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.     

β‐1,3‐Glucanases in wheat and resistance to the Russian wheat aphid

The Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko) is one of the most destructive insect pests of cereals world-wide. Although resistant cultivars have been bred, the biochemical mechanism of resistance is unknown. The aim of this work was to gain information on the mechanism of resistance which could contribute to more directed breeding of resistant cultivars in the future. The effect of RWA infestation on the inter- and intracellular β-1,3-glucanase activities was studied in different resistant wheat (Triticum aestivum L.) cultivars containing the Dn-1 gene for RWA resistance and corresponding near-isogenic susceptible cultivars. The activity was determined spectrophotometrically by measuring the release of glucose from laminarin. Infestation differentially induced the intra- and intercellular activities to much higher levels in resistant than susceptible cultivars within 48 h. According to immunological studies induced enzyme activities were due to increased protein levels. The intracellular β-1,3-glucanase contained about 8% exo-activity. The exo-activity made an insignificant contribution to the intercellular activity. The genetic background into which the resistance gene was bred did affect the level of activity that corresponded to the resistance performance. Seven apoplastic isoforms of β-1,3-glucanase, varying from acidic to basic, were resolved by isoelectric focusing. All isoenzymes were equally induced and no specific one could be linked to resistance or susceptibility. The RWA induced β-1,3-glucanase activity in resistant cultivars closely resembles defence responses during pathogenesis and seems to be part of a general defence response like the hypersensitive reaction (HR), which confers resistance to the RWA. This knowledge might be helpful in future to identify genes for RWA resistance. The increased β-1,3-glucanase activity after RWA infestation might serve as an additional measure to biochemically trace resistance in crosses during breeding.     

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.     

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.     

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.     

Some thaumatin-like proteins hydrolyse polymeric β-1,3-glucans

Thaumatin and 12 purified thaumatin-like (TL) proteins were surveyed for their capacity to hydrolyse beta-1,3-glucans by using an in-gel glucanase assay. Six TL proteins identified by N-terminal amino acid microsequencing were found to be active on carboxymethyl(CM)-pachyman: a barley leaf stress-related permatin, two tomato fruit osmotins, a cherry fruit and two tobacco stigma proteins. TL enzymes ranged in specific activity from 0.07 to 89 nkat mg-1 with CM-pachyman as substrate. Hydrolytic activities were not restricted to TL proteins strongly binding to water-insoluble beta-1,3-glucans since the two osmotins were active without tight binding to pachyman. Some TL proteins hydrolysed crude fungal walls and one barley TL enzyme even lysed fungal spores. No activity was observed on laminarin in the in-gel hydrolase assay. Thin-layer chromatography revealed that the six enzymes acted as endo-beta-1, 3-glucanases leading to the formation of various oligoglucosides. Thus far, the TL enzymes (EC 3.2.1.x) appeared different from the well-known beta-1,3-glucanases (EC 3.2.1.39). No activity was found with thaumatin, zeamatin, tobacco leaf PR-R protein and four stress-related TL proteins from barley and pea. This is the first demonstration that diverse TL proteins are enzymatically active. The functions of some TL proteins must be reassessed because they display endo-beta-1,3-glucanase activity on polymeric beta-1, 3-glucans.     

Intercellular chitinase and peroxidase activities associated with resistance conferred by geneLr35to leaf rust of wheat

The effect of leaf rust (Puccinia triticina) infection on intercellular chitinase (EC 3.2.1.14) and peroxidase (EC 1.11.1.7) activities was studied in resistant [RL 6082 (Thatcher/Lr35)] and susceptible (Thatcher) near isogenic wheat (Triticum aestivum L.) lines at seedling, stem elongation and flag leaf stages of plant growth. The levels of activity of these enzymes were low during the seedling and stem elongation stages. Resistant plants at the flag leaf stage, during which the Lr35 resistance gene was maximally expressed, exhibited high constitutive levels of chitinase and peroxidase activities, in contrast to the lower constitutive levels of susceptible plants. The results suggest that chitinase and peroxidase, constitutively present in the intercellular spaces of Thatcher/Lr35 wheat leaves, may play a role in Lr35 mediated resistance to leaf rust.     

Detection of quantitative trait loci associated with leaf rust resistance in bread wheat.

Leaf rust, caused by Puccinia recondita Rob. ex Desm., is a common disease in wheat. The objective of this study was to develop molecular markers associated with the quantitative trait loci (QTLs) putatively conferring durable leaf rust resistance in Triticum aestivum L. em. Thell. A population of 77 recombinant inbred lines (RILs) developed from 'Parula' (resistant) and 'Siete Cerros' (moderately susceptible) was used. Bulked segregant analysis was done using random amplified polymorphic DNAs (RAPDs) with DNA enriched for low-copy sequences using hydroxyapatite chromatography. Out of 400 decamer primers screened, 3 RAPD markers were identified between the bulk of the most resistant and the bulk of the most susceptible lines. These were cloned and used as probes on the RILs in Southern hybridizations. Two probes revealed two tightly linked loci. One-way analysis of variance showed that these two loci, and another revealed by the third probe, were linked to QTLs controlling leaf rust resistance based on data taken from 2 years of replicated field trials. Cytogenetic analysis placed the two tightly linked loci on the long arm of chromosome 7B. The third probe detected loci located on the short arms of chromosomes 1B and 1D. It is suggested that the QTL detected on 7BL may well be homoeoallelic to Lr34.     

