Parental transfer of the antimicrobial protein LBP/BPI protects Biomphalaria glabrata eggs against oomycete infections.

Vertebrate females transfer antibodies via the placenta, colostrum and milk or via the egg yolk to protect their immunologically immature offspring against pathogens. This evolutionarily important transfer of immunity is poorly documented in invertebrates and basic questions remain regarding the nature and extent of parental protection of offspring. In this study, we show that a lipopolysaccharide binding protein/bactericidal permeability increasing protein family member from the invertebrate Biomphalaria glabrata (BgLBP/BPI1) is massively loaded into the eggs of this freshwater snail. Native and recombinant proteins displayed conserved LPS-binding, antibacterial and membrane permeabilizing activities. A broad screening of various pathogens revealed a previously unknown biocidal activity of the protein against pathogenic water molds (oomycetes), which is conserved in human BPI. RNAi-dependent silencing of LBP/BPI in the parent snails resulted in a significant reduction of reproductive success and extensive death of eggs through oomycete infections. This work provides the first functional evidence that a LBP/BPI is involved in the parental immune protection of invertebrate offspring and reveals a novel and conserved biocidal activity for LBP/BPI family members.

Transcriptome dynamics of Arabidopsis thaliana root penetration by the oomycete pathogen Phytophthora parasitica.

BACKGROUND: Oomycetes are a group of filamentous microorganisms that includes both animal and plant pathogens and causes major agricultural losses. Phytophthora species can infect most crops and plants from natural ecosystems. Despite their tremendous economic and ecologic importance, few effective methods exist for limiting the damage caused by these species. New solutions are required, and their development will require improvements in our understanding of the molecular events governing infection by these pathogens. In this study, we characterized the genetic program activated during penetration of the plant by the soil-borne pathogen Phytophthora parasitica. RESULTS: Using all the P. parasitica sequences available in public databases, we generated a custom oligo-array and performed a transcriptomic analysis of the early events of Arabidopsis thaliana infection. We characterized biological stages, ranging from the appressorium-mediated penetration of the pathogen into the roots to the occurrence of first dead cells in the plant. We identified a series of sequences that were transiently modulated during host penetration. Surprisingly, we observed an overall down regulation of genes encoding proteins involved in lipid and sugar metabolism, and an upregulation of functions controlling the transport of amino acids. We also showed that different groups of genes were expressed by P. parasitica during host penetration and the subsequent necrotrophic phase. Differential expression patterns were particularly marked for cell wall-degrading enzymes and other proteins involved in pathogenicity, including RXLR effectors. By transforming P. parasitica with a transcriptional fusion with GFP, we showed that an RXLR-ecoding gene was expressed in the appressorium and infectious hyphae during infection of the first plant cell. CONCLUSION: We have characterized the genetic program activated during the initial invasion of plant cells by P. parasitica. We showed that a specific set of proteins, including effectors, was mobilized for penetration and to facilitate infection. Our detection of the expression of an RXLR encoding gene by the appressorium and infection hyphae highlights a role of this structure in the manipulation of the host cells.

The Phytophthora parasitica RXLR effector penetration-specific effector 1 favours Arabidopsis thaliana infection by interfering with auxin physiology.

Pathogenic oomycetes have evolved RXLR effectors to thwart plant defense mechanisms and invade host tissues. We analysed the function of one of these effectors (Penetration-Specific Effector 1 (PSE1)) whose transcript is transiently accumulated during penetration of host roots by the oomycete Phytophthora parasitica. Expression of PSE1 protein in tobacco (Nicotiana tabacum and Nicotiana benthamiana) leaves and in Arabidopsis thaliana plants was used to assess the role of this effector in plant physiology and in interactions with pathogens. A pharmacological approach and marker lines were used to charcterize the A. thaliana phenotypes. Expression of PSE1 in A. thaliana led to developmental perturbations associated with low concentrations of auxin at the root apex. This modification of auxin content was associated with an altered distribution of the PIN4 and PIN7 auxin efflux carriers. The PSE1 protein facilitated plant infection: it suppressed plant cell death activated by Pseudomonas syringae avirulence gene AvrPto and Phytophthora cryptogea elicitin cryptogein in tobacco and exacerbated disease symptoms upon inoculation of transgenic A. thaliana plantlets with P. parasitica in an auxin-dependant manner. We propose that P. parasitica secretes the PSE1 protein during the penetration process to favour the infection by locally modulating the auxin content. These results support the hypothesis that effectors from plant pathogens may act on a limited set of targets, including hormones.

