Cloning and Characterization of a Novel Wheat Glycoside Hydrolase Gene TaGlc2 Induced by Powdery Mildew Pathogen (Erysiphe graminis) Infection (original) (raw)
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Acta Agronomica Sinica, 2009
Fungal diseases cause serious yield losses in wheat (Triticum aestivum L.) worldwide. Numerous genes involved in the wheat-pathogen interactions have been identified, which include pathogenesis related (PR) genes and antifungal hydrolases, such as glucanase and chitinase genes. Recently, there has been increasing evidence of the potential involvement of 1,3--glucanase in defense against fungal infection. In this study, a cDNA encoding a 1,3--glucanase, designated TaGlc2, was identified from a wheat cDNA library. The deduced peptide sequence of TaGlc2 is similar to a glycoside hydrolase family 17. Using real-time PCR, the expression pattern of TaGlc2 in wheat seedlings inoculated with powdery mildew pathogen (Erysiphe graminis) was determined. The results showed that TaGlc2 is inducible in response to fungal infection. The 5' genomic region of TaGlc2 was isolated. This gene contains some cis-elements that are reported to be involved in pathogenesis response.
TAG Theoretical and Applied Genetics, 2001
Chitinases and β-1,3-glucanases are important components of plant defense in response to attack by pathogens. To identify specific chitinases and β-1,3-glucanases, we constructed a cDNA library using mRNA from wheat spikelets inoculated with conidia of Fusarium graminearum. Two chitinase and two β-1,3-glucanase clones were isolated using a rice chitinase Ia gene and barley cDNA clones for chitinase II and β-1,3-glucanase as probes. Sequence analysis showed that the cDNA clone SM194 encodes an acidic isoform of class-VII chitinase, the cDNA clone SM383 encodes a class-IV chitinase and the cDNA clones SM289 and SM638 encode two different acidic isoforms of β-1,3-glucanases. Nulli-tetrasomic analysis indicated that SM194 and SM383 were located on all of the group-2 chromosomes of wheat. Genetic mapping showed that at least three copies of class-IV and/or class-VII chitinase genes were clustered on the long arm of chromosome 2D of Aegilops tauschii and that they mapped genetically close to the centromere. SM289 and SM638 were located on all of the group 3 chromosomes of wheat by nulli-tetrasomic analysis, and to the β-1,3-glucanase clusters in the 3BL and 3DL chromosome arms of wheat by genetic mapping. Northern blot hybridization showed that the expression of these genes is induced upon infection with Fusarium graminearum. The accumulation of transcripts for these PR-proteins was more rapid in the resistant variety Sumai 3 than in its susceptible mutant during the first 24 h. This is the first report of the induction of class-IV and class-VII chitinases in cereals by a fungal pathogen.
Planta, 1997
Pathogenesis-related expression of the two antifungal hydrolases 13-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) was studied in wheat (Triticum aestivum L.) as part of the defence response to stem rust (Puccinia graminis f.sp. tritici; Pgt), mediated by the semi-dominantly acting resistance genes Sr5 and Sr24. Complete resistance (infection type 0), mediated by the Sr5 gene in cultivar Pre-Sr5, closely correlates with the hypersensitive response of penetrated cells at early stage of the interaction, when the first haustorium is formed. In contrast, cultivar Pre-Sr24 shows intermediate resistance (infection type 2 3) which is not directly linked to cell death. In both cases, the plant response included a rapid increase in 13-1,3-glucanase activity between 24 and 48 h after inoculation. One main extracellular 30-kDa isoform of 13-1,3-glucanase was present in both lines, as shown by polyacrylamide-gel electrophoresis. Two additional minor isoforms (32 and 23 kDa) were detected only in Pre-Sr24, and only at later time points. Increased enzyme activity and the appearance of new isoforms in the resistant lines was preceded by accumulation of mRNAs encoding [3-1,3glucanases and chitinases. However, there were no changes in chitinase activity or isoforms. A high constitutive level of chitinase activity was observed in all wheat genotypes. Serological studies indicated the presence of a class II chitinase of 26 kDa. Accumulation of 13-1,3-glucanase and chitinase transcripts was detected before the pathogen penetrated the leaves through stomata and approximately 16 h before the typical hypersensitive response was observed, indicating that signal(s) for defense gene activation were recognised by the host plant long before a tight contact between the pathogen and a host cell is established. A glycoprotein (Pgt elicitor) derived from hyphal walls, strongly induced [3-1,3-glucanase. We discuss the possible role Abbreviations: dpi = days post inoculation; HR = hypersensitive response; ICE = intracellular extract; IWF = intercellular washing fluid; Pgt = Puccinia graminis f.sp. tritici; PCR = polymerase chain reaction PR = pathogenesis-related Correspondence to: K.H. Kogel; FAX: 49 (241) 8888 182 of the elicitor in the early Signalling mediating Sr5-and Sr24-specified resistance in wheat.
