Fusicoccin effect on the in vitro interaction between plant 14-3-3 proteins and plasma membrane H+-ATPase

A 17-amino acid peptide was selectively cleaved from the highly variant C terminus of the 33-kDa 14-3-3 isoform occurring in fusicoccin receptor preparations from maize and was sequenced, The determined C-terminal sequence was identical to that of the already known maize 14-3-3 homolog GF14-6, thus prompting the use of recombinant GF14-6 in an in vitro protein-protein inter action study. The cDNA of GF14-6 was expressed in Escherichia coil as a P-32-phosphorylatable glutathione S-transferase fusion protein and was used as a probe in overlay experiments with HC-ATPase partially purified from maize roots. The results demonstrated that the recombinant protein specifically bound to H+-ATPase. The binding was dependent on Mg2+ and was strongly increased by fusicoccin. Controlled trypsin digestion of H+-ATPase abolished the association with GF14-6, a finding that was suggestive of an interaction with the C terminus of the enzyme, To confirm this result, the C-terminal domain of H+-ATPase was expressed as a glutathione S-transferase fusion peptide and was used in overlay experiments, GF14-6 was also able to bind to the isolated C terminus, but only in the presence of fusicoccin

Purification and photoaffinity labeling of fusicoccin receptors from maize

Crude soluble proteins from plasma membranes of maize shoots were purified (following the increase of fusicoccin-binding specificity) by using an original multi-step HPLC procedure. The method, based on a combination of adsorption, ion-exchange and gel-filtration chromatographies, is quick, efficient and does not damage the binding activity. It allows a 5000-fold increase of specific activity; SDS/PAGE of purified fractions shows two doublets that correspond to proteins with apparent molecular masses of 90 kDa and 30 kDa. Crude or partially purified material was irradiated for various periods in the presence of a tritiated azido analogue of fusicoccin. The electrophoretic analysis of the irradiated material shows that with a short irradiation time only the 90-kDa band is radiolabeled, whereas, as the irradiation time increases, a 30-kDa band becomes radiolabeled and less radioactivity is detected in the 90-kDa band. Irradiation of the crude material in the absence of the analogue results in a decrease of the binding capability of fusicoccin. The irradiated preparation also shows a decrease of photolabeling of the 90-kDa band. Our data suggest that the 90-kDa protein is the functional fusicoccin receptor. This conclusion is at variance with results of other authors who suggest the 30-kDa protein as the true receptor

Fusicoccin and its receptors - Perception and signal transduction

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

Purification and photoaffinity labeling of fusicoccin receptors from maize

Crude soluble proteins from plasma membranes of maize shoots were purified (following the increase of fusicoccin-binding specificity) by using an original multi-step HPLC procedure. The method, based on a combination of adsorption, ion-exchange and gel-filtration chromatographies, is quick, efficient and does not damage the binding activity. It allows a 5000-fold increase of specific activity; SDS/PAGE of purified fractions shows two doublets that correspond to proteins with apparent molecular masses of 90 kDa and 30 kDa. Crude or partially purified material was irradiated for various periods in the presence of a tritiated azido analogue of fusicoccin. The electrophoretic analysis of the irradiated material shows that with a short irradiation time only the 90-kDa band is radiolabeled, whereas, as the irradiation time increases, a 30-kDa band becomes radiolabeled and less radioactivity is detected in the 90-kDa band. Irradiation of the crude material in the absence of the analogue results in a decrease of the binding capability of fusicoccin. The irradiated preparation also shows a decrease of photolabeling of the 90-kDa band. Our data suggest that the 90-kDa protein is the functional fusicoccin receptor. This conclusion is at variance with results of other authors who suggest the 30-kDa protein as the true receptor

Fusicoccin and its receptors - Perception and signal transduction

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

Biochemical, sensorial and genomic profiling of traditional Italian tomato varieties

Fruit flavour of traditional tomato ecotypes differs from that of modern varieties, the latter being often blamed for lack of flavour. To respond to consumer complaints breeders should know the range of genetic variability available in tomato resources, including local ecotypes, for improving flavour. For this purpose, multitrait characterization, including biochemical, sensorial and molecular analyses, was performed on 12 Italian tomato ecotypes that belong to different types (San Marzano, Sorrento, and Vesuvio). The ecotypes showed a significant variation both in glucose and citric acid content. As for amino acid determination, lysine, aspartic acid and serine showed significant variation among the three types analyzed. Sensory analysis clearly evidenced differences among genotypes: the San Marzano ecotypes revealed sensory profiles that differ from the control processing variety in perception of sweetness and sourness, whereas the Sorrento and Vesuvio ecotype profiles were similar to the fresh market control. In particular, Vesuvio genotypes exhibited a good intensity of tomato aroma and sweetness and an intermediate level of acidity. Finally, molecular characterization performed through AFLP markers provided evidence for a very high level of polymorphism: an ecotype-specific AFLP pattern was identified for at least 11 genotypes, thus defining their molecular fingerprints

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 CD spectra were obtained for SRE bound to 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 snowbacterium 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