Biochemistry

The plant immune system is essential for plants to perceive and defend against bacterial, fungal and insect pests and pathogens. Induced systemic resistance (ISR) is a systemic immune response that occurs upon root colonization by beneficial microbes. A well-studied model for ISR is the association of specific beneficial strains of Pseudomonas spp. with the reference plant Arabidopsis thaliana. Here, we describe a robust, increased throughput, bioassay to study ISR against the bacterial pathogen Pseudomonas cannabina pv. alisalensis (formerly called Pseudomonas syringae pv. maculicola) strain ES4326 and the herbivore Trichoplusia ni by inoculating Pseudomonas simiae strain WCS417 (formerly called Pseudomonas fluorescens WCS417) on Arabidopsis plants grown in Jiffy-7^® peat pellets. While most commonly used for Pseudomonas-triggered ISR on Arabidopsis, this assay is effective for diverse rhizosphere bacterial strains, plant species, pathogens and herbivores.

In this protocol we describe a cell wall stress assay for the fungal pathogen F. oxysporum, based on exposure to the two anionic dyes Calcofluor White (CFW) and Congo Red (CR). Both compounds have been used to exert stress upon the fungal cell wall in vitro (Perez-Nadales and Di Pietro, 2015; Perez-Nadales and Di Pietro, 2011; Leach et al., 2012; Heilmann et al., 2013; Garcia et al., 2015). CFW perturbs chitin assembly, whereas CR interferes with β-glucan synthesis, resulting in cell wall-weakening and activation of the cell wall stress response (Ram and Klis, 2006; Kopecka and Gabriel, 1992; Roncero and Duran, 1985). Presumably, the signaling pathways and cell wall changes associated with this response reflect cell wall homeostasis during normal growth as well as cell wall remodeling events in response to stresses encountered during the fungus-host interactions. The conditions for preparation of CFW and CR culture medium specified in this protocol are based on the paper by Ram and Klis entitled “Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red”, published in Nature protocols (Ram and Klis, 2006). This paper established the optimum conditions for preparation of CFW and CR stock solutions and suggested maintaining the culture medium at a constant pH to avoid acidification, protonation and precipitation of these dyes. This cell wall stress assay has been widely used in our group for the characterization of F. oxysporum mutants in mitogen activated protein kinase (MAPK) signaling pathway genes involved in cell wall integrity (Perez-Nadales and Di Pietro, 2015; Perez-Nadales and Di Pietro, 2011; Turra et al., 2014).

XTT assay is a colorimetric method that uses the tetrazolium dye, 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) to quantify cell-mediated damage to fungi. Actively respiring fungal cells convert the water-soluble XTT to a water-soluble, orange colored formazan product (Meshulam et al., 1995). Here, we describe the protocol that measures the ability of plasmacytoid dendritic cells (pDCs) to exert antifungal activity. This approach was first established with human polymorphonuclear cells (PMN) by Meshulam et al. (1995) and then adapted to pDC by Ramirez-Ortiz et al. (2011) and Loures et al. (2015). It can be modified for use with other effector cells and to test compounds for antifungal activity.

Clotting times can be measured by using citrate plasma. The intrinsic pathway of coagulation is measured by the activated partial thromboplastin time (aPTT), the extrinsic pathway of coagulation, monitored by measuring the prothrombin time (PT), and thrombin-induced fibrin-network formation (thrombin clotting time; TCT).

A biofilm is a multicellular consortium of surface associated microbes surrounded by a hydrated, extracellular polymer matrix. The biofilm matrix plays a critical role in preventing desiccation, acquiring nutrients, and provides community protection from environmental assaults. Importantly, biofilms are significantly more resistant to antimicrobials relative to their free-swimming counterparts. The level of antimicrobial tolerance is influenced by a number of factors, including genetic/adaptive resistance mechanisms, stage of biofilm development, and pharmacokinetics of the antibiotic. Here, we describe an in vitro microtiter-based assay to quantify the minimal bactericidal concentration for biofilms (MBC-B) for short exposure times (2 h). This exposure period is significantly shorter than standard over-night and 24-hour treatments described in traditional protocols. This assay was developed to approximate the time an antibiotic is available during a one-time treatment before it is metabolized, sequestered by host proteins, or digested.

Many of the microbes including fungi produce metabolites possessing antifungal activity and in some cases fungal metabolites are main cause of antifungal activity resulted by fungus. To infer that antifungal activity is due to fungal metabolites, there is a need to develop a repeatable procedure to assay these metabolites. Here we are presenting the poisoned food technique for bioassay of extract from endophytic fungi, where culture media is supplemented with extract to testpathogen viability as proxy for antifungal activity.

Antibiosis is one of the possible modes of action shown by endophytic fungi having antifungal activity. To test if antifungal activity in endophytic fungi is due to antibiosis, assay of the metabolites of endophytic fungi was needed. To obtain metabolites for bioassay batch culture fermentation and extraction of metabolites was done. Fungus was multiplied on wickerham media at incubation temperature of 25 ± 2 °C for 4 weeks and then extracted with solvents of different polarity. All the solvent extracts were dried under vacuum rotary evaporator to get dried crude fungal extract, which was subjected to further fractionation and bioassay.