Maria Rosaria Fullone

The 30-kilodalton protein present in purified fusicoccin receptor preparations is a 14-3-3-like protein.

We have recently reported on the purification of the fusicoccin (FC) receptor from corn (Zea mays L.) and its identification by photoaffinity labeling (P. Aducci, A. Ballio, V. Fogliano, M.R. Fullone, M. Marra, N. Proietti [1993] Eur J Biochem 214: 339–345). Pure preparations of FC receptors, obtained under nondenaturing conditions, showed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis two doublets of proteins with apparent molecular masses of 30 and 90 kD. In the present paper we describe the isolation and identification of the primary structure of the 30-kD doublet proteins. Sequencing studies of peptides resulting from the digestion of the 30-kD protein showed a full identity with a 14–3–3like protein from corn, named GF14. The 14–3–3 family is a class of proteins that is widely distributed in eukaryotes and is known to play various regulatory roles. The 30-kD protein has been immunologically identified by specific antibodies prepared against a synthetic peptide based on the determined amino acid sequence. A similar protein is recognized in partially purified FC receptor preparations from bean and spinach leaves.

The H+-ATPase purified from maize root plasma membranes retains fusicoccin in vivo activation

The activity of ‘P‐type’ ATPases is modulated through the C‐terminal autoinhibitory domain. The molecular bases of this regulation are unknown. Their understanding demands functional and structural studies on the activated purified enzyme. In this paper the plasma membrane H+‐ATPase from maize roots activated in vivo by fusicoccin was solubilised and fractionated by anion‐exchange HPLC. Results showed that the H+‐ATPase separated from fusicoccin receptors retained fusicoccin activation and that it was more evident after enzyme insertion into liposomes. These data suggest that fusicoccin stimulation does not depend on a direct action of the fusicoccin receptor on the H+‐ATPase, but rather, fusicoccin brings about a permanent modification of the H+‐ATPase which very likely represents a general regulatory mechanism for ‘P‐type’ ATPases.

Entrapment into liposomes of fusicoccin binding sites

Abstract Fusicoccin (FC) binding sites solubilised from microsomal fractions of spinach leaves have been entrapped into soybean lecithin liposomes with an 80% yield. The investigation of the properties of these proteoliposomes has demonstrated that the rates of FC-binding and of exchange between radioactive and cold FC are intermediate between those observed with membrane-bound and with solubilised binding sites. It appears that the entrapped proteins are preferentially outside-oriented since they are inactivated by trypsin treatment; moreover, Triton X-100 permeabilization of the proteoliposomes demonstrated that one fifth of these sites are inside-oriented.

Insight into the structure–function relationship of the nonheme iron halogenases involved in the biosynthesis of 4‐chlorothreonine – Thr3 from Streptomyces sp. OH‐5093 and SyrB2 from Pseudomonas syringae pv. syringae B301DR

Molecular cloning of the biosynthetic gene cluster involved in the production of free 4‐chlorothreonine in Streptomyces sp. OH‐5093 showed the presence of six ORFs: thr1, thr2, thr3, orf1, orf2 and thr4. According to bioinformatic analysis, thr1, thr2, thr3 and thr4 encode a free‐standing adenylation domain, a carrier protein, an Fe(II) nonheme α‐ketoglutarate‐dependent halogenase and a thioesterase, respectively, indicating the role of these genes in the activation and halogenation of threonine and the release of 4‐chlorothreonine in a pathway closely reflecting the formation of this amino acid in the biosynthesis of the lipodepsipeptide syringomycin from Pseudomonas syringae pv. syringae B301DR. Orf1 and orf2 show sequence similarity with alanyl/threonyl‐tRNA synthetases editing domains and drug metabolite transporters, respectively. We show that thr3 can replace the halogenase gene syrB2 in the biosynthesis of syringomycin, by functional complementation of the mutant P. s. pv. syringae strain BR135A1 inactivated in syrB2. We also provide an insight into the structure–function relationship of halogenases Thr3 and SyrB2 using homology modelling and site‐directed mutagenesis.

Phospholipase A2 affects the activity of fusicoccin receptors

Biochemical properties of fusicoccin receptors are strongly influenced by the phospholipid environment. In this report we have studied the effect of different exogenous phospholipases on fusicoccin binding ability of both plasma membrane and solubilised receptors. Among the phospholipases tested only phospholipase A2; showed an inhibitory effect on fusicoccin binding. In particular, the influence of this enzyme on the time course and reversibility of the fusicoccin binding reaction was studied. The inhibitory effect of phospholipase A2 was the consequence of fatty acid release. The usual fatty acids of plasma membrane phospholipids were active in inhibiting the interaction of fusicoccin with its receptors. It is concluded that a phospholipid associated to the fusicoccin receptor might play a significant role in the modulation of binding.

Structure of the lipodepsipeptide syringomycin e in phospholipids and sodium dodecylsulphate micelle studied by circular dichroism, NMR spectroscopy and molecular dynamics

Syringomycin E (SRE) is a member of a family of lipodepsipeptides that characterize the secondary metabolism of the plant-associated bacteria Pseudomonas syringae pv. syringae. It displays phytotoxic, antifungal and haemolytic activities, due to the membrane interaction and ion channel formation. To gain an insight into the conformation of SRE in the membrane environment, we studied the conformation of SRE bound to SDS micelle, a suitable model for the membrane-bound SRE. In fact, highly similar circular dichroism (CD) spectra were obtained for SRE bound to sodium dodecylsulphate (SDS) and to a phospholipid bilayer, indicating the conformational equivalence of SRE in these two media, at difference with the CD spectrum of SRE in water solution. The structure of SDS-bound SRE was determined by NMR spectroscopy combined with molecular dynamics calculations in octane environment. The results of this study highlight the influence of the interaction with lipids in determining the three-dimensional structure of SRE and provide the basis for further investigations on structural determinants of syringomycin E-membrane interaction.

