A Common Toxin Fold Mediates Microbial Attack and Plant Defense

A common toxin fold mediates microbial attack and plant defense

Christian Ottmanna,b,1, Borries Luberackia,1,2, Isabell Ku¨ fnerc,1, Wolfgang Kocha, Fre´ de´ ric Brunnerc, Michael Weyandb, Laura Mattinend, Minna Pirhonend, Gregor Anderluhe, Hanns Ulrich Seitza,3, Thorsten Nu¨ rnbergerc,3, and Claudia Oeckinga,3

aCenter for Plant Molecular Biology–Plant Physiology, University of Tu¨bingen, D-72076 Tu¨bingen, Germany; bChemical Genomics Centre, D-44227 Dortmund, Germany; cCenter for Plant Molecular Biology–Plant Biochemistry, University of Tu¨bingen, D-72076 Tu¨bingen, Germany; dUniversity of Helsinki, Department of Applied Biology, FIN-00014, Helsinki, Finland; and eUniversity of Ljubljana, Department of Biology, SLO-1000 Ljubljana, Slovenia

Edited by Jeffery L. Dangl, The University of North Carolina, Chapel Hill, NC, and approved May 5, 2009 (received for review March 4, 2009) Many plant pathogens secrete toxins that enhance microbial vir- recognition of microbial patterns and effectors, plants also ulence by killing host cells. Usually, these toxins are produced by possess capacities to sense host-derived ‘‘damage’’ patterns that particular microbial taxa, such as bacteria or fungi. In contrast, originate, for example, from the degradation of the plant cell wall many bacterial, fungal and oomycete species produce necrosis and by microbial hydrolytic enzyme activities (10). Paradoxically, ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) that trig- some phytopathogenic microbe-derived cytolytic toxins have ger leaf necrosis and immunity-associated responses in various also been reported to elicit plant defenses, including Fusarium plants. We have determined the crystal structure of an NLP from spp.-derived fumonisin B1, Phomopsis amygdali-derived fusi- the phytopathogenic oomycete Pythium aphanidermatum to 1.35Å coccin, or the host-selective toxin, victorin, from Cochliobolus resolution. The protein fold exhibits structural similarities to cyto- victoriae (6, 11). However, for virtually all microbial toxins with lytic toxins produced by marine organisms (actinoporins). Compu- plant defense-stimulating potential, it is unknown whether ac- tational modeling of the 3-dimensional structure of NLPs from tivation of plant defenses results from toxin-induced cellular another oomycete, Phytophthora parasitica, and from the phyto- distress or, independently of toxin action, from recognition of pathogenic bacterium, Pectobacterium carotovorum, revealed a toxins as microbial patterns by plant pattern-recognition high extent of fold conservation. Expression of the 2 oomycete receptors. NLPs in an nlp-deﬁcient P. carotovorum strain restored bacterial NLPs constitute a superfamily of proteins that are produced virulence, suggesting that NLPs of prokaryotic and eukaryotic by various phytopathogenic micro-organisms, comprising both origins are orthologous proteins. NLP mutant protein analyses prokaryotic and eukaryotic organisms (3, 7, 12, 13). Necrosis and revealed that identical structural properties were required to cause ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) trigger plasma membrane permeabilization and cytolysis in plant cells, as well as to restore bacterial virulence. In sum, NLPs are conserved leaf necrosis that is genetically distinct from immunity- virulence factors whose taxonomic distribution is exceptional for associated programmed cell death (11) and stimulate immunity- microbial phytotoxins, and that contribute to host infection by associated defenses in all dicotyledonous plants tested, but not plasma membrane destruction and cytolysis. We further show that in monocotyledonous plants (3, 7). Hence, NLPs were proposed NLP-mediated phytotoxicity and plant defense gene expression to have dual functions in plant pathogen interactions, acting both share identical fold requirements, suggesting that toxin-mediated as triggers of immune responses and as toxin-like virulence interference with host integrity triggers plant immunity-associated factors (11). The broad taxonomic distribution of NLPs, in responses. Phytotoxin-induced cellular damage-associated activa- particular their occurrence in both prokaryotic and eukaryotic tion of plant defenses is reminiscent of microbial toxin-induced species, is quite unusual for known microbial phytotoxins, the production of which is restricted to a narrow range of microbial

