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. 2015 Oct 19;17(4):565–576. doi: 10.1111/mpp.12315

A mutational analysis of the cytosolic domain of the tomato Cf‐9 disease‐resistance protein shows that membrane‐proximal residues are important for Avr9‐dependent necrosis

Apratim Chakrabarti 1,2, Thilaga Velusamy 1, Choon Yang Tee 1, David A Jones 1,✉
PMCID: PMC6638541  PMID: 26315781

Summary

The tomato Cf‐9 gene encodes a membrane‐anchored glycoprotein that imparts race‐specific resistance against the tomato leaf mould fungus C ladosporium fulvum in response to the avirulence protein Avr9. Although the N‐terminal half of the extracellular leucine‐rich repeat (eLRR) domain of the Cf‐9 protein determines its specificity for Avr9, the C‐terminal half, including its small cytosolic domain, is postulated to be involved in signalling. The cytosolic domain of Cf‐9 carries several residues that are potential sites for ubiquitinylation or phosphorylation, or signals for endocytic uptake. A targeted mutagenesis approach was employed to investigate the roles of these residues and cellular processes in Avr9‐dependent necrosis triggered by Cf‐9. Our results indicate that the membrane‐proximal region of the cytosolic domain of Cf‐9 plays an important role in Cf‐9‐mediated necrosis, and two amino acids within this region, a threonine (T835) and a proline (P838), are particularly important for Cf‐9 function. An alanine mutation of T835 had no effect on Cf‐9 function, but an aspartic acid mutation, which mimics phosphorylation, reduced Cf‐9 function. We therefore postulate that phosphorylation/de‐phosphorylation of T835 could act as a molecular switch to determine whether Cf‐9 is in a primed or inactive state. Yeast two‐hybrid analysis was used to show that the cytosolic domain of Cf‐9 interacts with the cytosolic domain of tomato VAP27. This interaction could be disrupted by an alanine mutation of P838, whereas interaction with CITRX remained unaffected. We therefore postulate that a proline‐induced kink in the membrane‐proximal region of the cytosolic domain of Cf‐9 may be important for interaction with VAP27, which may, in turn, be important for Cf‐9 function.

Keywords: Cladosporium fulvum, endocytosis motif, leucine‐rich repeat receptor‐like protein, protein phosphorylation, protein trafficking, Solanum lycopersicum

Introduction

In tomato, leaf mould disease is caused by the biotrophic fungus Cladosporium fulvum. Resistance to this disease is race specific and conforms to the gene‐for‐gene hypothesis (Flor, 1971). Tomato plants harbouring the Cf‐9 gene are resistant to races of C. fulvum producing the avirulence protein Avr9 (Hammond‐Kosack and Jones, 1997; Joosten and de Wit, 1999). In the absence of either Cf‐9 or Avr9 or any other matching Cf/Avr gene pair, the fungus colonizes the plant, but, when both Cf‐9 and Avr9 are present, growth of the fungus is halted at the site of infection.

Cf‐9 (isolated by Jones et al., 1994) is the founding member of a large family of Hcr9 genes (Homologues of C. fulvum resistance gene Cf‐9), which also includes the Cf‐4 (Thomas et al., 1997), Cf‐4E (Takken et al., 1999), Cf‐9B (Panter et al., 2002), Cf‐9DC (Kruijt et al., 2004; Van der Hoorn et al., 2001a) and Cf‐ECP (Kruijt et al., 2005; Lauge et al., 1998) genes. These genes encode type I membrane‐anchored glycoproteins with large extracellular leucine‐rich repeat (eLRR) protein interaction domains, consistent with their roles as cell surface receptors (Fig. 1) (Jones et al., 1994; Kruijt et al., 2005; Rivas and Thomas, 2005). The Hcr9 proteins have domain structures similar to those of other plant receptor‐like proteins (RLPs) and receptor‐like kinases (RLKs), but, unlike RLKs, do not have cytosolic protein kinase domains (Jones et al., 1994; Rivas and Thomas, 2005; Wang et al., 2010). Typically, the majority of amino acid variations between members of the Hcr9 protein family are present in the N‐terminal halves of the proteins, including the mature N‐terminus and first 17 LRRs. Most of these variations occur among putative solvent‐exposed residues and have been proposed to play a role in Avr protein recognition (Jones and Jones, 1997; Parniske et al., 1997; Thomas et al., 1997). Extensive domain swap studies, as well as gene shuffling experiments, have narrowed down the region of Cf‐9 essential for Avr9 recognition to LRRs 13–16 (Chakrabarti et al., 2009; Wulff et al., 2001, 2009). Targeted mutation experiments identified five solvent‐exposed residues, C387 and Y389 in LRR13, E411 in LRR14, A433 in LRR15 and L457 in LRR16, as critical for Avr9 recognition (Wulff et al., 2001, 2009).

Figure 1.

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Cf‐9 structure and alignment of amino acids in the predicted cytosolic domains of Hcr9 family members. (A) Schematic diagram of the Cf‐9 protein showing seven predicted domains. Domain A is the signal peptide, absent from the mature protein. The leucine‐rich repeat (LRR) domain is subdivided into C1 (LRRs 1–23), C2 (loop out) and C3 (LRRs 23–27). Domain F is the transmembrane domain and domain G is the cytosolic C‐terminal domain. (B) Amino acids within domain G of the Hcr9 proteins are aligned to show their high degree of conservation. Serine and threonine residues (red) are potential sites for phosphorylation. Lysine residues (blue) are potential sites for ubiquitinylation on their ε‐NH2 groups. The YPAW and II motifs (green) are similar to the canonical Yxxϕ (x = any amino acid; ϕ = amino acid with bulky hydrophobic side chain) and LL endocytosis motifs.

In contrast with the variable N‐terminal halves, the C‐terminal halves of Hcr9 proteins are highly conserved and are thought to be involved in signal transduction. Despite the lack of any obvious signalling domain, the cytosolic domain of Cf‐9 carries several residues and sequence motifs that are conserved among Hcr9 proteins and thus may have a role in defence signalling (Fig. 1). The cytosolic domain of Cf‐9 carries six lysine (K) residues, five of which are highly conserved among members of the Hcr9 family. Two of these lysines form part of a functional C‐terminal endoplasmic reticulum (ER)‐retrieval motif (KKxx) (Benghezal et al., 2000), which may play a role in the quality control of protein folding (Benghezal et al., 2000; Piedras et al., 2000). Mutation of these two lysines to alanines led to only a slight loss of Cf‐9 activity, suggesting that this motif is not essential (Van der Hoorn et al., 2001b).

