生物科学

Plant growth–promoting rhizobacteria (PGPR) can be used as biofertilizers to enhance crop growth for better yield and soil fertility restoration. PGPR possesses certain traits such as nutrient solubilization, phytohormone production, and production of key enzymes for improved crop growth. These traits are also important for inhibiting the growth of plant root pathogens, improving root development, and conferring stress tolerance. However, the mere presence of PGPR traits in isolated bacteria may not directly reflect an improvement in plant growth, warranting researchers to evaluate phenotypic and physiological changes upon inoculation. The current manuscript provides a detailed step-by-step procedure for inoculating the PGPR Staphylococcus sciuri into seeds and seedlings of rice and tomato plants for visualizing the enhancement of root and shoot growth. The surface-sterilized seeds of rice and tomato plants are inoculated overnight with an actively grown log-phase culture of S. sciuri, and differences in growth and biomass of seedlings that emerged from the inoculated and uninoculated seeds are analyzed 10 days after germination. Plants grown in pots with sterile soil are also treated with PGPR S. sciuri by soil drenching. A remarkable increase in root and shoot growth is observed in inoculated plants. We suggest that treating seeds with bacteria and enriching the soil with bacterial inoculum provides an adequate load of PGPR that facilitates growth improvement. This method can be a reliable choice for screening and evaluating plant growth promotion by either isolated bacteria or bacterial consortia with plant-beneficial traits.

To prepare Hevea brasiliensis plantations, selected planting material is propagated by grafting using illegitimate seedlings as rootstocks, whose paternal genotype is unknown. Recent advances in rubber tree in vitro cloning propagation open the possibility of using these techniques to supply new planting material. Micrografting is a promising technique to speed up the preparation of plant material for rootstock–scion interaction studies. This article describes the implementation of an efficient micrografting technique from Hevea in vitro plants from clone PB 260. The procedure combines several conditions to preserve the root system and the grafted scion and to prevent any breakage of rootstock buds. This technique paves the way for clonal propagation and holds potential for further development on other rubber clones for further studies on the interaction between rootstock and scion.

Agrobacterium-mediated gene transformation method is a vital molecular biology technique employed to develop transgenic plants. Plants are genetically engineered to develop disease-free varieties, knock out unsettling traits for crop improvement, or incorporate an antigenic protein to make the plant a green factory for edible vaccines. The method’s robustness was validated through successful transformations, demonstrating its effectiveness as a standard approach for researchers working in plant biotechnology. It enables the introduction of foreign DNA into plant genomes. Conventionally, plant genetic transformation has relied on time-consuming, costly, and technically demanding procedures, such as electroporation and chimeric viruses or biolistic methods, which usually yield variable transformation efficiencies. This study presents a simple and fail-safe protocol that involves a modified freeze-thaw and heat-shock concoction method. This approach involves a streamlined plasmid miniprep procedure to isolate high-quality plasmid DNA from Escherichia coli K12 strain, followed by a target-specific transfer into A. tumefaciens EHA105 strain. The optimized method minimizes DNA degradation and maximizes uptake by Agrobacterium cells, making it a reproducible and accessible protocol for various genetic engineering applications. The transformation efficiency is consistently high, enhancing plasmid uptake while maintaining cell viability, requiring minimal specialized equipment and reagents. The proposed protocol offers significant advantages, including simplicity, reliability, and cost-effectiveness, positioning it as a valuable alternative to traditional techniques in the field of plant biotechnology.

