Acidic Pathogenesis-related Proteins Research Articles

Sulfated fucan oligosaccharides elicit defense responses in tobacco and local and systemic resistance against tobacco mosaic virus.

Sulfated fucans are common structural components of the cell walls of marine brown algae. Using a fucan-degrading hydrolase isolated from a marine bacterium, we prepared sulfated fucan oligosaccharides made of mono- and disulfated fucose units alternatively bound by alpha-1,4 and alpha-1,3 glycosidic linkages, respectively. Here, we report on the elicitor activity of such fucan oligosaccharide preparations in tobacco. In suspension cell cultures, oligofucans at the dose of 200 microg ml(-1) rapidly induced a marked alkalinization of the extracellular medium and the release of hydrogen peroxide. This was followed within a few hours by a strong stimulation of phenylalanine ammonia-lyase and lipoxygenase activities. Tobacco leaves treated with oligofucans locally accumulated salicylic acid (SA) and the phytoalexin scopoletin and expressed several pathogenesis-related (PR) proteins, but they displayed no symptoms of cell death. Fucan oligosaccharides also induced the systemic accumulation of SA and the acidic PR protein PR-1, two markers of systemic acquired resistance (SAR). Consistently, fucan oligosaccharides strongly stimulated both local and systemic resistance to tobacco mosaic virus (TMV). The use of transgenic plants unable to accumulate SA indicated that, as in the SAR primed by TMV, SA is required for the establishment of oligofucan-induced resistance.

A non-toxic pokeweed antiviral protein mutant inhibits pathogen infection via a novel salicylic acid-independent pathway.

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from Phytolacca americana, is characterized by its ability to depurinate the sarcin/ricin (S/R) loop of the large rRNA of prokaryotic and eukaryotic ribosomes. In this study, we present evidence that PAP is associated with ribosomes and depurinates tobacco ribosomes in vivo by removing more than one adenine and a guanine. A mutant of pokeweed antiviral protein, PAPn, which has a single amino acid substitution (G75D), did not bind ribosomes efficiently, indicating that Gly-75 in the N-terminal domain is critical for the binding of PAP to ribosomes. PAPn did not depurinate ribosomes and was non-toxic when expressed in transgenic tobacco plants. Unlike wild-type PAP and a C-terminal deletion mutant, transgenic plants expressing PAPn did not have elevated levels of acidic pathogenesis-related (PR) proteins. PAPn, like other forms of PAP, did not trigger production of salicylic acid (SA) in transgenic plants. Expression of the basic PR proteins, the wound-inducible protein kinase and protease inhibitor II, was induced in PAPn-expressing transgenic plants and these plants were resistant to viral and fungal infection. These results demonstrate that PAPn activates a particular SA-independent, stress-associated signal transduction pathway and confers pathogen resistance in the absence of ribosome binding, rRNA depurination and acidic PR protein production.

Spermine is a salicylate-independent endogenous inducer for both tobacco acidic pathogenesis-related proteins and resistance against tobacco mosaic virus infection

Intercellular spaces are often the first sites invaded by pathogens. In the spaces of tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco (Nicotiana tabacum L.) leaves, we found that an inducer for acidic pathogenesis-related (PR) proteins was accumulated. The induction activity was recovered in gel-filtrated fractions of low molecular mass with a basic nature, into which authentic spermine (Spm) was eluted. We quantified polyamines in the intercellular spaces of the necrotic lesion-forming leaves and found 20-fold higher levels of free Spm than in healthy leaves. Among several polyamines tested, exogenously supplied Spm induced acidic PR-1 gene expression. Immunoblot analysis showed that Spm treatment increased not only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein accumulation. Treatment of healthy tobacco leaves with salicylic acid (SA) caused no significant increase in the level of endogenous Spm, and Spm did not increase the level of endogenous SA, suggesting that induction of acidic PR proteins by Spm is independent of SA. The size of TMV-induced local lesions was reduced by Spm treatment. These results indicate that Spm accumulates outside of cells after lesion formation and induces both acidic PR proteins and resistance against TMV via a SA-independent signaling pathway.

Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco.

Transgenic tobacco deficient in the H2O2-removing enzyme catalase (Cat1AS) was used as an inducible and noninvasive system to study the role of H2O2 as an activator of pathogenesis-related (PR) proteins in plants. Excess H2O2 in Cat1AS plants was generated by simply increasing light intensities. Sustained exposure of Cat1AS plants to excess H2O2 provoked tissue damage, stimulated salicylic acid and ethylene production, and induced the expression of acidic and basic PR proteins with a timing and magnitude similar to the hypersensitive response against pathogens. Salicylic acid production was biphasic, and the first peak of salicylic acid as well as the peak of ethylene occurred within the first hours of high light, which is long before the development of tissue necrosis. Under these conditions, accumulation of acidic PR proteins was also seen in upper leaves that were not exposed to high light, indicating systemic induction of expression. Short exposure of Cat1AS plants to excess H2O2 did not cause damage, induced local expression of acidic and basic PR proteins, and enhanced pathogen tolerance. However, the timing and magnitude of PR protein induction was in this case more similar to that in upper uninfected leaves than to that in hypersensitive-response leaves of pathogen-infected plants. Together, these data demonstrate that sublethal levels of H2O2 activate expression of acidic and basic PR proteins and lead to enhanced pathogen tolerance. However, rapid and strong activation of PR protein expression, as seen during the hypersensitive response, occurs only when excess H2O2 is accompanied by leaf necrosis.

Regulation by Cytokinins of Endogenous Levels of Jasmonic and Salicylic Acids in Mechanically Wounded Tobacco Plants

Plants respond differentially to wounding and pathogens using distinct signaling pathways, so that wound signals are transmitted to jasmonic acid (JA) which induces basic pathogenesis-related (PR) proteins, whereas pathogenic signals cause, in addition to JA, accumulation of salicylic acid (SA) which stimulates production of acidic PR proteins. Transgenic tobacco plants expressing a gene for a small GTP binding protein respond abnormally to mechanical wounding to produce SA and consequently acidic PR proteins, suggesting that wound signals cross witli pathogen signaling pathways [Sano et al. (1994) Proc. Natl. Acad. Sci. USA 91: 10556]. This unusual signal crossing is associated with a highly sensitive wound-response of transgenic plants which, upon wounding, produce JA at least eighteen hours earlier than wild-type plants. When wildtype plants are wounded in the presence of the synthetic cytokinin, benzylaminopurine, production of JA begins six hours earlier than in untreated samples, and also SA begins to accumulate. The cytokinin antagonist, 2-chloro-4-cyclohexylamino-6-ethylamino-s-triazine, erases these effects. Because transgenic plants constitutively produce four- to six-fold higher amounts of endogenous cytokinins than wild-type plants, it is concluded that cytokinins are indispensable for control of endogenous levels of SA and JA.

Tobacco MAP kinase: a possible mediator in wound signal transduction pathways.

A complementary DNA encoding a mitogen-activated protein (MAP) kinase homolog has been isolated from tobacco plants. Transcripts of the corresponding gene were not observed in healthy tobacco leaves but began to accumulate 1 minute after mechanical wounding. In tobacco plants transformed with the cloned complementary DNA, trans inactivation of the endogenous homologous gene occurred, and both production of wound-induced jasmonic acid and accumulation of wound-inducible gene transcripts were inhibited. In contrast, the levels of salicylic acid and transcripts for pathogen-inducible, acidic pathogenesis-related proteins were increased upon wounding. These results indicate that this MAP kinase is part of the initial response of higher plants to mechanical wounding.