Lysine residues are also potential sites for ubiquitinylation, which is a reversible, post‐translational modification involved in protein degradation, membrane trafficking, signal transduction and host defence (MacGurn et al., 2012; Marino et al., 2012; Polo, 2012). A number of plant ubiquitin ligases have been documented as both positive and negative regulators of plant immunity. For example, direct ubiquitinylation of the flagellin receptor FLS2 by the ubiquitin ligases PUB12 and PUB13 attenuates the response to flagellin (Lu et al., 2011). Two ubiquitin ligases (ACRE74 and ACRE276) have been demonstrated to play a positive role in resistance to C. fulvum mediated by the Cf‐9 gene (González‐Lamothe et al., 2006).

Mono‐ubiquitinylation of one or more lysine residues is generally associated with endocytosis of membrane proteins (Polo, 2012), but polyubiquitinylation (attachment of multiple ubiquitin molecules linked together in a linear or branched fashion) generally marks proteins for proteasomal or vacuolar degradation. For example, polyubiquitinylation of the brassinosteroid receptor BRI1 marks it for endocytosis and targeting to the vacuole (Martins et al. 1997). Thus, the lysine residues in the cytosolic domain of Cf‐9 could potentially be involved in protein endocytosis and/or degradation.

Endocytosis may play a role in the regulation of defence signalling through the recycling of non‐activated receptors or the removal of activated receptors for degradation. For example, FLS2 is recycled in a pathway sensitive to Brefeldin A, an inhibitor of endocytic recycling of plasma membrane proteins via early endosomes, but, after flagellin binding, FLS2 is targeted to late endosomes and multivesicular bodies destined for vacuolar degradation in a pathway insensitive to Brefeldin A (Beck et al., 2012; Spallek et al., 2013). Whether endocytosis also plays a role in receptor activation and signalling from endosomal compartments remains unclear, but it should be noted that the brassinosteroid receptor BRI1, which plays a negative role in plant defence in addition to its primary role in plant development (Albrecht et al., 2012; Belkhadir et al., 2012), signals primarily from the plasma membrane rather than endosomes (Di Rubbo et al., 2013; Irani et al., 2012; Martins et al. 1997).

Endocytosis of membrane‐anchored receptor proteins can also occur through the recognition of cytosolic sorting motifs by the endocytic machinery without the need for ubiquitinylation. The best‐characterized endocytosis motifs are the Yxxϕ (where ϕ is any amino acid with a bulky hydrophobic side chain) and di‐leucine (LL) motifs (Bonifacino and Traub, 2003), and these motifs are present in a number of RLPs and RLKs important for plant development and defence signalling (Geldner and Robatzek, 2008; Zhang and Thomma, 2013). Endocytosis of the tomato eLRR‐RLP LeEIX2 is a crucial step in signalling triggered by EIX (ethylene‐inducing xylanase), and mutation of the Yxxϕ motif present in the cytosolic domain of LeEIX2 renders it ineffective (Ron and Avni, 2004). Cf‐9 has cytosolic YPAW and II motifs similar to the canonical Yxxϕ and LL endocytosis motifs (Fig. 1), but the role of these motifs in Cf‐9‐mediated defence signalling has never been studied.

The cytosolic domain of Cf‐9 also contains conserved serine (S) and threonine (T) residues, which are putative sites for phosphorylation/de‐phosphorylation, activities that play major roles in the regulation of almost all cellular processes. A wound‐induced protein kinase (WIPK), a salicylic acid‐induced protein kinase (SIPK) and a Ca‐dependent protein kinase (NtCDPK2) are all activated during Cf‐9‐mediated defence signalling (Romeis et al., 1999, 2000a, 2000b), suggesting a positive role for these protein kinases. Conversely, silencing of the protein phosphatase gene PP2Ac1 by virus‐induced gene silencing (VIGS) caused localized cell death in leaves and stems of silenced Nicotiana benthamiana plants, and enhanced the induction of Cf‐9/Avr9‐dependent necrosis (He et al., 2004), suggesting a negative role for protein phosphatase 2A. Furthermore, Cf‐9 interacts with the eLRR‐RLK SOBIR1 (Liebrand et al., 2013) and the C‐terminal domain of Cf‐9 protein interacts with an adaptor protein CITRX that engages an Avr9/Cf‐9‐induced kinase ACIK1 (Nekrasov et al., 2006; Rivas et al., 2004; Rowland et al., 2005). De novo phosphorylation after flagellin perception has been reported for FLS2 (Schulze et al., 2010). Mutation of S938, one of the possible phosphorylation sites in FLS2, to alanine severely reduced flagellin‐induced production of reactive oxygen species and callose deposition, but did not disrupt FLS2 interaction with its signalling partner BAK1 (Cao et al., 2013).

Phosphorylation can also regulate ubiquitinylation by influencing the interaction of ubiquitin ligases with target proteins, as well as regulating the activities of ubiquitinylating enzymes (Gao and Karin, 2005; Hunter, 2007). For example, ligand‐induced phosphorylation of epidermal growth factor (EGF) and platelet‐derived growth factor (PDGF) tyrosine kinase receptors promotes ubiquitinylation through the formation of a binding site for the ubiquitin ligase protein c‐Cbl (Hunter, 2007). Ubiquitinylation of activated receptor tyrosine kinases appears to target them for degradation (Lemmon and Schlessinger, 2010). In contrast, ubiquitin‐mediated degradation of FLS2 does not appear to be associated with FLS2 phosphorylation, but depends on the phosphorylation of the E3 ubiquitin ligase PUB12/PUB13 by the FLS2‐associated kinase BAK1 (Lu et al., 2011). Taken together, these various observations suggest that phosphorylation, ubiquitinylation and endocytosis, apart from their separate roles in maintaining cellular function, can also act in a concerted manner.

In the present study, the roles of ubiquitinylation, phosphorylation and endocytosis in Cf‐9 function have been addressed by mutating various conserved residues and/or motifs present in the C‐terminal domain of Cf‐9 which may be associated with these post‐translational modifications. The effect of these targeted mutation(s) on Cf‐9 function was assessed via induction of Cf‐9/Avr9‐dependent necrosis following agroinfiltration of N. benthamiana. We also studied how these mutations affect the interaction of the C‐terminal domain of Cf‐9 with two known interacting proteins: VAP27 (Laurent et al., 2000) and CITRX (Rivas et al., 2004). Although our findings suggest that ubiquitinylation or endocytosis of Cf‐9 may not be directly involved in Cf‐9‐mediated necrosis, we have identified a small membrane‐proximal region within the cytosolic domain of Cf‐9 that affects Avr9‐dependent necrosis and interaction of Cf‐9 with VAP27. We have also identified a threonine (T) residue and a proline (P) residue within this region which appear to be important for Cf‐9 function.

