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Published on: May 21, 2019
DspA/E Contributes to Apoplastic Accumulation of ROS in Non-host A. thaliana
Alban Launay1, Oriane Patrit2, Estelle Wénès3
1CNRS 3559, Institut Jean-Pierre Bourgin, INRA, AgroParisTech, ERL, Université Paris-SaclayVersailles, France; Université Paris-Sud-Université Paris-SaclayOrsay, France.
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.
Insights
Erwinia amylovora infection triggers hydrogen peroxide accumulation in plant cells and apoplasts. This apoplastic reactive oxygen species (ROS) accumulation is a defense response mediated by the type three effector DspA/E.
Area of Science:
- Plant pathology
- Bacterial pathogenesis
- Plant-microbe interactions
Background:
- Erwinia amylovora causes fire blight disease in Maleae, utilizing a type three-secretion system (T3SS) to inject effectors.
- T3SS-dependent reactive oxygen species (ROS) production is linked to E. amylovora pathogenicity, unusually associated with disease rather than resistance.
- Previous work showed transient E. amylovora multiplication in Arabidopsis thaliana with T3SS-dependent intracellular ROS production.
Purpose of the Study:
- To characterize the localization and source of hydrogen peroxide (H2O2) accumulation during E. amylovora infection.
- To investigate the role of the T3SS effector DspA/E in H2O2 accumulation.
- To determine the contribution of NADPH oxidase (RBOHD) to ROS production and its impact on bacterial multiplication and effector toxicity.
Main Methods:
- Transmission electron microscopy (TEM) to visualize H2O2 localization in infected Arabidopsis tissues.
- Analysis of wild-type and dspA/E-deficient E. amylovora strains.
- Use of transgenic lines expressing DspA/E and rbohD mutant plants.
- Co-inoculation experiments with cycloheximide (CHX) to assess its effect on ROS and bacterial growth.
Main Results:
- TEM revealed H2O2 accumulation in cytosol, plastids, peroxisomes, mitochondria, and apoplast.
- A dspA/E-deficient strain failed to induce apoplastic H2O2 accumulation, while DspA/E expression led to apoplastic ROS.
- The rbohD mutant showed significantly reduced H2O2 accumulation, but bacterial titers and DspA/E toxicity were unaffected.
- CHX treatment reduced apoplastic ROS but not intracellular ROS, while enhancing bacterial multiplication.
Conclusions:
- Apoplastic ROS accumulation is a component of the non-host defense response in Arabidopsis against E. amylovora.
- The type three effector DspA/E contributes to triggering apoplastic ROS.
- RBOHD is involved in E. amylovora-induced apoplastic ROS production.
- Apoplastic ROS accumulation, alongside callose deposition, represents a defense layer against E. amylovora, potentially modulated by factors like CHX.
