Structural-Functional Characterization and Physiological Significance of Ferredoxin-NADP+ Reductase from Xanthomonas axonopodis pv. citri

María Laura Tondo,Elena G Orellano,Eduardo A Ceccarelli,Matías A Musumeci,María Laura Delprato,Andreas Hofmann

doi:10.1371/journal.pone.0027124

María Laura Tondo, Elena G Orellano + Show 4 more

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https://doi.org/10.1371/journal.pone.0027124

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Abstract

Xanthomonas axonopodis pv. citri is a phytopathogen bacterium that causes severe citrus canker disease. Similar to other phytopathogens, after infection by this bacterium, plants trigger a defense mechanism that produces reactive oxygen species. Ferredoxin-NADP+ reductases (FNRs) are redox flavoenzymes that participate in several metabolic functions, including the response to reactive oxygen species. Xanthomonas axonopodis pv. citri has a gene (fpr) that encodes for a FNR (Xac-FNR) that belongs to the subclass I bacterial FNRs. The aim of this work was to search for the physiological role of this enzyme and to characterize its structural and functional properties. The functionality of Xac-FNR was tested by cross-complementation of a FNR knockout Escherichia coli strain, which exhibit high susceptibility to agents that produce an abnormal accumulation of •O2 -. Xac-FNR was able to substitute for the FNR in E. coli in its antioxidant role. The expression of fpr in X. axonopodis pv. citri was assessed using semiquantitative RT-PCR and Western blot analysis. A 2.2-fold induction was observed in the presence of the superoxide-generating agents methyl viologen and 2,3-dimethoxy-1,4-naphthoquinone. Structural and functional studies showed that Xac-FNR displayed different functional features from other subclass I bacterial FNRs. Our analyses suggest that these differences may be due to the unusual carboxy-terminal region. We propose a further classification of subclass I bacterial FNRs, which is useful to determine the nature of their ferredoxin redox partners. Using sequence analysis, we identified a ferredoxin (XAC1762) as a potential substrate of Xac-FNR. The purified ferredoxin protein displayed the typical broad UV-visible spectrum of [4Fe-4S] clusters and was able to function as substrate of Xac-FNR in the cytochrome c reductase activity. Our results suggest that Xac-FNR is involved in the oxidative stress response of Xanthomonas axonopodis pv. citri and performs its biological function most likely through the interaction with ferredoxin XAC1762.

Highlights

Xanthomonas axonopodis pv. citri is a Gram-negative obligate aerobic bacterium that is responsible for severe citrus canker disease, which affects most commercial citrus cultivars
The functionality of Xac-Ferredoxin-NADP+ reductases (FNRs) was initially tested by crosscomplementation of an E. coli fpr mutant
The E. coli fpr RR6A strain exhibited a high susceptibility to the bactericidal effects of methyl viologen (MV) [9]

Summary

Introduction

Xanthomonas axonopodis pv. citri is a Gram-negative obligate aerobic bacterium that is responsible for severe citrus canker disease, which affects most commercial citrus cultivars. The pathogen enters host plant tissues through the stomata or tissue wounds, and the infection is visualized as circular spots on the surface of the leaves. Pathogens need to prevent and overcome oxidative stress in order to establish and maintain infections [4]. Different studies have demonstrated the protective role that catalases and peroxidases perform in Xanthomonas spp during oxidative stress developed by the plant’s defence mechanisms [5,6]. In other Gram-negative bacteria, such as Escherichia coli and Pseudomonas putida, alternative mechanisms of response to oxidative stress have been reported where ferredoxin-NADP+ reductase (FNR) performs an important function [7,8,9,10,11,12]

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