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Bio-protocol May 2024-mediated transient gene expression in Nicotiana benthamiana is widely used to study gene function in plants. One dramatic phenotype that is frequently screened for is...
-mediated transient gene expression in Nicotiana benthamiana is widely used to study gene function in plants. One dramatic phenotype that is frequently screened for is cell death. Here, we present a simplified protocol for Agrobacterium-mediated transient gene expression by infiltration. Compared with current methods, the novel protocol can be done without a centrifuge or spectrometer, thereby suitable for K-12 outreach programs as well as rapidly identifying genes that induce cell death. Key features • The protocol simplifies the widely used -mediated transient gene expression assay [1] and can be completed within one week when plants are available. • Rice gene can induce a dramatic and easily identifiable cell death phenotype in • Allows identification of cell death-inducing genes and is suitable for teaching. • Compared to the currently used methods, our protocol omits the use of agroinfiltration buffer, pH meter, temperature-controlled growth chamber, centrifuge, and spectrophotometer. Graphical overview The photo demonstrates the method of agroinfiltration into the abaxial side of leaves using a needleless syringe.
PubMed: 38798979
DOI: 10.21769/BioProtoc.4987 -
Animals : An Open Access Journal From... May 2024The vaginal tract comprises commensal microorganisms, which play an essential role in the health of the reproductive tract. Any dysbiosis in the vaginal microenvironment...
The vaginal tract comprises commensal microorganisms, which play an essential role in the health of the reproductive tract. Any dysbiosis in the vaginal microenvironment may lead to severe urinary tract infections or even infertility. This study aimed to evaluate the aerobic bacterial flora isolated from vaginal samples from 100 lactating bitches in the antepartum period ( = 3), postpartum period ( = 80), and with ( = 17). Before vaginal swabs, all the bitches went through a gynecology consult, along with milk and blood sampling. Standard microbiological techniques were used for bacterial isolation. Among the 100 vaginal samples analyzed, 82% had a positive microbiological outcome, while 18% were negative. The microbiologic profile listed 17 different genera. The main isolated bacterial families were Micrococcaceae, Staphylococcaceae, Morganellaceae, Bacillaceae, and Rhizobiaceae. At the same time, strains like , , , , , or were isolated for the first time from the vaginal secretion of lactating bitches. The microbiological data demonstrates that lactating bitches' vaginal discharge is heterogeneous and may be affected by coitus, sampling season, age, and reproductive status.
PubMed: 38791718
DOI: 10.3390/ani14101501 -
Frontiers in Microbiology 2024Alterations in the microbial community significantly impact the yield and quality of ginseng. Yet, the dynamics of microbial community shifts within the root endophytes...
Alterations in the microbial community significantly impact the yield and quality of ginseng. Yet, the dynamics of microbial community shifts within the root endophytes of ginseng across varying cultivation periods remain inadequately understood. This study zeroes in on the microbial community variations within the xylem (M), phloem (R), and fibrous roots (X) of ginseng during the fourth (F4) and fifth (F5) years of cultivation, aiming to bridge this research gap. We assessed soil physicochemical properties, enzyme activities, and nine individual saponins, complemented by high-throughput sequencing techniques (16S rDNA and ITS) to determine their profiles. The results showed that cultivation years mainly affected the microbial diversity of endophytic bacteria in ginseng fibrous roots compartment: the ASVs number and α-diversity Chao1 index of bacteria and fungi in F5X compartment with higher cultivation years were significantly higher than those in F4X compartment with lower cultivation years. It is speculated that the changes of fibrous roots bacterial groups may be related to the regulation of amino acid metabolic pathway. Such as D-glutamine and D-glutamate metabolism D-glutamine, cysteine and methionine metabolism regulation. The dominant bacteria in ginseng root are Proteobacteria (relative abundance 52.07-80.35%), Cyanobacteria (1.97-42.52%) and Bacteroidota (1.11-5.08%). Firmicutes (1.28-3.76%). There were two dominant phyla: Ascomycota (60.10-93.71%) and Basidiomycota (2.25-30.57%). Endophytic fungi were more closely related to soil physicochemical properties and enzyme activities. AN, TK, OP, SWC and EC were the main driving factors of endophytic flora of ginseng root. decreased with the increase of cultivation years, and the decrease was more significant in phloem (F4R: 33.36%, F5R: 16.48%). The relative abundance of and in each ecological niche increased with the increase of cultivation years. The relative abundance of and in F5X increased by 8.35 and 9.29 times, respectively, and in F5M increased by 5.57 times. We found a variety of potential beneficial bacteria and pathogen antagonists related to ginseng biomass and saponins, such as , , and , which have good potential for practical application and development.
