Gene Function Validation Research Articles

Application of omics technology in ecotoxicology of arthropod in farmland

Abstract Arthropods, abundant in farmland, have unique biological traits that make them valuable for studying the ecotoxicological impacts of pollutants. Recent advancements in multi-omics technologies have enhanced their use in assessing pollution risks and understanding toxicity mechanisms. This paper reviews recent developments in applying omics technologies—genomics, transcriptomics, proteomics, metabolomics and meta-omics to ecotoxicological research on farmland arthropods. Agricultural arthropods manage genes and proteins like metallothioneins (MTs), antioxidant enzyme systems, heat shock proteins, cytochrome P450 (CYP450), carboxylesterases (CarEs), and glutathione S-transferases (GSTs), for detoxification and antioxidant purposes. They adjust amino acid, sugar, and lipid metabolism to counteract pollutant-induced energy drain and modify gut microbiota to aid in detoxification. This study advocates for enhanced analysis of compound pollution and emerging pollutants using multi-omics, especially meta-omics, to clarify the toxicological mechanisms underlying arthropod responses to these pollutants. Furthermore, it underscores the urgent need for subsequent gene function mining and validation to support biological control strategies and promote sustainable agricultural practices. The findings of this research provide significant insights into the toxicological impacts and mechanisms of pollutants within farmland ecosystems, thereby contributing to the preservation of arthropod diversity.

Integrating cotyledon-based virus-induced gene silencing with visual marker promises a rapid, highly effective validation of gene functions in Nepeta cataria.

Nepeta spp. generate volatile nepetalactone iridoids that have cat-attractant and insect-repellent activities. They differ from typical mint family (Lamiaceae) iridoids, which are non-volatile glucosides, and also vary from other species in the Nepetoideae sub-family, which do not generate iridoids. The chemistry and evolution of Nepeta make it suitable for further investigation. However, the lack of transgenic technology hampers the molecular and genetic investigations in Nepeta. Virus-induced gene silencing (VIGS) is a powerful tool to detect gene functions in vivo. Here, we constructed a modified VIGS method in Nepeta cataria, using cotyledon infiltration, with the gene silencing effect spreading to the first two pairs of true leaves. The VIGS efficiency reached as high as 84.4%, and the procedure takes only 3 weeks. We employed this method to validate the role of geraniol 8-hydroxylase in nepetalactone biosynthesis with ChlH as a visual marker in N. cataria. The method is also applicable to Nepeta mussinii. Thus, we developed an easy and effective VIGS approach, which will be advantageous for endogenous gene studies in two Nepeta species and holds the potential for application in other plants.

Ganoderma lucidum (Curtis) P. Karst. Immunomodulatory Protein Has the Potential to Improve the Prognosis of Breast Cancer Through the Regulation of Key Prognosis-Related Genes.

Background/Objectives: Breast cancer in women is the most commonly diagnosed and most malignant tumor. Although luminal A breast cancer (LumA) has a relatively better prognosis, it still has a persistent pattern of recurrence. Ganoderma lucidum (Curtis) P. Karst. is a kind of traditional Chinese medicine and has antitumor effects. In this study, we aimed to identify the genes relevant to prognosis, find novel targets, and investigate the function of the bioactive protein from G. lucidum, called FIP-glu, in improving prognosis. Methods: Gene expression data and clinical information of LumA breast cancer patients were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Using bioinformatics methods, a predictive risk model was constructed to predict the prognosis for each patient. The cell counting kit-8 (CCK8) and clone formation assays were used to validate gene function. The ability of FIP-glu to regulate RNA levels of risk genes was validated. Results: Six risk genes (slit-roundabout GTPase-activating protein 2 (SRGAP2), solute carrier family 35 member 2 (SLC35A2), sequence similarity 114 member A1 (FAM114A1), tumor protein P53-inducible protein 11 (TP53I11), transmembrane protein 63C (TMEM63C), and polymeric immunoglobulin receptor (PIGR)) were identified, and a prognostic model was constructed. The prognosis was worse in the high-risk group and better in the low-risk group. The receiver operating characteristic (ROC) curve confirmed the model's accuracy. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the differentially expressed genes (DEGs) between the high- and low-risk groups were significantly enriched in the immune responses. TMEM63C could promote tumor viability, growth, and proliferation in vitro. FIP-glu significantly regulated these risk genes, and attenuated the promoting effect of TMEM63C in breast cancer cells. Conclusions: SRGAP2, SLC35A2, FAM114A1, TP53I11, TMEM63C, and PIGR were identified as the potential risk genes for predicting the prognosis of patients. TMEM63C could be a potential novel therapeutic target. Moreover, FIP-glu was a promising drug for improving the prognosis of LumA breast cancer.

