PR-1a Protein Research Articles

Pru p 9, a new allergen eliciting respiratory symptoms in subjects sensitized to peach tree pollen.

Peach tree (PT) pollen sensitization is highly prevalent in subjects living in areas where this tree is widely cultivated. None of the allergens responsible for these sensitizations have been identified so far. Our aim was to identify the most relevant PT pollen allergens and analyze their capacity for inducing respiratory symptoms. We studied sixty-two individuals sensitized to PT pollen who developed symptoms after its exposure. The IgE binding profile on peach pollen extract by means of immunoblotting using sera from these subjects was analyzed. Protein extract was fractionated by anion-exchange chromatography and HPLC, fractions run in SDS-PAGE and proteins were identified from IgE-binding bands by mass spectrometry. Several allergenic proteins in the PT pollen extract were recognized by patients' IgE: a glucan endo-1,3-beta-glucosidase-like, a polygalacturonase, an UTP-glucose-1-phosphate uridylyltransferase and a PR-1a protein. This PR-1a protein is a novel allergen frequently recognized with a molecular mass of 18 kDa, named as Pru p 9 following the WHO-IUIS nomenclature. Skin Prick Test (SPT) performed with this allergen was positive in 41% of the PT pollen-sensitized clinical cases. Most of them had rhinitis or rhinoconjunctivitis, but a significant percentage experienced asthma with seasonal symptoms during the period of PT flowering. Nasal Provocation test (NPT) with Pru p 9 was positive in all cases with positive SPT to this new allergen eliciting nasal symptoms similar to those challenged with PT pollen. We demonstrate that PT pollen can induce sensitization and allergy in an exposed population, being Pru p 9 a relevant allergen responsible of respiratory symptoms. Considering the extensive peach worldwide production with a large number of people involved, our results add a great value for the diagnosis and management of subjects allergic to this pollen.

Induction of systemic disease resistance in Nicotiana benthamiana by the cyclodipeptides cyclo (l-Pro-l-Pro) and cyclo (d-Pro-d-Pro).

Cyclodipeptides, formed from two amino acids by cyclodehydration, are produced naturally by many organisms, and are known to possess a large number of biological activities. In this study, we found that cyclo (l-Pro-l-Pro) and cyclo (d-Pro-d-Pro) (where Pro is proline) could induce defence responses and systemic resistance in Nicotiana benthamiana. Treatment with the two cyclodipeptides led to a reduction in disease severity by Phytophthora nicotianae and Tobacco mosaic virus (TMV) infections compared with controls. Both cyclopeptides triggered stomatal closure, induced reactive oxygen species production and stimulated cytosolic calcium ion and nitric oxide production in guard cells. In addition, the application of cyclodipeptides significantly up-regulated the expression of the plant defence gene PR-1a and the PR-1a protein, and increased cellular salicylic acid (SA) levels. These results suggest that the SA-dependent defence pathway is involved in cyclodipeptide-mediated pathogen resistance in N. benthamiana. We report the systemic resistance induced by cyclodipeptides, which sheds light on the potential of cyclodipeptides for the control of plant diseases.

Homology Modeling and Functional Characterization of PR-1a Protein of Hordeum vulgare subsp. Vulgare.

Pathogenesis-related protein 1a of Hordeum vulgare subsp. Vulgare (HvPR-1a) is induced by various pathogens and stress related factors. It plays important roles in plant defense system. Since the discovery of HvPR-1a a great deal of research has been focused on its isolation and characterization. However, three dimensional structure of HvPR-1a is still unknown. 3D structure can be used for determining protein function, and identifying novel protein folds and potential targets for regulation. The protein model was developed using MODELLER 9v10. Physicochemical characterization and functional annotation of the model carried out with Expasy's ProtParam server and three different conserved domain finding programs including InterProScan, Proteins Families Database (Pfam), and NCBI Conserved Domains Database (NCBI-CDD). Applying validation programs revealed that the model has good quality and the RMSD value is 0.7. The predicted model submitted in Protein Model Database, PMDB for public use. This model will be used in wide range of studies for functional analysis and improvement activity of the protein.

Characterization of the Secretome of Suspension Cultures of Medicago Species Reveals Proteins Important for Defense and Development