Results

Role of cytosolic lysine residues in Cf‐9 function

The lysine (K) residues at positions 847 (K6), 855 (K5), 857 (K4), 858 (K3), 860 (K2) and 861 (K1) of Cf‐9 were cumulatively mutated to arginine (R) residues starting from K1 and K2 in K1‐2R through to mutations in all six lysine residues in K1‐6R. Plasmids encoding K1‐2R, K1‐4R, K1‐5R and K1‐6R mutations of haemagglutinin‐epitope‐tagged Cf‐9 protein (HA:Cf‐9) were co‐agroinfiltrated with pSLJ6201 (Avr9) into leaves of N. benthamiana. Co‐expression of HA:Cf‐9K1‐2R, HA:Cf‐9K1‐4R, HA:Cf‐9K1‐5R or HA:Cf‐9K1‐6R with Avr9 was as effective as HA:Cf‐9 (wild‐type) in causing necrosis (Fig. S1A, see Supporting Information). None of these constructs was active on its own in N. benthamiana. As ubiquitin is attached to the ε‐amino group of lysine residues, these results suggest that ubiquitinylation of Cf‐9 is not essential for Avr9‐dependent necrosis.

Role of the cytosolic YPAW and II motifs in Cf‐9 function

The putative endocytosis signal motifs of Cf‐9, YPAW and II were mutated either to destroy them (YPAW to APAW and II to AA) or to make them more similar to the canonical motifs Yxxϕ and LL reported in other systems (YPAW to YAAF and II to LL). Changing the II motif to either LL or AA (HA:Cf‐9LL and HA:Cf‐9AA) did not affect Avr9‐dependent necrosis, and the Cf‐9 mutant that combined the II to AA mutation with the Y837A mutation (HA:Cf‐9APAW/AA) showed Avr9‐dependent necrosis in N. benthamiana equivalent to HA:Cf‐9 (Fig. S1B), suggesting that neither Y837 nor the II motif is critical for Cf‐9 function. Surprisingly, necrotic responses were attenuated when mutations were introduced simultaneously to make the YPAW and II motifs better resemble endocytosis marker motifs (HA:Cf‐9YAAF/LL; Fig. S1B). Given that the II to LL mutation had no effect on Cf‐9 function, it was likely that mutations of either P838 or W840 or both caused the observed reduction in necrosis in the HA:Cf‐9YAAF/LL mutant. None of the four mutated Cf‐9 proteins caused necrosis in the absence of Avr9.

To further test their role within the YPAW motif, P838 and W840 were mutated both individually and together to alanine (A). W840 was also mutated individually to phenylalanine (F). Both HA:Cf‐9W840A and HA:Cf‐9W840F showed a wild‐type response, whereas the development of Avr9‐dependent necrosis was significantly reduced in HA:Cf‐9P838A and HA:Cf‐9P838A,W840A (Fig. 2A). None of these mutants was autoactive in N. benthamiana (Fig. 2A). These results suggest that P838 is essential for a wild‐type level of Cf‐9 activity.

Figure 2.

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Effects of mutations in the YPAW motif and S834 and T835 residues in the cytosolic domain of Cf‐9. (A) Binary vectors carrying HA:Cf‐9P838A, HA:Cf‐9P838A,W840A, HA:Cf‐9W840A and HA:Cf‐9W840F were assayed for necrosis induction in leaves of 8–10‐week‐old N icotiana benthamiana plants by co‐agroinfiltration with pSLJ6201 (Avr9) (top row). The control vector pCBJ310 (HA:Cf‐9) and vectors expressing mutated versions of HA:Cf‐9 were infiltrated in the left and right halves of each leaf, respectively. These vectors were also assayed for Avr9‐independent activity in N . benthamiana (bottom row). The leaves were photographed 5–7 days after infiltration. (B) Binary vectors carrying HA:Cf‐9S834D,T835D, HA:Cf‐9S834A,T835A, HA:Cf‐9S834D, HA:Cf‐9S834A, HA:Cf‐9T835D and HA:Cf‐9T835A were similarly assayed for Avr9‐dependent or ‐independent necrosis induction in N . benthamiana. (C) Binary vectors carrying HA:Cf‐9S834A and HA:Cf‐9T835A were additionally assayed for Avr9‐independent necrosis induction in N . benthamiana via co‐agroinfiltration with a 35S:p19 construct to enhance protein expression. (D) Western blot showing expression levels of haemagglutinin (HA)‐tagged Cf‐9 and mutants in N . benthamiana leaves 5 days after co‐agroinfiltration with 35S:p19. Bottom panel shows Rubisco bands on Ponceau‐stained membrane demonstrating approximately equal protein loading and transfer. –, leaves infiltrated with GV3101 cells carrying 35S:p19.

Role of cytosolic serine and threonine residues in Cf‐9 function

Serine (S) and threonine (T) residues in the cytosolic domain of Cf‐9 are potential sites for phosphorylation. These residues were either mutated to aspartate (D) to mimic phosphorylation or to alanine (A) to destroy their phosphorylation potential. When the Cf‐9 mutants HA:Cf‐9T853D,T854D, HA:Cf‐9T853A,T854A, HA:Cf‐9S842D and HA:Cf‐9S842A were tested, Avr9‐dependent necrosis remained unaffected and no autoactivity was observed (Fig. S1C). These observations suggest that phosphorylation of S842, T853 and T854, if it occurs, is not important for Avr9‐dependent necrosis.

Interestingly, when HA:Cf‐9S834D,T835D and HA:Cf‐9S834A,T835A were co‐expressed with Avr9, the necrosis observed with the HA:Cf‐9S834D,T835D mutant was reduced compared with the wild‐type HA:Cf‐9 (Fig. 2B), whereas HA:Cf‐9S834A,T835A demonstrated wild‐type activity. To examine the role of each residue, S834 and T835 were mutated individually to either D or A. Avr9‐dependent necrosis was not attenuated in HA:Cf‐9S834D, HA:Cf‐9S834A or HA:Cf‐9T835A, but was drastically reduced in HA:Cf‐9T835D (Fig. 2B). This suggests that mimicking phosphorylation at this position has a negative effect on Cf‐9 function.