VviMYB24 positively regulates flavonol biosynthesis in response to moderate drought stress in ‘Cabernet Sauvignon’ grape seedlings

Drought is a kind of adversity that has always severely restricted the growth of grapes. Flavonols, as flavonoid compounds, are abundant in grapes and have an indispensable function in resisting drought. Hereafter, the objectives of this investigation were to identify key drought-induced transcription factors in grape and explore their roles and regulatory networks in the flavonol-mediated drought response mechanism. In this study, one-year-old cutting seedlings of ‘Cabernet Sauvignon’ grape were used as experimental materials, cultivated in a solar greenhouse, and subjected to moderate drought treatment. The physiological and biochemical parameters related to stress and flavonol content in plant tissues were determined, and gene expression, functional validation, and transcriptional regulation relationships were explored. The results showed that the growth of grape plants was affected under drought conditions, the flavonol accumulation and the expression level of VviMYB24 were significantly induced by drought stress. Overexpression of VviMYB24 reduced the degree of cytoplasmic membrane damage and induced flavonol biosynthesis to confer drought tolerance in Arabidopsis plants and grape callus. Further study revealed that VviMYB24 can interact with the bHLH transcription factor VviMYC2b, which was positively responsive to drought, and they could activate VviFLS5 expression individually and/or in combination in the form of a complex. Interestingly, VviMYB24 could positively regulate VviMYC2b expression to form a transcriptional cascade, enhancing the function of the complex. Transient overexpression of VviMYB24, VviMYC2b, and VviFLS5 in grape leaves promoted the drought-responsive flavonol accumulation. Overall, the above data elucidated that the VviMYB24-VviMYC2b-VviFLS5 module positively regulates drought-induced flavonol biosynthesis in response to drought stress in grape, which contributes to further grape drought resistant breeding and variety improvement through biotechnology.

A dTALE approach demonstrates that induction of common bean OVATE Family Protein 7 promotes resistance to common bacterial blight.

Common bacterial blight (CBB) is a devastating seed-transmitted disease of common bean (Phaseolus vulgaris L.), caused by Xanthomonas phaseoli pv. phaseoli and Xanthomonas citri pv. fuscans. The genes responsible for CBB resistance are largely unknown. Moreover, the lack of a reproducible and universal transformation protocol limits the study of genetic traits in common bean. We produced X. phaseoli pv. phaseoli strains expressing artificially designed transcription-activator like effectors (dTALEs) to target 14 candidate genes for resistance to CBB based on previous transcriptomic data. In planta assays in a susceptible common bean genotype showed that induction of PvOFP7, PvAP2-ERF71, or PvExpansinA17 expression by dTALEs resulted in CBB symptom reduction. After PvOFP7 induction, in planta bacterial growth was reduced at early colonization stages, and RNA-seq analysis revealed up-regulation of cell wall formation and primary metabolism, together with major down-regulation of heat shock proteins. Our results demonstrated that PvOFP7 contributes to CBB resistance, and underlined the usefulness of dTALEs for functional validation of genes whose induction impacts Xanthomonas-plant interactions.

Research Progress on Genomic Regions and Candidate Genes Related to Milk Composition Traits of Dairy Goats Based on Functional Genomics: A Narrative Review.

Goat milk has gained global attention for its unique nutritional properties and potential health benefits. Advancements in functional genomic technologies have significantly progressed genetic research on milk composition traits in dairy goats. This review summarizes various research methodologies applied in this field. Genome-wide association studies (GWAS) have identified genomic regions associated with major milk components, with the diacylglycerol acyltransferase 1 (DGAT1) gene and casein gene cluster consistently linked to milk composition traits. Transcriptomics has revealed gene expression patterns in mammary tissue across lactation stages, while the role of non-coding RNAs (such as miRNAs and circRNAs) in regulating milk composition has been confirmed. Proteomic and metabolomic studies have not only helped us gain a more comprehensive understanding of goat milk composition characteristics but have also provided crucial support for the functional validation of genes related to milk components. The integration of multi-omics data has emerged as an effective strategy for elucidating complex regulatory networks from a systems biology perspective. Despite progress, challenges remain, including refining reference genomes, collecting large-scale phenotypic data, and conducting functional validations. Future research should focus on improving reference genomes, expanding study populations, investigating functional milk components, exploring epigenetic regulation and non-coding RNAs, and studying microbiome-host genome interactions. These efforts will inform more precise genomic and marker-assisted selection strategies, advancing genetic improvements in milk composition traits in dairy goats.

