Role of salicylic acid signaling in the biotrophy-necrotrophy transition of Xanthomonas campestris pv. campestris infection in Brassica napus

https://doi.org/10.1016/j.pmpp.2020.101578Get rights and content

Highlights

  • A time course-study revealed biotrophic and necrotrophic phases of Xcc infection.

  • Salicylic acid (SA)-mediated H2O2 accumulation seems to cause the necrosis.

  • Rapid changes in SA level causes transition from biotrophic to necrotrophic phase.

Abstract

To characterize the hormonal regulations in the transition between biotrophic and necrotrophic phases of Xanthomonas campestris pv. campestris (Xcc) infection, the endogenous phytohormone and reactive oxygen species (ROS) levels and glutathione (GSH) redox status of non-inoculated and Xcc-infected Brassica napus cv. Mosa leaves were measured at various points after inoculation. This time-course study revealed two distinct phases of the Xcc-infection, namely an early asymptomatic (biotrophic) phase that could be observed at 2 days post-inoculation (DPI) and a destructive necrotrophic phase that occurred at 7–24 DPI and was associated with V-shaped necrosis. The length of the V-shaped lesions increased significantly with the progression of infection, as did H2O2 accumulation and the expression of NADPH oxidase. Xcc-infections induced the gradual depression of the reduced/oxidized GSH/GSSG ratio with the progression of disease. Furthermore, Xcc inoculation reduced levels of salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and cytokinin (CK) during the biotrophic phase (at 2 DPI); increased SA level (9.5 fold) and the expression of both SA synthesis-related genes (EDS1 and ICS1) and signaling-related genes (NPR1 and PR1) from the 2 to7 DPI. ABA and CK levels gradually increased during the necrotrophic phase. Interestingly, JA level was either reduced or not significantly affected during the necrotrophic phase of infection. Altogether, these results indicate that, in B. napus, changes in SA-mediated ROS production regulate the transition from the symptomless biotrophic phase of Xcc infection to the destructive necrotrophic phase.

Introduction

As an agro-economically important crop, oilseed rape (Brassica napus, Brassicaceae) is an important source of edible oil and animal feed [1,2]. Xanthomonas campestris pv. campestris (Xcc) is a hemibiotrophic bacterial pathogen that causes black rot disease in brassicaceous crops and, thus, causes huge economic losses on an annual basis [3,4]. Infection by hemibiotrophic pathogens includes sequential biotrophic and necrotrophic phases. During the initial biotrophic phase, which is generally asymptomatic, the pathogen obtains nutrients from its host's living tissue, and during the following necrotrophic phase, which is relatively destructive, the pathogen damages or destroys host cells for nutrients [5,6]. The infection of plants with Xcc is also associated with the formation of V-shaped necrotic lesions on leaf margins and the induction of oxidative stress, including the accumulation of reactive oxygen species (ROS; [[7], [8], [9]].

Phytohormones including salicylic acid (SA) and jasmonic acid (JA) play leading roles in the activation of basal defenses against plant pathogens. For example, SA signaling mediates the resistance of plants against biotrophic pathogens, and JA-signaling regulates the resistance of plants against necrotrophs [7,10]. Moreover, SA and JA signaling are mutually antagonistic, and the stress-responsive hormone abscisic acid (ABA) antagonizes JA signaling, thereby inducing susceptibility to necrotrophs [11]. However, the plant growth hormone cytokinin (CK) synergistically interacts with SA to activate defense responses against biotrophic plant pathogens [10]. Previous studies have investigated variations in endogenous hormone levels in various B. napus cultivars, as well as the effects of signaling and hormone regulation on phenylpropanoid metabolism during the necrotrophic phase of Xcc infection [7,8]. Higher levels of SA and ABA, which are associated with higher SA/JA and ABA/JA ratios, were reported to regulate the development of disease symptoms during the necrotrophic phase of Xcc infection by enhanced the glutathione (GSH)-redox balance [7], and JA-mediated increase in phenolic accumulation was associated with Xcc resistance [8].

Hemibiotrophic pathogens manipulate endogenous hormone signaling in order to disrupt the basal defense responses of their hosts, i.e., to induce disease susceptibility [10]. However, little is known about Xcc-induced alterations in the phytohormonal signaling of infected plants during the pathogen's transition between biotrophy and necrotrophy. Investigating the alteration of hormone levels during different phases of Xcc infection may reveal new information about the complex hormonal changes that occur during biphasic plant-microbe interactions. Therefore, the aim of the present study was to monitor levels of endogenous hormones during both the biotrophic and necrotrophic phases of Xcc infection in B. napus and to characterize any physiological association of hormonal status, with ROS accumulation and GSH-redox homeostasis or other infection symptoms.

Section snippets

Plant culture and pathogen inoculation

Surface-sterilized seeds of Brassica napus cv. Mosa were grown in 0.6-L plastic pots and, after growing to the four-leaf phase, were divided into two treatment groups: a control group and an Xcc-inoculated group. The pathogenic bacterial (Xanthomonas campestris pv. campestris, Xcc) strain (KACC No-10377) was collected from the Korean Agricultural Culture Collection. The Xcc inoculum was prepared as described previously [7], bacterium inoculum was cultured for 48 h at 30 °C in Yeast Dextrose

Disease symptoms, H2O2 production and NADPH-oxidase gene expression

The Xcc-inoculated leaves exhibited two distinct phases of infection, namely an early asymptomatic, or biotrophic, phase that occurred at 2 DPI and a subsequent necrotrophic phase that occurred from 7 to 24 DPI and was associated with V-shaped, necrotic lesions (Fig. 1A and B). The length of the V-shaped lesions increased significantly with the progression of infection (Fig. 1B).

Meanwhile, level of H2O2, which was used as an indicator of oxidative stress, remained unchanged during the

Discussion

Plant pathogens are typically classified as biotrophic, necrotrophic, or hemibiotrophic, depending on their lifestyle. Biotrophic pathogens maintain a feeding relationship with living host cells, rather than killing them, whereas necrotrophs actively kill the host tissues to acquire nutrients from dead or dying cells, and hemibiotrophs exhibit both strategies of infection, generally shifting from an early biotrophic phase to a subsequent necrotrophic phase. In responses to these different

Author contributions

M.T. Islam and T.H. Kim designed the experiment and interpreted data; M.T. Islam and M. A. Mamun wrote the manuscript under the guidance of T.H. Kim.; M.T. Islam, M.A. Mamun, B.R. Lee and H.L. Van, performed the chemical and gene expression analyses; and D.W. Bae performed the phytohormone quantification. W.J. Jung participated in the critical reading of the manuscript.

Declaration of competing interest

The authors have no conflicts of interest to declare.

Acknowledgements

This work was supported by a grant from the National Research Foundation of South Korea (NRF-2019R1A2C1089340).

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