Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis>

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.     

Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. III. PR-protein gene expression and ethylene evolution in tomato cell lines transgenic for phytohormone-related bacterial genes

 Previous work carried out in our laboratory has shown that, in tomato, the alteration of endogenous phytohormone equilibria through the integration of Agrobacterium tumefaciens genes for auxin and cytokinin synthesis can modify the active defense response to Fusarium oxysporum f. sp. lycopersici. The susceptible cv ‘Red River’ acquires a stable competence for active defense, particularly when the phytohormone equilibrium is altered in favour of cytokinins. Here, we analyse the expression of genes involved in the defense response against pathogens, i.e. pathogenesis-related (PR)-protein genes, in the susceptible ‘Red River’ and resistant ‘Davis’ cultivars transgenic for the aforementioned genes. Fungal cell-wall components, glutathione, salicylic acid and the ethylene-forming ethephon are used as “probes” for the induction of defense processes, including ethylene production. The data obtained show that the extracellular PR-proteins (acidic chitinase and PR-1 protein) that were inducible in the control tissue of the resistant ‘Davis’ cultivar and not expressed in the susceptible ‘Red River’ cultivar became constitutive in the transgenic tissues of both. On the other hand, expression of the intracellular PR-proteins (basic chitinase and β-1,3-glucanase) was found to be constitutive in all cases, both in the control and in the transgenic cell lines of the resistant and the susceptible tomato cultivars. Ethylene production was higher in ‘Davis’ than in ‘Red River’, and significantly increased in the transgenic cell lines, particularly when cytokinin synthesis was altered. Received: 25 February 1998 / Accepted: 7 April 1998     

Effects of fungal infection and wounding on the expression of chitinases and β-1,3 glucanases in near-isogenic lines of barley

Chitinases (EC 3.2.1.14) and β -1.3 glucanases (EC 3.2.1.39) have been known to play a vital role in the defense of plants against fungal pathogens. The pattern of induction of these two enzymes subsequent to infection by powdery mildew was studied in 10 pairs of near-isogenic lines of barley ( Hordeum vulgare L.) which possess powdery mildew resistance genes. These isogenic lines have been grotiped according to their reaction to the fungus. The induction patterns varied between the resistant and the susceptible cultivars within each group and between different groups. More tsozymcs were induced in susceptible varieties of highly resistant groups and the overall levels and the number of isozymes of chitinases and β -1.3 glucanases were lower in groups with low resistance. The effect of powdery mildew infection and mechanical wounding on the cellular localization of chitinases and β -1.3 glucanases in barley leaves has also been studied. The 31 kDa leaf chitinase, L-CH2, and trace amounts of a 25 kDa chitinase. L-CH3. were present in healthy leaves. Wounding increased the levels of L-CH3 within I ft h. Powdery mildew infection increased the levels of L-CH3 both in intercellular fluid and in intracellular extract of leaves. A /3-I.3 glucanase. GH, also increased after infection and wounding. In infected barley leaves, GL-1 was present both in intercellular space and intracellular extract. It is concluded that powdery mildew resistance genes exhibit qualitative and quantitative differences in the expression of chitinases and β -1.3 glucanases. Further, chitinases and β -1.3 glucanases appear to be a response to active infection rather than the factors responsible for disease resistance.     

Characterization of chitinases and β-1,3-glucanases in grapefruit flavedo during fruit development

Chitinase (EC 3.2.1.14) and β-1,3-glucanase (EC 3.2.1.39) activities in the flavedo of grapefruit ( Citrus paradisi cv. Marsh) were determined at 17 times during the course of fruit development. Chitinase activity is initially high in flavedo, but drops rapidly and is low, although fairly constant throughout the remainder of fruit development. In contrast to chitinase, β-1,3-glucanase activity is lowest in young fruit and increases during development. Western blots of crude flavedo extracts following SDS-PAGE were probed with antibodies raised against purified citrus chitinase and β-1,3-glucanase. Results of immunostaining revealed that changes in the activities of chitinase and β-1,3-glucanase were reflected in the amount of chitinase and glucanase protein present in the extracts. Only a single chitinase band was detected on western blots of crude flavedo extracts, whereas one glucanase band was present in young fruit and a second one appeared later in older fruit. Partial purification of flavedo chitinases and glucanases was performed using extracts prepared from immature and mature fruit for the two enzymes, respectively. Acidic and basic forms of both enzymes were present in the extracts; acidic and basic forms of chitinase were present in nearly equal amounts whereas basic glucanases predominated (91% of total activity). Acidic and basic chitinases differed in substrate specificity as well as products of degradation indicating the heterogeneous nature of the enzymes. Both acidic and basic glucanases required the presence of β-1,3 linkages for activity, were active against both soluble and insoluble β-1,3 glucans and generated similar products.     