Activation of PKR by a short-hairpin RNA

Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54’s potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation.

Integrated Transcriptomics and Metabolomics Reveal Key Insights into Iridoid Biosynthesis in Gentiana crassicaulis Seeds during Germination.

Background:Gentiana crassicaulis Duthie ex Burk., a key species used in traditional Chinese medicine for treating rheumatic pain and stroke, contains iridoids as its primary active component. However, the biosynthetic mechanisms underlying iridoid production are not fully understood. Methods: This study focused on iridoid biosynthesis during the germination of G. crassicaulis seeds, integrating metabolomic and transcriptomic analyses to uncover the underlying pathways and key candidate genes. Results: 196,132 unigenes and 10 iridoid compounds were identified through RNA-seq and ultra performance liquid chromatography-quadrupole time of flight-mass spectrometer (UPLC-Q-TOF-MS), respectively. The intersection of results from Pearson correlation analysis and weighted gene co-expression network analysis (WGCNA) revealed a significant correlation between 26 genes and iridoid levels, suggesting their potential role in the iridoid metabolism. Notably, six highly expressed candidate genes (DL7H, SLS, CYP76, CYP72A2, CYP84A1, and 13-LOX3) and five iridoids (loganic acid, sweroside, swertiamarin, gentiopicroside, and 6'-O-β-D-glucosyl-gentiopicroside) responded to methyl jasmonate stimulation in G. crassicaulis seedlings. Conclusions: by combining the known functions of candidate gene families, It is hypothesized that the CYP716 and LOX families exert indirect influences on iridoid metabolism, while the CYP71, CYP81, CYP72, CYP76, CYP710 families, 2OG-FeII family, and the glucosyltransferase family are likely to play direct roles in the biosynthetic transformations of the five iridoids. This study provides a theoretical basis for further functional gene validation and metabolic engineering aimed at enhancing iridoid production. The insights gained could lead to improved iridoid production efficiency in medicinal plants, ultimately benefiting the quality and efficacy of medicinal materials.

A Fast, Efficient, and Tissue-Culture-Independent Genetic Transformation Method for Panax notoginseng and Lilium regale.

The Agrobacterium-based transgenic technique is commonly used for gene function validation and molecular breeding. However, it is not suitable for plants with a low regeneration capacity or a low transformation rate, such as Panax notoginseng (Burk) F.H. Chen and Lilium regale Wilson. In this study, a novel Agrobacterium transformation method based on injection in the meristems was developed using P. notoginseng and L. regale as experimental models. PCR analysis confirmed the successful integration of the reporter gene DsRed2 (Discosoma striata red fluorescence protein 2) into the genome of two experimental models. QRT-PCR and Western blot analysis demonstrated the transcriptional and translational expression of DsRed2. Additionally, laser confocal microscopy confirmed the significant accumulation of the red fluorescent protein in the leaves, stems, and roots of transformed P. notoginseng and L. regale. Most importantly, in the second year after injection, the specific bright orange fluorescence from DsRed2 expression was observed in the transgenic P. notoginseng and L. regale plants. This study establishes a fast, efficient, and tissue-culture-independent transgenic technique suitable for plants with a low regeneration capacity or a low transformation rate. This technique may improve the functional genomics of important medicinal and ornamental plants such as P. notoginseng and L. regale, as well as their molecular breeding.

Identification of hub genes associated with stripe rust disease in wheat through integrative transcriptome and gene-based association study

Stripe rust poses a severe menace to wheat cultivation worldwide, occurring as the most devastating disease threatening global wheat industry. The use of resistance genes has been considered as an effective method for management of rust diseases. However, for most of yellow rust (Yr) genes, the molecular mechanisms and pathways responsible for resistance response against Puccinia striiformis (Pst) infection are not well understood. In this study, transcriptome analysis was conducted in two cultivars viz. PBW343 (susceptible) and FLW30 (resistant) to the pathotype 46S119 of Pst in India. The inoculation with Pst pathotype 46S119 was followed by leaf sample collection at different hours per inoculation (hpi) viz.12, 48, and 72. The transcriptome analysis revealed a total of 18,482 differentially expressed genes (DEGs) across different time points. However, a set of 159 genes were identified to exhibit a genome-wide association with P ≤ 0.05 level of significance, which can be represented as Nominally Significant GWAS Geneset (NSGG). By utilizing Weighted Gene Co-expression Network Analysis (WGCNA), we identified 924 and 79 genes from the DEGs and NSGG dataset belonging to different modules. Functional validation of genes was done to confirm the transcriptional expression of four potential genes, including two hub genes associated with the defence mechanism against rust infection.

