植物科学

It has been discovered that many phytopathogenic fungi can absorb exogenous double-stranded RNAs (dsRNAs) to silence target genes, inhibiting fungal growth and pathogenicity for plant protection. In our recent report, the beneficial arbuscular mycorrhizal (AM) fungi are capable of acquiring external naked dsRNAs; however, whether the dsRNAs can be delivered into AM fungi through nanocarriers remains to be investigated. Here, we introduce a simple and advanced method for in vitro synthesizing chitosan (CS)/dsRNA polyplex nanoparticles (PNs) to silence the target gene in the AM fungus Rhizophagus irregularis. This method is straightforward, requiring minimal modifications, and is both efficient and eco-friendly, offering potential for rapid application in elucidating gene functions in AM fungi.

In many plant species, self-incompatibility (SI) is a mechanism that inhibits inbreeding. SI is gametophytic in the Solanaceae, with specificity determined by S-ribonucleases (S-RNases) in the pistil and S-locus F-box proteins (SLFs) in the pollen. The role of these proteins has been studied extensively in the Solanaceae, often using Petunia as a model. Using degenerate PCR and Sanger sequencing, this protocol identified three SLF sequences from self-incompatible diploid potato (Solanum okadae). While SLFs are well-characterized in model species like Petunia, there is limited sequence data and no standardized protocols for identifying SLFs in non-model species such as S. okadae, hindering broader insights into SI across the Solanaceae. This protocol fills that gap by using degenerate PCR and Sanger sequencing with primers designed from conserved Petunia SLF regions to identify SLF sequences in S. okadae. SLF sequences from 10 distinct Solanaceae members sharing maximum identity with the S2-haplotype of Petunia were used to design two pairs of primers targeting different regions of the target sequence. PCR amplification using designed degenerate primers yielded amplicons that were directly sequenced and joined together to get the partial SLF sequence. It was observed that the S. okadae shared an orthologous relation with the Petunia SLF according to the phylogenetic analysis. These SLFs could be used in future SI breakdown experiments via the competitive interaction route. This protocol, including the primer design, is novel for detecting SLF sequences in S. okadae.

Amyloplasts, non-photosynthetic plastids specialized for starch synthesis and storage, proliferate in storage tissue cells of plants. To date, studies of amyloplast replication in roots and the ovule nucelli from various plant species have been performed using electron and fluorescence microscopy. However, a complete understanding of amyloplast replication remains unclear due to the absence of experimental systems capable of tracking their morphology and behavior in living cells. Recently, we demonstrated that Arabidopsis ovule integument could provide a platform for live-cell imaging of amyloplast replication. This system enables precise analysis of amyloplast number and shape, including the behavior of stroma-filled tubules (stromules), during proplastid-to-amyloplast development in post-mitotic cells. Here, we provide technical guidelines for observing and quantifying amyloplasts using conventional fluorescence microscopy in wild-type and several plastid-division mutants of Arabidopsis.

In live-cell imaging, autofluorescence is often regarded as a negative factor that interferes with the accurate visualization of target fluorescence due to a phenomenon known as crosstalk. However, autofluorescence has also been effectively utilized as an organellar marker. For instance, the intense autofluorescence of chlorophyll in the red wavelength is widely used to visualize chloroplasts, the photosynthetic organelle in plants. Recently, we demonstrated that nuclei in plant cells emit phytochrome-derived autofluorescence in the red to infrared wavelength range, which can be visualized by a conventional confocal microscope equipped with a 640 nm laser. Here, we present protocols for growing plants and conducting confocal imaging of the near-infrared autofluorescence of nuclei in Arabidopsis thaliana.

Membranes are very complex and dynamic structures that are essential for plant cellular functions and whose lipidic composition can be influenced by numerous factors. Anionic phospholipids, which include phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphoinositides are key components of these membranes as they are involved in plant cell signaling and as even slight modifications in their quantities may largely impact the cell metabolism. However, the presence of these compounds in low amounts, as well as their poor stability during analysis by mass spectrometry, make their study very complicated. In addition, the precise quantification of all anionic phospholipid species is not possible by lipid separation using thin-layer chromatography followed by the analysis of their fatty acyl chains by gas chromatography. Here, we describe a straightforward strategy for the extraction and semi-quantification of all anionic phospholipid species from plant samples. Our method is based on the derivatization of the anionic phospholipids, and more especially on their methylation using trimethylsilyldiazomethane, followed by analysis by high-performance liquid chromatography coupled with a triple quadrupole mass spectrometer. This approach allows largely improving the sensitivity of the analysis of anionic phospholipids from plant samples, which will help to gain deeper insights into the functions and dynamics of these key parts of plant cellular signaling.

The ability to efficiently screen plant pathogen effectors is crucial for understanding plant–pathogen interactions and developing disease-resistant crops. Traditional methods are often labor-intensive and time-consuming. Here, we present a robust, high-throughput screening assay using the tobacco mosaic virus–green fluorescent protein (TMV-GFP) vector system. The screening system combines the TMV-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity (both activation and suppression). The biological function of these effectors can be easily evaluated within six days by observing the GFP fluorescence signal using a UV lamp. This protocol significantly reduces the time required for screening and increases the throughput, making it suitable for large-scale studies. The method is versatile, cost-effective, and can be adapted to effectors with immune interference activity from various pathogens.

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.

Antimicrobial peptides are effective agents against various pathogens, often targeting essential processes like protein translation to exert their antimicrobial effects. Traditional methods such as puromycin labeling have been extensively used to measure protein synthesis in mammalian and yeast systems; however, protocols tailored for plant pathogenic filamentous fungi, particularly those investigating translation inhibition by antifungal peptides, are lacking. This protocol adapts puromycin labeling to quantify translation inhibition in Botrytis cinerea germlings treated with antifungal peptides. Optimizing the method specifically for fungal germlings provides a precise tool to investigate peptide effects on fungal protein synthesis, advancing our understanding of translation dynamics during pathogen–host interactions in filamentous fungi.

Extracellular vesicles (EVs) are membrane-bound, non-replicating particles released by virtually all types of cells. EVs concentrate and deliver a plethora of biomolecules driving very important biological functions, including intercellular communication not only between cells of the same organism but also across different kingdoms. Plant extracellular vesicles (PEVs) are a promising alternative to mammalian EVs in biomedical applications. Here, we present an optimized and reproducible protocol for isolating PEVs from the hairy root (HR) cultures of medicinal plants Salvia dominica and S. sclarea. Our methodological approach introduces a significant advancement in the standardization of HR-EVs purification processes from plant biotechnological platforms, paving the way for their broader application across various sectors, including agriculture, pharmaceuticals, and nutraceuticals.

Plant proteases participate in a wide variety of biological processes, including development, growth, and defense. To date, numerous proteases have been functionally identified through genetic studies. However, redundancy among certain proteases can obscure their roles, as single-gene loss-of-function mutants often exhibit no discernible phenotype, limiting identification through genetic approaches. Here, we describe an efficient system for the identification of target proteases that cleave specific substrates in the Arabidopsis apoplastic fluid. The method involves using Arabidopsis-submerged culture medium, which contains apoplastic proteases, followed by native two-dimensional electrophoresis. Gel fractionation and an in-gel peptide cleavage assay with a fluorescence-quenching peptide substrate are then used to detect specific proteolytic activity. The active fraction is then subjected to mass spectrometry–based proteomics to identify the protease of interest. This method allows for the efficient and comprehensive identification of proteases with specific substrate cleavage activities in the apoplast.