植物科学

The root parasitic weed Striga hermonthica has a devastating effect on sorghum and other cereal crops in Sub-Saharan Africa. Available Striga management strategies are rarely sufficient or not widely accessible or affordable. Identification of soil- or plant-associated microorganisms that interfere in the Striga infection cycle holds potential for development of complementary biological control measures. Such inoculants should be preferably based on microbes native to the regions of their application. We developed a method to assess microbiome-based soil suppressiveness to Striga with a minimal amount of field-collected soil. We previously used this method to identify the mechanisms of microbe-mediated suppression of Striga infection and to test individual microbial strains. Here, we present protocols to assess the functional potential of the soil microbiome and individual bacterial taxa that adversely affect Striga parasitism in sorghum via three major known suppression mechanisms. These methods can be further extended to other Striga hosts and other root parasitic weeds.

The roots of herbaceous and woody plants growing in soil are complex structures that are affected by both natural and artificial fungal colonization to various extents. To obtain comprehensive information about the overall distribution of fungi or oomycetes inside a plant root system, rapid, effective, and reliable screening methods are required. To observe both fine roots, i.e., a common site for penetration of fungi and oomycetes, and mature roots, different techniques are required to overcome visual barriers, such as root browning or tissue thickening. In our protocol, we propose using fast, cost-effective, and non-harmful methods to localize fungal or oomycete structures inside plant roots. Root staining with a fluorescent dye provides a quick initial indication of the presence of fungal structures on the root surfaces. The protocol is followed by clearing and staining steps, resulting in a deeper insight into the root tissue positioning, abundance, and characteristic morphological/reproductive features of fungal or oomycete organisms. If required, the stained samples can be prepared by using freeze-drying for further observations, including advanced microscopic techniques.

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.