Section II. Plant-microorganisms interactions. Chapter 10: Hypersensitive response.

DAURELIO, L. D.; TONDO, M.L.; DUNGER, G.; GOTTIG, N.; OTTADO, J.; ORELLANO, E.G.

Book on Plant Bioassays

Studium Press, USA

Año: 2008;

Plants have evolved to defense themselves against a variety of pathogen as bacteria, viruses and fungi. Hypersensitive response (HR) is a rapid and localized cell death associated with disease resistance including: race-specific (explained by the classical R-Avr model) (Flor, 1971); race-non-specific and non-host. The HR was first described by Stakman in 1915 and is characterized by the rapid death of a limited number of cells in the vicinity of the invading pathogen (Heath, 2000) and its function is thought to restrict or delay further pathogen spread and eventually kill the pathogen. Then, subsequent signaling events induce local and systemic activation of a set of defense responses that play a role in resistance. One of the most rapid plants responses is associated with the oxidative burst, which constitutes the production and accumulation of active oxygen species (AOS), primarily superoxide (O2-) and hydrogen peroxide (H2O2), at the site of invasion (Lamb and Dixon, 1997) which can produce toxic effects via DNA damage, protein degradation and modification and lipid peroxidation (Imlay, Linn 1988, Science). Other physiological and molecular modifications correlated with the HR are ion leakage of the dead cell’s content into the apoplast (Mysore, K.S. and Ryu), the deposition of lignin and callose into the plant cell wall (Verma DP, Hong Z. (2001) and the production of phytoalexins, hydrolytic enzymes and pathogenesis-related proteins (PRs) (van Loon et al., 2006). Xanthomonas axonopodis pv. citri (Xac), the bacterium responsible for citrus canker, induces a non-host resistance with HR in non-host plants such as pepper, tobacco, tomato, bean and cotton (Dunger et al., 2005). This chapter describes a variety of techniques to evaluate the HR in the interaction between bacteria and non host plants using Xac and tobacco, pepper or cotton. A detailed description of plant growth and inoculation techniques is shown. Pathogen growth restriction in inoculated plant leaves is evaluated using growth curves. Also the AOS produced during the HR are biochemically detected. Hydrogen peroxide is detected using DAB (3,3´-Diaminobenzidine) staining that is oxidized by endogenous peroxidases in the presence of H2O2 , while superoxide production is characterized by NBT (Nitro-blue tetrazolium) reduction and the formation of insoluble blue staining. Ion leakage that reveals cellular membrane damage is determined by conductivity measurements and cell death can be monitored by Trypan blue staining since the dye only passes through membranes of dead cells. Finally, cell wall alterations revealed by callose deposition as a form of a non-host resistance is determined by staining with aniline blue, that selectively stains â-1-3 glycosidic linkages of the glucose units in callose.