Study Gene Function Research Articles

Methodological exploration of using gene editing technology to study gene function and disease mechanism

This paper discusses the application of gene editing technology, especially CRISPR-Cas9 system, in the study of gene function and disease mechanism. By modifying the genome sequence at a fixed point, gene editing technology uses specific nucleases to induce double strand breaks, trigger DNA repair mechanism, and realize accurate gene modification. This paper introduces the working principle of CRISPR-Cas9 and its application in establishing gene knock-out and knock-in models, and emphasizes the importance of this technology in revealing gene function and molecular mechanism of diseases. However, gene editing technology also faces challenges such as off-target effect and technical accuracy, and involves ethical issues. Therefore, the article puts forward that we should strengthen ethical review, improve laws and regulations, and enhance public awareness. In a word, gene editing technology provides a powerful tool for life science and medical research, and shows a broad application prospect.

From Random Perturbation to Precise Targeting: A Comprehensive Review of Methods for Studying Gene Function in Monascus Species.

Monascus, a genus of fungi known for its fermentation capability and production of bioactive compounds, such as Monascus azaphilone pigments and Monacolin K, have received considerable attention because of their potential in biotechnological applications. Understanding the genetic basis of these metabolic pathways is crucial for optimizing the fermentation and enhancing the yield and quality of these products. However, Monascus spp. are not model fungi, and knowledge of their genetics is limited, which is a great challenge in understanding physiological and biochemical phenomena at the genetic level. Since the first application of particle bombardment to explore gene function, it has become feasible to link the phenotypic variation and genomic information on Monascus strains. In recent decades, accurate gene editing assisted by genomic information has provided a solution to analyze the functions of genes involved in the metabolism and development of Monascus spp. at the molecular level. This review summarizes most of the genetic manipulation tools used in Monascus spp. and emphasizes Agrobacterium tumefaciens-mediated transformation and nuclease-guided gene editing, providing comprehensive references for scholars to select suitable genetic manipulation tools to investigate the functions of genes of interest in Monascus spp.

Wingless strain created using binary transgenic CRISPR/Cas9 alleviates concerns about mass rearing of Hermetia illucens

Larvae of the black soldier fly Hermetia illucens have potential as a natural waste recycler and subsequent use as protein-rich feed for livestock. A common question about the insect-farming processes is, what about the concerns of mass escape of insects from large populations? Here, we present a binary transgenic CRISPR/Cas9 system to generate wingless strain with the potential to address this issue. We identified gonad-specific promoters in vivo and evaluated use of the two strongest promoters, nanos and exuperantia, to drive Cas9 expression. We found that crossing the Hiexu-Cas9 with transgenic sgRNA-expressing insects resulted in higher knockout efficiency of the marker gene white. The Hiexu-Cas9 strain exhibited a maternal deposition of Cas9 that caused more effective knockout in the progeny of female Cas9-expressing individuals. Using this system, we generated wingless mutants lacking mating ability, which can be maintained in colony through a genetic cross of two single strain. These insects are less likely to escape and would be unable to successfully mate if they did escape. Taken together, this study validates effective genetic tools that can be used for gene function studies and industrial applications in black soldier fly and provides an approach to alleviate the concern about massive rearing.

Transgenic poplar for resistance against pest and pathogen attack in forests: an overview

