Abstract
The bacterium Erwinia amylovora is responsible for the fire blight disease of Maleae, which provokes necrotic symptoms on aerial parts. The pathogenicity of this bacterium in hosts relies on its type three-secretion system (T3SS), a molecular syringe that allows the bacterium to inject effectors into the plant cell. E. amylovora-triggered disease in host plants is associated with the T3SS-dependent production of reactive oxygen species (ROS), although ROS are generally associated with resistance in other pathosystems. We showed previously that E. amylovora can multiply transiently in the non-host plant Arabidopsis thaliana and that a T3SS-dependent production of intracellular ROS occurs during this interaction. In the present work we characterize the localization and source of hydrogen peroxide accumulation following E. amylovora infection. Transmission electron microscope (TEM) analysis of infected tissues showed that hydrogen peroxide accumulation occurs in the cytosol, plastids, peroxisomes, and mitochondria as well as in the apoplast. Furthermore, TEM analysis showed that an E. amylovora dspA/E-deficient strain does not induce hydrogen peroxide accumulation in the apoplast. Consistently, a transgenic line expressing DspA/E accumulated ROS in the apoplast. The NADPH oxidase-deficient rbohD mutant showed a very strong reduction in hydrogen peroxide accumulation in response to E. amylovora inoculation. However, we did not find an increase in bacterial titers of E. amylovora in the rbohD mutant and the rbohD mutation did not suppress the toxicity of DspA/E when introgressed into a DspA/E-expressing transgenic line. Co-inoculation of E. amylovora with cycloheximide (CHX), which we found previously to suppress callose deposition and allow strong multiplication of E. amylovora in A. thaliana leaves, led to a strong reduction of apoplastic ROS accumulation but did not affect intracellular ROS. Our data strongly suggest that apoplastic ROS accumulation is one layer of the non-host defense response triggered by the type three effector (T3E) DspA/E, together with callose deposition.
Highlights
One of the most common and earliest responses of plants to pathogens is reactive oxygen species (ROS) production (Lamb and Dixon, 1997)
In order to determine the localization of H2O2 accumulation in response to E. amylovora inoculation, we used diaminobenzidine (DAB), which forms a brown precipitate in the presence of H2O2
Five-weeks-old A. thaliana rosette leaves were infiltrated with E. amylovora and the accumulation of H2O2 was determined at 16 hpi
Summary
One of the most common and earliest responses of plants to pathogens is reactive oxygen species (ROS) production (Lamb and Dixon, 1997). DspA/E Contributes to Apoplastic Accumulation of ROS induces the production of hydrogen peroxide (H2O2) that is associated with disease (van Baarlen et al, 2007). It was shown that the NbrbohB-dependent production of H2O2 following infection by B. cinerea contributes to lesion development in Nicotiana benthamiana (Asai and Yoshioka, 2009). In the case of the necrotrophic bacterium Dickeya dadantii, strong oxidative stress was observed in Arabidopsis thaliana and it was shown that this accumulation contributes to defense (Fagard et al, 2007). In A. thaliana, RbohD was found to be the main source of ROS production to the avirulent strain of Pseudomonas syringae avrRpm (Torres et al, 2002) and to the bacterial necrotroph D. dadantii (Fagard et al, 2007). The current knowledge on RbohD, which encodes an NADPH oxidase, has been recently reviewed (Marino et al, 2012)
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