Inheritance of leaf rust and stem rust resistance in 'Roblin' wheat.

The Canadian common wheat (Triticum aestivum L.) cultivar 'Roblin' is resistant to both leaf rust (Puccinia recondita Rob. ex. Desm.) and stem rust (Puccinia graminis Pers. f. sp. tritici Eriks. and E. Henn.). To study the genetics of this resistance, 'Roblin' was crossed with 'Thatcher', a leaf rust susceptible cultivar, and RL6071, a stem rust susceptible line. A set of F6 random lines was developed from each cross. The random lines and the parents were grown in a field rust nursery artificially inoculated with a mixture of P. recondita and P. graminis isolates and scored for rust reaction. The same material was tested with specific races of leaf rust and stem rust. These data indicated that 'Roblin' has Lr1, Lr10, Lr13, and Lr34 for resistance to P. recondita and Sr5, Sr9b, Sr11, and possibly Sr7a and Sr12 for resistance to P. graminis. In a 'Thatcher' background, the presence of Lr34 contributes to improve stem rust resistance, which appears also to occur in 'Roblin'.     

Physical characterization of isozymes of endo-β-1,4-glucanase and β-1,4-glucosidase from Aspergillus species

Electrophoretic data revealed the presence of various isozymes of endoglucanase and beta-glucosidase, the number of which varied from one to three in various species of the genus Aspergillus. pH 5.0 was optimum for all the isozymes whereas metal ion treatment showed complete inhibition of almost all the isozymes by Hg2+ and partial inhibition by Ca2+ and Co2+ of isozymes of both the enzymes. An alteration in the electrophoretic mobility of isozymes of beta-glucosidase was also noticed in some species with Hg2+ treatment.     

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.     

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.     

β-Lactam resistance in Streptococcus pneumoniae: penicillin-binding proteins and non-penicillin-binding proteins

The beta-lactams are by far the most widely used and efficacious of all antibiotics. Over the past few decades, however, widespread resistance has evolved among most common pathogens. Streptococcus pneumoniae has become a paradigm for understanding the evolution of resistance mechanisms, the simplest of which, by far, is the production of beta-lactamases. As these enzymes are frequently plasmid encoded, resistance can readily be transmitted between bacteria. Despite the fact that pneumococci are naturally transformable organisms, no beta-lactamase-producing strain has yet been described. A much more complex resistance mechanism has evolved in S. pneumoniae that is mediated by a sophisticated restructuring of the targets of the beta-lactams, the penicillin-binding proteins (PBPs); however, this may not be the whole story. Recently, a third level of resistance mechanisms has been identified in laboratory mutants, wherein non-PBP genes are mutated and resistance development is accompanied by deficiency in genetic transformation. Two such non-PBP genes have been described: a putative glycosyltransferase, CpoA, and a histidine protein kinase, CiaH. We propose that these non-PBP genes are involved in the biosynthesis of cell wall components at a step prior to the biosynthetic functions of PBPs, and that the mutations selected during beta-lactam treatment counteract the effects caused by the inhibition of penicillin-binding proteins.     

Molecular mapping of stem and leaf rust resistance in wheat

Stem rust caused by Puccinia graminis f. sp. tritici Eriks and Henn and leaf rust caused by Puccinia triticina Rob. ex Desm. are major constraints to wheat production worldwide. In the present study, F₄-derived SSD population, developed from a cross between Australian cultivars ‘Schomburgk’ and ‘Yarralinka’, was used to identify molecular markers linked to rust resistance genes Lr3a and Sr22. A total of 1,330 RAPD and 100 ISSR primers and 33 SSR primer pairs selected ob the basis of chromosomal locations of these genes were used. The ISSR marker UBC 840₅₄₀ was found to be linked with Lr3a in repulsion at a distance of 6.0 cM. Markers cfa2019 and cfa2123 flanked Sr22 at a distance of 5.9 cM (distal) and 6.0 cM (proximal), respectively. The use of these markers in combination would predict the presence or absence of Sr22 in breeding populations. A previously identified PCR-based diagnostic marker STS638 linked to Lr20 was validated in this population. This marker showed a recombination value of 7.1 cM with Lr20.     

Molecular markers for leaf rust resistance genes in wheat

Over 100 genes of resistance to rust fungi: Puccinia recondita f. sp. tritici, (47 Lr - leaf rust genes), P. striiformis (18 Yr - yellow rust genes) and P. graminis f. sp. tritici (41 Sr - stripe rust genes) have been identified in wheat (Triticum aestivum L.) and its wild relatives according to recent papers. Sixteen Lr resistance genes have been mapped using restriction fragments length polymorphism (RFLP) markers on wheat chromosomes. More than ten Lr genes can be identified in breeding materials by sequence tagged site (STS) specific markers. Gene Lrk 10, closely linked to gene Lr 10, has been cloned and its function recognized. Available markers are presented in this review. The STS, cleaved amplified polymorphic sequence (CAPS) and sequence characterized amplified regions (SCAR) markers found in the literature should be verified using Triticum spp. with different genetic background. Simple sequence repeats (SSR) markers for Lr resistance genes are now also available.