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The tropical legume Sesbania rostrata provides its microsymbiont Azorhizobium caulinodans with versatile invasion strategies to allow nodule formation in temporarily flooded habitats. In aerated soils, the bacteria enter via the root hair curling mechanism. Submergence prevents this epidermal invasion by accumulation of inhibiting concentrations of ethylene and, under these conditions, the bacterial colonization occurs via intercellular cortical infection at lateral root bases. The transcriptome of both invasion ways was compared by cDNA-amplified fragment length polymorphism analysis. Clusters of gene tags were identified that were specific for either epidermal or cortical invasion or were shared by both. The data provide insight into mechanisms that control infection and illustrate that entry via the epidermis adds a layer of complexity to rhizobial invasion.

Background Biological nitrogen fixation is a prokaryotic process that plays an essential role in the global nitrogen cycle. Azorhizobium caulinodans ORS571 has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with Sesbania rostrata. The host is a fast-growing, submergence-tolerant tropical legume on which A. caulinodans can efficiently induce nodule formation on the root system and on adventitious rootlets located on the stem. Results The 5.37-Mb genome consists of a single circular chromosome with an overall average GC of 67% and numerous islands with varying GC contents. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct symbiosis region. The genome contains a plethora of regulatory and transporter genes and many functions possibly involved in contacting a host. It potentially encodes 4717 proteins of which 96.3% have homologs and 3.7% are unique for A. caulinodans. Phylogenetic analyses show that the diazotroph Xanthobacter autotrophicus is the closest relative among the sequenced genomes, but the synteny between both genomes is very poor. Conclusion The genome analysis reveals that A. caulinodans is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make A. caulinodans an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants.

Aquatic nodulation on the tropical legume Sesbania rostrata occurs at lateral root bases via intercellular crack-entry invasion. A gene was identified (Srprx1) that is transiently up-regulated during the nodulation process and codes for a functional class III plant peroxidase. The expression strictly depended on bacterial nodulation factors (NFs) and could be modulated by hydrogen peroxide, a downstream signal for crack-entry invasion. Expression was not induced after wounding or pathogen attack, indicating that the peroxidase is a symbiosis-specific isoform. In situ hybridization showed Srprx1 transcripts around bacterial infection pockets and infection threads until they reached the central tissue of the nodule. A root nodule extensin (SrRNE1) colocalized with Srprx1 both in time and space and had the same NF requirement, suggesting a function in a similar process. Finally, in mixed inoculation nodules that were invaded by NF-deficient bacteria and differed in infection thread progression, infection-associated peroxidase transcripts were not observed. Lack of Srprx1 gene expression could be one of the causes for the aberrant structure of the infection threads.

Upon submergence, Azorhizobium caulinodans infects the semiaquatic legume Sesbania rostrata via the intercellular crack entry process, resulting in lateral root-based nodules. A gene encoding a gibberellin (GA) 20-oxidase, SrGA20ox1, involved in GA biosynthesis, was transiently up-regulated during lateral root base nodulation. Two SrGA20ox1 expression patterns were identified, one related to intercellular infection and a second observed in nodule meristem descendants. The infection-related expression pattern depended on bacterially produced nodulation (Nod) factors. Pharmacological studies demonstrated that GAs were involved in infection pocket and infection thread formation, two Nod factor-dependent events that initiate lateral root base nodulation, and that they were also needed for nodule primordium development. Moreover, GAs inhibited the root hair curling process. These results show that GAs are Nod factor downstream signals for nodulation in hydroponic growth.