Plant genes involved in the establishment of an actinorhizal symbiosis

Actinorhizal multilobe nodules are induced by actinomycetes of the genus Frankia on the roots of several dicotyledenous species belonging to eight different plant families. Each nodule lobe is a modified lateral root, without a root cap, with a central vascular cylinder, and with infected and uninfected cortical cells.

To isolate plant genes involved in the establishment of an actinorhizal symbioses, we have differentially screened an A. glutinosa nodule cDNA library with nodule- and root cDNA, respectively. Several cDNAs, representing genes expressed at elevated levels in nodules compared to roots, as determined by RNA gel blot analysis, were isolated and sequenced. The localization of the corresponding mRNAs in the nodule was examined by in situ hybridization. Whenever necessary, model systems such as yeast and Arabidopsis were used to analyse the functions of the encoded proteins.

Two of the isolated cDNA clones corresponded to sucrose synthase and enolase, enzymes involved in carbon metabolism. The corresponding genes were expressed in all plant tissues but at markedly elevated levels in nodules. In situ hybridization showed that in nodules, both sucrose synthase and enolase were expressed at high levels in the infected cortical cells as well as in the pericycle of the vascular bundle.

Another cDNA clone, pAgthil, was shown to encode a homolog of yeast Thi4, which is involved in thiazole biosynthesis. The corresponding gene, agthil, was found to be expressed at high levels in nodules and shoot tips of A. glutinosa, while being expressed at low levels in roots, flowers, and developing fruits. In nodules, agthil mRNA was localized in the infected cortical cells and in the pericycle of the nodule vascular system. A homolog of this gene, ara6/tz was identified in Arabidopsis thaliana. ara6/tz maps in a region of chromosome 5 of Arabidopsis containing the tz locus. This is consistent with the observations that ara6/tz transcription was impaired in two out of five Arabidopsis tz mutant lines. ara6/tz is expressed at high levels in chloroplast-containing parenchymatic cells of leaves, inflorescence shoots and flowers of Arabidopsis, and at lower levels in the vascular system. The function of AgThi1 was demonstrated by yeast complementation studies, in which AgThi1 was able to rescue a yeast thi4 mutant.

cDNAs encoding glutamine synthetase (GS) and acetylomithine transaminase (AOTA), both involved in nitrogen metabolism, were isolated. GS is the enzyme responsible for ammonium assimilation, while AOTA is involved in the biosynthesis of citrulline, the nitrogen transport form in Alnus. GS mRNA was found in all tissues tested with the highest levels in nodules, where it was present in the infected cells as well as in the cells of the pericycle of the vascular system. AOTA transcripts were detected at very low levels in roots and at high levels in nodules, where it was confined to the infected cells. These data suggested that in A. glutinosa nodules, ammonium assimilation takes place in both the infected cells and in the pericycle of the vascular system, and citrulline biosynthesis occurs mainly in the infected cells. Ammonium assimilation in the pericycle is likely to be related to nitrogen transport.

One of the few nodule-specific genes, i.e., genes that are not expressed in roots, ag12, was shown to be expressed in nodules at the highest levels in infected cells before the onset of nitrogen fixation. Sequencing showed that ag12 encodes a serine protease of the subtilisin (EC 3.4.21.14) family. A homolog of ag12, ara12, was identified in Arabidopsis. ara12 was expressed in all organs, with the highest expression levels in the beginning of silique development. To assess the importance of this protease in other actinorhizal symbioses, the expression pattern of its homolog, cg12, was examined in nodules of Casuarina glauca and found to be similar to that of ag12. These results are discussed in view of the phylogenetical relationship of Alnus and Casuarina.