Chloroplast-generated reactive oxygen species play a key role in lesion development on tobacco leaves infected by Botrytis cinerea

ROSSI F., ; BÍSARO, F.; KRAPP A.R, CARRILLO N.; PIECKENSTAIN FL.

Santa Crruz

Simposio; XVII International Botrytis Symposium; 2016

Fac. de Cs. Agronómicas. UNacional de Chile

Chloroplast-generatedreactive oxygen species play a key role in lesion development on tobacco leaves infected by B. cinereaFranco Rossi1, Fabiana Bísaro2,Adriana Krapp2, Santiago Maiale1, Néstor Carrillo2, Fernando Pieckenstain1.1Instituto de Investigaciones Biotecnológicas- Instituto Tecnológico de Chascomús, (UNSAM-CONICET)Chascomús, 2Facultad de Ciencias Farmacéuticas y Bioquímicas, Universidad Nacional de Rosario,3Instituto de Biología Molecular y Celular de Rosario (CONICET) Rosario, ARGENTINA.E-mail: pieckenstain@intech.gov.arIn addition to phytotoxic metabolites and proteins, Botrytiscinerea produces reactive oxygen species (ROS) during host infection.Hydrogen peroxide derived from B. cinerea contributes to host cell death and favors fungal infection. However, ROS are not only derived from the fungus itself but also from host tissues, as part of defense responses. B. cinerea has been shown to manipulate host defenses by inducing the hypersensitive response, a process that involves ROS accumulation and causes cell death. In spite of the well-known role of ROS in the deployment of HR, the relative contribution of different subcellular ROS sources (apoplast, mitochondria, chloroplasts, peroxisomes) is not completely clear.Transgenic tobacco plants expressing a cyanobacterial flavodoxin gene in plastids (pfld lines) were previously shown to accumulate lower ROS levels in chloroplasts than wild-type (WT) plants in response to different stresses and exhibited increased resistance to multiple biotic and abiotic stresses. In the present work, pfld lines were used to analyze the contribution of chloroplastic ROS to host cell-death caused by B. cinerea infection. In planta fungal growth and tissue necrosis were significantly reduced in infected leaves of pfld lines, as compared to infected WT plants. ROS build-up triggered by fungal infection was also attenuated in pfld lines, as evidenced by 2?,7?-dichlorofluorescein diacetate staining. Confocal laser microscopy analysis of infected tissues revealed that in WT plants, ROS fluorescence was mainly detected in chloroplasts. Plastids of infected pfld plants exhibited a 70-80% decrease in ROS fluorescence, as compared to WT. Moreover, phytoalexin accumulation and expression of the defense genes β-1,3 glucanase, chitinase and PR-1 were induced to a lower degree in pfld plants than in WT. In addition, the impact of fungal infection on leaf photosynthetic activity was also lower in pfld lines than in WT plants.As a whole, results obtained in the present work demonstrate thatsuppression of ROS build-up in chloroplasts strongly reduces tissue necrosis. Chloroplastic ROS were thus found to play a fundamental role in the development of lesions during B. cinerea infection. Moreover, the present work demonstrates that modulation of chloroplastic ROS accumulation by means of the expression of a heterologous antioxidant protein can provide a significant level of protection against infection by B. cinerea, and probably other necrotrophic fungi.