Characterization of Nops, Nodulation Outer Proteins, Secreted Via the Type III Secretion System of NGR234

Abstract

The nitrogen-fixing symbiotic bacterium Rhizobium species NGR234 secretes, via a type III secretion system (TTSS), proteins called Nops (nodulation outer proteins). Abolition of TTSS-dependent protein secretion has either no effect or leads to a change in the number of nodules on selected plants. More dramatically, Nops impair nodule development on Crotalaria juncea roots, resulting in the formation of nonfixing pseudonodules. A double mutation of nopX and nopL, which code for two previously identified secreted proteins, leads to a phenotype on Pachyrhizus tuberosus differing from that of a mutant in which the TTSS is not functional. Use of antibodies and a modification of the purification protocol revealed that NGR234 secretes additional proteins in a TTSS-dependent manner. One of them was identified as NopA, a small 7-kDa protein. Single mutations in nopX and nopL were also generated to assess the involvement of each Nop in protein secretion and nodule formation. Mutation of nopX had little effect on NopL and NopA secretion but greatly affected the interaction of NGR234 with many plant hosts tested. NopL was not necessary for the secretion of any Nops but was required for efficient nodulation of some plant species. NopL may thus act as an effector protein whose recognition is dependent upon the hosts' genetic background.

Functional Analysis of the Type 3 Effector Nodulation Outer Protein L (NopL) from Rhizobium sp. NGR234

“…These findings were consistent with the observation that a nopL mutant of NGR234 (strain NGR⍀nopL) induced fewer nodules on the legume Flemingia congesta (22). Experiments with recombinant NopL protein expressed in Escherichia coli revealed that NopL could be phosphorylated at unknown sites by nondefined protein kinases from crude plant protein extracts (23,24).…”

“…Plants were either inoculated with ϳ10 9 bacteria of Rhizobium (Sinorhizobium) sp. strain NGR234 or its nopL mutant NGR⍀nopL (22). For complementation analysis, plasmid pLAFR-pnopL (supplemental Table S1) was constructed and mobilized from E. coli DH5␣ into NGR⍀nopL by a triparental mating procedure using the pRK2013 "helper" plasmid.…”

NopL, an Effector Protein ofRhizobiumsp. NGR234, Thwarts Activation of Plant Defense Reactions

“…Yet, because type-three secretion by NGR234 has little effect on nodulation of L. japonicus, it is not an optimal plant to assess the function of NopL. Future work will focus on the interaction between F. congesta and NGR234, in which nopL has been shown to promote nodule formation (Marie et al, 2003). It will be interesting to see whether induced systemic resistance and/or systemic acquired resistance interfere with the establishment of mutualistic symbioses.…”

“…This expression cassette was subcloned into the binary vector pPZP112 (Hajdukiewicz et al, 1994; yielding pPZP112nopL) and was transferred to A. tumefaciens by triparental matings using the helper plasmid pRK2013 (Figurski and Helinski, 1979). To produce recombinant protein, nopL was cloned into the pPROEX-1 vector (Invitrogen, Carlsbad, CA) to give pPROEX-1nopL (Bartsev et al, 2003;Marie et al, 2003). This vector added an in-frame hexa-His tag (6x-His) to the N terminus of NopL.…”

“…NGR234 secretes a number of proteins, among them nodulation outer protein L (NopL, formerly called y4xL) via its TTSS (Freiberg et al, 1997;Viprey et al, 1998). In comparison with wild-type bacteria, mutants lacking nopL induce fewer nodules on Flemingia congesta (Marie et al, 2003), indicating that NopL is a rhizobial "virulence factor" for this plant.…”