bZIP60 and Bax inhibitor 1 contribute IRE1-dependent and independent roles to potexvirus infection.

IRE1, BI-1, and bZIP60 monitor compatible plant-potexvirus interactions though recognition of the viral TGB3 protein. This study was undertaken to elucidate the roles of three IRE1 isoforms, the bZIP60U and bZIP60S, and BI-1 roles in genetic reprogramming of cells during potexvirus infection. Experiments were performed using Arabidopsis thaliana knockout lines and Plantago asiatica mosaic virus infectious clone tagged with the green fluorescent protein gene (PlAMV-GFP). There were more PlAMV-GFP infection foci in ire1a/b, ire1c, bzip60, and bi-1 knockout than wild-type (WT) plants. Cell-to-cell movement and systemic RNA levels were greater bzip60 and bi-1 than in WT plants. Overall, these data indicate an increased susceptibility to virus infection. Transgenic overexpression of AtIRE1b or StbZIP60 in ire1a/b or bzip60 mutant background reduced virus infection foci, while StbZIP60 expression influences virus movement. Transgenic overexpression of StbZIP60 also confers endoplasmic reticulum (ER) stress resistance following tunicamycin treatment. We also show bZIP60U and TGB3 interact at the ER. This is the first demonstration of a potato bZIP transcription factor complementing genetic defects in Arabidopsis. Evidence indicates that the three IRE1 isoforms regulate the initial stages of virus replication and gene expression, while bZIP60 and BI-1 contribute separately to virus cell-to-cell and systemic movement.

The ORFans' tale: new insights in plant biology.

Orphan genes (OGs) are protein-coding genes without a significant sequence similarity in closely related species. Despite their functional importance, very little is known about the underlying molecular mechanisms by which OGs participate in diverse biological processes. Here, we discuss the evolutionary mechanisms of OGs' emergence with relevance to species-specific adaptations. We also provide a mechanistic view of the involvement of OGs in multiple processes, including growth, development, reproduction, and carbon-metabolism-mediated immunity. We highlight the interconnection between OGs and the sucrose nonfermenting 1 (SNF1)-related protein kinases (SnRKs)-target of rapamycin (TOR) signaling axis for phytohormone signaling, nutrient metabolism, and stress responses. Finally, we propose a high-throughput pipeline for OGs' interspecies and intraspecies gene transfer through a transgenic approach for future biotechnological advances.

Preparation and Utilization of a Versatile GFP-Protein Trap-Like System for Protein Complex Immunoprecipitation in Plants.

Protein complex immunoprecipitation (co-IP) is an in vitro technique used to study protein-protein interaction between two or more proteins. This method relies on affinity purification of recombinant epitope-tagged proteins followed by western blotting detection using tag-specific antibodies for the confirmation of positive interaction. The traditional co-IP method relies on the use of porous beaded support with immobilized antibodies to precipitate protein complexes. However, this method is time-consuming, labor-intensive, and provides lower reproducibility and yield of protein complexes. Here, we describe the implementation of magnetic beads and high-affinity anti-green fluorescent protein (GFP) antibodies to develop an in vitro GFP-protein trap-like system. This highly reproducible system utilizes a combination of small sample size, versatile lysis buffer, and lower amounts of magnetic beads to obtain protein complexes and aggregates that are compatible with functional assays, Western blotting, and mass spectrometry. In addition to protein-protein interactions, this versatile method can be employed to study protein-nucleic acid interactions. This protocol also highlights troubleshooting and includes recommendations to optimize its application.

Cytotrap: An Innovative Approach for Protein-Protein Interaction Studies for Cytoplasmic Proteins.

