Symbiotic outcome modified by the diversification from 7 to over 700 nodule specific cysteine rich peptides

Legume-rhizobium symbiosis represents one of the most successfully co-evolved mutualisms. Within nodules, the bacterial cells undergo distinct metabolic and morphological changes and differentiate into nitrogen-fixing bacteroids. Legumes in the inverted repeat lacking clade (IRLC) employ an array of defensin-like small secreted peptides (SSPs), known as nodule-specific cysteine-rich (NCR) peptides, to regulate bacteroid differentiation and activity. While most NCRs exhibit bactericidal effects in vitro, studies confirm that inside nodules they target the bacterial cell cycle and other cellular pathways to control and extend rhizobial differentiation into an irreversible (or terminal) state where the host gains control over bacteroids. While NCRs are well established as positive regulators of effective symbiosis, more recent findings also suggest that NCRs affect partner compatibility. The extent of bacterial differentiation has been linked to species-specific size and complexity of the NCR gene family that varies even among closely related species, suggesting a more recent origin of NCRs followed by rapid expansion in certain species. NCRs have diversified functionally, as well as in their expression patterns and responsiveness, likely driving further functional specialisation. In this review, we evaluate the functions of NCR peptides and their role as a driving force underlying the outcome of rhizobial symbiosis, where the plant is able to determine the outcome of rhizobial interaction in a temporal and spatial manner

Cell specific analysis of Arabidopsis leaves using fluorescence activated cell sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated. To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6. FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence

Spatial dissection of the Arabidopsis thaliana transcriptional response to downy mildew using fluorescence activated cell sorting

Changes in gene expression form a crucial part of the plant response to infection. In the last decade, whole-leaf expression profiling has played a valuable role in identifying genes and processes that contribute to the interactions between the model plant Arabidopsis thaliana and a diverse range of pathogens. However, with some pathogens such as downy mildew caused by the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis (Hpa), whole-leaf profiling may fail to capture the complete Arabidopsis response encompassing responses of non-infected as well as infected cells within the leaf. Highly localized expression changes that occur in infected cells may be diluted by the comparative abundance of non-infected cells. Furthermore, local and systemic Hpa responses of a differing nature may become conflated. To address this we applied the technique of Fluorescence Activated Cell Sorting (FACS), typically used for analyzing plant abiotic responses, to the study of plant-pathogen interactions. We isolated haustoriated (Hpa-proximal) and non-haustoriated (Hpa-distal) cells from infected seedling samples using FACS, and measured global gene expression. When compared with an uninfected control, 278 transcripts were identified as significantly differentially expressed, the vast majority of which were differentially expressed specifically in Hpa-proximal cells. By comparing our data to previous, whole organ studies, we discovered many highly locally regulated genes that can be implicated as novel in the Hpa response, and that were uncovered for the first time using our sensitive FACS technique

Framework for quantification of the dynamics of root colonization by pseudomonas fluorescens isolate SBW25

Colonization of the root surface, or rhizoplane, is one of the first steps for soil-borne bacteria to become established in the plant microbiome. However, the relative contributions of processes, such as bacterial attachment and proliferation is not well characterized, and this limits our ability to comprehend the complex dynamics of microbial communities in the rhizosphere. The work presented here addresses this knowledge gap. A model system was developed to acquire quantitative data on the colonization process of lettuce (Lactuca sativa L. cultivar. All Year Round) roots by Pseudomonas fluorescens isolate SBW25. A theoretical framework is proposed to calculate attachment rate and quantify the relative contribution of bacterial attachment to colonization. This allows the assessment of attachment rates on the root surface beyond the short time period during which it can be quantified experimentally. All techniques proposed are generic and similar analyses could be applied to study various combinations of plants and bacteria, or to assess competition between species. In the future this could allow for selection of microbial traits that improve early colonization and maintenance of targeted isolates in cropping systems, with potential applications for the development of biological fertilizers

