Functional characterization of a defense-related class-III chitinase promoter from Lupinus albus, active in legume and monocot tissues

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.South African Agricultural Research Council (ARC) and the Protein Research Trust (PRT).http://link.springer.com/journal/106582017-12-31hb2016Plant Scienc

Analysis of orthologous secondary wall-associated nac domain1 (SND1) promotor activity in herbaceous and woody angiosperms

SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1) is a master regulator of fibre secondary wall deposition in Arabidopsis thaliana (Arabidopsis), with homologs in other angiosperms and gymnosperms. However, it is poorly understood to what extent the fibre-specific regulation of the SND1 promoter, and that of its orthologs, is conserved between diverged herbaceous and woody lineages. We performed a reciprocal reporter gene analysis of orthologous SND1 promoters from Arabidopsis (AthSND1), Eucalyptus grandis (EgrNAC61) and Populus alba P. grandidentata (PagWND1A) relative to secondary cell wall-specific Cellulose Synthase4 (CesA4) and CesA7 promoters, in both a non-woody (Arabidopsis) and a woody (poplar) system. -glucuronidase (GUS) reporter analysis in Arabidopsis showed that the SND1 promoter was active in vascular tissues as previously reported and showed interfascicular and xylary fibre-specific expression in inflorescence stems, while reporter constructs of the woody plant-derived promoters were partial to the (pro)cambium-phloem and protoxylem. In transgenic P. tremula P. alba plants, all three orthologous SND1 promoters expressed the GUS reporter similarly and preferentially in developing secondary xylem, ray parenchyma and cork cambium. Ours is the first study to reciprocally test orthologous SND1 promoter specificity in herbaceous and woody species, revealing diverged regulatory functions in the herbaceous system.The Department of Science and Technology, South Africa (Eucalyptus Genomics strategic grant), the National Research Foundation, South Africa (NRF Bioinformatics and Functional Genomics Programme grant UID 97911), with additional support through the Forest Molecular Genetics Programme by the Technologies and Human Resources for Industry Programme (THRIP), Mondi Ltd. and Sappi Ltd.http://www.mdpi.com/journal/ijmsam2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Functional characterization of a defense-related class-III chitinase promoter from Lupinus albus, active in legume and monocot tissues

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.South African Agricultural Research Council (ARC) and the Protein Research Trust (PRT).http://link.springer.com/journal/106582017-12-31hb2016Plant Scienc