When these mutants were tested for Avr9‐independent necrosis in N. benthamiana, only HA:Cf‐9S834A,T835A was able to cause sporadic patchy necrosis (Fig. 2B). To examine this autoactivity further, the four individual D or A mutants of S834 and T835 were also co‐expressed with the p19 silencing suppressor protein from tomato bushy stunt virus in order to enhance their expression in N. benthamiana (Voinnet et al., 2003). Only HA:Cf‐9S834A, and not any of the other S834 or T835 mutants of Cf‐9, caused autogenic necrosis when co‐expressed with p19 in N. benthamiana (Fig. 2C). Protein gel blot analysis indicated that these mutant proteins all accumulated to levels comparable with or greater than those of the wild‐type protein in N. benthamiana (Fig. 2D).

The role of the membrane‐proximal region of the cytosolic domain in Cf‐9 function

Taken together, the attenuation of Avr9‐dependent necrosis in T835D and P838A mutants, the weak auto‐activity of the S834A mutant and the apparent wild‐type activity for mutations made downstream of the P838 residue suggest that the membrane‐proximal region of the Cf‐9 cytosolic domain plays a more prominent role than the C‐terminal part in Cf‐9/Avr9‐mediated necrosis. To examine this further, we mutated Q836, F841 and R843 to alanine in order to complete a mutational scan of the region between S834A to R843, inclusive. Under our laboratory conditions, co‐expression of Cf‐9 and Avr9 in N. benthamiana elicited a stronger response than co‐expression in tobacco. Therefore, these nine mutants were tested in Avr9‐expressing N. tabacum plants to check for any subtle effects caused by these mutations (Fig. 3A). As expected, HA:Cf‐9P838A demonstrated a drastic reduction in necrosis induction as observed in co‐expression studies in N. benthamiana. Of the other mutations, only Q836A resulted in a consistent reduction in necrosis induction, whereas some minor reduction was occasionally observed with the HA:Cf‐9Y837A mutant. Q836A also reduced necrosis slightly when tested in N. benthamiana (Fig. S3, see Supporting Information). The Cf‐9S834A mutant consistently produced a faster and stronger necrosis compared with the wild‐type, reiterating the auto‐activity observed previously in N. benthamiana. Protein gel blot analysis indicated that the transiently expressed mutant proteins, including the loss‐of‐function mutants, were expressed at levels comparable with that of the wild‐type protein in N. benthamiana (Figs 2D and 3B). However, we cannot exclude the possibility that the reduced necrosis observed in N. tabacum for the P838A, Q836A and Y837A mutations was a consequence of reduced protein accumulation relative to wild‐type protein, rather than reduced protein activity. Interestingly, HA:Cf‐9T835A was consistently present at higher levels than wild‐type HA:Cf‐9 as judged by the relative band intensity. Overall, our analysis identified S834, T835, Q836 and P838, all of which are close to the transmembrane domain of Cf‐9, as residues important for Cf‐9 function.

Figure 3.

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Effects of mutations in the membrane‐proximal region of the cytosolic domain of Cf‐9. (A) Binary vectors carrying HA:Cf‐9S834A, HA:Cf‐9T835A, HA:Cf‐9Q836A, HA:Cf‐9Y837A, HA:Cf‐9P838A, HA:Cf‐9W840A, HA:Cf‐9F841A, HA:Cf‐9S842A, and HA:Cf‐9R843A constructs, together with the control vector carrying HA:Cf‐9, were assayed for necrosis induction in leaves of Avr9 tobacco plants by agroinfiltration. One leaf panel was used for each construct. The two left‐hand leaves show fully developed (21 days post‐infiltration) responses as evident from the opaque browny‐white necrotic patches in each responding leaf panel. The right‐hand leaf shows a developing (5 days post‐infiltration) response as evident from the grey translucent necrotic patches emerging in each responding leaf panel. It should be noted that necrosis is already fully confluent in the HA:Cf‐9S834A leaf panel, but still developing in the other leaf panels, indicating earlier and stronger necrosis induction in the case of HA:Cf‐9S834A. This differential was consistent within agroinfiltrated leaves, despite variation in responses between leaves. (B) Western blot showing expression levels of haemagglutinin (HA)‐tagged Cf‐9 and mutants in N icotiana benthamiana leaves 5 days after co‐agroinfiltration with 35S:p19. Bottom panel shows Rubisco bands on Ponceau‐stained membrane demonstrating approximately equal protein loading and transfer. –, leaves infiltrated with GV3101 cells carrying 35S:p19.

Effect of domain G mutations on interaction with VAP27 and CITRX

As the P838A mutation affected Cf‐9 function without reducing protein accumulation, we tested whether this mutation could perturb the interaction of Cf‐9 with the known cytosolic interactors VAP27 and CITRX. VAP27 was identified originally as a Nicotiana plumbaginifolia protein interacting in a yeast two‐hybrid assay with a truncated Cf‐9 protein carrying domains E, F and G (Laurent et al., 2000). The tomato homologue of VAP27 was polymerase chain reaction (PCR) amplified and tested for its interaction with the domains E, F and G of Cf‐9, as well as the cytosolic domain G by itself. VAP27 protein, and the EFG and G domains of Cf‐9, were expressed as fusion proteins to either the activation domain (AD) or the DNA‐binding domain (BD) of the GAL4 transcriptional activator in the histidine‐biosynthesis‐deficient Saccharomyces cerevisiae strain HF7c, and interaction was detected using a histidine biosynthetic gene responsive to the GAL4 transcriptional activator as a selectable marker. No significant growth on histidine‐deficient medium was observed for yeast transformants expressing GAL4 AD or BD, together with any of the test proteins fused to either AD or BD (Fig. S2, see Supporting Information). However, when tomato VAP27:AD was co‐expressed with either domain EFG:BD or domain G:BD, growth was detected on selective media (Fig. S2A), indicating that tomato VAP27 interacts with Cf‐9 and that domain G of Cf‐9 is sufficient for this interaction. No interaction was detected when tomato VAP27:BD was co‐expressed with domains EFG:AD or G:AD (Fig. S2A), consistent with the observations of Laurent et al. (2000). To investigate whether the transmembrane domain of VAP27 was responsible for this unidirectionality, the yeast two‐hybrid experiments with domain G were repeated using VAP27 lacking its transmembrane domain. The results were essentially the same (Fig. S2B), indicating that the transmembrane domain of VAP27 was not responsible.

