Tobacco Pathogenesis-related Protein Research Articles

Expression of pathogenesis-related proteins in transplastomic tobacco plants confers resistance to filamentous pathogens under field trials

Plants are continuously challenged by pathogens, affecting most staple crops compromising food security. They have evolved different mechanisms to counterattack pathogen infection, including the accumulation of pathogenesis-related (PR) proteins. These proteins have been implicated in active defense, and their overexpression has led to enhanced resistance in nuclear transgenic plants, although in many cases constitutive expression resulted in lesion-mimic phenotypes. We decided to evaluate plastid transformation as an alternative to overcome limitations observed for nuclear transgenic technologies. The advantages include the possibilities to express polycistronic RNAs, to obtain higher protein expression levels, and the impeded gene flow due to the maternal inheritance of the plastome. We transformed Nicotiana tabacum plastids to co-express the tobacco PR proteins AP24 and β-1,3-glucanase. Transplastomic tobacco lines were characterized and subsequently challenged with Rhizoctonia solani, Peronospora hyoscyami f.sp. tabacina and Phytophthora nicotianae. Results showed that transplastomic plants expressing AP24 and β-1,3-glucanase are resistant to R. solani in greenhouse conditions and, furthermore, they are protected against P.hyoscyami f.sp. tabacina and P. nicotianae in field conditions under high inoculum pressure. Our results suggest that plastid co- expression of PR proteins AP24 and β-1,3-glucanase resulted in enhanced resistance against filamentous pathogens.

Microperforated leaf blotting on polyvinylidene difluoride and nylon membranes to analyze spatial distribution of endogenous and viral gene expression in plant leaves

Leaf blotting to detect proteins and investigate their spatial distribution in leaves has so far mainly been used to detect viral coat proteins that accumulate abundantly in infected leaves, but rarely to detect endogenous plant proteins. We improved the method for detecting endogenous proteins. We found that microperforating leaves with bundled pins before blotting, then pressing leaves with a rolling pin onto polyvinylidene difluoride (PVDF) membranes enabled even blotting of sap. This microperforated leaf blotting (mPLB) was also suitable for use with nylon membranes to detect leaf RNA. The mPLB revealed that accumulation of two endogenous proteins, calmodulin-like rgs-CaM and actin, was respectively positively and negatively associated with that of viral coat protein in tobacco leaves infected with cucumber mosaic virus (CMV). When a tobacco plant primed with benzothiadiazole was inoculated with CMV, mPLB showed that the infection was restricted to some areas of the leaf and that in these areas the mRNA encoding tobacco pathogenesis-related protein 1, an indicator of salicylic acid-mediated immune responses, was induced. These results demonstrate the effectiveness of mPLB for investigating the spatial distribution of endogenous and viral gene expression in leaves.

High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid.

BackgroundLarge-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneously improve rice straw as an efficient biomass feedstock for conversion of cellulose to glucose.ResultsIn this study, the cellulose hydrolytic enzyme β-1, 4-endoglucanase (E1) gene, from the thermophilic bacterium Acidothermus cellulolyticus, was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial E1 gene in rice was driven by the constitutive Mac promoter, a hybrid promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer, with the signal peptide of tobacco pathogenesis-related protein for targeting the E1 protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial E1 enzyme were obtained that expressed the gene at high levels without severely impairing plant growth and development. However, some transgenic plants exhibited a shorter stature and flowered earlier than the wild type plants. The E1 specific activities in the leaves of the highest expressing transgenic rice lines were about 20-fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. A zymogram and temperature-dependent activity analyses demonstrated the thermostability of the E1 enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid for one hour at 39°C and another hour at 81°C yielded 43% more reducing sugars than wild type rice straw.ConclusionTaken together, these data suggest that transgenic rice can effectively serve as a bioreactor for the large-scale production of active, thermostable cellulose hydrolytic enzymes. As a feedstock, direct expression of large amount of cellulases in transgenic rice may also facilitate saccharification of cellulose in rice straw and significantly reduce the costs for hydrolytic enzymes.

