Gene Silencing In Plants Research Articles

A CC-NB-ARC-LRR Gene Regulates Bract Morphology in Cotton.

Bracts are leaf-like structures in flowering plants. They serve multiple functions such as attracting pollinators, aiding tolerance of abiotic stressors, and conducting photosynthesis. While previous studies extensively examine bract function, the molecular mechanisms underlying bract growth remain unknown. Here, the map-based isolation and characterization of a crucial factor responsible for cotton bract development, identified from a mutant known as frego bract (fg), discovered by Frego in 1945 are presented. This gene, named Ghfg, encodes a CC-NB-ARC-LRR (CNL) family protein. Through analysis of bract form in plants with virus-induced gene silencing (VIGS) and transgenic plants, this gene is confirmed to be the causal gene under the fg locus. Furthermore, high-resolution single-cell transcriptomic landscape of cotton bracts is generated, which reveals differences related to auxin in proliferating cells from TM-1 and T582; differences in auxin distribution and ROS accumulation are experimentally verified. These findings suggest that GhFG is in a self-activated state in the fg mutant, and its activity leads to ROS accumulation that impacts auxin distribution and transport. Finally, an island cotton variety with the frego bract trait is developed, demonstrating a novel solution for reducing the high impurity rate caused by bract remnants.

Epigenetic gene silencing in plants: Insights from triplet repeat expansion in Arabidopsis.

Epigenetic gene silencing in plants: Insights from triplet repeat expansion in Arabidopsis.

Gene silencing in broomrapes and other parasitic plants of the Orobanchaceae family: mechanisms, considerations, and future directions.

Holoparasites of the Orobanchaceae family are devastating pests causing severe damage to many crop species and are nearly impossible to control with conventional methods. During past decades RNA interference (RNAi) has been seen as a promising approach to control various crop pests. The exchange of small RNAs (sRNAs) between crops and parasitic plants has been documented indicating a potential for the development of methods to protect them via the delivery of the sRNAs to parasites, called host-induced gene silencing (HIGS). Here we describe various approaches used for gene silencing in plants and suggest solutions to improve the long-distance movement of the silencing triggers to elevate the HIGS efficiency in parasitic plants. We also investigate the important biological processes during parasites life cycle with a focus on broomrape species, providing several appropriate target genes that can be used in, especially, multiplex gene silencing experiments. We also touch on how the application of nanoparticles can improve the stability and delivery of the silencing triggers, highlighting its potential for parasitic plants control. Finally, suggestions for further research and possible directions for RNAi in parasitic plants are provided.

Analysis of CYP701A1 genes in Gossypium species and functional characterization through gene silencing

Gibberellins (GA) are known to play crucial roles in various aspects of plant growth and development. The cytochrome P450 enzyme family is recognized for its significance in plant metabolic processes. Specifically, CYP701s, a subgroup of CYP71, encode ent-kaurene oxidase in the gibberellin synthesis pathway. In this study, we analyzed genomic data from 30 Gossypium species, including nine allotetraploid genomes (AD1-AD7, with two each for AD1 and AD2), 21 diploid genomes (A-G, K, with two A-genomes and 12 D-genomes), and Gossypioides kirkii genome as an outgroup for evolutionary analysis, totaling 31 genomes. Subsequently, 40 CYP701A1 genes were identified from various genomes and conducted a comprehensive analysis of their structure and evolution. Virus-induced gene silencing (VIGS) technology was utilized to knock out the GhCYP701A1 gene in Gossypium hirsutum ac TM-1. Subsequent analysis revealed changes in hormone content, with decreased gibberellin levels and notable increases in auxin, cytokinin, and jasmonic acid contents. Conversely, salicylic acid content decreased, while the precursor for ethylene synthesis, 1-aminocyclopropane-1-carboxylic acid (ACC), remained relatively stable. Transcriptome analysis of the gene silencing plants identified 15,962 differentially expressed genes, including 8,376 upregulated and 7,586 downregulated genes. Enrichment analysis through KEGG pathway analysis highlighted 'Plant hormone signal transduction' as a prominent pathway with 234 differentially expressed genes. The study provided insights into the function and regulatory network of the gene.