Forests are potential habitats for immense terrestrial ecosystems and aquatic biodiversity, performing an essential role in ecological preservation and regulation of climate. The anthropogenic pressures on the forests lead to forest loss, fragmentation and degradation. Requirements for sustainable methodologies for forest protection are of utmost priority under the climate change regime. Among forest trees, poplar trees (Populus L.) have attracted attention in global forestry as a promising material for improving the quality and quantity of urban landscapes. These plants provide wood, which can be utilized as raw resources for the paper industry and as a potential source of biofuel. However, several biotic stresses, such as attacks by pests and pathogens, severely affect poplar production and productivity. The improvement of Populus trees through conventional tree breeding methods is restricted due to their long-life cycles and the lack of suitable donors with resistance genes. Populus has been utilized as a model plant for studying gene functions due to its highly efficient genetic transformation capabilities. The present review will provide a comprehensive overview of pest and pathogen attacks on poplar, focusing on their infection mechanisms, transmission routes, and control strategies. Additionally, it will examine the most widely used genetic transformation methods (gene gun-mediated, Agrobacterium tumefaciens-mediated, protoplast transformation, micro-RNA mediated and micro-RNA clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR-Cas) systems methods and RNA interference) for improving tolerance in poplar trees against pest and pathogens attack. Furthermore, it will delve into prospects, challenges, and recent advances in molecular biology tools and their safe application for genetic transformation to improve insect and pest resistance in poplar trees. Finally, the regeneration of transgenic poplar trees with enhanced resistance, developed through various genetic engineering techniques, is discussed.

A frameshift mutation in JAZ10 resolves the growth versus defense dilemma in rice

CRISPR-Cas9 genome editing systems have revolutionized plant gene functional studies by enabling the targeted introduction of insertion-deletions (INDELs) via the nonhomologous end-joining (NHEJ) pathway. Frameshift-inducing INDELs can introduce a premature termination codon and, in other instances, can lead to the appearance of new proteins. Here, we found that mutations in the rice jasmonate (JA) signaling gene OsJAZ10 by CRISPR-Cas9-based genome editing did not affect canonical JA signaling. However, a type of mutant with an INDEL that yielded a novel frameshift protein named FJ10 (Frameshift mutation of JAZ10), exhibited enhanced rice growth and increased resistance to brown planthopper attacks. Overexpression of FJ10 in wild-type plants phenocopies OsJAZ10 frameshift mutants. Further characterization revealed that FJ10 interacts with Slender Rice 1 (OsSLR1) and F-box/Kelch 16 (OsFBK16). These interactions disrupt the function of OsSLR1 in suppressing gibberellin-mediated growth and the function of OsFBK16 in repressing lignin-mediated defense responses, respectively. Field experiments with FJ10-expressing plants demonstrate that this protein uncouples the growth-defense tradeoff, opening broad avenues to obtain cultivars with enhanced yield without compromised defenses.

Effects of drought and salt stress on the root phenotype of wheat seedlings and underlying gene expression analysis.

In our previous study, three TaPSK genes highly expressed in the roots of wheat were screened. To explore the effects of adverse stresses on the wheat root phenotype and the expression of TaPSK3, TaPSK9 and TaPSK10, we measured the phenotypic parameters of the JM22 root system at the seedling stage after treatment with different concentrations of NaCl and PEG6000. Additionally, the relative expression levels of TaPSK3, TaPSK9, and TaPSK10 were analyzed via RT-qPCR within 72 h of treatment with 150 mM NaCl and 30% PEG6000. The results revealed that drought and salt stress significantly inhibited phenotypic parameters such as total root length, root surface area, root biomass distribution estimation and root tip number in wheat. Notably, salt stress causes wheat roots to germinate more root hairs. The expression of TaPSK3 did not change significantly during salt stress but was upregulated approximately five-fold at 12 h of drought stress. The gene expression levels of TaPSK9 and TaPSK10 were upregulated to varying degrees but gradually returned to normal at 72 h. These results show that when wheat encounters stresses, the expression of TaPSK genes is upregulated to promote root growth and ensure the normal growth and development of plants. This study provides data and theoretical support for further study of TaPSK gene function and cultivation of high-quality wheat plants with strong stress resistance.

Gcn2 rescues reprogramming in the absence of Hog1/p38 signaling in C. neoformans during thermal stress.

