Receptor kinases regulating rhizobial infection
Leguminous plants are able to establish a symbiotic interaction with nitrogen-fixing soil bacteria, generally called rhizobia. This host-specific interaction results in the formation of a completely new organ, the root nodule, where the bacteria are hosted intracellularly and are able to fix atmospheric nitrogen. A successful interaction requires the strict coordination of two processes: the formation of the nodule organ from root cortical cells and infection of this new organ by the bacteria through tubular structures, called infection threads, whose formation is started in the root hairs. To initiate these processes a molecular dialogue is required between the two partners. Upon perception of flavonoids secreted by the plant the rhizobia produce lipo-chitooligosaccharidic signalling molecules, the Nod factors, which are essential and in most cases sufficient for the induction of symbiotic responses in the host plant. These Nod factors have a common basic structure consisting of three to fiveb-1,4-linked N -acetyl-D-glucosamine subunits with an acyl chain linked to the non-reducing terminal sugar residue. Depending on the rhizobial species, the structure of the acyl chain can vary and specific decorations at the reducing and non-reducing terminal glucosamine residues can be present, which function as major determinants of the host-specificity of the interaction. Nod factors are biologically active at nano- to picomolar concentrations and their activity depends on their structure, which implies that they are perceived by specific receptors. The major goal of the research described in this thesis was to unravel the molecular basis of Nod factor perception. Therefore we focussed on the pea SYM2 gene, which was proposed to be involved in Nod factor perception. In the pea accession Afghanistana SYM2 A allele was identified, which controls rhizobial infection in a Nod factor structure dependent manner. Only Rhizobium leguminosarumbvviciae strains that contain the bacterial nodulation gene, nodX , which specifically acetylates the reducing terminal sugar residue of pentameric Nod factors, are allowed to successfully infect pea plants containing the SYM2 A allele fromAfghanistan. This suggests that SYM2 A is involved in recognizing these nodX modified Nod factors and it would be attractive to clone this gene. However, positional cloning in pea is severely hampered because of its large genome size (~5000 Mb) and it is difficult to transform this legume. Therefore we decided to use a synteny based positional cloning approach in the phylogenetically closely related model legume Medicago truncatula (Medicago).In Chapter 1 an overview is given of the different steps of nodulation with a focus on the molecular basis of Nod factor perception and transduction as it was known at the start of the research.In Chapter 2 the level of microsynteny between pea and Medicago was studied in the SYM2 region. This resulted in the delineation of a ~350 kb physical BAC contig representing the SYM2 orthologous region in Medicago.A conservedgene content was observed in the SYM2 orthologous regions, which supports the idea that Medicago can be used as intergenomic vehicle to clone pea genes.Because SYM2 represent a natural occurring variation in pea, sequence analysis could not be used to identify the SYM2 gene. Therefore we developed a reverse genetics approach in Medicago to identify genes involved in rhizobial infection. In Chapter 3 we report the effective use of RNA interference (RNAi) via Agrobacterium rhizogenes mediated root transformation as a reverse genetic tool to knock down gene expression in the roots of both Arabidopsis and Medicago. A. rhizogenes mediated root transformation has the advantage that it is a relatively fast method to generate genetically transformed roots. It is further shown that the silencing signal does not spread to non co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot. Furthermore, RNAi appeared to be cell-autonomous in the epidermis of Medicago roots.In Chapter 4 we used A. rhizogenes mediated RNAi to knock down candidate genes from the Medicago SYM2 othologous region and examined their role in rhizobial infection. We survey-sequenced the SYM2 orthologous region and strikingly many genes encoding receptor-like proteins appeared to be present. Knock down by RNAi of two genes, LYK3 and LYK4 , affected rhizobial infection. By using Sinorhizobiummeliloti strains that are mutated in their nod genes, a block of infection thread formation due to knock down of LYK3/4 was shown to be correlated to the structure of the produced Nod factors. The two identified genes encode LysM domain containing receptor-like kinases. The nature of the LysM domains together with the Nod factor structure dependent block of rhizobial infection strongly suggests that these genes represent Nod factor receptors.The formation of a functional root nodule requires the tight coordination of the infection process by the bacteria and the formation of the nodule organ. In all mutants currently characterized the loss of (all) Nod factor responses in the epidermis is always correlated with the loss of the cortical cell division (nodule primordium) response. This led to the hypothesis that Nod factor signalling in the epidermis results in the generation of a secondary signal that subsequently triggers cortical cell division. In Chapter 5 we show that the presence of an essential component of the Nod factor perception and transduction machinery, NODULATION RECEPTOR KINASE ( NORK ), exclusively in the epidermis is sufficient to trigger cortical cell divisions by external application of Nod factors. This shows that mitotic activation of cortical cells is triggered by Nod factors in a non cell-autonomous manner and implies the involvement of intercellular communication in Nod factor signalling.Several lines of evidence indicate that the Nod factor perception and transduction machinery also plays a role in the nodule. In Chapter 6 we show that NORK expression is highly upregulated in the nodule and the region where it is expressed coincides with rhizobial nod gene activity in the infection zone of the nodule where bacteria are released into the plant host cells. By mimicking allelic series of NORK via RNA interference and by expression of 35S::NORK constructs in the mutant background we show that NORK controls the switch from infection thread growth to release of bacteria in the nodule. This suggests a role for the Nod factor perception and signaling machinery in this process.Finally Chapter 7 summarizes and discusses the results obtained by the research described in this thesis.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
| Subjects: | genes, genetic regulation, nitrogen fixing bacteria, root nodules, signal transduction, genen, genetische regulatie, signaaltransductie, stikstofbindende bacteriën, wortelknolletjes, |
| Online Access: | https://research.wur.nl/en/publications/receptor-kinases-regulating-rhizobial-infection |
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