In vivo bioluminescence monitoring of defense gene expression in response to treatment with yeast cell wall extract

Previous studies indicated that Housaku Monogatari (HM), a plant activator made from yeast cell wall extract, is effective for control of various plant diseases. To investigate the effect of HM treatment on plant gene expression, we tested the expression of defense related gene induction by exploiting tobacco pathogenesis-related protein 1a (PR-1a) gene promoter- and the Arabidopsis plant defensin 1.2 (PDF1.2) gene promoter-luciferase fusions as reporter genes. Transgenic Arabidopsis plants harboring promoter-luciferase fusion genes were treated with HM and the promoter activity was monitored as changes in luciferase activity in planta. Results of bioluminescence monitoring assay indicated that the promoters were activated at different times after the treatment of test plants with HM. Maximum activation of the PR-1a promoter occurred 4 days, and of the PDF1.2 promoter 4 h, after treatment. These results suggest that HM might contain multiple microbe-associated molecular patterns (MAMPs) that activate systemic acquired resistance and induced systemic resistance signaling pathways at different times. This may explain the mechanisms involved in the induction of defense responses against multiple plant pathogens by HM treatment.

Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d

BackgroundAromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB) for coplanar aromatic motifs similar to those found in known glycan-binding proteins.ResultsThe proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO) enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192) in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry.ConclusionsBased on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

Heterologous Acidothermus cellulolyticus 1,4-β-endoglucanase E1 produced within the corn biomass converts corn stover into glucose

Commercial conversion of lignocellulosic biomass to fermentable sugars requires inexpensive bulk production of biologically active cellulase enzymes, which might be achieved through direct production of these enzymes within the biomass crops. Transgenic corn plants containing the catalytic domain of Acidothermus cellulolyticus E1 endo-1,4-beta glucanase and the bar bialaphos resistance coding sequences were generated after Biolistic (BioRad Hercules, CA) bombardment of immature embryo-derived cells. E1 sequences were regulated under the control of the cauliflower mosaic virus 35S promoter and tobacco mosaic virus translational enhancer, and E1 protein was targeted to the apoplast using the signal peptide of tobacco pathogenesis-related protein to achieve accumulation of this enzyme. The integration, expression, and segregation of E1 and bar transgenes were demonstrated, respectively, through Southern and Western blotting, and progeny analyses. Accumulation of up to 1.13% of transgenic plant total soluble proteins was detected as biologically active E1 by enzymatic activity assay. The corn-produced heterologous E1 could successfully convert ammonia fiber explosion-pretreated corn stover polysaccharides into glucose as a fermentable sugar for ethanol production, confirming that the E1 enzyme is produced in its active form.

Expression of the Human Hepatitis B Virus Large Surface Antigen Gene in Transgenic Tomato Plants

The original hepatitis B virus (HBV) large surface antigen gene was synthesized. In order to optimize the expression of this gene in tomato plants, the tobacco pathogenesis-related protein S signal peptide was fused to the 5' end of the modified gene and the sequence encoding amino acids S, E, K, D, E, and L was placed at the 3' end. The gene encoding the modified HBV large surface antigen under the control of a fruit-specific promoter was constructed and expressed in transgenic tomato plants. The expression of the antigen from transgenic plants was confirmed by PCR and reverse transcriptase PCR. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed that the maximal level of HBsAg was about 0.02% of the soluble protein in transgenic tomato fruit. The amount of HBsAg in mature fruits was found to be 65- to 171-fold larger than in small or medium fruits and leaf tissues. Examination of transgenic plant samples by transmission electron microscopy proved that HBsAg had been expressed and had accumulated. The HBsAg protein was capable of assembling into capsomers and virus-like particles. To our knowledge, this is the first time the HBV large surface antigen has been expressed in plants. This work suggests the possibility of producing a new alternative vaccine for human HBV.

Transient assay system for the analysis of PR-1a gene promoter in tobacco BY-2 cells.

In order to develop a rapid and versatile assay system suitable for the analysis of regulated expression of tobacco pathogenesis-related protein 1a (PR-1a) gene, we investigated the use of the transient gene expression system in tobacco BY-2 cells by microprojectile bombardment. Using dual luciferase assay as a reporter gene expression detection system, we observed significant induction of PR-1a promoter activity by salicylic acid (SA) treatment. On the other hand, treatment with 4-hydroxybenzoic acid (4-HBA) resulted in no detectable increase in luciferase activity. Co-expression of a trans-acting factor, the NPR1/NIM1 protein of Arabidopsis, resulted in the induction of higher expression levels of the PR-1a promoter. These results suggest that the assay system is applicable for the analysis of factors involved in the regulated expression of SA-inducible defense-related genes.

Salicylic acid and the hypersensitive response initiate distinct signal transduction pathways in tobacco that converge on the as-1-like element of the PR-1a promoter.

