The Lupinus albus class-III chitinase gene, IF3, is constitutively expressed in vegetative organs and developing seeds (original) (raw)
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European Journal of Plant Pathology, 2016
A class-III chitinase promoter was isolated from Lupinus albus. The region 5′ to the coding sequence of the IF3 gene was amplified by gene walking and sequenced. The proximal 2.0 kb sequence contains a predicted promoter site, including a TATA box, near the ATG start site. To test for minimal sequences needed for promoter activity, the region was restricted into fragments of 1.81, 1.51 and 1.13 kb and cloned into the pDM327 vector, upstream from the bar-gus fusion gene for Biolistic™ transformation. Transformation of lupin embryos, bean callus tissue, maize embryos and Ornithogalum callus demonstrated promoter activity for all fragments. In silico analysis identified putative cis-acting elements in the 1.81 kb fragment that could be important in controlling gene expression. Fungal elicitor activated-, woundinducible-and ethylene responsive elements were present in the 1.51 kb fragment. Myb elements and CAAT boxes that regulate responses to environmental factors and modulate promoter efficiency were identified in the 1.81 kb fragment. The 1.51 and 1.81 kb fragments were inserted upstream of the gus gene into the pBI121 vector for Agrobacterium tumefaciens transformation of tobacco. Quantitative GUS assays indicated that the promoter fragments are functional in planta and inducible by defense-related signals, wounding, as well as chemical elicitation. All important elements essential for Bion inducibility are present on the shorter (1.51 kb) promoter fragment, but both 5′ distal and proximal cis-elements are required for full functionality. The IF3 promoter is, thus, suitable for use in defense gene constructs prepared for the production of anthracnose resistant lupin.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1995
Complementary and genomic DNAs coding for a Brassica napus chitinase have been cloned and sequenced. The genomic DNA contains one intron and encodes a 322-amino acid basic chitinase with a 20-amino acid N-terminal signal peptide followed by a 40-amino acid cysteine-rich domain, linked by a hinge region to the main domain of the enzyme. The sequence of the cDNAs is identical to the exon sequence deduced from the genomic DNA. A probe derived from this gene identified a 1.2-kb transcript present in high amount in roots, moderate in floral tissues and low in stems and leaves. The synthesis of these transcripts is regulated during development and is induced in roots by wounding and ethephon. This type of chitinase is encoded by two sequences in Brassica napus, as shown either by Southern hybridizations or by genomic amplification and sequencing using the polymerase chain reaction. These genes are homologous to one sequence found in the Brassica oleracea genome.
Effect of chitinase antisense RNA expression on disease susceptibility of Arabidopsis plants
Plant Molecular Biology, 1994
Chitinases accumulate in higher plants upon pathogen attack are capable of hydrolyzing chitin-containing fungal cell walls and are thus implicated as part of the plant defense response to fungal pathogens. To evaluate the relative role of the predominate chitinase (class I, basic enzyme) ofArabidopsis thaliana in disease resistance, transgenic Arabidopsis plants were generated that expressed antisense RNA to the class I chitinase. Young plants or young leaves of some plants expressing antisense RNA had < 10~o of the chitinase levels of control plants. In the oldest leaves of these antisense plants, chitinase levels rose to 37-90~o of the chitinase levels relative to vector control plants, most likely because of accumulation and storage of the enzyme in vacuoles. The rate of infection by the fungal pathogen Botrytis cinerea was measured in detached leaves containing 7-15 ~o of the chitinase levels of control plants prior to inoculation. Antisense RNA was not effective in suppressing induced chitinase expression upon infection as chitinase levels increased in antisense leaves to 47~o of levels in control leaves within 24 hours after inoculation. Leaves from antisense plants became diseased at a slightly faster rate than leaves from control plants, but differences were not significant due to high variability. Although the tendency to increased susceptibility in antisense plants suggests that chitinases may slow the growth of invading fungal pathogens, the overall contribution of chitinase to the inducible defense reponses in Arabidopsis remains unclear.
Biologia Plantarum, 1996
cultivars suggests that these play a less important role in the thora capsici led to the accumulation of -1,3-glucanases in development of resistance although they appeared earlier and at greater intensity in the resistant cultivar than in the suscep-the stem tissues soon after inoculation. After the appearance of the symptoms on the pepper stems, -1,3-glucanase accu-tible one. Using degenerate primers and reverse-transcriptase polymerase chain reaction (RT-PCR), -1,3-glucanase mulation became much more pronounced in the resistant (Smith-5 [S-5]) than in the susceptible (Yolo Wonder [YW]) product of approximately 500 bp was obtained and cloned from cv. S-5 total RNA. This was used as a probe on northern cultivar. -1,3-Glucanase activity was also detected in the control stems (uninoculated) but only in the resistant cultivar.