After establishing a yeast two‐hybrid interaction between tomato VAP27 and domain G of Cf‐9, we tested whether this interaction was affected by the P838A mutation or some of the additional mutations in domain G (T835A, W840A and S842A). Co‐transformants carrying VAP27 and the T835A, T835D, W840A or S842A mutants of domain G showed growth on histidine‐deficient medium similar to that of the non‐mutated positive control, indicating that these mutations did not affect the interaction between tomato VAP27 and domain G of Cf‐9 (Figs 4A and S2C, D). However, growth of VAP27 co‐transformants carrying the P838A mutant of domain G was significantly reduced compared with that of the positive control (Figs 4A and S2C), indicating that the interaction of tomato VAP27 with Cf‐9 domain G was affected by the P838A mutation. Yeast co‐transformed with domain G:BD and empty AD vector (pGADT7) constructs or empty BD vector (pGBKT7) and VAP27:AD constructs were used as negative controls. No significant growth on histidine‐deficient medium was observed for any of the negative controls tested (Fig. S2C, D).

Figure 4.

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Yeast two‐hybrid analyses of the interactions between domain G mutants of Cf‐9 and VAP27 or CITRX. (A) Interaction between domain G mutants fused to the BD of the GAL4 transcription factor (T835A, W840A and S842A) and VAP27 fused to the AD of the GAL4 transcription factor (VAP27:AD), but no interaction for the P838A mutant of domain G. (B) Interaction between domain G mutants fused to the binding domain (BD) of the GAL4 transcription factor (T835A, P838A, W840A and S842A) and CITRX fused to the activation domain (AD) of the GAL4 transcription factor (CITRX:AD). Five independent yeast co‐transformants were used to test each interaction in (A) and (B).

The CITRX protein also interacts with domain G of Cf‐9 and negatively regulates Cf‐9 function (Rivas et al., 2004). Interaction between CITRX and domain G of Cf‐9 was confirmed by the growth of co‐transformants carrying CITRX:AD and domain G:BD fusions in the absence of histidine (Fig. S2C, D). Unlike VAP27, swapping of CITRX and domain G fusions between AD or BD did not affect this interaction (data not shown). None of the mutations tested, including P838A, affected the growth of CITRX co‐transformants on histidine‐deficient medium (Figs 4B and S2C, D). Interaction between the P838A mutant of domain G and CITRX indicates that a lack of interaction with VAP27 is unlikely to be a result of a lack of P838A domain G:BD fusion protein accumulation in yeast. No significant growth on histidine‐deficient medium was observed for any of the negative controls tested (Fig. S2C, D).

Discussion

Ubiquitinylation of Cf‐9 does not play a role in the Cf‐9 response to Avr9

In this study, we analysed the roles of several conserved amino acids and motifs in the cytosolic domain of Cf‐9 in defence signalling. Cf‐9 has six lysine residues in the cytosolic domain that are potential sites for ubiquitinylation. Mutation of all the lysines to arginines did not affect Cf‐9‐mediated necrosis in response to Avr9, suggesting that ubiquitinylation of these lysines, if it occurs, is not critical for Cf‐9 function. Previously, mutations of K860 and K861 to alanines were shown to slightly reduce Avr9‐dependent necrosis in transient expression assays in tobacco (Van der Hoorn et al., 2001b), and mutations of these two lysines to asparagines also reduced slightly Cf‐9‐mediated resistance to C. fulvum in transgenic tomato plants (D. A. Jones, unpublished data). The apparent lack of any effect of the lysine to arginine mutations in our study may reflect the more conserved nature of the changes we made.

As blocking of all the potential ubiquitinylation sites did not affect Cf‐9/Avr9‐dependent necrosis (HA:Cf‐9K1‐6R; Fig. S1A), ubiquitin‐dependent endocytosis or 26S proteasomal degradation of Cf‐9 does not seem to be required to trigger the Cf‐9‐dependent plant defence response. Whether Cf‐9 undergoes degradation via the ubiquitin/26S proteasome pathway cannot be ascertained from the present findings. However, Cf‐9/Avr9‐dependent necrosis was abolished in N. benthamiana plants silenced for NbSgt1, which encodes the ubiquitin ligase‐associated protein SGT1 (Peart et al., 2002), suggesting a role for ubiquitinylation at some point in Cf‐9/Avr9‐dependent necrosis. This may be mediated by the degradation or endocytosis of a protein involved in a shared pathway or by the regulation of assembly/disassembly of R protein complexes consistent with the proposed co‐chaperone role of SGT1 (Takahashi et al., 2003), rather than the degradation of Cf‐9 itself. The additional involvement of SGT1 in Cf‐4/Avr4‐, Rx/PVX‐CP‐ and Pto/AvrPto‐mediated resistance, and in both host and non‐host resistance (Peart et al., 2002), suggests a shared role.

Motifs resembling endocytosis signals do not play a role in Cf‐9 function consistent with endocytosis

The YPAW and II motifs present in the cytosolic domain of Cf‐9 are similar to the Yxxϕ and LL endocytosis motifs in animal systems (Bonifacino and Traub, 2003). Mutation of the II motif to either LL or AA did not affect Avr9‐dependent necrosis, suggesting that I851 and I852 are unlikely to play any major role in Cf‐9 function or endocytosis. Mutation of either Y837 or W840 in the YPAW motif had no effect on Avr9‐dependent necrosis in N. benthamiana (Fig. S1B), and only a slight reduction in necrosis was observed for the Y837A mutation when tested in Avr9 tobacco (Fig. 2). Alanine mutations of amino acid residues in the putative C‐terminal E/DxxxLϕ endocytosis motif or the conserved Y residue in the Yxxϕ motif in the cytosolic domain of Ve1 did not impair Ave1‐dependent necrosis in tobacco or resistance to Verticillium dahliae race 1 in transgenic Arabidopsis (Zhang et al., 2014). However, mutations of the Y residue in the YFTF motif of LeEIX2 completely inhibited EIX‐induced necrosis and endocytosis of LeEIX2 (Ron and Avni, 2004). The Y and ϕ residues at the Y + 3 position in the Yxxϕ endocytosis motif are critical for endocytosis as they bind to two hydrophobic pockets in the μ2 domain of the adaptor protein AP2. Correct binding of the Y and ϕ residues results in additional hydrogen bonding, ensuring strong binding (Owen and Evans, 1998). Thus, our findings suggest that either YPAW does not function as an endocytosis motif or it is not important for Cf‐9 function if it does. Generally, the ϕ position in the Yxxϕ motif is occupied by amino acids with bulky hydrophobic side chains: methionine, valine, leucine, isoleucine and phenylalanine (Marks et al., 1996). Tryptophan has a polar side chain and would therefore be unusual in this position. In the reported variations of the LL endocytosis motif, only one of the leucines can be substituted with isoleucine or methionine (Mousavi et al., 2004). The LL signals are also often associated with upstream acidic amino acid residues (D/ExxxLL/I, DxxLL) (Bonifacino and Traub, 2003; Evans and Owen, 2002), whereas the II motif in Cf‐9 is preceded by two basic residues at the corresponding positions. The experimental data obtained in this study are therefore consistent with the relatively poor fit to the canonical endocytosis motifs.

