Abstract
Clovers enter a symbiotic relationship with particular rhizobia through a highly specific and complex signal exchange culminating in the formation of nitrogen-fixing root nodules. Legumes exude flavonoids that are perceived by compatible rhizobia through a LysR-type regulator NodD. Activated NodD mediates expression of the nodulation (nod) genes, which produce a cocktail of signalling molecules known as Nod factors (NF). Recognition of NF by compatible legumes initiates nodule formation. Distinct strains of the clover-nodulating species, Rhizobium leguminosarum bv. trifolii (Rlt), vary in their ability to nodulate different clover species effectively, and to form nodules in competition with other strains. This is important agriculturally as indigenous strains often outcompete added inoculum strains that are superior at nitrogen fixation.
We investigated the genetic determinants of varying host responses between three Rlt strains that differed in their ability to form efficient symbioses on white, red and subterranean clovers. To determine whether differences in nod gene expression contributed to this host specificity or competitive ability, we investigated the induction of nodA and nodF promoters from the three strains. The studies revealed significant variation in nod gene expression in response to flavonoid 7,4’-dihydroxyflavone, due to both the host strain background and the particular promoter. The strength of promoter expression of nodA or nodF also appeared to correlate with the competitive ability of the Rlt strain. We also showed that expression of the nodF operon had a non-equal influence on symbiotic proficiency depending on the Rlt strain, and elevated nodF expression appeared to enhance nodulation of annual clover species.
Distinct phenological barriers exist that generally limit efficient Rlt symbiosis to either annual or perennial clover hosts. We found that Rlt nod gene expression profiles correlate with success on a specific type of host. We suggest that a two receptor system exists in clover, where following entry via a broad-spectrum receptor, a stringent cortical receptor distinguishes the NF profile produced, providing greater compatibility scrutiny by the host.
Previously Rhizobium leguminosarum species were considered to contain a single copy of nodD. However we showed that some strains of Rlt contain a second copy of nodD, designated nodD2. Single and double nodD1 and nodD2 markerless deletion mutants were constructed in Rlt strain TA1. The nodD1 deletion mutant revealed only a slight reduction in nodulation on white clover, whereas deletion of both copies of nodD resulted in abolished nodulation, which was restored by complementation with either nodD1 or nodD2. Together these results provided evidence of a functional NodD2 in some strains of Rlt. Moreover, we demonstrated that NodD1 and NodD2 show significant differences in their capacity to induce nod gene expression and infection thread (IT) formation. NodD1 and NodD2 protein structure predictions suggested variation in their flavonoid binding pockets, and furthermore NodD2 appeared to confer the ability to nodulate a broader range of hosts. Significantly, competition studies revealed an important role for NodD2 in competitive nodule formation on white clover.
nodD2 was found in the genomes of eight out of 13 strains of Rlt investigated, and was absent from the 13 strains with genome sequence available of another biovar of the same species, Rhizobium leguminosarum bv. viciae (Rlv). It appeared that nodD2 arose via two distinct genetic mechanisms, duplication in some strains and horizontal acquisition in others. Phylogenetic analysis of the Rlt NodD sequences revealed distinct clustering relating to the phenological distinction of the hosts. There was a correlation between the presence of NodD2 and the ability to form effective symbiosis with perennial clover hosts. We propose that NodD2 responds to a different inducer than NodD1 that is produced in the cortical cells of the host, and results in a specific NF profile that is perceived by the stringent cortical receptor, required for efficient and competitive infection of perennial clovers.
A pipeline for transcriptomic analysis of clover rhizosphere-grown Rlt was developed with promising early results. Despite a limited exploratory investigation, two of three genes chosen for investigation revealed a slight competitive disadvantage when deleted. This RNA-seq analysis has provided a basis for future large scale in-depth transcriptomic analysis to determine genetic traits contributing to rhizosphere competence.
In summary, we have determined several genetic traits to be utilised as selection criteria for highly competitive inoculant strains. Furthermore, the transcriptomic pipeline developed will enable identification of further genetic factors contributing to successful symbiosis to also be used as selection criteria for high-quality clover inoculants in NZ.