Crystal structure of a fungal elicitor secreted by Phytophthora cryptogea, a member of a novel class of plant necrotic proteins.

BACKGROUND: Elicitins form a novel class of plant necrotic proteins which are secreted by Phytophthora and Pythium fungi, parasites of many economically important crops. These proteins induce leaf necrosis in infected plants and elicit an incompatible hypersensitive-like reaction, leading to the development of a systemic acquired resistance against a range of fungal and bacterial plant pathogens. No crystal structures of this class of protein are available. The crystal structure determination of beta-cryptogein (CRY), secreted by Phytophthora cryptogea, was undertaken to identify structural features important for the necrotic activity of elicitins. RESULTS: The structure of CRY was determined using the multiwavelength anomalous diffraction technique and refined to 2.2 A resolution. The overall structure has a novel fold consisting of six alpha helices and a beak-like motif, whose sequence is highly conserved within the family, composed of an antiparallel two-stranded beta sheet and an omega loop. This motif is assumed to be a major recognition site for a putative receptor and/or ligand. Two other distinct binding sites seem to be correlated to the level of necrotic activity of elicitins. CONCLUSIONS: The determination of the crystal structure of a member of the elicitin family may make it possible to separate the activity that causes leaf necrosis from that inducing systemic acquired resistance to pathogens, making it feasible to engineer a non-toxic elicitin that only elicits plant defences. Such studies should aid the development of non-toxic agricultural pest control.

Cloning and expression of a queen pheromone-binding protein in the honeybee: an olfactory-specific, developmentally regulated protein.

Odorant-binding proteins (OBPs) are small abundant extracellular proteins thought to participate in perireceptor events of odor-pheromone detection by carrying, deactivating, and/or selecting odor stimuli. The honeybee queen pheromone is known to play a crucial role in colony organization, in addition to drone sex attraction. We identified, for the first time in a social insect, a binding protein called antennal-specific protein 1 (ASP1), which binds at least one of the major queen pheromone components. ASP1 was characterized by cDNA cloning, expression in Pichia pastoris, and pheromone binding. In situ hybridization showed that it is specifically expressed in the auxiliary cell layer of the antennal olfactory sensilla. The ASP1 sequence revealed it as a divergent member of the insect OBP family. The recombinant protein presented the exact characteristics of the native protein, as shown by mass spectrometry, and N-terminal sequencing and exclusion-diffusion chromatography showed that recombinant ASP1 is dimeric. ASP1 interacts with queen pheromone major components, opposite to another putative honeybee OBP, called ASP2. ASP1 biosynthetic accumulation, followed by nondenaturing electrophoresis during development, starts at day 1 before emergence, in concomitance with the functional maturation of olfactory neurons. The isobar ASP1b isoform appears simultaneously to ASP1a in workers, but only at approximately 2 weeks after emergence in drones. Comparison of in vivo and heterologous expressions suggests that the difference between ASP1 isoforms might be because of dimerization, which might play a physiological role in relation with mate attraction.

Determination of the primary and secondary structures of the dromedary (Camelus dromedarius) prolactin and comparison with prolactins from other species.

A non-glycosylated form of camel prolactin (camPRL), isolated from one-humped camel (Camelus dromedarius) pituitaries, was totally sequenced. A glycosylated form, separated by affinity chromatography on ConA-Sepharose, was partially sequenced. The comparison of the N-terminal amino acid sequences of the glycosylated and non-glycosylated forms showed that the only putative site of N-glycosylation (Asn-31) was indeed glycosylated. The far ultraviolet (UV) circular dichroism (CD) spectra of the two isohormones were identical, suggesting that the carbohydrate moiety had no effect on the global camPRL secondary structure. The far UV circular dichroism spectra of the two isohormones were analyzed in order to determine their relative proportions of periodic secondary structure, 60% of which was found to be in alpha-helix, as in prolactins of other species. The dromedary sequence was compared to those of other species and interpreted in term of evolutionary process. As already found for gonadotropins, the closest species to the dromedary was found to be the pig.

In vitro activation of glycoprotein hormones. Hybridization of subunits from thyrotropin, lutropin and human choriogonadotropin.

