The puroindoline proteins (PINA and PINB) of wheat (Triticum aestivum) are largely responsible for grain hardness (kernel texture), a property important in food technology and the wheat trade. PINs are small, basic, cysteine-rich proteins that possess antimicrobial properties, which make them very attractive for novel medical, pharmaceutical and food-industrial applications. Both PIN proteins contain a tryptophan-rich domain (TRD); the TRD of PINA includes five tryptophan (Trp) and three basic residues, while the PINB TRD contains only three Trp residues. Currently, the biochemical role of PIN proteins in endosperm texture as well as in antimicrobial functions remains unclear. In this report we describe the rapid transient expression of recombinant PINA and PINB proteins in Nicotiana benthamiana leaves using the deconstructed tobacco mosaic virus-based ‘magnICON’ plant expression system. Western blot analysis using a ‘monoclonal’ antibody was performed and confirmed the presence of recombinant PINA and PINB in the both dimeric (~16KDa) and oligomeric (~ 48KDa) forms. Maximum yields of recombinant proteins were obtained by directing protein expression to the chloroplast. Purified recombinant PINs exhibited antibacterial activity against gram-negative (Escherichia coli) and gram-positive bacteria (Staphylococcus aureus). To determine grain texture and antimicrobial functions, previous interaction studies of two PIN proteins by yeast two-hybrid systems have shown in vivo interactions between PINA and PINB. In this study, further interactions between PINA and PINB are investigated using Bimolecular Fluorescence Complementation (BiFC) in a plant system. The BiFC method is based on the complementation between fragments of enhanced Yellow Fluorescence Protein that have been attached to target PIN proteins. Expression and interaction of functional recombinant puroindolines in planta will enable further investigation of the key role for PINs on grain texture and antimicrobial abilities.