Protein-protein interaction mapping has gained immense importance in understanding protein functions in diverse biological pathways. There are various in vivo and in vitro techniques associated with the protein-protein interaction studies but generally, the focus is confined to understanding the protein interaction in the nucleus of the cell, and thus it limits the availability to explore protein interactions that are happening in the cytoplasm of the cell. Since posttranslational modification is a crucial step in signaling pathways and cellular protein interactions harnessing the cytoplasmic protein and evaluating the interaction in the cytoplasm, this protocol will provide more information about studying these types of protein interactions. Cytotrap is a type of yeast-two-hybrid system that differs in its ability to anchor along the membrane, thus directing the protein of interest to anchor along the membrane through the myristoylation signaling unit. The vector containing the target protein contains the myristoylation unit, called the prey, and the bait unit contains the protein of interest as a fusion with the hSos protein. In an event of interaction between the target and the protein of interest, the hSos protein unit will be localized to the membrane and the GDP/GTP exchange unit will trigger the activation of the Ras pathway that leads to the survival of the temperature-sensitive yeast strain at a higher temperature.

Dynamic Enrichment for Evaluation of Protein Networks (DEEPN): A High Throughput Yeast Two-Hybrid (Y2H) Protocol to Evaluate Networks.

Proteins are the building blocks of life, and a vast array of cellular processes is handled by protein-protein interactions (PPIs). The protein complexes formed via PPIs lead to tangled networks that, with their continuous remodeling, build up systematic functional units. Over the years, PPIs have become an area of interest for many researchers, leading to the development of multiple in vitro and in vivo methods to reveal these interactions. The yeast-two-hybrid (Y2H) system is a potent genetic way to map PPIs in both a micro- and high-throughput manner. Y2H is a technique that involves using modified yeast cells to identify protein-protein interactions. For Y2H, the yeast cells are engineered only to grow when there is a significant interaction between a specific protein with its interacting partner. PPIs are identified in the Y2H system by stimulating reporter genes in response to a restored transcription factor. However, Y2H results may be constrained by stringency requirements, as the limited number of colony screenings through this technique could result in the possible elimination of numerous genuine interactions. Therefore, DEEPN (dynamic enrichment for evaluation of protein networks) can be used, offering the potential to study the multiple static and transient protein interactions in a single Y2H experiment. DEEPN utilizes next-generation DNA sequencing (NGS) data in a high-throughput manner and subsequently applies computational analysis and statistical modeling to identify interacting partners. This protocol describes customized reagents and protocols through which DEEPN analysis can be utilized efficiently and cost-effectively.

The Lost and Found: Unraveling the Functions of Orphan Genes.

Orphan Genes (OGs) are a mysterious class of genes that have recently gained significant attention. Despite lacking a clear evolutionary history, they are found in nearly all living organisms, from bacteria to humans, and they play important roles in diverse biological processes. The discovery of OGs was first made through comparative genomics followed by the identification of unique genes across different species. OGs tend to be more prevalent in species with larger genomes, such as plants and animals, and their evolutionary origins remain unclear but potentially arise from gene duplication, horizontal gene transfer (HGT), or de novo origination. Although their precise function is not well understood, OGs have been implicated in crucial biological processes such as development, metabolism, and stress responses. To better understand their significance, researchers are using a variety of approaches, including transcriptomics, functional genomics, and molecular biology. This review offers a comprehensive overview of the current knowledge of OGs in all domains of life, highlighting the possible role of dark transcriptomics in their evolution. More research is needed to fully comprehend the role of OGs in biology and their impact on various biological processes.

Network Biology Analyses and Dynamic Modeling of Gene Regulatory Networks under Drought Stress Reveal Major Transcriptional Regulators in Arabidopsis.