In vitro assembly of thyrotropin alpha and beta subunits led to an increase in content of alpha helix and beta sheet very similar to that found for gonadotropins. This association-dependent active folding involved the burying of three tyrosine residues tentatively assigned to Tyr alpha 41, Tyr beta 37 and Tyr beta 59 and common to all studied glycoprotein hormones. In vitro hybridizations between alpha and beta subunits of various hormones (thyrotropin, lutropin and choriogonadotropin) from different species (ovine, bovine and human) triggered the same molecular events as assembly of homologous subunits: the burying of three tyrosine residues and the increase of periodic structure of the folding. These changes are slow, time-dependent processes. Rates and yields of hybrid formation measured by sedimentation analysis and difference spectroscopy of tyrosines are identical, within experimental error, with the rates and yields measured by the recovery of the biological activity either the stimulation of chick thyroids for thyrotropin-beta hybrids or binding to porcine testis receptors for gonadotropin-beta hybrids. Whatever the origin of the alpha subunit, the thyrotropin-beta hybrids were not able to bind to testis receptors although active on chick thyroids. Rates and yields of hybrid formation essentially depended on the origin of the beta subunit. All the hybrids could be dissociated at acid pH with rates similar to those of native hormone. The extension to thyrotropin and various hybrids of the structural features of the in vitro assembly already recognized for gonadotropins strengthens the hypothesis that one deals with a basic activation process which also occurs in vivo after the synthesis of the subunits.

Crystallization and preliminary X-ray diffraction studies of beta-cryptogein, a toxic elicitin secreted by the phytopathogenic fungus Phytophthora cryptogea.

Crystals of the basic elicitin secreted by Phytophthora cryptogea have been obtained by the hanging-drop method of vapor diffusion from sodium chloride solutions. The crystals belong to the tetragonal space group P4(1)22 (or enantiomorph P4(3)22), with unit cell dimensions a = b = 47 A, c = 137 A and probably contain two molecules per asymmetric unit. The crystals are very stable to X-rays and diffract to 2.2 A resolution on a synchrotron radiation source.

Three-dimensional solution structure of beta cryptogein, a beta elicitin secreted by a phytopathogenic fungus Phytophthora cryptogea.

Cryptogein belongs to a new family of 10-kDa proteins called elicitins. Elicitins are necrotic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive-like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The solution structure of beta cryptogein from Phytophthora cryptogea fungus was determined by using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. A set of 18 structures was calculated using 1360 NOE-derived distance restraints and 40 dihedral angle restraints obtained from 3JHNH alpha couplings. The RMS deviation from the mean structure is 0.87 +/- 0.14 A for backbone atoms and 1.34 +/- 0.14 A for all the non-hydrogen atoms of residues 2 to 98. The structure of beta cryptogein reveals a novel protein fold, with five helices and a double-stranded beta-sheet facing an omega-loop. One edge of the beta-sheet and the adjacent face of the omega-loop form a hydrophobic cavity. This cavity made of highly conserved residues represents a plausible binding site. Residue 13, which has been identified from directed mutagenesis and natural sequence comparison studies as a key amino acid involved in the differential control of necrosis, is surface exposed and could contribute to the binding to a ligand or a receptor. The solution structure is close to the X-ray structure, with slight differences lightly due to the crystal packing.

The 2.1 A structure of an elicitin-ergosterol complex: a recent addition to the Sterol Carrier Protein family.

Elicitins, produced by most of the phytopathogenic fungi of the genus Phytophthora, provoke in tobacco both remote leaf necrosis and the induction of a resistance against subsequent attack by various microorganisms. Despite the recent description of the three-dimensional crystal structure of cryptogein (CRY), the molecular basis of the interactions between Phytophthora and plants largely remains unknown. The X-ray crystal structure, refined at 2.1 A, of a ligand complexed, mutated CRY, K13H, is reported. Analysis of this structure reveals that CRY is able to encapsulate a ligand that induces only a minor conformational change in the protein structure. The ligand has been identified as an ergosterol by gas chromatographic analysis coupled with mass spectrometry analysis. This result is consistent with biochemical data that have shown that elicitins are a distinct class of Sterol Carrier Proteins (SCP). Data presented here provide the first structural description of the pertinent features of the elicitin sterol interaction and permit a reassessment of the importance of both the key residue 13 and the mobility of the omega loop for the accessibility of the sterol to the cavity. The biological implications thereof are discussed. This paper reports the first structure of a SCP/sterol complex.

Amino acid sequence and toxicity of the alpha elicitin secreted with ubiquitin by Phytophthora infestans.

A single alpha elicitin, phytotoxin secreted by Phytophthora infestans, responsible for systemic HR-like necroses in tobacco, was purified from the culture filtrate. Its sequence was compared to other alpha elicitins in correlation with toxicity. In the filtrate, we also found ubiquitin, whose biological function remains unclear.

Mapping the elicitor and necrotic sites of Phytophthora elicitins with synthetic peptides and reporter genes controlled by tobacco defense gene promoters.

