Epitope tagging of legume root nodule extensin modifies protein structure and crosslinking in cell walls of transformed tobacco leaves

Root nodule extensins (RNEs) are highly glycosylated plant glycoproteins localized in the extracellular matrix of legume tissues and in the lumen of Rhizobium-induced infection threads. In pea and other legumes, a family of genes encode glycoproteins of different overall length but with the same basic composition. The predicted polypeptide sequence reveals repeating and alternating motifs characteristic of extensins and arabinogalactan proteins. In order to monitor the behavior of individual RNE gene products in the plant extracellular matrix, the coding sequence of PsRNE1 from Pisum sativum was expressed in insect cells and in tobacco leaves. RNE products extracted from tobacco tissues were of high molecular weight (in excess of 80 kDa), indicating extensive glycosylation similar to that in pea tissues. Epitope-tagged derivatives of PsRNE1 could be localized in cell walls. However, the introduction of epitope tags at the C-terminus of RNE altered the behavior of RNE in the extracellular matrix, apparently preventing intermolecular crosslinking of RNE molecules and their covalent association with other cell wall components. These observations are discussed in the light of a computational model for the RNE glycoprotein that is consistent with an extended rod-like structure. It is proposed that RNE can undergo three classes of tyrosine-based crosslinking. Intramolecular crosslinking of vicinal Tyr residues is rod stiffening, end-to-end linkage is rod lengthening, and side-to-side intermolecular crosslinking is rod bundling. The control of these interconversions could have important implications for the biomechanics of infection thread growth.