Molecular events occurring in the plant apoplast contribute to important developmental and defense responses. To define the secretome of Medicago, we used suspension cultures to isolate and identify secreted proteins as a first step to determining their functions. Proteins in the extracellular medium of the suspension cultures were examined using SDS-PAGE, tandem mass spectrometry (MALDI-TOF/TOF) and bioinformatics tools. There were 39 proteins identified in the cultures derived from M. sativa, M. truncatula 2HA (an embryogenic line), and M. truncatula sickle (an ethylene-insensitive mutant). N-Terminal secretion signals were detected in 34 proteins and five other proteins were predicted to be secreted via a nonclassical (ER-independent) route. All samples possessed defense related proteins including pathogenesis related (PR) proteins. The glycoprotein, SIEP1L, was found only in M. sativa. Three secreted proteinases were identified in M. truncatula, including a serine carboxypeptidase detected only in 2HA. Some proteins were unique to a cell culture line. Quantitative real time RT-PCR was used to determine mRNA expression of selected genes corresponding to proteins found only in 2HA or sickle or in both. The results correlate well with the proteomic data. For instance, a GDSL-lipase gene known to be regulated by ethylene was found only in 2HA but not in the ethylene insensitive mutant. Similarly, the PR1a protein, expressed from a well recognized ethylene-regulated gene, was found in 2HA but not sickle. These experiments indicate that the suspension culture systems established here are useful to avoid contamination from cytoplasmic proteins and to identify secreted proteins in Medicago, and should have application in other plant systems.

Cloning of the subtilisin Pr1A gene from a strain of locust specific fungus, Metarhizium anisopliae, and functional expression of the protein in Pichia pastoris

The subtilisin-like protease Pr1A plays a role in insect cuticle breach and has been used in the development of advanced engineered biopesticides. We have identified and cloned the Pr1A gene from a locust specific Metarhizium anisopliae strain, CQMa102. The cDNA of Pr1A and its deduced protein sequence were deposited in GenBank (accession numbers EF627449 and ABR20899, respectively). Sequence analysis reveals that Pr1A belongs to the subtilisin-like serine protease family. Analysis of homologous species shows that the protein exhibits 99% identity with the subtilisin Pr1A from M. anisopliae var. acridum strain FI-985. The CQMa102 Pr1A protein was expressed in Pichia pastoris to verify its protease activity. Our results show that the Pr1A gene cloned from M. anisopliae strain CQMa102 has cuticle-degrading function and is a potential virulence factor for the development of engineered biopesticides.

Silencing of acidic pathogenesis-related PR-1 genes increases extracellular -(1->3)-glucanase activity at the onset of tobacco defence reactions

The class 1 pathogenesis-related (PR) proteins are thought to be involved in plant defence responses, but their molecular functions are unknown. The function of PR-1 was investigated in tobacco by generating stable PR-1a-silenced lines in which other acidic PR-1 genes (PR-1b and PR-1c) were silenced. Plants lacking extracellular PR-1s were more susceptible than wild-type plants to the oomycete Phytophthora parasitica but displayed unaffected systemic acquired resistance and developmental resistance to this pathogen. Treatment with salicylic acid up-regulates the PR-1g gene, encoding a basic protein of the PR-1 family, in PR-1-deficient tobacco, indicating that PR-1 expression may repress that of PR-1g. This shows that acidic PR-1s are dispensable for expression of salicylic acid-dependent acquired resistances against P. parasitica and may reveal a functional overlap in tobacco defence or a functional redundancy in the PR-1 gene family. The data also show that there is a specific increase in apoplastic beta-(1-->3)-glucanase activity and a decrease in beta-(1-->3)-glucan deposition in PR-1-silenced lines following activation of defence reactions. Complementation of the silencing by apoplastic treatment with a recombinant PR-1a protein largely restores the wild-type beta-(1-->3)-glucanase activity and callose phenotype. Taken together with the immunolocalization of PR-1a to sites of beta-(1-->3)-glucan deposition in wild-type plants, these results are indicative of a function for PR-1a in regulation of enzymatic activity of extracellular beta-(1-->3)-glucanases.

Salicylate Activity. 3. Structure Relationship to Systemic Acquired Resistance

Salicylic acid (2-hydroxybenzoic acid; SA) is a primary signal inducing plant defenses against pathogens. This plant disease resistance, known as systemic acquired resistance (SAR), is an attractive target for the development of new plant protection agents. SAR induction is a multistep process that includes accumulation of pathogenesis-related (PR) proteins. The structure-activity profile of salicylates and related compounds has been evaluated using an inducible PR protein (PR-1a) and plant resistance to tobacco mosaic virus (TMV) as markers. Among the 47 selected monosubstituted and multiple-substituted salicylate derivatives tested, all 8 derivatives that induced more PR-1a protein than SA were fluorinated or chlorinated in the 3- and/or 5-position (3,5-difluorosalicylate > 3-chlorosalicylate > 5-chlorosalicylate > 3,5-dichlorosalicylate > 3-chloro-5-fluorosalicylate > 3-fluorosalicylate > 3-fluoro-5-chlorosalicylate > 3,5-dichloro-6-hydroxysalicylate > SA). In general, substitutions for or on the 2-hydroxyl group or at the 4-position of the ring reduced or eliminated PR-1a protein induction. In contrast, substitutions in positions ortho (3-position) or para (5-position) to the hydroxyl group with electron-withdrawing groups other than chlorine or fluorine decreased induction, and electron-donating groups in these positions also had a deleterious effect on PR-1a induction. PR-1a protein accumulation and reduction in TMV lesion diameter exhibited a log-linear relationship. The seven salicylate derivatives that were the most active TMV resistance inducers were all halogenated in the 3- and/or 5-position (3-chlorosalicylate > 3,5-difluorosalicylate > 3,5-dichloro-6-hydroxysalicylate > 3,5,6-trichlorosalicylate > 5-chlorosalicylate > 5-fluorosalicylate > 3,5-dichlorosalicylate > 4-fluorosalicylate > 3-fluorosalicylate > 3-chloro-5-fluorosalicylate > 4-chlorosalicylate > SA). The correlation between PR-1a protein induction and resistance to TMV confirms the value of using PR-1a induction as a screening tool for developing new plant disease control agents.

