Members of the germin-like protein family in Brassica napus are candidates for the initiation of an oxidative burst that impedes pathogenesis of Sclerotinia sclerotiorum

Abstract

Germin-like proteins (GLPs) are defined by their sequence homology to germins from barley and are present ubiquitously in plants. Analyses of corresponding genes have revealed diverse functions of GLPs in plant development and biotic and abiotic stresses. This study describes the identification of a family of 14 germin-like genes from Brassica napus ( BnGLP) designated BnGLP1– BnGLP14 and investigated potential functions of BnGLPs in plant defense against the necrotrophic fungus Sclerotinia sclerotiorum. Sequence alignment and phylogenetic analyses classify the 14 BnGLPs into four groups, which were clearly distinguished from known germin oxalic acid oxidases. Transcriptional responses of the BnGLP genes to S. sclerotiorum infection was determined by comparing cultivars of susceptible B. napus ‘Falcon’ and partially resistant B. napus ‘Zhongshuang 9’. Of the 14 BnGLP genes tested, BnGLP3 was transcriptionally upregulated in both B. napus cultivars at 6h after S. sclerotiorum infection, while upregulation of BnGLP12 was restricted to resistant B. napus ‘Zhongshuang 9’. Biochemical analysis of five representative BnGLP members identified a H ₂O ₂-generating superoxide dismutase activity only for higher molecular weight complexes of BnGLP3 and BnGLP12. By analogy, H ₂O ₂ formation at infected leaf sites increased after 6h, with even higher H ₂O ₂ production in B. napus ‘Zhongshuang 9’ compared with B. napus ‘Falcon’. Conversely, exogenous application of H ₂O ₂ significantly reduced the susceptibility of B. napus ‘Falcon’. These data suggest that early induction of BnGLP3 and BnGLP12 participates in an oxidative burst that may play a pivotal role in defence of B. napus against S. sclerotiorum.