Perception and action of nod factors in Rhizobium-legume symbiosis

Rhizobium bacteria are able to invade the roots of their leguminous hosts and trigger the formation of a new organ, the root nodule. In these nodules the bacteria are hosted in the proper environment for fixing atmospheric nitrogen into ammonia, making plant growth independent of nitrogen compounds from the soil. The interaction between the bacterium and the host plant starts with a signal exchange, when flavonoids excreted by the plant induce the transcription of bacterial nodulation (nod) genes. Induction of these nod genes leads to the synthesis of specific lipo-oligosaccharides (Nod factors) required for inducing various root responses like root hair deformation, infection thread and nodule primordia formation. The aim of the research described in this thesis is to contribute to the insight in the mechanism by which Nod factors initiate root nodule formation. To address this issue it is essential to have a plant system in which both the morphological and molecular changes induced by Nod factors can be studied. We adapted a root hair deformation assay for Vicia sativa (vetch), growing the seedlings in Fahraeus slides, to study the activity and fate of the various Nod factors secreted by Rhizobium leguminosarum by viciae in a fast, simple and semiquantitative way (Chapter 2). Root hair deformation in this assay is limited to root hairs in a specific developmental stage and involves a reinitiation of tip growth which is visible within 3 hours after Nod factor application. In an attempt to identify genes the expression of which is induced in an immediate respons to Nod factors we used the differential RNA display method to compare the pattern of cDNAs expressed in the zone of the vetch root where root hair deformation occurs at 0, 1 and 3 hours after Nod factor addition. Surprisingly, the sequence of one of the isolated clones already expressed I hour after Nod factor application coded for leghemoglobin. This gene was used to show that gene expression and root hair deformation are not coupled and to investigate how NH4NO3* might block root hair deformation (Chapter 3). Root hair deformation induced by Nod factors involves a reinitiation of growth in the existing root hair tip, a mechanism reminiscent of ethylene induced tip growth leading to the formation of root hairs in the epidermis. Since ethylene is a potent inhibitor of cortical cell division there seems to be a paradox in its action during the initial interaction between rhizobia and its host. We showed that ethylene is not invoved in the root hair deformation process, but it is one of the factors involved in giving positional information determining where nodule primordia can be induced (Chapter 4). How Nod factors are perceived and induce the various responses in the root is poorly understood. Considering that Nod factors are active at very low concentrations it is likely that they are recognized by receptors. Besides a biochemical approach to search for such receptors it win be very important to isolate and analyse host mutants disturbed in the early staves of the interaction with Rhizobium. We chow to focus on the sym2 A gene originating from Afghanistan pea since the presence of this gene puts more stringent structural demands on the Nod facotrs secreted by Rhizobium leguminosarum by viciae. After detailed analysis of the phenotype conferred by sym2 A we propose that Sym2 controls the infection process in the epidermis, possibly representing a Nod factor receptor (Chapter 5).</p

Root developmental programs shape the Medicago truncatula nodule meristem

Nodules on the roots of legume plants host nitrogen-fixing Rhizobium bacteria. Several lines of evidence indicate that nodules are evolutionarily related to roots. We determined whether developmental control of the Medicago truncatula nodule meristem bears resemblance to that in root meristems through analyses of root meristem-expressed PLETHORA genes. In nodules, MtPLETHORA 1 and 2 are preferentially expressed in cells positioned at the periphery of the meristem abutting nodule vascular bundles. Their expression overlaps with an auxin response maximum and MtWOX5, which is a marker for the root quiescent center. Strikingly, the cells in the central part of the nodule meristem have a high level of cytokinin and display MtPLETHORA 3 and 4 gene expression. Nodule-specific knockdown of MtPLETHORA genes results in a reduced number of nodules and/or in nodules in which meristem activity has ceased. Our nodule gene expression map indicates that the nodule meristem is composed of two distinct domains in which different MtPLETHORA gene subsets are expressed. Our mutant studies show that MtPLETHORA genes function redundantly in nodule meristem maintenance. This indicates that Rhizobium has recruited root developmental programs for nodule formation

A Model for the Development of the Rhizobial and Arbuscular Mycorrhizal Symbioses in Legumes and Its Use to Understand the Roles of Ethylene in the Establishment of these two Symbioses

We propose a model depicting the development of nodulation and arbuscular mycorrhizae. Both processes are dissected into many steps, using Pisum sativum L. nodulation mutants as a guideline. For nodulation, we distinguish two main developmental programs, one epidermal and one cortical. Whereas Nod factors alone affect the cortical program, bacteria are required to trigger the epidermal events. We propose that the two programs of the rhizobial symbiosis evolved separately and that, over time, they came to function together. The distinction between these two programs does not exist for arbuscular mycorrhizae development despite events occurring in both root tissues. Mutations that affect both symbioses are restricted to the epidermal program. We propose here sites of action and potential roles for ethylene during the formation of the two symbioses with a specific hypothesis for nodule organogenesis. Assuming the epidermis does not make ethylene, the microsymbionts probably first encounter a regulatory level of ethylene at the epidermis–outermost cortical cell layer interface. Depending on the hormone concentrations there, infection will either progress or be blocked. In the former case, ethylene affects the cortex cytoskeleton, allowing reorganization that facilitates infection; in the latter case, ethylene acts on several enzymes that interfere with infection thread growth, causing it to abort. Throughout this review, the difficulty of generalizing the roles of ethylene is emphasized and numerous examples are given to demonstrate the diversity that exists in plants

