Search Results (18)

Plants have evolved a complex, multilayered immune system that integrates molecular recognition, signaling pathways, epigenetic regulation, and small RNA-mediated control. Recent studies have shown that DNA-level regulatory mechanisms, such as RNA-directed DNA methylation (RdDM), histone modifications, and chromatin remodeling, are critical for modulating immune gene expression, allowing for rapid and accurate pathogen-defense responses. The epigenetic landscape not only maintains immunological homeostasis but also promotes stress-responsive transcription via stable chromatin modifications. These changes contribute to immunological priming, a process in which earlier exposure to pathogens or abiotic stress causes a heightened state of preparedness for future encounters. Small RNAs, including siRNAs, miRNAs, and phasiRNAs, are essential for gene silencing before and after transcription, fine-tuning immune responses, and inhibiting negative regulators. These RNA molecules interact closely with chromatin features, influencing histone acetylation/methylation (e.g., H3K4me3, H3K27me3) and guiding DNA methylation patterns. Epigenetically encoded immune memory can be stable across multiple generations, resulting in the transgenerational inheritance of stress resilience. Such memory effects have been observed in rice, tomato, maize, and Arabidopsis. This review summarizes new findings on short RNA biology, chromatin-level immunological control, and epigenetic memory in plant defense. Emerging technologies, such as ATAC-seq (Assay for Transposase-Accessible Chromatin using Sequencing), ChIP-seq (Chromatin Immunoprecipitation followed by Sequencing), bisulfite sequencing, and CRISPR/dCas9-based epigenome editing, are helping researchers comprehend these pathways. These developments hold an opportunity for establishing epigenetic breeding strategies that target the production of non-GMO, stress-resistant crops for sustainable agriculture. Full article

Small RNAs (sRNAs) are pivotal in regulating gene expression and are involved in a diverse array of biological processes. Among these, microRNAs (miRNAs) and phased small interfering RNAs (phasiRNAs) have been extensively investigated over the past decades. We conducted an in-depth analysis of deep sequencing data from the gymnosperm Ginkgo biloba, encompassing sRNA, transcriptome, and degradome libraries. Our analysis identified a total of 746 miRNAs and 654 phasiRNA precursor (PHAS) loci, with 526 (80%) of the PHAS loci predicted to be triggered by 515 miRNAs (69%). Several miRNA-PHAS modules, particularly the miR159/miR319-PHAS module, were found to potentially regulate reproductive development by targeting GAMYB genes and triggering phasiRNA biogenesis. The miR390-PHAS module appears to be involved in flavonoid biosynthesis by targeting key enzyme genes such as chalcone synthase (CHS) and anthocyanin synthase (ANS). Through target gene identification and coexpression analysis, we uncovered two distinct models of complex regulatory networks: growth-related factors like ARF and GRF seem to be regulated exclusively by miRNAs (Model 1), while certain disease resistance-related genes are predicted to be regulated by both miRNAs and phasiRNAs (Model 2), indicating diverse regulatory mechanisms across different biological processes. Overall, our study provides a comprehensive annotation of miRNA and PHAS loci in G. biloba and elucidates a post-transcriptional regulatory network, offering novel insights into sRNA research in gymnosperms. Full article

Culex spp. mosquitoes transmit several pathogens concerning public health, including West Nile virus and Saint Louis encephalitis virus. Understanding the antiviral immune system of Culex spp. mosquitoes is important for reducing the transmission of these viruses. Mosquitoes rely on RNA interference (RNAi) to control viral replication. While the siRNA pathway in mosquitoes is heavily studied, less is known about the piRNA pathway. The piRNA pathway in mosquitoes has recently been connected to mosquito antiviral immunity. In Aedes aegypti, Piwi4 has been implicated in antiviral responses. The antiviral role of the piRNA pathway in Culex spp. mosquitoes is understudied compared to Ae. aegypti. Here, we aimed to identify the role of PIWI genes and piRNAs in Culex quinquefasciatus and Culex tarsalis cells during virus infection. We examined the effect of PIWI gene silencing on virus replication of two arboviruses and three insect-specific viruses in Cx. quinquefasciatus derived cells (Hsu) and Cx. tarsalis derived (CT) cells. We show that Piwi4 is antiviral against the La Crosse orthobunyavirus (LACV) in Hsu and CT cells, and the insect-specific rhabdovirus Merida virus (MERDV) in Hsu cells. None of the silenced PIWI genes impacted replication of the two flaviviruses Usutu virus (USUV) and Calbertado virus, or the phasivirus Phasi-Charoen-like virus. We further used small RNA sequencing to determine that LACV-derived piRNAs, but not USUV-derived piRNAs were generated in Hsu cells and that PIWI gene silencing resulted in a small reduction in vpiRNAs. Finally, we determined that LACV-derived DNA was produced in Hsu cells during infection, but whether this viral DNA is required for vpiRNA production remains unclear. Overall, we expanded our knowledge on the piRNA pathway and how it relates to the antiviral response in Culex spp mosquitoes. Full article

