Screen of Non-annotated Small Secreted Proteins of \u3ci\u3ePseudomonas syringae\u3c/i\u3e Reveals a Virulence Factor That Inhibits Tomato Immune Proteases

Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes

Screen of Non-annotated Small Secreted Proteins of \u3ci\u3ePseudomonas syringae\u3c/i\u3e Reveals a Virulence Factor That Inhibits Tomato Immune Proteases

Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes

Extracellular proteolytic cascade in tomato activates immune protease Rcr3

Proteolytic cascades regulate immunity and development in animals, but these cascades in plants have not yet been reported. Here we report that the extracellular immune protease Rcr3 of tomato is activated by P69B and other subtilases (SBTs), revealing a proteolytic cascade regulating extracellular immunity in solanaceous plants. Rcr3 is a secreted papain-like Cys protease (PLCP) of tomato that acts both in basal resistance against late blight disease (Phytophthora infestans) and in gene-for-gene resistance against the fungal pathogen Cladosporium fulvum (syn. Passalora fulva) Despite the prevalent model that Rcr3-like proteases can activate themselves at low pH, we found that catalytically inactive proRcr3 mutant precursors are still processed into mature mRcr3 isoforms. ProRcr3 is processed by secreted P69B and other Asp-selective SBTs in solanaceous plants, providing robust immunity through SBT redundancy. The apoplastic effector EPI1 of P. infestans can block Rcr3 activation by inhibiting SBTs, suggesting that this effector promotes virulence indirectly by preventing the activation of Rcr3(-like) immune proteases. Rcr3 activation in Nicotiana benthamiana requires a SBT from a different subfamily, indicating that extracellular proteolytic cascades have evolved convergently in solanaceous plants or are very ancient in the plant kingdom. The frequent incidence of Asp residues in the cleavage region of Rcr3-like proteases in solanaceous plants indicates that activation of immune proteases by SBTs is a general mechanism, illuminating a proteolytic cascade that provides robust apoplastic immunity

Balancing Selection at the Tomato RCR3 Guardee Gene Family Maintains Variation in Strength of Pathogen Defense

Coevolution between hosts and pathogens is thought to occur between interacting molecules of both species. This results in the maintenance of genetic diversity at pathogen antigens (or so-called effectors) and host resistance genes such as the major histocompatibility complex (MHC) in mammals or resistance (R) genes in plants. In plant-pathogen interactions, the current paradigm posits that a specific defense response is activated upon recognition of pathogen effectors via interaction with their corresponding R proteins. According to the''Guard-Hypothesis,'' R proteins (the ``guards'') can sense modification of target molecules in the host (the ``guardees'') by pathogen effectors and subsequently trigger the defense response. Multiple studies have reported high genetic diversity at R genes maintained by balancing selection. In contrast, little is known about the evolutionary mechanisms shaping the guardee, which may be subject to contrasting evolutionary forces. Here we show that the evolution of the guardee RCR3 is characterized by gene duplication, frequent gene conversion, and balancing selection in the wild tomato species Solanum peruvianum. Investigating the functional characteristics of 54 natural variants through in vitro and in planta assays, we detected differences in recognition of the pathogen effector through interaction with the guardee, as well as substantial variation in the strength of the defense response. This variation is maintained by balancing selection at each copy of the RCR3 gene. Our analyses pinpoint three amino acid polymorphisms with key functional consequences for the coevolution between the guardee (RCR3) and its guard (Cf-2). We conclude that, in addition to coevolution at the ``guardee-effector'' interface for pathogen recognition, natural selection acts on the ``guard-guardee'' interface. Guardee evolution may be governed by a counterbalance between improved activation in the presence and prevention of auto-immune responses in the absence of the corresponding pathogen

A Role in Immunity for Arabidopsis Cysteine Protease RD21, the Ortholog of the Tomato Immune Protease C14

Secreted papain-like Cys proteases are important players in plant immunity. We previously reported that the C14 protease of tomato is targeted by cystatin-like EPIC proteins that are secreted by the oomycete pathogen Phytophthora infestans (Pinf) during infection. C14 has been under diversifying selection in wild potato species coevolving with Pinf and reduced C14 levels result in enhanced susceptibility for Pinf. Here, we investigated the role C14-EPIC-like interactions in the natural pathosystem of Arabidopsis with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). In contrast to the Pinf-solanaceae pathosystem, the C14 orthologous protease of Arabidopsis, RD21, does not evolve under diversifying selection in Arabidopsis, and rd21 null mutants do not show phenotypes upon compatible and incompatible Hpa interactions, despite the evident lack of a major leaf protease. Hpa isolates express highly conserved EPIC-like proteins during infections, but it is unknown if these HpaEPICs can inhibit RD21 and one of these HpaEPICs even lacks the canonical cystatin motifs. The rd21 mutants are unaffected in compatible and incompatible interactions with Pseudomonas syringae pv. tomato, but are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinerea, demonstrating that RD21 provides immunity to a necrotrophic pathogen

Mutant <i>rd21</i> lines are not compromised in interactions with <i>Pst</i>DC3000.