In the P2X4 ATP‐gated ion channels in the rat, it was found that YxxGL can act as a signal for endocytosis (Royle et al., 2005). The additional glycine (G) is tolerated by a ‘kink’ in the extended β‐sheet structure formed by the YxxGL motif, allowing the leucine (L) at the Y + 4 position to fill the ϕ binding pocket of μ2. Cf‐9 has a kink‐forming proline (P) at the Y + 1 position, which is critical for Avr9‐dependent necrosis, and a phenylalanine (F) at the Y + 4 position. In Cf‐9, the presence of proline within the YPAWF motif could perhaps have provided the necessary ‘kink’ to bring the F into the required position to interact with the adaptor protein, potentially making YPAWF an extended endocytosis motif. However, the HA:Cf‐9F841A mutant was as active as the wild‐type Cf‐9 protein in Avr9‐dependent necrosis in both N. benthamiana and N. tabacum, suggesting that F841 is not critical for Cf‐9 function and that YPAWF is unlikely to be an extended endocytosis motif.

Although our findings suggest that YPAW and II are unlikely signature motifs for endocytosis, neither Avr9‐triggered internalization of Cf‐9 nor a role for endocytosis in Cf‐9 signalling can be ruled out. Over‐expression of the endocytosis inhibitor protein AtEHD2 in N. tabacum resulted in reduced Cf‐9/Avr9‐dependent necrosis (Bar and Avni, 2009; Bar et al., 2008), suggesting that endocytosis may play a role in Cf‐9 function. It is also possible that the cytosolic domain of Cf‐9 carries additional unknown endocytosis motifs which also need to be mutated to observe any major effect on Cf‐9 function. A study on endocytosis of derivatives of the type I transmembrane protein CD8 carrying randomly generated short sequences in their cytosolic domain suggested the presence of a more extensive repertoire of endocytosis signals than just the Yxxϕ, FxNPxY and LL motifs (Kozik et al., 2010).

Phosphorylation of a membrane‐proximal threonine residue may play a role in Cf‐9 function

The serine and threonine residues of the cytosolic domain of Cf‐9 were investigated as possible sites for phosphorylation. Only mutation of T835 to aspartate (D) resulted in a reduction in Avr9‐dependent necrosis in N. benthamiana, indicating that only a mutation mimicking phosphorylation at this position negatively affects Cf‐9 function. Consistent with this observation, T835 has the highest NetPhos score for predicted phosphorylation (http://www.cbs.dtu.dk/services/NetPhos/; Blom et al., 1999) of all the potential phosphorylation sites in the cytosolic domain of Cf‐9. Although we have not demonstrated that T835 is phosphorylated, it seems likely that phosphorylation of T835, if it did occur, would lead to the attenuation of the Cf‐9‐mediated defence response. Given that the Cf‐9‐interacting serine/threonine kinases ACIK1 (Rowland et al., 2005) and SOBIR1 (Liebrand et al., 2013) are positive regulators of Cf‐9 function, they are unlikely to be responsible for any phosphorylation of T835. Phosphorylation of T835 may be required to maintain Cf‐9 in an inactive state, and dephosphorylation of T835 may be necessary, but not sufficient, for activation of the Cf‐9 response pathway. We therefore hypothesize that phosphorylation/de‐phosphorylation of T835 could act as a molecular switch to determine whether Cf‐9 is in an inactive or primed state. Given that the T835A mutation of Cf‐9 consistently allowed the accumulation of more protein than did wild‐type Cf‐9, it is possible that phosphorylation of T835, if it occurs, could also be involved in the regulation of Cf‐9 protein abundance through a pathway for degradation other than lysine ubiquitinylation. However, the T835D mutation of Cf‐9 consistently allowed the accumulation of protein comparable with that of wild‐type Cf‐9, indicating that protein degradation by such a pathway, if it occurs, does not have a major effect on Cf‐9 abundance and does not explain the loss of function caused by this mutation.

In contrast with the loss of activity observed for the T835D mutation and the increased protein abundance observed for the T835A mutation, mutating the adjacent S834 to aspartate did not result in any loss of Cf‐9 activity or change in protein abundance. Instead, stronger necrosis was observed with the HA:Cf‐9S834A mutant than with wild‐type HA:Cf‐9 in N. tabacum, and over‐expression of HA:Cf‐9S834A, but not HA:Cf‐9T835A, in N. benthamiana resulted in weak Avr9‐independent necrosis. However, unlike T835, S834 has a very low NetPhos score for predicted phosphorylation, suggesting that the S834A mutation, rather than mimicking of dephosphorylation, may have had a more direct effect on Cf‐9 function. It is possible that the S834A mutation affected interaction with a negative regulator of Cf‐9, such as CITRX, resulting in enhanced activity of the Cf‐9S834A mutant. However, the observation that HA:Cf‐9S834A triggered stronger necrosis in the presence of Avr9 than in its absence indicates that HA:Cf‐9S834A is still negatively regulated to a large extent.

A structural kink in the membrane‐proximal region of the cytosolic domain of Cf‐9 may be more important for VAP27 interaction and Cf‐9 function than the exact nature of the residues involved

All the mutations that were found to affect Cf‐9 function (S834A, T835D, Q836A, Y837A and P838A) are located in the membrane‐proximal region of the C‐terminal cytosolic domain of Cf‐9. These mutations could have a number of possible effects, including a direct effect on interaction with proteins involved in signalling or proteins involved in the correct assembly of a signalling complex. They could also have an indirect effect on interactions with other proteins through a structural effect on the conformation of the adjacent transmembrane domain, which could be involved in the formation of helical bundles with co‐receptors or signalling partners, or on the cytosolic domain itself. Of these five mutations, only the P838A and T835D mutations were tested for an effect on interaction with the known Cf‐9‐interacting proteins CITRX and VAP27, and only the P838A mutation was found to affect interaction with the cytosolic domain of VAP27. Neither mutation affected interaction with CITRX. A functional requirement for VAP27 has not yet been established for Cf‐9, but it is possible that a lack of interaction with VAP27 may be responsible for the loss of Cf‐9 function observed for the P838A mutation, suggesting a positive role for VAP27 in Cf‐9 function. The role of VAP27 in Cf‐9 function is not known, but it may be required for correct assembly and trafficking of the Cf‐9 protein complex to the plasma membrane or for endocytosis of Cf‐9, if it occurs (Kruijt et al., 2005; Laurent et al., 2000).

