Combining the CowPEAsy web application with in planta agroinfiltration
for native promoter validation in Vigna unguiculata
Abstract
Cowpea ( Vigna unguiculata) is an important protein source in
Sub-Saharan Africa. Optimizing resilience and productivity through
genetic engineering in cowpea has been slow due in part to a lack of
defined species-specific regulatory elements and difficulty testing gene
function within the native system. In many plant species,
Agrobacterium-mediated transient gene expression is widely used
to validate constructs before investing in transgenic lines, but its
implementation in legumes has been challenging. In this study, we
optimized an in planta agroinfiltration assay in trifoliate
cowpea leaves using a betalain reporter. To demonstrate the “intact
plant” aspect of this system, we used this assay to characterize
drought-inducible promoters by challenging cowpea plants with drought
stress. Subsequently, to identify and broaden the pool of native
promoters known in cowpea, we developed a user-friendly web application,
CowPEAsy, allowing users to interrogate gene expression from our
canopy-level, developmental-series RNA-Seq dataset. Finally, using
CowPEAsy, we identified six promoters that showed constitutive
expression across all conditions and verified these promoters with our
transient system. This work provides an in vivo platform for
preliminary validation of regulatory elements in cowpea and other
legumes and enhances current genetic resources by identifying a suite of
physiologically relevant promoters of varying strengths.