Fungal-induced synthesis of pathogenesis-related proteins in germinating maize embryos

Changes in the pattern of protein synthesis in germinating maize embryos have been examined after infection with the fungus Fusarium moniliforme. Two-dimensional gel electrophoresis was used to compare changes in proteins formed in vivo and in an in vitro translation system, from non-infected and F. moniliforme-infected embryos. Moreover, the protein patterns obtained using acidic or neutral extraction buffers have been compared. In vivo labelling experiments show that infection with F. moniliforme results in the induction of the synthesis of two major acid-soluble polypeptides with mol. wts of 24 and 17 kDa and with highly basic isoelectric points. Induction of these basic proteins is followed by their accumulation in tissues of the infected embryos. In addition to this, the analysis of the proteins formed during a 3 h pulse labelling with 35S-methionine reveals the synthesis of a group of proteins with low mol. wts (below 30 kDa) and acidic isoelectric points in response to fungal infection or treatment with fungal elicitors. Translation of mRNAs obtained from uninfected and F. moniliforme-infected embryos in vitro confirms the accumulation of mRNAs coding for acidic low mol. wt proteins on infection. However, contrary to the behaviour of the fungal-induced basic proteins, the fungalinduced acidic proteins do not accumulate in embryo tissues during germination. We conclude that expression and/or accumulation of basic and acidic pathogenesis-related proteins in response to fungal infection are differentially regulated during germination of maize embryos. Mechanisms for the control of the expression of defence-related genes, other than transcriptional activation, may play an important role in defence responses against fungal infection during seed germination.

Expression of the gene for a small GTP binding protein in transgenic tobacco elevates endogenous cytokinin levels, abnormally induces salicylic acid in response to wounding, and increases resistance to tobacco mosaic virus infection.

Tobacco plants transformed with rgp1, a gene encoding a Ras-related small GTP binding protein, were previously shown to exhibit a distinct reduction in apical dominance with increased tillering. These abnormal pheno-types were later found to be associated with elevated levels of endogenous cytokinins (zeatin and zeatin riboside). Analysis of the expression of several genes known to be affected by cytokinins identified a clear increase in the mRNA levels of genes encoding acidic pathogenesis-related proteins in both transgenic plants and their progenies. This increase was directly attributable to elevated levels of the acidic pathogenesis-related protein inducers, salicylic acid (SA) and salicylic acid beta-glucoside, due to an abnormal and sensitive response of the transgenic plants to wounding. In contrast, mRNA levels of the gene for proteinase inhibitor II, which is normally induced by wounding, were generally suppressed in the same wounded plants, probably due to SA overproduction. The changes in SA and pathogenesis-related protein levels in the transgenic plants resulted in a distinct increase in their resistance to tobacco mosaic virus infection. In normal plants, the wound and pathogen-induced signal transduction pathways are considered to function independently. However, the wound induction of SA in the transgenic plants suggests that overexpression of this small GTP binding protein somehow interferes with the normal signal pathways, possibly by affecting cytokinin biosynthesis, and results in cross-signaling between these two transduction systems.

CDNA cloning, structure and expression of a novel pathogenesis-related protein in bean.

We have isolated cDNA clones spanning the full length of a transcript (PvPR3) encoding a novel pathogenesis-related (PR) protein in bean (Phaseolus vulgaris). The PvPR3 transcript accumulates gradually over 24 hr in elicitor-treated cell suspensions. This pattern of expression is distinct from those of previously reported elicitor-induced transcripts in bean. Specifically, transcripts encoding two recently described acidic bean PR proteins, phenylpropanoid pathway enzymes, accumulate to maximal levels by 4-8 hr, while hydroxyproline-rich glycoprotein mRNA accumulation is delayed by several hours. The PvPR3 mRNA also accumulates after wounding of hypocotyls with kinetics comparable to those of mRNA encoding phenylpropanoid pathway mRNAs. PvPR3 appears to exist as a single gene within a family of approximately 15 related genes in the bean genome. The PvPR3 protein deduced from the cDNA sequences (14,950 Da pI = 10.0) lacks a putative signal peptide suggesting a cytosolic localization. Amino acid sequence comparisons with databases revealed that PvPR3 represents a new class of PR proteins without significant sequence homology to previously characterized PR proteins or other proteins.

