From signal to form: Nod factor as a morhogenetic signal molecule to induce symbiotic responses in legume root hairs

In this thesis, research is presented which contributes to a better understanding of nod factor (NF) induced signalling in Iegume root hairs, leading to a successful symbiosis. We mainly use root hairs of the model Iegume Medicago truncatula ('barrel medic') as an experimental system. In the different chapters, different aspects of the NF induced changes in root hair morphology that are required for establishing a successful symbiosis between rhizobia and legumes are covered.Chapter 1 is a review article that describes the different roles of the actin cytoskeleton in Iegume root hairs: its different configurations in relation to root hair growth, its function as backbone of cytoplasmic strands and highway for cellular transport, and its target for NF-induced signalling.Chapter 2 describes a new experimental assay to test the effects of NF on legume root hairs. The advantage of this assay, in comparison with the classical global application assays, is that it better mimics the natural situation in which rhizobia are locally present on the hosts' root hairs. It tests a theoretical computer model explaining root hair curling around bacteria. With a microinjection needie, a small droplet of purified NF was applied on the side o1 the tip of growing root hairs. The result of this is that the root hair under study reoriented its growth axis - it curls, toward the site of NF application, and i1 expresses the early nodulin geneMutagenesis screens are nowadays widely performed to genetically dissec1 signal transduction pathways. In chapter 3, we studied the root hair phenotype in the non nodulating M. truncatula dmi2/N0RK mutant which was found in such a screen. This mutant, and its two orthologues in alfalfa and Lotus japonicus, appeared to exhibit an enhanced touch response to experimenta handling. When care was taken to not induce this touch response, the mutan1 root hairs responded morphologically like wild-type root hairs to NF application. A global application resulted in root hair deformation and NF spot application induced root hair reorientation or - branching, depending on the position of application on the root hair. In addition, dmi2/N0RK root hairs make 180° curls in the presence of rhizobia, but as soon as the root hair tip touches its own shank, the root hair stops growing/curling, and as such is unable to entrap bacteria in a three-dimensional pocket. Because dmi2 root hairs do not express the ProMtENOon-GUS reporter gene after NF application we propose a split in NF-induced signalling, with one branch to root hair curling, the other to ProutENOon-GUS expression.Pea plants can be successfully nodulated by certain strains of rhizobia that oroduce hardly detectable amounts of NF. In addition, very low concentrations of purified NF elicit changes in root hair morphology and gene expression in other legume species. Therefore, we tested what is the lowest NF concentration at which root hair reorientation and ProMtENooii-GUS expression.still occur. In chapter 4, we show the exciting result that one single NF molecule is sufficient to induce root hair reorientation and ProMtENoon-GUS expression,In chapter 5 we describe the results that we obtained after spot application of nod factor mixed with pharmacological agonists or antagonists of signal transduction pathways. As such, we show that NF-induced root hair reorientation can be blocked with gadolinium ions, ions that specifically block calcium influxes in plant cells. Moreover, we show that we can induce root hair reorientation in a number of hairs after spot application of a mixture of calcium ions and the ionophore A23187. Pertussis toxin specifically inhibits heterotrimeric G-proteins. Upon spot application of a mixture of NF and pertussis toxin, root hairs do reorient their growth axis, but do not express the ProMtENODii-GUS reporter gene. Spot application of mastoparan, a small peptide from wasp venom that activates heterotrimeric G-proteins, does not result in root hair reorientation, but does induce Pro^tENOon-GUS expression. Heterotrimeric G-proteins activate phospholipase C. Upon spot application of a mixture of NF and neomycin or U~73122, two known antagonists of phospholipase Ct ProMtENOon-GUS expression was inhibited, but root hair reorientation not Phospholipase C is an enzyme that cleaves phosphoinositolbisphosphate (PIP2) into diacylglycerolphosphate (DAG) and inositoltriphosphate (IP3). With microinjection of caged IP3 into growing M. truncatula root hairs and subsequent uncaging with an UV laser, we got expression of ProMtENooirGUS in a number of root hairs. This all shows that heterotrimeric G-protein coupled phosphoinositide signalling is involved in NF-induced Pr0MtEN0Di1-GUS expression in M. truncatula root hairs.Chapter 6 is a review which covers the current state of the art in the research of the Rhizobium-\egume symbiosis, with a special focus on signal transduction. It not only compares genetic dissection with pharmacological approaches, but also covers the cell biological aspects that are necessary to fully understand NF induced signal transduction.Chapter 7 is a chapter which describes that SHAGGY-kinase signalling is involved in root hair deformation in thale cress (Arabidopsis). This is a small weed that is used as a non~legume model plant. We show that application of lithium ions induces root hair deformation, and that in roots which lack a SHAGGY-kinase, a significant higher percentage of root hairs deform upon lithium application. In plant cells, lithium ions influence ethylene and phosphoinositide signalling, but with the use of specific agonists and antagonists of these pathways, we show that lithium induced root hair deformation in Arabidopsis is not caused by disrupted ethylene or phosphoinositide signalling.