The fungus Cryptococcus neoformans is an opportunistic pathogen of humans that reprograms its translatome to facilitate adaptation and virulence within the host. We studied the role of Hog1/p38 in reprogramming translation during thermal stress adaptation and found that this pathway acts on translation via crosstalk with the Gcn2 pathway, a well-studied regulator of general translation control. Using a combination of molecular assays and phenotypic analysis, we show that increased output from the Gcn2 pathway in a Hog1 deletion mutant is associated with rescue of thermal stress adaptation at both molecular and phenotypic scales. We characterize known outputs of the Hog1 pathway during thermal stress as either Gcn2-dependent or Gcn2-independent and demonstrate that Hog1 activation regulates the Gcn2 pathway even in the absence of thermal stress. Finally, we implicate this phenomenon in another Hog1-regulated process, morphogenesis, and recapitulate Hog1-Gcn2 crosstalk in the distantly related fungal pathogen, Candida albicans. Our results point to an important link between the stress response machinery and translation control and clarify the etiology of phenotypes associated with Hog1 deletion. More broadly, this study highlights complex interplay between core conserved signal transduction pathways and the utility of molecular assays to better understand how these pathways are connected.IMPORTANCECryptococcus neoformans is an opportunistic pathogen of humans that causes deadly cryptococcal meningitis, which is is responsible for an estimated 19% of AIDS-related mortality. When left untreated, cryptococcal meningitis is uniformly fatal, and in patients receiving the most effective antifungal regimens, mortality remains high. Thus, there is a critical need to identify additional targets that play a role in the adaptation to the human host and virulence. This study explores the role of the stress response kinases Hog1 and Gcn2 in thermoadaptation, which is a pre-requisite for virulence. Our results show that compensatory signaling occurs via the Gcn2 pathway when Hog1 is deleted, and that disruption of both pathways increases sensitivity to thermal stress. Importantly, our study highlights the insufficiency of using single-gene deletion mutants to study gene function, since many phenotypes associated with Hog1 deletion were driven by Gcn2 signaling in this background, rather than loss of direct Hog1 activity.

Rapid and efficient in planta genome editing in sorghum using foxtail mosaic virus-mediated sgRNA delivery.

The requirement of in vitro tissue culture for the delivery of gene editing reagents limits the application of gene editing to commercially relevant varieties of many crop species. To overcome this bottleneck, plant RNA viruses have been deployed as versatile tools for in planta delivery of recombinant RNA. Viral delivery of single-guide RNAs (sgRNAs) to transgenic plants that stably express CRISPR-associated (Cas) endonuclease has been successfully used for targeted mutagenesis in several dicotyledonous and few monocotyledonous plants. Progress with this approach in monocotyledonous plants is limited so far by the availability of effective viral vectors. We engineered a set of foxtail mosaic virus (FoMV) and barley stripe mosaic virus (BSMV) vectors to deliver the fluorescent protein AmCyan to track viral infection and movement in Sorghum bicolor. We further used these viruses to deliver and express sgRNAs to Cas9 and Green Fluorescent Protein (GFP) expressing transgenic sorghum lines, targeting Phytoene desaturase (PDS), Magnesium-chelatase subunit I (MgCh), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, orthologs of maize Lemon white1 (Lw1) or GFP. The recombinant BSMV did neither infect sorghum nor deliver or express AmCyan and sgRNAs. In contrast, the recombinant FoMV systemically spread throughout sorghum plants and induced somatic mutations with frequencies reaching up to 60%. This mutagenesis led to visible phenotypic changes, demonstrating the potential of FoMV for in planta gene editing and functional genomics studies in sorghum.

Efficient in situ epitope tagging of rice genes by nuclease-mediated prime editing.

In situ epitope tagging is crucial for probing gene expression, protein localization, and the dynamics of protein interactions within their natural cellular context. However, the practical application of this technique in plants presents considerable hurdles. Here, we comprehensively explored the potential of the CRISPR/Cas nuclease-mediated prime editing and different DNA repair pathways in epitope tagging of endogenous rice (Oryza sativa) genes. We found that a SpCas9 nuclease/microhomology-mediated end joining (MMEJ)-based prime editing (PE) strategy (termed NM-PE) facilitates more straightforward and efficient gene tagging compared to the conventional and other derivative PE methods. Furthermore, the PAM-flexible SpRY and ScCas9 nucleases-based prime editors have been engineered and implemented for the tagging of endogenous genes with diverse epitopes, significantly broadening the applicability of NM-PE in rice. Moreover, NM-PE has been successfully adopted in simultaneous tagging of the MAP kinase (MPK) genes OsMPK1 and OsMPK13 in rice plants with c-Myc and HA tags, respectively. Taken together, our results indicate great potential of the NM-PE toolkit in the targeted gene tagging for Rice Protein Tagging Project, gene function study and genetic improvement.

