Activation of stress-response genes by retrograde signaling-mediated destabilization of nuclear importin IMPα-9 and its interactor TPR2.

Stress-induced retrograde signal transmission from the plastids to the nucleus has long puzzled plant biologists. To address this, we performed a suppressor screen of the ceh1 mutant, which contains elevated 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate (MEcPP) levels, and identified the gain-of-function mutant impα-9, which shows reversed dwarfism and suppressed expression of stress-response genes in the ceh1 background despite heightened MEcPP. Subsequent genetic and biochemical analyses established that the accumulation of MEcPP initiates an upsurge in Arabidopsis SKP1-like 1 (ASK1) abundance, a pivotal component in the proteasome degradation pathway. This increase in ASK1 prompts the degradation of IMPα-9. Moreover, we uncovered a protein-protein interaction between IMPα-9 and TPR2, a transcriptional co-suppressor and found that a reduction in IMPα-9 levels coincides with a decrease in TPR2 abundance. Significantly, the interaction between IMPα-9 and TPR2 was disrupted in impα-9 mutants, highlighting the critical role of a single amino acid alteration in maintaining their association. Disruption of their interaction results in the reversal of MEcPP-associated phenotypes. Chromatin immunoprecipitation coupled with sequencing analyses revealed that TPR2 binds globally to stress-response genes and suggested that IMPα-9 associates with the chromatin. They function together to suppress the expression of stress-response genes under normal conditions, but this suppression is alleviated in response to stress through the degradation of the suppressing machinery. The biological relevance of our discoveries was validated under high light stress, marked by MEcPP accumulation, elevated ASK1 levels, IMPα-9 degredation, reduced TPR2 abundance, and subsequent activation of a network of stress-response genes. In summary, our study collectively unveils fresh insights into plant adaptive mechanisms, highlighting intricate interactions among retrograde signaling, the proteasome, and nuclear transport machinery.

Exploring the Genomic Landscape: An In-depth ChIP-seq Analysis Protocol for Uncovering Protein-DNA Interactions.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a widely employed technique for investigating protein-DNA interactions. However, the absence of a standardized and clear workflow necessitates researchers to independently assemble methodologies from diverse resources. This lack of uniformity hampers reproducibility and makes version control a complex endeavor, thereby limiting the accessibility of ChIP-seq analyses to individuals with extensive training in bioinformatics. In light of these challenges, we have developed an executable protocol that addresses these limitations. Our protocol encompasses all aspects of ChIP-seq analysis, ranging from quality control of raw reads to peak calling and downstream functional analyses. We have implemented two distinct approaches for peak calling, providing researchers with flexibility to choose the most suitable method for their specific experimental needs. This protocol will contribute to the scientific community by providing a standardized and clear resource that will enhance the reproducibility and accessibility of ChIP-seq analyses. © 2023 Wiley Periodicals LLC. Basic Protocol: ChIP-seq analysis workflow Alternative Protocol: Call differentially enriched peaks by using MACS3.

microRNA production in Arabidopsis.

In plants, microRNAs (miRNAs) associate with ARGONAUTE (AGO) proteins and act as sequence-specific repressors of target gene expression, at the post-transcriptional level through target transcript cleavage and/or translational inhibition. MiRNAs are mainly transcribed by DNA-dependent RNA polymerase II (POL II) and processed by DICER LIKE1 (DCL1) complex into 21∼22 nucleotide (nt) long. Although the main molecular framework of miRNA biogenesis and modes of action have been established, there are still new requirements continually emerging in the recent years. The studies on the involvement factors in miRNA biogenesis indicate that miRNA biogenesis is not accomplished separately step by step, but is closely linked and dynamically regulated with each other. In this article, we will summarize the current knowledge on miRNA biogenesis, including MIR gene transcription, primary miRNA (pri-miRNA) processing, miRNA AGO1 loading and nuclear export; and miRNA metabolism including methylation, uridylation and turnover. We will describe how miRNAs are produced and how the different steps are regulated. We hope to raise awareness that the linkage between different steps and the subcellular regulation are becoming important for the understanding of plant miRNA biogenesis and modes of action.