HNF4A and HNF1A exhibit tissue specific target gene regulation in pancreatic beta cells and hepatocytes

HNF4A and HNF1A encode transcription factors that are important for the development and function of the pancreas and liver. Mutations in both genes have been directly linked to Maturity Onset Diabetes of the Young (MODY) and type 2 diabetes (T2D) risk. To better define the pleiotropic gene regulatory roles of HNF4A and HNF1A, we generated a comprehensive genome-wide map of their binding targets in pancreatic and hepatic cells using ChIP-Seq. HNF4A was found to bind and regulate known (ACY3, HAAO, HNF1A, MAP3K11) and previously unidentified (ABCD3, CDKN2AIP, USH1C, VIL1) loci in a tissue-dependent manner. Functional follow-up highlighted a potential role for HAAO and USH1C as regulators of beta cell function. Unlike the loss-of-function HNF4A/MODY1 variant I271fs, the T2D-associated HNF4A variant (rs1800961) was found to activate AKAP1, GAD2 and HOPX gene expression, potentially due to changes in DNA-binding affinity. We also found HNF1A to bind to and regulate GPR39 expression in beta cells. Overall, our studies provide a rich resource for uncovering downstream molecular targets of HNF4A and HNF1A that may contribute to beta cell or hepatic cell (dys)function, and set up a framework for gene discovery and functional validation.

Development of a rapid and efficient system for CR genes identification based on hairy root transformation in Brassicaceae

Many economically important crops and vegetables belonging to the cruciferous family are heavily endangered by clubroot disease caused by Plasmodiophora brassicae infection. Breeding of clubroot resistant cultivars based on mapping and cloning of resistant genes is commonly regarded as the most cost-effective and efficient way to fight against this disease. The traditional way of R gene functional validation requires stable transformation that is both time- and labor-consuming. In this study, a rapid and efficient hairy-root transgenic protocol mediated by Agrobacterium rhizogenes was developed. The transformation positive rate was over 80% in Brassica napus showed by GUS reporter gene and this transformation only took 1/6 of the time compared with stable transformation. The system was applicable to different B. napus varieties and other cruciferous crops including Brassica rapa and Brassica oleracea. In particular, two known CR genes, CRA3.7.1 and CRA8.2.4 were used respectively, as example to show that the system works well for CR gene study combined with subsequent P. brassicae infection in B. napus. Most importantly, it works both in over-expression that led to disease resistance, as well as in RNAi which led to disease susceptible phenotype. Therefore, this system can be used in batch-wise identification of CR genes, and also offered the possibility of manipulating key genes within the P. brassicae genome that could improve our knowledge on host–pathogen interaction.

A high-quality chromosome-level genome assembly of the Chinese medaka Oryzias sinensis

Oryzias sinensis, also known as Chinese medaka or Chinese ricefish, is a commonly used animal model for aquatic environmental assessment in the wild as well as gene function validation or toxicology research in the lab. Here, a high-quality chromosome-level genome assembly of O. sinensis was generated using single-tube long fragment read (stLFR) reads, Nanopore long-reads, and Hi-C sequencing data. The genome is 796.58 Mb, and a total of 712.17 Mb of the assembled sequences were anchored to 23 pseudo-chromosomes. A final set of 22,461 genes were annotated, with 98.67% being functionally annotated. The Benchmarking Universal Single-Copy Orthologs (BUSCO) benchmark of genome assembly and gene annotation reached 95.1% (93.3% single-copy) and 94.6% (91.7% single-copy), respectively. Furthermore, we also use ATAC-seq to uncover chromosome transposase-accessibility as well as related genome area function enrichment for Oryzias sinensis. This study offers a new improved foundation for future genomics research in Chinese medaka.

