Differential in vitro DNA binding activity to a promoter element of the gn1β-1,3-glucanase gene in hypersensitively reacting tobacco plants

In a hypersensitive reaction to pathogen infection, expression of the β-1,3-glucanase gn1 gene is induced in cells surrounding the necrotic lesions. The 5′-flanking sequence of gn1 was examined to investigate the molecular basis controlling activation of gene expression during this plant defense response. Studies on transgenic tobacco plants containing gn1 promoter deletions fused to the β-glucuronidase reporter gene revealed the presence of negative and positive regulatory sequences mediating both the level and the spatial distribution of gn1 expression. Promoter sequences to ?138 bp were sufficient to confer increased gene expression around the necrotic lesions produced in response to Pseudomonas syringae pv. syringae inoculation. It is demonstrated by electrophoretic mobility shift assays that nuclear proteins in both healthy and hypersensitively reacting tobacco leaves interact with DNA sequences within the regulatory elements identified. Among the binding sequences characterized, the promoter region extending from ?250 to ?217 bp contained the DNA motif -GGCGGC- found to be conserved in most if not all promoters of genes encoding pathogenesis-related basic proteins. The activity bound by this promoter sequence was stronger in hypersensitively responding tissues than in healthy untreated tobacco leaves.     

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.     

Enhanced biocontrol activity of Trichoderma virens transformants constitutively coexpressing β-1,3- and β-1,6-glucanase genes

Evidence for the role of chitinases, proteases and β-1,3- and β-1,6-glucanases in mycoparasitism by Trichoderma species has been well documented. Moreover, constitutive over-expression of genes encoding individual cell-wall-degrading enzymes (CWDEs) has been shown to improve the potential of biological agents. In this study, we generated transformants of T. virens in which β-1,3- and β-1,6-glucanase genes, TvBgn2 and TvBgn3 , respectively, were constitutively coexpressed in the same genetic T. virens Gv29.8 wild-type background. The double over-expression transformants (dOEs) grow and sporulate slower than the wild-type (WT). However, the reduction in growth did not seem to affect their mycoparasitic and biocontrol capabilities, as dOEs displayed much higher levels of total β-1,3- and β-1,6-glucanase activity than the WT. This higher enzymatic activity of dOEs positively correlated with observed in vitro inhibition of Pythium ultimum and Rhizoctonia solani mycelia, and with enhanced bioprotection of cotton seedlings against P. ultimum , R. solani and Rhizopus oryzae . Besides effective biocontrol of all pathogens at an original inoculum level, the performance of dOEs was highly enhanced (up to 312% of WT performance) when pathogen pressure was greater (i.e. concentration of inoculum was higher or pathogens applied in combination). These results demonstrate that the strategy of introducing multiple lytic enzyme-encoding genes through transformation of a given biocontrol strain can be successfully used to achieve better biocontrol.     

Induction of chitinase and β-1,3-glucanase in Parthenocissus quinquefolia cells cultured in vitro

Chitin, chitosan and peptidoglycan induced chitinase (EC 3. 2. 1. 14) activity in Parthenocissus quinquefolia cells cultured in vitro, while cellulose did not. The real inducers seemed to be oligomers released from the large size polymers by hydrolytic enzymes secreted into the medium during the cell growth and division. This effect was mimicked by the addition to the medium of a partially purified Parthenocissus chitinase/lysozyme (EC 3. 2. 1. 17), which was also able to hydrolyse chitosan. Oligomers of chitin and of chitosan induced the activity to the same level and with the same time course, while peptidoglycan oligomers induced less activity. Oligomers also induced β-1,3-glucanase (EC 3. 2. 1. 6) activities. The changes with time of both activities and the relative effects of the three kinds of polymers suggested that the induction of both enzymes involves a common element early in the signal pathway.     

