Cladosporium Avr2 Inhibits Tomato Rcr3 Protease Required for Cf-2-Dependent Disease Resistance

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Science  17 Jun 2005:
Vol. 308, Issue 5729, pp. 1783-1786
DOI: 10.1126/science.1111404

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How plants recognize pathogens and activate defense is still mysterious. Direct interaction between pathogen avirulence (Avr) proteins and plant disease resistance proteins is the exception rather than the rule. During infection, Cladosporium fulvum secretes Avr2 protein into the apoplast of tomato leaves and, in the presence of the extracellular leucine-rich repeat receptor-like Cf-2 protein, triggers a hypersensitive response (HR) that also requires the extracellular tomato cysteine protease Rcr3. We show here that Avr2 binds and inhibits Rcr3 and propose that the Rcr3-Avr2 complex enables the Cf-2 protein to activate an HR.

Plant disease resistance (R) genes mediate race-specific recognition of pathogens via perception of avirulence (Avr) gene products (1). Tomato (Lycopersicon esculentum) Cf genes confer resistance to leaf mold caused by Cladosporium fulvum and encode transmembrane receptor-like proteins (RLPs) with extracellular leucine-rich repeats (LRRs) that mediate recognition of fungal Avrs secreted during infection (2). Cf-dependent perception of Avrs activates plant defense, including the HR, which results in host cell death at the site of penetration and limits pathogen ingress (3, 4). How Cf proteins enable tomato to perceive Avrs is unknown. So far, no direct interaction between Cf proteins and Avr proteins has been detected (5). A direct interaction has only been demonstrated for two Avrs and LRR-containing proteins (6, 7). The lack of a direct interaction led to the formulation of the guard hypothesis (8, 9), proposing that Avrs are virulence factors that interact with host targets to facilitate pathogen growth in the host and that R proteins monitor the status of these host targets (10).

Cf-2, which originates from the wild tomato variety L. pimpinellifolium, confers resistance to C. fulvum in tomato (11) on the basis of perception of Avr2, a cysteine-rich protein secreted by the fungus (12). Cf-2 function also requires Rcr3 (13), a secreted tomato cysteine protease (14) that is not required by other Cf genes, including the highly homologous Cf-5 gene (13, 14). The L. esculentum allele encodes the Rcr3esc protein that weakly activates Cf-2–dependent HR in tomato leaves in the absence of Avr2. The L. pimpinellifolium allele encodes Rcr3pim, required for Cf-2 to confer an Avr2 response (14).

We hypothesized that Rcr3 is a target of Avr2. To test this hypothesis, we produced Rcr3 as a C-terminal 6xHistidine (His)- and hemag-glutinin (HA)-tagged protein fusion (Rcr3-His-HA) both in Nicotiana benthamiana and in Pichia pastoris. Mature Rcr3 was recovered from intercellular fluid (IF) of N. benthamiana leaves by using the tags on the fusion protein (14). To monitor Rcr3 activity, we applied protease activity profiling at pH = 5 by using DCG-04, a biotinylated derivative of the irreversible cysteine protease inhibitor E-64 that has been used to profile cysteine protease activities from mammals (15), insects (16), and plants (17). DCG-04 treatment leads to irreversible labeling of cysteine proteases with biotin. Labeling of Rcr3 with 220 nM DCG-04 was assayed in the presence or absence of different concentrations of E-64 as a competitive inhibitor. After reaction with DCG-04, Rcr3-His-HA was precipitated with the use of Ni–nitrilotriacetic acid (NTA) (binding to His tag) or streptavidin (binding to biotin) beads. In the absence of E-64, DCG-04 biotinylates Rcr3, confirming that Rcr3 is a cysteine protease, whereas in the presence of 1120 nM E-64, Rcr3 is not biotinylated (Fig. 1A).

Fig. 1.