inﬂammasome activation in vertebrates and may thus constitute PLANT BIOLOGY another conserved element in animal and plant innate immunity. species (6, 8). Major open questions regarding the biological activities of crystal structure ͉ immunity ͉ pathogen ͉ plant immunity ͉ phytotoxin NLPs are as to whether (i) NLPs from various microbial origins are functionally conserved, orthologous proteins; (ii) the ne- illions of years of coevolution of plants and microbial crotic activities of NLP proteins contribute to microbial viru- Mpathogens have shaped both the abilities of microbial lence and infection; and (iii) the necrotic and defense- pathogens to overcome plant disease resistance and the abilities stimulating activities of NLP proteins are mechanistically linked. of plants to cope with microbial invasion (1). Phytopathogens To address these questions, NLPPya from the phytopathogenic from different taxonomic origins secrete structurally unrelated effectors into the apoplast and cytoplasm of plants to establish Author contributions: F.B., H.U.S., T.N., and C. Oecking designed research; C. Ottmann, B.L., infection and to suppress host defenses (2–4). In addition, I.K., M.W., and C. Oecking performed research; W.K., L.M., M.P., and G.A. contributed new phytopathogenic micro-organisms produce a wide range of reagents/analytic tools; C. Ottmann, B.L., I.K., W.K., F.B., M.W., H.U.S., T.N., and C. Oecking cytolytic toxins that function as key virulence determinants (5, analyzed data; and C. Ottmann, F.B., H.U.S., T.N., and C. Oecking wrote the paper. 6). In particular, micro-organisms preferring hemibiotrophic or The authors declare no conﬂict of interest. necrotrophic lifestyles produce host-selective and host- This article is a PNAS Direct Submission. nonselective toxins that contribute to microbial virulence by Freely available online through the PNAS open access option. facilitating the killing of plant tissues (6–8). Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, Microbial pattern recognition is a prerequisite for the initia- www.pdb.org (PDB ID code 3GNU). tion of antimicrobial defenses in all multicellular organisms, 1C. Ottmann, B.L., and I.K. contributed equally to this work. including plants. The bipartite plant immune system is based 2Present address: Gregor Mendel Institute, A-1030 Vienna, Austria. upon recognition of pathogen-associated molecular patterns by 3To whom correspondence may be addressed. E-mail: claudia.oecking@zmbp. pattern-recognition receptors (1, 9) as well as upon the activities uni-tuebingen.de, [email protected], or [email protected]. of resistance proteins that have evolved to recognize the pres- This article contains supporting information online at www.pnas.org/cgi/content/full/ ence or activities of microbial effectors (2). In addition to the 0902362106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902362106 PNAS ͉ June 23, 2009 ͉ vol. 106 ͉ no. 25 ͉ 10359–10364 Downloaded by guest on September 27, 2021 Fig. 1. Crystal structure of NLPPya. Secondary structure elements are shown in blue (helices) and green (␤-strands)(A–C). The bound magnesium atom is shown as a blue sphere (A, D–F). Residues involved in ion binding (sticks in E and F) or formation of the disulﬁde bond (sticks in A and F) are labeled. (A–C) Ribbon plot of NLPPya (A), the actinoporin, sticholysin (B), and the lectin, ABL (C). NGA, N-acetylgalactosamine; POC, phosphorylcholine. (D) Electrostatic surface of NLPPya with a color scale that varies from blue to red, representing positive and negative potential, respectively. (E) Electron density for the bound magnesium and its coordinating residues in NLPPya. The 2Fo-Fc density map (contoured at 1 ␴) is shown in black. (F) Comparison of NLPPya (orange) with modeled structures of NLPPp (blue) and NLPPcc (green).

