MicroRNAs (miRNAs) are a class of noncoding small RNAs, which play a crucial role in post-transcriptional gene regulation. Recently, various reports revealed that miRNAs could be transmitted between species to mediate cross-kingdom regulation by integrating into a specific target gene-mediated regulatory pathway to exert relevant biological functions. Some scholars and researchers have observed this as an attractive hypothesis that may provide a foundation for novel approaches in the diagnosis, prognosis, and treatment of disease. Meanwhile, others deem the mentioned results were obtained from a “false positive effect” of performed experiments. Here, we focus on several current studies concerning plant miRNA-mediated cross-kingdom regulation (from both fronts) and discuss the existing issues that need further consideration. We also discuss possible miRNA horizontal transfer mechanisms from one species to another and analyze the relationship between miRNA-mediated cross-kingdom regulation and coevolution during a long-term specific host⁻pathogen interaction.

Plant biotechnology traits provide a means to increase crop yields, manage weeds and pests, and sustainably contribute to addressing the needs of a growing population. One of the key challenges in developing new traits for plant biotechnology is the availability of expression elements for efficacious and predictable transgene regulation. Recent advances in genomics, transcriptomics, and computational tools have enabled the generation of new expression elements in a variety of model organisms. In this study, new expression element sequences were computationally generated for use in crops, starting from native Arabidopsis and maize sequences. These elements include promoters, 5' untranslated regions (5' UTRs), introns, and 3' UTRs. The expression elements were demonstrated to drive effective transgene expression in stably transformed soybean plants across multiple tissues types and developmental stages. The expressed transcripts were characterized to demonstrate the molecular function of these expression elements. The data show that the promoters precisely initiate transcripts, the introns are effectively spliced, and the 3' UTRs enable predictable processing of transcript 3' ends. Overall, our results indicate that these new expression elements can recapitulate key functional properties of natural sequences and provide opportunities for optimizing the expression of genes in future plant biotechnology traits.

Despite successful use of Plant Growth Promoting Rhizobacteria (PGPR) in agriculture, little is known about specific mechanisms of gene regulation facilitating the effective communication between bacteria and plants during plant colonization. Active PGPR strain Bacillus atrophaeus UCMB-5137 was studied in this research. RNA sequencing profiles were generated in experiments where root exudate stimulations were used to mimic interactions between bacteria and plants. It was found that the gene regulation in B. atrophaeus UCMB-5137 in response to the root exudate stimuli differed from the reported gene regulation at similar conditions in B. amyloliquefaciens FZB42, which was considered as a paradigm PGPR. This difference was explained by hypersensitivity of UCMB-5137 to the root exudate stimuli impelling it to a sessile root colonization behavior through the CcpA-CodY-AbrB regulation. It was found that the transcriptional factor DegU also could play an important role in gene regulations during plant colonization. A significant stress caused by the root exudates on in vitro cultivated B. atrophaeus UCMB-5137 was noticed and discussed. Multiple cases of conflicted gene regulations showed scantiness of our knowledge on the regulatory network in Bacillus. Some of these conflicted regulations could be explained by interference of non-coding RNA (ncRNA). Search through differential expressed intergenic regions revealed 49 putative loci of ncRNA regulated by the root exudate stimuli. Possible target mRNA were predicted and a general regulatory network of B. atrophaeus UCMB-5137 genome was designed.

The leaves of tea plants (Camellia sinensis) are used to produce tea, which is one of the most popular beverages consumed worldwide. The nutritional value and health benefits of tea are mainly related to three abundant characteristic metabolites; catechins, theanine and caffeine. Weighted gene co-expression network analysis (WGCNA) is a powerful system for investigating correlations between genes, identifying modules among highly correlated genes, and relating modules to phenotypic traits based on gene expression profiling. Currently, relatively little is known about the regulatory mechanisms and correlations between these three secondary metabolic pathways at the omics level in tea.

