WRKY48 negatively regulates plant acclimation to a combination of high light and heat stress.

Plants growing under natural conditions experience high light (HL) intensities that are often accompanied by elevated temperatures. These conditions could affect photosynthesis, reduce yield, and negatively impact agricultural productivity. The combination of different abiotic challenges creates a new type of stress for plants by generating complex environmental conditions that often exceed the impact of their individual parts. Transcription factors (TFs) play a key role in integrating the different molecular signals generated by multiple stress conditions, orchestrating the acclimation response of plants to stress. In this study, we show that the TF WRKY48 negatively controls the acclimation of Arabidopsis thaliana plants to a combination of HL and heat stress (HL + HS), and its expression is attenuated by jasmonic acid under HL + HS conditions. Using comparative physiological and transcriptomic analyses between wild-type and wrky48 mutants, we further demonstrate that under control conditions, WRKY48 represses the expression of a set of transcripts that are specifically required for the acclimation of plants to HL + HS, hence its suppression during the HL + HS stress combination contributes to plant survival under these conditions. Accordingly, mutants that lack WRKY48 are more resistant to HL + HS, and transgenic plants that overexpress WRKY48 are more sensitive to it. Taken together, our findings reveal that WRKY48 is a negative regulator of the transcriptomic response of Arabidopsis to HL + HS and provide new insights into the complex regulatory networks of plant acclimation to stress combination.

Diagnostic Utility of Genome-wide DNA Methylation Analysis in Genetically Unsolved Developmental and Epileptic Encephalopathies and Refinement of a CHD2 Episignature.

Sequence-based genetic testing currently identifies causative genetic variants in ∼50% of individuals with developmental and epileptic encephalopathies (DEEs). Aberrant changes in DNA methylation are implicated in various neurodevelopmental disorders but remain unstudied in DEEs. Rare epigenetic variations ("epivariants") can drive disease by modulating gene expression at single loci, whereas genome-wide DNA methylation changes can result in distinct "episignature" biomarkers for monogenic disorders in a growing number of rare diseases. Here, we interrogate the diagnostic utility of genome-wide DNA methylation array analysis on peripheral blood samples from 516 individuals with genetically unsolved DEEs who had previously undergone extensive genetic testing. We identified rare differentially methylated regions (DMRs) and explanatory episignatures to discover causative and candidate genetic etiologies in 10 individuals. We then used long-read sequencing to identify DNA variants underlying rare DMRs, including one balanced translocation, three CG-rich repeat expansions, and two copy number variants. We also identify pathogenic sequence variants associated with episignatures; some had been missed by previous exome sequencing. Although most DEE genes lack known episignatures, the increase in diagnostic yield for DNA methylation analysis in DEEs is comparable to the added yield of genome sequencing. Finally, we refine an episignature for CHD2 using an 850K methylation array which was further refined at higher CpG resolution using bisulfite sequencing to investigate potential insights into CHD2 pathophysiology. Our study demonstrates the diagnostic yield of genome-wide DNA methylation analysis to identify causal and candidate genetic causes as ∼2% (10/516) for unsolved DEE cases.

Therapeutic potential of rWnt5A in curbing Leishmania donovani infection.

In view of the antagonism of Wnt5A signaling toward microbial pathogens, we were interested in evaluating the therapeutic potential of recombinant Wnt5A (rWnt5A) in curbing Leishmania donovani infection. Initially, using L. donovani-infected RAW 264.7 and peritoneal macrophages, we demonstrated that application of rWnt5A as opposed to the vehicle control to the infected cells significantly dampens L. donovani infection. Inhibition of infection was associated with increase in cell-associated reactive oxygen species (ROS), and blocked by the ROS production inhibitor diphenylene iodonium chloride (DPI). rWnt5A, but not the vehicle control (PBS: phosphate-buffered saline) administration to L. donovani-infected mice appreciably reduced the infection load, and inhibited disease progression as evident from the preservation of splenic white pulp architecture. rWnt5A administration, moreover, led to elevation of both cell-associated ROS and the activation of splenic T cells. Substantial increase in T cell-associated Interleukin-2 (IL-2) and Granzyme B (GRB) upon exposure of splenic lymphocytes harvested from rWnt5A-treated mice to L. donovani-infected RAW 264.7 macrophages in vitro validated the occurrence of L. donovani-responsive T cell activation in vivo. In summary, this study unveils the therapeutic potential of rWnt5A in curbing L. donovani infection and the progression of experimental visceral leishmaniasis possibly through increase in cellular ROS and T cell activation. Accordingly, it opens up a new avenue of investigation into the use of rWnt5A as a therapeutic agent for restraining the progression of drug-resistant L. donovani infection.

