Characterization of vascular-specific RSs1 and rolC promoters for their utilization in engineering plants to develop resistance against hemipteran insect pests

Rice sucrose synthase1, RSs1 (isolated from rice) and rolC (isolated from Agrobacterium rhizogenes) promoters were evaluated by binding analyses of their respective cis-elements with host nuclear transcription factors. The expression profile of an insecticidal protein driven by these promoters in transgenic plants was monitored. Motif-search analysis with available phloem-specific promoter sequences revealed the presence of two BoxII elements in RSs1. An octopine synthase element, a stem-specific, a root-specific and a light-responsive element were found in the rolC promoter, whereas the ASL box, GATA and 13 bp motifs were detected in both promoters. Binding analysis of these cis-elements (both in native and mutant forms) with the trans-factors present in the nuclear extracts from rice, tobacco and chickpea, followed by electrophoretic mobility shift assay, documented a highly specific cis-trans interaction. Both promoters were utilized to express Allium sativum leaf agglutinin (ASAL) gene in the three aforementioned plant systems. By immunohistochemistry and immunohistofluorescence, specific patterns of ASAL accumulation were detected in vascular tissues of single copy transgenic plants. Transgenic plants expressing ASAL in a phloem-specific manner demonstrated about 60-65% more insecticidal activity than control plants. The two promoters, which evolved independently from two distinctly unrelated origins, were found to maintain their functionality in a conserved manner. They were able to express the insecticidal protein coding ASAL as transgene both in monocot and dicot hosts. Thus, the two promoters are valuable as prospective phloem-specific promoters for use in plant biotechnological programmes.