Virulent Agrobacterium tumefaciens strains integrate their T-DNA into the plant genome where the encoded agrobacterial oncogenes are expressed and cause crown gall disease. Essential for crown gall development are IaaH (indole-3-acetamide hydrolase), IaaM (tryptophan monooxygenase) and Ipt (isopentenyl transferase), which encode enzymes for the biosynthesis of auxin (IaaH, IaaM) and cytokinin (Ipt). Although these oncogenes are well studied as the tumor-inducing principle, nothing is known about the regulation of oncogene expression in plant cells. Our studies show that the intergenic regions (IGRs) between the coding sequences (CDS) of the three oncogenes function as promoters in plant cells. These promoters possess a eukaryotic sequence organization and cis-regulatory elements for the binding of plant transcription factors. WRKY18, WRKY40, WRKY60 and ARF5 were identified as activators of the Ipt promoter whereas IaaH and IaaM is constitutively expressed and no transcription factor further activates their promoters. Consistent with these results, the wrky triple mutant plants in particular, develops smaller crown galls than wild-type and exhibits a reduced Ipt transcription, despite the presence of an intact ARF5 gene. WRKY40 and WRKY60 gene expression is induced by A. tumefaciens within a few hours whereas the ARF5 gene is transcribed later during crown gall development. The WRKY proteins interact with ARF5 in the plant nucleus, but only WRKY40 together with ARF5 synergistically boosts the activation of the Ipt promoter in an auxin-dependent manner. From our data, we propose that A. tumefaciens initially induces WRKY40 gene expression as a pathogen defense response of the host cell. The WRKY protein is recruited to induce Ipt expression, which initiates cytokinin-dependent host cell division. With increasing auxin levels triggered by ubiquitous expression of IaaH and IaaM, ARF5 is activated and interacts with WRKY40 to potentiate Ipt expression and balance cytokinin and auxin levels for further cell proliferation.

In plant cells, most microRNAs (miRNAs) perform cleavages of target mature mRNAs in the cytoplasm. A recent report of a miRNA pathway involved in DNA methylation in the rice nucleus raises the possibility that plant miRNAs could cleave intron-containing pre-mRNAs (the precursor of messenger RNAs) located in the nucleus.

Fungi dominate the recycling of carbon sequestered in woody biomass. This process of organic turnover was first evolved among "white rot" fungi that degrade lignin to access carbohydrates and later evolved multiple times toward more efficient strategies to selectively target carbohydrates-"brown rot." The brown rot adaption was often explained by mechanisms to deploy reactive oxygen species (ROS) to oxidatively attack wood structures. However, its genetic basis remains unclear, especially in the context of gene contractions of conventional carbohydrate-active enzymes (CAZYs) relative to white rot ancestors. Here, we hypothesized that these apparent gains in brown rot efficiency despite gene losses were due, in part, to upregulation of the retained genes. We applied comparative transcriptomics to multiple species of both rot types grown across a wood wafer to create a gradient of progressive decay and to enable tracking temporal gene expression. Dozens of "decay-stage-dependent" ortho-genes were isolated, narrowing a pool of candidate genes with time-dependent regulation unique to brown rot fungi. A broad comparison of the expression timing of CAZY families indicated a temporal regulatory shift of lignocellulose-oxidizing genes toward early stages in brown rot compared to white rot, enabling the segregation of oxidative treatment ahead of hydrolysis. These key brown rot ROS-generating genes with iron ion binding functions were isolated. Moreover, transcription energy was shifted to be invested on the retained GHs in brown rot fungi to strengthen carbohydrate conversion. Collectively, these results support the hypothesis that gene regulation shifts played a pivotal role in brown rot adaptation.IMPORTANCE Fungi dominate the turnover of wood, Earth's largest pool of aboveground terrestrial carbon. Fungi first evolved this capacity by degrading lignin to access and hydrolyze embedded carbohydrates (white rot). Multiple lineages, however, adapted faster reactive oxygen species (ROS) pretreatments to loosen lignocellulose and selectively extract sugars (brown rot). This brown rot "shortcut" often coincided with losses (>60%) of conventional lignocellulolytic genes, implying that ROS adaptations supplanted conventional pathways. We used comparative transcriptomics to further pursue brown rot adaptations, which illuminated the clear temporal expression shift of ROS genes, as well as the shift toward synthesizing more GHs in brown rot relative to white rot. These imply that gene regulatory shifts, not simply ROS innovations, were key to brown rot fungal evolution. These results not only reveal an important biological shift among these unique fungi, but they may also illuminate a trait that restricts brown rot fungi to certain ecological niches.

While Brassica oleracea vegetables have been linked to cancer prevention, the exact mechanism remains unknown. Regulation of gene expression by cross-species microRNAs has been previously reported; however, its link to cancer suppression remains unexplored. In this study we address both issues. We confirm plant microRNAs in human blood in a large nutrigenomics study cohort and in a randomized dose-controlled trial, finding a significant positive correlation between the daily amount of broccoli consumed and the amount of microRNA in the blood. We also demonstrate that Brassica microRNAs regulate expression of human genes and proteins in vitro, and that microRNAs cooperate with other Brassica-specific compounds in a possible cancer-preventive mechanism. Combined, we provide strong evidence and a possible multimodal mechanism for broccoli in cancer prevention.

