Characterization and expression of β-1,3-glucanase genes in peach

Beta-1,3-glucanase is one of the pathogenesis-related (PR) proteins involved in plant defense responses. A peach beta-1,3-glucanase gene, designated PpGns1, has been isolated and characterized. The deduced amino acid sequence of the product of PpGns indicates that it is a basic isoform (pI 9.8), and contains a putative signal peptide of 38 amino acids but has no C-terminal extension. Amino acid sequence comparisons revealed that PpGns1 is 69% and 67% identical to citrus and soybean beta-1,3-glucanases, respectively. Southern analysis of total genomic DNA also indicates that at least three genes for beta-1,3-glucanases exist in peach, forming a small gene family. Characterization of four additional clones by PCR has identified a second beta-1,3-glucanase gene, PpGns2. PpGns2 has been partially sequenced, and when compared to PpGns1, it shows high sequence homology, 96% and 99% nucleotide identity in the first and (partial) second exons, respectively. The deduced partial sequence of the PpGns2 product displays only two differences from PpGns1 in the signal peptide and one in the (partial) mature protein (141 amino acids). The 5'-flanking promoter regions of these two genes share 90% identity in nucleotide sequences interrupted by five major gaps (4-109 nt long). The promoter region contains various sequences similar to cis-regulatory elements present in different stress-induced plant genes. In leaves and stems of peach shoot cultures grown in vitro, PpGns1 is induced within 12 h after exposure to a culture filtrate of Xanthomonas campestris pv. pruni or ethephon. However, it is not induced following treatment with mercuric chloride.     

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.     

Regulation of beta-1,3-glucanase synthesis in Penicillium italicum.

The filamentous fungus Penicillium italicum produced a certain level of beta-1,3-glucanase during active growth in a glucose-supplemented medium; however, at a low glucose concentration (2 to 10 mM), derepression took place and the specific activity of the enzyme increased significantly. Derepressed cells (incubated in a glucose-limited medium) accumulated a capacity for the synthesis of beta-1,3-glucanase, which led to a subsequent increase in the specific activity even when the cells were transferred to a medium with an excess of glucose (180 mM). Two protein synthesis inhibitors, cycloheximide and trichodermin, immediately stopped the increase in specific activity when added to derepressed cells. On the other hand, 8-hydroxyquinoline, an RNA a synthesis inhibitor, acted differently, since it permitted the specific activity to increase for some time after being added to depressed cells. Moreover, the concentration of glucose did not affect the 8-hydroxyquinoline-insensitive synthesis of beta-1,3-glucanase. It is concluded that the glucose repression effect on beta-1,3-glucanase production must be exerted at a pretranslational level that could be either mRNA synthesis or some stage of the process involved in its maturation or stabilization.     

Molecular characterization and expression in Escherichia coli of three beta-1,3-glucanase genes from Lysobacter enzymogenes strain N4-7

Lysobacter enzymogenes strain N4-7 produces multiple biochemically distinct extracellular beta-1,3-glucanase activities. The gluA, gluB, and gluC genes, encoding enzymes with beta-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of beta-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of gluA and gluC gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the gluC product. Analysis of the gluB gene product indicates that it is a member of family 64 glycoside hydrolases. Expression of each gene in Escherichia coli resulted in the production of proteins with beta-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45 degrees C and that GluB is most active between pH 4.5 and 5.0 at 41 degrees C. Activity of recombinant proteins against various beta-1,3 glucan substrates indicates that GluA and GluC are most active against linear beta-1,3 glucans, while GluB is most active against the insoluble beta-1,3 glucan substrate zymosan A. These data suggest that the contribution of beta-1,3-glucanases to the biocontrol activity of L. enzymogenes may be due to complementary activities of these enzymes in the hydrolysis of beta-1,3 glucans from fungal cell walls.     

Regulation of the beta-1,3-glucanase system in Penicillium italicum: glucose repression of the various enzymes.

The microscopic fungus Penicillium italicum when grown in a synthetic liquid medium produced at least three enzymes with beta-1,3-glucanase activity which were separated by diethylaminoethyl-Sephadex column chromatography. These were named beta-1,3-glucanases I, II, and III respective to their order of elution from the column. A tentative characterization of these three enzymes indicated that they have different modes of action; the first one is an endoglucanase, the second is an exoglucanase, and the third probably has both mechanisms of action. Glucose had a repressive effect on all three enzymes. Only small amounts of beta-1,3-glucanases II and III were present in the cells when they were actively growing in the presence of this sugar. However, when the cells were transferred to a medium low in glucose, a significant increase in the specific activity of beta-1,3-glucanase took place; this was due in part to a much more active production of beta-1,3-glucanases II and III and in part to the appearance of beta-1,3-glucanase I, which could only be detected after more than 12 h of incubation in this medium. The results are discussed in the context of possible beta-1,3-glucanase functions in the fungal cells.     