Bioactive lipopeptides of ice-nucleating snow bacterium Pseudomonas syringae strain 31R1

The production of secondary metabolite lipopeptides by ice-nucleating Pseudomonas syringae strain 31R1 was investigated. Pseudomonas syringae strain 31R1 is a rifampicin-resistant derivative of P. syringae no. 31 used for the commercial production of snow. It is shown that P. syringae strain 31R1 produces antifungal lipodepsipeptides, syringomycins E and G, and, in addition, a novel and unique lipopeptide, peptin31. Spectroscopic and spectrometric analyses revealed that peptin31 is a linear undecalipopeptide with sequence identities to N- and C-terminal portions but lacking 11 amino acids of known lipodepsipeptide syringopeptin SPPhv. Peptin31 displayed antifungal activities against Rhodotorula pilimanae, Rhizoctonia solani, and Trichoderma harzianum and also hemolytic and antibacterial activities. Extracts of P. syringae strain 31R1 grown in medium with chloride were fungicidal, but not when grown without chloride. The latter extracts lacked peptin 31 and contained des-chloro forms of syringomycins E and G with low antifungal activities. Thus, the three lipopeptides account for the fungicidal properties of P. syringae 31R1 extracts. The occurrence of these bioactive metabolites should be considered when P. syringae no. 31 and its derivatives are used in products for making artificial snow.

Lipodepsipeptides from Pseudomonas syringae are partially proteolyzed and are not absorbed by humans: an in vitro study.

There are some concerns about the use of Pseudomonas-based products as biocontrol agents because of the hemolytic activity shown by their metabolites. The effects of Pseudomonas lipodepsipeptides (LDPs) on mammals via ingestion and the LDP degradation during the digestion and intestinal permeability have not been evaluated. In this research, the susceptibility of different LDPs to degradation was assayed with enzymatic gastrointestinal digestion, and intestinal permeability to LDPs was investigated in an in vitro system based on an intestinal cell layer system. Results demonstrated that trypsin and chymotrypsin hydrolyze up to 50% of the various LDPs, and that proteolysis was further increased by pronase E treatment. A decrease in LDP hemolytic activity matched LDP degradation during the various steps of the digestion process. Moreover, it was shown that syringomycin E (SRE), the main known LDP, was not able to cross the intestinal cell layer, suggesting that SRE does not reach the bloodstream in vivo. It was concluded that the Pseudomonas-based biocontrol products do not represent a serious risk for consumer health. In fact, LDPs possibly present on biocontrol-treated agricultural commodities would likely be partially digested by gastrointestinal enzymes and would not be absorbed at the intestinal level.

Membrane Receptors for Fusicoccin: A Molecular Study

The phytotoxic metabolite of the fungus Fusicoccum amygdali Del. (Ballio, Chain, De Leo, Erlanger, Mauri, Tonolo, 1964) interacts with higher plant cells promoting cell extension growth and membrane transport processes (Marre, 1979). The interest of plant physiologists in this toxin has been increased by the finding that these effects are similar to those regulated by some plant hormones. It is now well established that all the hormone-like responses stimulated by fusicoccin (FC) depend on the activation of a protontranslocating plasmalemma ATPase, which is present in all higher plants and is necessary for nutrient transport. The stimulation of this enzyme represents the more direct FC action, but so far its mechanism is unclear. Many studies directed to elucidate the nature of the signal triggering the H+-ATPase showed that the specificity of FC interaction with plant cells depends on the presence of FC binding sites at the plasmalemma. Results obtained so far show that they can be considered true receptors (Aducci, Marra, Ballio, 1990). Their presence in higher plants which are not hosts of the fungus F. amygdali and therefore do not meet the toxin in natural conditions is explained by the occurrence, in several plants, of a physiological ligand mimicked by FC (Aducci, Crosetti, Federico, Ballio, 1980; Ballio and Aducci, 1987; Marra, Ballio, Aducci, 1988). It is very likely that the interaction of this ligand with FC receptors is the first step in a chain of reactions leading to the biological responses known to be promoted by FC. Therefore, the system recognised by FC can, under physiological conditions, play a crucial role in cell metabolism.

Fusicoccin and its receptors

Purified preparations of FC receptors from maize, obtained under non-denaturing conditions, showed in SDS-PAGE two doublets of proteins with an apparent molecular mass of 30 and 90 kDa. In this paper the isolation of the 30 kDa protein, its identification as a 14-3-3-like protein, as well as its immunological detection in partially-purified FC-receptor preparations from bean and spinach are described. The 14-3-3 proteins have biochemical properties consistent with potential signalling roles, and their presence in highly purified FC-receptor preparations suggests that they may be involved in FC- signal transduction. Photoaffinity labelling experiments demonstrating that the protein at 90 kDa binds FC are also presented. The evidence presented taken as a whole, suggests the occurrence in maize of a protein complex for FC perception and signal transduction.