Recommendations for naming plant pathogenesis-related proteins

Pathogenesis-related proteins (abbreviated PRs) are defined as plant proteins that are induced in pathological or related situations. We propose a unifying nomenclature for PRs based on their grouping into families sharing amino acid sequences, serological relationship, and/or enzymatic or biological activity. The nomenclature classifies novel proteins identified by electrophoresis or chromatography along with those established by other workers. The previously proposed system of the five well-established families from tobacco is extended to accommodate a further six families. Families are indicated by arabic numerals and individual members are named by lower case letters in the order in which they are described. Additional rules are proposed to deal with forms containing more than a single polypeptide and as yet unclassified PRs. PR genes whose sequences are conserved but whose designations are not based on function are to be designated Ypr in accordance with the recommendations of the Commission on Plant Gene Nomenclature.     

Identification,purification, and characterization of pathogenesis-related proteins from virus-infected Samsun NN tobacco leaves

Ten pH-3 soluble, low-molecular-weight pathogenesis-related proteins (PRs) were found to accumulate in leaves of tobacco cv. Samsun NN reacting hypersensitively to tobacco mosaic virus. Besides the previously characterized PRs 1a, 1b, 1c and 2, these proteins were provisionally designated N, O, P, Q, R, and S in order of decreasing electrophoretic mobility in native polyacrylamide gels. Two-dimensional gel electrophoresis indicated that the PRs consist of single polypeptides, except for R, which is composed of two components with slightly different molecular weights. Estimated molecular weights in SDS-containing gels were: PRs 1a and 1b 17 kD, 1c 16.5 kD, 2 31 kD, N 33 kD, O 35 kD, P 27 kD, Q 28 kD, R 13 and 15 kD, and S 25 kD. However, based on their elution from gel filtration columns and relative moblities in native gels of different acrylamide concentrations, P and Q appeared to have molecular weights similar to those of the PR 1 group. Upon chromatofocusing no additional components were resolved. The PRs were eluted between pH 7 and 4; except for R, their pIs, as judged from isoelectric focusing, appeared to lie in the range from pH 4 to 5.2. In the presence of 6 M urea PR 1a was split into two components, one of which was strongly retarded on gels, as were P and Q. None of the PRs was detected when gels were stained for glycoproteins.By combinations of gel filtration, DEAE-cellulose chromatography, and chromatofocusing, PRs 1a, 1b, 1c, 2 and N were purified, their amino acid compositions determined, and antisera raised against each of these components. By Western blotting, antisera against either PR 1a, 1b, or 1c reacted with each of the components of the PR 1 group, as well as with PR S. Similarly, the antisera against either PR 2 or N reacted with both 2 and N, as well as with O and R. On the basis of major similarities in molecular weight characteristics, amino acid compositions, and serological relationships, it is proposed to classify tobacco PRs into five groups: 1: PRs 1a, 1b, and 1c; 2: 2a (formerly 2), 2b (N), and 2c (O); 3: 3a (P), and 3b (Q); 4: 4a and 4b (the two components of R); and 5: PR 5 (S).     

Crystal structures of fusion proteins with large-affinity tags

The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest.     

Bet v 1 proteins, the major birch pollen allergens and members of a family of conserved pathogenesis-related proteins, show ribonuclease activity in vitro

The Bet v. 1 gene family of birch encodes the major pollen allergens as well as pathogenesis-related (PR) proteins that are induced by microbes in somatic tissues. These PR proteins belong to a group of conserved intracellular defense-related proteins that have been termed 'ribonuclease-like' PR proteins, on the basis of the partial sequence homology observed between PR1, a Bet v 1-homologue from parsley, and a recently characterized ginseng ribonuclease. However, this enzymatic activity has not yet been demonstrated, not for any of the members of this family of PR proteins, nor for the related pollen allergens. We have investigated the possible nuclease activity of Bet v 1 using apparently homogeneous preparations of natural Bet v 1 purified from birch pollen, and a recombinant non-fusion protein purified from E. coli extracts. We report here that Bet v 1 proteins indeed possess an intrinsic ribonucleolytic activity as they can digest different RNA substrates in vitro, but show no activity on single or double-stranded DNA.     

Structural and antifungal properties of a pathogenesis-related protein from wheat kernel

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 antifungal activity toward the wide-host-range pathogenBotrytis cinerea and the wheat-specific pathogenic fungi of wheatFusarium culmorum andFusarium 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.     

Two ABA-responsive proteins from pea (Pisum sativum L.) are closely related to intracellular pathogenesis-related proteins

We report here the isolation of cDNAs encoding two abscisic acid-responsive pea (Pisum sativum L.) proteins, ABR17 and ABR18, which are synthesized during late seed development in vivo. Southern blot analyses suggest that ABR17 cDNA corresponds to a single-copy gene, but ABR18 is one member of a family of closely related sequences in the pea genome. The deduced amino acid sequences of ABR17 and ABR18 cDNAs showed similarity to those of the pea disease resistance response proteins, to pathogenesis-related and to stress-induced proteins in other species and to the major birch pollen allergen Betvl.     

