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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.

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.

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.

PR proteins are soluble and host-coded molecules with antifungal activity induced by a variety of agents. Wheat contains several PR proteins and among them are those of the class 4 coded wheatwin1 and wheatwin2; the two native proteins have been isolated from wheat kernel and the coding cDNA clones have been recently characterized. Herein, we report the expression of recombinant wheatwin1 and wheatwin2 in Escherichia coli-insoluble fractions; a new protocol for the purification in high yields and correct processing of the two proteins was developed. The recombinant proteins have molecular weights identical to that of the native proteins, indicating that the removal of the N-terminal methionine and cyclization of glutamine to pyroglutamate was complete. Both recombinant proteins inhibited in vitro the growth of Fusarium culmorum exhibiting antifungal properties similar to those of the native proteins., (Copyright 2001 Elsevier Science.)

We have purified and characterized two new pathogenesis-related (PR) proteins from wheat belonging to the PR-4 family. We named the proteins wheatwin3 and wheatwin4 in analogy with the previously characterized wheatwin1 and wheatwin2. Their isoelectric points were 7.1 and 8.4, respectively. We determined the complete amino acid sequence of both proteins by a rapid approach based on the knowledge of the primary structures of the homologous wheatwin1 and wheatwin2. Wheatwin3 differs from wheatwin1 in one substitution at position 88, while wheatwin4 differs from wheatwin2 in one substitution at position 78. The secondary structure and solvent accessibility of these residues were determined on the three-dimensional model of wheatwinl. Residue 88 was very accessible and was located in a flexible region. Preliminary results indicate that, like wheatwin1 and wheatwin2, wheatwin3 and wheatwin4 have antifungal activity.

We set up a method to get rapid information on the three-dimensional structure of peptide and proteins of known sequence. Both native and alkylated polypeptide is hydrolyzed with a number of proteases at different digestion times and the resulting mixtures are compared by HPLC analysis to establish the differences in the hydrolysis pathways of the folded and unfolded molecule. Then, the unfractionated digestion mixtures of the native polypeptide are submitted to automatic sequence analysis to identify the hydrolysis sites. The sequence of each fragment present in the mixtures is reconstructed and its amount determined by quantitative data of the sequence analyses. We used this approach to determine the amino acid surface accessibility of wheatwin1, a pathogenesis-related protein from wheat, and constructed a predictive three-dimensional model based on the knowledge of the tertiary structure of barwin, a highly homologous protein from barley. The procedure allowed us to quickly identify and quantify the hydrolysis at the susceptible bonds which could be classified as exposed, partially hidden, or inaccessible. The results were useful to evidentiate and discuss concordances and differences between experimental and model predicted accessibilities of amino acid residues. Proteins 1999;36:192-204., (Copyright 1999 Wiley-Liss, Inc.)

Two cDNA clones encoding the previously characterised PR4 proteins wheatwin 1 and wheatwin2 from wheat (Triticum aestivum cv. S. Pastore) have been identified and named wPR4a and wPR4b, respectively. The clones have been isolated by screening a cDNA library with a specific cDNA probe obtained by RT-PCR. The wPR4a and wPR4b cDNAs contain open reading frames of 441 and 447 bp that encode for wheatwin1 and wheatwin2, respectively.

We designed a computer program for the assignment of protein disulphides using mass spectrometric data. All the theoretical linear peptides containing from one to three cysteines are generated on the basis of the protein sequence. Their combination ways are determined in order to create all the possible disulphide-bridged fragments containing from two to six cysteines and to calculate their molecular weight. One, two and three S-S bridges per linked fragment were considered, taking into account the possibility that the fragments contain unabridged residues. The mass data obtained from the spectral analysis of peptide mixtures of the digested protein are then associated to the fitting structures of disulphide-bridged peptides, giving rise to the primary output. This output can then be screened by using information on the specificity of the proteolytic agent(s) used and/or any further mass data provided by Edman degradation and/or carboxypeptidase treatment of the peptide mixtures. The need for such a computer aid is discussed and examples of application are given.

We have purified and characterized a protein from the water-soluble fraction of wheat kernel (Triticum aestivum cv. S. Pastore) consisting of a single polypeptide chain blocked at its N-terminus by a pyroglutamate residue; the complete amino acid sequence has been determined by automated sequence analysis performed on peptide fragments obtained by enzymatic hydrolyses of the protein. Homology studies have shown that this protein is very similar (97% sequence identity) to the previously characterized wheatwin1 as well as to other members of the pathogenesis-related (PR) proteins of class 4; in analogy with wheatwin1, we have termed this protein wheatwin2. Both wheatwin1 and wheatwin2 have specific antifugal activity toward the wide-host-range pathogen Botrytis cinerea and the wheat-specific pathogenic fungi of wheat Fusarium culmorum and Fusarium graminearum of groups 1 and 2. On the basis of their structural and functional properties, wheatwin1 and wheatwin2 can be classified as members of the PR4 protein family; this represents the first report concerning the presence of this kind of protein in wheat.

The amino acid sequence of wheatwin1, a monomeric protein of 125 residues isolated from wheat kernel (variety S. Pastore), is reported. Wheatwin1 is highly homologous (95%) to barwin, a protein from barley seed, which was shown to be related to the C-terminal domain of two proteins encoded by the wound-induced genes win1 and win2 in potato and to a protein encoded by the same domain of the hevein gene (hev1) in rubber tree. Similarly to barwin, wheatwin1 contains six cysteine residues all linked in disulfide bridges and the N-terminal residue is pyroglutamate. Moreover, structural studies performed on wheatwin1 and win1 protein by predictive methods demonstrated that these proteins and barwin are closely related in the secondary structure also. The high level of homology found with the product of win1, win2, and hev1 genes strongly suggests that barwin and wheatwin1 play a common role in the mechanism of plant defence.

The assignment of the five disulfide bridges in an alpha-amylase monomeric inhibitor from wheat kernel (coded 0.28) was achieved by combining fast-atom-bombardment mass spectrometry (FAB-MS) and automatic sequencing based on Edman degradation. Direct FAB-MS analysis of the native and reduced enzymatic digests of the protein allowed the assignment of three disulfide bridges out of five, including those involving two adjacent cysteine residues. The remaining two disulfide bridges were assigned by sequencing automatically the peptide clusters purified from the tryptic digest of the native protein.

The primary structure of an alpha-amylase inhibitor (coded 0.39) from wheat kernel was determined by fast atom bombardment mass spectrometry and Edman degradation. The sequence is similar to an extent of 97% compared to the other major component of the monomeric isoinhibitor family coded 0.28. The differences consist of the substitution of a single residue and the deletion of two residues in inhibitor 0.39. Neither heterogeneity nor polymorphism were observed in the primary structures of the inhibitors.

The amylase-protein amylase inhibitor system offers a unique model of specific and reversilbe protein-protein interaction. The monomeric and dimeric inhibitors, exhibiting closely related properties and interacting with the same amylase, also provide a convenient test to compare effects of monomer-monomer and monomer-dimer interactions between enzyme and inhibitor proteins.

An affinity column was devised for the purification of a large number of amylases inhibited by the albumin from wheat kernel. The procedure involved linking the protein inhibitors from wheat to Sepharose and then specifically eluting the amylase adsorbed to the gel with a high concentration of maltose. By this procedure, the amylases from Tenebrio molitor L. (yellow mealworm) larvae and chicken pancreas were purified to homogeneity with good yields for the first time, as shown by both alkaline and acidic electrophoresis. Human saliva alpha-amylase, purified by the same procedure, showed specific activity and electrophoretic patterns similar to those obtained by other workers with different techniques.