Nod factor signaling and infection in Rhizobium-legume symbiosis

Plants require nutrients in order to grow. Most of these are readily available, but a few, like the macronutrients nitrogen and phosphorous, are often limiting growth due to presence in low concentrations or in complexes that cannot be taken up by the plant root. To acquire these macronutrients plants can engage in a mutualistic symbiosis with other organisms. Most plants can interact with arbuscular mycorrhizal fungi that are able to solubilize complexed phosphorous. A subset of these plants, the legumes, can also interact with bacteria that can reduce atmospheric nitrogen into compounds plants can assimilate. Both interactions are so-called endosymbioses because the microsymbiont enters host cells. A symbiosis requires a good balance between costs and benefits. Therefore, infection is only initiated when the specific nutrient is scarce. Further, the host plant requires that the mutualistic microsymbionts produce signal molecules to identify themselves, and thereby prevents other (pathogenic) microorganisms to enter by similar mechanisms. The signal molecule(s) involved in mycorrhizal symbioses have not yet been identified. However, the rhizobial signal molecule, the so-called nodulation (Nod) factor has been identified and extensively studied. It has been intensively studied how Nod factors are perceived and how they induce Nod factor specific responses, including the formation of a completely new organ, the root nodule. In chapter one, we describe the state of the art (in 2005) regarding Nod factor signal transduction. Several approaches have been used to unravel Nod factor signaling in legumes, but the molecular-genetic approach has been most successful. More than 10 years after the first characterization of a Nod factor structure most genes essential for Nod factor signal transduction have been cloned and characterized. Additionally, the identification of these genes provides insight in how the Rhizobium-legume symbiosis evolved, because some of the identified Nod factor signaling genes are also essential to the arbuscular mycorrhizal symbiosis. The DMI genes, encoding a LRR receptor kinase, an ion channel, and a calcium/calmodulin dependent protein kinase, are required for both symbioses and are essential for most responses. The genes encoding the Nod factor receptors (NFR1, NFR5, LYK3, NFP), Nod factor response factors (NSP1, NSP2) and Nod factor responsive factors (NIN) show that besides the DMI genes additional genes are required to induce Nod factor specific responses. Therefore it is likely that the Rhizobium-legume symbiosis evolved from the more ancient mycorrhizal symbioses, but during evolution the nodulation specific genes have been recruited to induce Nod factor specific responses. In chapter two, we elaborate on two of the genes, NSP1 and DMI3, we introduced in chapter 1. We show in that chapter that NSP1 is a primary transcriptional regulator essential for the regulation of all Nod factor regulated genes, NSP1 is: a member of the GRAS transcription factor family; constitutively expressed; localized in the nucleus where also DMI3, acting upstream of NSP1, is being localized; essential for regulation of all known Nod factor regulated genes. NSP1 is a conserved member of the GRAS type protein family in the plant kingdom. Therefore, it is likely that NSP1, besides its normal function, gained a function in Nod factor signaling. In chapter three, we describe the identification of HCL, a gene required for root hair curling and infection, but not essential for other Nod factor-induced responses. We show by isolation of the weak hcl-4 mutant allele that HCL is also involved in infection thread formation. HCL encodes the previously identified Nod factor receptor LYK3. In hcl-4 infection thread growth, but not root hair curling, is dependent on the Nod factor structure excreted by the Rhizobium bacteria; only if the bacteria excrete the wild type Nod factor they are able to nodulate hcl-4 mutants. LYK3 is also essential for the regulation of a subset of Nod factor induced genes. However, NIN, a Nod factor regulated gene that is essential for both infection and nodule primordium formation, is not regulated by LYK3. The identification and characterization of hcl/lyk3 mutants is important as from previous work it was not clear whether LYK3 acts as a signaling or entry receptor. In this chapter, we clearly show that LYK3 acts as an infection receptor that regulates infection in a Nod factor structure dependent manner. In chapter three, we have shown that the infection receptor LYK3 is essential for specificity during infection. This receptor regulates bacterial growth in the infection thread. However, what mechanism the plant uses to prevent that other incompatible bacteria do grow along during infection? In chapter four we study bacterial growth in infection threads and show, using the fluorescent timer protein DsRED1-E5, that probably only at the tip of infection threads bacteria do grow. Therefore, during infection thread growth, most likely a continual selection of Nod factor producing Rhizobium bacteria takes place that will ultimately infect the nodule primordium established in the root cortex. This thesis is finalized (chapter five) by discussing new data obtained during the last couple of years, and draw new or readjust old conclusions. We discuss the following themes: how is the Nod factor signal transduced from the plasmamembrane into the nucleus; how did Nod factor signaling genes evolve; are there any new Nod factor signaling genes identified; is it useful to conduct more classical genetic screens for genes involved in root nodule symbiosis; and finally how Nod factor signal transduction plays a role in infection thread formation.