Pathogenesis-related acidic beta-1,3-glucanase genes of tobacco are regulated by both stress and developmental signals

Three pathogenesis-related (PR) proteins of tobacco are acidic isoforms of beta-1,3-glucanase (PR-2a, -2b, -2c). We have cloned and sequenced a partial cDNA clone (lambda FJ1) corresponding to one of the PR-2 beta-1,3-glucanases. A small gene family encodes the PR-2 proteins in tobacco, and similar genes are present in a number of plant species. We analyzed the stress and developmental regulation of the tobacco PR-2 beta-1,3-glucanases by using northern and western analyses and a new technique to assay enzymatic activity. Stress caused by both thiamine and tobacco mosaic virus (TMV) infection resulted in a dramatic increase in the levels of PR-2 mRNA, protein, and enzyme activities. The increased PR-2 gene expression in upper uninoculated leaves of plants infected with TMV also suggests a role in systemic acquired resistance. During floral development, a number of beta-1,3-glucanase activities were observed in all flower tissues. However, PR-2 polypeptides were observed only in sepal tissue. In contrast, an mRNA that hybridized to the PR-2 cDNA was present in stigma/style tissue and the sepals. Primer extension analysis confirmed the identity of the PR-2 mRNA in sepals, but indicated that the beta-1,3-glucanase gene expressed in the stigma/style of flowers was distinct from the PR-2 genes. The induction of PR-2 protein synthesis by both stress and developmental signals was accompanied by a corresponding increase in the steady-state levels of PR-2 mRNA, suggesting that PR-2 gene expression is regulated, in part, at the level of mRNA accumulation.     

Silencing of acidic pathogenesis-related PR-1 genes increases extracellular beta-(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.     

Cholera toxin elevates pathogen resistance and induces pathogenesis-related gene expression in tobacco.

In animals, plants and fungi, cholera toxin (CTX) can activate signalling pathways dependent on heterotrimeric GTP binding proteins (G-proteins). We transformed tobacco plants with a chimeric gene encoding the A1 subunit of CTX regulated by a light-inducible wheat Cab-1 promoter. Tissues of transgenic plants expressing CTX showed greatly reduced susceptibility to the bacterial pathogen Pseudomonas tabaci, accumulated high levels of salicylic acid (SA) and constitutively expressed pathogenesis-related (PR) protein genes encoding PR-1 and the class II isoforms of PR-2 and PR-3. In contrast, the class I isoforms of PR-2 and PR-3 known to be induced in tobacco by stress, by ethylene treatment and as part of the hypersensitive response to infection, were not induced and displayed normal regulation. In good agreement with these results, microinjection experiments demonstrated that CTX or GTP-gamma-S induced the expression of a PR1-GUS reporter gene but not that of a GLB-GUS reporter gene containing the promoter region of a gene encoding the class I isoform of PR-2. Microinjection and grafting experiments strongly suggest that CTX-sensitive G-proteins are important in inducing the expression of a subset of PR genes and that these G-proteins act locally rather than systemically upstream of SA induction.     

Tobacco genes encoding acidic and basic isoforms of pathogenesis-related proteins display different expression patterns

The induction by cytokinin stress and ethylene of nine different tobacco mosaic virus-inducible mRNA classes (termed A-I) encoding pathogenesis-related (PR) proteins was studied. The induced mRNA levels were compared to basal levels in healthy tobacco plants grown in tissue culture and in a greenhouse. Cytokinin stress and ethylene were found to induce different subsets of the mRNAs, indicating that ethylene is not the primary inducing signal in cytokinin-stressed shoots. mRNAs F, H and G encoding the basic hydrolytic enzymes chitinase, -1,3-glucanase and a basic equivalent of PR-1, respectively, were found to be expressed at high levels in roots of healthy plants. mRNAs D, I and B encoding the acidic equivalents of the proteins proved to be present at low levels in healthy plants. These results indicate that genes encoding basic and acidic isoforms of pathogenesis-related proteins are differentially regulated.     

