植物科学

We have established the Ustilago bromivora–Brachypodium spp. interaction as a new model pathosystem for biotrophic fungal plant infections of the head smut type (Rabe et al., 2016). In this protocol, the methodology used for comparing gene expression between saprophytic and in planta growth of the fungus is described. The experimental and analytical pipeline, how next generation RNA sequencing (Illumina RNA-Seq) analysis can be used to obtain lists of genes significantly up or down regulated in planta in comparison to axenic culture is given. Furthermore, different methods to identify functional categories that are over- or under-represented among specific classes of genes are presented.

Grapevine (Vitis vinifera L.) is susceptible to an array of diseases among them the grey mold caused by the necrotrophic fungus Botrytis cinerea that decreases grape productivity and quality. To ensure a satisfactory yield and harvest quality numerous chemical fungicides are required, but they have serious drawbacks. One alternative is the use of beneficial bacteria to improve plant health. Pseudomonas fluorescens has been shown to trigger a plant-mediated resistance response in aboveground plant tissues against fungal, oomycete, bacterial, and viral pathogens. Triggered plant resistance exploits mechanisms of the plant immune system through a priming state that provides plants with enhanced capacity for rapid and strong activation of defense responses after pathogen infection, resulting in a lower fitness-cost. The primed responses by beneficial bacteria include induced expression of defense-related genes, cell wall reinforcement, and the production of secondary metabolites after pathogen infection. In this protocol, we describe the experimental design to evaluate the priming state of grapevine plants by the beneficial bacterium Pseudomonas fluorescens PTA-CT2 and their resistance level to Botrytis cinerea according to Verhagen et al. (2011) and Gruau et al. (2015).

Damage to plant organs through both biotic and abiotic injury is very common in nature. Arabidopsis thaliana 5-day-old (5-do) seedlings represent an excellent system in which to study plant responses to mechanical wounding, both at the site of the damage and in distal unharmed tissues. Seedlings of wild type, transgenic or mutant lines subjected to single or repetitive cotyledon wounding can be used to quantify morphological alterations (e.g., root length, Gasperini et al., 2015), analyze the dynamics of reporter genes in vivo (Larrieu et al., 2015; Gasperini et al., 2015), follow transcriptional changes by quantitative RT-PCR (Acosta et al., 2013; Gasperini et al., 2015) or examine additional aspects of the wound response with a plethora of downstream procedures. Here we illustrate how to rapidly and reliably wound cotyledons of young seedlings, and show the behavior of two promoters driving the expression of β-glucuronidase (GUS) in entire seedlings and in the primary root meristem, following single or repetitive cotyledon wounding respectively. We describe two procedures that can be easily adapted to specific experimental needs.

Hyaloperonospora arabidopsidis (Hpa; formerly Peronospora parasitica or Hyaloperonospora parasitica) is an oomycete downy mildew pathogen of the model plant Arabidopsis. The pathosystem between Arabidopsis and Hpa has been extensively used to study host/pathogen co-evolution (Coates and Beynon, 2010). As Hpa is an obligate biotrophic pathogen, its host is absolutely required for survival. Thus, Hpa must be maintained on susceptible Arabidopsis accessions and mutants. Growth of Hpa is evaluated in two ways; counting conidiospores (Asai et al., 2014) or counting sporangiophores after trypan blue staining (Holt et al., 2005). Here, we describe how to do inoculation with Hpa and how to evaluate Hpa growth on Arabidopsis.

The mitogen activated protein kinase cascade is a highly conserved signal pathway in plants. The exogenous chemicals, like hormones, can trigger a series of signalling cascades, including MAPK pathway, to modulate the plant physiology. Upon activation, some MAPKs are phosphorylated. It is important to develop methods that can detect changes in the phosphorylation status of MAPKs in plants when they come in contact with external chemicals. This method describes the exogenous treatment of Arabidopsis protoplasts with Kinetin and subsequent detection of the activated MAPKs. This method is useful for studying the effect of exogenously applied chemical compounds on the MAPK signaling cascade in Arabidopsis.