Effects of SvAPETALA1-5 gene on floral organ development in Senecio vulgaris.

Asteraceae is a large class of eudicots with complex capitulum, and little is known regarding the molecular regulation mechanism of flower development. APETALA1(AP1) belongs to the MADS-box gene family and plays a key role in plant floral induction and floral organ development. In this study, the bioinformatics and tissue-specific expression of AP1 homologous gene SvAP1-5 in Senecio vulgaris were analyzed. Based on VIGS technology, SvAP1-5 gene silencing plants were created, and SvAP1-5 was overexpressed in Solanum nigrum. The results of bioinformatics analysis showed that SvAP1-5 gene had typical MADS-box and K-box structure, and contains FUL motif and paleoAP1 motif at the C-terminal. SvAP1-5 belongs to the euFUL branch of AP1 gene. qRT-PCR results showed that SvAP1-5 was expressed in bracts, petals and carpels, and was highly expressed in carpels. Compared with the control group, SvAP1-5 gene silencing resulted in irregular petal dehiscence, increased stigma division, and carpel dysplasia. The fruit development of SvAP1-5 overexpressing S.nigrum plants was abnormal, and the hyperplastic tissue similar to fruit appeared. In summary, SvAP1-5 gene may be involved in the development of petals and carpels and plays an important role during the development of S.vulgaris.

Editing of upstream regulatory elements advances plant gene silencing.

Editing of upstream regulatory elements advances plant gene silencing.

Transcription elongation of the plant RNA polymerase IV is prone to backtracking.

RNA polymerase IV (Pol IV) forms a complex with RNA-directed RNA polymerase 2 (RDR2) to produce double-stranded RNA (dsRNA) precursors essential for plant gene silencing. In the "backtracking-triggered RNA channeling" model, Pol IV backtracks and delivers its transcript's 3' terminus to RDR2, which synthesizes dsRNA. However, the mechanisms underlying Pol IV backtracking and RNA protection from cleavage are unclear. Here, we determined cryo-electron microscopy structures of Pol IV elongation complexes at four states of its nucleotide addition cycle (NAC): posttranslocation, guanosine triphosphate-bound, pretranslocation, and backtracked states. The structures reveal that Pol IV maintains an open DNA cleft and kinked bridge helix in all NAC states, loosely interacts with the nucleoside triphosphate substrate, and barely contacts proximal backtracked nucleotides. Biochemical data indicate that Pol IV is inefficient in forward translocation and RNA cleavage. These findings suggest that Pol IV transcription elongation is prone to backtracking and incapable of RNA hydrolysis, ensuring efficient dsRNA production by Pol IV-RDR2.

OsNAC121 regulates root development, tillering, panicle morphology, and grain filling in rice plant.

Transcription factors in coordination with phytohormones form an intricate regulatory network modulating vital cellular mechanisms like development, growth and senescence in plants. In this study, we have functionally characterized the transcription factor OsNAC121 by developing gene silencing and overexpressing transgenic rice plants, followed by detailed analyses of the plant architecture. Transgenic lines exhibited remodelling in crown root development, lateral root structure and density, tiller height and number, panicle and grain morphologies, underpinning the imbalanced auxin: cytokinin ratio due to perturbed auxin transportation. Application of cytokinin, auxin and abscisic acid increased OsNAC121 gene expression nearly 17-, 6- and 91-folds, respectively. qRT-PCR results showed differential expressions of auxin and cytokinin pathway genes, implying their altered levels. A 47-fold higher expression level of OsNAC121 during milky stage in untransformed rice, compared to 14-day old shoot tissue, suggests its crucial role in grain filling; as evidenced by a large number of undeveloped grains produced by the gene silenced lines. Crippled gravitropic response by the transgenic plants indicates their impaired auxin transport. Bioinformatics revealed that OsNAC121 interacts with co-repressor (TOPLESS) proteins and forms a part of the inhibitor complex OsIAA10, an essential core component of auxin signalling pathway. Therefore, OsNAC121 emerges as an important regulator of various aspects of plant architecture through modulation of crosstalk between auxin and cytokinin, altering their concentration gradient in the meristematic zones, and consequently modifying different plant organogenesis processes.