Local expression of enzymatically active class I β-1, 3-glucanase enhances symptoms of TMV infection in tobacco

Mutant tobacco plants deficient for class I beta-1,3-glucanase (GLU I) are decreased in their susceptibility to virus infection. This is correlated with delayed virus spread, a reduction in the size exclusion limit of plasmodesmata and increased cell-wall deposition of the beta-1,3-glucan callose. To further investigate a role of GLU I during cell-to-cell movement of virus infection, we inserted the GLU I coding sequence into TMV for overexpression in infected cells. Compared with the size of local lesions produced on plants infected with virus expressing either an enzymatically inactive GLU I or a frameshift mutant of the gene, the size of local lesions caused by infection with virus expressing active GLU I was consistently increased. Viruses expressing antisense GLU I constructs led to lesions of decreased size. Similar effects were obtained for virus spread using plants grown at 32 degrees C to block the hypersensitive response. Together, these results indicate that enzymatically active GLU I expressed in cells containing replicating virus can increase cell-to-cell movement of virus. This supports the view that GLU I induced locally during infection helps to promote cell-to-cell movement of virus by hydrolyzing callose. Moreover, our results provide the first direct evidence that a biological function of a plant beta-1,3-glucanase depends on its catalytic activity.     

Analysis of a maize α-tubulin gene promoter by transient expression and in transgenic tobacco plants

The pattern of expression directed by the promoter of the maize Tub α 1 gene was investigated by analysis of chloramphenicol acetyl transferase (CAT) and β-glucuronidase (GUS) activities in transient expression experiments of maize and tobacco protoplasts. The same promoter was also investigated by histochemical GUS analysis in transgenic tobacco plants containing promoter gene fusions. As determined by histochemical tests, the Tub α 1 promoter gene preferentially directs GUS expression in regenerating root tip meristems and pollen. This pattern corresponds to the distinctive features of natural expression of the gene in maize as determined by Northern analysis. However, no expression is observed in other meristematic tissues of the transgenic tobacco plants, as in shoot apex or in coleoptiles, which is weakly detected in maize. Analysis of the regulatory properties of 5' promoter deletions showed that the proximal region of the promoter, from positions −1410 or −449 to 15 bp upstream of the ATG, is sufficient to establish the qualitative pattern of expression in transgenic tobacco plants. Deletions to positions −352 or −117 abolished the expression in roots, but not in pollen, suggesting that upstream of these positions there are elements responsible for the pattern in root. Further deletions abolished all the promoter activity, suggesting that this promoter region contains the elements essential for expression in pollen. The different patterns and levels of transient and stable expression are discussed.     

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.     

Mapping of the gene for endo-β-1,3-1,4-glucanase of Bacillus subtilis

Abstract An integrating plasmid has been used to mutagenise the gene coding for endo-β-1,3-1,4-glucanase of Bacillus subtilis . The gene, named bgl , has been mapped by PBS-1 transduction to the sacA-pureA region of the B. subtilis chromosome and is closely linked to the hutP 1 locus. The order of markers in this region is sacA 321- thiC 5- bgl - hutP 1- purA 16.     

Antisense expression of chaperonin 60β in transgenic tobacco plants leads to abnormal phenotypes and altered distribution of photoassimilates

Chaperonins are a class of molecular chaperone, present in bacteria, mitochondria and chloroplasts, that are involved in protein folding and assembly in many organisms. Plastid α and β chaperonins have been suggested to be involved specifically in the assembly of Ribulose bisphosphate carboxylase/oxygenase. However, to date there is no direct evidence to confirm the in vivo role of plastid chaperonin 60 polypeptides as molecular chaperones. This paper reports on the production, by means of antisense technology, of transgenic tobacco plants with reduced levels of chaperonin 60β (Cpn60β). Antisense cpn 60β plants showed drastic phenotypic alterations including slow growth, delayed flowering, stunting and leaf chlorosis. The most extreme effect appeared to be lethality suggesting that cpn 60β functions are essential for viability. Cpn60β antisense plants accumulated Rubisco to specific activities equal to or higher than that of controls and had high plastid starch contents. These observations are discussed with respect to the suggestion that chaperonin is required for the assembly of active Rubisco in vivo . In addition, metabolic alterations in the antisense transgenic plants such as reduced soluble carbohydrate content as well as higher levels of starch in chloroplasts, suggest that Cpn60β may be required for import, assembly or membrane insertion of several chloroplast membrane proteins. These results are in agreements with the proposed role of Cpn60β as a molecular chaperone.     

