Legume AGP-Extensin (Root Nodule Extensin) and Infection Threads

Fig. 1 Computer- model for a legume AGP- Extensin (AGPE), previously termed root module extensin (RNE). These rod-shaped glycoproteins are intimately involved in the Rhizobium-infection process and subject to protein cross-linking in the presence of hydrogen peroxide. Centre: close-up of a repeating motif from AGPE with two glycosylated extensin motifs (SP4) flanking a His-rich region with alternating (hydroxy)-prolines. Alternating hydroxyprolines are targets for addition of large arabinogalactan (AG) blocks (not shown). Bottom: end-on view of extensin motif. AGPEs are a class of plant cell wall glycoprotein that is unique to legumes (Gucciardo et al. 2005).

Fig. 2 Electron micrograph showing a tubular infection thread traversing a legume host cell. Transcellular infection threads are tip-growing ingrowths of the plant cell wall. Rhizobium bacteria grow and divide within a plant-derived matrix containing root nodule extensins. In the micrograph, AGPE has been immunogold labelled with the monoclonal antibody MAC265 (Rathbun et al. 2002). Secretion of AGPE is apparently targeted to the tip of the infection thread lumen. Targeted secretion may be driven by a conserved 3'-UTR sequence containing multiple UUGU motifs that are putative targets for Pumilio-family (Puf) proteins.

AGPE – a unique component of the Rhizobium-legume infection process?

Cell wall remodelling is a key aspect of the Rhizobium infection process (Brewin, 2004). AGP-extensins are a legume-specific family of plant glycoproteins, previously termed legume Root Nodule Extensins (RNEs). They are abundant in the matrix of Rhizobium-induced infection threads (Rathbun et al. 2002). MtN12, a nodule enhanced transcript from M. truncatula shows close sequence homology with PsRNE1 the corresponding sequence derived from pea. Because AGPE glycoproteins apparently co-evolved with legumes, because they are structurally conserved in legumes and because they are always physically associated with rhizobial cells in the infection thread, it is suggested that they may play a role in regulating the infection process for unicellular nitrogen-fixing symbiotic bacteria secreting nod-factors (collectively termed rhizobia).

• AGP-extensin copolymer, unique to legumes (Rathbun et al. 2002);
• Related to Gum Arabic Glycoprotein from Acacia senegal (Brewin 2004);
• Component of intercellular and transcellular infection threads (VandenBosch et al. 1989);
• AGPEs from Pisum, Medicago, Lotus, Sesbania and all other legumes tested are recognised by rat monoclonal antibodies MAC265, MAC 236 and MAC 204;
• Secreted by legume root hairs soon after inoculation with Rhizobium (Rae et al. 1992);
• Attaches to bacterial surface (Bolanos et al. 2004), presumably because of high Lys content;
• Subject to cross-linking by peroxide (Wisniewski et al. 2000), presumably because of high Tyr content;
• Conserved 3’-UTR sequence containing multiple UUGU motifs that are putative targets for Pumilio-family (Puf) proteins involved in polarised transport of mRNA (Gerber et al. 2004).

Purification and properties of AGP-Extensin

AGPEs are highly glycosylated plant glycoproteins localized in the extracellular matrix of legume tissues and in the lumen of infection threads. An extended rod-shaped macromolecule is predicted from computational modelling studies (Fig. 1). They are recognised by a suite of monoclonal antibodies – MAC265, MAC236 and MAC204: these antibodies react strongly with AGPEs from Pea, Medicago, Lotus, Sesbania and all other legumes tested.

The predicted polypeptide sequence indicates repeating and alternating motifs characteristic of extensins and arabinogalactan (AGP) proteins. In PsRNE1, eleven extensin motifs (Ser-Prox) are predicted to carry relatively small arabinose glycosylations and six motifs with alternating proline residues (Pro-X-Pro-X-Pro) probably carry large glycan substitutions, each comprising a 1-3, beta-linked galactose backbone with two pentasaccharide side chains. PsRNE1 is just one of a family of closely related Pisum genes encoding glycoproteins of varying length and with varying numbers of the repeating structural motifs but with the same overall composition. Interspersal of AGP (gum) and extensin (resin) motifs within the same macromolecule is apparently a unique feature of legume glycoproteins and may indicate some unique physical properties associated with the Rhizobium infection process. It is also interesting to note that the 20-residue C-terminal sequence of RNE is highly conserved across many species of legumes (from Sesbania and Lupin to Soybean and Medicago), suggesting some important conservation of function.

Other interesting features of RNE sequences have implications for protein complex formation in symbiotic development. For example, PsRNE1 has nineteen Tyr-residues that offer scope for peroxide-based protein cross-linking, either through intramolecular isodityrosine residues or by intermolecular cross-bridges (Gucciardo et al. 2005; Held et al. 2004). Furthermore, there are 22 His-residues, often clustered, that seem to confer the ability to complex with Cu2+ and Ni2+ ions in the extracellular matrix. Ten basic residues (mainly Lys) confer a net positive charge to these rod-shaped macromolecules, facilitating attachment to bacterial cell surfaces (Bolanos et al. 2004).

