Rhizobium cellulase CelC2 is essential for primary symbiotic infection of legume host roots

*Departamento de Microbiología y Genética and Centro Hispano Luso de Investigaciones Agrarias, Universidad de Salamanca, 37007 Salamanca, Spain;
^†Grupo de Ecología Genética de la Rizosfera. Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain; and
^‡Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824

Communicated by James M. Tiedje, Michigan State University, East Lansing, MI, March 18, 2008 (received for review December 17, 2007)

Abstract

The rhizobia–legume, root-nodule symbiosis provides the most efficient source of biologically fixed ammonia fertilizer for agricultural crops. Its development involves pathways of specificity, infectivity, and effectivity resulting from expressed traits of the bacterium and host plant. A key event of the infection process required for development of this root-nodule symbiosis is a highly localized, complete erosion of the plant cell wall through which the bacterial symbiont penetrates to establish a nitrogen-fixing, intracellular endosymbiotic state within the host. This process of wall degradation must be delicately balanced to avoid lysis and destruction of the host cell. Here, we describe the purification, biochemical characterization, molecular genetic analysis, biological activity, and symbiotic function of a cell-bound bacterial cellulase (CelC2) enzyme from Rhizobium leguminosarum bv. trifolii, the clover-nodulating endosymbiont. The purified enzyme can erode the noncrystalline tip of the white clover host root hair wall, making a localized hole of sufficient size to allow wild-type microsymbiont penetration. This CelC2 enzyme is not active on root hairs of the nonhost legume alfalfa. Microscopy analysis of the symbiotic phenotypes of the ANU843 wild type and CelC2 knockout mutant derivative revealed that this enzyme fulfils an essential role in the primary infection process required for development of the canonical nitrogen-fixing R. leguminosarum bv. trifolii-white clover symbiosis.

Footnotes

^§To whom correspondence should be addressed. E-mail: pfmg{at}usal.es
Author contributions: J.M.D.-M., E.M.-M., and P.F.M. designed research; M.R., J.I.J.-Z., E.V., M.E.T., J.L.Z.-P., M.H.R.-B., B.R., F.D., and P.F.M. performed research; M.R., J.I.J.-Z., E.V., M.E.T., J.M.D.-M., F.D., E.M.-M., and P.F.M. analyzed data; and J.I.J.-Z., F.D., and P.F.M. wrote the paper.
The authors declare no conflict of interest.