Modular structure of HEL protein from Arabidopsis reveals new potential functions for PR-4 proteins.

Plants possess an innate immune system enabling them to defend themselves against pathogen attack.The accumulation of newly synthesized pathogenesis related proteins (PRs) is one of the most studied inducible plant defence response. In this paper, we report on the characterization of a class I PR4 vacuolar protein from Arabidopsis, named At HEL. The protein has a modular structure consisting of an N-terminal hevein-like domain(CB-HEL) and a C-terminal domain (CD-HEL) that are posttranslationally processed. Both domains show a strong antifungal activity, but they do not have chitinolitic properties.CD-HEL was found to be endowed with RNase, but not DNase activity. Molecular modeling carried out on both domains revealed that CB-HEL possesses a chitin binding site strictly conserved between hevein-type peptides and that the cavity involved in substrate interaction of CD-HEL do not show any residue substitution with respect to the orthologous wheatwin1 from wheat. Using a fishing for partners approach, CB-HEL was found to interact with a fungal fruiting body lectin. According to literature, we can hypothesize that CB-HEL could cross the pathogen hyphal membrane and that its interaction with a fungal lectin could knock out one of the weapons that the fungus uses.

Modular structure of HEL protein from Arabidopsis reveals new potential functions for PR-4 proteins.

Plants possess an innate immune system enabling them to defend themselves against pathogen attack. The accumulation of newly synthesized pathogenesis-related proteins (PRs) is one of the most studied inducible plant defence response. In this paper, we report on the characterization of a class I PR4 vacuolar protein from Arabidopsis, named AtHEL. The protein has a modular structure consisting of an N-terminal hevein-like domain (CB-HEL) and a C-terminal domain (CD-HEL) that are posttranslationally processed. Both domains show a strong antifungal activity, but they do not have chitinolitic properties. CD-HEL was found to be endowed with RNase, but not DNase activity. Molecular modeling carried out on both domains revealed that CB-HEL possesses a chitin binding site strictly conserved between hevein-type peptides and that the cavity involved in substrate interaction of CD-HEL do not show any residue substitution with respect to the orthologous wheatwin1 from wheat. Using a fishing for partners approach, CB-HEL was found to interact with a fungal fruiting body lectin. According to literature, we can hypothesize that CB-HEL could cross the pathogen hyphal membrane and that its interaction with a fungal lectin could knock out one of the weapons that the fungus uses.

Cloning and structural analysis of a haemocyanin from the Stonefly Perla grandis.

We characterized two subunits of a putative haemocyanin from the stonefly species Perla grandis. In particular, we cloned and sequenced the corresponding cDNAs and studied their expression in different insect stages. Moreover, using the deduced amino acid sequences, homology studies were performed both on their primary and tertiary structures. 3-D molecular modelling data showed that the residues involved in the oxygen transport and subunits contacts were located in spatial positions preserving the functionality of the molecule. Despite it was paradigmatically affirmed that insects do not have respiratory proteins, our data suggest that the haemocyanin could be involved in the respiratory mechanisms of P. grandis. As far as we know, this is the first haemocyanin 3-D structure described and analyzed in insects.

CysMap and CysJoin: database and tools for protein disulphides localisation.

We have developed a computer program able to make user-customised databases derived from the public PIR non-redundant reference protein database. When the database of interest has been created, the user will generate the map of all the possible linear peptides containing one and two cysteines for each protein and combine them to calculate the mass of all the possible clusters of linear peptides linked by a disulphide bridge with a cysteine pair. It is also possible to create selected maps corresponding to peptides formed by the action of specific proteases. In this way, mass spectrometric data obtained from the hydrolysis of proteins of unknown sequence can be related to that contained in the database for quick disulphide assignment and protein identification. To confirm signal attribution, the program will also furnish the expected mass of cluster peptides after performing a cycle of Edman degradation. The utility of the program is discussed and examples of application are given.

Wheat pathogenesis-related proteins of class 4 have ribonuclease activity.

We have demonstrated that wheatwin1, a wheat pathogenesis-related protein of class 4 (PR4), has ribonuclease activity. Both native and recombinant proteins hydrolyse RNA from wheat coleoptils and have antifungal activity. Sepharose-bound wheatwin1 is able to interact with either wheat or Fusarium culmorum RNA. 3D modelling studies showed that, like ribonucleases A and T1, the action mechanism should involve two His residues, an Arg residue and an Asp residue.

Deepening TOL and TOU catabolic pathways of Pseudomonas sp. OX1: cloning, sequencing and characterization of the lower pathways.