Elicitins are 10-kDa proteins secreted by Phytophthora and Pythium fungi that elicit a hypersensitive-like necrotic reaction, leading to resistance against fungal and bacterial plant pathogens. Induction of necrosis and resistance were previously shown to be borne by different sites of the molecule. Furthermore, sequence comparison indicated several potential residues necessary for necrosis. The role of one of these residues was previously evidenced with site-directed mutagenesis. In order to locate other necrosis-determining sites and reveal the defense-eliciting sites, we synthesized a series of synthetic peptides. Tests were performed on two types of transgenic tobacco plants, both transformed with a construction containing the beta-glucuronidase reporter gene, in one case controlled by the promoter of the multiple stimulus response gene str 246C and in the other by the promoter of the pathogenesis-related gene PR1a. We report that only certain peptides were found to be active. Whereas PR1a induction was consistently correlated with induction of necrosis, four peptides were observed to induce only str 246C expression without necrosis, which led to differentiate the defense-eliciting sites from the necrotic sites. From the structure-function relationship thus obtained, two different defense pathways were inferred to be independently induced by elicitins.

The relationships between the toxicity and the primary and secondary structures of elicitinlike protein elicitors secreted by the phytopathogenic fungus Pythium vexans.

Elicitins are toxic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive-like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. Such proteins were observed in the culture filtrate of another species of the Oomycete genus, Pythium vexans. Two alpha elicitinlike proteins were purified and sequenced. One of these novel elicitins (Vex2) exhibited a 100-residue sequence instead of 98 while the other (Vex1) had an N-glycosylation site, effectively glycosylated (equivalent of 16 hexose residues). In addition to the point mutations already observed in Phytophthora species, we found several novel amino acid changes. Furthermore, circular dichroism revealed some differences in their structure in solution compared with the Phytophthora elicitins that were correlated with specific point mutations. These sequences permitted the establishment of a phylogenic tree, suggesting that Pythium vexans is a species close to the Phytophthora genus. The toxicity of the Pythium vexans elictins to tobacco leaves was investigated and correlated with the occurrence of the carbohydrate moiety of one of the two isoforms, observed for the first time in an elicitin.

Amino acid sequence of cinnamomin, a new member of the elicitin family, and its comparison to cryptogein and capsicein.

The phytopathogenic fungi Phytophthora cinnamomi cause systemic leaf necrosis on its non-host tobacco; in culture, it secretes a protein, called cinnamomin, which elicits leaf necrosis and protects tobacco against the pathogen Phytophthora nicotianoe, in a way similar to cryptogein and different from capsicein, elicitins of known amino acid sequences. The cinnamomin sequence has been determined and compared to other elicitins. The differences in the 3 elicitin sequences were correlated to the biological activities: 2 lysines were ascribed as the key amino acids involved in the differential control of protection with respect to necrosis.

Characterization of elicitin-like phospholipases isolated from Phytophthora capsici culture filtrate.

The phytopathogenic oomycete Phytophthora capsici secretes in culture a phospholipase activity. Two enzyme isoforms exhibiting a high phospholipase B activity were isolated by chromatography and electrophoresis. They differ in their apparent molar masses (22 and 32 kDa). Both proteins are glycosylated and share the same N-terminal amino acid sequence up to the 39th residue with a high homology with capsicein, the P. capsici elicitin. Although devoid of phospholipase activity, capsicein was shown by circular dichroism to specifically interact with negatively charged phospholipids, suggesting that the membrane lipids could be a potential target for elicitins.

Amino acid sequence of locust neuroparsins.

Neuroparsins A and B were isolated from the nervous part of the corpus cardiaca of Locusta migratoria via a two-step purification procedure. Both consist of two polypeptide chains linked by disulfide bridges. The N-terminal sequence of both native neuroparsins was determined: the N-terminal end of neuroparsin B was unique while that of neuroparsin A showed three different sequences. These sequences were that of neuroparsin B and two others having five and two extra N-terminal residues. Neuroparsin B was found as a homodimer and the complete sequence of the monomer, determined from peptide fragments generated by treatment with cyanogen bromide and endoprotease Glu-C, comprises 78 residues.

Biochemical characterization, molecular cloning and localization of a putative odorant-binding protein in the honey bee Apis mellifera L. (Hymenoptera : Apidea).

A honey bee antennal water-soluble protein, APS2, was purified and characterized as the first Hymenoptera putative odorant-binding protein. Comparison of its measured Mr (13695.2+/-1.6) to that of the corresponding cDNA clone shows it does not undergo any post-translational modification other than a 19-residue signal peptide cleavage and formation of three disulfide bridges. These biochemical features are close to those of Lepidoptera odorant-binding proteins. In situ hybridization experiments demonstrated its specific expression in olfactory areas. Based on its higher expression in the worker than in the drone, ASP2 might be more involved in general odorant than in sex pheromone detection.

PCR cloning and expression analysis of a cDNA encoding a pectinacetylesterase from Vigna radiata L.