The Rcr3 cysteine protease of tomato is inhibited by Avr2 of C. fulvum. (A) Inhibition of Rcr3 produced in Nicotiana benthamiana by Avr2. IF was isolated from N. benthamiana expressing either the empty vector (pBin19) or Rcr3-His-HA (Rcr3). Protease activity profiling with 220 nM DCG-04 was performed in the absence of inhibitor (–) or in the presence of E-64, His-FLAG-Avr2 (Avr2), or His-FLAG-Avr4 (Avr4). Rcr3-His-HA was captured (pulled down) by Ni-NTA beads (left) or by streptavidin beads (right), electrophoresed on an SDS gel, and detected with streptavidin-HRP or His-specific antibodies (α-His) (28). Detection with streptavidin-HRP reveals that Rcr3 is not biotinylated in the presence of E-64 or Avr2, whereas biotinylation of Rcr3 occurs without inhibitor or with Avr4, indicating that, like E-64, Avr2 inhibits Rcr3 cysteine protease activity. α-His always detects Ni-NTA–captured Rcr3-His-HA irrespective of whether Rcr3 is inhibited or not (left), whereas α-His only detects biotinylated Rcr3-His-HA when Rcr3 is not inhibited by E-64 or Avr2 (right). No biotinylated cysteine proteases were detected in the empty vector control (pBin19). (B) Inhibition of Rcr3 by Avr2 is pH-dependent. IF from N. benthamiana containing Rcr3-His-HA was profiled with 220 nM DCG-04 in the absence of inhibitor (–), and in the presence of E-64 (1120 nM) or Avr2 (140 nM), over a pH range from 4.5 to 6.5. Rcr3-His-HA was captured by Ni-NTA beads and detected with streptavidin-HRP to demonstrate biotinylation (28). Rcr3 is biotinylated in the absence of inhibitor (–), with highest amounts of biotinylation at pH values between 5.0 and 6.0. Inhibition of biotinylation of Rcr3 by E-64 is complete at all pH values, whereas inhibition by Avr2 decreases at pH values above 6.0. (C) Inhibition of Rcr3 produced in Pichia pastoris by Avr2. Culture supernatant (CS) was isolated from P. pastoris cultures expressing either His-FLAG-Ecp4 (Ecp4) or Rcr3-His-HA (Rcr3), and protease activity was profiled with 220 nM DCG-04 in the absence of inhibitor (–) or in the presence of E-64, His-FLAG-Avr2 (Avr2) or His-FLAG-Avr4 (Avr4). Subsequently, Rcr3 was captured by Ni-NTA beads (left) or by streptavidin beads (right), electrophoresed on an SDS gel, and detected with streptavidin-HRP or HA-specific antibodies (α-HA-HRP) (28). Detection with streptavidin-HRP reveals that in the presence of E-64 or Avr2, Rcr3 is not biotinylated, whereas biotinylation of Rcr3 occurs in the absence of inhibitor or in the presence of Avr4, indicating that, similar to E-64, Avr2 inhibits Rcr3 cysteine protease produced in P. pastoris in a similar way as Rcr3 produced in N. benthamiana.

We tested whether Avr2 could inhibit biotinylation of Rcr3 by DCG-04. As a negative control, C. fulvum Avr4, which triggers Cf-4–dependent HR (18), was included. Both Avrs were expressed in P. pastoris as N-terminal His-FLAG-fusions and purified on a Ni-NTA column. In the presence of 140 nM Avr2, Rcr3 is not biotinylated (Fig. 1A), indicating that Avr2 inhibits Rcr3 activity (fig. S1). In the presence of Avr4, Rcr3 is biotinylated, showing that inhibition of Rcr3 by Avr2 is specific (Fig. 1A and fig. S1).

IF obtained from tomato has a pH of about 5 (19). To investigate the pH dependence of Rcr3 activity and its inhibition by Avr2, we incubated N. benthamiana IF containing Rcr3 with DCG-04 in the absence or presence of an excess of E-64 or Avr2 over a pH range from 4.5 to 6.5. Rcr3 activity is highest at pH of 5 to 6 and strongly decreases outside this range (Fig. 1B). Inhibition by E-64 is effective over the whole pH range, whereas inhibition by Avr2 is only effective below pH = 6 (Fig. 1B), indicating that the pH optimum for Rcr3 activity and its inhibition by Avr2 coincides with the pH of the apoplast of tomato (pH = 5). Rcr3 produced as a C-terminal His-HA fusion in P. pastoris is also inhibited by E-64 and Avr2 (Fig. 1C), indicating that Avr2 alone is sufficient to inhibit Rcr3 and that no additional plant factors are required. No biotinylation by DCG-04 of other cysteine proteases was observed in control IF from N. benthamiana nontransgenic for Rcr3 (Fig. 1A) or in control culture supernatant (CS) from P. pastoris nontransgenic for Rcr3 but expressing Ecp4, another protein secreted by C. fulvum (20) (Fig. 1C). This indicates that, at the DCG-04 concentration used (220 nM), no biotinylation of endogenous extracellular cysteine proteases could be detected.

