Differential expansion and retention patterns of LRR-RLK genes across plant evolution.

To maximize overall fitness, plants must accurately respond to a host of growth, developmental, and environmental signals throughout their life. Many of these internal and external signals are perceived by the leucine-rich repeat receptor-like kinases, which play roles in regulating growth, development, and immunity. This largest family of receptor kinases in plants can be divided into subfamilies based on the conservation of the kinase domain, which demonstrates that shared evolutionary history often indicates shared molecular function. Here we investigate the evolutionary history of this family across the evolution of 112 plant species. We identify lineage-specific expansions of the malectin-domain containing subfamily LRR subfamily I primarily in the Brassicales and bryophytes. Most other plant lineages instead show a large expansion in LRR subfamily XII, which in Arabidopsis is known to contain key receptors in pathogen perception. This striking asymmetric expansion may reveal a dichotomy in the evolutionary history and adaptation strategies employed by plants. A greater understanding of the evolutionary pressures and adaptation strategies acting on members of this receptor family offers a way to improve functional predictions for orphan receptors and simplify the identification of novel stress-related receptors.

Map of physical interactions between extracellular domains of Arabidopsis leucine-rich repeat receptor kinases.

Plants use surface receptors to perceive information about many aspects of their local environment. These receptors physically interact to form both steady state and signalling competent complexes. The signalling events downstream of receptor activation impact both plant developmental and immune responses. Here, we present a comprehensive study of the physical interactions between the extracellular domains of leucine-rich repeat receptor kinases (LRR-RKs) in Arabidopsis. Using a sensitized assay, we tested reciprocal interactions among 200 of the 225 Arabidopsis LRR-RKs for a total search space of 40,000 interactions. Applying a stringent statistical cut-off and requiring that interactions performed well in both bait-prey and prey-bait orientations resulted in a high-confidence set of 567 bidirectional interactions. Additionally, we identified a total of 2,586 unidirectional interactions, which passed our stringent statistical cut-off in only one orientation. These datasets will guide further investigation into the regulatory roles of LRR-RKs in plant developmental and immune signalling decisions.

Publisher Correction: An extracellular network of Arabidopsis leucine-rich repeat receptor kinases.

In this Letter, an incorrect version of the Supplementary Information file was inadvertently used, which contained several errors. The details of references 59-65 were missing from the end of the Supplementary Discussion section on page 4. In addition, the section 'Text 3. Y2H on ICD interactions' incorrectly referred to 'Extended Data Fig. 4d' instead of 'Extended Data Fig. 3d' on page 3. Finally, the section 'Text 4. Interaction network analysis' incorrectly referred to 'Fig. 1b and Extended Data Fig. 6' instead of 'Fig. 2b and Extended Data Fig. 7' on page 3. These errors have all been corrected in the Supplementary Information.

An extracellular network of Arabidopsis leucine-rich repeat receptor kinases.

The cells of multicellular organisms receive extracellular signals using surface receptors. The extracellular domains (ECDs) of cell surface receptors function as interaction platforms, and as regulatory modules of receptor activation. Understanding how interactions between ECDs produce signal-competent receptor complexes is challenging because of their low biochemical tractability. In plants, the discovery of ECD interactions is complicated by the massive expansion of receptor families, which creates tremendous potential for changeover in receptor interactions. The largest of these families in Arabidopsis thaliana consists of 225 evolutionarily related leucine-rich repeat receptor kinases (LRR-RKs), which function in the sensing of microorganisms, cell expansion, stomata development and stem-cell maintenance. Although the principles that govern LRR-RK signalling activation are emerging, the systems-level organization of this family of proteins is unknown. Here, to address this, we investigated 40,000 potential ECD interactions using a sensitized high-throughput interaction assay, and produced an LRR-based cell surface interaction network (CSILRR) that consists of 567 interactions. To demonstrate the power of CSILRR for detecting biologically relevant interactions, we predicted and validated the functions of uncharacterized LRR-RKs in plant growth and immunity. In addition, we show that CSILRR operates as a unified regulatory network in which the LRR-RKs most crucial for its overall structure are required to prevent the aberrant signalling of receptors that are several network-steps away. Thus, plants have evolved LRR-RK networks to process extracellular signals into carefully balanced responses.

A High-Sensitivity, Microtiter-Based Plate Assay for Plant Pattern-Triggered Immunity.

The first step in the plant immune response to pathogen challenge involves the perception of conserved epitopes, called microbe-associated molecular patterns (MAMPs), by cell-surface pattern recognition receptors (PRRs). Given the key roles that MAMPs and PRRs play in plant innate immunity, great effort has been expended to identify these molecules. Current methods for assaying these immune responses are often limited in their resolution and throughput, and consequently, there is a need for medium- to high-throughput methodologies. Here, we describe the development of a 96-well microtiter plate-based assay for plant pattern-triggered immunity that measures the activity of plant peroxidase (POX) enzymes produced in response to treatment with bacterial MAMPs. The system has been optimized to minimize both the amount of plant tissue and MAMPs required and displays up to three orders of magnitude greater sensitivity than the traditional luminol-based reactive oxygen species assay when measuring the plant response to treatment with the bacterial MAMP flg22, reaching detection limits in the picomolar range. This high sensitivity opens the possibility of evaluating the immune-eliciting effects of weaker elicitors. The throughput and material requirements of the assay make it ideal for screens involving quantitative measurement of the plant innate immune response to MAMPs.

