A Minimal Genetic Passkey to Unlock Many Legume Doors to Root Nodulation by Rhizobia

Abstract

On legume crops, formation of developmentally mature nodules is a prerequisite to efficient nitrogen fixation by populations of rhizobial bacteroids established inside nodule cells. Development of root nodules and concomitant microbial colonisation of plant cells are constrained by sets of recognition signals exchanged by infecting rhizobia and their legume hosts, with much of the specificity of symbiotic interactions being determined by the flavonoid cocktails released by legume roots and the strain-specific nodulation factors (NFs) secreted by rhizobia. Hence, much of Sinorhizobium fredii strain NGR234 symbiotic promiscuity was thought to stem from a family of >80 structurally diverse NFs and associated nodulation keys in the form of secreted effector proteins and rhamnose-rich surface polysaccharides. Here, we show instead that a mini-symbiotic plasmid (pMiniSym2) carrying only the nodABCIJ, nodS and nodD1 genes of NGR234 conferred promiscuous nodulation to ANU265, a derivative strain cured of the large symbiotic plasmid pNGR234a. The ANU265::pMiniSym2 transconjugant triggered nodulation responses on 12 of the 22 legumes we tested. On roots of Macroptilium atropurpureum, Leucaena leucocephala and Vigna unguiculata, ANU265::pMiniSym2 formed mature-like nodule and successfully infected nodule cells. While cowpea and siratro responded to nodule colonisation with defence responses that eventually eliminated bacteria, L. leucocephala formed leghemoglobin-containing mature-like nodules inside which the pMiniSym2 transconjugant established persistent intracellular colonies. This data shows seven nodulation genes of NGR234 suffice to trigger nodule formation on roots of many hosts and to establish chronic infections in Leucaena cells.

Keywords: legume-rhizobia symbioses; plant immune responses; symbiotic promiscuity; synthetic replicon.

Figure 1

Genetic maps of pMiniSym1, pMiniSym2 and pMiniSym2-Gus. In the linear genetic maps of pMiniSym1 (a), pMiniSym2 (b) and pMiniSym2-Gus (c), each of the assembled genetic modules is delimited by vertical dashed lines. pMiniSym1 carries functions for propagation (TrfA, oriV), selection (Km^R Omega interposon) and maintenance (spsAB) in E. coli and rhizobia, as well as a conjugative origin of transfer (oriT). In addition to pMiniSym1 functions, pMiniSym2 includes a small set of nodulation genes that confers to recipient bacteria, perception of plant flavonoids by NodD1, synthesis of pentameric methylated nodulation factor (NF) (by NodA, NodB, NodC and NodS) and their secretion (by NodI and NodJ), via a NodD1-dependent transcriptional activation mediated by the NB8 and NB12 nod boxes. pMiniSym2-Gus carries the uidA reporter gene for β-glucuronidase (Gus), which constitutive expression is controlled by the rpsL promoter (PrpsL) of NGR234 [59].

Figure 2

Representative examples of pseudo- or mature-like nodule structures formed by ANU265::pMiniSym2 (1) or NGR234 (2) on roots of, respectively: (a), Stylosanthes guianensis cv. Schofield; (b), Cajanus cajan cv. Lab22; (c), Tephrosia vogelii; (d), Vigna radiata cv. King; (e), Lablab purpureus; (f), Vigna unguiculata cv. Blackeye; and (g), Leucaena leucocephala. In (g1*) and (g2*), corresponding sections of the L. leucocephala nodules shown in (g1) and (g2), respectively. Scale bars in (a1), (a2), (b1) and (c1) are 0.5 mm. All other scale bars are 1 mm.

Figure 3

ANU265::pMiniSym2 formed persistent intracellular colonies in L. leucocephala. Micrographs of nodules harvested at 21 days post-infection with NGR234 in panels (a–d) and ANU265::pMiniSym2 in panels (e–g). Light micrographs (a,e) were of 500-nm thick sections whereas electron micrographs were of 85 nm thick sections of the same nodules. Sectors observed at higher magnifications are framed in white rectangles, with for example panels (c,d) corresponding to magnified areas of the nodule cell shown in (b). Higher magnification of NGR234 (d) and ANU265::pMiniSym2 (g) intracellular nodule bacteria. (c), cross section of one transcellular infection-thread. Poly-β-hydroxybutyrate (PHB) seen as electron-transparent droplets in bacteria. More than six nodules collected on several roots were processed per treatment. Scale bars are 50 µm in (a,e), 5 µm in (b,f) and 1 µm in panels (c,d,g).