Leveraging comparative genomics to uncover alien genes in bacterial genomes.

A significant challenge in bacterial genomics is to catalogue genes acquired through the evolutionary process of horizontal gene transfer (HGT). Both comparative genomics and sequence composition-based methods have often been invoked to quantify horizontally acquired genes in bacterial genomes. Comparative genomics methods rely on completely sequenced genomes and therefore the confidence in their predictions increases as the databases become more enriched in completely sequenced genomes. Recent developments including in microbial genome sequencing call for reassessment of alien genes based on information-rich resources currently available. We revisited the comparative genomics approach and developed a new algorithm for alien gene detection. Our algorithm compared favourably with the existing comparative genomics-based methods and is capable of detecting both recent and ancient transfers. It can be used as a standalone tool or in concert with other complementary algorithms for comprehensively cataloguing alien genes in bacterial genomes.

Phytochrome B regulates reactive oxygen signaling during abiotic and biotic stress in plants.

Reactive oxygen species (ROS) and the photoreceptor protein phytochrome B (phyB) play a key role in plant acclimation to stress. However, how phyB that primarily functions in the nuclei impacts ROS signaling mediated by respiratory burst oxidase homolog (RBOH) proteins that reside on the plasma membrane, during stress, is unknown. Arabidopsis thaliana and Oryza sativa mutants, RNA-Seq, bioinformatics, biochemistry, molecular biology, and whole-plant ROS imaging were used to address this question. Here, we reveal that phyB and RBOHs function as part of a key regulatory module that controls apoplastic ROS production, stress-response transcript expression, and plant acclimation in response to excess light stress. We further show that phyB can regulate ROS production during stress even if it is restricted to the cytosol and that phyB, respiratory burst oxidase protein D (RBOHD), and respiratory burst oxidase protein F (RBOHF) coregulate thousands of transcripts in response to light stress. Surprisingly, we found that phyB is also required for ROS accumulation in response to heat, wounding, cold, and bacterial infection. Our findings reveal that phyB plays a canonical role in plant responses to biotic and abiotic stresses, regulating apoplastic ROS production, possibly while at the cytosol, and that phyB and RBOHD/RBOHF function in the same regulatory pathway.

Wnt5A Signaling Regulates Gut Bacterial Survival and T Cell Homeostasis.

In light of the demonstrated antagonism of Wnt5A signaling toward the growth of several bacterial pathogens, it was important to study the influence of Wnt5A on gut-resident bacteria and its outcome. Here, we demonstrate that in contrast to inhibiting the survival of the established gut pathogen Salmonella enterica, Wnt5A clearly promotes the survival of the common gut commensals Enterococcus faecalis and Lactobacillus rhamnosus within macrophages through a self-perpetuating Wnt5A-actin axis. A Wnt5A-actin axis furthermore regulates the subsistence of the natural bacterial population of the Peyer's patches, as is evident from the diminution in the countable bacterial CFU therein through the application of Wnt5A signaling and actin assembly inhibitors. Wnt5A dependency of the gut-resident bacterial population is also manifested in the notable difference between the bacterial diversities associated with the feces and Peyer's patches of Wnt5A heterozygous mice, which lack a functional copy of the Wnt5A gene, and their wild-type counterparts. Alterations in the gut commensal bacterial population resulting from either the lack of a copy of the Wnt5A gene or inhibitor-mediated attenuation of Wnt5A signaling are linked with significant differences in cell surface major histocompatibility complex (MHC) II levels and regulatory versus activated CD4 T cells associated with the Peyer's patches. Taken together, our findings reveal the significance of steady state Wnt5A signaling in shaping the gut commensal bacterial population and the T cell repertoire linked to it, thus unveiling a crucial control device for the maintenance of gut bacterial diversity and T cell homeostasis. IMPORTANCE Gut commensal bacterial diversity and T cell homeostasis are crucial entities of the host innate immune network, yet the molecular details of host-directed signaling pathways that sustain the steady state of gut bacterial colonization and T cell activation remain unclear. Here, we describe the protective role of a Wnt5A-actin axis in the survival of several gut bacterial commensals and its necessity in shaping gut bacterial colonization and the associated T cell repertoire. This study opens up new avenues of investigation into the role of the Wnt5A-actin axis in protection of the gut from dysbiosis-related inflammatory disorders.