An Alternative Strategy for Trypanosome Survival in the Mammalian Bloodstream Revealed through Genome and Transcriptome Analysis of the Ubiquitous Bovine Parasite Trypanosoma (Megatrypanum) theileri.

There are hundreds of Trypanosoma species that live in the blood and tissue spaces of their vertebrate hosts. The vast majority of these do not have the ornate system of antigenic variation that has evolved in the small number of African trypanosome species, but can still maintain long-term infections in the face of the vertebrate adaptive immune system. Trypanosoma theileri is a typical example, has a restricted host range of cattle and other Bovinae, and is only occasionally reported to cause patent disease although no systematic survey of the effect of infection on agricultural productivity has been performed. Here, a detailed genome sequence and a transcriptome analysis of gene expression in bloodstream form T. theileri have been performed. Analysis of the genome sequence and expression showed that T. theileri has a typical kinetoplastid genome structure and allowed a prediction that it is capable of meiotic exchange, gene silencing via RNA interference and, potentially, density-dependent growth control. In particular, the transcriptome analysis has allowed a comparison of two distinct trypanosome cell surfaces, T. brucei and T. theileri, that have each evolved to enable the maintenance of a long-term extracellular infection in cattle. The T. theileri cell surface can be modeled to contain a mixture of proteins encoded by four novel large and divergent gene families and by members of a major surface protease gene family. This surface composition is distinct from the uniform variant surface glycoprotein coat on African trypanosomes providing an insight into a second mechanism used by trypanosome species that proliferate in an extracellular milieu in vertebrate hosts to avoid the adaptive immune response.

Genomic screens identify a new phytobacterial microbe-associated molecular pattern and the cognate Arabidopsis receptor-like kinase that mediates its immune elicitation.

BACKGROUND: The recognition of microbe-associated molecular patterns during infection is central to the mounting of an effective immune response. In spite of their importance, it remains difficult to identify these molecules and the host receptors required for their perception, ultimately limiting our understanding of the role of these molecules in the evolution of host-pathogen relationships. RESULTS: We employ a comparative genomics screen to identify six new immune eliciting peptides from the phytopathogenic bacterium Pseudomonas syringae. We then perform a reverse genetic screen to identify Arabidopsis thaliana leucine-rich repeat receptor-like kinases required for the recognition of these elicitors. We test the six elicitors on 187 receptor-like kinase knock-down insertion lines using a high-throughput peroxidase-based immune assay and identify multiple lines that show decreased immune responses to specific peptides. From this primary screen data, we focused on the interaction between the xup25 peptide from a bacterial xanthine/uracil permease and the Arabidopsis receptor-like kinase xanthine/uracil permease sensing 1; a family XII protein closely related to two well-characterized receptor-like kinases. We show that xup25 treatment increases pathogenesis-related gene induction, callose deposition, seedling growth inhibition, and resistance to virulent bacteria, all in a xanthine/uracil permease sensing 1-dependent manner. Finally, we show that this kinase-like receptor can bind the xup25 peptide directly. These results identify xup25 as a P. syringae microbe-associated molecular pattern and xanthine/uracil permease sensing 1 as a receptor-like kinase that detects the xup25 epitope to activate immune responses. CONCLUSIONS: The present study demonstrates an efficient method to identify immune elicitors and the plant receptors responsible for their perception. Further exploration of these molecules will increase our understanding of plant-pathogen interactions and the basis for host specificity.

Peptides and small molecules of the plant-pathogen apoplastic arena.

Plants reside within an environment rich in potential pathogens. Survival in the presence of such threats requires both effective perception of, and appropriate responses to, pathogenic attack. While plants lack an adaptive immune system, they have a highly developed and responsive innate immune system able to detect and inhibit the growth of the vast majority of potential pathogens. Many of the critical interactions that characterize the relationship between plants and pathogens are played out in the intercellular apoplastic space. The initial perception of pathogen invasion is often achieved through specific plant receptor-like kinases that recognize conserved molecular patterns presented by the pathogen or respond to the molecular debris caused by cellular damage. The perception of either microbial or damage signals by these receptors initiates a response that includes the production of peptides and small molecules to enhance cellular integrity and inhibit pathogen growth. In this review, we discuss the roles of apoplastic peptides and small molecules in modulating plant-pathogen interactions.

Immunomodulation by the Pseudomonas syringae HopZ type III effector family in Arabidopsis.