In-locus gene silencing in plants using genome editing.

Gene silencing is crucial in crop breeding for desired trait development. RNA interference (RNAi) has been used widely but is limited by ectopic expression of transgenes and genetic instability. Introducing an upstream start codon (uATG) into the 5'untranslated region (5'UTR) of a target gene may 'silence' the target gene by inhibiting protein translation from the primary start codon (pATG). Here, we report an efficient gene silencing method by introducing a tailor-designed uATG-containing element (ATGE) into the 5'UTR of genes in plants, occupying the original start site to act as a new pATG. Using base editing to introduce new uATGs failed to silence two of the tested three rice genes, indicating complex regulatory mechanisms. Precisely inserting an ATGE adjacent to pATG achieved significant target protein downregulation. Through extensive optimization, we demonstrated this strategy substantially and consistently downregulated target protein expression. By designing a bidirectional multifunctional ATGE4, we enabled tunable knockdown from 19% to 89% and observed expected phenotypes. Introducing ATGE into Waxy, which regulates starch synthesis, generated grains with lower amylose, revealing the value for crop breeding. Together, we have developed a programmable and robust method to knock down gene expression in plants, with potential for biological mechanism exploration and crop enhancement.

Genome-wide identification and analysis of DEAD-box RNA helicases in Gossypium hirsutum

DEAD-box RNA helicases, a prominent subfamily within the RNA helicase superfamily 2 (SF2), play crucial roles in the growth, development, and abiotic stress responses of plants. This study identifies 146 DEAD-box RNA helicase genes (GhDEADs) and categorizes them into four Clades (Clade A-D) through phylogenetic analysis. Promoter analysis reveals cis-acting elements linked to plant responses to light, methyl jasmonate (MeJA), abscisic acid (ABA), low temperature, and drought. RNA-seq data demonstrate that Clade C GhDEADs exhibit elevated and ubiquitous expression across different tissues, validating their connection to leaf development through real-time quantitative polymerase chain reaction (RT-qPCR) analysis. Notably, over half of GhDEADs display up-regulation in the leaves of virus-induced gene silencing (VIGS) plants of GhVIR-A/D (members of m6A methyltransferase complex, which regulate leaf morphogenesis). In conclusion, this study offers a comprehensive insight into GhDEADs, emphasizing their potential involvement in leaf development.

Extracellular perception of multiple novel core effectors from the broad host-range pear anthracnose pathogen Colletotrichum fructicola in the nonhost Nicotiana benthamiana.

Colletotrichum fructicola is emerging as a devastating pathogenic fungus causing anthracnose in a wide range of horticultural crops, particularly fruits. Exploitation of nonhost resistance (NHR) represents a robust strategy for plant disease management. Perception of core effectors from phytopathogens frequently leads to hypersensitive cell death and resistance in nonhost plants; however, such core effectors in C. fructicola and their signaling components in non-hosts remain elusive. Here, we found a virulent C. fructicola strain isolated from pear exhibits non-adaptation in the model plant Nicotiana benthamiana. Perception of secreted molecules from C. fructicola appears to be a dominant factor in NHR, and four novel core effectors-CfCE4, CfCE25, CfCE61, and CfCE66-detected by N. benthamiana were, accordingly, identified. These core effectors exhibit cell death-inducing activity in N. benthamiana and accumulate in the apoplast. With a series of CRISPR/Cas9-edited mutants or gene-silenced plants, we found the coreceptor BAK1 and helper NLRs including ADR1, NRG1, and NRCs mediate perceptions of these core effectors in N. benthamiana. Concurrently, multiple N. benthamiana genes encoding cell surface immune receptors and intracellular immune receptors were greatly induced by C. fructicola. This work represents the first characterization of the repertoire of C. fructicola core effectors responsible for NHR. Significantly, the novel core effectors and their signaling components unveiled in this study offered insights into a continuum of layered immunity during NHR and will be helpful for anthracnose disease management in diverse horticultural crops.