Preassembled complexes of hAgo2 and ssRNA delivered by nanoparticles: A novel silencing gene expression approach overcoming the absence of the canonical pathway of siRNA processing in the apicomplexan parasite Babesia microti, blood parasite of veterinary and zoonotic importance

Due to the lack of efficacy of the currently used chemical drugs, poor tick control, and lack of effective vaccines against Babesia, novel control strategies are urgently needed. In this regard, searching for anti-Babesia gene therapy may facilitate the control of this infection. Following this pattern, small interfering RNAs (siRNAs) are widely used to study gene function and hence open the way to control the parasite. However, the primary constraint of this approach is the lack of Babesia to RNA-induced silencing complex (RISC) enzymes, making siRNA impractical. In this study, we preassembled complexes with the human enzyme argonaute 2 (hAgo2) and a small interfering RNA (siRNA)/single-stranded RNA (ssRNA) against B. gibsoni and B. microti metabolite transporters. The assembled complexes were generated by developing a gene delivery system with chitosan dehydroascorbic acid nanoparticles. The delivery system effectively protected the loaded RNAi and targeted Babesia-infected RBCs with a relatively high internalization rate. The assembled complexes were successfully transfected into live parasites for specific slicing of Babesia targets. We demonstrated a reduction in the expression of target genes at the mRNA level. Furthermore, this silencing inhibited Babesia growth in vitro and in vivo. For the first time, we used this method to confirm the role of the assembled complexes in manipulating the noncanonical pathway of RNAi in Babesia parasites. This novel method provides a means of silencing Babesia genes to study their role in host-parasite interactions and as potential targets for gene therapy and control.

Genome-Wide Identification and Expression Analysis of Pseudo-Response Regulators (PRRs) in the Tea Plant Camellia sinensis (L.) O. Kuntze

The circadian clock plays a vital role in facilitating plant adaptation to rhythmically changing environmental factors. Pseudo-response regulators (PRRs) are key components regulating the plant circadian clock and have been extensively characterized in model plants. However, the PRRs in the tea plant have not been comprehensively studied. In this study, seven CsPRRs were cloned from the tea plant. Domain, phylogenetic evolution, gene structure, motifs, and cis-acting element analysis revealed their sequence characters and suggested that the first subgroup members, CsPRR1a, 1b, 5a, 5b, 7, and 37, may be responsible for circadian rhythm regulation and abiotic stress responses, while the second subgroup member, CsPRR2, may be involved in development and chloroplast function regulation. Most CsPRRs showed relatively higher expression in flowers, implying their potential roles in photoperiod-regulated flower induction. Moreover, rhythmic expression of CsPRR7, 5b, 5a, 37, 1b, and 1a was observed under long-day conditions in a sequential manner. Additionally, CsPRRs were differently induced/inhibited by cold, heat, and drought stresses in tissue-specific and photoperiod-related manners. A stronger cold induction of CsPRRs was observed under long-day conditions than under short-day conditions. And, among the two tested tissues, changes in the expression of CsPRRs caused by various stresses were more obvious in young shoots. Studies using a floriferous cultivar (FDDB) and an oliganthous cultivar (PYTZ) implied that CsPRRs also played crucial roles in tea-plant flower induction. This study presents the first comprehensive analysis of CsPRRs in the tea plant, providing vital information for further elucidation of CsPRR functions. It also suggests that tissue type and photoperiod conditions should be taken into consideration when conducting gene function studies in the tea plant.

An Evolutionary Framework Exploiting Virologs and Their Host Origins to Inform Poxvirus Protein Functions.