Pseudomonas syringae employs a type III secretion system to inject 20-30 different type III effector (T3SE) proteins into plant host cells. A major role of T3SEs is to suppress plant immune responses and promote bacterial infection. The YopJ/HopZ acetyltransferases are a superfamily of T3SEs found in both plant and animal pathogenic bacteria. In P. syringae, this superfamily includes the evolutionarily diverse HopZ1, HopZ2 and HopZ3 alleles. To investigate the roles of the HopZ family in immunomodulation, we generated dexamethasone-inducible T3SE transgenic lines of Arabidopsis for HopZ family members and characterized them for immune suppression phenotypes. We show that all of the HopZ family members can actively suppress various facets of Arabidopsis immunity in a catalytic residue-dependent manner. HopZ family members can differentially suppress the activation of mitogen-activated protein (MAP) kinase cascades or the production of reactive oxygen species, whereas all members can promote the growth of non-virulent P. syringae. Localization studies show that four of the HopZ family members containing predicted myristoylation sites are localized to the vicinity of the plasma membrane while HopZ3 which lacks the myristoylation site is at least partially nuclear localized, suggesting diversification of immunosuppressive mechanisms. Overall, we demonstrate that despite significant evolutionary diversification, all HopZ family members can suppress immunity in Arabidopsis.

Targeting cattle-borne zoonoses and cattle pathogens using a novel trypanosomatid-based delivery system.

Trypanosomatid parasites are notorious for the human diseases they cause throughout Africa and South America. However, non-pathogenic trypanosomatids are also found worldwide, infecting a wide range of hosts. One example is Trypanosoma (Megatrypanum) theileri, a ubiquitous protozoan commensal of bovids, which is distributed globally. Exploiting knowledge of pathogenic trypanosomatids, we have developed Trypanosoma theileri as a novel vehicle to deliver vaccine antigens and other proteins to cattle. Conditions for the growth and transfection of T. theileri have been optimised and expressed heterologous proteins targeted for secretion or specific localisation at the cell interior or surface using trafficking signals from Trypanosoma brucei. In cattle, the engineered vehicle could establish in the context of a pre-existing natural T. theileri population, was maintained long-term and generated specific immune responses to an expressed Babesia antigen at protective levels. Building on several decades of basic research into trypanosomatid pathogens, Trypanosoma theileri offers significant potential to target multiple infections, including major cattle-borne zoonoses such as Escherichia coli, Salmonella spp., Brucella abortus and Mycobacterium spp. It also has the potential to deliver therapeutics to cattle, including the lytic factor that protects humans from cattle trypanosomiasis. This could alleviate poverty by protecting indigenous African cattle from African trypanosomiasis.

A soluble factor from Trypanosoma cruzi inhibits transforming growth factor-ß-induced MAP kinase activation and gene expression in dermal fibroblasts.

The protozoan parasite Trypanosoma cruzi, which causes human Chagas' disease, exerts a variety of effects on host extracellular matrix (ECM) including proteolytic degradation of collagens and dampening of ECM gene expression. Exposure of primary human dermal fibroblasts to live infective T. cruzi trypomastigotes or their shed/secreted products results in a rapid down-regulation of the fibrogenic genes collagenIα1, fibronectin and connective tissue growth factor (CTGF/CCN2). Here we demonstrate the ability of a secreted/released T. cruzi factor to antagonize ctgf/ccn2 expression in dermal fibroblasts in response to TGF-ß, lysophosphatidic acid or serum, where agonist-induced phosphorylation of the mitogen-activated protein (MAP) kinases Erk1/2, p38 and JNK was also inhibited. Global analysis of gene expression in dermal fibroblasts identified a discrete subset of TGF-ß-inducible genes involved in cell proliferation, wound repair, and immune regulation that are inhibited by T. cruzi secreted/released factors, where the genes exhibiting the highest sensitivity to T. cruzi are known to be regulated by MAP kinase-activated transcription factors. Consistent with this observation, the Ets-family transcription factor binding site in the proximal promoter region of the ctgf/ccn2 gene (-91 bp to -84 bp) was shown to be required for T. cruzi-mediated down-regulation of ctgf/ccn2 reporter expression. The cumulative data suggest a model in which T. cruzi-derived molecules secreted/released early in the infective process dampen MAP kinase signaling and the activation of transcription factors that regulate expression of fibroblast genes involved in wound repair and tissue remodelling, including ctgf/ccn2. These findings have broader implications for local modulation of ECM synthesis/remodelling by T. cruzi during the early establishment of infection in the mammalian host and highlight the potential for pathogen-derived molecules to be exploited as tools to modulate the fibrogenic response.

The role of host cell lysosomes in Trypanosoma cruzi invasion.

The cell-invasive, trypomastigote form of Trypanosoma cruzi exhibits a unique relationship with lysosomes in target host cells. In contrast to many intracellular pathogens that are adept at avoiding contact with lysosomes, T. cruzi requires transient residence within this acidic organelle for productive infection. The low pH environment of lysosomes facilitates parasite egress from the vacuole and delivery into the host cytosol, a critical step in the T. cruzi developmental program. Recent studies also suggest that early lysosome fusion with invading or recently internalized parasites is critical for cellular retention of parasites. To ensure targeting to host cell lysosomes, T. cruzi trypomastigotes exploit two distinct modes of invasion that rapidly converge in the cell. In this chapter, we summarize the recent progress and changing views regarding the role of host cell lysosomes in the T. cruzi infection process where our discussion is limited to invasion of nonprofessional phagocytic cells.