Secreted hen lysozyme in transgenic tobacco: recovery of bound enzyme and in vitro growth inhibition of plant pathogens

Abstract

Less than 1% of secreted hen egg white lysozyme (HEWL) was isolated in intercellular fluid neutral phosphate buffer extracts of transgenic tobacco ( Nicotiana tabacum L.). When foliar tissue was infiltrated with 50 mM calcium chloride or 1% histamine, 10–20% of HEWL was isolated when compared to HEWL obtained in tissue homogenates boiled in the presence of sodium dodecyl sulfate. The presence of mature HEWL was shown by N-terminal amino acid microsequencing of the major electrophoretic form (14.4 kDa). A minor electrophoretic form (17 kDa) having the same N-terminus end (KVFGRC) as the major mature from was also found. Tissue imprinting of transgenic tobacco cut stem, petiole and leaf tissue for detecting lysis of Micrococcus luteus cells embedded as lysozyme substrate in polyacrylamide gels could be performed in the presence of histamine for release of HEWL. Despite tight binding of HEWL in transgenic tobacco, some HEWL activity could be detected around growing transgenic tobacco seedling rootlets. Moreover, HEWL recovered from transgenic tobacco extracellular extracts was shown to inhibit the growth of some bacterial and fungal plant pathogens.