N. tabacum plants severely suffered from the PVX infection such that it was impossible to obtain sufficient amounts of AF and were, therefore, discarded from the assay. Similar as for tomato, for N. benthamiana, N. clevelandi, and N. glutinosa, detection of Avr2 using the FLAG-specific antibodies failed while detection using Avr2-specific antibodies resulted in specific signals (data not shown).
In addition, an attempt was made to assess the stability of affinity-tagged C. fulvum effector proteins in a non-Solanaceous plant species. To this end the model plant Arabidopsis thaliana was chosen. Since PVX infection cannot be employed in Arabidopsis, P. pastoris produced His6-FLAG-tagged Avr2 was incubated for 1, 2, or 4 h in AF isolated from Arabidopsis as well as tomato (Fig. 6). This experiment clearly demonstrated that after incubation for 1 h in tomato AF, the His6-FLAG-tagged Avr2 was already partly degraded. This degradation did not occur upon incubation in Arabidopsis AF (Fig. 6). Since affinity-tagged effectors appear stable upon incubation in Arabidopsis AF, an attempt was made to perform affinity purification in Arabidopsis. Arabidopsis plants were stably transformed (Clough and Bent, 1998) allowing expression of His6-FLAG-tagged Ecp2 driven by the constitutive CaMV 35S promoter and using the tobacco PR-1a signal sequence for extracellular targeting. First generation transformants were selected on 50 µM kanamycin and subsequently transferred to soil. These plants were used for the isolation of AF as well as total leaf extract. Affinity purification based on the presence of the His6-tag indeed resulted in successful recovery of affinity-tagged Ecp2 in amounts that are clearly visible on silver-stained Tricine gel (Fig. 7A). The identity of the band was confirmed in western analysis using anti-FLAG to detect His6-FLAG-tagged Ecp2 (Fig. 7B). This experiment clearly shows that the affinity-tag remains on the fusion proteins expressed in Arabidopsis in contrast to fusion proteins expressed in Solanaceous plants.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Now that full-genome sequences are increasingly becoming available for more and more organisms, the major focus in research has shifted from the presence and transcription of specific genes towards the functions of their encoded products. Therefore, the interest in biochemical functions of proteins, their interacting partners, and their post-translational modifications is growing. Several methods are available to study proteins in vitro, but the question remains whether results obtained with in vitro methods represent the in vivo situation. Therefore, in vivo experiments are preferred above in vitro experiments. The production of recombinant proteins carrying epitope-tags that allow proteins of interest to be detected and purified through affinity purification are frequently used to find interacting partners within crude extracts of complex biological materials (Hearn and Acosta, 2001
; Terpe, 2003; Witte et al., 2004; Lichty et al., 2005).
To identify in planta virulence targets of the effector proteins that are secreted by C. fulvum during infection of its host, recombinant C. fulvum effector proteins were expressed in planta as fusions with an affinity-tag. Many protein-tags are available for such studies, each with their own characteristics. Because the C. fulvum effector proteins are rather small (ranging between 3 and 15 kDa), peptide affinity-tags were chosen (Table 2) that are likely to exert minimal or no effect on the tertiary structure and biological activity of the effector proteins. This is a major concern as in some cases it has indeed been reported that an affinity-tag can interfere with a biologically relevant target binding site (Goel et al., 2000).
To study effector proteins without the interference of additional C. fulvum effectors, in planta expression of single effector proteins was pursued rather than exploiting C. fulvum itself to express recombinant effectors. A systemic PVX expression system was chosen, allowing expression of the recombinant affinity-tagged fusion proteins throughout the whole plant, thus anticipating on low abundant interactors. In several studies, this PVX expression system was successfully employed for the in planta expression of secreted C. fulvum proteins (Luderer et al., 2002; Takken et al., 2000; Westerink et al., 2004). Also in this study, inoculation with PVX constructs resulted in the production of biologically active proteins that were correctly targeted towards the tomato leaf apoplast (Fig. 2).
Based on the results obtained in this study, it can be concluded that the stability of the affinity-tag that is fused to the C. fulvum effector proteins is rather low due to cleavage of the tag in the tomato apoplast. The C. fulvum effector proteins themselves are very stable due to their disulphide bridges that render them less prone to degradation by plant proteases (Kooman-Gersmann et al., 1997; Luderer et al., 2001; van den Hooven et al., 2001; van den Burg et al., 2003). Indeed, for Avr4 it has been shown that disruption of individual disulphide bridges causes the protein to be sensitive to proteolysis (van den Burg et al., 2003). Interestingly, it has been demonstrated for the C. fulvum effector peptide Avr9 that proteolytic processing by plant factors leads to the trimming of the 34 amino acid Avr9 precursor into the 28 amino acid mature Avr9 peptide that acts as an elicitor of plant defence (van den Ackerveken et al., 1993b). This trimming stops in close proximity to the first structural cysteine residue that is involved in a disulphide bridge (van den Hooven et al., 2001).
Since the protection of proteins by a compact tertiary structure seems to be crucial in the tomato leaf apoplast, it is anticipated that any stretch of linear amino acids that is added to an extracellular protein is sensitive to proteolytic degradation. This might explain why all the different tags that were tested, both as an N-terminal of C-terminal fusion, are proteolytically removed from the fusion protein. To overcome this problem, several strategies could be pursued. One such strategy could be to include the affinity-tag at such a position in the protein that it is protected by the tertiary structure. However, it is not unlikely that the addition of a number of amino acids in the core of a protein would interfere with its biological activity. An alternative strategy could be to develop affinity-tags for N- or C-terminal fusions that adopt a tertiary structure and thus might be more resistant to proteolytic cleavage. However, it is anticipated that due to their size such tags might interfere with the biological activity of the protein of interest.
Interestingly, the stability of the affinity-tagged effector proteins does not seem to be a major issue in the apoplast of Arabidopsis. Affinity purification using stable Arabidopsis transformants expressing His6-FLAG-tagged Ecp2 resulted in successful recovery of the tagged effector protein. Although C. fulvum is not a pathogen of Arabidopsis, it can be anticipated that part of the host defence responses that are targeted by its effector proteins might also exist in this non-host species. Therefore, an attempt will be made to isolate targets for C. fulvum effector proteins in Arabidopsis by exploiting overexpression of recombinant affinity-tagged fusions of C. fulvum effector proteins. Once virulence targets in Arabidopsis are identified, homologues of these targets might be found in the tomato genome. These homologues can be used for detailed functional analysis.
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Acknowledgements |
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BPHJT is supported by a VENI grant of the Research Council for Earth and Life sciences (ALW) of the Netherlands Organization for Scientific Research (NWO). This project was (co)financed by the Centre for BioSystems Genomics (CBSG) which is part of the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research (NWO). We thank Bert Essenstam, Henk Smit, and Dirk van Dam at Unifarm for excellent plant care. The PVX expression vector pGR106 was kindly provided by Dr D Baulcombe, Sainsbury Laboratory, Norwich, UK.
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Footnotes |
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* Both authors contributed equally.
Abbreviations: Avr, avirulence protein; AF, apoplastic fluid; Cf, tomato resistance gene against Cladosporium fulvum; Ecp, extracellular protein; HR, hypersensitive response; PVX, potato virus X.
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