Genetic constraints that determine rhizobium-root nodule formation in Parasponia andersonii

Bacteria of the genus Rhizobium play a very important role in agriculture by inducing nitrogen-fixing nodules on the roots of legumes. Root nodule symbiosis enables nitrogen‐fixing bacteria (Rhizobium) to convert atmospheric nitrogen into a form that is directly available for plant growth. This symbiosis can relieve the requirements for added nitrogenous fertilizer during the growth of leguminous crops. Research on legume-rhizobium symbioses has emphasized fitness benefits to plants but in our research, we take a different vantage point, focusing on the <em>Parasponia-rhizobium</em> symbiosis.<em> Parasponia </em>is the only non-legume plant capable of establishing mutualistic relation with rhizobia. This study will provide background knowledge for use in applied objectives as well as yielding a wealth of fundamental knowledge with wide implications from rhizobium symbiosis evolution. This thesis describes my research on genetic constrains that determine rhizobium-root nodule formation. To identify these constraints we used <em>Parasponia anadersnii</em> as only non-legume capable to establish nitrogen fixing rhizobium symbiosis. Our main attempt in this thesis was to find the genetic constraints using <em>Parasponia</em> as a key and reconstruct an auto active symbiotic signaling cascade in the non- legume plants. In line with this, a simple and efficient hairy root transformation method was established in this thesis. To determine the genetic elements that underlie the rhizobium symbiosis, we aimed to compare <em>Parasponia </em>with closest non nodulating specious, <em>Trema tomentosa</em>. To do so, we also developed an efficient genetic transformation method for <em>Trema</em> mediated by <em>Agrobacterium tumefaciens.</em><em> In different attempt </em>we implemented in a physiological study on symbiotic response of <em>Parasponia</em> to nitrate. This research opened a novel view on the <em>Parasponia-rhizobium</em> symbiosis by discovering a different mechanism that control root nodule formation in <em>Parasponia</em> in compare with legumes. We discovered that <em>Parasponai-rhizbium</em> symbiosis is not evolved to regulate the nodule number in presence of the nitrate. According to the fact that <em>Parasponia</em> and legumes are remotely related, it was hypothesized that, <em>Parasponia-rhizobium</em> symbiosis evolved independently. Therefore we put forward our attempt to determine the genes required for nodule formation in <em>Parasponia, </em>by extending our research on symbiotic genes which are available in non nodulating plants with different function, namely <em>NSP1</em> and <em>NSP2</em>. We showed that <em>NSP1 </em>and <em>NSP2</em> are involved in both nodulation and mycorrhization. This result highlight the idea that RN and AM symbiosis are conserved in part of the pathway and probably bifurcates into two branches by NSP transcription factor allowing specific activation of nodulation or mycorrhization. Aiming to know the role of hormones in symbiotic behavior, we focused on ethylene as a negative regulator of nodule formation in legumes. We found the negative effect of ethylene on root nodulation of <em>Parasponia</em>. For the first time we reported a hyper nodulation (20 fold nodule number in compare with control plants) phenotype in <em>Parasponia</em> by performing knocked down mutant of <em>EIN2</em> gene. Finally, the results obtained in this study provide new insight into the fact that rhizobium symbiosis are under tight genetic constraints that guide endosymbiosis in remotely evolved host plants, legumes and <em>Parasponia</em>.