1997
Near-isogenic resistant and susceptible lines of barley (Hordeum ulgare L.) were inoculated with a virulent race of the causal agent of leaf scald, Rhynchosporium secalis, and the expression patterns of specific barley (143)-β-glucan endohydrolase (EC 3;2;1;39) isoenzymes monitored. Induction of (143)-β-glucanase activity occurred 1 day earlier and to higher levels in the resistant backcross line BC-200 than in the susceptible parent line. Thus, the resistant plants of this backcross line responded more rapidly to pathogen invasion than susceptible lines. The increased (143)-βglucanase activity was attributable predominantly to a 2-3 day period in which mRNA transcripts of genes that encode isoenzymes GII and GI accumulated. These genes may therefore be important in the defence responses of barley backcross line BC-200 to pathogen attack. A second scald-resistant backcross line, BC-30, developed levels of (143)-β-glucanase similar to BC-200 upon infection, but maximal levels were reached later than in BC-200. The third resistant backcross line, BC-35, showed the same pattern of expression as the susceptible parent cultivar, Clipper. The differences in expression of (143)-β-glucanase between the resistant backcross lines clearly show that there are physiologically distinct modes of resistance to the scald fungus in barley, and that at least one of these is accompanied by (143)-β-glucanase induction.
Plant Cell Reports, 2004
Two-step PCR (RT-PCR and nested PCR) was used to detect gene expression in powdery mildew pathogen-infected cells of detached inner epidermis of barley coleoptiles. Cellular contents of infected cells were microscopically suctioned with a micropipette and subjected to PCR. Triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase genes involved in the glycolytic pathway and a stimulus-induced endochitinase gene were targeted, and their expression was determined by detecting cDNAs derived from spliced transcripts. The two gycolysis-related genes were constantly expressed in the tissue irrespective of pathogen inoculation. In contrast, chitinase gene expression was induced in non-infected inner epidermis after detachment. After inoculation, this expression was selectively suppressed in pathogen-invaded cells, in spite of continuous expression in non-invaded cells of the same epidermis. Thus, the present method enabled us to directly analyze transcripts in individual cells at the infection site and assess the capability of the pathogen to regulate host gene expression.
Molecular Assesment of Chitinase Activity in Transgenic Wheat
Egyptian Journal of Genetics and Cytology, 2016
Fungal diseases especially rust of common wheat (Triticum aestivum) and durum (T. turgidum subsp. durum), historically was one of the most destruc- tive wheat diseases. Significant losses occurred in the past when the disease de- veloped into epidemic proportions in wheat crops (Roelfs, 1978). Plants natu- rally respond to fungal attack by a com- plex network of defense mechanisms, which are activated upon perception of a pathogen and designed to limit its pene- tration and development. Defense responses include structural and biochemical responses like reinforcement of the plant cell wall, accumulation of phytox- lexins with microbial toxicity, ribosome- inactivating proteins (RIPs) that inhibit protein synthesis, antimicrobial peptides and the synthesis of other pathogenesis- related (PR) proteins (Yang et al., 1997). Some PR proteins, such as chitinase and glucanases, have hydrolytic activities against structural components of fungal cell walls and may exhibit strong antifun- gal...