Drought is one of the most serious abiotic stressors in the environment, restricting agricultural production by reducing plant growth, development, and productivity. To investigate such a complex and multifaceted stressor and its effects on plants, a systems biology-based approach is necessitated, entailing the generation of co-expression networks, identification of high-priority transcription factors (TFs), dynamic mathematical modeling, and computational simulations. Here, we studied a high-resolution drought transcriptome of Arabidopsis. We identified distinct temporal transcriptional signatures and demonstrated the involvement of specific biological pathways. Generation of a large-scale co-expression network followed by network centrality analyses identified 117 TFs that possess critical properties of hubs, bottlenecks, and high clustering coefficient nodes. Dynamic transcriptional regulatory modeling of integrated TF targets and transcriptome datasets uncovered major transcriptional events during the course of drought stress. Mathematical transcriptional simulations allowed us to ascertain the activation status of major TFs, as well as the transcriptional intensity and amplitude of their target genes. Finally, we validated our predictions by providing experimental evidence of gene expression under drought stress for a set of four TFs and their major target genes using qRT-PCR. Taken together, we provided a systems-level perspective on the dynamic transcriptional regulation during drought stress in Arabidopsis and uncovered numerous novel TFs that could potentially be used in future genetic crop engineering programs.

A genome-wide comparative evolutionary analysis of zinc finger-BED transcription factor genes in land plants.

Zinc finger (Zf)-BED proteins are a novel superfamily of transcription factors that controls numerous activities in plants including growth, development, and cellular responses to biotic and abiotic stresses. Despite their important roles in gene regulation, little is known about the specific functions of Zf-BEDs in land plants. The current study identified a total of 750 Zf-BED-encoding genes in 35 land plant species including mosses, bryophytes, lycophytes, gymnosperms, and angiosperms. The gene family size was somewhat proportional to genome size. All identified genes were categorized into 22 classes based on their specific domain architectures. Of these, class I (Zf-BED_DUF-domain_Dimer_Tnp_hAT) was the most common in the majority of the land plants. However, some classes were family-specific, while the others were species-specific, demonstrating diversity at different classification levels. In addition, several novel functional domains were also predicated including WRKY and nucleotide-binding site (NBS). Comparative genomics, transcriptomics, and proteomics provided insights into the evolutionary history, duplication, divergence, gene gain and loss, species relationship, expression profiling, and structural diversity of Zf-BEDs in land plants. The comprehensive study of Zf-BEDs in Gossypium sp., (cotton) also demonstrated a clear footprint of polyploidization. Overall, this comprehensive evolutionary study of Zf-BEDs in land plants highlighted significant diversity among plant species.

The potential of Bacillus subtilis and phosphorus in improving the growth of wheat under chromium stress.

AIM: Hexavalent chromium (Cr+6 ) is one of the most toxic heavy metals that have deteriorating effects on the growth and quality of the end product of wheat. Consequently, this research was designed to evaluate the role of Bacillus subtilis and phosphorus fertilizer on wheat facing Cr+6 stress. METHODS AND RESULTS: The soil was incubated with Bacillus subtilis and phosphorus fertilizer before sowing. The statistical analysis of the data showed that the co-application of B. subtilis and phosphorus yielded considerably more significant (p < 0.05) results compared with an individual application of the respective treatments. The co-treatment improved the morphological, physiological and biochemical parameters of plants compared with untreated controls. The increase in shoot length, root length, shoot fresh weight and root fresh weight was 38.17%, 29.31%, 47.89% and 45.85%, respectively, compared with untreated stress-facing plants. The application of B. subtilis and phosphorus enhanced osmolytes content (proline 39.98% and sugar 41.30%), relative water content and stability maintenance of proteins (86.65%) and cell membranes (66.66%). Furthermore, augmented production of antioxidants by 67.71% (superoxide dismutase), 95.39% (ascorbate peroxidase) and 60.88% (catalase), respectively, were observed in the Cr+6 - stressed plants after co-application of B. subtilis and phosphorus. CONCLUSION: It was observed that the accumulation of Cr+6 was reduced by 54.24%, 59.19% and 90.26% in the shoot, root and wheat grains, respectively. Thus, the combined application of B. subtilis and phosphorus has the potential to reduce the heavy metal toxicity in crops. SIGNIFICANCE AND IMPACT OF THE STUDY: This study explored the usefulness of Bacillus subtilis and phosphorus application on wheat in heavy metal stress. It is a step toward the combinatorial use of plant growth-promoting rhizobacteria with nutrients to improve the ecosystems' health.