Reconstructing horizontal gene flow network to understand prokaryotic evolution.

Horizontal gene transfer (HGT) is a major source of phenotypic innovation and a mechanism of niche adaptation in prokaryotes. Quantification of HGT is critical to decipher its myriad roles in microbial evolution and adaptation. Advances in genome sequencing and bioinformatics have augmented our ability to understand the microbial world, particularly the direct or indirect influence of HGT on diverse life forms. Methods for detecting HGT can be classified into phylogenetic-based and parametric or composition-based approaches. Here, we exploited the complementary strengths of both the approaches to construct a high confidence horizontal gene flow network. Our network is unique in its ability to detect the transfer of native genes of a genome to genomes from other taxa, thus establishing donor and recipient organisms (taxa), rather than through a post hoc analysis as is the practice with several other approaches. The scale-free horizontal gene flow network presented here provides new insights into modes of transfer for the exchange of genetic information and also illuminates differential gene flow across phyla.

The Arabidopsis gene co-expression network.

Identifying genes that interact to confer a biological function to an organism is one of the main goals of functional genomics. High-throughput technologies for assessment and quantification of genome-wide gene expression patterns have enabled systems-level analyses to infer pathways or networks of genes involved in different functions under many different conditions. Here, we leveraged the publicly available, information-rich RNA-Seq datasets of the model plant Arabidopsis thaliana to construct a gene co-expression network, which was partitioned into clusters or modules that harbor genes correlated by expression. Gene ontology and pathway enrichment analyses were performed to assess functional terms and pathways that were enriched within the different gene modules. By interrogating the co-expression network for genes in different modules that associate with a gene of interest, diverse functional roles of the gene can be deciphered. By mapping genes differentially expressing under a certain condition in Arabidopsis onto the co-expression network, we demonstrate the ability of the network to uncover novel genes that are likely transcriptionally active but prone to be missed by standard statistical approaches due to their falling outside of the confidence zone of detection. To our knowledge, this is the first A. thaliana co-expression network constructed using the entire mRNA-Seq datasets (>20,000) available at the NCBI SRA database. The developed network can serve as a useful resource for the Arabidopsis research community to interrogate specific genes of interest within the network, retrieve the respective interactomes, decipher gene modules that are transcriptionally altered under certain condition or stage, and gain understanding of gene functions.

A Protocol for Phylogenetic Reconstruction.

The similarity of biological functions and molecular mechanisms in living organisms suggests their common origin. The inference of evolutionary relationships among the extant organisms is primarily based on structural, functional, and sequence data of biomolecules, such as DNA, RNA, and protein, and their relative changes over the course of time. To decipher evolutionary relationships, a variety of data can be used. The exponential growth of genomic data, spurred by advances in DNA sequencing, has enabled biologists to reconstruct the tree or network of life for a vast number of organisms dwelling in the earth. In addition of organismal relationships, phylogenetic analysis is often performed to characterize gene families, specifically to identify the orthologs and paralogs of a gene of interest and understand their varied functions in light of evolution. In this chapter, we describe a protocol for reconstructing a phylogenetic tree using maximum-likelihood approach. We demonstrate using an example dataset and a suite of publicly available programs.

The impact of multifactorial stress combination on plant growth and survival.

Climate change-driven extreme weather events, combined with increasing temperatures, harsh soil conditions, low water availability and quality, and the introduction of many man-made pollutants, pose a unique challenge to plants. Although our knowledge of the response of plants to each of these individual conditions is vast, we know very little about how a combination of many of these factors, occurring simultaneously, that is multifactorial stress combination, impacts plants. Seedlings of wild-type and different mutants of Arabidopsis thaliana plants were subjected to a multifactorial stress combination of six different stresses, each applied at a low level, and their survival, physiological and molecular responses determined. Our findings reveal that, while each of the different stresses, applied individually, had a negligible effect on plant growth and survival, the accumulated impact of multifactorial stress combination on plants was detrimental. We further show that the response of plants to multifactorial stress combination is unique and that specific pathways and processes play a critical role in the acclimation of plants to multifactorial stress combination. Taken together our findings reveal that further polluting our environment could result in higher complexities of multifactorial stress combinations that in turn could drive a critical decline in plant growth and survival.

