A Simple and Rapid Assay for Measuring Phytoalexin Pisatin, an Indicator of Plant Defense Response in Pea (Pisum sativum L.)

Background

Plants have host resistance and nonhost resistance depending upon the nature of plant-pathogen interactions. Host resistance is mostly controlled by R genes and less durable, whereas nonhost resistance is generally a multi-gene trait and more durable in comparison with host resistance (Gill et al., 2015; Lee et al., 2017). The pea plant has served as a model system for research on the signals that trigger the nonhost defense response when challenged by incompatible pathogens that fall outside that species’ host range (Hadwiger, 2008). An indicator of this response in peas is the induction of a secondary metabolism to the isoflavonoid, pisatin. Pisatin has strong antifungal properties but its presence is a valuable indicator of plant defense response. Pisatin can be quantitated in various ways from classical organic chemistry procedures (Schwochau and Hadwiger, 1969) to Mass-spec analysis (Seneviratne et al., 2015). However, a procedure with high reproducibility and simplicity is described herein that can easily handle large numbers of treatments. The targeted tissue is the inside layer of an immature pea pod, called endocarp. This pristine cuticle-free tissue is capable of responding rapidly to candidate microbes or elicitor compounds to generate the pea defense response. The exposed epidermal layer of cells can be monitored for light microscope-visible or stained cellular component changes. The overall changes that culminate in pisatin accumulations can be determined by immersing the pod half in 5 ml of hexane for 4 h in the dark and subsequently allowing the decanted hexane to evaporate in the air flow of a hood. The residue remaining is dissolved in 1 ml of 95% alcohol and read at OD₃₀₉ using a spectrophotometer: 1 OD₃₀₉ = 43.8 µg pisatin/ml in 1 cm pathlength (Cruickshank and Perrin, 1961; Perrin and Cruickshank, 1965; Teasdale et al., 1974). This reading minus the background control tissue and the characteristic UV spectrum are essentially free from other hexane soluble components of the pea tissue. This protocol was used in our recent publications (Hadwiger and Tanaka, 2014 and 2017; Tanaka and Hadwiger, 2017).