Abstract
Little is known of how plant disease resistance (R) proteins recognize pathogens and activate plant defenses. Rcr3 is specifically required for the function of Cf-2, aLycopersicon pimpinellifolium gene bred into cultivated tomato (Lycopersicon esculentum) for resistance toCladosporium fulvum. Rcr3 encodes a secreted papain-like cysteine endoprotease. Genetic analysis shows Rcr3 is allelic to the L. pimpinellifolium Ne gene, which suppresses the Cf-2–dependent autonecrosis conditioned by its L. esculentum allele, ne (necrosis).Rcr3 alleles from these two species encode proteins that differ by only seven amino acids. Possible roles of Rcr3 in Cf-2–dependent defense and autonecrosis are discussed.
Plant disease R proteins activate defense mechanisms upon perception of pathogen-derived molecules. Intracellular and extracellular race-specific elicitors are recognized by structurally distinct classes of R proteins (1, 2). Tomato Cf- genes confer resistance to the fungus Cladosporium fulvum. During infection numerous peptides are secreted into the apoplast (3), and some are products of fungal avirulence (Avr) genes. Cf- genes encode transmembrane proteins with extracellular leucine-rich repeats (LRRs) and short (23 to 36 amino acid) cytoplasmic domains (1, 2). In tomato, Avr peptide recognition activates a defense reaction dependent on Cf- genes, the hypersensitive response (HR), which results in localized cell death and the arrest of pathogen ingress. In tobacco cells expressing Cf-9, elicitation with Avr9 leads within 5 to 15 min to reactive oxygen production, protein kinase activation and novel gene expression (4). How Cf proteins activate defense responses is unknown.
Cf-2 confers Avr2-dependent resistance toC. fulvum. Mutations in Rcr3 suppressCf-2 function (2). Rcr3 is unlikely to be a component shared by multiple Cf- signaling pathways, because it is dispensable for the function of Cf-9 and evenCf-5, an ortholog of Cf-2 (5).
We isolated Rcr3 by positional cloning (6). Rcr3 encodes a protein of 344 amino acids that is 43% identical to papain from Carica papaya (Fig. 1A). Rcr3 expressed from its own promoter restores Cf-2–dependent resistance torcr3 mutants (Fig. 1B). Rcr3 contains conserved amino acid residues of the active site of eukaryotic thiol proteases (C154, H286, and N307(7), Fig. 1A), and six cysteines that form three putative cystine bridges. Single nucleotide exchanges were found in allrcr3 mutants (6) resulting in single amino acid exchanges in rcr3-1 and rcr3-2, and premature translation stop codons in rcr3-3 and rcr3-4(Fig. 1A). The rcr3-1 mutation, a C to S substitution at position 151 (C151S), does not completely compromiseCf-2 function (2), and serine is found at a similar position in other cysteine proteases (PROSITE:PDOC00126). The G314V substitution in rcr3-2 results in complete loss of function.
Rcr3 characterization. (A) Alignment of Rcr3 from L. pimpinellifolium (Rcr3pim),L. esculentum (Rcresc), and papain. Identical amino acids are shown in bold, allele-specific differences in red, and rcr3 mutant residues in blue. Asterisks indicate the first amino acid after the predicted signal peptide and prodomain cleavage sites, and triangles indicate the protease catalytic residues. (B) Complementation of Cf2 rcr3-3 mutants withpRcr3:Rcr3pim . The abaxial surface of tomato leaves is shown 16 days after infection with C. fulvum race 5 that expresses Avr2. (a) Disease-sensitive Cf2 rcr3-3 control; (b) Cf2 rcr3-3 transformed with pRcr3:Rcr3. (C) Expression of Rcr3. Upper panel, reverse-transcriptase–polymerase chain reaction (RT-PCR) products ofRcr3 and actin as a control for RNA prepared from Cf2 plants of different ages. Lane 1, 3-week-old plant, lanes 2 to 4, 12-week-old plant, RNA was prepared from the leaf indicated. Lower panel, RT-PCR products of Rcr3 and actin in 2-week-old plants inoculated with C. fulvum race 5 in compatible (Cf0) or incompatible interactions (Cf2, Cf9) at 3, 6, 9, and 12 days postinfection (dpi).
Rcr3 expression is regulated developmentally and in response to C. fulvum infection. Rcr3 mRNA levels are elevated in older plants irrespective of leaf age (Fig. 1C). After infection with C. fulvum, Rcr3 expression is elevated in compatible (disease causing) interactions, but much more rapidly in incompatible (resistant) interactions mediated byCf-2 or Cf-9 (Fig. 1C). The regulation ofRcr3 expression resembles that of pathogenesis-related genes (8).