Because Avr2 inhibits Rcr3, we expected a physical interaction between the two proteins. This was investigated by co-immunoprecipitation studies. His-FLAG-Avr2 or His-FLAG-Avr4 was added to N. benthamiana IF or culture supernatant (CS) of P. pastoris containing Rcr3-His-HA and immunoprecipitated with a FLAG-specific antibody (α-FLAG). As a control, Rcr3-His-HA was also preincubated with E-64 to block the active site before adding His-FLAG-Avr2. After immunoprecipitation, the samples were run on SDS gels and blotted, and Avr proteins and Rcr3 were detected with use of α-FLAG and an Rcr3-specific antibody (α-Rcr3), respectively (Fig. 2). Rcr3 co-immunoprecipitates with Avr2 but not with Avr4 (Fig. 2), indicating a specific interaction between Avr2 and Rcr3. Blocking the active site of Rcr3 by E-64 eliminates this interaction (Fig. 2). In the presence of Avr2, α-FLAG co-immunoprecipitates Rcr3 irrespective of the source of Rcr3, again indicating that the interaction between Avr2 and Rcr3 is independent of additional plant factors (Fig. 2). No signals were detected on blots probed with α-Rcr3 after immunoprecipitation of proteins from control N. benthamiana IF (marked pBin19 in Fig. 2) or control CS of P. pastoris nontransgenic for Rcr3 (marked Ecp4 in Fig. 2), indicating that α-Rcr3 is specific.

Fig. 2.

Rcr3 and Avr2 physically interact. Rcr3-His-HA (Rcr3) was incubated with His-FLAG-Avr2 (Avr2), with or without pretreatment with E-64 or His-FLAG-Avr4 (Avr4), and then immunoprecipitated (IP) with FLAG-specific antibody (α-FLAG). IF from N. benthamiana expressing either the empty vector (pBin19) or Rcr3, or CS from P. pastoris expressing either His-FLAG-Ecp4 (Ecp4) or Rcr3, was incubated without inhibitor (–) or with an excess of E-64 before adding Avr2 or Avr4. After immunoprecipitation with α-FLAG beads, the Avr proteins were detected with α-FLAG, whereas co-immunoprecipitated Rcr3 was detected with α-Rcr3 (28). Rcr3 is only co-immunoprecipitated in the presence of Avr2 without preincubation with E-64.

We tested whether native Rcr3 in tomato IF can be detected and inhibited by E-64 and Avr2. IF (6 ml) from different Cf tomato plants producing Rcr3 were labeled with 2.2 μM DCG-04 in the presence or absence of E-64 (28.6 μM) or Avr2 (6.9 μM). Biotinylated proteins were captured on streptavidin beads, run on an SDS gel, and probed with α-Rcr3 or streptavidin–horseradish peroxidase (HRP) (fig. S2). Native Rcr3 is detected by α-Rcr3 in Cf0 and Cf2 tomato lines, and its biotinylation by DCG-04 is inhibited by Avr2, whereas Rcr3 is absent in Cf-2/rcr3-3 plants (13, 14) (fig. S2; upper panel). In addition to Rcr3, several other apoplastic cysteine proteases are biotinylated that can be inhibited by Avr2 (fig. S2; lower panel).

To determine whether inhibition of Rcr3 by Avr2 is sufficient to trigger Cf-2–dependent HR, we infiltrated Rcr3 produced in N. benthamiana, either alone or in combination with Avr2, E-64, or E-64 and Avr2, or we infiltrated Avr2 alone, into Cf-2/rcr3-3 tomato leaves (Fig. 3). Infiltration of Avr2 or Rcr3 alone or Rcr3 incubated with E-64 does not trigger an HR, whereas infiltration of Rcr3 incubated with Avr2 does. However, Rcr3 preincubated with an excess of E-64 to saturate the active site, and subsequently incubated with Avr2, does not trigger Cf-2–mediated HR, indicating that Cf-2 specifically recognizes the Rcr3-Avr complex. Similar results were obtained with P. pastoris–produced Rcr3 pretreated with the same compounds (21).

Fig. 3.