Intercellular proteins and β-1,3-glucanase activity associated with leaf rust resistance in wheat

To investigate biochemical aspects of resistance conferred by the Lr35 gene for adult-plant resistance in wheat ( Triticum aestivum L.) to leaf rust, pathogen development was related to intercellular protein composition and β -1,3-glucanase (EC 3.2.1.39) activities at three growth stages in infected and uninfected resistant (RL6082 [Thatcher/ Lr35 ]) and susceptible (Thatcher) plants. Leaf rust symptoms produced by pathotype UVPrt9 of Puccinia recondita f. sp. tritici showed that resistance conferred by Lr35 was most effective at the flag leaf stage. Furthermore, fluorescence microscopy indicated that resistance was strongly associated with hypersensitive cell death of invaded tissue. According to polypeptide profiles, intercellular proteins with molecular masses of 35, 33, 31 and 26 kDa were constitutively present at higher levels in resistant than in susceptible plants at the flag leaf stage. Four intercellular proteins (35, 33, 32 and 31 kDa) serologically related to β -1,3-glucanase were present in resistant and susceptible genotypes during all stages of plant growth. Resistance was associated with high constitutive levels of β -1,3-glucanase activity. Susceptibility on the other hand was associated with low constitutive levels of β -1,3-glucanase, while high levels were induced by infection during more advanced stages of colonization. Our results suggest that β -1,3-glucanase is involved in the defense response controlled by the Lr35 gene.     

Chitinase and β-1,3-glucanase activities in chickpea (Cicer arietinum). Induction of different isoenzymes in response to wounding and ethephon

Chitinase and β-1,3-glucanase activities were assayed in roots, stems and leaves of 12-day-old chickpea ( Cicer arietinum L.) plants. While glucanase activity was higher in roots than in the aerial parts of the plant, leaves had higher Chitinase activity. Both glucanase and chitinase activities were induced in roots and stems in response to wounding (excision into 1-cm pieces), with activity increasing 6 h after treatment, reaching a maximum between 24 and 48 h, and thereafter remaining nearly constant up to 72 h. Ethephon treatment also induced β-1,3-glucanase and chitinase activities in stems but not in roots. Both enzymes occurred in root and stem tissues as a complex mixture of isoenzymes. At least four different peaks with glucanase and chitinase activities could be resolved by gel filtration chromatography on Sephacryl S-200 and chromatofocusing on PBE 94 (pH 4–7). Following ammonium sulfate precipitation and ion exchange on CM- and DEAE-Trisacryl, three β-1,3-glucanase and chitinase fractions, referred to as basic, neutral and acidic, were separated on the basis of their chromatographic behaviour. Most of the total protein (75%) of stem extracts was found in the acidic fraction, whereas the major glucanase (53%) and chitinase (62%) activities were in the basic and neutral fractions, respectively. While wounding resulted in an increase in the neutral glucanase and chitinase activities, the activities of the acidic fractions were promoted by ethephon.     

Oxidative responses of resistant and susceptible cereal leaves to symptomatic and nonsymptomatic cereal aphid (Hemiptera: Aphididae) feeding.

The impact of the leaf-chlorosis-eliciting Russian wheat aphid, Diuraphis noxia (Mordvilko), and the nonchlorosis-eliciting bird cherry-oat aphid, Rhopalosiphum padi (L.), feeding on D. noxia-susceptible and -resistant cereals was examined during the period (i.e., 3, 6, and 9 d after aphid infestation) that leaf chlorosis developed. After aphid number, leaf rolling and chlorosis ratings, and fresh leaf weight were recorded on each sampling date, total protein content, peroxidase, catalase, and polyphenol oxidase activities of each plant sample were determined spectrophotometrically. Although R. padi and D. noxia feeding caused significant increase of total protein content in comparison with the control cereal leaves, the difference in total protein content between R. padi and D. noxia-infested leaves was not significant. Although R. padi-feeding did not elicit any changes of peroxidase specific activity in any of the four cereals in comparison with the control leaves, D. noxia feeding elicited greater increases of peroxidase specific activity only on resistant 'Halt' wheat (Triticum aestivum L.) and susceptible 'Morex' barley (Hordeum vulgare L.), but not on susceptible 'Arapahoe' and resistant 'Border' oat (Avena sativa L.). D. noxia-feeding elicited a ninefold increase in peroxidase specific activity on Morex barley and a threefold on Halt wheat 9 d after the initial infestation in comparison with control leaves. Furthermore, D. noxia feeding did not elicit any differential changes of catalase and polyphenol oxidase activities in comparison with either R. padi feeding or control leaves. The findings suggest that D. noxia feeding probably results in oxidative stress in plants. Moderate increase of peroxidase activity (approximately threefold) in resistant Halt compared with susceptible Arapahoe wheat might have contributed to its resistance to D. noxia, whereas the ninefold peroxidase activity increase may have possibly contributed to barley's susceptibility. Different enzymatic responses in wheat, barley, and oat to D. noxia and R. padi feeding indicate the cereals have different mechanisms of aphid resistance.     