There are several different roles that P838 could play in the interaction with VAP27. Proline is generally considered as a helix breaker in water‐soluble proteins (MacArthur and Thornton, 1991). The pyrrolidine ring in proline restricts its backbone dihedral phi angle to around −60°. This imparts an exceptional conformational rigidity to proline compared with other amino acids and makes proline a breaker of regular secondary structures, such as α helices, as well as the most preferred amino acid for turns. As a result, proline residues are generally solvent exposed and potentially involved in protein–protein interactions. Hydrogen atoms on the pyrrolidine ring of proline can engage in hydrogen bonding with the π electron cloud in the aromatic ring of either adjacent or intermolecular tyrosine (Y), phenylalanine (F), tryptophan (W) or histidine (H) residues (Bhattacharyya and Chakrabarti, 2003). P838 is flanked by Y837, W838 and F839, providing ample opportunity for cis interaction, perhaps contributing to a protein turn rather than interaction with another protein. Whether interacting with these residues or not, it can be argued that the observed effect of the P838A mutation on Cf‐9 function is consistent with a major disruptive effect on the structure of the cytosolic domain of Cf‐9. The less severe effects observed for mutations in residues surrounding P838 are consistent with an accessory role in maintaining/stabilizing the structure of the cytosolic domain that could potentially involve interaction with P838.

Interestingly, although S834, T835, Q836, Y837 and P838 are conserved among the tomato Hcr9 proteins, only S834 and T835 are conserved in the tomato Cf‐2 and Cf‐5 resistance proteins or members of the Hcr2 family and other closely related members of the eLRR‐RLP family in tomato (Fig. S4, see Supporting Information). Conservation is often indicative of functional importance. However, W839 and R843 are also conserved across this range of proteins, yet alanine mutations of these residues in Cf‐9 had no effect on Avr9‐induced necrosis. It is possible that these residues work in conjunction with each other or with other conserved residues (L848, E849, H850, I851, K857 and K858), such that mutations affecting two or more of these residues may be required to see any effect on Cf‐9 function, or that they play a different role in tomato than in tobacco. In contrast, in spite of its apparent importance for Cf‐9 function, P838 is replaced by a leucine residue in many of these homologues, but, in each case, there is also a glycine residue present in place of Q836. Glycine has the flexibility to allow tight turns and could conceivably compensate for the loss of proline in these variants. We therefore hypothesize that a proline‐ or glycine‐induced kink in the membrane‐proximal region of the cytosolic domain of these eLRR‐RLPs, as exemplified by Cf‐9, is important for interaction with VAP27 and for resistance protein function as a consequence.

Experimental Procedures

Materials

Plasmid pCBJ109, carrying a gene encoding Cf‐9 with an N‐terminal triple‐HA tag (HA:Cf‐9) (M. Benghezal and D. A. Jones, unpublished data), was used as the source of HA‐tagged Cf‐9 for our experiments (Fig. S5, see Supporting Information). Plasmid pCBJ314 containing the 3′ coding region and 3′ untranslated region (UTR) of Cf‐9 was used for all the site‐directed mutagenesis experiments (Fig. S5). The binary vectors used to express the mutated versions of HA:Cf‐9 were all constructed using pCBJ306, a derivative of pGREENII (Hellens et al., 2000, 2005), containing the Cauliflower mosaic virus (CaMV) 35S promoter and Tobacco mosaic virus (TMV) omega leader sequences (Chakrabarti et al., 2009). The binary vector pSLJ6201 contains the Avr9 gene (Hammond‐Kosack et al., 1994). The plasmid p35S:p19 expresses the p19 gene encoding a suppressor of gene silencing, under the control of the CaMV 35S promoter (Voinnet et al., 2003).

Site‐directed mutagenesis

Exsite or Quick change site‐directed mutagenesis kits (both from Stratagene, La Jolla, CA, USA) were used for site‐directed mutagenesis of Cf‐9 sequences in pCBJ314 according to the manufacturer's instructions (Fig. S5). The primers used for mutagenesis are listed in Table S1 (see Supporting Information). Depending on the distance between the targeted amino acids, multiple modifications were performed, either simultaneously or in successive rounds, or a combination of both. All six lysine (K) residues (designated K1 to K6, starting from the most C‐terminal lysine residue K861) were mutated to arginine (R) in a cumulative fashion. In the first round, K860 (K2) and K861 (K1) were mutated simultaneously. The resulting plasmid was used for the next round of mutagenesis to generate one in which K857 (K4) and K858 (K3) were also mutated. The resulting plasmid was then used to convert K855 (K5) to R and, finally, K847 (K6) to R in sequential reactions. Thus, the final plasmid in this series, in which all six lysine residues are mutated, addresses the possibility of redundancy, which could have contributed to a lack of any major effect for mutations of K1 and K2 alone, as observed by Van der Hoorn et al. (2001b). Similarly, the II motif was initially mutated to AA and LL and the resulting plasmids were used to convert the YPAW motif to APAW and YAAF, respectively. All the intermediate plasmids carrying the mutations were sequence verified. S834 and T835, and T853 and T854, were mutated as pairs in single mutagenesis experiments. The S834A + T835A and S834D + T835D double mutants were then used to generate individual S834A, T835A, S834D and T835D mutations.

Construction of binary vectors for transient expression

Binary vector construction

A ClaI‐XbaI fragment from pCBJ109, containing the coding region of Cf‐9 together with the HA tag and the 3′ UTR, was inserted into the multiple cloning site of pCBJ306 downstream of the 35S promoter and TMV omega leader sequences to generate pCBJ310 (Fig. S5). To construct binary vectors expressing mutated versions of HA:Cf‐9, HindIII‐XbaI fragments from the mutated pCBJ314‐derived intermediate plasmids (containing Cf‐9 coding sequences from the internal HindIII site to the stop codon) were ligated together with the ClaI‐HindIII fragment from pCBJ109 (containing the HA tag and Cf‐9 coding sequence between the start codon and the internal HindIII site) into ClaI‐XbaI‐digested pCBJ306 (Fig. S5). The binary vectors were co‐electroporated with pSOUP (Hellens et al., 2000) into electrocompetent Agrobacterium tumefaciens GV3101.