Genome sequence and annotation of Periconia digitata a hopeful biocontrol agent of phytopathogenic oomycetes.

The Periconia fungal genus belongs to the phylum Ascomycota, order Pleosporales, family Periconiaceae. Periconia are found in many habitats, but little is known about their ecology. Several species from this genus produce bioactive molecules. Periconia digitata extracts were shown to be deadly active against the pine wilt nematode. Furthermore, P. digitata was shown to inhibit the plant pathogenic oomycete Phytophthora parasitica. Because P. digitata has great potential as a biocontrol agent and high quality genomic resources are still lacking in the Periconiaceae family, we generated long-read genomic data for P. digitata. Using PacBio Hifi sequencing technology, we obtained a highly-contiguous genome assembled in 13 chromosomes and totaling ca. 39 Mb. In addition, we produced a reference transcriptome, based on 12 different culture conditions, and proteomic data to support the genome annotation. Besides representing a new reference genome within the Periconiaceae, this work will contribute to our better understanding of the Eukaryotic tree of life and opens new possibilities in terms of biotechnological applications.

BABA-induced pathogen resistance: a multi-omics analysis of the tomato response reveals a hyper-receptive status involving ethylene.

Prior exposure to microbial-associated molecular patterns or specific chemical compounds can promote plants into a primed state with stronger defence responses. ß-aminobutyric acid (BABA) is an endogenous stress metabolite that induces resistance protecting various plants towards diverse stresses. In this study, by integrating BABA-induced changes in selected metabolites with transcriptome and proteome data, we generated a global map of the molecular processes operating in BABA-induced resistance (BABA-IR) in tomato. BABA significantly restricts the growth of the pathogens Oidium neolycopersici and Phytophthora parasitica but not Botrytis cinerea. A cluster analysis of the upregulated processes showed that BABA acts mainly as a stress factor in tomato. The main factor distinguishing BABA-IR from other stress conditions was the extensive induction of signaling and perception machinery playing a key role in effective resistance against pathogens. Interestingly, the signalling processes and immune response activated during BABA-IR in tomato differed from those in Arabidopsis with substantial enrichment of genes associated with jasmonic acid (JA) and ethylene (ET) signalling and no change in Asp levels. Our results revealed key differences between the effect of BABA on tomato and other model plants studied until now. Surprisingly, salicylic acid (SA) is not involved in BABA downstream signalization whereas ET and JA play a crucial role.

Imbalanced lignin biosynthesis promotes the sexual reproduction of homothallic oomycete pathogens.

Lignin is incorporated into plant cell walls to maintain plant architecture and to ensure long-distance water transport. Lignin composition affects the industrial value of plant material for forage, wood and paper production, and biofuel technologies. Industrial demands have resulted in an increase in the use of genetic engineering to modify lignified plant cell wall composition. However, the interaction of the resulting plants with the environment must be analyzed carefully to ensure that there are no undesirable side effects of lignin modification. We show here that Arabidopsis thaliana mutants with impaired 5-hydroxyguaiacyl O-methyltransferase (known as caffeate O-methyltransferase; COMT) function were more susceptible to various bacterial and fungal pathogens. Unexpectedly, asexual sporulation of the downy mildew pathogen, Hyaloperonospora arabidopsidis, was impaired on these mutants. Enhanced resistance to downy mildew was not correlated with increased plant defense responses in comt1 mutants but coincided with a higher frequency of oomycete sexual reproduction within mutant tissues. Comt1 mutants but not wild-type Arabidopsis accumulated soluble 2-O-5-hydroxyferuloyl-L-malate. The compound weakened mycelium vigor and promoted sexual oomycete reproduction when applied to a homothallic oomycete in vitro. These findings suggested that the accumulation of 2-O-5-hydroxyferuloyl-L-malate accounted for the observed comt1 mutant phenotypes during the interaction with H. arabidopsidis. Taken together, our study shows that an artificial downregulation of COMT can drastically alter the interaction of a plant with the biotic environment.

Ecosystem screening approach for pathogen-associated microorganisms affecting host disease.