Induction of β-1,3-glucanase and chitinase in Vigna aconitifolia inoculated with Macrophomina phaseolina

Pathogenesis-related (PR) proteins are induced in response to pathogen attack. In the present study, the induction of PR proteins in response to the fungal pathogen Macrophomina phaseolina was investigated in 15-day- and 1-month-old plants of Vigna aconitifolia with resistant and susceptible cultivars. Inoculation of the fungal pathogen resulted in the enzyme activity gradually increased throughout the experimental period of 168 h compared to control. However, the activation of β-1,3-glucanase and chitinase was more rapid and to a greater extent in the resistant FMM-96 cultivar as compared to susceptible RM0-40 and CZM-3 cultivars. Furthermore, the western blot analysis revealed the presence of 33- and 30-kDa bands of β-1,3-glucanase and chitinase in induced moth bean plants, respectively. The possible implications of these findings as part of the general defense response of moth bean plants against the fungal pathogen (M. phaseolina) have been discussed.     

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.     

Variability in antifungal proteins in the grains of maize, sorghum and wheat

Crude protein extracts were made from kernels of 12 cultivars each of maize, sorghum and wheat. These preparations were fractionated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and subjected to Western blot analyses. Bands corresponding to chitinases and β-glucanases were identified immunologically (Western blots) and on activity gels. Ribosome Inactivating Protein(s) (RIP) and permatins were identified immunologically. In maize, two chitinase bands (25–29 kDa) were seen in all cultivars, and a third band of about 23 kDa was detected in 7 of the 12 cultivars. Two or three β-glucanase bands of sizes between 24 and 36 kDa (probably a mixture of 1,3–β- and 1,3–1,4-β-glucanases) were detected in blots of SDS gels, and one band was detected in activity gels (1,3-β-glucanase). In sorghum, one chitnase band of approximately 29 kDa, and two or three additional bands ranging in size from 21–24 kDa were observed. Only one β-glucanase band was identified, with an estimated molecular weight of 30 kDa. All bands that appeared on Western blots of SDS gels corresponded to bands detected on activity gels. In wheat, one chitmase band of around 20 kDa, one β-glucanase band of about 30 kDa and one RIP band of about 30 kDa were identified. Permatins (molecular weight about 22 kDa) were identified in maize, sorghum and wheat, with the different cultivars having varying amounts of permatins.     

Evidence for a role of β-1,3-glucanase in dicot seed germination

Class I β-1,3-glucanases are antifungal vacuolar proteins implicated in plant defense that show developmental, hormonal, and pathogenesis-related regulation. The expression was studied in germinating tobacco seeds of a chimeric β-glucuronidase (GUS) reporter gene fused to 1.6 kb of the 5' flanking sequence of the tobacco class I β-1,3-glucanase B (GLB) promoter. Histological staining for GUS activity showed that expression of the GLB promoter is highly localized in a specific zone of the endosperm in germinating seeds. The temporal and spatial patterns of GUS and β-1,3-glucanase activity found, suggest a novel function for class I β-1,3-glucanases during seed germination in a dicotyledonous plant.     

Evidence for secretion of vacuolar α-mannosidase, class I chitinase, and class I β-1,3-glucanase in suspension cultures of tobacco cells

We have investigated the possibility that vacuolar proteins can be secreted into the medium of cultured cells of Nicotiana tabacum L. Time-course and balance-sheet experiments showed that a large fraction, up to ca. 19%, of vacuolar α-mannosidase (EC 3.2.1.24) and vacuolar class I chitinase (EC 3.2.1.14) in suspension cultures accumulated in the medium within one week after subculturing. This effect was most pronounced in media containing 2,4-dichlorophenoxyacetic acid (2,4-D). Under comparable conditions only a small fraction, 1.8–5.1% of the total protein and ca. 1% of malate dehydrogenase (EC 1.1.1.37), which is localized primarily in the mitochondria and cytoplasm, accumulated in the medium. Pulse-chase experiments showed that newly synthesized vacuolar class I isoforms of chitinase and β-1,3-glucanase (EC 3.2.1.39) were released into the medium. Post-translational processing, but not the release of these proteins, was delayed by the secretion inhibitor brefeldin A. Only forms of the proteins present in the vacuole, i.e. mature chitinase and pro-β-1,3-glucanase and mature β-1,3-glucanase, were chased into the medium of tobacco cell-suspension cultures. Our results provide strong evidence that vacuolar α-mannosidase, chitinase and β-1,3-glucanase can be secreted into the medium. They also suggest that secretion of chitinase and β-1,3-glucanase might be via a novel pathway in which the proteins pass through the vacuolar compartment. Received: 3 September 1997 / Accepted: 30 October 1997     