Arabidopsis RBV is a conserved WD40 repeat protein that promotes microRNA biogenesis and ARGONAUTE1 loading.

MicroRNAs (miRNAs) play crucial roles in gene expression regulation through RNA cleavage or translation repression. Here, we report the identification of an evolutionarily conserved WD40 domain protein as a player in miRNA biogenesis in Arabidopsis thaliana. A mutation in the REDUCTION IN BLEACHED VEIN AREA (RBV) gene encoding a WD40 domain protein led to the suppression of leaf bleaching caused by an artificial miRNA; the mutation also led to a global reduction in the accumulation of endogenous miRNAs. The nuclear protein RBV promotes the transcription of MIR genes into pri-miRNAs by enhancing the occupancy of RNA polymerase II (Pol II) at MIR gene promoters. RBV also promotes the loading of miRNAs into AGO1. In addition, RNA-seq revealed a global splicing defect in the mutant. Thus, this evolutionarily conserved, nuclear WD40 domain protein acts in miRNA biogenesis and RNA splicing.

Secrets of the MIR172 family in plant development and flowering unveiled.

In plants, conserved microRNAs (miRNAs) tend to be encoded by gene families with multiple members. Two recent studies interrogated the functions of the 5-member MIR172 family in Arabidopsis and revealed complexities and intricacies of gene regulatory networks underlying floral transition.

Linking key steps of microRNA biogenesis by TREX-2 and the nuclear pore complex in Arabidopsis.

Unlike in metazoans, the stepwise biogenesis of microRNAs (miRNAs) in plants occurs within the nucleus. Whether or how the major steps in miRNA biogenesis are coordinated is largely unknown. Here we show that the plant TREX-2 complex promotes multiple steps in miRNA biogenesis, including transcription, processing and nuclear export. THP1 and SAC3A-the core subunits of TREX-2-interact and colocalize with RNA polymerase II to promote the transcription of MIR genes in the nucleoplasm. TREX-2 interacts with the microprocessor component SERRATE and promotes the formation of dicing bodies in the nucleoplasm. THP1 also interacts and colocalizes with the nucleoporin protein NUP1 at the nuclear envelope. NUP1 and THP1 promote the nuclear export of miRNAs and ARGONAUTE1. These results suggest that TREX-2 coordinates the transcription, processing and export steps in miRNA biogenesis to ensure efficient miRNA production.

A Phytophthora Effector Suppresses Trans-Kingdom RNAi to Promote Disease Susceptibility.

RNA silencing (RNAi) has a well-established role in anti-viral immunity in plants. The destructive eukaryotic pathogen Phytophthora encodes suppressors of RNAi (PSRs), which enhance plant susceptibility. However, the role of small RNAs in defense against eukaryotic pathogens is unclear. Here, we show that Phytophthora infection of Arabidopsis leads to increased production of a diverse pool of secondary small interfering RNAs (siRNAs). Instead of regulating endogenous plant genes, these siRNAs are found in extracellular vesicles and likely silence target genes in Phytophthora during natural infection. Introduction of a plant siRNA in Phytophthora leads to developmental deficiency and abolishes virulence, while Arabidopsis mutants defective in secondary siRNA biogenesis are hypersusceptible. Notably, Phytophthora effector PSR2 specifically inhibits secondary siRNA biogenesis in Arabidopsis and promotes infection. These findings uncover the role of siRNAs as antimicrobial agents against eukaryotic pathogens and highlight a defense/counter-defense arms race centered on trans-kingdom gene silencing between hosts and pathogens.

The Arabidopsis MOS4-Associated Complex Promotes MicroRNA Biogenesis and Precursor Messenger RNA Splicing.

