Plant Science

Maize is one of the most important crops in the world, and ensuring its successful growth and productivity is crucial for global food security. One way to enhance maize growth and productivity is by improving the colonization of its roots by beneficial microorganisms. In this regard, Serendipita indica, a plant growth–promoting fungus, has gained attention for its ability to enhance plant growth and productivity, especially in cereal crops and medicinal plants. Previous studies have shown that S. indica can colonize various plant species, including maize, but the efficiency of the colonization process in maize seedlings has not been extensively characterized. This protocol outlines a method for efficient colonization of maize seedlings with the beneficial fungus S. indica. The protocol includes the preparation of stock solutions, maintenance and growth of S. indica, surface sterilization and germination of seeds, preparation of S. indica chlamydospores, and colonization of maize plants with S. indica. The advantages of this protocol include the use of surface sterilization techniques that minimize contamination, the production of a large number of viable chlamydospores, and efficient colonization of maize seedlings with S. indica. This protocol may be useful for researchers studying the role of S. indica in promoting plant growth and combating biotic and abiotic stress. Additionally, this protocol may be used in the development of biofertilizers using S. indica as a means of increasing crop yields and reducing dependence on synthetic fertilizers. Overall, this protocol offers a reliable and efficient method for colonizing maize seedlings with S. indica and may have potential applications in the agricultural industry. This study also provides a valuable tool for researchers interested in studying plant–microbe interactions in maize and highlights the potential of S. indica as a biocontrol agent to enhance maize productivity under adverse conditions.

Key features

• This protocol builds upon the method developed by Narayan et al. (2022), and its application optimized for the root endophytic symbiotic fungus S. indica.

• This protocol also allows for histochemical analysis to visualize the colonized fungal spores in the root cells of host plant species.

• This protocol helps in mathematical calculation of the percent colonization or efficiency of colonization.

• This protocol utilizes readily available laboratory equipment, including a light microscope, autoclave, and laminar flow hood, ensuring ease of reproducibility in other research laboratories.

Graphical overview

Barley (Hordeum vulgare) is one of the most important agricultural crops in the world, but pathogen infections regularly limit its annual yield. A major threat is the infection with the biotrophic leaf rust fungus, Puccinia hordei. Rust fungi have a complex life cycle, and existing resistances can be easily overcome. To address this problem, it is crucial to develop barley varieties with improved and durable resistance mechanisms. An essential step towards this goal is a simple and reproducible infection protocol to evaluate potential resistance phenotypes in the lab. However, available protocols sometimes lack detailed procedure or equipment information, use spore application methods that are not suitable for uniform spore dispersion, or require special mineral oils or engineered fluids. In addition, they are often optimized for pathogen-dedicated greenhouses or phytochambers, which may not be available to every research institute. Here, we describe an easy and user-friendly procedure to infect barley with Puccinia hordei on a small laboratory scale. This procedure utilizes inexpensive and simple tools to evenly split and apply spores to barley leaves. The treated plants are incubated in affordable and small phytocabinets. Our protocol enables a quick and reproducible infection of barley with leaf rust, a method that can easily be transferred to other rust fungi, including stripe rust, or to other plant species.

Key features

• Step-by-step infection protocol established for barley cv. Golden Promise, the gold standard genotype for genetic transformation

• Plant age–independent protocol

• Precise spore application by using inexpensive pipe cleaners for uniform symptom formation and increased reproducibility

• No specialized equipment needed

• Includes simple spore harvesting method

• Protocol is applicable to other biotrophic pathogens (stripe rust or powdery mildew) and other plants (e.g., wheat)

• Protocol is also applicable for a detached leaf assay

Graphical overview

Arabidopsis thaliana-Pseudomonas syringae pathosystem has been used as an important model system for studying plant-microbe interactions, leading to many milestones and breakthroughs in the understanding of plant immune system and pathogenesis mechanisms. Bacterial infection and plant disease assessment are key experiments in the studies of plant-pathogen interactions. The hypersensitive response (HR), which is characterized by rapid cell death and tissue collapse after inoculation with a high dose of bacteria, is a hallmark response of plant effector-triggered immunity (ETI), one layer of plant immunity triggered by recognition of pathogen-derived effector proteins. Here, we present a detailed protocol for bacterial disease and hypersensitive response assays applicable to studies of Pseudomonas syringae interaction with various plant species such as Arabidopsis, Nicotiana benthamiana, and tomato.