Development of a model for infection thread growth

Based on our own studies of root nodule extensin (Wisniewski et al. 2000) and some complementary studies by Hérouart et al. (2002) concerning the important role for hydrogen peroxide in the regulation of infection thread development in Medicago, we have proposed a general model for apical growth and propagation of a transcellular infection thread (Brewin 2004). This model (Fig. 3) is based on the concept that fluid-to-solid transitions of RNE in the infection thread lumen may control the biophysics of infection thread growth. The fluid phase promotes bacterial growth, the solid phase prevents it. Further work is now needed to identify the functional roles of the molecules and motifs that might be involved. Because it seems that many of the genes encoding specialist components of the plant extracellular matrix (such as RNE) may be functionally redundant, it follows that single mutations may not result in a symbiotically defective phenotype. Future research should involve biochemistry, cell biology and molecular genetics to investigate the function(s) of Root Nodule Extensins in infection thread development.

Fig. 3. Tyrosine cross-linking involving RNE molecules seems to control the dynamics of infection thread growth Biochemical model illustrating potential mechanisms of peroxide-driven tyrosine cross-linking for RNE in the extracellular matrix (adapted from Wisniewski et al. 2000). Possible sources of peroxide are from diamine oxidase (a copper-containing enzyme that is abundant in the extracellular matrix) and NADPH oxidase. The distribution of tyrosine residues in RNE sequences (Fig. 1) indicates the potential for three types of tyrosine cross-linking. Intramolecular cross-linking is rod-strengthening; end-to-end conjugation is rod lengthening; side-to-side intermolecular cross-linking is rod bundling and may depend on prior removal of the AG blocks (Gucciardo et al. 2005).

Evidence for a plant locasome in the establishment of cell polarity

As indicated in the attached sequence, there are multiple UUGU binding sites for Pumilio family proteins in the 3’-UTR of MtN12. This gene encodes legume AGP-Extensin, a plant glycoprotein targeted to the extracellular matrix at the apical growing point of infection threads during the invasion phase of the Rhizobium-legume symbiosis. AGPE is recognised by rat monoclonal antibody MAC265.

Pumilio (PUF) protein family members are often involved in the establishment of cell polarity, e.g. in budding yeast (Puf6p / ASH1 mRNA) and the Drosophila embryo: (Pumilio/ oscar mRNA), but a role in plant cells has not previously been demonstrated. PUF proteins bind to the 3’-UTR sequence of target mRNA carrying the UUGU motif. Before exit from the nucleus, the RNA-protein dimer becomes associated with a larger ribonucleoprotein complex. In budding yeast, this complex is called the “locasome”, comprising She2p, She3p, Myo4p components that repress protein translation and promote translocation of the RNA along the polarised actin cytoskeleton towards a predefined region of the cell (Gerber et al. 2004).

In the case of the Rhizobium-legume synthesis, PUF association could account for the polarised apical growth of infection threads. A yeast three-hybrid system could be used to identify the PUF proteins that interact with the conserved 3’-UTR of RNA encoding legume AGP-Extensin.

Key References

Gucciardo S et al. (2005) Epitope tagging of legume root nodule extensin modifies protein structure and cross-linking in cell walls of transformed tobacco leaves. MPMI 18: 24-32.

Brewin, N.J. (2004) Cell Wall Remodelling in the Rhizobium-legume symbiosis. Crit Rev. Plant Sci. 23: 1-24.

Bolanos L. et al. (2004) Cell surface interactions of Rhizobium and other bacterial strains with symbiosomal and peribacteroid membrane components from pea nodules. MPMI. 17: 216-223.

Gerber A.P., et al. (2004). Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast. PLoS Biol. 2 (3): 0342-0354.

Held M. A. et al (2004) Di-isodityrosine is the intermolecular cross-link of isodityrosine-rich extensin analogues cross-linked in vitro. J. Biol Chem. 279: 13156-13165

Hérouart D et al. (2002) A key role for ROS in the establishment of the legume-Rhizobium symbiosis? Plant Physiol. Biochem. 40: 619-624

Rathbun E. A., et al. (2002) Identification of a family of extensin-like glycoproteins in the lumen of Rhizobium -induced infection threads in pea root nodules. MPMI 15: 350-359.

Wisniewski J-P., Rathbun E. A., Knox J. P., Brewin N. J. (2000) Involvement of diamine oxidase and peroxidase in insolubilization of the extracellular matrix: implications for pea nodule initiation by Rhizobium leguminosarum. MPMI 13: 413-420.

Brewin, N. J. (1998) Tissue and cell invasion by Rhizobium: the structure and development of infection threads and symbiosomes. In: The Rhizobaceae, pp. 417-429. Spaink, H. P. Eds., Kluwer, Dordrecht.

Rae, A L, et al (1992)  Structure and growth of infection threads in the legume symbiosis with Rhizobium leguminosarum. Plant Journal 2, 385-395.

VandenBosch, K A, et al (1989) Common components of the infection thread matrix and the intercellular space identified by immunocytochemical analysis of pea nodules and uninfected roots. EMBO Journal. 8: 335-342.

John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK

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