2020
Bread wheat (Triticum aestivum L.) is the most widely grown crop worldwide. Powdery mildew caused by fungal pathogen Blumeria graminis is one of the most devastating diseases of wheat. The present study aimed to identify differentially expressed genes and investigate their expression in response to B. graminis in susceptible (Bolani) and resistant (KC2306) wheat genotypes, using publicly available microarray data set and qRT-PCR analysis. A total of 5760 and 5315 probe sets were detected which 5427 and 4630 by adjusted P-value < 0.05 and 168 and 144 genes based on e-value < 1 × 10–5 cut-off were differentially expressed in susceptible and resistant wheat genotypes, respectively. Among exclusively up regulated genes in the resistant genotype 12 hpi compared to its control, fifteen potential genes that may be responsible for B. graminis inoculation resistance were detected. The results of real time PCR for the candidate genes showed that the genes were upregulated in the resista...
Physiological and Molecular Plant Pathology, 1992
A cDNA library of RNA from barley leaves inoculated with Ervsiphe graminis was screened using labelled cDNA enriched for specific sequences by subtractive hybridization against RNA from non-inoculated leaves. This resulted in isolation of several clones representing pathogen induced genes. By cross-hybridization and sequence analysis, one of the cDNAs (pBT6-3) was found to be a partial clone representing a putative peroxidase, for which a full-length eDNA clone (pBH6-301) was subsequently isolated. The predicted amino acid sequence revealed a 21 amino acid signal peptide and a 294 amino acid mature protein I3I kDa1 and shows 56 t 0 amino acid identity to a basic peroxidase from turnip, 89 % to a putative peroxidase from wheat, but only 38 % to the amino acid sequence derived from the eDNA clone 1pcD1311) of a second putative barley peroxidase expressed in leaves. Northern blot analysis showed that the pBT6-3 (pBH6-301) transcript is elevated as early as 4 h after inoculation with E. graminis f. sp. harder and that two maxima in transcript levels appear, which can be correlated with penetration attempts by the fungus. The amount of the pcD 131 1 transcript was also found to increase in inoculated leaves but at a later time point .
BMC biotechnology, 2024
Fusarium head blight (FHB) is a devastating fungal disease affecting different cereals, particularly wheat, and poses a serious threat to global wheat production. Chitinases and β-glucanases are two important proteins involved in lysing fungal cell walls by targeting essential macromolecular components, including chitin and β-glucan micro fibrils. In our experiment, a transgenic wheat (Triticum aestivum) was generated by introducing chitinase and glucanase genes using Biolistic technique and Recombinant pBI121 plasmid (pBI-ChiGlu (-)). This plasmid contained chitinase and glucanase genes as well as nptII gene as a selectable marker. The expression of chitinase and glucanase was individually controlled by CaMV35S promoter and Nos terminator. Immature embryo explants from five Iranian cultivars (Arta, Moghan, Sisun, Gascogen and A-Line) were excised from seeds and cultured on callus induction medium to generate embryonic calluses. Embryogenic calluses with light cream color and brittle texture were selected and bombarded using gold nanoparticles coated with the recombinant pBI-ChiGlu plasmid. Bombarded calluses initially were transferred to selective callus induction medium, and later, they were transfferd to selective regeneration medium. The selective agent was kanamycin at a concentration of 25 mg/l in both media. Among five studied cultivars, A-Line showed the highest transformation percentage (4.8%), followed by the Sisun, Gascogen and Arta in descending order. PCR and Southern blot analysis confirmed the integration of genes into the genome of wheat cultivars. Furthermore, in an in-vitro assay, the growth of Fusarium graminearum was significantly inhibited by using 200 μg of leaf protein extract from transgenic plants. According to our results, the transgenic plants (T 1) showed the resistance against Fusarium when were compared to the non-transgenic plants. All transgenic plants showed normal fertility and no abnormal response was observed in their growth and development.