NJ Brewin & EA Rathbun, John Innes Centre, Norwich UK

Rhizobium leguminosarum normally gains access to pea host cells through tubular cell wall ingrowths termed infection threads. The infection thread initiates from a kink in a deformed root hair cell. Polar growth through the root hair cell is guided by transcellular cytoplasmic bridges. Having traversed the epidermal cell, the infection thread fuses with the underlying cell wall. Bacteria are released into the extracellular matrix and the process of infection thread initiation repeats itself in the underlying cortical cell. Occasionally, infection threads abort.

Infection threads revealed using a rhizobial strain carrying a gene encoding glucuronidase

A major component of the infection thread lumen is an extensin-like glycoprotein, first recognised by monoclonal antibody MAC265 through a carbohydrate epitope. We hypothesise that targeted secretion of root nodule extensin into the lumen of infection threads may be an important factor governing infection thread growth. Furthermore, a peroxide-driven insolubilisation of the extracellular matrix could control the biophysics of inward infection thread growth in opposition to the turgour pressure of the host cell. The relative rates of secretion and solidification of root nodule extensin may also determine whether or not an infection thread aborts.

Immunogold staining of root nodule extensin in the lumen of an infection thread

Over 30 cDNA clones have been isolated from pea root nodule transcripts. Sequence analysis reveals a family of extensin-like glycoproteins with repetitive (hydroxy)proline-rich motifs.

Consensus of 30 translated cDNA sequences encoding root nodule extensins.
The leader peptide is highlighted. Conserved N-terminal, Central and C-terminal domains are shown in bold. Other motifs are repeated to a variable extent in different clones.
Interesting structural features of root nodule extensin are: -

1. the repetitive blocks of Ser-Pro_4/5 characteristic of extensins);
2. the presence of two Tyrosines between each Ser-Pro_4/5 block (a target for peroxide-driven cross-linking);
3. the presence of double basic amino acids (Lys-Lys, or Asn-Gln), which could be involved in binding to plant or bacterial cell surface components.

Root nodule extensins have a highly conserved C-terminal domain. The C-terminal residue is apparently different in different host legumes.

C-terminal sequence of legume root nodule extensins from different legumes

There is also conservation in the 3’-UTR region.

Comparison of 3’-UTR sequence for root nodule extensin cDNAs derived from pea (Ext3.2) and Medicago truncatula (MtN12).

Our model for promotion and prevention of infection thread growth is based on the dynamic balance between targetted secretion of root nodule extensin into infection threads and the peroxide-driven cross-linking of this material in the infection thread lumen. Another component of this process may be diamine oxidase, which uses putrescine as a source of peroxide in the extracellular matrix. The biophysical role for root nodule extensin in the nodulation process may be further modified by interaction with other cell wall glycoproteins (early nodulins) that are expressed transiently during infection by Rhizobium. Examples include ENOD11 and ENOD12, both of which are associated with infection thread development in host cells of Medicago truncatula.

Sequence of mature polypeptides for MtENOD11 and MtENOD12, showing pentapeptide motifs and comparison with root nodule extensin, using the same highlighting symbols.

Sequence analysis of the 3’-UTR region of root nodule extensin clones indicates that many clones share strong sequence homology. There is even strong sequence homology between 3’-UTR sequences from Pisum sativum and those of Medicago truncatula. The conserved sequence motifs in the 3’-UTR of root nodule extensin are associated with secondary structure that is also conserved.

Predicted fold structure for the 3’-UTR region for pea root nodule extensin 3.2p