Transformed Hairy Roots of Discaria trinervis: A tool for studying actinorhizal symbiosis in the context of intercellular infection

IMANISHI L; VAYSSIERES A; FRANCHE C; BOGUSZ D; SVISTOONOFF S; WALL LG

Porto

Congreso; 16 International meeting on Frankia and actinorhizal plants; 2010

The mechanisms and genetics by which actinorhiza forming plants recognize Frankia symbionts, before and during infection, remain unknown. Actinorhizal plant species are distributed within the orders Fagales, Cucuribtales and Rosales. The most studied model systems, in terms of molecular biology and genetics, belong to Fagales (Alnus spp and Casuarina spp) which at the same time are examples of the root hair infection pathway. Discaria trinervis, a Patagonean Rhamnaceae shrub, is an example of actinorhizal plants belonging to Rosales which has been described to be infected via intercellular pathway. We have set up a genetic transformation of Discaria trinervis root system based on Agrobacterium rhizogenes, comparing in vitro transformation system and ex-vitro one. Composite plants with transgenic roots can be specifically and efficiently nodulated allowing for functional nitrogen fixing symbiosis by inoculation with Frankia BCU110501. We studied the expression in Discaria trinervis of some symbiotic marker genes from legumes (Enod11) or other actinorhizal plants as Casuarina glauca (CgAux1 and Cg12), which have been previously characterized in root hair infected actinorhizal plants. Prior to Frankia inoculation no GUS expression was detected in plants transformed with ProCg12::GUS whereas the promoters of CgAux1 and MtEnod11 were strongly active in the root vasculature and root tips of plants. Upon the inoculation by Frankia BCU110501, strong activation of ProCg12 and ProMtEnod11 was detected in the root cortex in places where small nodule primordia were starting to appear. The expression was localized in the apical zone of the developing nodule corresponding to the infection zone. Upon the inoculation by Frankia BCU110501, strong activation of ProCg12 and ProMtEnod11 was detected in the root cortex in places where small nodule primordia were starting to appear. The expression was localized in the apical zone of the developing nodule corresponding to the infection zone. ProCg12 appear to be a good reporter for the study of intercellular root invasion. In contrast to ProCg12 and ProMtEnod11, ProCgaux1 was not active during Frankia intercellular infection. GUS expression was observed in young and mature nodular lobes, related to infection zone and initially infected hyperplasic cells but no GUS crystals were detected in infected cells with vesicle differentiation. These promising results prompt us to look for the orthologous genes in Discaria trinervis. Dt12 has been detected and seem to be specifically induced in nodules and not in roots. Its cloning is under progress. The developed strategy would now enable the study of the intercellular infection pathway and the molecular mechanisms of the interaction of Frankia with actinorhizal plant belonging to the order Rosales.