Plant Molecular Biology, 2003
Pathogenesis-related (PR) proteins are plant proteins that are induced in response to pathogen attack. PR proteins are grouped into independent families based on their sequences and properties. The PR-4 family comprises class I and class II chitinases. We have isolated a full-length cDNA encoding a chitinase from maize which shares a high degree of nucleotide and amino acid sequence homology with the class II chitinases of the PR-4 family of PR proteins. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by the fungus Fusarium moniliforme, increase the level of ZmPR4 mRNA. In situ mRNA hybridization analysis in sections obtained from fungus-infected germinating embryos revealed that ZmPR4 mRNA accumulation occurs in those cell types that first establish contact with the pathogen. ZmPR4 mRNA accumulation is also stimulated by treatment with silver nitrate whereas the application of the hormones gibberellic acid or acetylsalicylic acid has no effect. Wounding, or treatment with abscisic acid or methyl jasmonate, results in accumulation of ZmPR4 mRNA in maize leaves. Furthermore, the ZmPR4 protein was expressed in Escherichia coli, purified and used to obtain polyclonal antibodies that specifically recognized ZmPR4 in protein extracts from fungus-infected embryos. Accumulation of ZmPR4 mRNA in fungus-infected maize tissues was accompanied by a significant accumulation of the corresponding protein. The possible implications of these findings as part of the general defence response of maize plants against pathogens are discussed.
Physiological and Molecular Plant Pathology, 2011
We have cloned three cDNAs encoding chitinases from oil palm, EgCHI1, EgCHI2, and EgCHI3. The abundance of transcripts encoding all three chitinases was relatively higher in oil palm root tissues treated with either Ganoderma boninense or Trichoderma harzianum singly compared to that of untreated oil palm root tissues at 5 week post inoculation (wpi). The expression of EgCHI1 and EgCHI2 was also up-regulated in oil palm roots treated with a combination of G. boninense and T. harzianum at 2, 5 and 8 wpi. The up-regulation of chitinases understudied was likely a universal response of host plant to fungal attack.► Association of host plants and fungi increased expression of defense-related enzymes. ► Chitinases are important PR proteins that degrade fungal cell wall. ► Fungi increased transcripts encoding plant chitinases. ► Universal response of host plant against fungi.
Pathogenesis-Related Proteins for the Plant Protection
Fungi are far more complex organisms than viruses or bacteria and can developed numerous diseases in plants that cause loss of big portion of the crop every year. Plants have developed various mechanisms to defend themselves against these fungi which include the production of low molecular weight secondary metabolites, proteins and peptides having antifungal activity. In this review, brief information like biochemistry, source, regulation of gene expression, mode of action of defense mechanism of various pathogenesis-related proteins is given. Proteins include pathogenesis-related protein 1, â-glucanases, chitinases, chitin binding protein, thaumatine like protein, glycine-histidine rich proteins, ribosome inactivating protein, and some newly discovered antifungal proteins.
The role of plant defence proteins in fungal pathogenesis
Molecular Plant Pathology, 2007
It is becoming increasingly evident that a plant-pathogen interaction may be compared to an open warfare, whose major weapons are proteins synthesized by both organisms. These weapons were gradually developed in what must have been a multimillionyear evolutionary game of ping-pong. The outcome of each battle results in the establishment of resistance or pathogenesis. The plethora of resistance mechanisms exhibited by plants may be grouped into constitutive and inducible, and range from morphological to structural and chemical defences. Most of these mechanisms are defensive, exhibiting a passive role, but some are highly active against pathogens, using as major targets the fungal cell wall, the plasma membrane or intracellular targets. A considerable overlap exists between pathogenesis-related (PR) proteins and antifungal proteins. However, many of the now considered 17 families of PR proteins do not present any known role as antipathogen activity, whereas among the 13 classes of antifungal proteins, most are not PR proteins. Discovery of novel antifungal proteins and peptides continues at a rapid pace. In their long coevolution with plants, phytopathogens have evolved ways to avoid or circumvent the plant defence weaponry. These include protection of fungal structures from plant defence reactions, inhibition of elicitor-induced plant defence responses and suppression of plant defences. A detailed understanding of the molecular events that take place during a plant-pathogen interaction is an essential goal for disease control in the future. Comparison of three pathogenesis-related proteins from plants of two cultivars of tobacco infected with TMV. J. Gen. Virol. 47, 79-87. Argos, P., Narayana, S.V. and Nielsen, N.C. (1985) Structural similarity between legumin and vicilin storage proteins from legumes. EMBO J. 4, 1111-1117. Arie, M., Hikichi, K., Takahashi, K. and Esaka, M. (2000) Characterization of a basic chitinase which is secreted by cultured pumpkin cells. Physiologia Plantarum, 110, 232-239. Ary, M.B., Richardson, M. and Shewry, P.R. (1989) Purification and characterization of an insect alpha-amylase inhibitor/endochitinase from seeds of Job's Tears (Coix lachryma-jobi ). Biochim. Biophys. Acta, 999, 260-266. Localized changes in peroxidase activity accompany hydrogen peroxide generation during the development of a nonhost hypersensitive reaction in Lettuce. Plant Physiol. 118, 1067-1078. Blackwell, J. (1988) Physical methods for the determination of chitin. structure and conformation. Methods Enzymol. 161, 435-442. Blilou, I., Ocampo, J.A. and Garcia-Garrido, J.M. (2000) Induction of Ltp (lipid transfer protein) and Pal (phenylalanine ammonia-lyase) gene expression in rice roots colonized by the arbuscular mycorrhizal fungus Glomus mosseae. HPLC analysis of grapevine phytoalexins coupling photodiode array detection and fluorometry. Anal. Chem. 69, 5172-5177. Jeandet, P., Douillet-Breuil, A.C., Bessis, R., Debord, S., Sbaghi, M. and Adrian, M. (2002) Phytoalexins from the Vitaceae: biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism. Constitutive expression of a celery mannitol dehydrogenase in tobacco enhances resistance to the mannitol-secreting fungal pathogen Alternaria alternata. Plant J. 32, 41-49. Jones, D.A. and Takemoto, D. (2004) Plant innate immunity-direct and indirect recognition of general and specific pathogen-associated molecules. Curr. Opin. Immunol. 16, 48-62. Jongedijk, E., Tigelaar, H., Roekel, J.S.C., Bres-Vloemans, S.A., Dekker, I., Elzen, P.J.M., Cornelissen, B.J.C. and Melchers, L.S. (1955) Synergistic activity of chitinases and beta-1,3-glucanases enhances fungal resistance in transgenic tomato plants. Euphytica, 85, 173-180. Jordá, L., Conejero, V. and Vera, P. (2000) Characterization of P69E and P69F, two differentially regulated genes encoding new members of the subtilisin-like proteinase family from tomato plants. Plant Physiol. 122, 67-74. Joshi, B.N., Sainani, M.N., Bastawade, K.B., Gupta, V.S. and Ranjekar, P.K. (1998) Cysteine protease inhibitor from pearl millet: a new class of antifungal protein. A novel yeast gene, RHK1, is involved in the synthesis of the cell wall receptor for the HM-1 killer toxin that inhibits beta-1,3-glucan synthesis. N-glycosylation is involved in the sensitivity of Saccharomyces cerevisiae to HM-1 killer toxin secreted from Hansenula mrakii IFO 0895. Appl. Microbiol. Biotechnol. 51, 176-184. Kitajima, S. and Sato, F. (1999) Plant pathogenesis-related proteins: molecular mechanisms of gene expression and protein function.
Biotechnology reports (Amsterdam, Netherlands), 2017
Chitinases are the hydrolytic enzymes which belong to the pathogenesis-related (PR) protein family and play an important role not only in plant defense but also in various abiotic stresses. However, only a limited number of chitinase genes have been characterised in B. juncea. In this study, we have characterised B. juncea class IV chitinase gene (accession no EF586206) in response to fungal infection, salicylic acid (SA), jasmonic acid (JA) treatments and wounding. Gene expression studies revealed that the transcript levels of Bjchitinase (BjChp) gene increases significantly both in local and distal tissues after Alternaria infection. Bjchitinase gene was also induced by jasmonic acid and wounding but moderately by salicylic acid. A 2.5 kb class IV chitinase promoter of this gene was isolated from B. juncea by Genome walking (accession no KF055403.1). In-silico analysis of this promoter revealed a number of conserved cis-regulatory elements related to defense, wounding and signalli...
2003
Chitinase catalyses the hydrolysis of β-1,4-N-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin and is involved in the inducible defenses of plants. The aim of this research was to isolate and clone chitinase cDNAs from the seed of winged bean. Chitinase gene fragments were isolated from a winged bean seed cDNA library using two sets of degenerate primers corresponding to the conserved regions of chitinase class I and IV. The poly A + mRNA was reversed transcribed and further amplified using RT-PCR. A 1.1 Kb fragment was selected, cloned and sequenced. A nucleotide se- quence comparison identified the fragment as a Class I basic chitinase cDNA; this fragment was subsequently used as a probe to screen for a full length transcript from the cDNA library. Library screening resulted in the isolation of a 1324 bp clone designated CHRZP; encoding a polypeptide of 289 amino acids containing the diagnostic N-terminal cysteine-rich domain of class 1 chitinases. CHRZP showed...