In Arabidopsis thaliana, the MOS4-ASSOCIATED COMPLEX (MAC) is required for defense and development. The evolutionarily conserved, putative RNA helicase MAC7 is a component of the Arabidopsis MAC, and the human MAC7 homolog, Aquarius, is implicated in pre-mRNA splicing. Here, we show that mac7-1, a partial loss-of-function mutant in MAC7, and two other MAC subunit mutants, mac3a mac3b and prl1 prl2 (pleiotropic regulatory locus), exhibit reduced microRNA (miRNA) levels, indicating that MAC promotes miRNA biogenesis. The mac7-1 mutant shows reduced primary miRNA (pri-miRNA) levels without affecting miRNA gene (MIR) promoter activity or the half-life of pri-miRNA transcripts. As a nuclear protein, MAC7 is not concentrated in dicing bodies, but it affects the localization of HYPONASTIC LEAVES1 (HYL1), a key protein in pri-miRNA processing, to dicing bodies. Immunoprecipitation of HYL1 retrieved 11 known MAC subunits, including MAC7, indicating association between HYL1 and MAC. We propose that MAC7 links MIR transcription to pri-miRNA processing. RNA-seq analysis showed that downregulated genes in MAC subunit mutants are mostly involved in plant defense and stimulus responses, confirming a role of MAC in biotic and abiotic stress responses. We also discovered global intron retention defects in mutants in three subunits of MAC, thus linking MAC function to splicing in Arabidopsis.

Reactivity toward Bifidobacterium longum and Enterococcus hirae demonstrate robust CD8+ T cell response and better prognosis in HBV-related hepatocellular carcinoma.

Recent studies suggest that several bacterial species are involved in tumor immunosurveillance and antitumor immunity. The role of bacteria in immune responses in HBV-related hepatocellular carcinoma (HCC) patients is still unknown. In this study, we examined the bacteria-reactive CD8+ T cell response in patients with HBV-related HCC. We found that circulating CD8+ T cells from healthy individuals demonstrated minimal or zero specificity toward a series of commensals and bacteria previously associated with antitumor effects, including Escherichia coli, Enterococcus faecium, Bifidobacterium longum, Bacteroides fragilis, and Enterococcus hirae. In contrast, the circulating CD8+ T cells from HBV-related HCC patients presented significantly elevated bacteria-reactive responses, albeit with high variations among different HCC individuals. Reactivity toward bacteria was also identified in tumor-infiltrating CD8+ T cells. These bacteria-reactive responses were not primarily induced by TLR ligand, but were dependent on the presence of antigen-presenting monocytes, and were MHC class I-restricted. Interestingly, we observed that the CD8+ T cell-to-Foxp3+ regulatory T cell ratio was positively correlated with the proportions of Bifidobacterium longum-reactive and Enterococcus hirae-reactive CD8+ T cells, while the frequency of PD-1+ CD8+ T cells was negatively correlated with the frequency of Enterococcus hirae-reactive CD8+ T cells. Furthermore, the disease-free survival time of HCC patients after tumor resection was positively correlated with the frequencies of Bifidobacterium longum-reactive and Enterococcus hirae-reactive CD8+ T cells. Together, these results suggested that certain bacterial species might present valuable antitumor effects.

Biogenesis of phased siRNAs on membrane-bound polysomes in Arabidopsis.

Small RNAs are central players in RNA silencing, yet their cytoplasmic compartmentalization and the effects it may have on their activities have not been studied at the genomic scale. Here we report that Arabidopsis microRNAs (miRNAs) and small interfering RNAs (siRNAs) are distinctly partitioned between the endoplasmic reticulum (ER) and cytosol. All miRNAs are associated with membrane-bound polysomes (MBPs) as opposed to polysomes in general. The MBP association is functionally linked to a deeply conserved and tightly regulated activity of miRNAs - production of phased siRNAs (phasiRNAs) from select target RNAs. The phasiRNA precursor RNAs, thought to be noncoding, are on MBPs and are occupied by ribosomes in a manner that supports miRNA-triggered phasiRNA production, suggesting that ribosomes on the rough ER impact siRNA biogenesis. This study reveals global patterns of cytoplasmic partitioning of small RNAs and expands the known functions of ribosomes and ER.

Evolution and protein interactions of AP2 proteins in Brassicaceae: Evidence linking development and environmental responses.