A TIReless battle: TIR domains in plant-pathogen interactions.

Toll/interleukin-1 receptor (TIR) domain-containing proteins are conserved across kingdoms, and their mechanistic understanding holds promise for basic plant biology and agriculture. Here, we discuss the novel enzymatic TIR domain functions of nucleotide-binding leucine-rich repeat receptors (NLRs) in cell death, and posit how TIR domain-containing effectors mechanistically subvert host immune systems.

The interplay of GTP-binding protein AGB1 with ER stress sensors IRE1a and IRE1b modulates Arabidopsis unfolded protein response and bacterial immunity.

In eukaryotic cells, the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) results in ER stress that induces a cascade of reactions called the unfolded protein response (UPR). In Arabidopsis, the most conserved UPR sensor, Inositol-requiring enzyme 1 (IRE1), responds to both abiotic- and biotic-induced ER stress. Guanine nucleotide-binding proteins (G proteins) constitute another universal and conserved family of signal transducers that have been extensively investigated due to their ubiquitous presence and diverse nature of action. Arabidopsis GTP-binding protein ß1 (AGB1) is the only G-protein ß-subunit encoded by the Arabidopsis genome that is involved in numerous signaling pathways. Mounting evidence suggests the existence of a crosstalk between IRE1 and G protein signaling during ER stress. AGB1 has previously been shown to control a distinct UPR pathway independently of IRE1 when treated with an ER stress inducer tunicamycin. Our results obtained with combinatorial knockout mutants support the hypothesis that both IRE1 and AGB1 synergistically contribute to ER stress responses chemically induced by dithiothreitol (DTT) as well as to the immune responses against a phytopathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000. Our study highlights the crosstalk between the plant UPR transducers under abiotic and biotic stress.

A Quantitative Arabidopsis IRE1a Ribonuclease-Dependent in vitro mRNA Cleavage Assay for Functional Studies of Substrate Splicing and Decay Activities.

The unfolded protein response (UPR) is an adaptive eukaryotic reaction that controls the protein folding capacities of the endoplasmic reticulum (ER). The most ancient and well-conserved component of the UPR is Inositol-Requiring Enzyme 1 (IRE1). Arabidopsis IRE1a (AtIRE1) is a transmembrane sensor of ER stress equipped with dual protein kinase and ribonuclease (RNase) activities, encoded by its C-terminal domain. In response to both physiological stresses and pathological perturbations, AtIRE1a directly cleaves bZIP60 (basic leucine zipper 60) mRNA. Here, we developed a quantitative in vitro cleavage assay that combines recombinant AtIRE1a protein that is expressed in Nicotiana benthamiana and total RNA isolated from Arabidopsis leaves. Wild-type AtIRE1a as well as its variants containing point mutations in the kinase or RNase domains that modify its cleavage activity were employed to demonstrate their contributions to cleavage activity levels. We show that, when exposed to total RNA in vitro, the AtIRE1a protein cleaves bZIP60 mRNA. Depletion of the bZIP60 transcript in the reaction mixture can be precisely quantified by a qRT-PCR-mediated assay. This method facilitates the functional studies of novel plant IRE1 variants by allowing to quickly and precisely assess the effects of protein mutations on the substrate mRNA cleavage activity before advancing to more laborious, stable transgenic approaches in planta. Moreover, this method is readily adaptable to other plant IRE1 paralogs and orthologs, and can also be employed to test additional novel mRNA substrates of plant IRE1, such as transcripts undergoing degradation through the process of regulated IRE1-dependent decay (RIDD). Finally, this method can also be modified and expanded to functional testing of IRE1 interactors and inhibitors, as well as for studies on the molecular evolution of IRE1 and its substrates, providing additional insights into the mechanistic underpinnings of IRE1-mediated ER stress homeostasis in plant tissues.

