Transformed Hairy Roots of Discaria trinervis: A Valuable Tool for Studying Actinorhizal Symbiosis in the Context of Intercellular Infection

Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized. Intercellular infection has been described in Discaria trinervis, an actinorhizal plant belonging to the Rosales order. To decipher the molecular mechanisms underlying intercellular infection with Frankia bacteria, we set up an efficient genetic transformation protocol for D. trinervis based on Agrobacterium rhizogenes. We showed that composite plants with transgenic roots expressing green fluorescent protein can be specifically and efficiently nodulated by Frankia strain BCU110501. Nitrogen fixation rates and feedback inhibition of nodule formation by nitrogen were similar in control and composite plants. In order to challenge the transformation system, the MtEnod11 promoter, a gene from Medicago truncatula widely used as a marker for early infection-related symbiotic events in model legumes, was introduced in D. trinervis. MtEnod11::GUS expression was related to infection zones in root cortex and in the parenchyma of the developing nodule. The ability to study intercellular infection with molecular tools opens new avenues for understanding the evolution of the infection process in nitrogen-fixing root nodule symbioses.

Diffusible factors from Frankia modify nodulation kinetics in Discaria trinervis, an intercellular root-infected actinorhizal symbiosis

Frankia BCU110501 induces nitrogen-fixing root nodules in Discaria trinervis (Gillies ex Hook. & Arn.) Reiche (Rhamnaceae) via intercellular colonisation, without root hair deformation. It produces diffusible factors (DFs) that might be involved in early interactions with the D. trinervis roots, playing a role in the nodulation process. The induction of root nodule development in actinorhizal symbiosis would depend on the concentration of factors produced by the bacteria and the plant. A detailed analysis of nodulation kinetics revealed that these DFs produce changes at the level of initial rate of nodulation and also in nodulation profile. Diluted Frankia BCU110501 inoculum could be activated in less than 96 h by DFs produced by Frankia BCU110501 cells that had been previously washed. Biochemical characterisation showed that Frankia BCU110501 DFs have a molecular weight of <12 kDa, are negatively charged at pH 7.0 and seem to contain a peptide bond necessary for their activity. Frankia BCU110501, belonging to Frankia Clade 3, does not induce nodules in Alnus acuminata H.B.K. ssp. acuminata but is able to deform root hairs, as do Frankia strains from Clade 1. The root hair deforming activity of Frankia BCU110501 DFs show the same biochemical characteristics of the DFs involved in nodulation of D. trinervis. These results suggest that Frankia symbiotic factors have a basic structure regardless of the infection pathway of the host plant.

Diffusible factors involved in early interactions of actinorhizal symbiosis are modulated by the host plant but are not enough to break the host range barrier

Nodulation kinetics were analysed in two nitrogen-fixing actinorhizal symbioses that show different pathways for infection: Alnus acuminata H. B. K., which is infected by Frankia ArI3, and Discaria trinervis (Hooker et Arnot) Reiche, which is infected by Frankia BCU110501. Both pairs are incompatible in cross-inoculation experiments. The dose–response effects in nodulation were studied in A. acuminata seedlings using different concentrations of compatible and incompatible bacteria in co-inoculation experiments. Restriction fragment length polymorphism PCR analysis and plant-trapping analysis showed no co-occupation in A. acuminata nodules when plants were co-inoculated with Frankia BCU110501 and Frankia ArI3. Despite the lack of co-occupation, the noninfective BCU110501 could modify the nodulation parameters of the non-host A. acuminata when infective ArI3 was present in the inoculum. The results suggest that although BCU110501 was not able to induce nodulation in A. acuminata, its interaction with the plant could induce autoregulation as if some level of infection or partial recognition could be achieved. We explored the possibility that physiological complementation of the heterologous Frankia BCU110501 for nodulation of A. acuminata originated in the homologous Frankia ArI3 in the presence of compatible root exudates. Despite the possibility of full activation between bacteria and the host, there was no co-infection of Frankia BCU110501 in Alnus or of Frankia ArI3 in Discaria either. These negative results suggest a physical recognition barrier in actinorhizal symbiosis that operates after early interactions, involving something other than root exudates and diffusible factors of bacterial or plant origin, regardless of the infection pathway.

Reproductive ecology of Ochetophila trinervis in Northwest Patagonia

Native actinorhizal species Ochetophila trinervis (Gillies ex Hook. & Arn.) Poepp. ex Miers, also known as Discaria trinervis (Kellermann et al. 2005), grows along watercourses and distant from them, along a rainfall gradient in north-west Patagonia. We studied the reproductive ecology of this species under different macro- and micro-environmental conditions, in three zones in a rainfall gradient (western, intermediate and eastern) with two sites for each one, near and distant to a watercourse (riparian and dry-land). We performed field studies and germination trials. Plant size, reproductive effort (seed abundance per branch) and seed bank size of O. trinervis, were favoured by the proximity of streams in the drier environments of the gradient. The abundance of seedlings and saplings in the field was very low, which was in agreement with a lack of germination in the field, despite the good germination capacity of seeds. Sexual reproduction of O. trinervis was affected by the low abundance of seedlings. Lower rainfall, higher temperatures and a longer growing season along the gradient favour the potential regeneration of the species. Because of its nitrogen-fixing capacity and other features, O. trinervis has potential for reclamation of eroded lands in Patagonia. Patterns of seed biology and regeneration presented in this study will aid in the use of O. trinervis in the reclamation of disturbed lands.