The microbial community in which a pathogen evolves is fundamental to disease outcome. Species interacting with a pathogen on the host surface shape the distribution, density, and genetic diversity of the inoculum, but the role of these species is rarely determined. The screening method developed here can be used to characterize pathogen-associated species affecting disease. This strategy involves three steps: (i) constitution of the microbial community, using the pathogen as a trap; (ii) community selection, using extracts from the pathogen as the sole nutrient source; and (iii) molecular identification and the screening of isolates focusing on their effects on the growth of the pathogen in vitro and host disease. This approach was applied to a soilborne plant pathogen, Phytophthora parasitica, structured in a biofilm, for screening the microbial community from the rhizosphere of Nicotiana tabacum (the host). Two of the characterized eukaryotes interfered with the oomycete cycle and may affect the host disease. A Vorticella species acted through a mutualistic interaction with P. parasitica, disseminating pathogenic material by leaving the biofilm. A Phoma species established an amensal interaction with P. parasitica, strongly suppressing disease by inhibiting P. parasitica germination. This screening method is appropriate for all nonobligate pathogens. It allows the definition of microbial species as promoters or suppressors of a disease for a given biotope. It should also help to identify important microbial relationships for ecology and evolution of pathogens.

(Homo)glutathione depletion modulates host gene expression during the symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti.

Under nitrogen-limiting conditions, legumes interact with symbiotic rhizobia to produce nitrogen-fixing root nodules. We have previously shown that glutathione and homoglutathione [(h)GSH] deficiencies impaired Medicago truncatula symbiosis efficiency, showing the importance of the low M(r) thiols during the nodulation process in the model legume M. truncatula. In this study, the plant transcriptomic response to Sinorhizobium meliloti infection under (h)GSH depletion was investigated using cDNA-amplified fragment length polymorphism analysis. Among 6,149 expression tags monitored, 181 genes displayed significant differential expression between inoculated control and inoculated (h)GSH depleted roots. Quantitative reverse transcription polymerase chain reaction analysis confirmed the changes in mRNA levels. This transcriptomic analysis shows a down-regulation of genes involved in meristem formation and a modulation of the expression of stress-related genes in (h)GSH-depleted plants. Promoter-beta-glucuronidase histochemical analysis showed that the putative MtPIP2 aquaporin might be up-regulated during nodule meristem formation and that this up-regulation is inhibited under (h)GSH depletion. (h)GSH depletion enhances the expression of salicylic acid (SA)-regulated genes after S. meliloti infection and the expression of SA-regulated genes after exogenous SA treatment. Modification of water transport and SA signaling pathway observed under (h)GSH deficiency contribute to explain how (h)GSH depletion alters the proper development of the symbiotic interaction.

Silencing of acidic pathogenesis-related PR-1 genes increases extracellular beta-(1->3)-glucanase activity at the onset of tobacco defence reactions.

The class 1 pathogenesis-related (PR) proteins are thought to be involved in plant defence responses, but their molecular functions are unknown. The function of PR-1 was investigated in tobacco by generating stable PR-1a-silenced lines in which other acidic PR-1 genes (PR-1b and PR-1c) were silenced. Plants lacking extracellular PR-1s were more susceptible than wild-type plants to the oomycete Phytophthora parasitica but displayed unaffected systemic acquired resistance and developmental resistance to this pathogen. Treatment with salicylic acid up-regulates the PR-1g gene, encoding a basic protein of the PR-1 family, in PR-1-deficient tobacco, indicating that PR-1 expression may repress that of PR-1g. This shows that acidic PR-1s are dispensable for expression of salicylic acid-dependent acquired resistances against P. parasitica and may reveal a functional overlap in tobacco defence or a functional redundancy in the PR-1 gene family. The data also show that there is a specific increase in apoplastic beta-(1-->3)-glucanase activity and a decrease in beta-(1-->3)-glucan deposition in PR-1-silenced lines following activation of defence reactions. Complementation of the silencing by apoplastic treatment with a recombinant PR-1a protein largely restores the wild-type beta-(1-->3)-glucanase activity and callose phenotype. Taken together with the immunolocalization of PR-1a to sites of beta-(1-->3)-glucan deposition in wild-type plants, these results are indicative of a function for PR-1a in regulation of enzymatic activity of extracellular beta-(1-->3)-glucanases.

Strategies of attack and defense in plant-oomycete interactions, accentuated for Phytophthora parasitica Dastur (syn. P. Nicotianae Breda de Haan).