Characterization of a 29-kDa beta-1,3-glucanase from Trichoderma harzianum

A beta-1,3-glucanase, from culture filtrates of Trichoderma harzianum, was purified in sequential steps by gel filtration, hydrophobic interaction and ion exchange chromatography. A typical procedure provided 69-fold purification with 0.32% yield. The molecular mass of the protein was found to be approximately 29 kDa, as estimated by SDS-PAGE on a 10% slab gel. The K(M) and V(max) values for beta-1,3-glucanase, using laminarin as substrate, were 1. 72 mg ml(-1) and 3.10 U ml(-1), respectively. The pH optimum for the enzyme was pH 4.4 and maximum activity was obtained at 50 degrees C. The enzyme was strongly inhibited by HgCl(2) and SDS. These results suggest that each beta-1,3-glucanase produced by T. harzianum is different and is probably encoded by different genes.     

Analysis of the beta-1,3-Glucanolytic System of the Biocontrol Agent Trichoderma harzianum

The biocontrol agent Trichoderma harzianum IMI206040 secretes beta-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of beta-1,3-glucanase activity secreted was found to be proportional to the amount of glucan present in the inducer. The fungus produces at least seven extracellular beta-1,3-glucanases upon induction with laminarin, a soluble beta-1,3-glucan. The molecular weights of five of these enzymes fall in the range from 60,000 to 80,000, and their pIs are 5.0 to 6.8. In addition, a 35-kDa protein with a pI of 5.5 and a 39-kDa protein are also secreted. Glucose appears to inhibit the formation of all of the inducible beta-1,3-glucanases detected. A 77-kDa glucanase was partially purified from the laminarin culture filtrate. This enzyme is glycosylated and belongs to the exo-beta-1,3-glucanase group. The properties of this complex group of enzymes suggest that the enzymes might play different roles in host cell wall lysis during mycoparasitism.     

Diversity and evolution of a non-TIR-NBS sequence family that clusters to a chromosomal ’’hotspot” for disease resistance genes in soybean

In soybean, genes controlling resistance to numerous diseases have been shown to cluster to regions on several chromosomes. One such vital chromosomal region is on the soybean molecular linkage group (MLG) F flanked by the RFLP markers K644 and B212. Here, genes controlling resistance to bacterial blight, Phytophthora root rot, and several viral diseases, as well as QTLs conditioning resistance to corn earworm, root knot nematode, and white mold have been mapped. We have previously identified two classes (b and j) of disease resistance-related nucleotide binding site (NBS) sequences that localize to this cluster of genes. Using both cDNA and genomic analyses, we have studied one multi-gene family of sequences representing the previously reported class j NBS of soybean. This class of NBS resembles the RPS2-like NBS sequences. RPS2 and similar resistance genes are referred to as non-TIR because they do not encode motifs homologous to the Toll-Interleukin-1 region (TIR). By designing PCR primers that specifically target these non-TIR-NBS encoding sequences, we have amplified at least six class j sequence members from soybean. In addition, we have conducted genomic and cDNA library screenings to identify additional class j members. In all, we have characterized 12 class j NBS sequence members. These members have been mapped within a 2-cM region of the soybean F linkage group. We have also identified homoeologous chromosomal regions on linkage groups A2 and E that contain class j NBS sequences. A BLAST search of the GenBank database has shown that non-TIR NBS sequences are present across the legume family. We have compared these non-TIR sequences from other legumes with the soybean clones to assess the level of diversity within this class of disease resistance-related sequences.     

Induction of beta-1,3-glucanase in barley in response to infection by fungal pathogens.