Engineered dsRNA-protein nanoparticles for effective systemic gene silencing in plants.

Long-distance transport or systemic silencing effects of exogenous biologically active RNA molecules in higher plants have not been reported. Here, we report that cationized bovine serum albumin (cBSA) avidly binds double-stranded beta-glucuronidase RNA (dsGUS RNA) to form nucleic acid-protein nanocomplexes. In our experiments with tobacco and poplar plants, we have successfully demonstrated systemic gene silencing effects of cBSA/dsGUS RNA nanocomplexes when we locally applied the nanocomplexes from the basal ends of leaf petioles or shoots. We have further demonstrated that the cBSA/dsGUS RNA nanocomplexes are highly effective in silencing both the conditionally inducible DR5-GUS gene and the constitutively active 35S-GUS gene in leaf, shoot, and shoot meristem tissues. This cBSA/dsRNA delivery technology may provide a convenient, fast, and inexpensive tool for characterizing gene functions in plants and potentially for in planta gene editing.

Genome-wide identification of the GRF family in sweet orange (Citrus sinensis) and functional analysis of the CsGRF04 in response to multiple abiotic stresses

BackgroundCitrus is one of the most valuable fruits worldwide and an economic pillar industry in southern China. Nevertheless, it frequently suffers from undesirable environmental stresses during the growth cycle, which severely restricts the growth, development and yield of citrus. In plants, the growth-regulating factor (GRF) family of transcription factors (TF) is extensively distributed and plays an vital part in plant growth and development, hormone response, as well as stress adaptation. However, the systematic identification and functional analysis of GRF TFs in citrus have not been reported.ResultsHere, a genome-wide identification of GRF TFs was performed in Citrus sinensis, 9 members of CsGRFs were systematically identified and discovered to be scattered throughout 5 chromosomes. Subsequently, physical and chemical properties, phylogenetic relationships, structural characteristics, gene duplication events, collinearity and cis-elements of promoter were elaborately analyzed. In particular, the expression patterns of the CsGRF genes in response to multiple phytohormone and abiotic stress treatments were investigated. Predicated on this result, CsGRF04, which exhibited the most differential expression pattern under multiple phytohormone and abiotic stress treatments was screened out. Virus-induced gene silencing (VIGS) technology was utilized to obtain gene silenced plants for CsGRF04 successfully. After the three stress treatments of high salinity, low temperature and drought, the CsGRF04-VIGS lines showed significantly reduced resistance to high salinity and low temperature stresses, but extremely increased resistance to drought stress.ConclusionsTaken together, our findings systematically analyzed the genomic characterization of GRF family in Citrus sinensis, and excavated a CsGRF04 with potential functions under multiple abiotic stresses. Our study lay a foundation for further study on the function of CsGRFs in abiotic stress and hormone signaling response.

Establishment and application of a root wounding-immersion method for efficient virus-induced gene silencing in plants.

In the post-genomic era, virus-induced gene silencing (VIGS) has played an important role in research on reverse genetics in plants. Commonly used Agrobacterium-mediated VIGS inoculation methods include stem scratching, leaf infiltration, use of agrodrench, and air-brush spraying. In this study, we developed a root wounding-immersion method in which 1/3 of the plant root (length) was cut and immersed in a tobacco rattle virus (TRV)1:TRV2 mixed solution for 30 min. We optimized the procedure in Nicotiana benthamiana and successfully silenced N. benthamiana, tomato (Solanum lycopersicum), pepper (Capsicum annuum L.), eggplant (Solanum melongena), and Arabidopsis thaliana phytoene desaturase (PDS), and we observed the movement of green fluorescent protein (GFP) from the roots to the stem and leaves. The silencing rate of PDS in N. benthamiana and tomato was 95-100%. In addition, we successfully silenced two disease-resistance genes, SITL5 and SITL6, to decrease disease resistance in tomatoes (CLN2037E). The root wounding-immersion method can be used to inoculate large batches of plants in a short time and with high efficiency, and fresh bacterial infusions can be reused several times. The most important aspect of the root wounding-immersion method is its application to plant species susceptible to root inoculation, as well as its ability to inoculate seedlings from early growth stages. This method offers a means to conduct large-scale functional genome screening in plants.