oomycete Pythium aphanidermatum (14) was selected for tertiary produced by sea anemones (Actinia equina equinatoxin II, structure studies. Stichodactyla helianthus sticholysin) (20–22) are most similar to NLPPya (see Fig. 1 A–C). Like NLPPya, these proteins are small, Results single-domain polypeptides exhibiting a central ␤-sandwich ar- The NLP Fold Is Highly Conserved and Is Distantly Related to Cytolytic chitecture surrounded by helices. Superimposition of actino- Actinoporins. Recombinant NLPPya (lacking the N-terminal sig- porins and lectins suggests a high degree of structural similarity nal peptide for secretion) [supporting information (SI) Fig. S1] (score 10.0–10.8), whereas NLPPya represents a more distantly was produced in Escherichia coli, purified under denaturing related protein (see Table S3). Actinoporins and lectins are conditions and reconstituted into its biologically active form soluble proteins that target specific components of membrane (15). The refolded protein crystallized in orthorhombic crystals bilayers via a surface-exposed cavity (see Fig. 1 B and C and Fig. whose structure was solved at a resolution of 1.35 Å. The S2). Moreover, actinoporins are cytolytic toxins that form trans- corresponding crystal structure (crystallography statistics given membrane pores via their flexible N-terminal regions (22). This in Table S1) reveals a single-domain molecule with a fold N-terminal region is absent in lectins (see Fig. 1C), but is found consisting of a central ␤-sandwich, with 3 strands in the first in NLPPya to be required for NLP-induced necrosis and plant sheet and a 5-stranded antiparallel second sheet. Three helices defense activation (16). This suggests that NLPs and actino- encompass the second sheet at the top of the sandwich, giving porins share a cytolytic, membrane-disintegrating mode of action. rise to a flat surface (Fig. 1A, and see Fig. S1). In contrast, an The NLPPya fold was used as template to model the 3-dimen- uneven surface is established at the base of the polypeptide sional structure of NLPPcc from the phytopathogenic bacterium, mainly by 3 broad loops (L1–L3). Formation of an intramolec- Pectobacterium carotovorum (23, 24), and NLPPp from the ular disulfide bridge between 2 conserved cysteine residues that oomycete Phytophthora parasitica (16). Structurally outstanding are essential for NLP activities (16) appears to be crucial to regions of these proteins match to a high extent, despite of anchor loop L1 to the central sheet core (see Fig. 1A and Fig. differences in primary sequences (Fig. 1F and see Fig. S1). In S1). A cavity exhibiting a strong negative net charge is formed particular, the positions of residues constituting the cation- above loops L2/L3 at the surface of the rigid globular fold, with binding site and the disulfide bridge in NLPPya are conserved a divalent cation bound therein (Fig. 1D). Residues D93 (␤5), (see Fig. 1F). The high degree of tertiary structure conservation D104 (␤6), E106 (␤6), and H159 (main chain carbonyl group, among NLPs suggests that these proteins are functionally ␤10) are directly involved in cation binding, whereas H101 (␤6) homologous. stabilizes the corresponding water network (see Fig. 1E and Fig. S1). NLPs are Orthologous Virulence-Promoting Cytolytic Phytotoxins. To Tertiary structure-based comparison of the NLPPya fold test the aforementioned hypothesis, we transformed the NLP- [DALI-program (17), http:///www.ebi.ac.uk/dali/] revealed 35 deficient Pcc nlp– strain that was recently shown to exhibit proteins that scored over the default significance level for reduced aggressiveness on potato (23, 24), with NLPPya or similarity (DALI Z scores 3.5–2.0) (Tables S2 and S3). Based NLPPp-encoding sequences. Both strains showed partial resto- upon visual inspection of any structural superimposition and in ration (30–40%) in bacterial virulence and potato tuber mac- consideration of the physiological context, fungal lectins (Agar- eration relative to a control strain complemented with the icus bisporus, Xerocomus chrysenteron) (18, 19) and actinoporins endogenous NLPPcc gene (Fig. 2A). Quantitative differences in