The pathogenic fungus Ophiostoma novo-ulmi spreads within the secondary xylem vessels of infected elm trees, causing the formation of vessel plugs due to tyloses and gels, which ultimately result in Dutch elm disease. Foliage discoloration, wilting and falling from the tree are typical external leaf symptoms of the disease followed by the subsequent death of sensitive trees. Cellulolytic enzymes produced by the fungus are responsible for the degradation of medium molecular weight macromolecules of cellulose, resulting in the occurrence of secondary cell wall ruptures and cracks in the vessels but rarely in the fibers (Ďurkovič et al., 2014). The goal of this procedure is to evaluate the extent of cellulose degradation by a highly aggressive strain of O. novo-ulmi ssp. americana × novo-ulmi. Size-exclusion chromatography (SEC) compares molecular weight distributions of cellulose between the infected and the non-infected elm trees, and reveals changes in the macromolecular traits of cellulose, including molecular weights, degree of polymerization, and polydispersity index. ¹³C magic angle spinning nuclear magnetic resonance (¹³C MAS NMR) spectra help to identify and also to quantify the loss of both crystalline and non-crystalline cellulose regions due to degradation. The procedure described herein can also be easily used for other woody plants infected with various cellulose-degrading fungi.

Unbiased screening approaches are powerful tools enabling identification of novel players in biological processes. Chemical genetic screening refers to the technique of using a reporter response, such as expression of luciferase driven by a promoter of interest, to discover small molecules that affect a given process when applied to plants. These chemicals then act as tools for identification of regulatory components that could not otherwise be detected by forward genetic screens due to gene family redundancy or mutant lethality.

This protocol describes a chemical genetic screen using Arabidopsis thaliana seedlings, which has led to recognition of novel players in the plant general stress response.

Inducible gene expression systems offer researchers the opportunity to synchronize target gene expression at particular developmental stages and in particular tissues. The glucocorticoid receptor (GR), a vertebrate steroid receptor, has been well adopted for this purpose in plants. To generate steroid-inducible plants, a construct of GAL4-binding domain-VP16 activation domain-GR fusion (GVG) with the target gene under the control of upstream activation sequence (UAS) has been developed and extensively used in plant research.

Immune receptors perceive conserved molecular patterns secreted by pathogens and initiate robust immune responses. The rice immune receptor, XA21, recognizes a molecular pattern highly conserved in all sequenced genomes of Xanthomonas, and confers robust resistance to X. oryzae pv. oryzae (Xoo). However, identifying genes downstream of XA21 has been hindered because of the restrained lesion and thus limited defense response region in the plants expressing Xa21. Inducible expression allows for a synchronized immune response across a large amount of rice tissue, well suited for studying XA21-mediated immunity by genome-wide approaches such as transcriptomics and proteomics. In this protocol, we describe the use of this GVG system to synchronize Xa21 expression.

Studying the biochemical interaction of ligands with their corresponding receptors requires highly sensitive detection and monitoring of the bound ligand. Classically, radioactively labelled ligands have been widely used as highly sensitive tools for such binding measurements. Disadvantages of radiolabelling include instability of products, high costs and risks of working with radioactivity. Thus, assays using chemiluminescent probes offer convenient, highly sensitive alternatives. Here we suggest acridinium esters as suitable conjugates to label ligands of interest. Chemical oxidation of acridinium esters triggers chemiluminescence, allowing quantitation of this compound down to amol concentrations in standard luminometers. The first report about acridinium esters in immunoassays date back to 1983 (Weeks et al., 1983) and demonstrated the ability to conjugate acridinium to peptides, followed by using such peptides to measure receptor – peptide ligand interactions (Joss and Towbin, 1994).

Recently, this binding assay was adapted for studying derivatives of the plant peptide IDA (INFLORESCENCE DEFICIENT IN ABSCISSION) and their interaction with the corresponding receptor HSL2 (HAESA-LIKE 2) was reported (Butenko et al., 2014). Here we describe how this sensitive, nonradioactive binding approach can be used to reveal receptor-ligand binding in plant material.

We developed this protocol to assay the extent of proliferation of Pseudomonas syringae pv. glycinea in soybean leaves. This method specifically enables accurate pathogenesis assays of soybean plants at V2/V3 (2^nd/3^rd trifoliate) or higher stages of growth. The leaves of soybean plants at these growth stages are not amenable to bacterial infiltration using routine needleless syringe infiltration due to the high number of trichomes on these mature leaves. This method enables efficient infiltration of bacteria into the epidermal cells of mature leaves using a pressure pump.