Unraveling the involvement of WRKY TFs in regulating plant disease defense signaling.

This review article explores the intricate role, regulation, and signaling mechanisms of WRKY TFs in response to biotic stress, particularly emphasizing their pivotal role in the trophism of plant-pathogen interactions. Transcription factors (TFs) play a vital role in governing both plant defense and development by controlling the expression of various downstream target genes. Early studies have shown the differential expression of certain WRKY transcription factors by microbial infections. Several transcriptome-wide studies later demonstrated that diverse sets of WRKYs are significantly activated in the early stages of viral, bacterial, and fungal infections. Furthermore, functional investigations indicated that overexpression or silencing of certain WRKY genes in plants can drastically alter disease symptoms as well as pathogen multiplication rates. Hence the new aspects of pathogen-triggered WRKY TFs mediated regulation of plant defensecanbeexplored. The already recognized roles of WRKYs include transcriptional regulation of defense-related genes, modulation of hormonal signaling, and participation in signal transduction pathways. Some WRKYs have been shown to directly bind to pathogen effectors, acting as decoys or resistance proteins. Notably, the signaling molecules like salicylic acid, jasmonic acid, and ethylene which are associated with plant defense significantly increase the expression of several WRKYs. Moreover, induction of WRKY genes or heightened WRKY activities is also observed during ISR triggered by the beneficial microbes which protect the plants from subsequent pathogen infection. To understand the contribution of WRKY TFs towards disease resistance and their exact metabolic functions in infected plants, further studies are required. This review article explores the intrinsic transcriptional regulation, signaling mechanisms, and hormonal crosstalk governed by WRKY TFs in plant disease defense response, particularly emphasizing their specific role against different biotrophic, hemibiotrophic, and necrotrophic pathogen infections.

Endocellulase SlGH9-21 significantly improves drought resistance and storage capacity of tomato

Drought, one of the most important abiotic factors affecting plant growth, causes a large reduction in tomato production every year. Tomato, a berry fruit and vegetable that can be eaten fresh or cooked, has rich nutritional value. However, most of ripe fruits show softening of pericarp, which leads to damage easily during transportation, and shorten the shelf life. Fruit mechanical strength is important to the texture, transport resistance and shelf life of tomato fruit. We found a natural tomato mutant in the field, which showed excellent drought resistance ability and late ripening characteristics. The target gene Solyc09g075360 (SlGH9-21) was obtained through transcriptome data screening. After the gene was silenced, tomatoes improved their drought resistance by reducing stomatal aperture and increasing stem vessel diameter. We also found that silencing of the SlGH9-21 gene delayed the ripening of tomato fruit and improved fruit firmness. Compared with the control, the ripening of the fruit of SlGH9-21 gene-silenced plants was delayed by approximately 10 days, which increased the fruit firmness and storage tolerance and reduced the water loss rate of fruit. Overall, the study of tomato stress response mechanisms and the identification of key genes regulating fruit hardness are beneficial to the improvement of tomato resistance. It is also conducive to the improvement of tomato quality. The research results can lay a foundation for the enhancement of fruit storage tolerance and stress resistance.

Transgene-free, virus-based gene silencing in plants by artificial microRNAs derived from minimal precursors.

