Early detection of bacterium-induced basal resistance in tobacco leaves with diaminobenzidine and dichlorofluorescein diacetate

The present study demonstrate that in tobacco leaves the diaminobenzidine (DAB) and 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining is a useful indicator of the basal (also known as general or innate) defence-associated reactions, especially of the early developing form of basal resistance (EBR). DAB and DCFH-DA, in the presence of H2O2 and peroxidase converts to a brown polymer and fluorescent DCF respectively. In the present study, the hypersensitive response (HR)-inducing avirulent Pseudomonas syringae pv. syringae 61, its HR-negative hrp/hrc mutants and even non-pathogenic bacteria such as P. fluorescens and Escherichia coli caused DAB and DCFH-DA staining, if the dyes were injected 3-4 h after bacterial inoculation into tobacco leaves. The conditions that enable the staining of plant leaves infiltrated with HR-negative bacteria were persisted for 1 to several days depending on the physiological state of the plant, and plant activity was required to the development of the staining. The live virulent P. syringae pv. tabaci was able to suppress the development of the staining reaction. Bacteria that induced more intensive staining reaction triggered stronger local resistance response, which was verified by its ability to inhibit the HR by challenging avirulent bacteria and by expression analysis of genes that are activated during the basal defence response. The peroxidase enzyme activity increased in bacterially treated tobacco tissue, and inhibition of peroxidase activity blocked the development of the staining. The results showed that in tobacco leaves the staining reactions were associated with the general recognition and basal defence reaction of tobacco plant and can be used as markers in tobacco leaves for testing the occurrence of this type of defence.