Vitamin a potentiates sheep myoblasts myogenic differentiation through BHLHE40-modulated ID3 expression

BackgroundVitamin A and retinoic acid (RA, a metabolite of vitamin A), are inextricably involved to the development of skeletal muscle in animals. However, the mechanisms regulating skeletal muscle development by vitamin A remain poorly reported. The current study designed to investigate the underlying mechanism of vitamin A affecting myogenic differentiation of lamb myoblasts through transcriptome sequencing (RNA-Seq) and gene function validation experiments. It provides a theoretical basis for elucidating the regulation of vitamin A on skeletal muscle development as well as for improving the economic benefits of the mutton sheep industry.ResultsNewborn lambs were injected with 7,500 IU vitamin A, and longissimus dorsi (LD) muscle tissue was surgically sampled for RNA-Seq analysis and primary myoblasts isolation at 3 weeks of age. The results showed that a total of 14 down-regulated and 3 up-regulated genes, were identified between control and vitamin A groups. Among them, BHLHE40 expression was upregulated in vitamin A group lambs. Furthermore, BHLHE40 expression is significantly increased after initiation of differentiation in myoblasts, and RA addition during differentiation greatly promoted BHLHE40 mRNA expression. In vitro, RA inhibited myoblasts proliferation and promoted myoblasts myogenic differentiation through BHLHE40. Moreover, BHLHE40 was proved to inhibit the expression of the DNA binding inhibitor 3 (ID3), and meanwhile, ID3 could effectively promote myoblasts proliferation and inhibit myoblasts myogenic differentiation.ConclusionsTaken together, our results suggested that vitamin A inhibited myoblasts proliferation and promoted myoblasts myogenic differentiation by inhibiting ID3 expression through BHLHE40.

Aging-Induced Reduction in Safflower Seed Germination via Impaired Energy Metabolism and Genetic Integrity Is Partially Restored by Sucrose and DA-6 Treatment.

Seed storage underpins global agriculture and the seed trade and revealing the mechanisms of seed aging is essential for enhancing seed longevity management. Safflower is a multipurpose oil crop, rich in unsaturated fatty acids that are at high risk of peroxidation as a contributory factor to seed aging. However, the molecular mechanisms responsible for safflower seed viability loss are not yet elucidated. We used controlled deterioration (CDT) conditions of 60% relative humidity and 50 °C to reduce germination in freshly harvested safflower seeds and analyzed aged seeds using biochemical and molecular techniques. While seed malondialdehyde (MDA) and fatty acid content increased significantly during CDT, catalase activity and soluble sugar content decreased. KEGG analysis of gene function and qPCR validation indicated that aging severely impaired several key functional and biosynthetic pathways including glycolysis, fatty acid metabolism, antioxidant activity, and DNA replication and repair. Furthermore, exogenous sucrose and diethyl aminoethyl hexanoate (DA-6) treatment partially promoted germination in aged seeds, further demonstrating the vital role of impaired sugar and fatty acid metabolism during the aging and recovery processes. We concluded that energy metabolism and genetic integrity are impaired during aging, which contributes to the loss of seed vigor. Such energy metabolic pathways as glycolysis, fatty acid degradation, and the tricarboxylic acid cycle (TCA) are impaired, especially fatty acids produced by the hydrolysis of triacylglycerols during aging, as they are not efficiently converted to sucrose via the glyoxylate cycle to provide energy supply for safflower seed germination and seedling growth. At the same time, the reduced capacity for nucleotide synthesis capacity and the deterioration of DNA repair ability further aggravate the damage to DNA, reducing seed vitality.

Genome-Wide Identification of the GhANN Gene Family and Functional Validation of GhANN11 and GhANN4 under Abiotic Stress.

Annexins (ANNs) are a structurally conserved protein family present in almost all plants. In the present study, 27 GhANNs were identified in cotton and were unevenly distributed across 14 chromosomes. Transcriptome data and RT-qPCR results revealed that multiple GhANNs respond to at least two abiotic stresses. Similarly, the expression levels of GhANN4 and GhANN11 were significantly upregulated under heat, cold, and drought stress. Using virus-induced gene silencing (VIGS), functional characterization of GhANN4 and GhANN11 revealed that, compared with those of the controls, the leaf wilting of GhANN4-silenced plants was more obvious, and the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) were lower under NaCl and PEG stress. Moreover, the expression of stress marker genes (GhCBL3, GhDREB2A, GhDREB2C, GhPP2C, GhRD20-2, GhCIPK6, GhNHX1, GhRD20-1, GhSOS1, GhSOS2 and GhSnRK2.6) was significantly downregulated in GhANN4-silenced plants after stress. Under cold stress, the growth of the GHANN11-silenced plants was significantly weaker than that of the control plants, and the activities of POD, SOD, and CAT were also lower. However, compared with those of the control, the elasticity and orthostatic activity of the GhANN11-silenced plants were greater; the POD, SOD, and CAT activities were higher; and the GhDREB2C, GhHSP, and GhSOS2 expression levels were greater under heat stress. These results suggest that different GhANN family members respond differently to different types of abiotic stress.