Differential induction of acquired resistance and PR gene expression in tobacco by virus infection, ethephon treatment, UV light and wounding

Genes for acidic, extracellular and basic, intracellular pathogenesis-related (PR) proteins of tobacco were studied for their response to tobacco mosaic virus (TMV) infection, ethephon treatment, wounding and UV light. The genes encoding the acidic PR proteins (PR-1, PR-2, PR-3, PR-4 and PR-5) responded similarly to the different forms of stress. They appeared to be highly inducible by TMV, moderately inducible by ethephon treatment and UV light and not inducible by wounding. The genes for the basic counterparts of PR-1, PR-2, PR-3 and PR-5 also displayed a common stress response. However, this response was different from that of the acidic PR proteins. Here, the highest induction was obtained upon ethephon treatment, while the other stress conditions resulted in somewhat lower levels of expression. Most genes for acidic PR proteins are systemically induced in the uninfected upper leaves of TMV-infected plants, whereas the genes encoding the basic PR proteins are not. Increased levels of resistance to TMV, comparable to resistance obtained by pre-infection with the virus, were found in UV-irradiated leaves but not in wounded or ethephon-treated leaves. This indicates that the basic PR proteins are not involved in resistance to TMV infection. Tobacco phenylalanine ammonia-lyase genes were not inducible by the various stress conditions. The implications of these findings in relation to the phenomenon of acquired resistance are discussed.

Pathogenesis-related protein 4 is structurally homologous to the carboxy-terminal domains of hevein, Win-1 and Win-2

The extracellular, acidic pathogenesis-related protein, PR-4, was purified to homogeneity from leaves of Nicotiana tabacum infected with tobacco mosaic virus (TMV) and characterized by partial amino acid sequencing. Complementary DNA clones encoding PR-4 were isolated using an oligonucleotide probe based on the sequence of one of the peptides. The deduced PR-4 protein sequence was found to be related to a family of proteins including hevein and Win-1, which have an amino-terminal lectin domain and a carboxy-terminal domain of unknown function. PR-4 is homologous to the carboxy-terminus of these proteins but does not contain the lectin domain. Thus, the organization of the PR-4 family of proteins is similar to that of the plant chitinase family, in that both contain structural subclasses characterized by the presence or absence of an amino-terminal lectin domain. This observation is consistent with the proposal that the DNA encoding the lectin domain may be capable of transposing to form new genes encoding proteins of more complex, multi-domain structure. The expression of PR-4 mRNA was found to increase dramatically in response to TMV infection and the time course of RNA accumulation was similar to that of other PR proteins.

Subcellular localization of acidic and basic PR proteins in tobacco mosaic virus-infected tobacco

Infection of Samsum NN tobacco with tobacco mosaic virus (TMV) results in the induction of the synthesis of acidic and basic isoforms of many pathogenesis-related (PR) proteins. By immunogold-electromicroscopy we have shown that PR proteins accumulate mainly in cells around the necrotic spots of TMV-induced lesions. The acidic chitinases, beta-(1,3)-glucanases and thaumatin-like proteins were found to accumulate in extracellular "pocket-like" vesicles while the basic chitinases were found in electron dense inclusion bodies in the vacuoles. These structures were not detectable in PR-containing leaves devoid of virus nor in healthy plants.

Identification of a basic pathogenesis-related, thaumatin-like protein of virus-infected tobacco as osmotin

A basic PR protein was isolated and characterized from Samsun NN tobacco leaves infected with tobacco mosaic virus. The protein is serologically-related to the pathogenesis-related (PR) proteins R and S, the thaumatin-like (TL) proteins so-called because of their high level of homology with thaumatin, a sweet-tasting protein from the West African shrub Thaumatococcus daniellii Benth. Its amino acid composition and its NH 2 -terminal sequence indicate that this PR protein is in fact osmotin, a protein known to accumulate in tobacco cells in response to osmotic stress. A specific serum was obtained and used in immunoblotting experiments to study the serological relationships of TL-proteins of tobacco and to compare the induction of osmotin and acidic PR proteins R and S during the hypersensitive reaction of tobacco to tobacco mosaic virus.

