Aspartate Oxidase Plays an Important Role in Arabidopsis Stomatal Immunity

Perception of pathogen-associated molecular patterns (PAMPs), such as bacterial flagellin (or the peptide flg22), by surface-localized receptors activates defense responses and subsequent immunity. In a previous forward-genetic screen aimed at the identification of Arabidopsis (Arabidopsis thaliana) flagellin-insensitive (fin) mutants, we isolated fin4, which is severely affected in flg22-triggered reactive oxygen species (ROS) bursts. Here, we report that FIN4 encodes the chloroplastic enzyme ASPARTATE OXIDASE (AO), which catalyzes the first irreversible step in the de novo biosynthesis of NAD. Genetic studies on the role of NAD have been hindered so far by the lethality of null mutants in NAD biosynthetic enzymes. Using newly identified knockdown fin alleles, we found that AO is required for the ROS burst mediated by the NADPH oxidase RBOHD triggered by the perception of several unrelated PAMPs. AO is also required for RBOHD-dependent stomatal closure. However, full AO activity is not required for flg22-induced responses that are RBOHD independent. Interestingly, although the fin4 mutation dramatically affects RBOHD function, it does not affect functions carried out by other members of the RBOH family, such as RBOHC and RBOHF. Finally, we determined that AO is required for stomatal immunity against the bacterium Pseudomonas syringae. Altogether, our work reveals a novel specific requirement for AO activity in PAMP-triggered RBOHD-dependent ROS burst and stomatal immunity. In addition, the availability of viable mutants for the chloroplastic enzyme AO will enable future detailed studies on the role of NAD metabolism in different cellular processes, including immunity, in Arabidopsis

An evolutionary conserved region (ECR) in the human dopamine receptor D4 gene supports reporter gene expression in primary cultures derived from the rat cortex

<p>Abstract</p> <p>Background</p> <p>Detecting functional variants contributing to diversity of behaviour is crucial for dissecting genetics of complex behaviours. At a molecular level, characterisation of variation in exons has been studied as they are easily identified in the current genome annotation although the functional consequences are less well understood; however, it has been difficult to prioritise regions of non-coding DNA in which genetic variation could also have significant functional consequences. Comparison of multiple vertebrate genomes has allowed the identification of non-coding evolutionary conserved regions (ECRs), in which the degree of conservation can be comparable with exonic regions suggesting functional significance.</p> <p>Results</p> <p>We identified ECRs at the dopamine receptor D4 gene locus, an important gene for human behaviours. The most conserved non-coding ECR (D4ECR1) supported high reporter gene expression in primary cultures derived from neonate rat frontal cortex. Computer aided analysis of the sequence of the D4ECR1 indicated the potential transcription factors that could modulate its function. D4ECR1 contained multiple consensus sequences for binding the transcription factor Sp1, a factor previously implicated in DRD4 expression. Co-transfection experiments demonstrated that overexpression of Sp1 significantly decreased the activity of the D4ECR1 <it>in vitro</it>.</p> <p>Conclusion</p> <p>Bioinformatic analysis complemented by functional analysis of the DRD4 gene locus has identified a) a strong enhancer that functions in neurons and b) a transcription factor that may modulate the function of that enhancer.</p

The transcriptional regulator BZR1 mediates trade-off between plant innate immunity and growth

The molecular mechanisms underlying the trade-off between plant innate immunity and steroid-mediated growth are controversial. Here, we report that activation of the transcription factor BZR1 is required and sufficient for suppression of immune signaling by brassinosteroids (BR). BZR1 induces the expression of several WRKY transcription factors that negatively control early immune responses. In addition, BZR1 associates with WRKY40 to mediate the antagonism between BR and immune signaling. We reveal that BZR1-mediated inhibition of immunity is particularly relevant when plant fast growth is required, such as during etiolation. Thus, BZR1 acts as an important regulator mediating the trade-off between growth and immunity upon integration of environmental cues

Competitive Index: Mixed Infection-Based Virulence Assays for Genetic Analysis in Pseudomonas syringae-Plant Interactions.

When studying bacterial plant pathogens, the genetic analysis of the contribution of virulence factors to the infection process has traditionally been hindered by their high degree of functional redundancy. In recent years, it has become clear that the use of competitive index in mixed infections provides an accurate and sensitive manner of establishing virulence phenotypes for mutants for which other assays have failed. Such increases in sensitivity and accuracy are due to the direct comparison between the respective growths of the co-inoculated strains within the same infection, each strain replicating as they would in individual infections. Interferences between the co-inoculated strains must be therefore avoided using the appropriate experimental settings. In this chapter, we will present the optimal experimental conditions to achieve maximum sensitivity on virulence assays using the phytopathogenic bacterium Pseudomonas syringae, as well as some additional considerations to ensure the correct interpretations of the results

The C4 protein from Tomato yellow leaf curl virus can broadly interact with Plant receptor-like kinases