Sequential and structural homology between intracellular pathogenesis-related proteins and a group of latex proteins

The intracellular pathogenesis-related proteins have been identified in a broad range of flowering plants. Some display quite different patterns of expression, in many cases unrelated to the pathogenic response. Nevertheless, these proteins are all very similar and in most cases share more than 35% sequence identity. In this report we investigate the significance of a rather weak similarity between the intracellular pathogenesis-related (IPR or PR-10) proteins and a group of proteins identified in the latex of opium poppy and in Arabidopsis, among others. A sequence analysis held together with the recently published three-dimensional structure of Bet v 1, an IPR protein from birch pollen, strongly suggests sequential and structural homology between the two protein families.     

Accumulation of pathogenesis-related type-5 like proteins in phytoplasma-infected garland chrysanthemum Chrysanthemum coronarium

Soluble proteins extracted from leaves, apical shoots, axillary shoots, and stems of garland chrysanthemum plants infected by onion yellows phytoplasma were analyzed by two-dimensional gel electrophoresis. Computerized matching analysis revealed that at least six soluble proteins were accumulated specifically in phytoplasma-infected garland chrysanthemum. N-terminal amino acids sequences of these soluble     

Immunity functions of Arabidopsis pathogenesis-related 1 are coupled but not confined to its C-terminus processing and trafficking

The pathogenesis-related 1 (PR1) proteins are members of the cross-kingdom conserved CAP superfamily (from Cysteine-rich secretory protein, Antigen 5, and PR1 proteins). PR1 mRNA expression is frequently used for biotic stress monitoring in plants; however, the molecular mechanisms of its cellular processing, localization, and function are still unknown. To analyse the localization and immunity features of Arabidopsis thaliana PR1, we employed transient expression in Nicotiana benthamiana of the tagged full-length PR1 construct, and also disrupted variants with C-terminal truncations or mutations. We found that en route from the endoplasmic reticulum, the PR1 protein transits via the multivesicular body and undergoes partial proteolytic processing, dependent on an intact C-terminal motif. Importantly, only nonmutated or processing-mimicking variants of PR1 are secreted to the apoplast. The C-terminal proteolytic cleavage releases a protein fragment that acts as a modulator of plant defence responses, including localized cell death control. However, other parts of PR1 also have immunity potential unrelated to cell death. The described modes of the PR1 contribution to immunity were found to be tissue-localized and host plant ontogenesis dependent.     

Temporal and spatial expression of mRNAs encoding pathogenesis-related proteins during ethylene-promoted leaflet abscission in Sambucus nigra*

Differential screening of a cDNA library generated from RNA extracted from ethylene-treated leaflet abscission zones of Sambucus nigra resulted in the isolation of 20 abscission-related clones. These clones could be grouped into seven families. Sequencing of members of these families revealed that the majority encoded pathogenesis-related (PR) proteins, and these could be identified by sequence homology as a polyphenol oxidase (PPO), a PR-1 type protein, a Chial type chitinase, a PR-4 type protein similar to the potato win peptides, a PR-6 type proteinase inhibitor, a Chia4 type chitinase and a metallothionein-like protein (Coupe, Taylor & Roberts 1995, Planta 197, 442–447). Northern analysis revealed that these mRNAs were not expressed in freshly excised material but accumulated primarily in the abscission zone tissue after 18 h of exposure to ethylene at a time when abscission of the leaflet explants had reached 70%. Expression of the PPO and the Chia4-type chitinase was ethylene-dependent while that of the PR-4 type was up-regulated in the abscission zone tissue in the absence of the gas. The characterization of these mRNAs and their encoded proteins is presented and their possible roles during abscission are discussed.     

Isolation and amino acid sequence of two new PR-4 proteins from wheat

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 wheatwin1. 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.     

Molecular modeling of pathogenesis-related proteins of family 5

The family of pathogenesis-related (PR) 5 proteins have diverse functions, and some of them are classified as thaumatins, osmotins, and inhibitors of α-amylase or trypsin. Although the specific function of many PR5 in plants is unknown, they are involved in the acquired systemic resistance and response to biotic stress, causing the inhibition of hyphal growth and reduction of spore germination, probably by a membrane permeabilization mechanism or by interaction with pathogen receptors. We have constructed three-dimensional models of four proteins belonging to the Rosaceae and Fagaceae botanical families by using the technique of comparative molecular modelling by homology. There are four main structural differences between all the PR5, corresponding to regions with replacements of amino acids. Folding and the secondary structures are very similar for all of them. However, the isoelectric point and charge distributions differ for earch protein.     

Preparation of recombinant RNase single-chain antibody fusion proteins

This article describes the construction, expression, and purification of RNase single-chain antibody fusion proteins. To construct a fusion protein, the gene for each moiety, the RNase and the binding ligand, is modified separately to contain complementary DNA encoding a 13 amino acid spacer that separates the RNase from the binding moiety. Appropriate restriction enzyme sites for cloning into the vector are also added. The modified DNA is combined and fused using the PCR technique of splicing by overlap extension (1). The resulting DNA construct is expressed in inclusion bodies in BL21(DE3) bacteria that are specifically engineered for the expression of toxic proteins (2). After isolation and purification of the inclusion bodies, the fusion protein is solubilized, denatured, and renatured. The renatured RNase fusion protein mixture is purified to homogeneity by two chromatography steps. The first column, a CM-Sephadex C-50 or a heparin Sepharose column, eliminates the majority of contaminating proteins while the second column, an affinity column (Ni²⁺-NTA agarose), results in the final purification of the RNase fusion protein.     