Being sessile in nature, plants have to withstand various adverse environmental stress conditions including both biotic and abiotic stresses. Comparatively, abiotic stresses such as drought, salinity, high temperature, and cold pose major threat to agriculture by negatively impacting plant growth and yield worldwide. Rice is one of the most widely consumed staple cereals across the globe, the production and productivity of which is also severely affected by different abiotic stresses. Therefore, several crop improvement programs are directed toward developing stress tolerant rice cultivars either through marker assisted breeding or transgenic technology. Alternatively, some known rhizospheric competent bacteria are also known to improve plant growth during abiotic stresses. A plant growth promoting rhizobacteria (PGPR), Bacillus amyloliquefaciens NBRI-SN13 (SN13) was previously reported by our lab to confer salt stress tolerance to rice seedlings. However, the present study investigates the role of SN13 in ameliorating various abiotic stresses such as salt, drought, desiccation, heat, cold, and freezing on a popular rice cv. Saryu-52 under hydroponic growth conditions. Apart from this, seedlings were also exogenously supplied with abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA) and ethephon (ET) to study the role of SN13 in phytohormone-induced stress tolerance as well as its role in abiotic and biotic stress cross-talk. All abiotic stresses and phytohormone treatments significantly affected various physiological and biochemical parameters like membrane integrity and osmolyte accumulation. SN13 also positively modulated stress-responsive gene expressions under various abiotic stresses and phytohormone treatments suggesting its multifaceted role in cross-talk among stresses and phytohormones in response to PGPR. To the best of our knowledge, this is the first report on detailed analysis of plant growth promotion and stress alleviation by a PGPR in rice seedlings subjected to various abiotic stresses and phytohormone treatments for 0, 1, 3, 10, and 24 h.

Many plant species possess compounds with juvenile hormone disruptor (JHD) activity. In some plant species, such activity has been attributed to diterpene secondary metabolites. Plant JHD diterpenes disrupt insect development by interfering with the juvenile hormone (JH)-mediated formation of JH receptor complexes. Here, we demonstrate that a plant extract and a diterpene from Lindera erythrocarpa (methyl lucidone) interfere with the formation of both methoprene-tolerant (Met)/Taiman and Germ cell-expressed (GCE)/Taiman heterodimer complexes in yeast two-hybrid assays in vitro. In addition to the in vitro JHD activity, the diterpene and the plant extract from L. erythrocarpa also disrupt the development of larvae and pupae in Drosophila melanogaster. Comparing the transcriptomes of juvenile hormone analog (JHA, methoprene)- and JHD (methyl lucidone)-fed wandering third-instar larvae revealed a large number of genes that were coregulated by JHA and JHD. Moreover, most (83%) of the genes that were repressed by methyl lucidone were significantly activated by methoprene, indicating that JHDs and JHAs have opposing effects on the transcriptional regulation of many JH-dependent genes. Gene ontology analysis also suggested that some of the genes activated-by-JHA/repressed-by-JHD play roles in spermatogenesis. Affymetrix microarray-based analysis indicated that the expression of genes activated-by-JHA/repressed-by-JHD was testis-specific. Together, these results suggest that JH is involved in testis-specific gene expression and that plant JHD diterpenes function as JH antagonists in such JHA-mediated gene regulation.