Tobacco pathogenesis-related protein 1a (PR-1a) is induced in plants during the hypersensitive response (HR) after exposure of plants to salicylic acid (SA) and by developmental cues. Gene activation by these diverse stimuli is mediated via an as-1-like element in the PR-1a upstream region. To further analyze the significance of this cis-acting sequence, an authentic as-1 element from the cauliflower mosaic virus 35S RNA promoter was inserted into the PR-1a promoter in place of the as-1-like motif. Reporter gene analysis in transgenic tobacco plants demonstrated that as-1 can functionally replace the as-1-like element in the PR-1a promoter in response to all stimuli. However, reporter gene induction from the as-1 carrying promoter was enhanced in response to SA compared to the wild-type promoter, and the ratio of reporter gene activities in SA treated leaf tissue to tissue exhibiting the HR increased with the as-1 promoter construct. Our findings support a model where PR-1a gene expression relies on at least two distinct signal transduction pathways initiated by SA and by a yet unknown signal produced during the HR, that promote different, albeit related, transcription complexes on the PR-1a as-1-like element. Analysis of PR-1 proteins in plants expressing salicylate hydroxylase yielded additional evidence that an HR dependent pathway leads to high level PR-1 gene induction in tobacco.

Overexpression of a cell wall glycoprotein in Fusarium oxysporum increases virulence and resistance to a plant PR‐5 protein

Fusarium oxysporum f. sp. nicotianae is a causal agent for vascular wilt disease in tobacco. It is sensitive to osmotin, a tobacco pathogenesis-related protein (PR-5) that is implicated in plant defense against phytopathogenic fungi. We show that osmotin susceptibility of F. oxysporum f. sp. nicotianae was reduced by overexpression of the heterologous cell wall glycoprotein Saccharomyces cerevisiae protein containing inverted repeats (PIR2), a member of the PIR family of fungal cell wall glycoproteins that protect S. cerevisiae from the toxic action of osmotin. S. cerevisiae PIR2 was targeted to the cell wall of F. oxysporum. Disease severity and fungal growth were increased in tobacco seedlings inoculated with F. oxysporum transformed with PIR2 compared to seedlings infected with untransformed F. oxysporum or that transformed with vector, although accumulation of transcript and protein of defense genes was similar. The results show that fungal cell wall components can increase resistance to plant defense proteins and affect virulence.

Combined use of regulatory elements within the cDNA to increase the production of a soluble mouse single-chain antibody, scFv, from tobacco cell suspension cultures.

In order to facilitate production and secretion of a soluble form of a small, single-chain antibody ScFv (32 kDa) in tobacco cell suspension culture, several modifications were made simultaneously to the antibody cDNA that included elements that have been shown to regulate the expression of proteins in plants. The scFv cDNA was initially ligated into a binary vector under the control of the CaMV 35S promoter and the T7 terminator for expression in tobacco suspension culture. Subsequently, modifications were engineered into the cDNA for enhancement of scFv production. These included the following: (i) the signal peptide (SP) of the tobacco pathogenesis-related protein PR1a which was added in-frame to the N-terminal end of scFv cDNA; (ii) a 5'-nontranslated region from the tobacco etch virus (TEV leader sequence), which was fused to the N-terminal end of the SP; and (iii) the endoplasmic reticulum retention signal peptide KDEL, which was added to the C-terminal end of the scFv protein. Using a modified disruption method involving pectinase, the highest expression of total scFv (344 ng scFv/g cell) occurred when the plant leader sequence, the TEV sequence, and the KDEL peptide were all present in the expression construct. Although the addition of the KDEL sequence significantly increased the total yield of protein 5.4-fold, it did not increase the overall amount of protein secreted. These studies indicate that while the SP is very important in promoting secretion of the scFv, it had little influence on increasing scFv secretion levels even when both the TEV and the KDEL sequences significantly increased overall protein levels.

Accumulation of extracellular pathogenesis-related proteins in rose leaves following inoculation of in vitro shoots with Diplocarpon rosae

Rose shoots var. ‘Iris Gee’ grown in vitro infected with Diplocarpon rosae (blackspot causal agent) led to the accumulation of the pathogenesis-related (PR) proteins in the intercellular space of leaves. Electrophoresis of intercellular fluids by SDS-PAGE and 2D-PAGE revealed that extracellular PR proteins accumulated strongly in infected leaves starting at day 3 after inoculation and increased in abundance with disease development. Sixteen proteins, which were absent or present at a low level in the healthy leaves, have been detected in the intercellular fluid extracted from the infected rose leaves 7 days after inoculation with D. rosae. Western-blot analysis showed that rose PR proteins are serologically related to tobacco PR proteins. Infection of D. rosae resulted not only in strong accumulation of PR-2, PR-3, and PR-5 proteins in the intercellular spaces of rose leaves but also induction of PR-1 protein at a later stage of infection. However, no systemic induction of PR-1 protein was detected in upper newly expanded and uninfected leaves.

