Transgenic alfalfa secretes a fungal endochitinase protein to the rhizosphere

Transgenic plants containing a chimeric gene construct that facilitates the exudation of proteins from roots offer novel approaches for modification of the rhizosphere and production of relatively pure recombinant proteins. The aim of this study was to develop alfalfa (Medicago sativa L.) plants that exude a heterologous recombinant protein into the rhizosphere. Alfalfa transformed with a fungal endochitinase (ech42) cDNA fused in frame to the signal peptide of a white lupin acid phosphatase and under the control of the cassava vein mosaic virus (CsVMV) promoter expressed increased chitinase activity in vegetative organs and root exudates. Chitinase activity in root exudates of transgenic alfalfa was 7.5–25.7 times higher than in the untransformed Regen-SY plants. Chitinase enzyme activity was accompanied by increased synthesis of mRNA and protein in transformed plants. By comparison, untransformed and vector only transformed plants displayed no expression of recombinant protein and mRNA. A single band of the expected molecular weight was present only in western blots of root exudates of transgenic alfalfa plants. The secreted endochitinase enzyme not only retained its lytic activity against glycol chitin but also showed antifungal activity by inhibition of spore germination of two fungal pathogens. Exudation of recombinant proteins from roots may offer alternative uses for alfalfa in the production of value-added biopharmaceuticals and may influence microbes or modify soil nutrient availability near plant roots.Mesfin Tesfaye, Matthew D. Denton, Deborah A. Samac & Carroll P. Vanc