TRANSGENIC ALFALFA SECRETES A FUNGAL ENDOCHITINASE PROTEIN TO THE RHIZOSPHERE

Authors: Gebeyaw, Mesfin; DENTON, MATTHEW - UNIVERSITY OF MINNESOTA; Samac, Deborah - Debby; Vance, Carroll

Submitted to: Plant and Soil
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/4/2004
Publication Date: 2/1/2005
Citation: Tesfaye, M., Denton, M.D., Samac, D.A., Vance, C.P. 2005. Transgenic alfalfa secretes a fungal endochitinase protein to the rhizosphere. Plant and Soil Journal. 269:233-243.

Interpretive Summary: Roots of all plants export a number of compounds into the surrounding soil to aid in plant growth. The roots of white lupin plants export large amounts of an enzyme called acid phosphatase to capture phosphorus, a nutrient required for growth of all plants. A portion of the enzyme, the signal peptide, directs the plant cell to export the enzyme outside of the root. The ability of this signal peptide to direct the export of a foreign enzyme, called endochitinase, was compared to the native signal peptide from the enzyme. Endochitinase enzymes break down the cell walls of fungi and have been shown to help protect plants from fungal diseases. Transgenic alfalfa plants were generated that expressed the endochitinase gene either with or without the white lupin signal peptide. Export of the endochitinase was observed for plants with the white lupin and the fungal signal peptide. Plants with the white lupin signal peptide produced more chitinase than plants with the fungal signal peptide. The secreted enzyme was fully functional and had the expected enzymatic activity. In addition, it inhibited the germination of spores from two different fungal pathogens of alfalfa. These results show that the white lupin signal peptide can successfully direct the export of foreign proteins from alfalfa roots into the surrounding medium. Export of proteins from roots could be useful in production of high-value proteins because purification of proteins would be simpler and more economical than if proteins were retained inside of plant cells. The export of an antifungal protein from roots may be useful in protecting plants from disease-causing organisms thereby increasing plant yields and reducing production costs.

Technical Abstract: Transgenic alfalfa (Medicago sativa L.) plants that express ech42, an endochitinase cDNA from the biocontrol fungus Trichoderma harzianum, under the control of a constitutive cassava vein mosaic virus promoter were produced. The native endochitinase signal peptide (FSP) or an acid phosphatase signal peptide (APSP) from white lupin fused at the N-terminal region of the mature endochitnase protein was used for protein targeting into the rhizosphere. The transcription and expression of the endochitinase transgenes in alfalfa was accompanied by increased chitinase activity in leaf and root tissues of transgenic alfalfa, but not in untransformed and vector only transformed control plants. Chitinase activity in root exudates of transgenic alfalfa expressing the ech42-FSP and ech42-APSP constructs was from 7.5 to 25.7 times higher than the untransformed Regen-SY plants. A single band of the expected molecular weight was present only in western blots of root exudates of alfalfa expressing the ech42-FSP and ech42-APSP constructs, although silver staining of SDS-PAGE revealed the presence of additional secreted proteins in root exudates of control and transgenic alfalfa. The secreted endochitinase enzyme retained its lyctic activity against glycol chitin and also showed antifungal activity by inhibition of spore germination of two fungal pathogens. At comparable protein concentrations, transgenic alfalfa with the ech42-APSP construct showed higher endochitinase polypeptide levels and chitinase activity in root tissues compared to the alfalfa line containing the ech42-FSP construct. Results demonstrate the potential of the white lupin APase signal peptide for directing the exudation of a recombinant protein to the alfalfa rhizosphere.