Elicitation of Hypersensitive Cell Death by Extracellularly Targeted HrpZPsph Produced In Planta

The ability of the Pseudomonas syringae pv. phaseolicola harpin (HrpZPsph) to elicit hypersensitive response was investigated in three Nicotiana genotypes. The hrpZPsph gene was placed under chemical regulation (tetracycline induction) in TetR+ Nicotiana tabacum cv. Wisconsin 38 (W38) or was transiently expressed in N. benthamiana following infection with a PVX-derived vector and in three Nicotiana genotypes by agroinfiltration. The constructs were designed to express either the canonical form of harpin (HrpZPsph) or an N-terminally extended version of the protein carrying the signal peptide portion of the tobacco pathogenesis-related protein PR1a (SP-HrpZPsph). Stable transformants of N. tabacum cv. W38 did not develop necrosis upon induction with tetracycline, probably as a result of insufficient harpin accumulation. In contrast, N. benthamiana plants infected with the PVX constructs produced high concentrations of harpin in biologically active form, but only those expressing the secretable form of harpin developed necrotic symptoms. These symptoms were less severe than those caused by PVX::avrPto; however, they were accompanied by induction of hsr203J, a hypersensitive response-specific gene transcript. These results suggest that the plant cellular receptor(s) for harpin is extracellular.

An HR-induced tobacco peroxidase gene is responsive to spermine, but not to salicylate, methyl jasmonate, and ethephon.

In Tobacco mosaic virus (TMV)-infected tobacco plants carrying the N resistance gene, a hypersensitive reaction or response (HR) occurs to enclose the virus in the infected tissue. Although a contribution of peroxidases to the resistance has been proposed, no evidence has been presented that tobacco peroxidase genes respond to HR. Here, we describe the HR-induced expression of a tobacco peroxidase gene (tpoxC1) whose induction kinetics were slightly different from those of acidic and basic tobacco pathogenesis-related (PR) protein genes. Interestingly, tpoxC1 was insensitive to the inducers of PR genes such as salicylic acid, methyl jasmonate, and ethephon. Spermine activated tpoxC1 gene expression at a low level and both acidic and basic PR gene expression at a considerably higher level. These results indicate that the induced expression of tpoxC1 is regulated differently from that of classical tobacco PR genes in the N gene-mediated self-defense system in tobacco plants.

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox gene OSH1 lead to altered morphology of leaves in transgenic tobacco.

Transgenic tobacco plants were generated carrying a rice homeobox gene, OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicates arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotrophic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly, OHS1 expression was undetectable in mature leaves with the aberrant morphological features. Thus, OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate that PR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopic OSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. The OSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.

Fine structure and function of the osmotin gene promoter

The gene encoding osmotin, a tobacco pathogenesis-related protein, has been shown to be regulated by an array of hormonal and environmental signals. The osmotin promoter fragment -248 to -108 upstream of the transcription start site (fragment A), was sufficient to direct reporter gene expression when fused to a minimal CaMV 35S promoter in transient assays using microprojectile bombardment. This was consistent with previous 5'-deletion analyses of the osmotin promoter which showed that the promoter sequence from -248 to -108 is absolutely required for reporter gene activity. Nuclear protein factors from salt-adapted tobacco cells, ABA-treated unadapted cells, and young cultured tobacco leaves were shown to interact with fragment A by gel mobility-shift assays. DNase I footprinting revealed that three conserved promoter elements in fragment A interact specifically with nuclear factors. These elements are: (1) a cluster of G-box-like sequences (G sequence); (2) an AT-1 box-like sequence, 5'-AATTATTTTATG-3' (AT sequence); (3) a sequence highly conserved in ethylene-induced PR gene promoters, 5'-TAAGA/CGCCGCC-3' (PR sequence). Transient expression assays performed with fragment A deletions fused to GUS indicated that osmotin promoter activity correlated with the presence of these elements. UV cross-linking analysis showed that the protein complex bound to fragment A consisted of at least four individual proteins with approximate molecular masses of 28, 29, 40 and 42 kDa. One component of this protein complex, which was associated with the G sequence, was a 14-3-3 like protein.