Poxviruses represent evolutionary successful infectious agents. As a family, poxviruses can infect a wide variety of species including humans, fish, and insects. While many other viruses are species-specific, an individual poxvirus species is often capable of infecting diverse hosts and cell types. For example, the prototypical poxvirus, vaccinia, is well known to infect numerous human cell types but can also infect cells from divergent hosts like frog neurons. Notably, poxvirus infections result in both detrimental human and animal diseases. The most infamous disease linked to a poxvirus is smallpox caused by variola virus. Poxviruses are large double-stranded DNA viruses, which uniquely replicate in the cytoplasm of cells. The model poxvirus genome encodes ~200 nonoverlapping protein-coding open reading frames (ORFs). Poxvirus gene products impact various biological processes like the production of virus particles, the host range of infectivity, and disease pathogenesis. In addition, poxviruses and their gene products have biomedical application with several species commonly engineered for use as vaccines and oncolytic virotherapy. Nevertheless, we still have an incomplete understanding of the functions associated with many poxvirus genes. In this chapter, we outline evolutionary insights that can complement ongoing studies of poxvirus gene functions and biology, which may serve to elucidate new molecular activities linked to this biomedically relevant class of viruses.

Genome editing in almond using hairy root transformation system

Woody plant species are often challenging to transform, which complicates gene function studies using molecular biology techniques. To develop an effective gene editing tool for almond (Prunus dulcis, syn. Prunus amygdalus), we established a new method for inducing hairy roots in almond seedlings based on the injection of agrobacterial culture. We generated composite plants comprising wild-type shoots and transgenic hairy roots. This approach was also successful in almond x peach hybrids. The hairy root transformation system was used alongside Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 gene editing to target two transcription factor genes (ETHYLENE RESPONSE FACTOR 74 and GIBBERELLIC ACID INSENSITIVE). We successfully generated hairy roots with knockouts of the target genes and evaluated the efficiency of guide RNAs for gene editing in almond. Our transgenic root method could be a valuable tool for routine gene function studies in almond.

Establishment and Validation of an Efficient Agrobacterium Tumefaciens-Mediated Transient Transformation System for Salix Psammophila.

Salix psammophila, C. Wang & Chang Y. Yang, a desert-adapted shrub, is recognized for its exceptional drought tolerance and plays a vital role in ecosystem maintenance. However, research on S. psammophila has been limited due to the lack of an efficient and reliable genetic transformation method, including gene functional studies. The Agrobacterium-mediated transient overexpression assay is a rapid and powerful tool for analyzing gene function in plant vivo. In this study, tissue culture seedlings of S. psammophila were utilized as the recipient materials, and the plant expression vector pCAMBIA1301, containing the GUS reporter gene, was transferred into the seedlings via an Agrobacterium-mediated method. To enhance the efficiency of the system, the effects of secondary culture time, Agrobacterium concentration, infection time, and co-culture duration on the transient transformation efficiency of S. psammophila were explored. The optimal combination for the instantaneous transformation of S. psammophila tissue culture seedlings mediated by Agrobacterium was determined as follows: a secondary culture time of 30 d, a value of OD600 of 0.8, an infection time of 3 h, and a co-culture duration of 48 h. Subsequently, the effectiveness of the transformation system was validated using the S. psammophila drought response gene SpPP2C80. To further confirm the accuracy of the system, SpPP2C80-overexpressing Arabidopsis was constructed and drought resistance analysis was performed. The results were consistent with the transient overexpression of SpPP2C80 in S. psammophila tissue culture seedlings, indicating that this system can be effectively employed for studying gene function in S. psammophila. These findings provide essential information for investigating gene function in non-model plants and pave the way for advancements in molecular biology research in S. psammophila.

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function.

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.

