Cell death-mediated antiviral effect of chitosan in tobacco.
The antiviral activity induced by chitosan (CHT), and the mechanisms underlying it, were studied in a tobacco-tobacco necrosis necrovirus (TNV) pathosystem. Treatments with 0.1% CHT enhanced tobacco inducible defenses against TNV, reducing significantly the virus-induced necrotic lesions (in a range from 32% to 83%). In planta, this resistance was associated with a network of callose deposits, micro-oxidative bursts and micro-hypersensitive responses (micro-HRs), as assessed, respectively, by aniline blue, 3,3'-diaminobenzidine (DAB) and Evans blue staining. In order to verify if CHT-elicited cell death could be regarded as an apoptotic process, tobacco bright yellow 2 (BY2) cell cultures were treated with different CHT concentrations, ranging from 0.01% to 0.1%. After 6 h about half of the cultured cells incubated in 0.05% CHT were Evans blue positive, showing some typical morphological features of apoptosis, such as cytoplasm shrinkage and nuclear chromatin condensation. The latter was checked by 4',6-diamino-2-phenylindole (DAPI) and ethidium bromide nuclear staining and was visible already at 2 h after treatment. Moreover, the cell death kinetic induced by CHT was delayed by Verapamil(R), a calcium channel blocker. Finally, electrophoresis of genomic DNA extracted from cultured cell after 48 h treatment showed internucleosomal fragmentation, visualized as a distinct ladder of DNA bands corresponding to oligonucleosomal units.
Iriti
M
M
Istituto di Patologia Vegetale, Università di Milano, Milano, Italy.
Sironi
M
M
Gomarasca
S
S
Casazza
A P
AP
Soave
C
C
Faoro
F
F
D000998:Antiviral Agents / Q000494:pharmacology*; D002614:Chelating Agents / Q000494:pharmacology; D048271:Chitosan / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects*; D018744:DNA, Plant / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D010935:Plant Diseases / Q000821:virology; D010942:Plant Viruses / Q000378:metabolism*; D014026:Nicotiana / Q000166:cytology / Q000378:metabolism* / Q000821:virology
D000998:Antiviral Agents; D002614:Chelating Agents; D018744:DNA, Plant; D048271:Chitosan