Cf-2–mediated HR requires physical interaction between Rcr3 and Avr2. Fully expanded leaves of 5-week-old Cf-2/rcr3-3 tomato were infiltrated with Rcr3-His-HA (Rcr3) produced in N. benthamiana (either alone or in combination with His-FLAG-Avr2 (Avr2), E-64, or E-64 and Avr2) or infiltrated with Avr2 alone (28). Leaves were photographed 3 days postinfiltration. The infiltrated sectors are outlined and the infiltrated compounds indicated. The HR is only triggered when Rcr3 and Avr2 can interact, whereas the HR is blocked when interaction of Rcr3 with Avr2 is prevented by preincubation of Rcr3 with E-64. Rcr3 produced in P. pastoris treated with the same compounds gave similar results (21).

Inhibition of Rcr3 activity by Avr2 could be caused by either Avr2 acting solely as an inhibitor of Rcr3 or Avr2 being both a substrate and an inhibitor. However, we observed no degradation of Avr2 upon incubation with Rcr3 (Fig. 2), suggesting that Avr2 is not a substrate for Rcr3. Furthermore, if processing of Avr2 by Rcr3 were required for Cf-2–mediated HR, then Avr2 present in IF from Cf0 tomato plants (containing Rcr3) infected by Avr2-producing C. fulvum strains would induce an HR in Cf-2/rcr3-3 tomato. This was not observed (21), indicating that Avr2 is an inhibitor, not a substrate, of Rcr3. However, inhibition of Rcr3 activity is not sufficient to initiate Cf-2–mediated HR (Fig. 3). Therefore, we propose that inhibition of Rcr3 by Avr2 induces a conformational change in Rcr3 that triggers the Cf-2 protein to activate HR. This model is consistent with the observation that the Rcr3esc protein alone provokes a weak Cf-2–dependent, Avr2-independent HR (14). We suggest that Rcr3esc, which differs from Rcr3pim in one amino acid deletion and six amino acid changes, constitutively mimics the conformational change imposed on Rcr3pim (present in Cf-2 plants) by Avr2 binding and weakly activates Cf-2–dependent HR in the absence of Avr2.

The role of Rcr3 cysteine protease activity for tomato and the importance of its inhibition by Avr2 for C. fulvum during infection are unknown, but secreted plant cysteine proteases possibly have antimicrobial activity. Rcr3 transcription is induced faster and transcripts accumulate to higher concentrations in incompatible compared with compatible interactions between tomato and C. fulvum (14), as do transcripts for pathogenesis-related proteins after infection by this fungus (22). Rcr3 is also induced in the absence of Cf-2, consistent with a role for Rcr3 in basal host defense (14). Furthermore, in addition to Rcr3, several other apoplastic cysteine proteases are inhibited by Avr2, suggesting that Avr2 is a general virulence factor facilitating growth of C. fulvum in the apoplast. Recently, it has been shown that a protease inhibitor from Phytophthora infestans interacts with and inhibits the plant serine protease P69B, which is induced during infection of tomato by this pathogen (23). Thus, inhibition of plant proteases may represent a general counterdefense used by invading pathogens.

The role of Rcr3 in the perception of Avr2 by Cf-2 is consistent with the guard hypothesis. The Rcr3-Avr2 complex, but not other Avr2-cysteine protease complexes, activates Cf-2. So far, all bacterial pathogens colonizing the apoplast of plants deliver their effector proteins into the plant cell by the type III secretion system where they interact with cytoplasmic virulence targets (10, 2427). In the case of RPM1- and RPS2-mediated resistance in Arabidopsis, the action of the Avr proteins AvrB, AvrRpm1, and AvrRpt2 on the guardee RIN4 is thought to trigger the activation of RPM1 (resistance to Pseudomonas syringae p. maculicola expressing AvrRpm1) or RPS2 (resistance to P. syringae pv. tomato expressing AvrRpt2) proteins (2427). Similarly, in RPS5-mediated resistance in Arabidopsis the cysteine protease activity of the Avr protein, AvrPphB, on the guardee PBS1 is required to trigger the HR (28). Such indirect interactions between pathogen Avrs and plant R proteins may be more difficult for the pathogen to circumvent without a virulence penalty than direct interactions (8, 9). In addition to Rcr3, other tomato cysteine proteases are inhibited by Avr2 that are not guarded by known Cf proteins. Characterization and functional analysis of these proteases will be the subject of future studies.

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