Efficient genome editing using CRISPR/Cas9 technology and its application for identifying Sesquiterpene synthases involved in the biosynthesis of Steperoxides in Steccherinum ochraceum

CRISPR technology has been widely used for gene editing in various species，but the genetic manipulation in basidiomycete mushrooms is still notoriously difficult for unknown endogenous promoters and inefficient DNA delivery. Steccherinum ochraceum is a white rot basidiomycete fungus with abundant secondary metabolites and plays an important ecological role worldwide. To facilitate the study of gene function in S. ochraceum, an effective CRISPR/Cas9 system was successfully developed by identifying highly efficient endogenous promoters, and utilizing the Agrobacterium-transformation method. Two efficient endogenous RNA polymerase II promoters (Psogpd and Psotef1) and one efficient RNA polymerase III promoter (Pu6-d) were identified and characterized, with an editing efficiency of 61.5 % at the ura3 locus. Using this optimized system, the sesquiterpene gene A0064, which could produce 10 possible sesquiterpenes in the heterologous expression system of A. oryzae, was knocked out to obtain A0064 knockout strain S. ochraceum (∆A0064). Steperoxide A could not be detected in S. ochraceum (∆A0064), demonstrating that A0064 was the only enzyme responsible for the biosynthesis of β-chamigrene (the sesquiterpene skeleton of steperoxide A) in S. ochraceum. This efficient system will enable precise targeting and multiplex editing of S. ochraceum genes, facilitating functional studies of genes involved in lignin degradation and natural product biosynthesis in S. ochraceum, and providing some valuable guidance for gene editing in tens of thousands of macrofungi.

Silencing of the MP Gene via dsRNA Affects Root Development and Growth in the Invasive Weed Mikania micrantha.

Mikania micrantha ("mile-a-minute" weed) is a global invasive alien weed that can cause severe damage to agroforestry ecosystems and significant agricultural losses worldwide. Although chemical, manual, or mechanical control methods are widely used to control M. micrantha, RNA interference (RNAi)-based biocontrol methods have rarely been reported for this species. The MONOPTEROS (MP) gene, encoding an auxin response factor, plays an essential role in embryonic root initiation in Arabidopsis thaliana. In this study, we identified the MP gene from M. micrantha via orthologous gene analysis. A total of 37 MP orthologous genes was identified in 4 plants, including 9 MP candidate genes in M. micrantha, 13 in Helianthus annuus, 6 in Chrysanthemum nankingense, and 9 in Lactuca sativa. Phylogenetic analysis revealed that an MP candidate gene in M. micrantha (Mm01G000655, named MmMP) was clustered into one clade with the MP gene in A. thaliana (AtMP). In addition, both MmMP and AtMP contain a B3-DNA binding domain that is shared by transcription factors that regulate plant embryogenesis. To study gene function, dsRNA against MmMP (dsMmMP) was applied to the roots of M. micrantha. Compared with those of the controls, the expression of MmMP was reduced by 43.3%, 22.1%, and 26.2% on the first, third, and fifth days after dsMmMP treatment, respectively. The dsMmMP-treated plants presented several morphological defects, mostly in the roots. Compared with water-treated plants, the dsMmMP-treated plants presented reduced developmental parameters, including root length, number of adventitious roots, root fresh and dry weights, plant height, and aboveground biomass. Additionally, safety assessment suggested that this dsMmMP treatment did not silence MP genes from non-target plants, including rice and tomato; nor did it inhibit root growth in those species. Collectively, these results suggest that MmMP plays an important role in root development in M. micrantha and provides a potential target for the development of species-specific RNAi-based herbicides.

Development and evaluation of RNA microsphere-based RNAi approaches for managing the striped flea beetle (Phyllotreta striolata), a globally destructive pest of Cruciferae crops.