Most terrestrial plants are associated with symbiotic Glomeromycotina fungi, commonly known as arbuscular mycorrhizal (AM) fungi. AM fungi increase plant biomass in phosphate-depleted conditions by allocating mineral nutrients to the host; therefore, host roots actively exude various specialized metabolites and orchestrate symbiotic partners. The hyphal branching activity induced by strigolactones (SLs), a category of plant hormones, was previously discovered using an in vitro assay system. For this bioassay, AM fungi of the Gigaspora genus (Gigasporaeae) are commonly used due to their linear hyphal elongation and because the simple branching pattern is convenient for microscopic observation. However, many researchers have also used Glomeraceae fungi, such as Rhizophagus species, as the symbiotic partner of host plants, although they often exhibit a complex hyphal branching pattern. Here, we describe a method to produce and quantify the hyphal branches of the popular model AM fungus Rhizophagus irregularis. In this system, R. irregularis spores are sandwiched between gels, and chemicals of interest are diffused from the surface of the gel to the germinating spores. This method enables the positive effect of a synthetic SL on R. irregularis hyphal branching to be reproduced. This method could thus be useful to quantify the physiological effects of synthesized chemicals or plant-derived specialized metabolites on R. irregularis.

Toxoplasma gondii is a zoonotic protozoan parasite and one of the most successful foodborne pathogens. Upon infection and dissemination, the parasites convert into the persisting, chronic form called bradyzoites, which reside within cysts in muscle and brain tissue. Despite their importance, bradyzoites remain difficult to investigate directly, owing to limited in vitro models. In addition, the need for new drugs targeting the chronic stage, which is underlined by the lack of eradicating treatment options, remains difficult to address since in vitro access to drug-tolerant bradyzoites remains limited. We recently published the use of a human myotube-based bradyzoite cell culture system and demonstrated its applicability to investigate the biology of T. gondii bradyzoites. Encysted parasites can be functionally matured during long-term cultivation in these immortalized cells and possess many in vivo–like features, including pepsin resistance, oral infectivity, and antifolate resistance. In addition, the system is scalable, enabling experimental approaches that rely on large numbers, such as metabolomics. In short, we detail the cultivation of terminally differentiated human myotubes and their subsequent infection with tachyzoites, which then mature to encysted bradyzoites within four weeks at ambient CO₂ levels. We also discuss critical aspects of the procedure and suggest improvements.

Key features

• This protocol describes a scalable human myotube-based in vitro system capable of generating encysted bradyzoites featuring in vivo hallmarks.

• Bradyzoite differentiation is facilitated through CO₂ depletion but without additional artificial stress factors like alkaline pH.

• Functional maturation occurs over four weeks.

Graphical overview

Campylobacter jejuni, a zoonotic foodborne pathogen, is the worldwide leading cause of acute human bacterial gastroenteritis. Biofilms are a significant reservoir for survival and transmission of this pathogen, contributing to its overall antimicrobial resistance. Natural compounds such as essential oils, phytochemicals, polyphenolic extracts, and D-amino acids have been shown to have the potential to control biofilms formed by bacteria, including Campylobacter spp. This work presents a proposed guideline for assessing and characterizing bacterial biofilm formation in the presence of naturally occurring inhibitory molecules using C. jejuni as a model. The following protocols describe: i) biofilm formation inhibition assay, designed to assess the ability of naturally occurring molecules to inhibit the formation of biofilms; ii) biofilm dispersal assay, to assess the ability of naturally occurring inhibitory molecules to eradicate established biofilms; iii) confocal laser scanning microscopy (CLSM), to evaluate bacterial viability in biofilms after treatment with naturally occurring inhibitory molecules and to study the structured appearance (or architecture) of biofilm before and after treatment.

Drug biotransformation by the host microbiome can impact the therapeutic success of treatment. In the context of cancer, drug degradation can take place within the microenvironment of the targeted tumor by intratumor bacteria. In pancreatic cancer, increased chemo-resistance against the frontline chemotherapy gemcitabine is thought to arise from drug degradation by the tumor microbiome. This bacterial–drug interaction highlights the need for developing rapid assays for monitoring bacterial gemcitabine breakdown. While chemical approaches such as high-performance liquid chromatography are suitable for this task, they require specialized equipment and expertise and are limited in throughput. Functional cell-based assays represent an alternate approach for performing this task. We developed a functional assay to monitor the rate of bacterial gemcitabine breakdown using a highly sensitive bacterial reporter strain. Our method relies on standard laboratory equipment and can be implemented at high throughput to monitor drug breakdown by hundreds of strains simultaneously. This functional assay can be readily adapted to monitor degradation of other drugs.