Isolation and characterization of cDNA clones encoding pathogenesis-related proteins from tobacco mosaic virus infected tobacco plants.

Infection of the tobacco cultivar Samsun NN by tobacco mosaic virus (TMV) results in a hypersensitive response. During this defense reaction several host encoded proteins, known as pathogenesis-related proteins (PR-proteins), are induced. Poly(A)+ RNA from TMV infected tobacco plants was used to construct a cDNA library. Thirty two cDNA clones were isolated and after digestion with different restriction endonucleases, twenty clones were found to code for PR-1a, six clones for PR-1b, and four clones for PR-1c. Two independent cDNA clones of each class were further characterized by DNA sequence analysis. All clones analyzed contained the 138 amino acid coding regions of their respective mature proteins, but only partial sequences of the signal peptides. Minor differences between the nucleotide sequences for clones belonging to the same class were detected. Comparison of the amino acid sequence for PR-1a deduced from its nucleotide sequence with published data obtained by Edman degradation of the protein showed four differences. Analysis of the 3' ends of the cDNA clones indicates that various alternate poly(dA) addition sites are used. Southern blot analysis using these cDNAs as probes suggests the presence of multiple PR-protein genes in the genomes of tobacco and tomato plants.     

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

Summary 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.     

Structure of tobacco genes encoding pathogenesis-related proteins from the PR-1 group.

Infection of Samsun NN tobacco with tobacco mosaic virus (TMV) was found to induce the synthesis of mRNA encoding a basic protein with a 67% amino acid sequence homology to the known acidic pathogenesis-related (PR) proteins 1a, 1b and 1c. By Southern blot hybridization it was shown that the tobacco genome contains at least eight genes for acidic PR-1 proteins and a similar number of genes encoding the basic homologues. Clones corresponding to three of the genes for acidic PR-1 proteins were isolated from a genomic library of Samsun NN tobacco. The nucleotide sequence of these genes and their flanking sequences were determined. One clone was found to correspond to the PR-1a gene; the two other clones do not correspond to known TMV-induced PR-1 mRNA's and may represent silent genes. Compared to the PR-1a gene, these genes contain an insertion or deletion in the putative promoter region and mutations affecting the PR-1 reading frame.     

Purification and serological characterization of three basic 15-kilodalton pathogenesis-related proteins from tomato

In tomato (Lycopersicon esculentum) several acidic and basic apoplastic pathogenesis-related (PR) proteins are induced upon inoculation with virulent or avirulent races of Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif). One of the most predominant and best characterized tomato PR proteins is P14, a basic protein that shows homology to the tobacco (Nicotiana tabacum) PR-1 protein family. To investigate whether, by analogy with these tobacco PR-1 proteins, P14 also belongs to a family of differently charged isomers, the abundantly occurring PR proteins with molecular masses around 15 kilodaltons (kD) were purified from apoplastic fluids isolated from C. fulvum-infected tomato. Three basic proteins migrating similarly to P14 on sodium dodecyl sulfate polyacrylamide gels were purified to homogeneity by gel filtration followed by high resolution liquid chromatography. Two proteins (15.5 kD, isoelectric point [pl] 10.9 and 10.7 appeared to be serologically related to each other and to the tobacco PR-1 proteins. A third protein (15 kD, pl 10.4) was not serologically related to any other tomato PR protein but was found to be related to PR-R from tobacco.     

Pathogenesis-related PR-1 proteins are antifungal. Isolation and characterization of three 14-kilodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophthora infestans.

