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Inducible promoters

A technology of promoters and nucleic acid molecules, applied in the field of systemic acquired resistance phenomena, can solve problems such as inability to induce SAR gene expression or disease resistance, blocking pathways, etc.

Inactive Publication Date: 2007-04-25
SYNGENTA PARTICIPATIONS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although nim1 is able to accumulate SA after pathogen infection, it cannot induce SAR gene expression or disease resistance, suggesting that the mutation blocks pathways downstream of SA

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Example 1: NIM1 two-hybrid screening

[0129] The Arabidopsis NIM1 gene (Ryals et al., 1997) was used as bait in the yeast two-hybrid screen. The screening system used was based on the LexA version of the two-hybrid screening system originally developed by Fields and Song (1989) and later modified by Gyuris et al. (1993). The full-length NIM1 gene sequence was cloned into the bait plasmid pEG202 (Gyuris et al., 1993) as a fusion with the LexA DNA-binding domain using PCR products (primers NIM5'RI and NIM3'SalI, see Table 1 below). Another bait (NIM450) was constructed using the nucleotide sequence encoding the first 450 amino acids of the NIM1 protein (Ryals et al., 1997) and primers NIM5'RI and NIMtruncNCOI3'. A third bait containing only the C-terminal portion of the NIM1 gene encoding amino acids 366-594 of the NIM1 protein (Ryals et al., 1997) was cloned using primers NIMloop5'RI and NIM3'SalI. These baits were transformed into yeast strain EGY188 c...

Embodiment 2

[0132] Example 2: Induction of NI16 mRNA

[0133] 1. Salicylic acid and BTH induce NI16 mRNA

[0134] 4-week-old Wassilewskija (WS -0) Plant RNA preparation. Ward et al. (1991) and Lawton et al. (1996) have demonstrated that both SA and BTH activate SAR and induce pathogenesis-related (PR) gene expression in Arabidopsis. RNA extraction and Northern blot hybridization were performed as previously described by Ausubel et al. (1987). NI16 RNA was induced 5-10 fold within 15 minutes of SA or BTH induction and peaked at approximately 50 fold induction within 2 hours of SA treatment and 25 fold induction with BTH treatment. Transcripts were highly induced for up to 24 hours after SA treatment and remained highly induced for at least 48 hours after BTH treatment.

[0135] 2. NI16 mRNA is induced in response to pathogen infection but not in NahG or nim1-4 plants

[0136] WS-0 plants were infected with the avirulent pathogen Pseudomonas syringae pv tomato (P.s.t.) cont...

Embodiment 3

[0137] Example 3: Genomic Cloning of NI16 and Related Promoter Sequences

[0138] A genomic copy of NI16 was cloned from the lambda ZapII library (Stratagene) into the vector pBluescript according to the manufacturer's protocol. Two separate clones were obtained from the library. Clone 4-1 contained 3.1 kb of NI16 upstream sequence and NI16 coding region up to the internal EcoRI site (see SEQ ID NO: 1). The second clone, 5-1, contained the 3' sequence of the EcoRI site and 3.1 kb downstream of NI16. A complete genomic clone was obtained by first deleting the 1.87 kb BglII / BamHI fragment from clone 4-1 and then ligating the 350 bp EcoRI / EcoRV fragment from clone 5-1 into the remaining EcoRI and EcoRV sites of clone 4-1. The genomic clone was sequenced to obtain the NI16 genomic sequence shown as SEQ ID NO:3. The promoter element is located in the 5' upstream region adjacent to the NI16 genomic coding region (see SEQ ID NO: 3). The E. coli strain DH5α containing the pl...

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PUM

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Abstract

The Arabidopsis NI16 gene was isolated in a yeast 2-hybrid screen via its interaction with the NIM1 protein and encodes a protein involved in the regulation of SAR gene expression in plants. NI16 is strongly induced in NTM1-overexpressing plants treated with benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH). The nucleic acid sequence of the Arabidopsis NI16 promoter is disclosed herein.

Description

field of invention [0001] The present invention relates to broad-spectrum disease resistance in plants, including the phenomenon of systemic acquired resistance (SAR). More specifically, the present invention relates to the identification, isolation, and characterization of genes encoding proteins involved in the regulation of SAR gene expression in plants. Background of the invention [0002] Plants are constantly challenged by a wide variety of pathogenic organisms, including viruses, bacteria, fungi, and nematodes. Crops are particularly vulnerable because they are often grown as genetically uniform monocrops; when diseases strike, the losses can be devastating. However, most plants have their own innate defense mechanisms against pathogenic organisms. Natural variations in plant pathogen resistance have been identified by plant breeders and pathologists and bred into many crops. These natural disease resistance genes often confer high levels of resistance or immunity ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H5/10
CPCC12N15/8239C12N15/8237
Inventor R·M·凯德R·A·迪特里希K·A·劳顿
Owner SYNGENTA PARTICIPATIONS AG
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