Plants have evolved a large number of transcription factors (TF), which are enriched among duplicate genes, highlighting their roles in complex regulatory networks. The APETALA2/EREBP-like genes constitute a large plant TF family and participate in development and stress responses. To probe the conservation and divergence of AP2/EREBP genes, we analyzed the duplication patterns of this family in Brassicaceae and identified interacting proteins of representative Arabidopsis AP2/EREBP proteins. We found that many AP2/EREBP duplicates generated early in Brassicaceae history were quickly lost, but many others were retained in all tested Brassicaceae species, suggesting early functional divergence followed by persistent conservation. In addition, the sequences of the AP2 domain and exon numbers were highly conserved in rosids. Furthermore, we used 16 A. thaliana AP2/EREBP proteins as baits in yeast screens and identified 1,970 potential AP2/EREBP-interacting proteins, with a small subset of interactions verified in planta. Many AP2 genes also exhibit reduced expression in an anther-defective mutant, providing a possible link to developmental regulation. The putative AP2-interacting proteins participate in many functions in development and stress responses, including photomorphogenesis, flower development, pathogenesis, drought and cold responses, abscisic acid and auxin signaling. Our results present the AP2/EREBP evolution patterns in Brassicaceae, and support a proposed interaction network of AP2/EREBP proteins and their putative interacting proteins for further study.

Arabidopsis TOE proteins convey a photoperiodic signal to antagonize CONSTANS and regulate flowering time.

Plants flower in an appropriate season to allow sufficient vegetative development and position flower development in favorable environments. In Arabidopsis, CONSTANS (CO) and FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) promote flowering by inducing FLOWER LOCUS T (FT) expression in the long-day afternoon. The CO protein is present in the morning but could not activate FT expression due to unknown negative mechanisms, which prevent premature flowering before the day length reaches a threshold. Here, we report that TARGET OF EAT1 (TOE1) and related proteins interact with the activation domain of CO and CO-like (COL) proteins and inhibit CO activity. TOE1 binds to the FT promoter near the CO-binding site, and reducing TOE function results in a morning peak of the FT mRNA. In addition, TOE1 interacts with the LOV domain of FKF1 and likely interferes with the FKF1-CO interaction, resulting in partial degradation of the CO protein in the afternoon to prevent premature flowering.

RceIF5A, encoding an eukaryotic translation initiation factor 5A in Rosa chinensis, can enhance thermotolerance, oxidative and osmotic stress resistance of Arabidopsis thaliana.

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein known to contain the unusual amino acid hypusine. It is a highly conserved protein found in all eukaryotic organisms. Although originally identified as a translation initiation factor, recent studies suggest that eIF5A is mainly involved in translation elongation, mRNA turnover and decay, cell proliferation, and programmed cell death. However, the precise cellular function of eIF5A remains largely unknown, especially in plants. Here, we report the identification and characterization of RceIF5A from Rosa chinensis. RceIF5A expression is up-regulated in Rosa chinensis under high temperature, and oxidative and osmotic stress conditions. We produced transgenic Arabidopsis that constitutively enhanced or suppressed expression of RceIF5A. The RceIF5A over-expression plants exhibited increased resistance to heat, and oxidative and osmotic stresses, while the suppressed expression plants (three AteIF5A isoforms in Arabidopsis were down-regulated) showed more susceptibility to these stresses. These results reveal a new physiological role for eIF5A in plants and contribute to the elucidation of the molecular mechanisms involved in the stress response pathway.

One novel mitochondrial citrate synthase from Oryza sativa L. can enhance aluminum tolerance in transgenic tobacco.

Rice exhibits the greatest aluminum (Al) tolerance compared with other cereals such as wheat, barley, maize, etc. A full-length gene, OsCS1, encoding citrate synthase, which is highly induced by aluminum toxicity in rice (Oryza sativa L.), was isolated. Sequence analysis and the sub-cellular localization of OsCS1 in yeast revealed that it is a mitochondrial citrate synthase. OsCS1 was induced by Al toxicity. Several independent transgenic tobacco lines expressing OsCS 1 exhibited increased citrate efflux and extraordinary Al tolerance. Possible outlook for OsCS1 to be applied to enhance plant tolerance to Al toxicity was also discussed.