Induction of pathogenesis-related proteins by spermidine exogenously supplied to detached tobacco leaves

Continuous treatment with spermidine or 1-aminocyclopropane-1-carboxylic acid stimulated ethylene production and ethylene-forming enzyme activity and accelerated chlorophyll breakdown in detached tobacco leaves. The treatments also induced the production of eleven major acidic pathogenesis-related proteins, which were also produced during the hypersensitive reaction to tobacco necrosis virus. A delay between the onset of the stimulated ethylene increase and the detection of PR-proteins was found; ethylene production was stimulated after a few hours of treatment, whereas one, three and all the eleven PR-proteins were detected by polyacrylamide gel electrophoresis of fluid extracts after 2, 4 and 6 days of treatments, respectively. The possible causal relationship between stimulation of ethylene production and PR-protein accumulation is discussed.

Two‐dimensional polyacrylamide gel electrophoresis of extracellular soybean pathogenesis‐related proteins using PhastSystem

Acidic and basic pathogenesis-related proteins (PR-Ps) were extracted from the intercellular fluid (IF) of soybean leaves, locally infected with tobacco necrosis virus and showing necrotic local lesions. Proteins were detected by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using PhastSystem and precast commercially available gels. Extracts from healthy leaves were run as controls. PR-Ps were first run under native PAGE conditions or isoelectric focusing (IEF), the gels stained with Coomassie Blue, then run under sodium dodecyl sulfate (SDS)-denaturing conditions and finally stained with silver. Ten major acidic PR-Ps were separated; their Mr's were close to those found by conventional PAGE. Their isoelectric points ranged from 3.5 to 5.0. Ten basic PR-Ps were separated and their Mr's estimated. None of these acidic or basic soybean PR-Ps was a glycoprotein. PAGE with PhastSystem and precast gels gives reliable results, comparable with those from conventional 2D-PAGE, with simpler experimental procedures. By electrophoresing Coomassie-stained gels with SDS in the second dimension, we were able to control the first-dimensional separation and to avoid laborious protocols generally adopted with unstained gels.

Localization of pathogenesis-related proteins in infected and non-infected leaves of samsun NN tobacco during the hypersensitive reaction to tobacco mosaic virus

The ten major, acidic pathogenesis-related proteins (PRs) accumulating in Samsun NN tobacco during the hypersensitive reaction to tobacco mosaic virus (TMV) were present in the same relative proportions in leaf extracts and in intercellular fluid washed out infected leaves. About 60% of the PRs present in the leaves was recovered in the intercellular fluid. Up to 65% of the PRs was washed out of upper, non-infected leaves of plants of which the lower leaves had been inoculated, indicating that the presence of PRs in the apoplast was not the result of tissue necrosis. Protoplasts from these tissues retained at most 5% of of the PRs, suggesting that upon synthesis they are all transported across the plasmalemma. In non-infected leaves the relative proportions of the ten PRs differed from those in infected leaves. PR1c was virtually absent, whereas PRs 2a, 2b and 3a were relatively abundant and the amount of PR 5 was low. Infected leaves contained relatively small amounts of up to seven additional basic proteins that were preferentially solubilized at pH 3. While three of these proteins were relatively abundant in intercellular fluid, less than 5% of the others was recovered in this way, suggesting that they are intracellular proteins.