Plant receptor-like kinases (RLKs) exert an essential function in the transduction of signals from the cell exterior to the cell interior, acting as important regulators of plant development and responses to environmental conditions. A growing body of evidence suggests that RLKs may play relevant roles in plant-virus interactions, although the details and diversity of effects and underlying mechanisms remain elusive. The C4 protein from different geminiviruses has been found to interact with RLKs in the CLAVATA 1 (CLV1) clade. However, whether C4 can interact with RLKs in other subfamilies and, if so, what the biological impact of such interactions might be, is currently unknown. In this work, we explore the interaction landscape of C4 from the geminivirus Tomato yellow leaf curl virus within the Arabidopsis RLK family. Our results show that C4 can interact with RLKs from different subfamilies including, but not restricted to, members of the CLV1 clade. Functional analyses of the interaction of C4 with two well-characterized RLKs, FLAGELLIN SENSING 2 (FLS2) and BRASSINOSTEROID INSENSITIVE 1 (BRI1), indicate that C4 might affect some, but not all, RLK-derived outputs. The results presented here offer novel insight on the interface between RLK signaling and the infection by geminiviruses, and point at C4 as a potential broad manipulator of RLK-mediated signaling

The C2 Protein from the Geminivirus Tomato Yellow Leaf Curl Sardinia Virus Decreases Sensitivity to Jasmonates and Suppresses Jasmonate-Mediated Defences

An increasing body of evidence points at a role of the plant hormones jasmonates (JAs) in determining the outcome of plant-virus interactions. Geminiviruses, small DNA viruses infecting a wide range of plant species worldwide, encode a multifunctional protein, C2, which is essential for full pathogenicity. The C2 protein has been shown to suppress the JA response, although the current view on the extent of this effect and the underlying molecular mechanisms is incomplete. In this work, we use a combination of exogenous hormone treatments, microarray analysis, and pathogen infections to analyze, in detail, the suppression of the JA response exerted by C2. Our results indicate that C2 specifically affects certain JA-induced responses, namely defence and secondary metabolism, and show that plants expressing C2 are more susceptible to pathogen attack. We propose a model in which C2 might interfere with the JA response at several levels

Application of an improved global-scale groundwater model for water table estimation across New Zealand

Many studies underline the importance of groundwater assessment at the larger, i.e. global, scale. The groundwater models used for these assessments are dedicated to the global scale and therefore not often applied for studies in smaller areas, e.g. catchments, because of their simplifying assumptions. In New Zealand, advanced numerical groundwater flow models have been applied in several catchments. However, that application is piecemeal: only for a limited amount of aquifers and through a variety of groundwater model suites, formats, and developers. Additionally, there are large areas where groundwater models and data are sparse. Hence, an inter-catchment, inter-regional, or nationwide overview of important groundwater information, such as the water table, does not exist. The investment needed to adequately cover New Zealand with high-resolution groundwater models in a consistent approach would be significant and is therefore not considered possible at this stage. This study proposes a solution that obtains a nationwide overview of groundwater that bridges the gap between the (too-)expensive advanced local models and the (too-)simple global-scale models. We apply an existing, global-scale, groundwater flow model and improve it by feeding in national input data of New Zealand terrain, geology, and recharge, and by slight adjustment of model parametrisation and model testing. The resulting nationwide maps of hydraulic head and water table depths show that the model points out the main alluvial aquifers with fine spatial detail (200&thinsp;m grid resolution). The national input data and finer spatial detail result in better and more realistic variations of water table depth than the original, global-scale, model outputs. In two regional case studies in New Zealand, the hydraulic head shows excellent correlation with the available groundwater level data. Sensitivity and other analyses of our nationwide water tables show that the model is mostly driven by recharge, model resolution, and elevation (gravity), and impeded by the geology (permeability). The use of this first dedicated New Zealand-wide model can aid in provision of water table estimates in data-sparse regions. The national model can also be used to solve inconsistency of models in areas of trans-boundary aquifers, i.e. aquifers that cover more than one region in New Zealand. Comparison of the models, i.e. the national application (National Water Table model: NWT) with the global model (Equilibrium Water Table model: EWT), shows that most improvement is achieved by feeding in better and higher-resolution input data. The NWT model still has a bias towards shallow water tables (but less than the EWT model because of the finer model resolution), which could only be solved by feeding in a very high resolution terrain model that incorporates drainage features. Although this is a model shortcoming, it can also be viewed as a valuable indicator of the pre-human water table, i.e. before 90&thinsp;% of wetlands were drained for agriculture since European settlement in New Zealand. Calibration to ground-observed water level improves model results but can of course only work where there are such data available. Future research should therefore focus on both model improvements and more data-driven, improved estimation of hydraulic conductivity, recharge, and the digital elevation model. We further surmise that the findings of this study, i.e. successful application of a global-scale model at smaller scales, will lead to subsequent improvement of the global-scale model equations.</p