10360 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902362106 Ottmann et al. Downloaded by guest on September 27, 2021 dye, calcein, to track membrane damage by measuring dye release (25). All NLPs tested disintegrated vesicles made of tobacco or Arabidopsis plasma membranes within 5 min upon application (Fig. 2C) at similar concentrations (EC50 values determined for tobacco: NLPPya, 0.4 nM; NLPPp, 3.5 nM; NLPPcc, 13.4 nM). In contrast, vesicles made of the monocot Commelina communis proved resistant to NLP treatment (see Fig. 2C), suggesting that NLPs constitute necrosis-promoting proteins that target dicot plasma membrane-specific surface structures and, subsequently, cause membrane disruption. Comparison of NLP primary sequences found in public da- tabases revealed strong conservation of a central hepta-peptide motif ‘‘GHRHDWE’’ (7) (amino acids 100–106 in NLPPya) (see Fig. S1). Remarkably, this region is part of the negatively charged cavity exposed at the protein surface (see Fig. 1 D and E). Three amino acid residues of this motif (H101, D104, E106) and another highly conserved residue (D93) are implicated in coordination of a divalent cation within this cavity (see Figs. 1E,3A, and Fig. S1), suggesting that this domain is important for the biological activities of NLP proteins. To scrutinize this hypothesis, a total of 8 highly conserved amino acid residues (K92, D93, H101, R102, H103, D104, E106, S126) (see Fig. 3A and Fig. S1) were individually exchanged for alanine in both NLPPcc and NLPPp. The mutant proteins were analyzed for their ability to (i) – complement the reduced virulence of the Pcc nlp strain (NLPPcc mutant derivatives only) (Fig. 3B), (ii) trigger necrosis in tobacco leaves (Fig. 3C and Fig. S3), and (iii) permeabilize tobacco plasma membranes (Fig. 3D and see Fig. S3). We observed a strict qualitative and quantitative correlation of NLP activities. Mutant proteins that were unable to restore P. carotovorum virulence on potato (see Fig. 3B: D93A, H101A, D104A, E106A) failed to cause leaf necrosis (at concentrations up to 5 ␮M, not shown) (see Fig. 3C: concentration 0.5 ␮M) and to trigger calcein release from tobacco plasma membrane vesicles (see Fig. 3D). Likewise, mutants that retained the ability to complement the Pcc nlp– mutation (see Fig. 3B: R102A, H103A, S126A) caused necrosis (see Fig. 3C) and disturbed plasma membrane integrity (see Fig. 3D). All mutant proteins were shown to be produced and secreted from Pcc nlp– strains in similar size and amount (Fig. S4). Moreover, circular dichroism analyses of all mutant proteins Fig. 2. NLPs are conserved virulence factors. (A) Complementation of the tested and crystallization of 2 inactive NLPPya mutants (D93A and reduced virulence of the NLP-deficient P. carotovorum strain (Pcc nlp–)by H101A) (see Fig. S4) revealed no significantly altered tertiary expression of either NLPPcc or the oomycete-derived NLPPp and NLPPya, respec- structures within the mutant proteins, suggesting that the observed ϩ – tively. The wild-type strain (Pcc nlp ) and Pcc nlp transformed with the empty differences in biological activities of the mutants are the result of PLANT BIOLOGY vector are shown for comparison. Bacterial growth (Upper) (average ϩ/– SD) as well as soft rot disease development on potato tuber disks (Lower) inocu- subtle changes in the close vicinity of the mutated residues. lated with the corresponding strains is indicated. (B) Plant necrosis-promoting Measurement of necrosis by electrolyte leakage assays (see activity of NLPs. Leaves from tobacco (Upper)orA. thaliana (Lower) were Fig. 3C) suggested that the cytolytic activity of the active mutants infiltrated with buffer as control or 0.5 ␮M NLP. Lesion formation 3 days after (R102A, H103A, S126A) was reduced. However, this reduction infiltration is