Katharina Pawlowski, Department of Ecology, Environment and Plant Sciences, Plant Physiology Unit, SU


Two types of nitrogen-fixing root nodules are known, between unicellular soil bacteria collectively called rhizobia and legumes, and between filamentous soil bacteria from the genus Frankia and a diverse group of plants, collectively called actinorhizal plants. Swedish examples of actinorhizal plants are Alnus sp. (al) and Hippophae rhamnoides (havtorn). In contrast with rhizobia, Frankia strains can fix nitrogen in the free-living state under aerobic conditions. Rhizobia cannot protect their nitrogenase enzyme complex from destruction by oxygen, but Frankia strains can.
The Alnus microsymbiont ACN14 a (Normand et al. 2007) was chosen as model organism. It shall be used to inoculate three different plant species:
- a close relative of its host plant, Betula pendula
- a dicot species belonging to a clade close to the nitrogen-fixing clade, tobacco
- an agriculturally relevant monocot species, barley

Experimental steps:
• Co-cultivation in soil will be used for different time periods to allow the Frankia strain to enter the plant roots. Then total DNA will be isolated from surface sterilized plant parts (roots, stems, leaves) and the presence of the microbe will be analysed using PCR with Frankia-specific primers.
• In case of positive results, Frankia hyphae/vesicles can be visualized microscopically in paraffin-embedded plant tissue. Attempts will be made to develop a protocol for staining Frankia structures in cleared plant roots using features of the Gram-positive cell walls.
• To find out whether endophytic Frankia fixes nitrogen, expression of the nitrogenase structural gene nifH will be examined using RT-PCR
• To quantify root and shoot colonization by Frankia, and nifH expression, real time PCR (qPCR) methods will be used


Techniques involved:
• Bacterial culture
• Plant culture
• Light microscopy
• DNA- and RNA isolation
• PCR and RT-PCR, later RT-qPCR


Normand et al. (2007) Genome characteristics of facultatively symbiotic Frankia sp. strains reflect host range and host plant biogeography. Genom Res. 17, 7-15.