Study of β-1,3-glucanase activity during autolysis of Aspergillus nidulans by FPLC ion-exchange chromatography

The behaviour of β-1,3-glucanase activity during Aspergillus nidulans autolysis was studied in a basal medium and in the same medium supplemented with 0.5 g l^-1 of microcrystalline cellulose, laminarin, pectin, seedling of Lycopersicum esculentum extract, chitin and xylan respectively. In any case β-1,3-glucanase activity was detected in the culture fluid before the onset of the autolysis, but afterwards a progressive increase of β-1,3-glucanase activity took place with incubation time. In the media supplemented with pectin and seedling of Lycopersicum esculentum extract higher activity in the first days of autolysis was found. The activity at the end of the studied process by sample was 2.5, 2.1, 2.5, 1.9, 2.2, 2.3 and 2.3 U, and the specific activity 83, 53, 85, 55, 64, 90 and 53 mU mg^-1 of protein for each medium respectively. The β-1,3-glucanase activity in Aspergillus nidulans seems to be related to autolysis and not to the presence of different substances in the culture medium. The behaviour of β-1,3-glucanase activity during the degradative process was followed by FPLC ion-exchange chromatography. Three proteins (I, II, III) with β-1,3-glucanase activity were separated and quantified. These proteins have similar behaviour in all the media. Proteins I and II increase progressively with incubation time but protein III is only present at the first and last days of autolysis.     

The expression of a heat-inducible chimeric gene in transgenic tobacco plants

Summary A chimeric gene under the control of the hsp70 promoter of Drosophila is heat regulated in roots, stems and leaves, but not in pollen of transgenic tobacco plants. For these and other parameters, it behaves similarly to plant heat-shock genes.     

Variability of organ-specific gene expression in transgenic tobacco plants

Variability of expression of introduced marker genes was analysed in a large number of tobacco regenerants from anAgrobacterium-mediated transformation. In spite of standardization of sampling, considerable variation of GUS and NPTII expression was observed between individual transformants at different times of analysis and in different parts of the same plant. Organ-specificity of root versus leaf expression conferred by the par promoter from the haemoglobin gene ofParasponia andersonii in front of thegus gene showed a continuous spectrum. GUS expression in roots was found in 128 out of 140 plants; expression in leaves was found in 46 plants, and was always lower than in the corresponding roots. NPTII expression regulated by the nos promoter also showed a continuous spectrum. Expression levels were generally higher in roots than in leaves. Plants with high GUS expression in leaves showed high NPTII activity as well. A positive correlation between the level of NPTII expression and the numbers of integrated gene copies was noted. Chromosomal position effects and physiological determination are suggested as triggers for the variations. The transformed regenerated tobacco plants were largely comparable to clonal variants.     

Characterization, induction by wounding and salicylic acid, and activity against Botrytis cinerea of chitinases and β‐1,3‐glucanases of ripening grape berries

In order to better understand the defense strategy of grape berries ( Vitis vinifera L. cv. Pinot noir) as they mature, the activities of the defense‐related proteins, chitinase (CHV, EC 3.2.1.14) and β‐1,3‐glucanase (laminarinase, EC 3.2.1.39) were first estimated in berries at different maturation stages. Chitinase levels rose proportionally to the berry reducing sugar content, an indicator of the berry ripening degree, up to values 10 times higher than the ones seen in resting grapevine leaves. This rise in activity was due to the accumulation of two isoforms, CHV 5 and CHV 11. One more chitinase isoform, CHV 12, appeared in senescent berries. Conversely, no glucanase activity could be detected in berries at any maturation stage. Accumulation of chitinases and (β‐1,3‐glucanases could be stimulated by wounding the berry peduncle. Adding salicylic acid to the wounded berries only potentiated the wounding effect on the berry chitinase activity. The most active chitinase isoform, CHV 5, was purified to homogeneity. It represented about 40% of the total extractable protein content of a ripe berry. Its molecular mass was estimated to be 31 kDa. The peptide sequencing of four of its tryptic fragments revealed strong homologies to several class IV chitinases. Finally, it was shown to inhibit the germination of conidia of Botrytis cinerea by 50% at a concentration of 7.5 µg ml⁻¹.