Sexuality and apomixis are interconnected plant reproductive routes possibly behaving as polyphenic traits under the influence of the environment. In the subtropical grass Paspalum notatum, one of the controllers of apospory, a main component of gametophytic apomixis reproduction, is TRIMETHYLGUANOSINE SYNTHASE 1 (TGS1), a multifunctional gene previously associated with RNA cleavage regulation (including mRNA splicing as well as rRNA and miRNA processing), transcriptional modulation and the establishment of heterochromatin. In particular, the downregulation of TGS1 induces a sexuality decline and the emergence of aposporous-like embryo sacs. The present work was aimed at identifying TGS1 target RNAs expressed during reproductive development of Paspalum notatum. First, we mined available RNA databases originated from spikelets of sexual and apomictic plants, which naturally display a contrasting TGS1 representation, to identify differentially expressed mRNA splice variants and miRNAs. Then, the role of TGS1 in the generation of these particular molecules was investigated in antisense tgs1 sexual lines. We found that CHLOROPHYLL A-B BINDING PROTEIN 1B-21 (LHC Ib-21, a component of the chloroplast light harvesting complex), QUI-GON JINN (QGJ, encoding a MAP3K previously associated with apomixis) and miR2275 (a meiotic 24-nt phasi-RNAs producer) are directly or indirectly targeted by TGS1. Our results point to a coordinated control exercised by signal transduction and siRNA machineries to induce the transition from sexuality to apomixis. Full article

Plant small RNAs (sRNAs) are a heterogeneous group of noncoding RNAs with a length of 20–24 nucleotides that are widely studied due to their importance as major regulators in various biological processes. sRNAs are divided into two main classes—microRNAs (miRNAs) and small interfering RNAs (siRNAs)—which differ in their biogenesis and functional pathways. Their identification and enrichment with new structural variants would not be possible without the use of various high-throughput sequencing (NGS) techniques, allowing for the detection of the total population of sRNAs in plants. Classifying sRNAs and predicting their functional role based on such high-performance datasets is a nontrivial bioinformatics task, as plants can generate millions of sRNAs from a variety of biosynthetic pathways. Over the years, many computing tools have been developed to meet this challenge. Here, we review more than 35 tools developed specifically for plant sRNAs over the past few years and explore some of their basic algorithms for performing tasks related to predicting, identifying, categorizing, and quantifying individual sRNAs in plant samples, as well as visualizing the results of these analyzes. We believe that this review will be practical for biologists who want to analyze their plant sRNA datasets but are overwhelmed by the number of tools available, thus answering the basic question of how to choose the right one for a particular study. Full article

Crop domestication, which gives rise to a number of desirable agronomic traits, represents a typical model system of plant evolution. Numerous genomic evidence has proven that noncoding RNAs such as microRNAs and phasiRNAs, as well as protein-coding genes, are selected during crop domestication. However, limited data shows plant long noncoding RNAs (lncRNAs) are also involved in this biological process. In this study, we performed strand-specific RNA sequencing of cultivated rice Oryza sativa ssp. japonica and O. sativa ssp. indica, and their wild progenitor O. rufipogon. We identified a total of 8528 lncRNAs, including 4072 lncRNAs in O. rufipogon, 2091 lncRNAs in japonica rice, and 2365 lncRNAs in indica rice. The lncRNAs expressed in wild rice were revealed to be shorter in length and had fewer exon numbers when compared with lncRNAs from cultivated rice. We also identified a number of conserved lncRNAs in the wild and cultivated rice. The functional study demonstrated that several of these conserved lncRNAs are associated with domestication-related traits in rice. Our findings revealed the feature and conservation of lncRNAs during rice domestication and will further promote functional studies of lncRNAs in rice. Full article