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. Due to their particular physiological characteristics, no efficient treatments against diseases caused by these microorganisms are presently available. To develop such treatments, it appears essential to dissect the molecular mechanisms that determine the interaction between Phytophthora species and host plants. Available data are scarce, and genomic approaches were mainly developed for the two species, Phytophthora infestans and Phytophthora sojae. However, these two species are exceptions from, rather than representative species for, the genus. P. infestans is a foliar pathogen, and P. sojae infects a narrow range of host plants, while the majority of Phytophthora species are quite unselective, root-infecting pathogens. To represent this majority, Phytophthora parasitica emerges as a model for the genus, and genomic resources for analyzing its interaction with plants are developing. The aim of this review is to assemble current knowledge on cytological and molecular processes that are underlying plant-pathogen interactions involving Phytophthora species and in particular P. parasitica, and to place them into the context of a hypothetical scheme of co-evolution between the pathogen and the host.

Quantification of Salicylic Acid (SA) and SA-glucosides in Arabidopsis thaliana.

Homeostasis between the cytoplasmic plant hormone salicylic acid (SA) and its' inactive, vacuolar storage forms, SA-2-O-ß-D-glucoside (SAG) and SA-ß-D-Glucose Ester (SGE), regulates the fine-tuning of defense responses to biotrophic pathogens in Arabidopsis thaliana. This protocol describes a simplified, optimized procedure to extract and quantify free SA and total hydrolyzable SA in plant tissues using a classical HPLC-based method.

Modulation of the biological activity of a tobacco LTP1 by lipid complexation.

Plant lipid transfer proteins (LTPs) are small, cysteine-rich proteins secreted into the extracellular space. They belong to the pathogenesis-related proteins (PR-14) family and are believed to be involved in several physiological processes including plant disease resistance, although their precise biological function is still unknown. Here, we show that a recombinant tobacco LTP1 is able to load fatty acids and jasmonic acid. This LTP1 binds to specific plasma membrane sites, previously characterized as elicitin receptors, and is shown to be involved in the activation of plant defense. The biological properties of this LTP1 were compared with those of LTP1-linolenic and LTP1-jasmonic acid complexes. The binding curve of the LTP1-linolenic acid complex to purified tobacco plasma membranes is comparable to the curve obtained with LTP1. In contrast, the LTP1-jasmonic acid complex shows a strongly increased interaction with the plasma membrane receptors. Treatment of tobacco plants with LTP1-jasmonic acid resulted in an enhancement of resistance toward Phytophthora parasitica. These effects were absent upon treatment with LTP1 or jasmonic acid alone. This work presents the first evidence for a biological activity of a LTP1 and points out the crucial role of protein-specific lipophilic ligand interaction in the modulation of the protein activity.

Accessibility of tobacco lipid transfer protein cavity revealed by 15N NMR relaxation studies and molecular dynamics simulations.

Plant LTP1 are small helical proteins stabilized by four disulfide bridges and are characterized by the presence of an internal cavity, in which various hydrophobic ligands can be inserted. Recently, we have determined the solution structure of the recombinant tobacco LTP1_1. Unexpectedly, despite a global fold very similar to the structures already known for cereal seed LTP1, its binding properties are different: Tobacco LTP1_1 is able to bind only one monoacylated lipid, whereas cereal LTP1 can bind either one or two. The 3D structure of tobacco LTP1_1 revealed the presence of a hydrophobic cluster, not observed on cereal LTP1 structures, which may hinder one of the two entrances of the cavity defined for wheat LTP1. To better understand the mechanism of lipid entrance for tobacco LTP1_1 and to define the regions of the protein monitoring the accessibility of the cavity, we have complemented our structural data by the study of the internal dynamics of tobacco LTP1_1, using (15)N magnetic relaxation rate data and MD simulations at room and high temperatures. This work allowed us to define two regions of the protein experiencing the largest motions. These two regions delineate a portal that opens up during the simulation constituting a unique entrance of the hydrophobic cavity, in contrast with wheat LTP1 where two routes were detected. The hydrophobic interactions resulting from a few point mutations are strong enough to completely block the second portal so that the accessibility of the cavity is restricted to one entrance, explaining why this particular LTP1 binds only one lipid molecule.

From elicitins to lipid-transfer proteins: a new insight in cell signalling involved in plant defence mechanisms.

