Rhizobium leguminosarum bv. trifolii NodD2 Enhances Competitive Nodule Colonization in the Clover-Rhizobium Symbiosis

Abstract

Establishment of the symbiotic relationship that develops between rhizobia and their legume hosts is contingent upon an interkingdom signal exchange. In response to host legume flavonoids, NodD proteins from compatible rhizobia activate expression of nodulation genes that produce lipochitin oligosaccharide signaling molecules known as Nod factors. Root nodule formation commences upon legume recognition of compatible Nod factor. <i>Rhizobium leguminosarum</i> was previously considered to contain one copy of <i>nodD</i>; here, we show that some strains of the <i>Trifolium</i> (clover) microsymbiont <i>R. leguminosarum</i> bv. trifolii contain a second copy designated <i>nodD2. nodD2</i> genes were present in 8 out of 13 strains of <i>R. leguminosarum</i> bv. trifolii, but were absent from the genomes of 16 <i>R. leguminosarum</i> bv. viciae strains. Analysis of single and double <i>nodD1</i> and <i>nodD2</i> mutants in <i>R. leguminosarum</i> bv. trifolii strain TA1 revealed that NodD2 was functional and enhanced nodule colonization competitiveness. However, NodD1 showed significantly greater capacity to induce <i>nod</i> gene expression and infection thread formation. Clover species are either annual or perennial and this phenological distinction is rarely crossed by individual <i>R. leguminosarum</i> bv. trifolii microsymbionts for effective symbiosis. Of 13 strains with genome sequences available, 7 of the 8 effective microsymbionts of perennial hosts contained <i>nodD2</i>, whereas the 3 microsymbionts of annual hosts did not. We hypothesize that NodD2 inducer recognition differs from NodD1, and NodD2 functions to enhance competition and effective symbiosis, which may discriminate in favor of perennial hosts.<b>IMPORTANCE</b> Establishment of the rhizobium-legume symbiosis requires a highly specific and complex signal exchange between both participants. Rhizobia perceive legume flavonoid compounds through LysR-type NodD regulators. Often, rhizobia encode multiple copies of <i>nodD</i>, which is one determinant of host specificity. In some species of rhizobia, the presence of multiple copies of NodD extends their symbiotic host-range. Here, we identified and characterized a second copy of <i>nodD</i> present in some strains of the clover microsymbiont <i>Rhizobium leguminosarum</i> bv. trifolii. The second <i>nodD</i> gene contributed to the competitive ability of the strain on white clover, an important forage legume. A screen for strains containing <i>nodD2</i> could be utilized as one criterion to select strains with enhanced competitive ability for use as inoculants for pasture production.