Bois noir is the most widespread phytoplasma grapevine disease in Europe. It is associated with ‘Candidatus Phytoplasma solani’, but molecular interactions between the causal pathogen and its host plant are not well understood. In this work, we combined the analysis of high-throughput RNA-Seq and sRNA-Seq data with interaction network analysis for finding new cross-talks among pathways involved in infection of grapevine cv. Zweigelt with ‘Ca. P. solani’ in early and late growing seasons. While the early growing season was very dynamic at the transcriptional level in asymptomatic grapevines, the regulation at the level of small RNAs was more pronounced later in the season when symptoms developed in infected grapevines. Most differentially expressed small RNAs were associated with biotic stress. Our study also exposes the less-studied role of hormones in disease development and shows that hormonal balance was already perturbed before symptoms development in infected grapevines. Analysis at the level of communities of genes and mRNA-microRNA interaction networks revealed several new genes (e.g., expansins and cryptdin) that have not been associated with phytoplasma pathogenicity previously. These novel actors may present a new reference framework for research and diagnostics of phytoplasma diseases of grapevine. Full article

MicroRNAs (miRNAs) are a class of small RNAs (sRNAs) that repress gene expression via high complementary binding sites in target mRNAs (messenger RNAs). Many miRNAs are ancient, and their intricate integration into gene expression programs have been fundamental for plant life, controlling developmental programs and executing responses to biotic/abiotic cues. Additionally, there are many less conserved miRNAs in each plant species, raising the possibility that the functional impact of miRNAs extends into virtually every aspect of plant biology. This Special Issue of Plants presents papers that investigate the function and mechanism of miRNAs in controlling development and abiotic stress response. This includes how miRNAs adapt plants to nutrient availability, and the silencing machinery that is responsible for this. Several papers profile changes in miRNA abundances during stress, and another study raises the possibility of circular RNAs acting as endogenous decoys to sequester and inhibit plant miRNA function. These papers act as foundational studies for the more difficult task ahead of determining the functional significance of these changes to miRNA abundances, or the presence of these circular RNAs. Finally, how miRNAs trigger the production of secondary sRNAs is reviewed, along with the potential agricultural impact of miRNAs and these secondary sRNA in the exemplar crop maize. Full article

Potato virus Y (PVY) isolate PVY^C-to induces growth reduction and foliar symptoms in tomato, but new vegetation displays symptom recovery at a later stage. In order to investigate the role of micro(mi)RNA and secondary small(s)RNA-regulated mechanisms in tomato defenses against PVY, we performed sRNA sequencing from healthy and PVY^C-to infected tomato plants at 21 and 30 days post-inoculation (dpi). A total of 792 miRNA sequences were obtained, among which were 123 canonical miRNA sequences, many isomiR variants, and 30 novel miRNAs. MiRNAs were mostly overexpressed in infected vs. healthy plants, whereas only a few miRNAs were underexpressed. Increased accumulation of isomiRs was correlated with viral infection. Among miRNA targets, enriched functional categories included resistance (R) gene families, transcription and hormone factors, and RNA silencing genes. Several 22-nt miRNAs were shown to target R genes and trigger the production of 21-nt phased sRNAs (phasiRNAs). Next, 500 phasiRNA-generating loci were identified, and were shown to be mostly active in PVY-infected tissues and at 21 dpi. These data demonstrate that sRNA-regulated host responses, encompassing miRNA alteration, diversification within miRNA families, and phasiRNA accumulation, regulate R and disease-responsive genes. The dynamic regulation of miRNAs and secondary sRNAs over time suggests a functional role of sRNA-mediated defenses in the recovery phenotype. Full article