shown. (C) Membrane permeabilization triggered by NLPPp corresponded to partial virulence complementation and mem- (blue), NLPPya (orange), or NLPPcc (green). The NLP-induced release of calcein brane disintegration activities of these NLP variants. In this from plasma membrane vesicles obtained either from N. tabacum (closed regard, the NLP mutant protein K92A exhibits residual, albeit circles), A. thaliana (open squares), or C. communis (open circles) is shown as significantly reduced activity (Fig. 3 B and D) and, consequently, the percentage of the maximal release obtained at the end of the assay by still triggers lesion formation at higher concentrations (5 ␮M; addition of the detergent Triton X-100. A representative experiment of 3 yielding similar results is shown. not shown). All together, our findings support the view that NLPs are toxins whose contribution to microbial virulence is based upon their abilities to permeabilize dicotyledonous plant plasma membranes, thereby causing cytolysis and necrosis. complementation efficiencies might be a result of NLP /NLP Pp Pya Residues D93, H101, D104, and E106 are important for the protein expression levels that were lower than that of NLPPcc cytolytic activity of NLPs and for the coordination of a Mg2ϩ ion (not shown). All together, our data suggest that NLPs are within the acidic cavity (see Figs. 1E,3A, and Fig. S3). To orthologous virulence factors. investigate the importance of a negative charge therein, we NLPs trigger necrosis upon infiltration into leaves of dicoty- replaced D104 either by a noncharged (D104N), a cationic ledonous plants (Fig. 2B). We have previously shown that (D104K), or another anionic residue (D104E). All but one targeting of NLPPp exclusively to the extracellular side of plant (D104E) abolished the cytolytic activities of the NLP mutant plasma membranes results in lesion formation (11). To explore proteins (Fig. S5), suggesting that coordination of a cationic 2ϩ similarities in the molecular mode of action of actinoporins and ligand is crucial. The Mg -occupancy in NLPPya crystals is not NLPs we have used a vesicle-leakage assay. Highly enriched complete (Ϸ60–70%), despite of high Mg2ϩ-concentration in plant-plasma membrane vesicles were loaded with a fluorescent the crystallization buffer (500 mM). This indicates that Mg2ϩ

Ottmann et al. PNAS ͉ June 23, 2009 ͉ vol. 106 ͉ no. 25 ͉ 10361 Downloaded by guest on September 27, 2021 Fig. 3. Structural requirements for the virulence-promoting cytolytic and plant defense stimulating activity of NLPs are identical. (A) Residues mutated in NLPPcc/NLPPp. Detailed view of the region between the ␤-sheet core and loops L1–L3. Selected residues that were individually mutated are shown as sticks. Parts of the semitransparent solid surface have been removed for clarity. (B) Complementation of the reduced virulence of the NLP-deficient P. carotovorum strain – (Pcc nlp ) by expression of NLPPcc wild-type versus mutant proteins. See Fig. 2A for details. ‘‘*’’ (P-value Ͻ0.05) and ‘‘**’’ (P-value Ͻ0.005) indicates significant differences (Student’s t test) from control values. (C) Plant necrosis-promoting activity of NLPPcc variants visualized by lesion formation (Upper) and electrolyte leakage (Lower) upon infiltration into tobacco leaves. See Fig. 2B for details. Labels are indicated in (D). Data in the lower panel refer to average values from 3 experiments ϩ/– SD. (D) Permeabilization of tobacco plasma membrane vesicles triggered by NLPPcc variants. See Fig. 2C for details. (E) Early defense-related gene expression mediated by NLPPcc variants. Quantitative real-time RT-PCR analysis of HIN1 and PAD3 expression in tobacco leaves harvested 4 h after infiltration of 0.5 ␮M NLPPcc variants. The gene induction (fold change) by NLP infiltration was compared to the expression level of buffer infiltration (control). The data presented are the average of 3 determinations ϩ/– SD per experiment. The experiment was done 3 times with similar results.