PubMed: 38756733
DOI: 10.3389/fmicb.2024.1402921 -
Plant Signaling & Behavior Dec 2024The purpose of this study was to analyze the role of transcription factor in , proving that the transcription factor was related to the plant phenotypes of - cv....
The purpose of this study was to analyze the role of transcription factor in , proving that the transcription factor was related to the plant phenotypes of - cv. 'GuangYaoDa1' and it could be used in molecular-assisted breeding. 'GuangYaoDa1' was used as the material and its DNA was the template to clone DsWRKY6, the transgenic line was constructed by agrobacterium tumefaciens‑mediated transformation. Transgenic was cultivated to study phenotype and physiological and biochemical indexes. Phenotypic observation showed that transgenic had a faster growth rate while compared with the control group, they had longer lengths of main stem, lateral branches of cauline leaves, and root, but a lower number of cauline leaves and lateral branches of cauline leaves. And it also showed that their flowering and fruiting periods were advanced. The results of physiological and biochemical indexes showed that the relative expressions of increased and the abscisic acid content significantly increased in transgenic compared with the control group. According to the above results, could regulate the advancing of flowering and fruiting periods caused by the improvement of abscisic acid content, and expression of the transcription factor might be the cause of the upright growth of 'GuangYaoDa1'.
Topics: Arabidopsis; Plants, Genetically Modified; Cloning, Molecular; Plant Proteins; Transcription Factors; Gene Expression Regulation, Plant; Fabaceae; Phenotype; Abscisic Acid; Genes, Plant
PubMed: 38743594
DOI: 10.1080/15592324.2024.2349868 -
BMC Plant Biology May 2024'Candidatus Phytoplasma mali', the causal agent of apple proliferation disease, exerts influence on its host plant through various effector proteins, including SAP11...
'Candidatus Phytoplasma mali' SAP11-Like protein modulates expression of genes involved in energy production, photosynthesis, and defense in Nicotiana occidentalis leaves.
BACKGROUND
'Candidatus Phytoplasma mali', the causal agent of apple proliferation disease, exerts influence on its host plant through various effector proteins, including SAP11 which interacts with different TEOSINTE BRANCHED1/ CYCLOIDEA/ PROLIFERATING CELL FACTOR 1 and 2 (TCP) transcription factors. This study examines the transcriptional response of the plant upon early expression of SAP11. For that purpose, leaves of Nicotiana occidentalis H.-M. Wheeler were Agrobacterium-infiltrated to induce transient expression of SAP11 and changes in the transcriptome were recorded until 5 days post infiltration.
RESULTS
The RNA-seq analysis revealed that presence of SAP11 in leaves leads to downregulation of genes involved in defense response and related to photosynthetic processes, while expression of genes involved in energy production was enhanced.
CONCLUSIONS
The results indicate that early SAP11 expression might be important for the colonization of the host plant since phytoplasmas lack many metabolic genes and are thus dependent on metabolites from their host plant.
Topics: Nicotiana; Phytoplasma; Plant Leaves; Photosynthesis; Gene Expression Regulation, Plant; Plant Diseases; Bacterial Proteins; Energy Metabolism
PubMed: 38741080
DOI: 10.1186/s12870-024-05087-4 -
Plants (Basel, Switzerland) Apr 2024Increasing the ultraviolet radiation (UV) level, particularly UV-B due to damage to the stratospheric ozone layer by human activities, has huge negative effects on plant...
Increasing the ultraviolet radiation (UV) level, particularly UV-B due to damage to the stratospheric ozone layer by human activities, has huge negative effects on plant and animal metabolism. As a widely grown cool-season forage grass and turfgrass in the world, perennial ryegrass () is UV-B-sensitive. To study the effects of miR164, a highly conserved microRNA in plants, on perennial ryegrass under UV stress, both Osa overexpression (OE164) and target mimicry (MIM164) transgenic perennial ryegrass plants were generated using agrobacterium-mediated transformation, and UV-B treatment (~600 μw cm) of 7 days was imposed. Morphological and physiological analysis showed that the gene affected perennial ryegrass UV tolerance negatively, demonstrated by the more scorching leaves, higher leaf electrolyte leakage, and lower relative water content in OE164 than the WT and MIM164 plants after UV stress. The increased UV sensitivity could be partially due to the reduction in antioxidative capacity and the accumulation of anthocyanins. This study indicated the potential of targeting and/or its targeted genes for the genetic manipulation of UV responses in forage grasses/turfgrasses; further research to reveal the molecular mechanism underlying how miR164 affects plant UV responses is needed.