Elicitins and lipid-transfer proteins are small cysteine-rich lipid-binding proteins secreted by oomycetes and plant cells, respectively, that share some structural and functional properties. In spite of intensive work on their structure and diversity at the protein and genetic levels, the precise biological roles of lipid-transfer proteins remains unclear, although the most recent data suggest a role in somatic embryogenesis, in the formation of protective surface layers and in defence against pathogens. By contrast, elicitins are known elicitors of plant defence, and recent work demonstrating that elicitins and lipid-transfer proteins share the same biological receptors gives a new perspective to understand the role played by lipid binding proteins, mainly the early recognition of intruders in plants.

Solution structure of a tobacco lipid transfer protein exhibiting new biophysical and biological features.

Plant lipid transfer proteins are small soluble extracellular proteins that are able to bind and transfer a variety of lipids in vitro. Recently, it has been proposed that lipid transfer proteins may play a key role in plant defence mechanisms, especially during the induction of systemic acquired resistance. However, very little is known about the proteins expressed in developing plants and tissues, since almost all the biophysical and structural data available to date on lipid transfer proteins originate from proteins present in storage tissues of monocot cereal seeds. In this paper, we report the structural and functional characteristics of a lipid transfer protein (named LTP1_1) constitutively expressed in young aerial organs of Nicotiana tabacum (common tobacco). The unlabelled and uniformly labelled proteins were produced in the yeast Pichia pastoris, and we determined the three-dimensional (3D) structure of LTP1_1 using nuclear magnetic resonance (NMR) spectroscopy and molecular modeling techniques. The global fold of LTP1_1 is very close to the previously published structures of LTP1 extracted from cereal seeds, including an internal cavity. However, the chemical shift variations of several NMR signals upon lipid binding show that tobacco LTP1_1 is able to bind only one LysoMyristoylPhosphatidylCholine (LMPC), while wheat and maize LTPs can bind either one or two. Titration experiments using intrinsic tyrosine fluorescence confirm this result not only with LMPC but also with two fatty acids. These differences can be explained by the presence in tobacco LTP1_1 of a hydrophobic cluster closing the second possible access to the protein cavity. This result suggests that LTP1 lipid binding properties could be modulated by subtle changes in a conserved global structure. The biological significance of this finding is discussed in the light of the signalling properties of the tobacco LTP1_1-jasmonate complex described elsewhere.

A tobacco S-like RNase inhibits hyphal elongation of plant pathogens.

Ribonuclease (RNase) NE gene expression is induced in tobacco leaves in response to Phytophthora parasitica. Using antibodies directed against RNase NE, we demonstrate that RNase NE is extracellular at the early steps of the interaction, while the fungal tip growth is initiated in the apoplastic compartment. After production in Pichia pastoris and biochemical purification, we show that the S-like RNase NE inhibits hyphal growth from P. parasitica zoospores and from Fusarium oxysporum conidia in vitro. Conversion into an enzymatically inactive form after mutagenesis of the active site-histidine 97 residue to phenylalanine leads to the suppression of this activity, suggesting that RNase NE inhibits the elongation of germ tubes by degradation of microbial RNAs. Exogenous application of RNase NE in the extracellular space of leaves inhibits the development of P. parasitica. Based on its induction by inoculation, its localization, and its activity against two plant pathogens, we propose that RNase NE participates in tobacco defense mechanisms by a direct action on hyphal development in the extracellular space. The RNase activity-dependent antimicrobial activity of the S-like RNase NE shares similarities with the only other biological activity demonstrated for plant RNases, the inhibition of elongation of pollen tubes by the S-RNase in gametophytic self-incompatibility, suggesting a functional link between self and nonself interactions in plants.

Characterization of PPMUCL1/2/3, three members of a new oomycete-specific mucin-like protein family residing in Phytophthora parasitica biofilm.