Calcium signaling is an emerging mechanism by which bacteria respond to environmental cues. To measure the intracellular free-calcium concentration in bacterial cells, [Ca²⁺]_i, a simple spectrofluorometric method based on the chemical probe Fura 2-acetoxy methyl ester (Fura 2-AM) is here presented using Pseudomonad bacterial cells. This is an alternative and quantitative method that can be completed in a short period of time with low costs, and it does not require the induction of heterologously expressed protein-based probes like Aequorin. Furthermore, it is possible to verify the properties of membrane channels involved in Ca²⁺ entry from the extracellular matrix. This method is in particular valuable for measuring [Ca²⁺]_i in the range of 0.1-39.8 µM in small cells like those of prokaryotes.

Bacteria blight diseases of rice due to several genera of pathogenic bacteria are one of the major constraints worldwide for rice production. The disease can be best managed through host plant resistance sources. For most of these bacteria such as Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, Pseudomonas fuscovaginae, Burkholderia glumae, Burkholderia gladioli and Acidovorax avenae subsp. avenae, specific diagnostic techniques that include molecular and pathogenicity tests have been developed.

However, for Pantoea spp., information on pathogenicity assay is very limited and protocols used are not uniform. Most authors use the leaf clipping method. In this paper, we describe the protocol for mechanical inoculation of rice seedlings aged 35 days. The method consists of infiltrating bacterial suspensions at concentrations of 10⁸ CFU/ml, with a needleless syringe into the intercellular and interveinal spaces of rice leaves underside at about 4-5 cm below the leaf tip.

This method can be used for a standardized pathogenicity assessment, germplasm resistance evaluation for identifying and characterizing resistance sources.

Potato virus Y (PVY), the type member of the genus Potyvirus (family Potyviridae), is the most widespread virus affecting potato and is included in the top five most economically detrimental plant viruses. Recently, the structure of the PVY virion has been determined by cryo-electron microscopy, which has opened the doors to functional studies that explore the involvement of selected amino acids in different stages of the viral cycle. The only way to functionally challenge in planta the role of particular amino acids in the coat protein of PVY, or in other viral proteins, is by using cDNA clones. The use and manipulation of PVY cDNA clones, unlike those of other potyviruses, has been traditionally impaired by the toxicity that certain sequences within the PVY genome pose to Escherichia coli. Here, we describe the use of a published PVY cDNA clone, which harbours introns that overcome the aforementioned toxicity, to explore the effects of different coat protein modifications on viral infection. The protocol includes manipulation of the cDNA clone in E. coli, biolistic inoculation of plants with the constructed clones, observation of the biological effects on plants, quantification of cDNA clones by reverse transcription quantitative PCR, and confirmation of virion formation by transmission electron microscopy. Future possibilities involve the use of PVY cDNA clones tagged with fluorescent protein reporters to allow further insights into the effects of coat protein mutations on the cell-to-cell movement of PVY virions.

The mechanisms of virulence and immunity are often governed by molecular interactions between pathogens and host proteins. The study of these interactions has major implications on understanding virulence activities, and how the host immune system recognizes the presence of pathogens to initiate an immune response. Frequently, the association between pathogen molecules and host proteins are assessed using qualitative techniques. As small differences in binding affinity can have a major biological effect, in vitro techniques that can quantitatively compare the binding between different proteins are required. However, these techniques can be manually intensive and often require large amounts of purified proteins. Here we present a simplified Surface Plasmon Resonance (SPR) protocol that allows a reproducible side-by-side quantitative comparison of the binding between different proteins, even in cases where the binding affinity cannot be confidently calculated. We used this method to assess the binding of virulence proteins (termed effectors) from the blast fungus Magnaporthe oryzae, to a domain of a host immune receptor. This approach represents a rapid and quantitative way to study how pathogen molecules bind to host proteins, requires only limited quantities of proteins, and is highly reproducible. Although this method requires the use of an SPR instrument, these can often be accessed through shared scientific services at many institutions. Thus, this technique can be implemented in any study that aims to understand host-pathogen interactions, irrespective of the expertise of the investigator.