Three distinct basic 14-kD proteins, P14a, P14b, and P14c, were isolated from tomato (Lycopersicon esculentum Mill. cv Baby) leaves infected with Phytophthora infestans. They exhibited antifungal activity against P. infestans both in vitro (inhibition of zoospore germination) and in vivo with a tomato leaf disc assay (decrease in infected leaf surface). Serological cross-reactions and amino acid sequence comparisons showed that the three proteins are members of the PR-1 group of pathogenesis-related (PR) proteins. P14a and P14b showed high similarity to a previously characterized P14, whereas P14c was found to be very similar to a putative basic-type PR-1 from tobacco predicted from isolated DNA clones. This protein, named PR-1 g, was purified from virus-infected tobacco (Nicotiana tabacum Samsun NN) leaves and characterized by amino acid microsequencing, along with the well-known acidic tobacco PR-1a, PR-1b, and PR-1c. The various tomato and tobacco PR-1 proteins were compared for their biological activity and found to display differential fungicidal activity against P. infestans in both the in vitro and in vivo assays, the most efficient being the newly characterized tomato P14c and tobacco PR-1g.     

The Comparative Analysis of Osmotins and Osmotin-Like PR-5 Proteins

Abstract: One of the ways that plants respond to biotic and/or abiotic stress factors is the accumulation of pathogenesis-related proteins of class 5 (PR-5), which are evolutionary conserved in the plant kingdom. Within the PR-5 family, a distinct subgroup of osmotin and closely related proteins has been characterized. In contrast to the extracellular forms of PR-5 proteins, osmotins presumably accumulate in the vacuole of the cell. They contain a C-terminal propeptide that is considered to be a determinant for vacuolar targeting. The comparison of the three-dimensional structure of tobacco PR-5 d with the sequences of some osmotins showed that the proteins consist of three conserved domains, with the acidic cleft between domains I and II. Besides the constitutive species and tissue-specific presence, the osmotins are also induced by several abiotic and biotic stresses. Among them, fungal infections can elicit osmotin gene expression, and most known proteins from the family have antifungal activity in in vitro assays. In agreement with the osmotin structure and data on the activity of similar proteins, a two-step mechanism, which involves reaction of osmotins with the fungal wall and the permeabilization of fungal membranes, is discussed.     

Purification and characterization of 8 of the pathogenesis-related proteins in tobacco leaves reacting hypersensitively to tobacco mosaic virus

Summary Leaves of tobacco plants (Nicotiana tabacum cv. Samsun NN) which are reacting hypersensitively to infection with tobacco mosaic virus contain 10 major pathogenesis-related (PR) proteins which are absent, or present in small amounts in uninfected leaves. We describe here a preparative procedure of purification of the tobacco PR-proteins which involves a combination of conventional and high-performance liquid chromatography. The separation and isolation of the proteins were based on differences in net charge at different pH values, in isoelectric point and in apparent molecular weight. This procedure led to the purification to homogeneity of 8 PR-proteins, as shown by polyacrylamide slab gel electrophoresis (PAGE) of the purified proteins under denaturing and non-denaturing conditions. These were the 3 well-known proteins PR-1a,-1b and-1c, and 5 other major PR-proteins, called PR-2,-N,-O,-P and-Q, according to the nomenclature of Van Loon (39). None of the purified PR-proteins gave a positive Schiff reaction for carbohydrate content. Molecular weight determinations from gel permeation chromatography and from sodium dodecyl sulphate (SDS)-PAGE indicated that all 8 PR-proteins were monomers and that three groups could be distinguished among them. The first group is the PR-1 group containing PR-1a,-1b and-1c (12000 MW), the second consists of PR-P and PR-Q (14000 MW) and the third of PR-2, PR-N and PR-O (25000 MW). In the PR-1 group, PR-1a can be distinguished clearly from the two other members on denaturing slab gels containing both SDS and urea.     