The impact of water deficit and heat stress combination on the molecular response, physiology, and seed production of soybean.

A combination of drought and heat stress, occurring at the vegetative or reproductive growth phase of many different crops can have a devastating impact on yield. In soybean (Glycine max), a considerable effort has been made to develop genotypes with enhanced yield production under conditions of drought or heat stress. However, how these genotypes perform in terms of growth, physiological responses, and most importantly seed production, under conditions of drought and heat combination is mostly unknown. Here, we studied the impact of water deficit and heat stress combination on the physiology, seed production, and yield per plant of two soybean genotypes, Magellan and Plant Introduction (PI) 548313, that differ in their reproductive responses to heat stress. Our findings reveal that although PI 548313 produced more seeds than Magellan under conditions of heat stress, under conditions of water deficit, and heat stress combination its seed production decreased. Because the number of flowers and pollen germination of PI 548313 remained high under heat or water deficit and heat combination, the reduced seed production exhibited by PI 548313 under the stress combination could be a result of processes that occur at the stigma, ovaries and/or other parts of the flower following pollen germination.

MYB30 Orchestrates Systemic Reactive Oxygen Signaling and Plant Acclimation.

Systemic acquired acclimation (SAA) is a key biological process essential for plant survival under conditions of abiotic stress. SAA was recently shown to be controlled by a rapid systemic signaling mechanism termed the reactive oxygen species (ROS) wave in Arabidopsis (Arabidopsis thaliana). MYB30 is a key transcriptional regulator mediating many different biological processes. MYB30 was found to act downstream of the ROS wave in systemic tissues of Arabidopsis in response to local high light (HL) stress treatment. However, the function of MYB30 in systemic signaling and SAA is unknown. To determine the relationship among MYB30, the ROS wave, and systemic acclimation in Arabidopsis, the SAA response to HL stress of myb30 mutants and wild-type plants was determined. Although myb30 plants were found to display enhanced rates of ROS wave propagation and their local tissues acclimated to the HL stress, they were deficient in SAA to HL stress. Compared to wild type, the systemic transcriptomic response of myb30 plants was also deficient, lacking in the expression of over 3,500 transcripts. A putative set of 150 core transcripts directly associated with MYB30 function during HL stress was determined. Our study identifies MYB30 as a key regulator that links systemic ROS signaling with systemic transcriptomic responses, SAA, and plant acclimation to HL stress. In addition, it demonstrates that plant acclimation and systemic ROS signaling are interlinked and that the lack of systemic acclimation drives systemic ROS signaling to occur at faster rates, suggesting a feedback mechanism (potentially involving MYB30) between these two processes.

Systemic signaling during abiotic stress combination in plants.

Extreme environmental conditions, such as heat, salinity, and decreased water availability, can have a devastating impact on plant growth and productivity, potentially resulting in the collapse of entire ecosystems. Stress-induced systemic signaling and systemic acquired acclimation play canonical roles in plant survival during episodes of environmental stress. Recent studies revealed that in response to a single abiotic stress, applied to a single leaf, plants mount a comprehensive stress-specific systemic response that includes the accumulation of many different stress-specific transcripts and metabolites, as well as a coordinated stress-specific whole-plant stomatal response. However, in nature plants are routinely subjected to a combination of two or more different abiotic stresses, each potentially triggering its own stress-specific systemic response, highlighting a new fundamental question in plant biology: are plants capable of integrating two different systemic signals simultaneously generated during conditions of stress combination? Here we show that plants can integrate two different systemic signals simultaneously generated during stress combination, and that the manner in which plants sense the different stresses that trigger these signals (i.e., at the same or different parts of the plant) makes a significant difference in how fast and efficient they induce systemic reactive oxygen species (ROS) signals; transcriptomic, hormonal, and stomatal responses; as well as plant acclimation. Our results shed light on how plants acclimate to their environment and survive a combination of different abiotic stresses. In addition, they highlight a key role for systemic ROS signals in coordinating the response of different leaves to stress.

