Early Detection of Bacterium‐induced Basal Resistance in Tobacco Leaves with Diaminobenzidine and Dichlorofluorescein Diacetate

Abstract

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 H 2 O 2 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.www.blackwell-synergy.com

Identification of Harpins in Pseudomonas syringae pv. tomato DC3000, Which Are Functionally Similar to HrpK1 in Promoting Translocation of Type III Secretion System Effectors

“…Because our complementation experiments with the ⌬hrpK1 quadruple harpin gene polymutant suggest that HrpK1 and three of the harpins have overlapping functions, it is important to assess the evidence that HrpK1 is a translocator. An overriding problem in this regard is that P. syringae hrpK1 mutants of various pathovars have been reported to have only variably reduced or conditional phenotypes (6,7,16,45,54). A recent report by Petnicki-Ocwieja et al (54) showed that P. syringae pv.…”

Real-time in vivo visualization of oxidative stress in duckweed (Lemna minor L.)

“…Most of the plant ROS visualization studies were otherwise performed on cell cultures [7,9]. The main drawback of these studies is dubious extrapolation of results to intact higher plants where the cells originated from.…”

“…Therefore, one could consider using the more expensive, carboxylated, form of H 2 DCFDA (carboxy-H 2 DCFDA) because of its better membrane permeability and better retention in the intracellular space [11]. According to our results, it could be argued that yeast cells [8] or plant cell cultures [7,9] are probably better test subjects than L. minor plants for intracellular ROS generation studies. This is primarily because of their big surface-to-volume ratio that enables faster dye uptake and thus short loading periods.…”

“…Dichlorofluorescin (H 2 DCF) is a fluorescent marker widely used for determination of H 2 O 2 [6,7], and for studies of oxidative stress at the cellular level [8,9]. In cellular studies, dichlorofluorescein (H 2 DCF) is given as a diacetate ester (dichlorofluorescein diacetate; H 2 DCFDA), which permeates the plasma membrane and is hydrolyzed inside the cells by unspecific esterases yielding polar products, supposedly trapped within the cell.…”

Quantitative X‐ray microanalysis of hydrogen peroxide within plant cells

“…To address the role of H 2 O 2 in stress responses, it is not only important to study its subcellular localization but also to investigate changes in its concentration in the different compartments. Histochemical techniques have been widely used for the detection of H 2 O 2 generated in plant cells during plant-pathogen interactions (Alvarez et al, 1998;Bestwick et al, 1997;Bozsó et al, 2005;Iwano et al, 2002;Melillo et al, 2006;Musetti et al, 2004Musetti et al, , 2005Renard-Merlier et al, 2007;Soylu et al, 2005;Thordal-Christensen et al, 1997;Waetzig et al, 1999), in response to wounding (Le-Deunff et al, 2004;Orozco-Cá rdenas and Ryan, 1999;Orozco-Cá rdenas et al, 2001), salinity (Herná ndez et al, 2001;Menezes-Benavente et al, 2004;Wi et al, 2006a), gamma irradiation (Wi et al, 2006b(Wi et al, , 2007, water stress (Hu et al, 2006), ozone stress (Pellinen et al, 1999(Pellinen et al, , 2002, and plant growth regulators, e.g., abscisic acid, jasmonic acid, salicylic acid, and ethylene precursor (Bóka et al, 2007;De Cnodder et al, 2005;Hu et al, 2005). The cytochemical dye 3,3-diaminobenzidine (DAB) or alternatively, the more sensitive CeCl 3 , have been used to assess the subcellular localization of H 2 O 2 .…”