Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis

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Highlights

  • Transcriptome profile in myb51 were investigated and compared with wild type.

  • MYB51 was possibly involved in most resistance processes.

  • MYB51 had a positive feedback effect on its important upstream regulators.

Abstract

Indole glucosinolates are known to play essential and diverse roles in Arabidopsis immunity to pathogens. However, a complete understanding of the function of these compounds in plant immunity remains unclear. In this study, we investigated the transcriptome profile in loss-of-function mutant of MYB51, the key transcription factor that controls the biosynthesis of indole glucosinolates. Upon treatment with flg22 (a 22-amino acid peptide derived from bacterial flagellin), the genes that responded in a MYB51-dependent manner were analyzed. The results suggested that MYB51 was possibly implicated in most resistance processes, including pathogen recognition, signal transduction and PR protein activation. Of note, several genes in the ethylene pathway and the WRKY family, including WRKY33, were induced by flg22 in a MYB51-dependent manner. WRKY33 and ethylene were demonstrated to be crucial regulators in plant immunity defense and are functionally upstream of MYB51 during MAMP triggered immunity (MTI). This result suggested a “positive feedback loop” between MYB51 and its upstream regulators.

Introduction

Glucosinolates are nitrogen and sulfur containing secondary metabolites derived from amino acids. Depending on their amino acid precursors, they are classified as aliphatic, aromatic and indole glucosinolates [1]. In Arabidopsis, tryptophan derived indole glucosinolates and their metabolic products were found to be essential for the defense response to various biotic stresses, especially pathogens [[2], [3], [4], [5], [6], [7]]. Therefore, these compounds have been widely investigated and the mechanisms by which indole glucosinolates are functional in pathogen resistance have been gradually revealed. Besides their direct antimicrobial activity [8], indole glucosinolates are also involved in triggering highly conserved immune responses in the plant kingdom. In Arabidopsis, the degradation of indole glucosinolate are essential for bacteria-triggered callose deposition [9,10], and can also modulate pathogen-induced hypersensitive programmed cell death (PCD) [11,12]. Indole glucosinolate breakdown product, indole-3-carbinol (I3C), can act as an auxin antagonist [13] and can affect production and localization of the auxin transporters [14]. In addition, indolic glucosinolates were reported to be induced systemically at the uninfected tissue when challenged by bacterial pathogen and contributed to systemic acquired resistance (SAR) [15,16].

These studies all suggest that indole glucosinolate metabolism has a profound impact on pathogen resistance through various molecular mechanisms. Although the functions of indole glucosinolates and their breakdown products are attracting extensive attention, the global view of the exact role and precise mechanism of indole glucosinolates in immunity remains elusive.

In this study, we used flg22 to simulate a pathogen-triggered immune response and compared these responses between wild type and an indole glucosinolate deficient mutant. The metabolism and regulation of indole glucosinolates have been clearly identified in Arabidopsis, and the MYB51, MYB34 and MYB122 transcription factors are known to be regulators of indole glucosinolates [17,18]. MYB122 is considered to play an accessory role in the biosynthesis of indole glucosinolates, while both MYB51 and MYB34 are important transcription factors that activate the production of indole glucosinolates. Since the indole glucosinolate biosynthesis intermediate product IAOx, is also a precursor of the predominate auxin IAA (indole-3-acetic acid), the metabolism of indole glucosinolates is closely related with IAA production. The biosynthesis of indole glucosinolates in both myb51 and myb34 is largely defective [17], however, MYB34 significantly affects the homeostasis of IAA and in contrast, neither overexpression nor deficiency of MYB51 lead to altered IAA level [17,18]. To avoid pleiotropic effects and focus on obtaining information related to indole glucosinolate dependent immunity response, myb51 was chosen as the indole glucosinolate defective mutant.

To provide a full view of indole glucosinolate functions during the Arabidopsis response to pathogen invasion, high-throughput Illumina sequencing was performed to characterize the transcriptomes in wild-type and myb51 plants. Analysis of MYB51 dependent flg22-response genes showed that MYB51 might participate in most of the pathogen resistance processes including pathogen recognition, signal transduction and activation of PR proteins. WRKY transcription factors and the ethylene (ET) signaling pathway have been demonstrated to be functionally upstream of MYB51 in response to flg22. Interestingly, the responses of these upstream regulatory genes to flg22 were impaired in myb51, suggesting that there might be a positive feedback loop among MYB51 and these important regulators. Our study shed light on the diversity and complexity of indole glucosinolate-mediated pathogen resistance.

Section snippets

Plant materials and growth conditions

Arabidopsis ecotype Columbia (Col-0) was used in this study. Seeds of wild type and a T-DNA insertion mutant for MYB51 (SALK_059771) were purchased from the Arabidopsis Biological Resource Center (ABRC). The seeds were sown on 1/2 Murashige and Skoog (1/2MS) basal medium under 24 °C, a light regime of 16 h light and 8 h dark, 70% relative humidity and a constant illumination of 100 μmol m−2 s−1.

Genotyping and RT-PCR analysis of myb51

Genomic DNA was isolated using an EasyPure Plant Genomic DNA Kit (TransGen, Beijing, China). Primers

Illumina sequencing and assembly

First, the myb51 was confirmed to be homozygous and have no detectable transcripts for MYB51 (Fig. S1). RNA samples from 12-day-old wild-type seedlings without flg22 treatment (represented as WT), wild type with flg22 treatment (represented as WT + flg22), myb51 without flg22 treatment (represented as myb51), and myb51 with flg22 treatment (represented as myb51 + flg22) were used to generate cDNA libraries, yielding 45,250,494, 47,172,134, 52,202,996 and 59,359,844 clean reads for WT, myb51,

Discussion

Indole glucosinolates plays a variety of roles in plant immune responses, including systemic acquired resistance, hypersensitive response and callose accumulation [10,12,15]. The function of these metabolites seems to exceed our expectations. To more comprehensively understand the functions of these indolic secondary metabolites in plant immunity, high-throughput RNA-sequencing of wild type and myb51 following treatment with flg22 were performed.

As shown in the analysis of MYB51-dependent

Funding

This work was supported by the National Natural Science Foundation of China (NSFC) 31370334 and Natural Science Foundation of Heilongjiang Province C2017031.

Declaration of competing interest

Authors declare that there is no conflict of interest.

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    These authors have contributed equally to this work.

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