Colonization of Transgenic Tobacco Constitutively Expressing Pathogenesis-Related Proteins by the Vesicular-Arbuscular Mycorrhizal Fungus Glomus mosseae

We studied the effect of constitutive expression of pathogenesis-related proteins (PRs) in tobacco plants on vesicular-arbuscular mycorrhiza. Tobacco lines genetically transformed to express various PRs constitutively under the control of the cauliflower mosaic virus 35S promoter of tobacco were examined. Immunoblot analysis and activity measurements demonstrated high levels of expression of the PRs in the root systems of the plants. Constitutive expression of the following acidic isoforms of tobacco PRs did not affect the time course or the final level of colonization by the vesicular-arbuscular mycorrhizal fungus Glomus mosseae: PR-1a, PR-3 (=PR-Q), PR-Q(prm1), PR-4, and PR-5. Similarly, constitutive expression of an acidic cucumber chitinase, of a basic tobacco chitinase with and without its vacuolar targeting peptide, of a basic (beta)-1,3-glucanase, and of combinations of PR-Q and PR-Q(prm1) or basic chitinase and basic (beta)-1,3-glucanase did not affect colonization by the mycorrhizal fungus. A delay of colonization by G. mosseae was observed in tobacco plants constitutively expressing the acidic isoform of tobacco PR-2, a protein with (beta)-1,3-glucanase activity.

A novel pathogen- and wound-inducible tobacco (Nicotiana tabacum) protein with antifungal activity.

A novel pathogen- and wound-inducible antifungal protein of 20 kD was purified from tobacco (Nicotiana tabacum) Samsun NN leaves inoculated with tobacco mosaic virus (TMV). The protein, designated CBP20, was purified by chitin-affinity chromatography and gel filtration. In vitro assays demonstrated that CBP20 exhibits antifungal activity toward Trichoderma viride and Fusarium solani by causing lysis of the germ tubes and/or growth inhibition. In addition it was shown that CBP20 acts synergistically with a tobacco class I chitinase against F. solani and with a tobacco class I beta-1,3-glucanase against F. solani and Alternaria radicina. Analysis of the protein and corresponding cDNAs revealed that CBP20 contains an N-terminal chitin-binding domain that is present also in the class I chitinases of tobacco, the putative wound-induced (WIN) proteins of potato, WIN1 and WIN2, and several plant lectins. The C-terminal domain of CBP20 showed high identity with tobacco pathogenesis-related (PR) proteins, PR-4a and PR-4b, tomato PR-P2, and potato WIN1 and WIN2. CBP20 is synthesized as a preproprotein, which is processed into the mature protein by the removal of an N-terminal signal peptide and a C-terminal propeptide, most likely involved in the vacuolar targeting of the protein. The intracellular localization of CBP20 and its induction upon TMV infection and wounding indicate that CBP20 is the first class I PR-4 type protein purified.

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.

Sunflower (Helianthus annuus L.) Pathogenesis-Related Proteins (Induction by Aspirin (Acetylsalicylic Acid) and Characterization).

Sunflower leaf discs floated on a solution containing aspirin (acetylsalicylic acid) produced a set of new proteins extractable at pH 5.2 and excreted into the intercellular space. More than 80% of the proteins found in the intercellular fluids of induced leaf discs have been identified as pathogenesis-related (PR) proteins by their immunological relationship with tobacco PR proteins. Members of the four major classes of PR proteins have been characterized. Sunflower PR proteins of type 1 (PR1) and of type 3 (PR3) were found to have acidic isoelectric points, whereas the induced PR protein of type 2 (PR2) had a basic isoelectric point. Members of the type 5 PR proteins (PR5), known in tobacco as thaumatin-like proteins, showed a more complex pattern. Multiple sunflower PR5 isomers of similar molecular weight but of different isoelectric points were excreted from the cells in response to the aspirin treatment. PR2 and PR3 proteins were found at very low basal levels in untreated leaves, whereas PR1 and PR5 proteins could not be detected at all in the same extracts. Glucanase and chitinase activities were always associated with PR2 and PR3 proteins in partially purified sunflower extracts. All of these data indicate that, in response to aspirin treatment, sunflower plants produce a complete set of PR proteins characterized by an apparently exclusively extracellular localization.