Transient expression assays

Agrobacterium tumefaciens GV3101 cells lacking a binary vector and cells harbouring the binary vector pSLJ6201 (Avr9) or binary vectors expressing wild‐type or mutant Cf‐9 proteins were all grown overnight in liquid culture, washed twice with infiltration buffer (Chakrabarti et al., 2009) and resuspended in infiltration buffer to an optical density at 600 nm (OD600) of 1.00. Cells were mixed in a 4 : 5 : 1 ratio of GV3101 : pSLJ6201 : Cf‐9 or Cf‐9 mutant. These mixtures were infiltrated into leaves of 8–10‐week‐old N. benthamiana plants to look for differences in the induction of Cf‐9/Avr9‐dependent necrosis. To minimize inter‐leaf variation, control and mutated HA:Cf‐9 were expressed on the left and right halves of the same leaf, respectively, and leaves were photographed 5–7 days after infiltration. To test for Avr9‐independent necrosis induction, GV3101 cells with binary vectors expressing the HA:Cf‐9 mutants were infiltrated separately into N. benthamiana leaves and co‐infiltrated with p35S:p19. To look for differences in Avr9‐dependent necrosis induction in tobacco, GV3101 cells with binary vectors expressing HA:Cf‐9 or its variants were resuspended in infiltration buffer to OD600 = 0.5 and infiltrated into leaves of budding SLJ6201 tobacco plants expressing Avr9. One leaf panel for each construct was used with a random arrangement of constructs on each leaf. At least three plants and three leaves on each plant were used for each experiment and each experiment was repeated at least three times.

Protein gel blots

For protein gel blot analysis, HA:Cf‐9 or its mutants were expressed together with the p19 suppressor of silencing by co‐agroinfiltration of N. benthamiana leaves as described above. p19 was used because we found Cf‐9 difficult to detect following agroinfiltration and because transgene silencing further reduced the level of protein expression over time. The use of p19 is possible in N. benthamiana, but not in N. tabacum, because p19 triggers necrosis in N. tabacum. Samples were collected 5 days after infiltration. One hundred milligrams of leaf tissue were ground in 200 μL Laemmli buffer [5 m urea, 2% sodium dodecylsulfate (SDS), 0.24 m Tris‐Cl, pH 6.8, 30% glycerol and 1 m β‐mercaptoethanol]. Samples were boiled for 5 min and centrifuged at maximum speed for 10 min at 4 °C to obtain protein extract (supernatant). Protein concentration was determined by the Bradford assay and 15 μg of total protein for each sample were size separated by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) (10% polyacrylamide) and electroblotted onto nitrocellulose membrane (Bio‐Rad, Hercules, CA, USA). Protein blots were probed with rat high‐affinity anti‐HA primary antibody (Roche, Basel Switzerland monoclonal antibody clone 3F10, 1 : 100), followed by horseradish peroxidase‐conjugated mouse anti‐rat secondary antibody (Roche; 1 : 10 000), and visualized using Supersignal West Pico chemiluminescent substrate as described by the manufacturer (Pierce, Waltham, MA, USA).

Yeast two‐hybrid assays

CITRX and VAP27 cDNAs from tomato (Solanum lycopersicum) were PCR amplified from the corresponding cDNA clones, FA18BC01 and LA22AH01, obtained from Kazusa DNA Research Institute, Chiba, Japan. Cf‐9 sequence encoding domains EFG and G were amplified from pCBJ109, whereas domain G mutants were amplified from the corresponding plasmids. The primers used for amplification of CITRX and VAP27 and the introduction of 5′ EcoRI and 3′ BamHI cloning sites are listed in Table S2 (see Supporting Information). PCR products were cloned into pCR2.1 (Invitrogen, Carlsbad, CA, USA) and sequence verified before being excised using EcoRI and BamHI and cloned into pGBKT7 and/or pGADT7 (Clontech, Mountain View, CA, USA) carrying the GAL4 BD and AD, respectively. Yeast transformation was carried out and interactions were visualized by growth on synthetic media lacking histidine as described in the Clontech Yeast Protocols Handbook.

Supporting information

Fig. S1 Effects on Cf‐9 function of mutations in residues and motifs potentially involved in ubiquitinylation, endocytosis and phosphorylation.

Fig. S2 Yeast two‐hybrid analyses of the interactions between VAP27 and Cf‐9 and between CITRX and Cf‐9.

Fig. S3 Effect of the Q836A mutation on Cf‐9 function in N. benthamiana.

Fig. S4 Alignment of Cf‐9 with Cf‐2, Cf‐5 and other Hcr2 proteins, and related tomato extracellular leucine‐rich repeat receptor‐like proteins (eLRR‐RLPs).

Fig. S5 Construction of binary vectors for transient expression of wild‐type and mutated HA:Cf‐9 proteins.

Table S1 Primers used for site‐directed mutagenesis of Cf‐9 domain G.

Table S2 Primer sequences used to amplify Cf‐9, VAP27 and CITRX sequences for yeast two‐hybrid analysis.

Click here for additional data file. (51.1MB, docx)

Acknowledgements

Apratim Chakrabarti was supported by an Australian National University (ANU) PhD scholarship. We thank the Research School of Biology (RSB) Plant Culture Staff for horticultural assistance.

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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 Effects on Cf‐9 function of mutations in residues and motifs potentially involved in ubiquitinylation, endocytosis and phosphorylation.

Fig. S2 Yeast two‐hybrid analyses of the interactions between VAP27 and Cf‐9 and between CITRX and Cf‐9.

Fig. S3 Effect of the Q836A mutation on Cf‐9 function in N. benthamiana.

Fig. S4 Alignment of Cf‐9 with Cf‐2, Cf‐5 and other Hcr2 proteins, and related tomato extracellular leucine‐rich repeat receptor‐like proteins (eLRR‐RLPs).

Fig. S5 Construction of binary vectors for transient expression of wild‐type and mutated HA:Cf‐9 proteins.

Table S1 Primers used for site‐directed mutagenesis of Cf‐9 domain G.

Table S2 Primer sequences used to amplify Cf‐9, VAP27 and CITRX sequences for yeast two‐hybrid analysis.

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