may not be a physiological ligand. NLPs act at the plant cell stimulating activities of NLPs, we tested NLP mutant proteins surface in a Ca2ϩ-rich environment. Indeed, scavenging extra- for their abilities to trigger immunity-associated gene expression cellular calcium by a membrane-impermeable Ca2ϩ-chelator, in intact tobacco plants. Quantitative RT-PCR analysis of HIN1 BAPTA, abolished the plasma membrane-disintegrating activity and PAD3 transcript accumulation (28) revealed that mutant of NLPs (see Fig. S5), suggesting that coordinative binding of a proteins R102A, H103A, and S126A trigger gene expression. In Ca2ϩ ion is crucial for NLP function. Ca2ϩ may thus, as in the contrast, D93A, H101A, D104A, and E106A mutants are com- case of the C2 membrane binding domain of several extrinsic pletely inactive, whereas K92A shows residual activity (Fig. 3E). membrane proteins (26, 27), mediate docking of NLPs to target Importantly, the same activity pattern of mutants was observed membranes, or might alternatively be required for the mem- in virulence complementation, leaf cell cytolysis, and plasma B D brane-permeabilizing activity. membrane destabilization assays (see Fig. 3 – ). Altogether, these data suggest that a common fold of a cytolytic toxin is required for both microbial pathogenicity and activation of plant Identical Fold Requirements for NLP-Mediated Virulence and Plant immune responses. Thus, NLP-mediated plant-defense re- Immunity. Various microbial phytotoxins have been shown to sponses are likely the consequence of NLP-mediated cytolysis. trigger plant immunity-associated responses (6). In virtually all cases, however, it is unknown whether the toxin mode of action Discussion is responsible for the activation of plant defenses. To uncover a We have combined tertiary structure and mutagenesis-based causal relationship between the cytolytic and defense- analyses with genetic and biochemical technologies to show that

10362 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902362106 Ottmann et al. Downloaded by guest on September 27, 2021 microbial NLPs constitute positive virulence factors accelerating microbial toxin-induced innate immunity is also known from disease and pathogen growth in host plants through disintegra- animal systems. Cytolytic bacterial (pneumolysin), fungal (ni- tion of the plasma membrane and subsequent cytolysis. NLPs are gericin), or marine (maitotoxin) toxins are triggers of the distinguished from other known phytotoxins by their wide mammalian inflammasome (32, 33). Toxin-mediated activation distribution across taxa and their broad spectrum of activity of mammalian immune responses is based upon the recognition against dicotyledonous plants. In particular, the production of of endogenous, host-derived compounds that are released upon orthologous cytolytic toxins in prokaryotic and eukaryotic phy- toxin-induced host cell damage and subsequent formation of topathogenic microbes is without precedence and suggests that damage-associated molecular patterns (34–36). Consequently, NLPs constitute an evolutionary ancient toxin fold that has been mammalian inflammasome activation is now considered to be retained, preferably in organisms exhibiting a hemibiotrophic or activated not only upon perception of microbial patterns, but necrotrophic lifestyle. This view is supported by published also by the action of toxins (32, 33, 37, 38). We suggest that plant reports of NLPs from at least 10 different organisms (including cells are also capable of sensing toxin-induced cellular changes. bacterial, oomycete, and genuine fungal species) that have been NLP-driven membrane disruption may result in the release of demonstrated experimentally to possess cytolytic activities host-derived molecules that serve as endogenous damage- against dicotyledonous plant cells (7, 11). Importantly, by using associated molecular patterns. Alternatively, NLP-induced dis- Ϸ the NLPPp sequence as query in BLAST searches, 130 se- turbance of the cellular ion homeostasis or membrane potential quences were retrieved from GenBank with E-values higher than may signal activation of plant defenses. Remarkably, NLP- ϩ ϩ ϩ that of NLPPcc [P. carotovorum (4e-16)], for which the cytolytic induced Ca2 and H influx, as well as K efflux, were reported activity was demonstrated. This suggests that numerous, yet (16, 39) that mimicked synthetic ionophore-induced ion fluxes in untested NLPs harbor a similar cytolytic activity as NLPPp, plant cells, which themselves were shown to trigger plant de- NLPPya,orNLPPcc. In addition, the high sequence conservation fense-associated responses in a nonreceptor-mediated manner of the heptapeptide motif (GHRHDWE, see Fig. S1) required (40). Our structure-based analyses suggest that NLPs are cyto- for all NLP activities further implies a widespread usage of lytic toxins that trigger plant immunity-associated defenses cytolytic NLPs by taxonomically diverse microbial organisms. In through interference with plant tissue integrity. Hence, dis- the future, it can be expected that phylogeny-based relationship turbed host integrity as a common signal for the activation of studies will facilitate analyses on the evolutionary origins of immune defenses adds to the list of conceptual similarities in the NLP. Such analyses may further reveal possible horizontal organization of innate immunity in the animal and plant lineage. gene-transfer events that may have resulted in the transfer of prokaryotic sequences into unicellular eukaryotic microbes and Materials and Methods that may be the reason for the unprecedented broad occurrence Recombinant NLP Purification and 3-Dimensional Structure Determination of of NLP sequences in phylogenetically different taxa. NLPPya. For functional studies, secretory expression of NLPs was performed Accumulating evidence suggests that NLP gene sequences either in Pichia pastoris GS115 (secretory expression plasmid pPIC9K, Multi- have also undergone functional diversification. NLP gene fam- Copy Pichia Expression Kit Instructions, Invitrogen) or in the NLP-deficient P. ilies from various Phytophthora species are exceptionally large (Ͼ carotovoum subsp. carotovorum SCC3200 strain (Pcc nlp–) (23). For crystalli- 20 putative NLP-encoding genes) compared to those of other zation of NLPPya and mutants D93A and H101A, recombinant proteins were species (7, 11), suggesting that not all NLP proteins may harbor produced without signal peptides as described in ref. 15. Further experimental cytolytic activity. Indeed, of 3 different NLPs from P. infestans details on recombinant protein expression, protein structure elucidation and tested, only one (PiNPP1.1) was found to cause necrosis when analysis can be found in SI Materials and Methods. expressed in plants (29). Importantly, the 2 noncytolytic NLPs Plant Material and Growth Conditions. Nicotiana tabacum cv. Samsun NN, carry mutations in an aspartate residue that corresponds to Commelina communis L., and Arabidopsis thaliana Col-0 were grown at 22 °C NLPPya D104, which is essential for the negative charge of the (15 h light). Solanum tuberosum cv. Atica tubers were obtained from Sa Ka GHRHDWE motif and for the cytolytic activity of NLPs (see Pflanzenzucht GbR. Fig. 3 and Fig. S3). Moreover, the genomes of the fungal pathogens Magnaporthe grisea and Mycosphaerella graminicola Leaf Infiltration and Electrolyte Leakage Assay. Purified NLPs (0,5 ␮M, 10 ␮l) or PLANT BIOLOGY (30, 31) encode NLP sequences. This is important because the mock solution were infiltrated abaxialy into leaves of N. tabacum (4–6 weeks host range of both pathogens is restricted to monocots that are old) or A. thaliana (3–4 weeks old). For quantification of necrosis, 1 leaf disc known to be resistant to the cytolytic activities of NLPs (11). In (6-mm diameter) per plant (3 different plants) was removed immediately after agreement with that finding, the M. graminicola NLP caused infiltration and washed in 1 ml of distilled water. After 30 min, the discs were cytolysis and plant defense activation in Arabidopsis, but not in transferred to 1 ml of fresh water and electrolyte leakage was measured with wheat (31), suggesting that these proteins might have either lost a conductivity meter (Q cond 2,200). Alternatively, lesion formation was documented 3 days after infiltration of the various NLP proteins. their physiological importance to the fungus [inactivation of the sole NLP in M. graminicola did not affect its virulence (31)] or Pathogenicity Assay. The NLP-deficient Pcc nlp– strain expressing wild-type might have adopted a yet elusive novel activity. The latter NLPPp, NLPPya, NLPPcc, or mutant versions of NLPPcc was grown in liquid culture assumption