A nodule-specific gene encoding a subtilisin-like protease is expressed in early stages of actinorhizal nodule development

To identity genes specifically expressed during early stages of actinorhizal nodule development a cDNA library made from poly(A) RNA from root nodules of Alnus glutinosa was screened differentially with nodule and root cDNA respectively. Seven nodule-enhanced and four nodule-specific cDNA clones were isolated. By using in situ hybridization two of the nodule-specific cDNAs were shown to be expressed at the highest levels in infected cells before the onset of nitrogen fixation; one of them ag12 (A. glutinosa) was examined in detail. Sequencing showed that ag12 codes for a serine protease of the subtilisin (EC 3.4.21.14) family. Subtilisins previously appeared to be limited to microorganisms. However, subtilisin-like serine proteases have recently been found in archaebacteria, fungi and yeasts as well as in mammals; a plant subtilisin has also been sequenced. In yeast and mammals subtilases are responsible for processing peptide hormones. A homolog of ag12 ara12 was identified in Arabidopsis; it was expressed in all organs and its expression levels were highest during silique development. Hence our study shows that subtilases are also involved in both symbiotic and nonsymbiotic processes in plant development