Plant Science 161 (2001) 1161 – 1170 Temporal and spatial expression analyses of TrEnod40, TrEnod5 and a novel early nodulin in white clover roots and nodules Martin A. Crockard a, *, Anthony J. Bjourson a,b , Maria Gabriella Pulvirenti b , James E. Cooper a,b a Applied Plant Science Diision, Department of Agriculture and Rural Deelopment (NI), Newforge Lane, Belfast BT95PX, UK b Department of Applied Plant Science, Queen’s Uniersity Belfast, Newforge Lane, Belfast BT95PX, UK Received 6 July 2001; received in revised form 20 August 2001; accepted 3 September 2001 Abstract 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. Keywords: Early nodulins; Differential display; RT-PCR; In situ hybridization analyses; Trifolium repens www.elsevier.com/locate/plantsci 1. Introduction During the complex association between legumes and their soil-borne rhizobial microsymbionts, regions of the legume root, specifically in the region of root hair emergence, undergo a series of physiological changes, including unscheduled mitosis of cortical cells, which result in the formation of a new organ, the root nodule [1]. In general, rhizobia interact with almost all emerg- ing root hairs in legumes, but only about 25% of these form the characteristic shepherd’s crook [2]. Changes in cell wall architecture result in the formation of a plant- derived infection thread (IT), originating from the site of rhizobial attachment at the shepherd’s crook [3]. Through this IT, the colonizing rhizobia enter nodule primordium cells in the cortex and are released by swelling and blebbing off of the infection thread ends, thereby encapsulating them by phagocytosis in the peribacteroid membrane (PBM, [1,4]). These PBM-en- closed rhizobia, released into the plant cell cytoplasm of expanding cortical cells, eventually differentiate into bacteroids, capable of fixing atmospheric nitrogen within the nodule central tissue, which can be se- questered by the plant [5]. In temperate legumes, such as clover, vetch and pea, the root nodules are indeter- minate, possessing a persistent meristem. As the nodule develops, a distinct zonation of the central tissue oc- curs, first described by Vasse et al. [6], from the meris- tem (I), through the infection zone (II), interzone (II – III, early symbiotic zone), nitrogen fixing zone (III, symbiotic zone) and, in older nodules, the senescent zone (IV). Throughout the symbiotic interaction, induced plant genes known as nodulins drive changes in plant mor- phology and assimilation of fixed nitrogen [1,7]. Early nodulins are those expressed before the onset of nitro- * Corrosponding author. Tel.: +44-2890255481; fax: +44- 2890255007. E-mail address: martin.crockard@dardni.gov.uk (M.A. Crockard). 0168-9452/01/$ - see front matter Published by Elsevier Science Ireland Ltd. PII:S0168-9452(01)00525-8