Evidence that the caterpillar salivary enzyme glucose oxidase provides herbivore offense in solanaceous plants

The insect salivary enzyme glucose oxidase (GOX) can inhibit wound-inducible nicotine production in tobacco, Nicotiana tabacum. We examined whether salivary gland extracts of Helicoverpa zea lacking active GOX could still suppress nicotine in tobacco, Nicotiana tabacum, and whether GOX could suppress wound-inducible defenses of another Solanaceous plant, tomato Lycopersicon esculentum. Tobacco leaves were wounded with a cork borer and treated with water, salivary gland extracts with active GOX (SxG), or salivary gland extracts with inactive GOX (SxI). After three days, leaves treated with SxG had significantly less nicotine than all other wounded treatments. Neonates that fed on the terminal leaves of tobacco plants treated with SxG had significantly higher survival than neonates that fed on leaves treated with either SxI or water. This evidence supports the assertion that GOX is the salivary factor responsible for the suppression of tobacco plant nicotine production by H. zea saliva. Results for the NahG tobacco plants, which lack salicylic acid (SA) due to a transgene for bacterial SA hydroxylase, indicate that suppression of nicotine by GOX does not require SA. However, tobacco leaves that were wounded and treated with SxG had significantly higher levels of the SA-mediated PR-1a protein than leaves treated with SxI or water. Leaves of tomato plants wounded with scissors and then treated with SxG had trypsin inhibitor levels that were moderately lower than plants wounded and treated with purified GOX, water, or SxI. However, all the wounded tomato leaves irrespective of treatment resulted in lower caterpillar growth rates than the non-wounded tomato leaves. Glucose oxidase is the first insect salivary enzyme shown to suppress wound-inducible herbivore defenses of plants.

Cell Defense Responses Associated with Localized and Systemic Resistance to Phytophthora parasitica Induced in Tomato by an Arbuscular Mycorrhizal Fungus

The arbuscular mycorrhizal fungus Glomus mosseae is able to confer bioprotection against Phytophthora parasitica in tomato roots. Localized and induced systemic resistance (ISR) have been demonstrated to be involved in pathogen control in mycorrhizal and nonmycorrhizal roots with a split root experimental system. Decreased pathogen development in mycorrhizal and nonmycorrhizal parts of mycorrhizal root systems is associated with accumulation of phenolics and plant cell defense responses. G. mosseae-containing cortical cells in the mycorrhizal tissues are immune to the pathogen and exhibit a localized resistance response with the formation of cell wall appositions reinforced by callose adjacent to intercellular hyphae. The systemically induced resistance in nonmycorrhizal root parts is characterized by elicitation of host wall thickenings containing non-esterified pectins and PR-1a protein in reaction to intercellular pathogen hyphae, and by the formation of callose-rich encasement material around P. parasitica hyphae that are penetrating root cells. PR-la protein is detected in the pathogen wall only in these tissues. None of these cell reactions are observed in nonmycorrhizal pathogen-infected root systems, where disease development leads to host cell death. The cellular and molecular basis of bioprotection by an arbuscular mycorrhizal fungus is discussed in relation to that induced by other nonpathogenic microorganisms.

The suppression of mannitol induced pathogenesis-related protein accumulation in tobacco by antibiotics

Antibiotics suppressed the induction of PR1a protein in tobacco leaf discs by mannitol but not by salicylic acid. Mannitol suppressed the induction of PR1a protein by salicylic acid, increasing concentrations of mannitol progressively inhibiting PR1a protein accumulation. This suppression was probably due to an inhibition of total protein synthesis by mannitol. It is probable that the previously reported induction of pathogenesis-related proteins by mannitol is due to the activity of contaminating micro-organisms present either in the mannitol solution or within the plant.