The sequence of a partial cDNA clone corresponding to an mRNA induced in leaves of barley (Hordeum vulgare) by infection with fungal pathogens matched almost perfectly with that of a cDNA clone coding for beta-1,-3-glucanase isolated from the scutellum of barley. Western blot analysis of intercellular proteins from near-isogenic barley lines inoculated with the powdery mildew fungus (Erysiphe graminis f. sp. hordei) showed a strong induction of glucanase in all inoculated lines but was most pronounced in two resistant lines. These data were confirmed by beta-1,3-glucanase assays. The barley cDNA was used as a hybridization probe to detect mRNAs in barley, wheat (Triticum aestivum), rice (oryza sativus), and sorghum (Sorghum bicolor), which are induced by infection with the necrotrophic pathogen Bipolaris sorokiniana. These results demonstrate that activation of beta-1,3-glucanase genes may be a general response of cereals to infection by fungal pathogens.     

Disease-resistance related sequences in common bean.

Primers based on a conserved nucleotide binding site (NBS) found in several cloned plant disease resistance genes were used to amplify DNA fragments from the genome of common bean (Phaseolus vulgaris). Cloning and sequence analysis of these fragments uncovered eight unique classes of disease-resistance related sequences. All eight classes contained the conserved kinase 2 motif, and five classes contained the kinase 3a motif. Gene expression was noted for five of the eight classes of sequences. A clone from the SB3 class mapped 17.8 cM from the Ur-6 gene that confers resistance to several races of the bean rust pathogen Uromyces appendiculatus. Linkage mapping identified microclusters of disease-resistance related sequence in common bean, and sequences mapped to four linkage groups in one population. Comparison with similar sequences from soybean (Glycine max) revealed that any one class of common bean disease-resistance related sequences was more identical to a soybean NBS-containing sequence than to the sequence of another common bean class.     

绿色木霉LTR-2 β-1, 3-葡聚糖酶基因的克隆及其在 毕赤酵母中的表达

根据从GenBank中检索到的木霉菌β-1,3-葡聚糖酶基因序列设计引物,以高产β-1,3-葡聚糖酶菌株--绿色木霉LTR-2的cDNA为模板,采用PCR方法扩增得到内切β-1,3-葡聚糖酶基因(glu).将glu克隆至载体pMD18-T上,进行了全序列测定.序列分析表明该基因由2289个核苷酸残基组成,含有一个开放阅读框架,可以编码762个氨基酸,与报道基本相同.翻译后的氨基酸序列含有两个β-1,3-葡聚糖酶的保守区RVVYIPPGTY和AASQNKVAYF.基因与已发表的木霉β-1,3-葡聚糖酶基因有较高的同源性,其中和哈茨木霉bgn3.1和绿木霉bgn13.1的同源性达到93%.序列已经提交GenBank,登录号为EF176582.将glu基因插入到巴斯德毕赤酵母(Pichia pastoris)穿梭载体pPIC9K中,获得重组质粒pGLU14,经线性化后转化毕赤酵母菌株KM71.经大量平板筛选,获得能有效分泌表达β-1,3-葡聚糖酶的毕赤酵母工程菌株KGLU14,菌落PCR扩增证实了glu基因已经整合到酵母基因组中.SDS电泳结果表明其β-1,3-葡聚糖酶的分子量大约为80kDa,和理论推测值大致相同.摇瓶发酵结果表明,培养基中β-1,3-葡聚糖酶的活力可达889U/mL.     

Characterization of a chitinase and an endo-beta-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii

Of 24 Trichoderma isolates, T harzianum Rifai (T24) showed a potential for control of the phytopathogenic basidiomycete Sclerotium rolfsii. When T24 was grown on different carbon sources, growth inhibition of S. rolfsii by the T24 culture filtrate correlated with the activity of extracellular chitinase and beta-1,3-glucanase. The 43-kilodalton (kDa) chitinase and the 74-kDa beta-1,3-glucanase were purified from the T24 culture filtrate in two and three steps, respectively, using ammonium sulphate precipitation followed by hydrophobic interaction chromatography (phenyl-Sepharose) and gel filtration (beta-1,3-glucanase). Km and Kcat were 3.8 g l(-1) and 0.71 s(-1) for the chitinase (chitin) and 1.1 g(-1) and 52 s(-1) for the beta-1,3-glucanase (laminarin). The chitinase showed higher activity on chitin than on less-acetylated substrate analogues (chitosan), while the beta-1,3-glucanase was specific for beta-1,3-linkages in polysaccharides. Both enzymes were stable at 30 degrees C, while at 60 degrees C the chitinase and the beta-1,3-glucanase were rapidly inactivated, showing half-lives of 15 and 20 min, respectively. The enzymes inhibited growth of S. rolfsii in an additive manner showing a promising ED50 (50% effective dose) value of 2.7 microg/ml.     