A strobilurin fungicide enhances the resistance of tobacco against tobacco mosaic virus and Pseudomonas syringae pv tabaci

The strobilurin class of fungicides comprises a variety of synthetic plant-protecting compounds with broad-spectrum antifungal activity. In the present study, we demonstrate that a strobilurin fungicide, F 500 (Pyraclostrobin), enhances the resistance of tobacco (Nicotiana tabacum cv Xanthi nc) against infection by either tobacco mosaic virus (TMV) or the wildfire pathogen Pseudomonas syringae pv tabaci. F 500 was also active at enhancing TMV resistance in NahG transgenic tobacco plants unable to accumulate significant amounts of the endogenous inducer of enhanced disease resistance, salicylic acid (SA). This finding suggests that F 500 enhances TMV resistance in tobacco either by acting downstream of SA in the SA signaling mechanism or by functioning independently of SA. The latter assumption is the more likely because in infiltrated leaves, F 500 did not cause the accumulation of SA-inducible pathogenesis-related (PR)-1 proteins that often are used as conventional molecular markers for SA-induced disease resistance. However, accumulation of PR-1 proteins and the associated activation of the PR-1 genes were elicited upon TMV infection of tobacco leaves and both these responses were induced more rapidly in F 500-pretreated plants than in the water-pretreated controls. Taken together, our results suggest that F 500, in addition to exerting direct antifungal activity, may also protect plants by priming them for potentiated activation of subsequently pathogen-induced cellular defense responses.     

Crystal structure of tobacco PR-5d protein at 1.8 A resolution reveals a conserved acidic cleft structure in antifungal thaumatin-like proteins

The crystal structure of tobacco PR-5d, an antifungal thaumatin-like protein isolated from cultured tobacco cells, was determined at the resolution of 1.8 A. The structure consists of 208 amino acid residues and 89 water molecules with a crystallographic R-factor of 0.169. The model has good stereochemistry, with respective root-mean-square deviations from the ideal values for bond and angle distances of 0.007 A and 1.542 degrees. Of the homologous PR-5 proteins, only those with antifungal activity had a common motif, a negatively charged surface cleft. This cleft is at the boundary between domains I and II, with a bottom part consisting of a three-stranded antiparallel beta-sheet in domain I. The acidic residues located in the hollow of the cleft form the beta-sheet region. Sequence and secondary structure analyses showed that the amino acid residues comprising the acidic cleft of PR-5d are conserved among other antifungal PR-5 proteins. This is the first report on the high-resolution crystal structure of an antifungal PR-5 protein. This structure provides insight into the function of pathogenesis-related proteins.     

Lupinus luteus pathogenesis-related protein as a reservoir for cytokinin

Plant pathogenesis-related (PR) proteins of class 10 (PR-10) are small and cytosolic. The main feature of their three-dimensional structure is a large cavity between a seven-stranded antiparallel β-sheet and a long C-terminal α-helix. Although PR-10 proteins are abundant in plants, their physiological role remains unknown. Recent data have indicated ligand binding as their possible biological function. The article describes the structure of a complex between a classic PR-10 protein (yellow lupine LlPR-10.2B) and the plant hormone, trans-zeatin. Previously, trans-zeatin binding has been reported in a structurally related cytokinin-specific binding protein, which has a distant sequence relation with classic PR-10 proteins. In the present 1.35 Å resolution crystallographic model, three perfectly ordered zeatin molecules are found in the binding cavity of the protein. The fact that three zeatin molecules are bound by the protein when only a fourfold molar excess of the ligand was used indicates an unusual type of affinity for this ligand and suggests that LlPR-10.2B, and perhaps other PR-10 proteins as well, acts as a reservoir of cytokinin molecules in the aqueous environment of the cell.     

Salicylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco.

