Early nodulins in root nodule development

The symbiotic interaction between bacteria of the genus Rhizobium and leguminous plants leads to the formation of root nodules, which are specific nitrogen-fixing organs on the roots of plants. Bacteria enter the root by infection threads, and concomitantly cell divisons are induced in the root cortex, which lead to the formation of a meristern. From this meristern the different tissues of the root nodule originate. In the nodule bacteria are released in plant cells and then differentiate into the endosymbiotic bacteroids. These bacteroids are capable of nitrogen fixation.The formation of root nodules involves expression of both bacterial and plant genes. Rhizobium genes involved in nodule formation are the nodulation ( nod ) genes. Nodulespecific plant genes are termed nodulin genes. According to their timing of expression they can be divided into early and late nodulin genes. Early nodulin genes are expressed well before the onset of nitrogen fixation, at the time that the nodule tissue is formed and the roots become infected by bacteria, while expression of late nodulin genes starts shortly before the onset of nitrogen fixation, when the nodule structure has been formed. Therefore only early nodulins can be involved in the infection process and in nodule development. Early nodulin genes expressed during the pea ( Pisum sativum L.) - Rhizobium leguminosarum bv. viciae interaction are the subject of this thesis. Several cDNA clones representing pea early nodulin genes have been isolated and they have been used to study root nodule development and the communication between bacteria and host plant.In chapter 2 we review general aspects of plant development. Recent progresses in understanding the molecular mechanisms underlying animal development are listed, and the possible significance of such mechanisms for plant development is discussed. The features of the root nodule formation system that make it suitable to study particular questions on the molecular basis of plant development are put forward.In chapter 3 the pea early nodulin cDNA clone pPsENOD2 is characterized. The nature of the encoded polypeptide is compared with that of the soybean early nodulin described before. ENOD2 transcripts are localized both in pea and soybean root nodules throughout successive stages of development by in situ hybridization. Data on the primary structure of the ENOD2 protein and localization data are then combined to hypothesise that the function of this early nodulin is to create an oxygen barrier in the root nodule.In chapter 4 the early nodulin ENOD12 is described. The spatial distribution of the corresponding transcript throughout root nodule development is depicted to demonstrate the involvement of ENOD12 in the infection process. We describe the primary structure of the ENOD12 protein and we examine whether ENOD12 gene expression is related to a defense respons. Using a sensitive detection method based on the polymerase chain reaction (PCR) we demonstrate that ENOD12 gene expression is induced by excreted Rhizobium factors and that bacterial nod genes are involved. ENOD12 transcripts found in flower and stem tissue are compared to the ENOD12 mRNAs in nodules using, among other techniques, a novel adaptation of RNase mapping to determine whether the same genes are expressed in these different tissues or not.In chapter 5 it is demonstrated that the accumulation pattern of the transcripts corresponding to the pPsENOD5, pPsENOD3 and pPsENOD14 cDNA clones differs from that of ENOD2 and ENOD12 mRNA. The distribution of the former three transcripts is compared with the distribution of ENOD12 mRNA and the late nodulin leghemoglobin transcript. It is shown that the different transcripts are present at successive stages of development of the infected cell type. The primary structure of the ENOD5, ENOD3 and ENOD14 early nodulins is determined and these data are combined with the localization data of the transcripts to speculate on functions of these proteins, The involvement of different factors to induce expression of different early and late nodulin genes is discussed.In chapter 6 the results described in the previous three chapters are summarized and some additional data on early nodulins are presented. The significance of the availability of early nodulin gene probes to elucidate the mechanisms of communication between rhizobia and legumes, which underly the process of root nodule formation, is discussed. Finally, in chapter 7, the value of the obtained information on early nodulins for studying both specific and general aspects of root nodule development is discussed.