PubMed: 38732457
DOI: 10.3390/plants13091242 -
PloS One 2024Sunflower is one of the four major oil crops in the world. 'Zaoaidatou' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle,...
Sunflower is one of the four major oil crops in the world. 'Zaoaidatou' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.
Topics: Helianthus; Transformation, Genetic; Agrobacterium tumefaciens; Plants, Genetically Modified; Tissue Culture Techniques; Plant Roots; Plant Breeding; Crops, Agricultural
PubMed: 38722945
DOI: 10.1371/journal.pone.0298299 -
Frontiers in Plant Science 2024In the post-genomic era, virus-induced gene silencing (VIGS) has played an important role in research on reverse genetics in plants. Commonly used -mediated VIGS...
In the post-genomic era, virus-induced gene silencing (VIGS) has played an important role in research on reverse genetics in plants. Commonly used -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 and successfully silenced , tomato (), pepper ( L.), eggplant (), and phytoene desaturase (), and we observed the movement of green fluorescent protein (GFP) from the roots to the stem and leaves. The silencing rate of in and tomato was 95-100%. In addition, we successfully silenced two disease-resistance genes, and , 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.
PubMed: 38708388
DOI: 10.3389/fpls.2024.1336726 -
Plant Signaling & Behavior Dec 2024The hairy root induction system was used to efficiently investigate gene expression and function in plant root. Cucumber is a significant vegetable crop worldwide, with...
The hairy root induction system was used to efficiently investigate gene expression and function in plant root. Cucumber is a significant vegetable crop worldwide, with shallow roots, few lateral roots, and weak root systems, resulting in low nutrient absorption and utilization efficiency. Identifying essential genes related to root development and nutrient absorption is an effective way to improve the growth and development of cucumbers. However, genetic mechanisms underlying cucumber root development have not been explored. Here, we report a novel, rapid, effective hairy root transformation system. Compared to the in vitro cotyledon transformation method, this method shortened the time needed to obtain transgenic roots by 13 days. Furthermore, we combined this root transformation method with CRISPR/Cas9 technology and validated our system by exploring the expression and function of , a pivotal gene associated with root development and nutrient uptake. The hairy root transformation system established in this study provides a powerful method for rapidly identifying essential genes related to root development in cucumber and other horticultural crop species. This advancement holds promise for expediting research on root biology and molecular breeding strategies, contributing to the broader understanding and improvements crop growth and development.
Topics: Plant Roots; Cucumis sativus; Plant Proteins; Plants, Genetically Modified; Gene Expression Regulation, Plant; Transformation, Genetic; CRISPR-Cas Systems
PubMed: 38686613
DOI: 10.1080/15592324.2024.2345983 -
Open Life Sciences 2024Allene oxide synthase (AOS) is a key enzyme involved in the jasmonic acid (JA) synthesis pathway in plants. To explore its function on the regulatory mechanism of JA...
Allene oxide synthase (AOS) is a key enzyme involved in the jasmonic acid (JA) synthesis pathway in plants. To explore its function on the regulatory mechanism of JA synthesis, we screened and identified two genes and in qingke. Both HvnAOS1 and HvnAOS2 contained conserved heme-binding motif, which is most closely related to AtsAOS2, indicating controlled dehydration of fatty acid hydroperoxides to allene oxides. Molecular docking simulations identified the key amino acid sites that were important for heme binding and interaction with 13()-HPOT, respectively. The expression pattern also indicated that and were highly induced by JA, abscisic acid, and salicylic acid. Subcellular localization of and using transient expression of showed the green fluorescent protein signal in the cell cytoplasm of the . leaves. Overexpression of and in mutant restored male fertility and plant resistance to , indicating that and can restore the functions of in mutant.
PubMed: 38681731
DOI: 10.1515/biol-2022-0855