We sequenced Rcr3 in several Lycopersicon species and in L. esculentum near-isogenic lines. Analysis ofRcr3 from L. pimpinellifolium(Rcr3pim ) revealed nucleotide changes leading to a deletion of one amino acid and six amino acid exchanges compared with the L. esculentum allele (Rcr3esc ;Fig. 1A). The Cf2 line contains Rcr3pim (Fig. 1A), suggesting it was cointrogressed with Cf-2 even though the genes are unlinked (2). Another breeding line containingCf-2 (Ontario 7620) also containsRcr3pim . During introgression of Cf-2into tomato, a second L. pimpinellifolium gene (Ne) was required to suppress Cf-2–dependent autonecrosis conditioned by its L. esculentum allele (ne, necrosis) (9). The F2progeny from a L. esculentum × L. pimpinellifolium (Cf-2) cross, which carryCf-2 and are homozygous for ne, are autonecrotic. This phenotype first appears in older leaves after the onset of flowering but eventually spreads to all leaves.
We investigated whether Rcr3pim andNe are allelic by testing if rcr3 mutants had lost Ne function. When Cf2 rcr3-2 and Cf0 (ne/ne) lines were crossed, the F1 progeny showed necrotic lesions on older leaves comparable to those observed in Cf-2/+,ne/ne plants (Fig. 2A). Therefore, mutations inRcr3pim abolish Ne function. The phenotypes of Cf2 rcr3-3 transgenic plants transformed with the clone p28L2 (10) that contains Rcr3esc were also analyzed, and those plants that exhibitedCf-2–dependent resistance to C. fulvum infection also developed autonecrotic lesions (Fig. 2A). Thus,Rcr3esc actively confersCf-2–dependent necrosis. We also characterized twoCf-9/Cf-2 chimeras (11). In the pCf-9:Cf-2chimera, Cf-2 is expressed from the Cf-9promoter (Fig. 2B). The Cf-2/9 chimera encodes a protein with the 34 NH2-terminal LRRs of Cf-2 fused to three COOH-terminal LRRs and COOH-terminal sequences of Cf-9, expressed from the Cf-2 promoter (Fig. 2B). Both chimeras conferAvr2- and Rcr3-dependent resistance to C. fulvum infection (12). Because the resistance conferred by Cf-2/9 requires Rcr3, when Cf-9does not (2), this requirement must be a property of the extracellular LRR domain of Cf-2.
Rcr3pim is Ne,Rcr3esc is ne. (A) Autonecrosis is Rcr3esc -dependent. Leaflets of 12-week-old plants with the following genotypes; (a)Cf-2/+, ne/ne; (b) Cf0 × Cf2 rcr3-3 F1; (c) Cf2 rcr3-3 expressing theRcr3esc transgene on p28L2; (d) transgenic Cf0 expressing pCf-9:Cf-2. (B) Schematic representation of Cf-9, Cf-2, and two chimeras. Fragments of genomic clones of Cf-9 and Cf-2 were exchanged (11). 5′ signifies 5′ flanking DNA; 3′ signifies 3′ flanking DNA. Cf-2/9 contains 5′ coding sequences fromCf-2 fused to the 3′ coding region of Cf-9. InpCf-9:Cf-2, Cf-2 is expressed from theCf-9 promoter.
Cf0 plants expressing a Cf-2 transgene (13) or the Cf-2/9 chimera are not autonecrotic. Therefore,Cf-2 expression may be necessary but not always sufficient for autonecrosis. However, Rcr3esc -dependent autonecrosis was observed in Cf0 transgenic plants expressingpCf-9:Cf-2 (14) (Fig. 2D). The Cf-9promoter is stronger than that of Cf-2 (15). In older tomato plants, increased levels of Rcr3 mRNA are observed (Fig. 1C). Elevated expression of pathway components may inappropriately activate Cf-2–dependent plant defenses. Together these data demonstrate that Rcr3 is required forCf-2–dependent resistance to C. fulvuminfection, and that Rcr3pim (Ne) can suppress Rcr3esc (ne)-dependent autonecrosis.
The papain class of cysteine proteases is translated into a prepro form, with a signal peptide for localization to the vacuole or extracellular space and a prodomain that blocks the active site until proteolytic removal (16). To localize Rcr3 and to assay protease activity, Rcr3 was expressed transiently inNicotiana benthamiana (17). Leaves were infiltrated with Agrobacterium tumefaciens carrying a 35S:histidine (His)- and hemagglutinin (HA)-taggedRcr3 fusion. A protein of the predicted size for the secreted, unprocessed Rcr3 (38 kD) was observed in total extracts using the HA antibody (Fig. 3B). In the apoplastic intercellular fluid (18), a second band that corresponds to the predicted size of Rcr3 lacking its prodomain was detected (Fig. 3B).