RNA interference (RNAi) technology has emerged as a promising strategy for species-specific management of agricultural pests. However, the application of this technology has been significantly hindered by the instability of the interfering RNA molecules in the insect body after ingestion leading to variations in the susceptibility to the RNA triggers across different taxonomic groups of insects. Therefore, it is necessary to develop new approaches that will overcome these challenges associated with the use of RNAi-based insect pest management strategies. This study explored the use of RNA microspheres (RMS) synthesized via rolling-circle transcription (RCT) technology as a potential method for managing striped flea beetle (Phyllotreta striolata), a globally destructive pest of Cruciferae crops. The synthesized RMS against the genes encoding reticulocalbin (RMS-PsRCN) and ribosomal RNA (RMS-PsrRNA) were highly effective in both silencing their target genes and causing increased P. striolata adult mortality. Relative expression levels of the target genes RMS-PsRCN and RMS-PsrRNA were decreased by 74.9% and 68.92%, respectively, in RMS fed adults, compared with the control adults fed RMS-EGFP. Consequently, the adult mortalities were 81.7% and 73.3% when fed RMS-PsRCN and RMS-PsrRNA, respectively, compared with 8.3% in the control adults. Furthermore, movements of adults fed RMS-PsRCN and RMS-PsrRNA were decreased by 70.2% and 55.7%, respectively, compared with the control adults. This study shows the potential of using RMS to suppress the expression of target genes and subsequently produce significant mortality rates and behavioral changes in RMS-fed adult P. striolata. These findings underscore the promises and viability of using RMS as an effective strategy for gene function studies and species-specific management of agricultural important insect pests. © 2024 Society of Chemical Industry.

Genome-Wide Analysis of CSL Family Genes Involved in Petiole Elongation, Floral Petalization, and Response to Salinity Stress in Nelumbo nucifera.

Lotus (Nelumbo nucifera), a perennial aquatic plant, endures various environmental stresses. Its diverse ornamental traits make it an ideal model for studying multigene family functional differentiation and abiotic stress responses. The cellulose synthase-like (CSL) gene family includes multiple subfamilies and holds potentially pivotal roles in plant growth, development, and stress responses. Thus, understanding this family is essential for uncovering the attributes of ancient dicotyledonous lotus species and offering new genetic resources for targeted genetic improvement. Herein, we conducted a genome-wide NnCSL gene identification study, integrating tissue-specific expression analysis, RNA-seq, and qRT-PCR validation. We identified candidate NnCSL genes linked to petiole elongation, floral petalization, salinity stress responses, and potential co-expressed TFs. 22 NnCSL genes were categorized into six subfamilies: NnCSLA, NnCSLB, NnCSLC, NnCSLD, NnCSLE, and NnCSLG. Promoter regions contain numerous cis-acting elements related to growth, development, stress responses, and hormone regulation. Nineteen NnCSL genes showed specific differential expression in LPA (large plant architecture) versus SPA (small plant architecture): petioles, petalized carpels (CP) and normal carpels (C), and petalized stamens (SP) and normal stamens (S). Notably, most NnCSLC, NnCSLA, and NnCSLB subfamily genes play diverse roles in various aspects of lotus growth and development, while NnCSLE and NnCSLG are specifically involved in carpel petalization and petiole elongation, respectively. Additionally, 11 candidate NnCSL genes responsive to salinity stress were identified, generally exhibiting antagonistic effects on growth and developmental processes. These findings provide an important theoretical foundation and novel insights for the functional study of NnCSL genes in growth, development, and stress resistance in lotus.

A new cell culture resource for investigations of reptilian gene function.

The establishment of CRISPR/Cas9 gene editing in Anolis sagrei has positioned this species as a powerful model for studies of reptilian gene function. To enhance this model, we developed an immortalized lizard fibroblast cell line (ASEC-1) for the exploration of reptilian gene function in cellular processes. We demonstrate the use of this cell line by scrutinizing the role of primary cilia in lizard Hedgehog (Hh) signaling. Using CRISPR/Cas9 mutagenesis, we disrupted the ift88 gene, which is required for ciliogenesis in diverse organisms. We determined that loss of itf88 from lizard cells leads to an absence of primary cilia, a partial derepression of gli1 transcription, and an inability of the cells to respond to the Smoothened agonist, SAG. Through a cross-species analysis of SAG-induced transcriptional responses in cultured limb bud cells, we further determined that ∼46% of genes induced as a response to Hh pathway activation in A. sagrei are also SAG responsive in Mus musculus limb bud cells. Our results highlight conserved and diverged aspects of Hh signaling in anoles and establish a new resource for investigations of reptilian gene function.