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Salicylate pathway gene and its use for induction of resistance in plants

A gene and resistance technology, applied in the field of salicylic acid biosynthesis pathway, can solve problems such as can not weaken allergic reactions

Inactive Publication Date: 2001-05-16
杰尼克莫根有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, alternative pathways of hypersensitive response induction clearly exist, and overexpression of nahG in tomato does not attenuate hypersensitive responses that occur after challenge of Cf9 or Cf2 plants with Avr9 and Avr2-containing Cladosporium fulvum species, respectively

Method used

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  • Salicylate pathway gene and its use for induction of resistance in plants
  • Salicylate pathway gene and its use for induction of resistance in plants
  • Salicylate pathway gene and its use for induction of resistance in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Purification of periwinkle ICS

[0066] Periwinkle (L.) was cultured in MS medium (Murashige and Skoog, 1962) supplemented with 30 g / l sucrose as previously described (Moreno, P. et al., Plant Cell Reports 12, 702-705, 1993). G. Don cell culture. Cell cultures were challenged with Pythium aphanisermatum (CBS, Baarn, Netherlands) filtrate as described by Moreno et al. (1993). After 24 hours of stimulation, the cells were collected by suction, washed once with water, immediately frozen in liquid nitrogen, and stored at -80°C. 600 g of frozen cells were homogenized in a Waring Blender equipped with a stainless steel bucket. Add 1ml extraction buffer (0.1M Tris-HCl pH7.5, 10% glycerol (v / v), 1mM DTT, 0.2mM PMSF, 10μM leupeptin and 1mM EDTA) and 50mg polyvinylpyrrolidone per gram of fresh weight . After thawing, the homogenate was centrifuged at 10,000g for 30 minutes to remove cell debris. The supernatant is called crude extract. The following operat...

Embodiment 2

[0070] Cloning of ICS Gene of Vinca

[0071] The ICSII-containing protein band was isolated from a native PAGE gel and digested with trypsin to yield approximately 50 peptides. Five of these peptides were isolated and sequenced. One of these peptides shows high homology to the bacterial isochoristate synthase sequence. Therefore, degenerate primers against this peptide were prepared. A PCR reaction against the primed Vinca cell culture cDNA library using this primer and the T7 primer of pBluescript generated a 520 bp fragment. This fragment was cloned and sequenced. A cDNA library of primed periwinkle cell cultures was screened with the 440 bp amplified DNA fragment. Screening of 450,000 plaques identified 52 independent positive plaques. Twelve of them were isolated and screened a second time with the same 440bp probe. This resulted in the identification of 7 independent positive plaques. They were dissected and partially sequenced in vivo. The longest...

Embodiment 3

[0077] EntC / orfD constructs

[0078]The entC coding sequence was isolated using a PCR strategy on E. coli genomic DNA (Ozenberger et al., J. Bacteriol. 171, 775-783, 1989). For this, primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2) were used. This will amplify the entire coding region of entC and add additional BamHI sites to both ends. This fragment was cloned into vector pMOG843 in which a BamHI site was introduced into a HindIII site via a linker sequence. The resulting pMOG834B-entC contained the entC coding sequence bound to the 35S CaMV promoter, followed by the potato PI-II 3' untranslated sequence. The 35S-entC-PI cassette was then transferred into pIC20H by XbaI digestion and cloning into the XbaI site of pIC20H. A partial XbaI digestion of pIC20H was used. Thus, the cassette is located in the XbaI site flanked by EcoRV sites. The resulting vector was named pIC20H-entC.

[0079] A chloroplast transit peptide (called ss) was isolated from tob...

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Abstract

This invention describes a method to induce pathogen resistance in plants, characterized in that plants are transformed with an expression cassette harboring a gene coding for an isochorismate synthase. More specifically, this method is characterized in that the gene coding for isochorismate synthase is selected from a group consisting of entC, orfA, pchA and ICS, this last gene preferably the ICS gene from Catharantus roseus. Another embodiment of the invention is a method according to the method described above, characterized in that plants are additionally transformed with an expression cassette harboring a gene coding for an isochorismate pyruvate lyase, preferably on the same expression cassette as the gene coding for isochorismate synthase. The gene coding for isochorismate pyruvate lyase is preferably selected from the group consisting of orfD and pchB. A further aspect of the invention is a protein having isochorismate synthase activity which is isolated from Catharantus roseus. Still a further aspect of the invention is the nucleotide sequence comprising the 5' regulatory region which is naturally found to regulate the expression of the ICS gene in Catharantus roseus.

Description

field of invention [0001] The present invention relates to genes in the biosynthetic pathway of salicylic acid, more specifically the salicylic acid pathway via isochorismic acid, and their use in induction of resistance by salicylic acid in plants. More specifically, the present invention relates to the use of the isochoristate synthase (ICS) gene in the production of salicylic acid, in particular through a novel plant isochoristate synthase gene, more specifically the removal of isochoristate synthase In addition to enzymes, the application of isochoristate pyruvate lyase is also involved. In addition, the present invention also relates to the application of the promoter of the novel plant isochoristate synthase gene as a pathogen-inducible promoter. Background technique [0002] Following pathogen attack, plants can respond by initiating defense mechanisms that act locally as well as systemically. In hypersensitivity reactions (HR), local reactions include, inter alia, ...

Claims

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Application Information

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IPC IPC(8): A01H5/00C12N1/21C12N5/10C12N9/88C12N9/90C12N15/09C12N15/61C12N15/82C12N15/84
CPCC12N9/90C12N9/88C12N15/8282C12N15/8283C12N15/8239
Inventor H·J·M·林瑟斯特R·沃普尔特M·C·沃勃恩P·R·H·莫莱诺L·J·P·范泰戈兰G·J·乌勒姆斯A·F·克罗斯M·H·斯退弗J·H·H·V·库斯特斯L·H·希蒙斯L·S·摩尔切尔斯J·F·波尔
Owner 杰尼克莫根有限公司
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