Although the regulatory function of miRNAs and their targets have been characterized in model plants, a possible underlying role in the cotton response to herbivore infestation has not been determined. To investigate this, we performed small RNA and degradome sequencing between resistant and susceptible cotton cultivar following infestation with the generalist herbivore whitefly. In total, the 260 miRNA families and 241 targets were identified. Quantitative-PCR analysis revealed that several miRNAs and their corresponding targets exhibited dynamic spatio-temporal expression patterns. Moreover, 17 miRNA precursors were generated from 29 long intergenic non-coding RNA (lincRNA) transcripts. The genome-wide analysis also led to the identification of 85 phased small interfering RNA (phasiRNA) loci. Among these, nine PHAS genes were triggered by miR167, miR390, miR482a, and two novel miRNAs, including those encoding a leucine-rich repeat (LRR) disease resistance protein, an auxin response factor (ARF) and MYB transcription factors. Through combined modeling and experimental data, we explored and expanded the miR390-tasiARF cascade during the cotton response to whitefly. Virus-induced gene silencing (VIGS) of ARF8 from miR390 target in whitefly-resistant cotton plants increased auxin and jasmonic acid (JA) accumulation, resulting in increased tolerance to whitefly infestation. These results highlight the provides a useful transcriptomic resource for plant-herbivore interaction. Full article

Small RNA (sRNA) population in plants comprises of primarily micro RNAs (miRNAs) and small interfering RNAs (siRNAs). MiRNAs play important roles in plant growth and development. The miRNA-derived secondary siRNAs are usually known as phased siRNAs, including phasiRNAs and tasiRNAs. The miRNA and phased siRNA biogenesis mechanisms are highly conserved in plants. However, their functional conservation and diversification may differ in maize. In the past two decades, lots of miRNAs and phased siRNAs have been functionally identified for curbing important maize agronomic traits, such as those related to developmental timing, plant architecture, sex determination, reproductive development, leaf morphogenesis, root development and nutrition, kernel development and tolerance to abiotic stresses. In contrast to Arabidopsis and rice, studies on maize miRNA and phased siRNA biogenesis and functions are limited, which restricts the small RNA-based fundamental and applied studies in maize. This review updates the current status of maize miRNA and phased siRNA mechanisms and provides a survey of our knowledge on miRNA and phased siRNA functions in controlling agronomic traits. Furthermore, improvement of those traits through manipulating the expression of sRNAs or their targets is discussed. Full article

Background: Cold tolerance is important for plants’ geographical distribution and survival in extreme seasonal variations of climate. However, Populus simonii × P. nigra shows wide adaptability and strong cold resistance. Transcriptional and post-transcriptional regulation of cold-responsive genes is crucial for cold tolerance in plants. To understand the roles of regulatory RNAs under cold induction in Populus simonii × P. nigra, we constructed cDNA and small RNA libraries from leaf buds treated or not with −4 °C for 8 h for analysis. Results: Through high-throughput sequencing and differential expression analysis, 61 miRNAs and 1229 DEGs were identified under cold induction condition in Populus simonii × P. nigra. The result showed that miR167a, miR1450, miR319a, miR395b, miR393a-5p, miR408-5p, and miR168a-5p were downregulated, whereas transcription level of miR172a increased under the cold treatment. Thirty-one phased-siRNA were also obtained (reads ≥ 4) and some of them proceeded from TAS3 loci. Analysis of the differentially expressed genes (DEGs) showed that transcription factor genes such as Cluster-15451.2 (putative MYB), Cluster-16493.29872 (putative bZIP), Cluster-16493.29175 (putative SBP), and Cluster-1378.1 (putative ARF) were differentially expressed in cold treated and untreated plantlets of Populus simonii × P. nigra. Integrated analysis of miRNAs and transcriptome showed miR319, miR159, miR167, miR395, miR390, and miR172 and their target genes, including MYB, SBP, bZIP, ARF, LHW, and ATL, were predicted to be involved in ARF pathway, SPL pathway, DnaJ related photosystem II, and LRR receptor kinase, and many of them are known to resist chilling injury. Particularly, a sophisticated regulatory model including miRNAs, phasiRNAs, and targets of them was set up. Conclusions: Integrated analysis of miRNAs and transcriptome uncovered the complicated regulation of the tolerance of cold in Populus simonii × P. nigra. MiRNAs, phasiRNAs, and gene-encoded transcription factors were characterized at a whole genome level and their expression patterns were proved to be complementary. This work lays a foundation for further research of the pathway of sRNAs and regulatory factors involved in cold tolerance. Full article