Induction of β-1,3-glucanase and chitinase activity in the defense response of Eruca sativa plants against the fungal pathogen Alternaria brassicicola

Plants have developed many mechanisms to protect themselves against most potential microbial pathogens and diseases. Pathogenesis-related proteins are produced as a part of the active defenses to prevent attack. In this study, the induction of PR proteins in Eruca sativa in response to fungal pathogen Alternaria brassicicola was investigated in 10 days and one-month-old plants. Induction of pathogen resulted in a much marked increase in the activities of β-1,3-glucanase and chitinase in resistant cultivar (RTM-2002) as compared to susceptible (T-27) one. The enzyme activity gradually increased throughout the experimental period of 168 h compare to control. However, the activation of β-1,3-glucanase and chitinase was more rapid and to a greater extent in plants of RTM-2002 than in T-27. western blot analysis revealed the presence of 33 and 32 kDa β-1,3-glucanase and chitinase in induced arugula plants, respectively. The biochemical approach described in this article with E. sativa provide the basis for further efforts concentrating on the isolation and characterization of elements involved in perception and in the early steps of intracellular signal transduction.     

Pathogenesis-related proteins protect extrafloral nectar from microbial infestation

Plants in more than 300 genera produce extrafloral nectar (EFN) to attract carnivores as a means of indirect defence against herbivores. As EFN is secreted at nectaries that are not physically protected from the environment, and contains carbohydrates and amino acids, EFN must be protected from infestation by micro-organisms. We investigated the proteins and anti-microbial activity in the EFN of two Central American Acacia myrmecophytes ( A. cornigera and A. hindsii ) and two related non-myrmecophytes ( A. farnesiana and Prosopis juliflora ). Acacia myrmecophytes secrete EFN constitutively at high rates to nourish the ants inhabiting these plants as symbiotic mutualists, while non-myrmecophytes secrete EFN only in response to herbivore damage to attract non-symbiotic ants. Thus, the quality and anti-microbial protection of the EFN secreted by these two types of plants were likely to differ. Indeed, myrmecophyte EFN contained significantly more proteins than the EFN of non-myrmecophytes, and was protected effectively from microbial infestation. We found activity for three classes of pathogenesis-related (PR) enzymes: chitinase, β-1,3-glucanase and peroxidase. Chitinases and β-1,3-glucanases were significantly more active in myrmecophyte EFN, and chitinase at the concentrations found in myrmecophyte EFN significantly inhibited yeast growth. Of the 52 proteins found in A. cornigera EFN, 28 were annotated using nanoLC-MS/MS data, indicating that chitinases and glucanases contribute more than 50% of the total protein content in the EFN of this myrmecophyte. Our study demonstrates that PR enzymes play an important role in protecting EFN from microbial infestation.     

Kinetics of drought-induced pathogenesis-related proteins and its physiological significance in white clover leaves

To investigate the responses of pathogenesis-related (PR) proteins to the intensity of drought stress and their physiological significance in white clover ( Trifolium repens L.), the change of enzyme activity and its relationship with some physiological parameters were assessed for 28 days under well-watered (control) and water-deficit conditions. Water-deficit treatment gradually decreased leaf water potential (Ψ_w) to −2.33 MPa at day 28. Dry matter significantly decreased from 21 days of water-deficit treatment, while proline and ammonia concentration increased within 7 days. The increase in PR-protein activity was closely related with the decrease in Ψ_w. The β-1,3-glucanase (EC 3.2.1.39) activity in water-deficit leaves rapidly increased for the first 14 days (Ψ_w ≥ −1.67) and then slightly decreased, while the chitinase (EC 3.2.1.14) and cellulase (EC 3.2.1.4) activity continued to increase throughout the experimental period. The enhanced activation of β-1,3-glucanase, chitinase and cellulase for the period of days 0–14 was significantly ( P  ≤ 0.01) related to the increase of proline and ammonia concentrations. The results indicate that the enhanced activity of β-1,3-glucanase, cellulase and chitinase for the early period might be an act of transient tolerance to drought stress, but the activation of these enzymes during terminal stress might be a drought-stress-induced injurious symptom.