Crystal structures of SnoaL2 and AclR: two putative hydroxylases in the biosynthesis of aromatic polyketide antibiotics

SnoaL2 and AclR are homologous enzymes in the biosynthesis of the aromatic polyketides nogalamycin in Streptomyces nogalater and cinerubin in Streptomyces galilaeus, respectively. Evidence obtained from gene transfer experiments suggested that SnoaL2 catalyzes the hydroxylation of the C-1 carbon atom of the polyketide chain. Here we show that AclR is also involved in the production of 1-hydroxylated anthracyclines in vivo. The three-dimensional structure of SnoaL2 has been determined by multi-wavelength anomalous diffraction to 2.5A resolution, and that of AclR to 1.8A resolution using molecular replacement. Both enzymes are dimers in solution and in the crystal. The fold of the enzyme subunits consists of an alpha+beta barrel. The dimer interface is formed by packing of the beta-sheets from the two subunits against each other. In the interior of the alpha+beta barrel a hydrophobic cavity is formed that most likely binds the substrate and harbors the active site. The subunit fold and the architecture of the active site in SnoaL2 and AclR are similar to that of the polyketide cyclases SnoaL and AknH; however, they show completely different quaternary structures. A comparison of the active site pockets of the putative hydroxylases AclR and SnoaL2 with those of bona fide polyketide cyclases reveals distinct differences in amino acids lining the cavity that might be responsible for the switch in chemistry. The moderate degree of sequence similarity and the preservation of the three-dimensional fold of the polypeptide chain suggest that these enzymes are evolutionary related. Members of this enzyme family appear to have evolved from a common protein scaffold by divergent evolution to catalyze reactions chemically as diverse as aldol condensation and hydroxylation.     

Oxalic acid-induced resistance to Rhizoctonia solani in rice is associated with induction of phenolics,peroxidase and pathogenesis-related proteins

Abstract

Oxalic acid (1 mM) when applied as a foliar spray to rice plants induced resistance to challenge infection with Rhizoctonia solani, the rice sheath blight pathogen. Maximum reduction in sheath blight incidence was observed when the plants were sprayed with oxalic acid three days before inoculation with the fungus. The biochemical alterations in rice plants treated with oxalic acid was also investigated. When rice plants were treated with oxalic acid, a two-fold increase in phenolic content in leaf sheaths was recorded three days after treatment. Phenylalanine ammonia-lyase and peroxidase activities increased significantly starting from two days after treatment. Peroxidase (PO) isozyme analysis indicated that PO-3 and PO-4 were induced two days after treatment with oxalic acid. Western blot analysis revealed that two chitinases (28 and 35 kDa) and two β-1,3-glucanases (30 and 32 kDa) were strongly induced in rice sheaths four to six days after treatment with oxalic acid. Immunoblot analysis of protein extracts from oxalic acid-treated plants demonstrated the induction of a 23 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antibody. These results suggest that the enhanced activities of defense enzymes and defense-related compounds in oxalic acid-treated rice plants may contribute to resistance against R. solani.     

Differential expression of pathogenesis-related proteins and defense enzymes in banana: interaction between endophytic bacteria,Banana bunchy top virus and Pentalonia nigronervosa

Banana bunchy top disease caused by Banana bunchy top virus is the most serious viral disease of banana and plantain worldwide. The virus is transmitted by the aphid vector Pentalonia nigronervosa in a persistent manner. This paper deals with the effect of the interaction between plant growth promoting endophytic bacteria, Banana bunchy top virus, and the banana aphid Pentalonia nigronervosa in the expression of Pathogenesis-related proteins (PR-proteins) and defense enzymes in banana. The existence of virus in the aphids was confirmed by ELISA, DIBA and PCR. PCR could amplify 1100-bp replicase gene of BBTV from viruliferous aphids. A significant increase in the enzymatic activity of all measured PR proteins and defense enzymes, as compared to control plants, was seen in the plants inoculated with endophytic bacteria and challenged with viruliferous aphids. Native gel electrophoresis revealed expression of more isoforms of PR proteins viz., peroxidase and chitinase in the banana plants challenged with mixtures of plant growth promoting endophytic bacteria and viruliferous aphids. Enhanced activity of a PR-2 protein viz., β-1,3-glucanase was also noticed in the viruliferous aphids infested plants. Some of the defense-related enzymes viz., Polyphenol oxidase and Phenylalanine ammonia lyase and phenolic compounds were also upregulated, up to 5 days after aphid infestation and thereafter there was a reduction in the enzymatic activity. Thus, there exist a differential accumulation of PR proteins and defense-related enzymes, when there is tri-tropic interaction between endophytic bacteria, virus, and insect and the role of the endophytic bacteria in the defense mechanisms against insect pests needs to be elucidated.