Uridine diphosphate glucosyltransferases are involved in spinosad resistance in western flower thrips Frankliniella occidentalis (Pergande)

Uridine diphosphate glucosyltransferases (UGTs) play crucial roles in the insect detoxification system and are associated with pesticide resistance. Our previous transcriptomic analysis of spinosad-susceptible (Ivf03) and resistant (NIL-R) Frankliniella occidentalis revealed numerous upregulated UGT genes in the NIL-R strain, suggesting their potential contribution to spinosad resistance. To investigate this hypothesis, here we conducted UGT activity assays and spinosad induction experiments, employing RNA interference (RNAi) techniques for gene function validation. We found significantly elevated UGT activity in the NIL-R strain compared to Ivf03, with 5-nitrouracil showing a substantial synergistic effect on the resistant strain. Eighteen UGT genes were identified in F. occidentalis, with gene expansion and duplication observed within families UGT466, 467, and 468. Ten out of the eighteen UGTs exhibited higher expression levels in NIL-R, specifically FoUGT466B1, FoUGT468A3, and FoUGT468A4 consistently being upregulated across nymphs, males, and females. RNAi-based functional validation targeting these three UGT genes led to increased susceptibility to spinosad in a life stage-, sex-, and dose-dependent manner. These results indicate that UGTs are indeed involved in spinosad resistance in F. occidentalis, and the effects are dependent on life stage, sex, and dose. Therefore, sustainable control for F. occidentalis resistance should always consider these differential responses.

Ethylene enhances resistance to cucumber green mottle mosaic virus via the ClWRKY70-ClACO5 module in watermelon plants.

Ethylene (ET) is involved in plant responses to viral infection. However, its molecular mechanisms and regulatory network remain largely unknown. In the present study, we report that cucumber green mottle mosaic virus (CGMMV) in watermelon (Citrullus lanatus) triggers ET production by inducing the expression of ClACO5, a key gene of the ET biosynthesis pathway through transcriptome data analysis and gene function validation. The knock-down of ClACO5 expression through virus-induced gene silencing in watermelon and overexpressing ClACO5 in transgenic Nicotiana benthamiana indicated that ClACO5 positively regulates CGMMV resistance and ET biosynthesis. The salicylic acid-responsive transcription factor gene ClWRKY70 shares a similar expression pattern with ClACO5. We demonstrate that ClWRKY70 directly binds to the W-box cis-element in the ClACO5 promoter and enhances its transcription. In addition, ClWRKY70 enhances plant responses to CGMMV infection by regulating ClACO5 expression in watermelon. Our results demonstrate that the ClWRKY70-ClACO5 module positively regulates resistance to CGMMV infection in watermelon, shedding new light on the molecular basis of ET accumulation in watermelon in response to CGMMV infection.

An ex vitro hairy root system from petioles of detached soybeanleaves for in planta screening of target genes and CRISPR strategies associated with nematode bioassays.

The ex vitro hairy root system from petioles of detached soybeanleaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybeanleaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the gallsweresuccessfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.

The Development of Plant Genome Sequencing Technology and Its Conservation and Application in Endangered Gymnosperms.

Genome sequencing is widely recognized as a fundamental pillar in genetic research and legal studies of biological phenomena, providing essential insights for genetic investigations and legal analyses of biological events. The field of genome sequencing has experienced significant progress due to rapid improvements in scientific and technological developments. These advancements encompass not only significant improvements in the speed and quality of sequencing but also provide an unparalleled opportunity to explore the subtle complexities of genomes, particularly in the context of rare species. Such a wide range of possibilities has successfully supported the validation of plant gene functions and the refinement of precision breeding methodologies. This expanded scope now includes a comprehensive exploration of the current state and conservation efforts of gymnosperm gene sequencing, offering invaluable insights into their genomic landscapes. This comprehensive review elucidates the trajectory of development and the diverse applications of genome sequencing. It encompasses various domains, including crop breeding, responses to abiotic stress, species evolutionary dynamics, biodiversity, and the unique challenges faced in the conservation and utilization of gymnosperms. It highlights both ongoing challenges and the unveiling of forthcoming developmental trajectories.