Wnt5A-Mediated Actin Organization Regulates Host Response to Bacterial Pathogens and Non-Pathogens.

Wnt5A signaling facilitates the killing of several bacterial pathogens, but not the non-pathogen E. coli DH5α. The basis of such pathogen vs. non-pathogen distinction is unclear. Accordingly, we analyzed the influence of Wnt5A signaling on pathogenic E. coli K1 in relation to non-pathogenic E. coli K12-MG1655 and E. coli DH5α eliminating interspecies variability from our study. Whereas cell internalized E. coli K1 disrupted cytoskeletal actin organization and multiplied during Wnt5A depletion, rWnt5A mediated activation revived cytoskeletal actin assembly facilitating K1 eradication. Cell internalized E. coli K12-MG1655 and E. coli DH5α, which did not perturb actin assembly appreciably, remained unaffected by rWnt5A treatment. Phagosomes prepared separately from Wnt5A conditioned medium treated K1 and K12-MG1655 infected macrophages revealed differences in the relative levels of actin and actin network promoting proteins, upholding that the Wnt5A-Actin axis operates differently for internalized pathogen and non-pathogen. Interestingly, exposure of rWnt5A treated K1 and K12-MG1655/DH5α infected macrophages to actin assembly inhibitors reversed the scenario, blocking killing of K1, yet promoting killing of both K12-MG1655 and DH5α. Taken together, our study illustrates that the state of activation of the Wnt5A/Actin axis in the context of the incumbent bacteria is crucial for directing host response to infection.

Expression of a dominant-negative AtNEET-H89C protein disrupts iron-sulfur metabolism and iron homeostasis in Arabidopsis.

Iron-sulfur (Fe-S) clusters play an essential role in plants as protein cofactors mediating diverse electron transfer reactions. Because they can react with oxygen to form reactive oxygen species (ROS) and inflict cellular damage, the biogenesis of Fe-S clusters is highly regulated. A recently discovered group of 2Fe-2S proteins, termed NEET proteins, was proposed to coordinate Fe-S, Fe and ROS homeostasis in mammalian cells. Here we report that disrupting the function of AtNEET, the sole member of the NEET protein family in Arabidopsis thaliana, triggers leaf-associated Fe-S- and Fe-deficiency responses, elevated Fe content in chloroplasts (1.2-1.5-fold), chlorosis, structural damage to chloroplasts and a high seedling mortality rate. Our findings suggest that disrupting AtNEET function disrupts the transfer of 2Fe-2S clusters from the chloroplastic 2Fe-2S biogenesis pathway to different cytosolic and chloroplastic Fe-S proteins, as well as to the cytosolic Fe-S biogenesis system, and that uncoupling this process triggers leaf-associated Fe-S- and Fe-deficiency responses that result in Fe over-accumulation in chloroplasts and enhanced ROS accumulation. We further show that AtNEET transfers its 2Fe-2S clusters to DRE2, a key protein of the cytosolic Fe-S biogenesis system, and propose that the availability of 2Fe-2S clusters in the chloroplast and cytosol is linked to Fe homeostasis in plants.

Jasmonic Acid Is Required for Plant Acclimation to a Combination of High Light and Heat Stress.

In the field, plants experience high light (HL) intensities that are often accompanied by elevated temperatures. Such conditions are a serious threat to agriculture production, because photosynthesis is highly sensitive to both HL intensities and high-temperature stress. One of the potential cellular targets of HL and heat stress (HS) combination is PSII because its degree of photoinhibition depends on the balance between the rate of PSII damage (induced by light stress), and the rate of PSII repair (impaired under HS). Here, we studied the responses of Arabidopsis (Arabidopsis thaliana) plants to a combination of HL and HS (HL+HS) conditions. Combined HL+HS was accompanied by irreversible damage to PSII, decreased D1 (PsbA) protein levels, and an enhanced transcriptional response indicative of PSII repair activation. We further identified several unique aspects of this stress combination that included enhanced accumulation of jasmonic acid (JA) and JA-Ile, elevated expression of over 2,200 different transcripts that are unique to the stress combination (including many that are JA-associated), and distinctive structural changes to chloroplasts. A mutant deficient in JA biosynthesis (allene oxide synthase) displayed enhanced sensitivity to combined HL+HS and further analysis revealed that JA is required for regulating several transcriptional responses unique to the stress combination. Our study reveals that JA plays an important role in the acclimation of plants to a combination of HL+HS.