Key features

• Quantification of gemcitabine breakdown by incubating bacteria that degrades the drug and subsequently testing the growth of a reporter strain on filtered supernatant.

• Use of an optimized reporter strain that was genetically engineered to be a non-degrader strain and highly sensitive to gemcitabine.

• A high-throughput assay performed in microplates that can be adjusted for identifying bacteria with a fast or slow gemcitabine degradation rate.

• The assay results can be compared to results from a standard curve with known drug concentrations to quantify degradation rate.

Graphical overview

Protocol overview. (1) Bacteria are incubated with gemcitabine for a set period of time. (2) Samples are removed from co-incubated suspensions and filtered to remove bacteria to halt gemcitabine degradation. (3) A gemcitabine-sensitive reporter strain is then added to the conditioned supernatant and is supplemented with growth media. (4) Growth of the reporter strain is monitored over time. (5) Results from the growth experiments are used to infer the concentration of gemcitabine in the co-culture supernatant and the drug degradation rate.

Ubiquitination is a post-translational modification conserved across eukaryotic species. It contributes to a variety of regulatory pathways, including proteasomal degradation, DNA repair, and cellular differentiation. The ubiquitination of substrate proteins typically requires three ubiquitination enzymes: a ubiquitin-activating E1, a ubiquitin-conjugating E2, and an E3 ubiquitin ligase. Cooperation between E2s and E3s is required for substrate ubiquitination, but some ubiquitin-conjugating E2s are also able to catalyze by themselves the formation of free di-ubiquitin, independently or in cooperation with a ubiquitin E2 variant. Here, we describe a method for assessing (i) di-ubiquitin formation by an E1 together with an E2 and an E2 variant, and (ii) the cooperation of an E3 with an E1 and E2 (with or without the E2 variant). Reaction products are assessed using western blotting with one of two antibodies: the first detects all ubiquitin conjugates, while the second specifically recognizes K63-linked ubiquitin. This allows unambiguous identification of ubiquitinated species and assessment of whether K63 linkages are present. We have developed these methods for studying ubiquitination proteins of Leishmania mexicana, specifically the activities of the E2, UBC2, and the ubiquitin E2 variant UEV1, but we anticipate the assays to be applicable to other ubiquitination systems with UBC2/UEV1 orthologues.

The human immunodeficiency virus 1 (HIV-1) consists of a viral membrane surrounding the conical capsid. The capsid is a protein container assembled from approximately 1,500 copies of the viral capsid protein (CA), functioning as a reaction and transport chamber for the viral genome after cell entry. Transmission electron microscopy (TEM) is a widely used technique for characterizing the ultrastructure of isolated viral capsids after removal of the viral membrane, which otherwise hinders negative staining of structures inside the viral particle for TEM. Here, we provide a protocol to permeabilize the membrane of HIV-1 particles using a pore-forming toxin for negative staining of capsids, which are stabilized with inositol hexakisphosphate to prevent premature capsid disassembly. This approach revealed the pleomorphic nature of capsids with a partially intact membrane surrounding them. The permeabilization strategy using pore-forming toxins can be readily applied to visualize the internal architecture of other enveloped viruses using TEM.

Graphical abstract:

Cryptococcus neoformans is a human pathogenic fungus that can cause pulmonary infections and meningitis in both immunocompromised and otherwise healthy individuals. Limited treatment options and a high mortality rate underlie the necessity for extensive research of the virulence of C. neoformans. Here we describe a detailed protocol for using the Galleria mellonella (Greater Wax Moth) larvae as a model organism for the virulence analysis of the cryptococcal infections. This protocol describes in detail the evaluation of G. mellonella larvae viability and the alternatives for troubleshooting the infection procedure. This protocol can be easily modified to study different inocula or fungal species, or the effects of a drug or antifungal agent on fungal disease within the larvae. We describe modified alternative versions of the protocol that allow using G. mellonella to study fungal diseases with different inocula and at different temperatures.