Determinants of host range specificity in Legume-Rhizobia symbiosis

Leguminous plants possess the almost unique ability to enter symbiosis with soil-resident, nitrogen fixing bacteria called rhizobia. During this symbiosis, the bacteria physically colonize specialized organs on the roots of the host plant called nodules, where they reduce atmospheric nitrogen into forms that can be assimilated by the host plant and receive photosynthates in return. In order for nodule development to occur, there is extensive chemical cross-talk between both parties during the formative stages of the symbiosis. The vast majority of the legume family are capable of forming root nodules and typically rhizobia are only able to fix nitrogen within the context of this symbiotic association. However, many legume species only enter productive symbiosis with a few, or even single rhizobial species or strains, and vice-versa. Permitting symbiosis with only rhizobial strains that will be able to fix nitrogen with high efficiency is a crucial strategy for the host plant to prevent cheating by rhizobia. This selectivity is enforced at all stages of the symbiosis, with partner choice beginning during the initial communication between the plant and rhizobia. However, it can also be influenced even once nitrogen-fixing nodules have developed on the root. This review sets out current knowledge about the molecular mechanisms employed by both parties to influence host range during legume-rhizobia symbiosis

Ca2+ signatures in symbiosis : another level of dynamism for this key messenger

This article comments on: Binci F, Offer E, Crosino A, Sciascia I, Kleine-Vehn J, Genre A, Giovannetti M, Navazio L. 2024. Spatially and temporally distinct Ca2+ changes in Lotus japonicus roots orient fungal-triggered signalling pathways towards symbiosis or immunity. Journal of Experimental Botany 75,605–619

Plant–environment response pathway regulation uncovered by investigating non-typical legume symbiosis and nodulation

Nitrogen is an essential element needed for plants to survive, and legumes are well known to recruit rhizobia to fix atmospheric nitrogen. In this widely studied symbiosis, legumes develop specific structures on the roots to host specific symbionts. This review explores alternate nodule structures and their functions outside of the more widely studied legume–rhizobial symbiosis, as well as discussing other unusual aspects of nodulation. This includes actinorhizal-Frankia, cycad-cyanobacteria, and the non-legume Parasponia andersonii-rhizobia symbioses. Nodules are also not restricted to the roots, either, with examples found within stems and leaves. Recent research has shown that legume–rhizobia nodulation brings a great many other benefits, some direct and some indirect. Rhizobial symbiosis can lead to modifications in other pathways, including the priming of defence responses, and to modulated or enhanced resistance to biotic and abiotic stress. With so many avenues to explore, this review discusses recent discoveries and highlights future directions in the study of nodulation

Root architecture and rhizosphere–microbe interactions

Plant roots fulfil crucial tasks during a plant’s life. As roots encounter very diverse conditions while exploring the soil for resources, their growth and development must be responsive to changes in the rhizosphere, resulting in root architectures that are tailor-made for all prevailing circumstances. Using multi-disciplinary approaches, we are gaining more intricate insights into the regulatory mechanisms directing root system architecture. This Special Issue provides insights into our advancement of knowledge on different aspects of root development and identifies opportunities for future research

Legal Rights and Issues Surrounding Conception, Pregnancy, and Birth

Advances in medicine are reported almost daily in the media. Medical researchers have developed and are continuing to develop new methods of creating, saving, and prolonging life. This Special Project examines the impact that rapidly advancing medical technology has on the law governing conception, pregnancy, and birth. Although medical techniques have advanced rapidly during the past decades, state and federal legislatures have responded in-adequately to the legal consequences of these new birth technologies. The resulting lag between technology and the law has forced courts to confront new situations that do not fit neatly into the statutory framework created to deal with past fact situations. For example, courts have applied statutes prohibiting child bartering to surrogate parenting cases and statutes prohibiting fetal experimentation to artificial insemination cases although it is clear that the legislators never considered such fact patterns when passing the statutes. A lag is inevitable because the law can only respond to, rather than predict, emerging medical developments. Nonetheless, legislators must respond promptly by confronting the new legal issues that result from new medical technologies. One impediment to prompt legislative response to the lag between medical technology and the law is the controversial nature of the legal problems posed. Abortion continues to be an extremely controversial issue thirteen years after the Supreme Court legalized it in the landmark decision Roe v. Wade. The Baby Doe issue of whether to force hospitals and parents of severely deformed newborns to provide medical care is another extremely controversial issue. Baby Doe has become highly politicized as the Reagan administration, Congress, right-to-life groups, disability groups, medical professionals, and other groups have taken stances. Surrogate parenting also has produced controversial situations. In one incident, a New York couple contracted with a California surrogate mother. When the surrogate mother breached the agreement, the couple brought suit. The court discovered that the couple consisted of a man and a transsexual, thus raising the issue of whether transsexuals or homosexuals should be allowed to adopt children by contracting with surrogate mothers