Sequence-specific binding of protein factors to two independent promoter regions of the acidic tobacco pathogenesis-related-1 protein gene (PR-1)

Gel shift mobility analysis, using the proximal 0.3 kb fragment of the tobacco pathogenesis-related protein 1a gene (PR-1a) and nuclear extracts from healthy Samsun NN tobacco leaves, which do not produce PR-1 proteins, showed a broad shifted signal with low mobility. This signal was not detected with nuclear proteins from the interspecific hybrid of Nicotiana glutinosa x Nicotiana debneyi, which constitutively produces the PR-1a protein. Similar shifted signals were detected with both proximal and distal regions of the 0.3 kb fragment using nuclear proteins from healthy Samsun NN tobacco, but not with proteins from the interspecific hybrid. Further experiments, performed using 5' or 3' truncated fragments of the 0.3 kb fragment, identified two independent binding sites: a distal site between -179 and -168 bp from the transcription start site, and a proximal site between -61 and -37 bp. Footprint analysis revealed two protected sequences, a distal region between -184 and -172 bp, and a proximal region between -68 and -51 bp. These results indicate the presence of regulatory factor(s) for expression of the acidic PR-1a gene. The possibility of negative regulation of the gene is discussed.

Immunocytochemical localization of pathogenesis-related PR-1 proteins in tobacco root tissues infected in vitro by the black root rot fungus Chalara elegans

Subcellular localization of the pathogenesis-related PR-1 proteins of unknown function was studied in roots of resistant Nicotiana tabacum cv Xanthi nc uninfected or infected in vitro by the black root rot fungus Chalara elegans , using polyclonal or monoclonal antibodies raised against PR-1a (or b 1) protein. In healthy tobacco roots, the PR-1 proteins were found to be present in low amounts in intercellular space material, over cell walls and over secondary thickenings of xylem vessels. All these cell compartments were significantly enriched in the PR-1 proteins in infected tobacco root tissues. Cell wall outgrowths, typically induced in hypodermal cells by Chalara elegans infection and wall appositions formed in cortical parenchymal cells in response to infection, were also the sites of PR-1 protein accumulation. In contrast, very little occurred in electron-translucent intercellular spaces. PR-1 proteins were also detected in the wall of both inter- and intracellular fungal hyphae invading root tissues, but not in axenically cultured Chalara elegans or hyphae developing outside roots. These results are discussed in relation to the possible role of the PR-1 proteins in defence mechanisms in roots.

Induced expression of a chimeric gene construct in transgenic lettuce plants using tobacco pathogenesis-related protein gene promoter region

The expression of a stress- and salicylic acidinducible protein gene from tobacco, PR1a protein gene, was determined after its Introduction to lettuce (Lactuca sativa L.) plants. The 5' flanking 2.4 Kb fragment from PR1a gene was joined to the bacterial β -glucuronidase (GUS) gene (PR-GUS) and introduced into lettuce cotyledons by Agrobacterium-mediated gene transfer using a binary vector containing a kanamycin-resistance gene as a selectable marker. As a control with constitutive expression, the chimeric gene consisting of CaMV 35S RNA promoter and GUS gene (35S-GUS) was used. An improved method for shoot formation directly from lettuce cotyledons was used effectively for transformation, shortening the time for regeneration. In 70% or more of kanamycin-resistant regenerated lettuce plants, into which PR-GUS or 35S-GUS was introduced, high GUS activity and integration of the chimeric gene into the lettuce genome were detected. By treatment with salicylic acid, GUS activity increased 3- to 50-fold in PR-GUS transformants, however, no increase was detected in 35S-GUS plants. These results showed that the promoter of the stress-inducible tobacco PR1a protein gene was introduced into lettuce plants, and the introduced chimeric gene was expressed normally under the regulated control of the PRla promoter.

Analysis of regulatory elements involved in the induction of two tobacco genes by salicylate treatment and virus infection.

Tobacco genes encoding the PR-1a protein and a glycine-rich protein are expressed after treatment of plants with salicylate or infection with tobacco mosaic virus. Upstream sequences of these genes were fused to reporter genes, and these constructs were used to transform tobacco. Upstream sequences of the PR-1a gene of 689 base pairs or longer were sufficient for induction of the reporter gene in tobacco mosaic virus-inoculated leaves, systemically induced leaves from infected plants, and leaves treated with salicylate. No such induction was found with upstream sequences of 643 base pairs or shorter of the PR-1a gene. When the PR-1a upstream sequence from nucleotides -625 to -902 was fused to the cauliflower mosaic virus 35S core promoter, a construct was obtained that conferred tobacco mosaic virus and salicylate inducibility to the reporter gene in transgenic plants. This confirmed the localization of tobacco mosaic virus- and salicylate-responsive elements between positions -643 and -689 in the PR-1a promoter. With the glycine-rich protein gene, an upstream sequence of 645 base pairs was sufficient for tobacco mosaic virus and salicylate inducibility of the reporter gene, whereas constructs containing 400 base pairs or fewer of the glycine-rich protein promoter were largely inactive.