Pseudomonas sp. OX1 is able to metabolize toluene and o-xylene through the TOU catabolic pathway, whereas its mutant M1 strain was found to be able to use m- and p-xylene as carbon and energy source, using the TOL catabolic pathway. Here we report the complete nucleotide sequence of the phe lower operon of the TOU catabolic pathway, and the sequence of the last four genes of the xyl-like lower operon of the TOL catabolic pathway. DNA sequence analysis shows the gene order within the operons to be pheCDEFGHI (phe operon) and xyl-likeQKIH (xyl-like operon), identical to the order found for the isofunctional genes of meta operons in the toluene/xylene pathway of TOL plasmid pWW0 from Pseudomonas putida mt-2 and the phenol/methylphenol pathway of pVIl50 from Pseudomonas sp. CF600. The nucleotide and the deduced amino acid sequences are homologous to the equivalent gene and enzyme sequences from other Pseudomonas meta pathways. Recombinant 2-hydroxymuconic semialdehyde dehydrogenase (HMSD) and 2-hydroxymuconic semialdehyde hydrolase (HMSH), coded by pheCD genes, respectively, and ADA and HOA enzymes from both phe and xyl operons were expressed in E. coli and steady-state kinetic analysis was carried out. The analysis of the kinetic parameters of HMSD and HMSH showed that the enzymes from Pseudomonas sp. OX1 are more specialized to channel metabolites into the two branches of the lower pathway than homologous enzymes from other pseudomonads. The kinetics parameters of recombinant ADA from phe and xyl-like operon were found to be similar to those of homologous enzymes from other Pseudomonas strains. In addition, the enzyme from xyl-like operon showed a substrate affinity three times higher than the enzyme from phe operon.

Crosstalk between salicylic acid and jasmonate in Arabidopsis investigated by an integrated proteomic and transcriptomic approach.

Resistance conferred by biotrophic pathogens often requires salicylic acid (SA) signaling, whereas necrotrophic pathogens or wounding mainly activate the jasmonate/ethylene (JA/ET)-dependent pathway. Crosstalk connections between these two independent signaling pathways may lead to synergistic or antagonistic behavior. In order to shed some light on the crosstalk between these two hormones in Arabidopsis plants, a proteomic approach combined with a transcriptomic analysis has been used to identify molecules differentially expressed upon single or simultaneous treatment with both phytohormones. Twenty-five nonredundant differential proteins were revealed upon treatment with SA or JA alone or in combination, which are involved in general metabolic processes as well as in response to stress, in developmental processes, in protein metabolism and transport. Interestingly, gene expression study, carried out on genes involved in oxidative stress and in biotic and/or abiotic stress, highlighted the correspondence between proteomic and transcriptomic approaches, performed here by RT-PCR. Our data clearly demonstrate that almost all genes/proteins involved in oxidative stress as well as in biotic and/or abiotic stress are mainly induced upon JA treatment and only a few of them are overexpressed upon SA treatment. Moreover, we found that a substantially negative crosstalk is established upon the combined action of the two hormones and that generally SA exerts a negative crosstalk compared to the JA pathway. Our study corroborates the hypothesis that the combination of both phytohormones induces reprogramming of the plant transcriptome, but at the same time highlights the presence of a complex network of signaling that is far to be completely elucidated.

Molecular characterization of a wheat protein induced by vernalisation.

Using a PCR strategy we isolated from winter wheat (Triticum aestivum L. cv. Bolero) the ver2 gene coding for a modular protein constituted by an N-terminal domain called "dirigent", found in several defence-related genes, and a C-terminal domain related to jacalin-related lectin (JRL). ver2 transcript as well as native Ver2 levels increased during vernalisation and upon methyl jasmonate treatment of young seedlings. ver2 transcript levels were kept constant either in infected tissues or in wounded samples indicating that Ver2 is not directly involved in plant defence mechanisms. The Ver2 protein was expressed in bacteria as a recombinant GST-Ver2 fusion protein. The purified recombinant protein was further characterized using an affinity chromatography approach based on its interaction with mannose-agarose beads. GST-Ver2 tightly bound to the matrix. Molecular modelling of the jacalin domain and mannose docking confirmed that Ver2 possesses D: -mannose binding capacity.

Structural basis of the antifungal activity of wheat PR4 proteins.

PR4 proteins possess antifungal activity against several pathogenic fungi suggesting a pivotal role in defence reactions against plant pathogen attack. We already showed that wheatwin1, a wheat PR protein of class 4, is endowed with ribonuclease activity. In this study we produced three mutants altering the active site and performed comparative analysis with the native protein also in the presence of the ribonuclease inhibitor 5'-ADP. We characterized the RNA binding site and its interaction with 5'-ADP by 3D modelling and docking studies. Moreover, in vitro antifungal assays have been carried out in order to study the relationship between antifungal and ribonuclease activities. Finally, localization of wheatwin1 in Fusarium culmorum spores was evaluated using fluorescence light microscope.