Dynamic Regulatory Event Mining by iDREM in Large-Scale Multi-omics Datasets During Biotic and Abiotic Stress in Plants.

The system-wide complexity of genome regulation encoding the organism phenotypic diversity is well understood. However, a major challenge persists about the appropriate method to describe the systematic dynamic genome regulation event utilizing enormous multi-omics datasets. Here, we describe Interactive Dynamic Regulatory Events Miner (iDREM) which reconstructs gene-regulatory networks from temporal transcriptome, proteome, and epigenome datasets during stress to envisage "master" regulators by simulating cascades of temporal transcription-regulatory and interactome events. The iDREM is a Java-based software that integrates static and time-series transcriptomics and proteomics datasets, transcription factor (TF)-target interactions, microRNA (miRNA)-target interaction, and protein-protein interactions to reconstruct temporal regulatory network and identify significant regulators in an unsupervised manner. The hidden Markov model detects specialized manipulated pathways as well as genes to recognize statistically significant regulators (TFs/miRNAs) that diverge in temporal activity. This method can be translated to any biotic or abiotic stress in plants and animals to predict the master regulators from condition-specific multi-omics datasets including host-pathogen interactions for comprehensive understanding of manipulated biological pathways.

UPR signaling at the nexus of plant viral, bacterial, and fungal defenses.

In recent years there have been significant advances in our understanding of the ER stress responses in plants that are associated with virus infection, as well as bacterial and fungal diseases. In plants, ER stress induced by virus infection includes several signaling pathways that include the unfolded protein response (UPR) to promote the expression of chaperone proteins for proper protein folding. Understanding how facets of ER stress signaling broadly engage in pathogen responses, as well as those that are specific to virus infection is important to distinguishing features essential for broad cellular defenses and processes that may be specifically linked to viral infectivity and disease.

Probing natural variation of IRE1 expression and endoplasmic reticulum stress responses in Arabidopsis accessions.

The environmental effects shape genetic changes in the individuals within plant populations, which in turn contribute to the enhanced genetic diversity of the population as a whole. Thus, individuals within the same species can acquire and accumulate genetic differences in their genomes depending on their local environment and evolutionary history. IRE1 is a universal endoplasmic reticulum (ER) stress sensor that activates an evolutionarily conserved signalling cascade in response to biotic and abiotic stresses. Here, we selected nine different Arabidopsis accessions along with the reference ecotype Columbia-0, based on their geographical origins and differential endogenous IRE1 expression under steady-state conditions to investigate the natural variation of ER stress responses. We cloned and analysed selected upstream regulatory regions of IRE1a and IRE1b, which revealed differential levels of their inducibility. We also subjected these accessions to an array of biotic and abiotic stresses including heat, ER stress-inducing chemical tunicamycin, phytohormone salicylic acid, and pathogen infection. We measured IRE1-mediated splicing of its evolutionarily conserved downstream client as well as transcript accumulation of ER-resident chaperones and co-chaperones. Collectively, our results illustrate the expression polymorphism of a major plant stress receptor and its relationship with molecular and physiological ER stress sensitivity.

Multilevel regulation of endoplasmic reticulum stress responses in plants: where old roads and new paths meet.

The sessile lifestyle of plants requires them to cope with a multitude of stresses in situ. In response to diverse environmental and intracellular cues, plant cells respond by massive reprogramming of transcription and translation of stress response regulators, many of which rely on endoplasmic reticulum (ER) processing. This increased protein synthesis could exceed the capacity of precise protein quality control, leading to the accumulation of unfolded and/or misfolded proteins that triggers the unfolded protein response (UPR). Such cellular stress responses are multilayered and executed in different cellular compartments. Here, we will discuss the three main branches of UPR signaling in diverse eukaryotic systems, and describe various levels of ER stress response regulation that encompass transcriptional gene regulation by master transcription factors, post-transcriptional activities including cytoplasmic splicing, translational control, and multiple post-translational events such as peptide modifications and cleavage. In addition, we will discuss the roles of plant ER stress sensors in abiotic and biotic stress responses and speculate on the future prospects of engineering these signaling events for heightened stress tolerance.