Systemic induction of pathogenesis-related (PR) proteins in tobacco, which occurs during the hypersensitive response to tobacco mosaic virus (TMV), may be caused by a minimum 10-fold systemic increase in endogenous levels of salicylic acid (SA). This rise in SA parallels PR-1 protein induction and occurs in TMV-resistant Xanthi-nc tobacco carrying the N gene, but not in TMV-susceptible (nn) tobacco. By feeding SA to excised leaves of Xanthi-nc (NN) tobacco, we have shown that the observed increase in endogenous SA levels is sufficient for the systemic induction of PR-1 proteins. TMV infection became systemic and Xanthi-nc plants failed to accumulate PR-1 proteins at 32 degrees C. This loss of hypersensitive response at high temperature was associated with an inability to accumulate SA. However, spraying leaves with SA induced PR-1 proteins at both 24 and 32 degrees C. SA is most likely exported from the primary site of infection to the uninfected tissues. A computer model predicts that SA should move rapidly in phloem. When leaves of Xanthi-nc tobacco were excised 24 hr after TMV inoculation and exudates from the cut petioles were collected, the increase in endogenous SA in TMV-inoculated leaves paralleled SA levels in exudates. Exudation and leaf accumulation of SA were proportional to TMV concentration and were higher in light than in darkness. Different components of TMV were compared for their ability to induce SA accumulation and exudation: three different aggregation states of coat protein failed to induce SA, but unencapsidated viral RNA elicited SA accumulation in leaves and phloem. These results further support the hypothesis that SA acts as an endogenous signal that triggers local and systemic induction of PR-1 proteins and, possibly, some components of systemic acquired resistance in NN tobacco.     

Differential targeting of the tobacco PR-1 pathogenesis-related proteins to the extracellular space and vacuoles of crystal idioblasts.

Several biochemical and localization studies have shown that the acidic isoforms of the tobacco pathogenesis-related (PR) proteins, PR-1a, -1b and -1c are secreted to the extracellular spaces of leaves in response to pathogen infection or chemical treatment. Here we report the differential accumulation of these proteins within the vacuoles of specialized cells known as crystal idioblasts. In situ hybridization analysis indicated that crystal idioblasts expressed the PR-1 genes at the mRNA level and suggested that PR-1 proteins were synthesized by these cells. Transgenic plants which constitutively express a chimeric gene encoding an acidic PR-1b isoform also accumulated PR-1 protein in the extracellular spaces and within crystal idioblast vacuoles. Analysis of mRNA derived from these transgenic plants indicated that expression of the introduced PR-1b gene was responsible for the accumulation of PR-1 protein in these two distinct locations. The synthesis and accumulation within crystal idioblasts of PR-1 proteins, which are secreted by other cell types, indicates that idioblasts sort these proteins in a unique manner. Moreover, this suggests that protein sorting in higher plants may be modulated in a cell specific manner.     

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.     

Protein dephosphorylation mediates salicylic acid-induced expression of PR-1 genes in tobacco

Several lines of evidence suggest that salicylic acid (SA) is an endogenous signal for the activation of several plant defense responses, including the expression of genes encoding pathogenesis-related (PR) proteins such as the acidic PR-1 proteins. During recent years, studies have suggested that interaction of SA with catalase and ascorbate peroxidase leads to two signals in tobacco - elevated H₂O₂ levels and lipid peroxides. However, to date, relatively little is known about the molecular and biochemical mechanisms that mediate transduction beyond these signals or through other SA-effector proteins. Using protein kinase and phosphatase inhibitors, this study demonstrates that PR-1 gene induction can be mediated by dephosphorylation of serine/threonine residue(s) of two or more unidentified phosphoproteins. The protein phosphatase inhibitors, okadaic acid and calyculin A blocked SA-mediated induction of PR-1 genes, implying the involvement of a phosphoprotein downstream of SA. The protein kinase inhibitors K-252a and staurosporine induced PR-1 gene expression. PR-1 gene induction by K-252a was suppressed by okadaic acid. Surprisingly, this induction was also suppressed in NahG transgenic tobacco plants which convert SA to catechol. Moreover, K-252a stimulated production of SA and its glucoside, suggesting that another phosphoprotein acts upstream of SA. Taken together, these results suggest that there are two (or more) phosphoproteins which function in the same signal transduction pathway leading to PR-1 gene induction. The SA-inducible acidic PR-2 genes were similarly affected by the inhibitors, while the genes for actin and phenylalanine ammonia lyase were not.