Temporal and spatial expression analyses of TrEhod40, TrEnod5 and a novel early nodulin in white clover roots and nodules

Using differential display RT-PCR on mRNA populations from white clover roots challenged with a mock-inoculation or the microsymbiont, Rhizobium leguminosarum biovar trifolii ANU843, a number of expressed sequence tags (ESTs), specific to the early stages of symbiosis, were identified. Similarity searches of many of these ESTs identified them as novel, with no significant sequence homologues. Of the white clover cDNA fragments with homologies to sequences in EMBL databases, several early nodulins were identified and further characterized by RT-PCR and in situ hybridization analyses to provide detailed temporal and spatial expression profiles. Here, we report on the analyses of a clover Enod40 homologue and a new early nodulin precursor (DD17), with 3' homology to Enod8, previously only identified in Medicago species. An arabinogalactan-like protein, highly homologous to previously reported Enod5s from other legumes. was also characterized and used as a standard for all expression analyses, following its isolation through degenerate-primer RT-PCR. Published by Elsevier Science Ireland Ltd.