In plants, proper development and response to abiotic and biotic stimuli requires an orchestrated regulation of gene expression. Small RNAs (sRNAs) are key molecules involved in this process, leading to downregulation of their target genes. Two main classes of sRNAs exist, the small interfering RNAs (siRNAs) and microRNAs (miRNAs). The role of the latter class in plant development and physiology is well known, with many examples of how miRNAs directly impact the expression of genes in cells where they are produced, with dramatic consequences to the life of the plant. However, there is an aspect of miRNA biology that is still poorly understood. In some cases, miRNA targeting can lead to the production of secondary siRNAs from its target. These siRNAs, which display a characteristic phased production pattern, can act in cis, reinforcing the initial silencing signal set by the triggering miRNA, or in trans, affecting genes that are unrelated to the initial target. In this review, the mechanisms and implications of this process in the gene regulation mediated by miRNAs will be discussed. This work will also explore techniques for gene silencing in plants that are based on this unique pathway. Full article

Small RNA mediated responses are essential for antiviral defence in mosquitoes, however, they appear to differ per virus-vector combination. To further investigate the diversity of small RNA responses against viruses in mosquitoes, we applied a small RNA deep sequencing approach on five mosquito cell lines: Culex tarsalis CT cells, Aedes albopictus U4.4 and C6/36 cells, Ae. aegypti Aag2 cells (cleared from cell fusing agent virus and Culex Y virus (CYV) by repetitive dsRNA transfections) and Ae. pseudoscutellaris AP-61 cells. De novo assembly of small RNAs revealed the presence of Phasi Charoen-like virus (PCLV), Calbertado virus, Flock House virus and a novel narnavirus in CT cells, CYV in U4.4 cells, and PCLV in Aag2 cells, whereas no insect-specific viruses (ISVs) were detected in C6/36 and AP-61 cells. Next, we investigated the small RNA responses to the identified ISVs and to acute infection with the arthropod-borne West Nile virus (WNV). We demonstrate that AP-61 and C6/36 cells do not produce siRNAs to WNV infection, suggesting that AP-61, like C6/36, are Dicer-2 deficient. CT cells produced a strong siRNA response to the persistent ISVs and acute WNV infection. Interestingly, CT cells also produced viral PIWI-interacting (pi)RNAs to PCLV, but not to WNV or any of the other ISVs. In contrast, in U4.4 and Aag2 cells, WNV siRNAs, and pi-like RNAs without typical ping-pong piRNA signature were observed, while this signature was present in PCLV piRNAs in Aag2 cells. Together, our results demonstrate that mosquito small RNA responses are strongly dependent on both the mosquito cell type and/or the mosquito species and family of the infecting virus. Full article

Mycoviruses belonging to the family Hypoviridae cause persistent infection of many different host fungi. We previously determined that the white mold fungus, Sclerotinia sclerotiorum, infected with Sclerotinia sclerotiorum hypovirus 2-L (SsHV2-L) exhibits reduced virulence, delayed/reduced sclerotial formation, and enhanced production of aerial mycelia. To gain better insight into the cellular basis for these changes, we characterized changes in mRNA and small RNA (sRNA) accumulation in S. sclerotiorum to infection by SsHV2-L. A total of 958 mRNAs and 835 sRNA-producing loci were altered after infection by SsHV2-L, among which >100 mRNAs were predicted to encode proteins involved in the metabolism and trafficking of carbohydrates and lipids. Both S. sclerotiorum endogenous and virus-derived sRNAs were predominantly 22 nt in length suggesting one dicer-like enzyme cleaves both. Novel classes of endogenous small RNAs were predicted, including phasiRNAs and tRNA-derived small RNAs. Moreover, S. sclerotiorum phasiRNAs, which were derived from noncoding RNAs and have the potential to regulate mRNA abundance in trans, showed differential accumulation due to virus infection. tRNA fragments did not accumulate differentially after hypovirus infection. Hence, in-depth analysis showed that infection of S. sclerotiorum by a hypovirulence-inducing hypovirus produced selective, large-scale reprogramming of mRNA and sRNA production. Full article