CONSTITUTIVE EXPRESSION OF AN INDUCIBLE BETA-1,3-GLUCANASE IN ALFALFAREDUCES DISEASE SEVERITY CAUSED BY THE OOMYCETE PATHOGEN PHYTOPHTHORA-MEGASPERMA F-SP MEDICAGINIS, BUT DOES NOT REDUCE DISEASE SEVERITY OF CHITIN-CONTAINING FUNGI

cDNA sequences coding for an acidic glucanase (Aglu1) that is expresse d in elicited alfalfa cell suspension cultures, and a rice basic chiti nase (RCH10) that is induced by elicitor and wounding, were placed int o constitutive expression cassettes under control of the cauliflower m osaic virus 35S promoter or 35S enhancer sequences, and introduced in alfalfa plants of the regenerable cultivar Regen SY by Agrobacterium-m ediated transformation. Southern and northern blot analysis confirmed stable incorporation and transcription, respectively, of the chimaeric genes in the transgenic plants. Active rice chitinase was expressed i n alfalfa leaves, and leaves of plants transformed with the Aglu1 sequ ence exhibited increased glucanase activity and the appearance of an a dditional glucanase band on activity gels. A glucanase of similar nati ve electrophoretic mobility was constitutively present in root extract s of non-transformed alfalfa plants, and was induced in pathogen-infec ted leaves, presumably reflecting the expression pattern of the endoge nous Aglu1 gene. Thus, expression of the chimaeric Aglu1 transgene inc reased the amount, and broadened the tissue-type constitutive expressi on, of the Aglu1 protein compared to control plants. Transgenic alfalf a plants containing a binary vector with both chimaeric genes in tande m expressed each gene to a much lesser extent than transgenic plants c ontaining a single chimaeric gene. Expression of RCH10 in transgenic a lfalfa did not appear to affect negatively the Rhizobium/alfalfa inter action. Analysis of primary transformants for response to several fung al pathogens of alfalfa indicated statistically significant symptom re duction only in the case of Phytophthora megasperma f sp. medicaginis (Pmm), and only in plants overexpressing Aglu1. Resistance against Pmm segregated with glucanase expression ina cross between transgenic Reg en SY and the commercial alfalfa cultivar Apollo.