Root hydrotropism is controlled via a cortex-specific growth mechanism

Plants can acclimate by using tropisms to link the direction of growth to environmental conditions. Hydrotropism allows roots to forage for water, a process known to depend on abscisic acid (ABA) but whose molecular and cellular basis remains unclear. Here, we show that hydrotropism still occurs in roots after laser ablation removed the meristem and root cap. Additionally, targeted expression studies reveal that hydrotropism depends on the ABA signalling kinase, SnRK2.2, and the hydrotropism-specific MIZ1, both acting specifically in elongation zone cortical cells. Conversely, hydrotropism, but not gravitropism, is inhibited by preventing differential cell-length increases in the cortex, but not in other cell types. We conclude that root tropic responses to gravity and water are driven by distinct tissue-based mechanisms. In addition, unlike its role in root gravitropism, the elongation zone performs a dual function during a hydrotropic response, both sensing a water potential gradient and subsequently undergoing differential growth

Probing the roles of LRR RLK genes in Arabidopsis thaliana roots using a custom T-DNA insertion set

Leucine-rich repeat receptor-like protein kinases (LRR RLKs) represent the largest group of Arabidopsis RLKs with approximately 235 members. A minority of these LRR RLKs have been assigned to diverse roles in development, pathogen resistance and hormone perception. Using a reverse genetics approach, a collection of homozygous T-DNA insertion lines for 69 root expressed LRR RLK genes was screened for root developmental defects and altered response after exposure to environmental, hormonal/chemical and abiotic stress. The obtained data demonstrate that LRR RLKs play a role in a wide variety of signal transduction pathways related to hormone and abiotic stress responses. The described collection of T-DNA insertion mutants provides a valuable tool for future research into the function of LRR RLK genes

Different reprogramming propensities in plants and mammals: Are small variations in the core network wirings responsible?

Although the plant and animal kingdoms were separated more than 1,6 billion years ago, multicellular development is for both guided by similar transcriptional, epigenetic and posttranscriptional machinery. One may ask to what extent there are similarities and differences in the gene regulation circuits and their dynamics when it comes to important processes like stem cell regulation. The key players in mouse embryonic stem cells governing pluripotency versus differentiation are Oct4, Sox2 and Nanog. Correspondingly, the WUSCHEL and CLAVATA3 genes represent a core in the Shoot Apical Meristem regulation for plants. In addition, both systems have designated genes that turn on differentiation. There is very little molecular homology between mammals and plants for these core regulators. Here, we focus on functional homologies by performing a comparison between the circuitry connecting these players in plants and animals and find striking similarities, suggesting that comparable regulatory logics have been evolved for stem cell regulation in both kingdoms. From in silico simulations we find similar differentiation dynamics. Further when in the differentiated state, the cells are capable of regaining the stem cell state. We find that the propensity for this is higher for plants as compared to mammalians. Our investigation suggests that, despite similarity in core regulatory networks, the dynamics of these can contribute to plant cells being more plastic than mammalian cells, i.e. capable to reorganize from single differentiated cells to whole plants-reprogramming. The presence of an incoherent feed-forward loop in the mammalian core circuitry could be the origin of the different reprogramming behaviour.This work was supported Swedish Research Council, grant VR 621-2013-4547 to CP; the Swedish Foundation for Strategic Research, grant A3 04 159p to CP; the Gatsby Charitable Foundation (GB), grant GAT3395-PR4 to HJ and the Swedish Research Council, grant VR 621- 2013-4632 to HJ

Digging out Roots: Pattern Formation, Cell Division, and Morphogenesis in Plants

The analysis of plant development by genetic, molecular, and surgical approaches has accumulated a large body of data, and yet it remains a challenge to uncover the basic mechanisms that are operating. Early steps of development, when the zygote and its daughter cells organize the embryonic plant, are poorly understood despite considerable efforts toward the identification of relevant genes. Reported cases of genetic redundancy suggest that the difficulty in uncovering patterning genes may reflect overlapping gene activities. Our current knowledge on plant embryo development still leaves open whether mechanisms for axis formation and subsequent pattern formation are fundamentally different in animals and plants. Axis formation may follow the general principle of establishing a peripheral asymmetric cue and mobilizing the cytoskeleton toward this cue-in the case of plants possibly located in the cell wall-but the molecules involved may be entirely different. Embryonic pattern formation involves the establishment of different domains, but although there are candidates, it is not clear whether genes that define these domains are identified yet. Pattern formation continues postembryonically in the meristem, and the flexibility of this process may be explained by a feed-forward system of patterning cues originating from more mature cells. Control of cell division and differentiation, which is important in the meristems-regions of continuous development-has been studied intensively and appears to involve short-range signaling and transmembrane receptor kinase activation. Finally, although high importance of control of cell division rates and planes for plant morphogenesis have been often inferred, recent genetic studies as well as comparative morphological data point to a less decisive role of cell division and to global controls of as yet unknown nature

The PLETHORA gene regulatory network guides growth and cell differentiation in Arabidopsis roots

Organ formation in animals and plants relies on precise control of cell state transitions to turn stem cell daughters into fully differentiated cells. In plants, cells cannot rearrange due to shared cell walls. Thus, differentiation progression and the accompanying cell expansion must be tightly coordinated across tissues. PLETHORA (PLT) transcription factor gradients are unique in their ability to guide the progression of cell differentiation at different positions in the growing Arabidopsis root, which contrasts with well-described transcription factor gradients in animals specifying distinct cell fates within an essentially static context. To understand the output of the PLT gradient, we studied the gene set transcriptionally controlled by PLTs. Our work reveals how the PLT gradient can regulate cell state by region-specific induction of cell proliferation genes and repression of differentiation. Moreover, PLT targets include major patterning genes and autoregulatory feedback components, enforcing their role as master regulators of organ development