Detection of β‐ 1,3‐glucanase activity after native polyacrylamide gel electrophoresis: Application to tobacco pathogenesis‐related proteins

Beta-1,3-Glucanase (laminarinase) activity was detected after polyacrylamide gel electrophoresis under native conditions by using laminarin as substrate. Following incubation of gels, laminarin was stained with Aniline Blue. Under UV illumination, lysis zones appeared as dark bands against a fluorescent background. As low as 0.001 unit of commercial Penicillium laminarinase could be observed after incubating the polyacrylamide gel for 45 min at pH 5.0. Extracts of commercial Penicillium laminarinase exhibited four bands with lytic activity towards laminarin. Analysis of intercellular fluid extracts of tobacco mosaic virus-infected tobacco leaves revealed four beta-1,3-glucanases corresponding to three acidic pathogenesis-related proteins, b4 (2), b5 (N) and b6b (0), and one basic protein. The presence of laminarin in gels retarded the migration of some proteins with beta 1,3-glucanase activity. This change in electrophoretic mobility could be used as a complementary affinity test for identifying proteins with beta-1,3-glucanase activity.

Serological relationships between pathogenesis-related leaf proteins from four Nicotiana species, Solanum tuberosum, and Chenopodium amaranticolor

Leaves of Nicotiana tabacum L. cv. Xanthi-nc, Nicotiana clevelandii Gray, Nicotiana rustica L., and Chenopodium amaranticolor Coste & Reyn. were infected with the U1 strain of tobacco mosaic virus and leaves of Nicotiana sylvestris Speg. & Comes and Solanum tuberosum L. cv. Kennebec were infected with the U2 strain. After 10 days, pathogenesis-related (PR) proteins from intercellular fluid extracts were separated by nondenaturing polyacrylamide gel electrophoretic (PAGE) systems for acidic or basic proteins, sometimes followed by denaturating PAGE with lithium dodecyl sulfate. PR proteins were then transferred to nitrocellulose to determine serological relationships. Using an antiserum directed against native PR protein b4 from 'Xanthi-nc' tobacco, serologically reacting acidic PR proteins could be detected in all plant species. Except for potato, related basic PR proteins were also found in all species. Serological relationships were further studied between 'Xanthi-nc' tobacco and 'Kennebec' potato PR proteins separated in PAGE gels under native conditions. Potato basic PR proteins with Rfs of 0.83, 0.85, and 1.00 were serologically related to tobacco acidic PR proteins of the b4 group. Potato acidic PR proteins with Rfs of 0.56, 0.58, 0.61, and 0.64 were related to tobacco acidic PR proteins of the b4 group. Potato acidic PR proteins with Rfs of 0.18 and 0.21 reacted with an antiserum against tobacco PR protein b9. Three potato acidic peroxidases were also serologically related to tobacco peroxidases b6a and b7a. Analysis with denaturing PAGE systems gave results difficult to interpret because bovine carbonic anhydrase, for example, cross-reacted with an antiserum against tobacco PR protein b4. A similar relationship could not be detected in PAGE systems under native conditions.

Homology between chitinases that are induced by TMV infection of tobacco

Recently, four chitinases have been detected in tobacco mosaic virus (TMV) infected tobacco: two acidic chitinases that were identified as pathogenesis-related (PR) proteins P and Q and two basic chitinases (Legrand et al., Proc.Natl. Acad. Sci. USA, in press). Here, it was shown that P and Q are closely serologically related but not related to other known acidic tobacco PR proteins. Antisera to P and Q were used to characterize translation products of TMV-induced mRNAs that were hybrid-selected with cDNA clones described previously (Hooft van Huijsduijnen et al., EMBO J 5: 2057-2061, 1986). In this way cDNA clones corresponding to the acidic and basic chitinases were identified. The partial amino acid sequences of the acidic and basic tobacco chitinases that were represented in the clones, showed an approximately 70% homology to each other and to the sequence of a bean chitinase. Although the acidic and basic chitinases differ in apparent molecular weight, they were found to have homologous C-termini.Hybridization of cDNA probes to genomic blots indicated that the acidic and basic chitinases are each encoded by two to four genes in the amphidiploid genome of Samsun NN tobacco. A similar complexity was found for the genes encoding the tobacco PR protein that is homologous to the sweet-tasting protein thaumatin and to the bifunctional trypsin/α-amylase inhibitor from maize.