Arabidopsis GCN2 kinase contributes to ABA homeostasis and stomatal immunity.

General Control Non-derepressible 2 (GCN2) is an evolutionarily conserved serine/threonine kinase that modulates amino acid homeostasis in response to nutrient deprivation in yeast, human and other eukaryotes. However, the GCN2 signaling pathway in plants remains largely unknown. Here, we demonstrate that in Arabidopsis, bacterial infection activates AtGCN2-mediated phosphorylation of eIF2α and promotes TBF1 translational derepression. Consequently, TBF1 regulates a subset of abscisic acid signaling components to modulate pre-invasive immunity. We show that GCN2 fine-tunes abscisic acid accumulation and signaling during both pre-invasive and post-invasive stages of an infection event. Finally, we also demonstrate that AtGCN2 participates in signaling triggered by phytotoxin coronatine secreted by P. syringae. During the preinvasive phase, AtGCN2 regulates stomatal immunity by affecting pathogen-triggered stomatal closure and coronatine-mediated stomatal reopening. Our conclusions support a conserved role of GCN2 in various forms of immune responses across kingdoms, highlighting GCN2's importance in studies on both plant and mammalian immunology.

NPR1 in JazzSet with Pathogen Effectors.

NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) is a master regulator of salicylic acid (SA)-mediated systemic acquired resistance (SAR), a broad-spectrum disease resistance mechanism in plants. NPR1 controls approximately 90% of SA-dependent transcriptome in Arabidopsis. Here, we discuss how pathogen effectors manipulate NPR1 functions in different cellular compartments to establish disease.

Pathogen Tactics to Manipulate Plant Cell Death.

Cell death is a vital process for multicellular organisms. Programmed cell death (PCD) functions in a variety of processes including growth, development, and immune responses for homeostasis maintenance. In particular, plants and animals utilize PCD to control pathogen invasion and infected cell populations. Despite some similarity, there are a number of key differences between how these organisms initiate and regulate cell death. In contrast to animals, plants are sessile, lack a circulatory system, and have additional cellular structures, including cell walls and chloroplasts. Plant cells have the autonomous ability to induce localized cell death using conserved eukaryotic pathways as well as unique plant-specific pathways. Thus, in order to successfully infect host cells, pathogens must subvert immune responses and avoid detection to prevent PCD and allow infection. Here we discuss the roles of cell death in plant immune responses and the tactics pathogens utilize to avert cell death.

Endoplasmic Reticulum Stress Signaling in Plant Immunity--At the Crossroad of Life and Death.

Rapid and complex immune responses are induced in plants upon pathogen recognition. One form of plant defense response is a programmed burst in transcription and translation of pathogenesis-related proteins, of which many rely on ER processing. Interestingly, several ER stress marker genes are up-regulated during early stages of immune responses, suggesting that enhanced ER capacity is needed for immunity. Eukaryotic cells respond to ER stress through conserved signaling networks initiated by specific ER stress sensors tethered to the ER membrane. Depending on the nature of ER stress the cell prioritizes either survival or initiates programmed cell death (PCD). At present two plant ER stress sensors, bZIP28 and IRE1, have been described. Both sensor proteins are involved in ER stress-induced signaling, but only IRE1 has been additionally linked to immunity. A second branch of immune responses relies on PCD. In mammals, ER stress sensors are involved in activation of PCD, but it is unclear if plant ER stress sensors play a role in PCD. Nevertheless, some ER resident proteins have been linked to pathogen-induced cell death in plants. In this review, we will discuss the current understanding of plant ER stress signaling and its cross-talk with immune signaling.