is supported by the fact that the obligate biotroph supplemented with ampicillin until the logarithmic phase was reached. The oomycete, Hyaloperonospora arabidopsidis, produces at least 3 OD546 was then adjusted to 0.07. Potato tuber slices were inoculated with 15 NLP proteins, none of which proved to be cytolytic (A. Cabral, ␮l of the bacterial suspension and incubated in water-filled, sealed boxes on G. van den Ackerveken, personal communication). While the a grate 2-cm above the water surface. Rotting symptoms were documented function of these NLPs is currently unclear, it is tempting to after 3 days. To analyze bacterial growth at that time, 3 potato tuber slices per speculate that the NLP fold, which is also similar to that of fungal transgenic strain were pooled and stirred in 25 ml 10 mM MgCl2. Dilutions of lectins (see Fig. 1C and Fig. S2), has been reshaped during this extract were plated on solid LB medium supplemented with ampicillin, chloramphenicol, and cycloheximide to determine colony forming units. evolution to serve functions in microbial attachment to host – plant surfaces. Growth of the various transgenic Pcc nlp strains was calculated relative to the wild-type strain (Pcc nlpϩ) or the NLP -complemented strain. Our data further imply that toxin-induced interference with Pcc cell integrity may culminate in plant immune responses. While Preparation and Permeabilization of Calcein-Loaded Plasma Membrane Vesicles. the plant defense-stimulating potential of a number of myco- Plant plasma membranes were prepared by phase partitioning of microsomal toxins is known for some time (6, 11), our data uniquely fractions (41) using Dextran and PEG in a concentration of 6.4% (wt/wt) and demonstrate that the virulence activity of a microbial toxin is 3 mM KCl. For preparation of calcein-loaded vesicles, plasma membranes (500 also the cause for stimulation of plant defenses. Importantly, ␮g protein) were sonified (30 min on ice, 20-sec pulse on, 20-sec pulse off,

Ottmann et al. PNAS ͉ June 23, 2009 ͉ vol. 106 ͉ no. 25 ͉ 10363 Downloaded by guest on September 27, 2021 amplitude 25%) in the presence of calcein (60 mM, pH 7.0 with NaOH). The real-time PCR amplification was carried out in the presence of SYBR Green external calcein was removed by gel filtration using Sephadex G-75 (Sigma) (Bio-Rad) with an iQ5 iCycler (Bio-Rad). Amplification of actin served as medium column equilibrated in 20 mM Tris pH 8.5, 140 mM NaCl, and 1 mM internal standard. Data analysis was performed according to the 2-⌬⌬CT- EDTA. Permeabilization of the vesicles (1 ng protein/␮l) induced by 20 nM NLPs method (42). Gene induction (fold change) by NLP was presented as the (0.04 ng/␮l) was assayed at room temperature in 20 mM Mes pH 5.8, 140 mM average of 3 determinations plus or minus standard deviation and compared NaCl by measuring fluorescence (excitation 485 nm, emission 520 nm) in a to the expression level of mock infiltration. microplate reader (Sirius HT Injector, MWG). The percentage of calcein release ϭ (R) was calculated according to the equation R (Fmeas–Finit)/(Fmax–Finit)*100, Coordinates. Crystallographic coordinates have been deposited in the Protein where F , F , and F are the measured, initial, and maximal fluorescence, meas init max Data Bank under the accession code PDB ID 3GNU. respectively. Fmax was obtained by the addition of Triton X-100 to 0.5% (vol/vol) final concentration at the end of each measurement. The experi- ACKNOWLEDGMENTS. We thank to Mark Gijzen, Georg Felix and Sophien ments were repeated 3 times with similar results. Kamoun for useful comments on the manuscript, the staff of the Swiss Light Source, Villingen, Switzerland, Ursula Neu for technical support, and Ilme ␮ RNA Isolation and RT-PCR. Tobacco leaves infiltrated with 0.5 M NLPPcc Schlichting, Wulf Blankenfeldt, Andrea Scrima, and Nils Schrader for data variants or mock solutions were harvested after 4 h. RNA was isolated using collection. This work was supported by Grant DFG Se 229/19–3 from the the Tri Reagent method (Sigma). Synthesis of cDNA was performed by means German Federal Ministry of Education and Research (to H.U.S.) and Grant SFB of the RevertAidTM MuLV reverse transcriptase (Fermentas). Quantitative 446 from the German Research Foundation (to C. Oecking and T.N.).

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