In multicellular organisms, development, growth and reproduction require coordinated expression of numerous functional and regulatory genes. Insects, in addition to being the most speciose animal group with enormous biological and economical significance, represent outstanding model organisms for studying regulation of synchronized gene expression due to their rapid development and reproduction. Disease-transmitting female mosquitoes have adapted uniquely for ingestion and utilization of the huge blood meal required for swift reproductive events to complete egg development within a 72-h period. We investigated the network of regulatory factors mediating sequential gene expression in the fat body, a multifunctional organ analogous to the vertebrate liver and adipose tissue, of the female Aedes aegypti mosquito. Transcriptomic and bioinformatics analyses revealed that ~7500 transcripts are differentially expressed in four sequential waves during the 72-h reproductive period. A combination of RNA-interference gene-silencing and in-vitro organ culture identified the major regulators for each of these waves. Amino acids (AAs) regulate the first wave of gene activation between 3 h and 12 h post-blood meal (PBM). During the second wave, between 12 h and 36 h, most genes are highly upregulated by a synergistic action of AAs, 20-hydroxyecdysone (20E) and the Ecdysone-Receptor (EcR). Between 36 h and 48 h, the third wave of gene activation-regulated mainly by HR3-occurs. Juvenile Hormone (JH) and its receptor Methoprene-Tolerant (Met) are major regulators for the final wave between 48 h and 72 h. Each of these key regulators also has repressive effects on one or more gene sets. Our study provides a better understanding of the complexity of the regulatory mechanisms related to temporal coordination of gene expression during reproduction. We have detected the novel function of 20E/EcR responsible for transcriptional repression. This study also reveals the previously unidentified large-scale effects of HR3 and JH/Met on transcriptional regulation during the termination of vitellogenesis and remodeling of the fat body.

Identifying genes that are differentially expressed in response to social interactions is informative for understanding the molecular basis of social behavior. To address this question, we described changes in gene expression as a result of differences in the extent of social interactions. We housed threespine stickleback (Gasterosteus aculeatus) females in either group conditions or individually for one week, then measured levels of gene expression in three brain regions using RNA-sequencing. We found that numerous genes in the hindbrain/cerebellum had altered expression in response to group or individual housing. However, relatively few genes were differentially expressed in either the diencephalon or telencephalon. The list of genes upregulated in fish from social groups included many genes related to neural development and cell adhesion as well as genes with functions in sensory signaling, stress, and social and reproductive behavior. The list of genes expressed at higher levels in individually-housed fish included several genes previously identified as regulated by social interactions in other animals. The identified genes are interesting targets for future research on the molecular mechanisms of normal social interactions.

An important capacity of genes is the rapid change of expression levels to cope with the environment, known as expression responsiveness or plasticity. Elucidating the genomic mechanisms determining expression plasticity is critical for understanding the molecular basis of phenotypic plasticity, fitness and adaptation. In this study, we systematically quantified gene expression plasticity in four metazoan species by integrating changes of expression levels under a large number of genetic and environmental conditions. From this, we demonstrated that expression plasticity measures a distinct feature of gene expression that is orthogonal to other well-studied features, including gene expression level and tissue specificity/broadness. Expression plasticity is conserved across species with important physiological implications. The magnitude of expression plasticity is highly correlated with gene function and genes with high plasticity are implicated in disease susceptibility. Genome-wide analysis identified many conserved promoter cis-elements, trans-acting factors (such as CTCF), and gene body histone modifications (H3K36me3, H3K79me2 and H4K20me1) that are significantly associated with expression plasticity. Analysis of expression changes in perturbation experiments further validated a causal role of specific transcription factors and histone modifications. Collectively, this work reveals the general properties, physiological implications and multivariable regulation of gene expression plasticity in metazoans, extending the mechanistic understanding of gene regulation.

Cannabis, an important industrial crop, has a high sensitivity to photoperiods. The flowering time of cannabis is one of its important agronomic traits, and has a significant effect on its yield and quality. The CONSTANS-like (COL) gene plays a key role in the regulation of flowering in this plant. However, the specific roles of the COL gene family in cannabis are still unknown.

Most organisms use daily light/dark cycles as timing cues to control many essential physiological processes. In plants, growth rates of the embryonic stem (hypocotyl) are maximal at different times of day, depending on external photoperiod and the internal circadian clock. However, the interactions between light signaling, the circadian clock, and growth-promoting hormone pathways in growth control remain poorly understood. At the molecular level, such growth rhythms could be attributed to several different layers of time-specific control such as phasing of transcription, signaling, or protein abundance. To determine the transcriptional component associated with the rhythmic control of growth, we applied temporal analysis of the Arabidopsis thaliana seedling transcriptome under multiple growth conditions and mutant backgrounds using DNA microarrays. We show that a group of plant hormone-associated genes are coexpressed at the time of day when hypocotyl growth rate is maximal. This expression correlates with overrepresentation of a cis-acting element (CACATG) in phytohormone gene promoters, which is sufficient to confer the predicted diurnal and circadian expression patterns in vivo. Using circadian clock and light signaling mutants, we show that both internal coincidence of phytohormone signaling capacity and external coincidence with darkness are required to coordinate wild-type growth. From these data, we argue that the circadian clock indirectly controls growth by permissive gating of light-mediated phytohormone transcript levels to the proper time of day. This temporal integration of hormone pathways allows plants to fine tune phytohormone responses for seasonal and shade-appropriate growth regulation.