The plant pathogen Phytophthora parasitica forms a biofilm on the host surface. The biofilm transcriptome is characterized by the expression of PPMUCL1/2/3 (PHYTOPHTHORA PARASITICA MUCIN-LIKE) genes, which we report here to be members of a new, large mucin-like gene family restricted to the oomycete lineage. These genes encode secreted proteins organized into two domains. The NH2-terminal domain is highly conserved, but of unknown function. The second domain is a mucin-like domain enriched in threonine and serine residues, with a large number of putative O-glycosylation sites and a repeated motif defining 15 subgroups among the 315 members of the family. The second domain was found to be glycosylated in the recombinant rPPMUCL1 and rPPMUCL2 proteins. An analysis of PPMUCL1/2/3 gene expression indicated that these genes were expressed in a specific and coordinated manner in the biofilm. A novel cis-motif (R) bound to nuclear proteins, suggesting a possible role in PPMUCL1/2/3 gene regulation. Immunohistochemical staining revealed that the PPMUCL1/2 proteins were secreted and accumulated on the surface of the biofilm. Our data demonstrate that PPMUCL1/2/3 belong to a new oomycete-specific family of mucin-like proteins playing a structural role in the biofilm extracellular matrix.

Structure of sylvaticin, a new alpha-elicitin-like protein from Pythium sylvaticum.

The structure of sylvaticin, a 10 kDa major pythin protein excreted by the parasitic oomycete Pythium sylvaticum, has been determined. Although closely related to alpha-elicitins in its biological response, toxicity and overall structure, sylvaticin presents a number of structural features that make it an unusual member of the elicitin class. Elicitins possess a large hydrophobic cavity and the mechanism of the systemic acquired resistance induced in planta is known to proceed through lipid transport and complexation within this cavity. Unlike other elicitins, sylvaticin contains tryptophan residues, one of which points inwards towards the central cavity, thus limiting access to sterols. In the case of sylvaticin, the sterol-transport mechanism is likely to be of less importance compared with other members of the elicitin family and still remains to be fully characterized.

Specific adduction of plant lipid transfer protein by an allene oxide generated by 9-lipoxygenase and allene oxide synthase.

Lipid transfer proteins (LTPs) are ubiquitous plant lipid-binding proteins that have been associated with multiple developmental and stress responses. Although LTPs typically bind fatty acids and fatty acid derivatives in a non-covalent way, studies on the LTPs of barley seeds have identified an abundantly occurring covalently modified form, LTP1b, the lipid ligand of which has resisted clarification. In the present study, this adduct was identified as the alpha-ketol 9-hydroxy-10-oxo-12(Z)-octadecenoic acid. Further studies on the formation of LTP1b demonstrated that the ligand was introduced by nucleophilic attack of the free carboxylate group of the Asp-7 residue of the protein at carbon-9 of the allene oxide fatty acid 9(S),10-epoxy-10,12(Z)-octadecadienoic acid. This reactive oxylipin was produced in barley seeds by oxygenation of linoleic acid by 9-lipoxygenase followed by dehydration of the resulting hydroperoxide by allene oxide synthase. The generation of protein-oxylipin adducts represents a new function for plant allene oxide synthases, enzymes that have earlier been implicated mainly in the biosynthesis of the jasmonate family of plant hormones. Additionally, the LTP-allene oxide synthase interaction opens new perspectives regarding the roles of LTPs in the signaling of plant defense and development.

Construction of cryptogein mutants, a proteinaceous elicitor from Phytophthora, with altered abilities to induce a defense reaction in tobacco cells.

We prepared a series of cryptogein mutants, an elicitor from Phytophthora cryptogea, with altered abilities to bind sterols and fatty acids. The induction of the early events, i.e., synthesis of active oxygen species and pH changes, in suspension tobacco cells by these mutated proteins was proportional to their ability to bind sterols but not fatty acids. Although the cryptogein-sterol complex was suggested to be a form triggering a defense reaction in tobacco, some proteins unable to bind sterols induced the synthesis of active oxygen species and pH changes. The modeling experiments showed that conformational changes after the introduction of bulky residues into the omega loop of cryptogein resemble those induced by sterol binding. These changes may be necessary for the ability to trigger the early events by elicitins. However, the ability to stimulate necrosis in suspension tobacco cells and the expression of defense proteins in tobacco plants were linked neither to the lipid binding capacity nor to the capacity to provoke the early events. On the basis of these experiments and previous results, we propose that elicitins could stimulate two signal pathways. The first one induces necroses and the expression of pathogen-related proteins, includes tyrosine protein kinases and mitogen-activated protein kinases, and depends on the overall structure and charge distribution. The second type of interaction is mediated by phospholipase C and protein kinase C. It triggers the synthesis of active oxygen species and pH changes. This interaction depends on the ability of elicitins to bind sterols.