The interaction between the host plant Arabidopsis thaliana (Arabidopsis) and the oomycete Hyaloperonospora arabidopsidis (Hpa) is an established model system for the study of an obligate biotrophic downy mildew interaction. The evaluation of the developmental success of Hpa is often based on the quantification of reproductive structures that are formed on the surface of leaves, such as the sporangiophores or the conidiospores they carry. However, the structural basis of this interaction lies within the plant tissue and, in particular, the haustoria that form inside plant cells. Therefore, valuable additional information about the performance and compatibility of the downy mildew interaction can be gained by light microscopical inspection of the hyphal and haustorial shape inside the plant tissue and within plant cells respectively. Here we describe a protocol for the visualization and quantification of morphological phenotypes inside the plant. While we focus specifically on the quantification of haustorial shape variants, the protocol can easily be adapted for the quantification of other morphological features such as hyphal deformations, or oogonia frequency. By including and refining already existing protocols from a variety of sources, we assembled the entire experimental pipeline for the Arabidopsis Hpa bioassay to provide a practical guide for the initial setup of this system in the laboratory. This pipeline includes the following steps: A) growing Arabidopsis, B) Hpa propagation and strain maintainance C) Hpa inoculation and incubation D) staining of plant tissues for visualization of the pathogen and E) an introduction of the Keyence VHX microscope and Fiji plugin of ImageJ for the quantification of structures of interest. While described here for Arabidopsis and Hpa, the protocol steps B-E should be easily adjustable for the study of other plant-oomycete pathosystems.

Plants recognize a wide variety of microbial molecules to detect and respond to potential invaders. Recognition of Microbe-Associated Molecular Patterns (MAMPs) by cell surface receptors initiate a cascade of biochemical responses that include, among others, ion fluxes across the plasma membrane. A consequence of such event is a decrease in the concentration of extracellular H⁺ ions, which can be experimentally detected in plant cell suspensions as a shift in the pH of the medium. Thus, similarly to reactive oxygen species (ROS) accumulation, phosphorylation of MAP kinases and induction of defense-related genes, MAMP-induced medium alkalinization can be used as a proxy for the activation of plant immune responses. Here, we describe a detailed protocol for the measurement of medium alkalinization of tobacco BY-2 cell suspensions upon treatment with two different MAMPs: chitohexamers derived from fungal cell walls (NAG6; N-acetylglucosamine) and the flagellin epitope flg22, found in the bacterial flagellum. This method provides a reliable and fast platform to access MAMP-Triggered Immunity (MTI) in tobacco cell suspensions and can be easily adapted to other plant species as well as to other MAMPs.

Yeasts such as Aureobasidium pullulans are unicellular fungi that occur in all environments and play important roles in biotechnology, medicine, food and beverage production, research, and agriculture. In the latter, yeasts are explored as biocontrol agents for the control of plant pathogenic fungi (e.g., Botrytis cinerea, Fusarium sp.); mainly on flowers and fruits. Eventually, such yeasts must be evaluated under field conditions, but such trials require a lot of time and resources and are often difficult to control. Experimental systems of intermediate complexity, between in vitro Petri dish assays and field trials, are thus required. For pre- and post-harvest applications, competition assays on fruits are reproducible, economical and thus widely used. Here, we present a general protocol for competition assays with fruits that can be adapted depending on the biocontrol yeast, plant pathogen, type of assay or fruit to be studied.