Research on regulation of cellulases and hemicellulases gene expression may be very useful for increasing the production of these enzymes in their native producers. Mechanisms of gene regulation of cellulase and hemicellulase expression in filamentous fungi have been studied, mainly in Aspergillus and Trichoderma. The production of these extracellular enzymes is an energy-consuming process, so the enzymes are produced only under conditions in which the fungus needs to use plant polymers as an energy and carbon source. Moreover, production of many of these enzymes is coordinately regulated, and induced in the presence of the substrate polymers. In addition to induction by mono- and oligo-saccharides, genes encoding hydrolytic enzymes involved in plant cell wall deconstruction in filamentous fungi can be repressed during growth in the presence of easily metabolizable carbon sources, such as glucose. Carbon catabolite repression is an important mechanism to repress the production of plant cell wall degrading enzymes during growth on preferred carbon sources. This manuscript reviews the recent advancements in elucidation of molecular mechanisms responsible for regulation of expression of cellulase and hemicellulase genes in fungi.

Eukaryotic organisms possess a complex RNA-directed gene expression regulatory network allowing the production of unique gene expression patterns. A recent addition to the repertoire of RNA-based gene regulation is miRNA target decoys, endogenous RNA that can negatively regulate miRNA activity. miRNA decoys have been shown to be a valuable tool for understanding the function of several miRNA families in plants and invertebrates. Engineering and precise manipulation of an endogenous RNA regulatory network through modification of miRNA activity also affords a significant opportunity to achieve a desired outcome of enhanced plant development or response to environmental stresses. Here we report that expression of miRNA decoys as single or heteromeric non-cleavable microRNA (miRNA) sites embedded in either non-protein-coding or within the 3' untranslated region of protein-coding transcripts can regulate the expression of one or more miRNA targets. By altering the sequence of the miRNA decoy sites, we were able to attenuate miRNA inactivation, which allowed for fine regulation of native miRNA targets and the production of a desirable range of plant phenotypes. Thus, our results demonstrate miRNA decoys are a flexible and robust tool, not only for studying miRNA function, but also for targeted engineering of gene expression in plants. Computational analysis of the Arabidopsis transcriptome revealed a number of potential miRNA decoys, suggesting that endogenous decoys may have an important role in natural modulation of expression in plants.

Major secondary metabolites, including flavonoids, caffeine, and theanine, are important components of tea products and are closely related to the taste, flavor, and health benefits of tea. Secondary metabolite biosynthesis in Camellia sinensis is differentially regulated in different tissues during growth and development. Until now, little was known about the expression patterns of genes involved in secondary metabolic pathways or their regulatory mechanisms. This study aimed to generate expression profiles for C. sinensis tissues and to build a gene regulation model of the secondary metabolic pathways.

Argonaute (AGO) is the core component of RNA-induced silencing complex. The AGO gene family has been analyzed in various plant species; however, there is no report about AGOs in the well-known Traditional Chinese Medicine (TCM) plant, Salvia miltiorrhiza.

Considering the relevance of insect α-amylases and natural α-amylase inhibitors present in plants to protect against insect damage, we investigated the effect of white bean and rapeseed protein extracts on digestive α-amylase gene expression of the Colorado potato beetle, Leptinotarsa decemlineata (Say). For this purpose, in vitro and in vivo trials were performed to determine the inhibitory activity of seed proteins on the third and fourth instar larvae. In both trials, the significant inhibitory effect of each extracts on the third and fourth instar larval α-amylase activity and considerable mortality in treatments were observed compared to control trials. In the RT-qPCR, expression ratio demonstrated that the α-amylase gene of two different larval stages grown on both proteins treated leaves had significantly differentiated expression and was up-regulated in third instar larvae and down-regulated in fourth instar larvae compared to control. Results suggest that the hyper-production of α-amylase in third instar larvae is elicited to compensate for the enzyme activity inhibition at an earlier stage and also down-regulation suggests the existence of a negative feedback of plant proteins on the last instar larvae via impaired food intake and digestive α-amylase activity in Colorado potato beetle. Therefore, disruption of the insect's digestive physiology by plant defensive proteins can be considered in the development of innovative controlling methods of this crucial potato pest.