Artificial microRNAs (amiRNAs) are highly specific, 21-nucleotide (nt) small RNAs designed to silence target transcripts. In plants, their application as biotechnological tools for functional genomics or crop improvement is limited by the need of transgenically expressing long primary miRNA (pri-miRNA) precursors to produce the amiRNAs in vivo. Here, we analyzed the minimal structural and sequence requirements for producing effective amiRNAs from the widely used, 521-nt long AtMIR390a pri-miRNA from Arabidopsis thaliana. We functionally screened in Nicotiana benthamiana a large collection of constructs transiently expressing amiRNAs against endogenous genes and from artificially shortened MIR390-based precursors and concluded that highly effective and accurately processed amiRNAs can be produced from a chimeric precursor of only 89 nt. This minimal precursor was further validated in A. thaliana transgenic plants expressing amiRNAs against endogenous genes. Remarkably, minimal but not full-length precursors produce authentic amiRNAs and induce widespread gene silencing in N. benthamiana when expressed from an RNA virus, which can be applied into leaves by spraying infectious crude extracts. Our results reveal that the length of amiRNA precursors can be shortened without affecting silencing efficacy, and that viral vectors including minimal amiRNA precursors can be applied in a transgene-free manner to induce whole-plant gene silencing.

Application and Expansion of Virus-Induced Gene Silencing for Functional Studies in Vegetables

Increased consumption of vegetables has been recommended worldwide as a part of a healthy diet; therefore, determining gene function among breeding materials is crucial for vegetable improvement to meet the sustainable development of new vegetable varieties. However, genetic transformation is time-consuming and laborious, which limits the exploration of gene function for various vegetable crops. Virus-Induced Gene Silencing (VIGS) can perform large-scale and rapid gene silencing in plants due to a reduction in the experimental period and its independence from the stable genetic transformation, providing an excellent opportunity for functional research. VIGS can accelerate model plant research and make it easier to analyze gene function and validation in vegetable crops. Moreover, with the advent of technologies such as virus-mediated heterologous protein expression and the development of CRISPR/Cas9 technology, virus-mediated genetic tools have ushered in a new era in genetics and crop improvement. This study summarizes recent achievements in VIGS and Virus-Induced Gene Editing (VIGE) in vegetables. We also identify several challenges in the current state of VIGS technology in vegetables, serving as a guide for future research.

A high-efficiency gene silencing in plants using two-hit asymmetrical artificial MicroRNAs.

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in gene regulation. They are produced through an enzyme-guided process called dicing and have an asymmetrical structure with two nucleotide overhangs at the 3' ends. Artificial microRNAs (amiRNAs or amiRs) are designed to mimic the structure of miRNAs and can be used to silence specific genes of interest. Traditionally, amiRNAs are designed based on an endogenous miRNA precursor with certain mismatches at specific positions to increase their efficiency. In this study, the authors modified the highly expressed miR168a in Arabidopsis thaliana by replacing the single miR168 stem-loop/duplex with tandem asymmetrical amiRNA duplexes that follow the statistical rules of miRNA secondary structures. These tandem amiRNA duplexes, called "two-hit" amiRNAs, were shown to have a higher efficiency in silencing GFP and endogenous PDS reporter genes compared to traditional "one-hit" amiRNAs. The authors also demonstrated the effectiveness of "two-hit" amiRNAs in silencing genes involved in miRNA, tasiRNA, and hormone signalling pathways, individually or in families. Importantly, "two-hit" amiRNAs were also able to over-express endogenous miRNAs for their functions. The authors compare "two-hit" amiRNA technology with CRISPR/Cas9 and provide a web-based amiRNA designer for easy design and wide application in plants and even animals.

A gene silencing screen uncovers diverse tools for targeted gene repression in Arabidopsis

DNA methylation has been utilized for target gene silencing in plants. However, it is not well understood whether other silencing pathways can be also used to manipulate gene expression. Here we performed a gain-of-function screen for proteins that could silence a target gene when fused to an artificial zinc finger. We uncovered many proteins that suppressed gene expression through DNA methylation, histone H3K27me3 deposition, H3K4me3 demethylation, histone deacetylation, inhibition of RNA polymerase II transcription elongation or Ser-5 dephosphorylation. These proteins also silenced many other genes with different efficacies, and a machine learning model could accurately predict the efficacy of each silencer on the basis of various chromatin features of the target loci. Furthermore, some proteins were also able to target gene silencing when used in a dCas9-SunTag system. These results provide a more comprehensive understanding of epigenetic regulatory pathways in plants and provide an armament of tools for targeted gene manipulation.