A cDNA clone encoding a pectinacetylesterase (PAE) was isolated from 3-day-old mung bean seedlings using PCR-based techniques. Degenerate oligonucleotide primers were designed according to the N-terminus and internal peptides from the purified PAE. The full-length clone of 1453 bp codes for a signal peptide of 24 amino acids and a mature protein of 375 amino acids. The Mr and the pI of the cDNA-deduced amino acid sequence agree with the values estimated for the purified enzyme. No significant sequence identity between the PAE and any known protein could be found in the databases. Northern analysis revealed developmentally regulated expression of the mRNA in mung been seedlings.

High homology between a trophoblastic protein (trophoblastin) isolated from ovine embryo and alpha-interferons.

Ovine trophoblastic protein B (oTPB), an embryonic protein, is a 20 kDa secretory protein which is synthesized by the ovine conceptus from days 12 to 22 of pregnancy. oTPB was purified by HPLC using ion-exchange chromatography on a DEAE column and was subsequently chromatographed on a reversed-phase column. Automated Edman degradation was then used to determine the N-terminal amino acid sequence up to 45 residues. The sequence data reveal a significant homology between oTPB and bovine interferons alpha of class II: 64% of the amino acids are identical and 75% are homologous. A highly conserved region including residues 23-44 exhibits 82% homology. Identity between oTPB and either HuIFN-alpha.9 or MuIFN alpha.1 is 55%. These alignments between oTPB and IFNs occur at the N-terminus of the mature proteins and proceed without deletion. These results suggest that oTPB is an embryonic interferon.

Odorant and pheromone binding by aphrodisin, a hamster aphrodisiac protein.

Aphrodisin is a soluble glycoprotein of hamster vaginal discharges, which stimulates male copulatory behavior. Natural aphrodisin was purified and its post-translational modifications characterized by MALDI-MS peptide mapping. To evaluate its ability to bind small volatile ligands, the aphrodisiac protein was expressed in the yeast Pichia pastoris as two major isoforms differing in their glycosylation degree, but close in conformation to the natural protein. Dimeric recombinant aphrodisins were equally able to efficiently bind odors (2-isobutyl-3-methoxypyrazine and methyl thiobutyrate) and a pheromone (dimethyl disulfide), suggesting that they could act as pheromone carriers instead of, or in addition to, direct vomeronasal neuron receptor activators.

N-terminal sequences of oat avenins compared to other cereal prolamins.

Like the alcohol-soluble seed storage proteins (also called prolamins) of other cereals, avenins, the oat prolamins, are a series of polymorphic molecules belonging to a multigenic family stored within the protein bodies of the starchy endosperm. Nevertheless, they exhibit some pecularities: among the seed storage proteins, their proportion is low compared to prolamins from other cereal species; their net charge is higher; the amount of Gln + Pro only reaches 49 mol%; they are less polymorphic. We have isolated and purified several avenins and sequenced their N-terminal end. The microheterogeneity and the pecularity of avenins are revealed by the comparison of the N-terminal sequences. Like other prolamins, they exhibit tandem repeats; these repetitive peptides are slightly different from those of other prolamins of the Festucoideae, and the repetition begins earlier in the sequence. As for prolamins from other species, their predicted secondary structure reveals successive beta-turns which might be arranged in a pseudo-helix structure.

Biochemical aspect of photoassimilated carbon partitioning at late kernel fill in maize under climatic stress.

The aim of this work was to describe the incorporation of 14CO2 into maize at the late kernel fill under chilling and the subsequent movement of the photoassimilated 14C out the fed ear leaf. Cool temperatures were observed to decrease the photosynthetic rate and to alter the operation of the carbon assimilation pathway with 14C accumulation in alpha-alanine. They were shown also to affect the rate of photoassimilated carbon out of the fed area, and especially by delaying the seed import processes.

High-level expression of recombinant porcine LH receptor in baculovirus-infected insect cells or caterpillars.

Porcine LH receptor ectodomain was overexpressed in insect cells and lepidopteran larvae using the recombinant baculovirus expression system. A low multiplicity of infection yielded the largest active production, of approximately 10(7) receptors/cell or 3 micrograms active receptor/mg total protein in infected cells. The truncated ectodomain solubilized with Triton X-100 bound its ligand with a high affinity which was comparable with that of the native membrane receptor. Increasing the multiplicity of infection resulted in an optimum protein production of 0.6 mg receptor/mg total protein in infected cells. This receptor was largely inactive, probably trapped within aggregation pools. Active receptor could be recovered by dilution of the samples. No secretion of recombinant receptor was ever observed whatever the conditions of infection. Expression of the recombinant receptor in insect larvae was also tested. This low-cost system failed both to increase the amount of active receptor and to induce secretion into the haemolymph. Two methods remain for producing sizeable amounts of active receptor with this baculovirus/insect cell system. One relies on immunoaffinity purification of the active protein and requires large-scale production, and the other is based on the purification of overexpressed inactive receptor followed by renaturation.