Mobile Health Application and e-Health Literacy: Opportunities and Concerns for Cancer Patients and Caregivers.

Health literacy is critical for cancer patients as they must understand complex procedures or treatment options. Caregivers' health literacy also plays a crucial role in caring for cancer patients. Low health literacy is associated with low adherence to medications, poor health status, and increased health care costs. There is a growing interest in the use of mobile health applications (apps) to improve health literacy. Mobile health apps can empower underserved cancer patients and their caregivers by providing features or functionalities to enhance interactive patient-provider communication and to understand medical information more readily. Despite the potentiality of improving health literacy through mobile health apps, there exist several related concerns: no equal access to mobile technology, no familiarity or knowledge of using mobile health apps, and privacy and security concerns. These elements should be taken into account for health policy making and mobile apps design and development. Importantly, mobile apps should be developed with the goal of achieving a high range of user access by considering all health literacy level and various cultural and linguistic needs.

Rapid Responses to Abiotic Stress: Priming the Landscape for the Signal Transduction Network.

Plants grow and reproduce within a highly dynamic environment that can see abrupt changes in conditions, such as light intensity, temperature, humidity, or interactions with biotic agents. Recent studies revealed that plants can respond within seconds to some of these conditions, engaging many different metabolic and molecular networks, as well as rapidly altering their stomatal aperture. Some of these rapid responses were further shown to propagate throughout the entire plant via waves of reactive oxygen species (ROS) and Ca2+ that are possibly mediated through the plant vascular system. Here, we propose that the integration of these signals is mediated through pulses of gene expression that are coordinated throughout the plant in a systemic manner by the ROS/Ca+2 waves.

Identification and characterization of a core set of ROS wave-associated transcripts involved in the systemic acquired acclimation response of Arabidopsis to excess light.

Systemic acquired acclimation (SAA) plays a key role in optimizing growth and preventing damage associated with fluctuating or abrupt changes in the plant environment. To be effective, SAA has to occur at a rapid rate and depend on rapid signaling pathways that transmit signals from affected tissues to all parts of the plant. Although recent studies have identified several different rapid systemic signaling pathways that could mediate SAA, very little information is known about the extent of their involvement in mediating transcriptomic responses. Here we reveal that the systemic transcriptomic response of plants to excess light stress is extensive in its context and involves an early (2 min) and transient stage of transcript expression that includes thousands of genes. This early response is dependent on the respiratory burst oxidase homolog D protein, and the function of the reactive oxygen species (ROS) wave. We further identify a core set of transcripts associated with the ROS wave and suggest that some of these transcripts are involved in linking ROS with calcium signaling. Priming of a systemic leaf to become acclimated to a particular stress during SAA involves thousands of transcripts that display a rapid and transient expression pattern driven by the ROS wave.

Phylogenetic analysis of the CDGSH iron-sulfur binding domain reveals its ancient origin.

The iron-sulfur (2Fe-2S) binding motif CDGSH appears in many important plant and animal proteins that regulate iron and reactive oxygen metabolism. In human it is found in CISD1-3 proteins involved in diabetes, obesity, cancer, aging, cardiovascular disease and neurodegeneration. Despite the important biological role of the CDGSH domain, its origin, evolution and diversification, are largely unknown. Here, we report that: (1) the CDGSH domain appeared early in evolution, perhaps linked to the heavy use of iron-sulfur driven metabolism by early organisms; (2) a CISD3-like protein with two CDGSH domains on the same polypeptide appears to represent the ancient archetype of CDGSH proteins; (3) the origin of the human CISD3 protein is linked to the mitochondrial endosymbiotic event; (4) the CISD1/2 type proteins that contain only one CDGSH domain, but function as homodimers, originated after the divergence of bacteria and archaea/eukaryotes from their common ancestor; and (5) the human CISD1 and CISD2 proteins diverged about 650-720 million years ago, and CISD3 and CISD1/2 share their descent from an ancestral CISD about 1-1.1 billion years ago. Our findings reveal that the CDGSH domain is ancient in its origin and shed light on the complex evolutionary path of modern CDGSH proteins.