Modeling the 3-D structure of a recombinant laccase from Trametes trogii active at a pH close to neutrality.

A cDNA encoding a novel laccase from the white-rot fungus Trametes trogii was cloned and expressed in Pichia pastoris. The recombinant protein (Lcc2) exhibited kinetic parameters for both phenolic and non phenolic substrates that were different from the previously described Lcc1, the main laccase isoform expressed by T. trogii; in addition, the pH/activity profiles for phenolic substrates of Lcc2 were shifted upward by 1-1.5 pH units towards neutrality as compared to Lcc1. Comparative modeling of the two laccases (69.2% identity) showed that the overall fold of Lcc2 is very similar to Lcc1 and other laccases. The substrate cavity of Lcc2 contains the Asp residue which is thought to mediate the laccase activity at acidic pHs, whereas two hydrophobic residues (Phe, Ile) on the cavity orifice of Lcc2 replace the two polar residues (Thr, Ser) of Lcc1. These structural differences may be responsible for the unique kinetic performances of Lcc2.

Molecular and functional analysis of new members of the wheat PR4 gene family.

Five new genes belonging to the pathogenesis-related (PR) 4 family have been cloned and characterised in Triticum aestivum. Two full-length genes, named wPR4e and wPR4f-b, were isolated by library screening, demonstrating the presence of a small intron only in wPR4f-b. Two other PR4 genes (wPR4f-a and wPR4f-c) were isolated by PCR, showing very high sequence identity with wPR4f-b and constituting a new sub-family. Transcription start analysis was performed by RLM-RACE, leading to the isolation of a fifth gene, named wPR4g, that is highly homologous to wPR4e; both encode putative vacuolar PR4 proteins (Wheatwin7 and Wheatwin5, respectively). wPR4e and wPR4f sub-family genes are induced by F. culmorum infection, by chemicals that lead to systemic acquired resistance and by wounding, showing different spatial and temporal induction pathways. In silico analysis of the 5' untranslated regions of wPR4e and wPR4f-b revealed the presence of several abiotic and biotic stress-responsive elements. wPR4e and wPR4f-b putative promoters were fused to the beta-glucuronidase (GUS) reporter gene, and transient and stable expression assays demonstrated that both are able to drive expression of GUS. Characterisation of these new PR4 genes and particularly of their 5' untranslated regions, as well as the determination of their expression patterns, will contribute to our understanding of the responsiveness of this gene family to various stress conditions and of its role in plant defence.

Comparing the modeled structures of PR-4 proteins from wheat.

We have constructed three-dimensional models of four pathogenesis-related (PR) proteins from wheat (wheatwins) belonging to the PR-4 family. All the models were based on the knowledge of the tertiary structure of barwin, a highly homologous protein from barley. Wheatwin1 and wheatwin2 differ in two amino acid residues (positions 62 and 68) out of 125. Wheatwin4 differs from wheatwin2 in one residue at position 78, while wheatwin3 differs from wheatwin1 in one residue at position 88. The global folding and the secondary structures were very similar through all the sequences, including the regions of the amino acid substitutions. The main differences were found in the traits 15-21, 84-86 and 91-93. Trait 15-21 was predicted as ss-sheet in wheatwin4 and random-coil in the other proteins. Trait 84-86 was predicted as ss-sheet in wheatwin3 and random-coil in the other proteins. Trait 91-93 was predicted as random coil in wheatwin1 and wheatwin3 and ss-sheet in the other two proteins. Traits 15-21 and 84-86 were exposed, while trait 91-93 was quite hidden in all the proteins. The antifungal activities of the four proteins towards the specific pathogenic fungus Fusarium culmorum were distinct and well correlated to the structural differences. These results suggest that these regions may have a role in the action mechanism, which is still unknown.

Structural properties of the protein SV-IV.

We have investigated the molecular mechanisms that produce different structural and functional behavior in the monomeric and trimeric forms of seminal vesicle protein no. 4, a protein with immunomodulatory, anti-inflammatory, and procoagulant activity secreted from the rat seminal vesicle epithelium. The monomeric and trimeric forms were characterized in solution by CD. Details of the self-association process and structural changes that accompany aggregation were investigated by different experimental approaches: trypsin proteolysis, sequence analysis, chemical modification, and computer modeling. The self-association process induces conformational change mainly in the 1-70 region, which appears to be without secondary structure in the monomer but contains alpha-helix in the trimer. In vivo, proteolysis of seminal vesicle protein no. 4 generates active peptides and this is affected by the monomer/trimer state, which is regulated by the concentration of the protein. The information obtained shows how conformational changes between the monomeric and trimeric forms represent a crucial aspect of activity modulation.