Enzyme Production by the Mycoparasite Verticillium biguttatum against Rhizoctonia solani

Verticillium biguttatum, a mycoparasite of the ubiquitous soil-borne plant pathogen Rhizoctonia solani, excreted chitinase and beta-1,3-glucanase into liquid medium when grown on laminarin and chitin, respectively. Neither chitinase nor beta-1,3-glucanase was produced by the mycoparasite when grown on cell walls of two isolates of R. solani representing anastomosis groups (AG)-3 and AG-8. Extracellular protease was induced by growth on cell walls of the pathogen, whereas beta-1,3-glucanase and chitinase were produced bound to the cell wall of V. biguttatum. This is the first report of chitinase, beta-1,3-glucanase and protease production by V. biguttatum. These enzymes may play a previously unforeseen role in dissolving and penetrating the cell walls of R. solani.     

Genomic analysis of a region encompassing QRfs1 and QRfs2: genes that underlie soybean resistance to sudden death syndrome.

Candidate genes were identified for two loci, QRfs2 providing resistance to the leaf scorch called soybean (Glycine max (L.) Merr.) sudden death syndrome (SDS) and QRfs1 providing resistance to root infection by the causal pathogen Fusarium solani f.sp. glycines. The 7.5 +/- 0.5 cM region of chromosome 18 (linkage group G) was shown to encompass a cluster of resistance loci using recombination events from 4 near-isogenic line populations and 9 DNA markers. The DNA markers anchored 9 physical map contigs (7 are shown on the soybean Gbrowse, 2 are unpublished), 45 BAC end sequences (41 in Gbrowse), and contiguous DNA sequences of 315, 127, and 110 kbp. Gene density was high at 1 gene per 7 kbp only around the already sequenced regions. Three to 4 gene-rich islands were inferred to be distributed across the entire 7.5 cM or 3.5 Mbp showing that genes are clustered in the soybean genome. Candidate resistance genes were identified and a molecular basis for interactions among the disease resistance genes in the cluster inferred.     

Structure, chromosome location, and expression of the human gamma-actin gene: differential evolution, location, and expression of the cytoskeletal beta- and gamma-actin genes.

The accumulation of the cytoskeletal beta- and gamma-actin mRNAs was determined in a variety of mouse tissues and organs. The beta-isoform is always expressed in excess of the gamma-isoform. However, the molar ratio of beta- to gamma-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. We conclude that, whereas the cytoskeletal beta- and gamma-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human gamma-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike beta-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the beta- and gamma-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human beta- and gamma-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the beta-actin gene but are conserved between the human gamma-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of beta- and gamma-actin or to the unique regulation and function of the gamma-actin gene. Finally, we demonstrate that the human gamma-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human gamma-actin gene is appropriately regulated.     

Cloning and sequence analysis of genes encoding xylanases and acetyl xylan esterase from Streptomyces thermoviolaceus OPC-520.

Three genes encoding two types of xylanases (STX-I and STX-II) and an acetyl xylan esterase (STX-III) from Streptomyces thermoviolaceus OPC-520 were cloned, and their DNA sequences were determined. The nucleotide sequences showed that genes stx-II and stx-III were clustered on the genome. The stx-I, stx-II, and stx-III genes encoded deduced proteins of 51, 35.2, and 34.3 kDa, respectively. STX-I and STX-II bound to both insoluble xylan and crystalline cellulose (Avicel). Alignment of the deduced amino acid sequences encoded by stx-I, stx-II, and stx-III demonstrated that the three enzymes contain two functional domains, a catalytic domain and a substrate-binding domain. The catalytic domains of STX-I and STX-II showed high sequence homology to several xylanases which belong to families F and G, respectively, and that of STX-III showed striking homology with an acetyl xylan esterase from S. lividans, nodulation proteins of Rhizobium sp., and chitin deacetylase of Mucor rouxii. In the C-terminal region of STX-I, there were three reiterated amino acid sequences starting from C-L-D, and the repeats were homologous to those found in xylanase A from S. lividans, coagulation factor G subunit alpha from the horseshoe crab, Rarobacter faecitabidus protease I, beta-1,3-glucanase from Oerskovia xanthineolytica, and the ricin B chain. However, the repeats did not show sequence similarity to any of the nine known families of cellulose-binding domains (CBDs). On the other hand, STX-II and STX-III contained identical family II CBDs in their C-terminal regions.