Rcr3 is a secreted protease. (A) Coomassie blue–stained gel of N. benthamiana total cellular protein, intercellular fluid (IF) protein and affinity-purified Rcr3. Lanes 1 to 3 were loaded with total plant extracts, lanes 4 to 6 with IF and lanes 7 to 9 with the eluate from a TALON column. Lanes 1, 4, and 7 show proteins from plants expressing empty vector control; lanes 2, 5, and 8 show proteins from plants expressing pMWBin19Rcr3:His and lanes 3, 6, and 9 show proteins from plants expressing pMWBin19Rcr3:His:HA. (B) Western blots of samples shown above were probed with the antibodies as indicated on the right of the panels. (C) Lane numbers correspond to samples shown above. In-gel assay of total IF and affinity-purified proteins using gelatin as substrate detects protease activity at approximately 34 kD under these conditions. (D) Protease assay using FTC-casein as sub- strate. Incubation was for 16 hours at 30°C in the presence (black columns) or absence (white columns) of 5 μM E-64, a cysteine protease inhibitor. Total intercellular fluid and affinity-purified proteins (eluate) from plants expressing empty vector (–) or pMWBin19Rcr3:His:HA (Rcr3).
These smaller forms of Rcr3-His and Rcr3-His-HA proteins were affinity-purified from intercellular fluid using TALON beads (Clontech) (6) and tested for protease activity in gels containing gelatin (19). A zone of clearing was detected within the gel due to gelatin degradation. This zone was specific for intercellular fluid containing epitope-tagged Rcr3 and was also detected in the TALON eluate (Fig. 3C). Thus, mature Rcr3 can utilize gelatin as a substrate. We also used a protease assay that detects cleavage of fluorescein thiocarbamoyl (FTC)– labeled casein (Fig. 3D). Protease activity in intercellular fluid was partially inhibited and, in affinity-purified preparations, was almost completely inhibited by E-64, a specific inhibitor of papainlike cysteine proteases (20).
We show here that Rcr3 is a positive regulator ofCf-2–dependent resistance and thatRcr3esc is also a positive regulator ofCf-2–dependent autonecrosis. The molecular mechanism forRcr3pim suppression ofRcr3esc -dependent autonecrosis remains to be determined. Because Rcr3 is a secreted cysteine protease and it has a specific role in Cf-2–mediated resistance, it likely functions upstream of Cf-2.
Plant proteases are involved in a multitude of cellular processes (21–23). Several roles for a protease inCf-2–dependent resistance can be envisaged. Rcr3 might process Avr2 to generate a mature ligand. Several C. fulvumavirulence proteins, including Avr4 and Avr9, are proteolytically processed in planta (3). Processing of a peptide ligand is exemplified by the generation of the spätzle ligand for theDrosophila Toll receptor that is generated via a serine protease cascade (24). In mature flies, Toll also functions in immunity to fungal pathogens, and derepression of this pathway, due to the absence of the Spn43Ac protease inhibitor, also results in necrosis (25).
Generating a mature ligand is unlikely to be the sole function of Rcr3, because rcr3 mutants do not exhibit an HR when infiltrated with intercellular fluid preparations that contain Avr2 isolated from infected tomato plants (2).
Alternatively, Rcr3 could process Cf-2 or another plant protein. Differences in the substrate specificity or activity of Rcr3pim and Rcr3esc might explain why Rcr3esc induces Avr2-independent necrosis. An extracellular protease is also required for brassinosteroid perception inArabidopsis (26). Overexpression of the serine carboxypeptidase BRS1 suppresses extracellular domain mutants of the BRI1 LRR-receptor kinase. BRS1 may process a protein that forms part of the BRI1 ligand (26). Alternatively, Rcr3 might be a plant defense component that is inhibited by Avr2. Avr2 could also inhibit other cysteine proteases, either by binding to or by modifying them. Whether Rcr3 has a role in defense is not established. The Cf2 Rcr3 mutant lines do not appear more susceptible to C. fulvum than Cf0, but tomato encodes many different cysteine proteases. It is interesting that the C. fulvum Avr9 protein shows significant structural homology to several protease inhibitors (27).
It is possible that Avr2 and Rcr3 together constitute a complex ligand that is recognized by Cf-2. It has been suggested that R proteins act as ‘guards‘ for specific proteins targeted by pathogen Avr proteins during infection (1, 2). Cf-2 may guard Rcr3 and trigger a defense response upon perception of an Rcr3/Avr2 complex. In rcr3 mutants, no Avr2-independent signaling would occur either because no Rcr3pim/Cf-2 complex is formed or because the complex does not activate defense signaling. A subtle structural difference in Rcr3esc (Fig. 1A) may result in activation of an Avr2-independent response upon binding to Cf-2.
With the recent isolation of the Avr2 gene (28), it will be possible to determine whether the Avr2, Rcr3, and Cf-2 proteins can interact. This should further increase our understanding of the molecular mechanism of ligand perception by this unique class of R proteins.
↵* To whom correspondence should be addressed. E-mail jonathan.jones{at}bbsrc.ac.uk