<p><b>A,</b> Compatible <i>Pst</i> interactions. (Mutant) Arabidopsis plants were spray-inoculated with <i>Pst</i>DC3000 (vir) and bacterial populations were measured at 0 and 3 dpi. Error bars represent SD of 5 independent bacterial extractions. This experiment was repeated three times with similar results. <b>B,</b> Incompatible <i>Pst</i> interactions. (Mutant) Arabidopsis plants were spray-inoculated with <i>Pst</i>DC3000 avrRpm1 (avr) and bacterial populations were measured at 0 and 3 dpi. Error bars represent SD of 5 independent bacterial extractions. This experiment was repeated three times with similar results.</p

EPIC-like proteins from <i>Hpa</i>.

<p><b>A,</b> Phylogenetic relationship between EPIC proteins. One of 1,000 most parsimonious trees showing the relationship between <i>Pinf</i> and <i>Hpa</i> EPICs. This tree was obtained by heuristic search with bootstrap support. A cystatin from <i>Albugo laibachii</i> was used as outgroup. <b>B,</b> Protein sequence alignment of <i>Hpa</i>EPIC-B and -C with <i>Pinf</i>EPIC1, -2B and 3. *, functionally important residues; NT, N-terminus; L1, loop-1; L2, loop-2. Triangles indicate amino acids at variant codons. <b>C,</b> Distribution of four variant codons found in <i>HpaEPIC-B</i> and <i>-C</i> sequences of various <i>Hpa</i> isolates. The variant codons are indicated with grey and black lines and the amino acid encoding the codons are indicated on the top and bottom. <b>D, </b><i>HpaEPIC-B</i> and <i>-C</i> are expressed during infection. RNA was isolated from Arabidopsis plants infected with <i>Hpa</i> isolates Noco2, Cala2 or Emoy2 at 5 dpi and used as template for RT-PCR with specific primers for <i>HpaEPIC-B</i> and <i>-C</i>.</p

RD21 structure and knock-out lines.

<p><b>A,</b> Gene structure of <i>RD21A</i> (At1g47128). The <i>RD21A</i> gene consists of 5 exons. Mutants <i>rd21-1</i> (SALK_90550) and <i>rd21-2</i> (SALK_65256) contain T-DNA insertions in the third and first introns, respectively. <b>B,</b> Domains encoded by the <i>RD21A</i> open reading frame. The RD21 protein consists of a signal peptide (sp, left), an autoinhibitory prodomain (pro), a protease domain with catalytic cysteine (white stripe), and a granulin domain (right). RD21 exists in two active isoforms: the granulin-containing intermediate (i) RD21, and the mature (m) RD21 lacking the granulin domain. <b>C,</b> The <i>rd21-1</i> line is a null mutant. The <i>rd21-1</i> mutant lacks iRD21 and mRD21 proteins (left) and the major upper signals in the protease activity profile (right). Leaf extracts of Col-0 and <i>rd21-1</i> mutant plants were labelled with DCG-04 and proteins were detected with RD21 antibody and streptavidin-HRP. A remaining signal at 30 kDa is sometimes visible in the <i>rd21</i> mutant lines (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029317#pone.0029317.s006" target="_blank">Figure S6</a></b>), and can contain CTB3, XCP2 and XCP1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029317#pone.0029317-VanderHoorn1" target="_blank">[20]</a>.</p

Mutant <i>rd21</i> lines are unaltered in their interactions with <i>Hpa</i>.

<p><b>A,</b> Compatible <i>Hpa</i> interactions. (Mutant) Arabidopsis seedlings were infected with <i>Hpa</i> isolate Noco2, which is virulent on Col-0 but not on Ws. Spores were counted at 7 dpi in triplicate. Error bars represent standard deviation (SD) of three independent spore isolations. This experiment is repeated once with similar results. <b>B,</b> Incompatible <i>Hpa</i> interactions. (Mutant) Arabidopsis seedlings were infected with <i>Hpa</i> isolate Emwa1, which is virulent on Ws-0 but not on Col-0. Spores were counted at 7 dpi in triplicate. Error bars represent SD of three independent spore isolations.</p

Screen of Non-annotated Small Secreted Proteins of \u3ci\u3ePseudomonas syringae\u3c/i\u3e Reveals a Virulence Factor That Inhibits Tomato Immune Proteases

Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes