Induction of a tomato anionic peroxidase gene (tap1) by wounding in transgenic tobacco and activation of tap1/GUS and tap2/GUS chimeric gene fusions in transgenic tobacco by wounding and pathogen attack.
The anionic peroxidase genes of tomato, tap1 and tap2, are induced by wounding in tomato fruits and by elicitor treatment in cell suspension cultures. These homologous genes code for anionic peroxidases that are postulated to cause polymerization of the phenolic residues into wall polymers in wound-healing and pathogen-infected tissues. An expression construct containing the entire TAP1 gene with its 5' and 3' flanking sequences was introduced into tobacco by Agrobacterium tumefaciens-mediated gene transfer. Also, constructs containing the 5' upstream regions of tap1 and tap2 including sequences coding for their respective putative leader peptides fused translationally to the beta-glucuronidase (GUS) reporter gene were made and introduced into tobacco. Northern blot analysis of transcripts from wound-healing leaf tissues of transformants containing tap1 showed that the introduced gene was being transcribed in the heterologous host. The induction of tap1 transcripts in the wound-healing transgenic tobacco tissues was observed by 48 h and increased over time period of 84 h. Wounding also led to expression of GUS in tap1/GUS and tap2/GUS transformants and GUS activity was localized to the wound site. Activation of the tap1 and tap2 promoters in wound-healing transgenic tobacco tissues showed a GUS expression profile that correlated with the postulated role for anionic peroxidases in phenolic polymerization in suberizing tissues. Inoculation of tap1/GUS and tap2/GUS transformant leaves with fungal conidia from Fusarium solani f. sp. pisi caused expression of GUS in locally inoculated regions, and GUS expression increased over a period of four days.
Mohan
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R
Ohio State Biotechnology Center and Biochemistry Program, Ohio State University, Columbus 43210.
Bajar
A M
AM
Kolattukudy
P E
PE
D015152:Blotting, Northern; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D010544:Peroxidases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010944:Plants / Q000201:enzymology / Q000235:genetics*; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013997:Time Factors; D014026:Nicotiana / Q000235:genetics*; D014158:Transcription, Genetic; D014947:Wounds and Injuries
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D012313:RNA; D010544:Peroxidases; D005966:Glucuronidase