Identification and validation of the wound and insect bite early inducible promoter from Arabidopsis thaliana.

A wound-inducible promoter facilitates the regulated gene expression at the targeted site during the time of mechanical stress or infestation by the pathogen. The present work has aimed to identify a wound-inducible promoter that expresses at early time points preceding wound-stress treatment in Arabidopsis thaliana. The computational analysis of microarray data (GSE5627) resulted in the identification of five early inducible genes, viz., AT1G17380, AT1G80440, AT2G43530, AT3G48360, and AT5G13220. The RT-PCR analysis showed AT5G13220 (JASMONATE-ASSOCIATED 1) gene induced at a significantly higher level post 30 min of wounding. Thus, the promoter of the highly induced and early expressed wound-inducible gene, AT5G13220 (named PW220), was characterized by fusing with ß-glucuronidase (gusA) reporter or Cry1EC genes. The fluorometric analysis and histochemical staining of the gusA gene and quantitative estimation of Cry1EC protein in Nicotiana tabacum transgenic lines confirmed wound-induced expression characteristic of the selected promoter. Insect bioassay suggested that wound-inducible and constitutive expression of Cry1EC protein in transgenic lines showed a similar level of protection against different instar Spodoptera litura larvae. Furthermore, we identified that abscisic acid influenced the wound-specific expression of the selected PW220 promoter in the transgenic lines, which correlates with the presence of conserved cis-regulatory elements associated with dehydration and abscisic acid responses. Altogether, our results suggested that the wound-inducible promoter PW220 provides an excellent alternative for developing insect-tolerant transgenic crops in the future. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03143-0.

Whitefly and aphid inducible promoters of Arabidopsis thaliana L.

Lack of regulated expression and tissue specificity are the major drawbacks of plant and virus-derived constitutive promoters. A precise tissue or site-specific expression, facilitate regulated expression of proteins at the targeted time and site. Publically available microarray data on whitefly and aphid infested Arabidopsis thaliana L. was used to identify whitefly and aphid-inducible genes. The qRT-PCR further validated the inducible behaviour of these genes under artificial infestation. Promoter sequences of genes were retrieved from the Arabidopsis Information Resources database with their corresponding 5'UTR and cloned from the A. thaliana genome. Promoter reporter transcriptional fusions were developed with the beta-glucuronidase (GUS) gusA gene in a binary expression vector to validate the inducible behaviour of these promoters in eight independent transgenic Nicotiana tabaccum lines. Histochemical analysis of the reporter gene in T2 transgenic tobacco lines confirmed promoter driven expression at the sites of aphid and whitefly infestation. The qRT-PCR and GUS expression analysis of transgenic lines revealed that abscisic acid largely influenced the expression of both aphid and whitefly inducible promoters. Further, whitefly-specific promoter respond to salicylic acid and jasmonic acid (JA), whereas aphid-specific promoters to JA and 1-aminocyclopropane carboxylic acid. The response of promoters to phytohormones correlated to the presence of corresponding conserved cis-regulatory elements.