A 3R-MYB transcription factor is involved in Methyl Jasmonate-Induced disease resistance in Agaricus bisporus and has implications for disease resistance in Arabidopsis

Abstract

Introduction: Methyl jasmonate (MeJA) and MYB transcription factors (TFs) play important roles in pathogen resistance in several plants, but MYB TFs in conjunction with MeJA-induced defense against Pseudomonas tolaasii in edible mushrooms remain unknown.

Objectives: To investigate the role of a novel 3R-MYB transcription factor (AbMYB11) in MeJA-induced disease resistance of Agaricus bisporus and in the resistance of transgenic Arabidopsis to P. tolaasii.

Methods: Mushrooms were treated with MeJA alone or in combination with phenylpropanoid pathway inhibitors, and the effects of the treatments on the disease-related and physiological indicators of the mushrooms were determined to assess the role of MeJA in inducing resistance and the importance of the phenylpropanoid pathway involved. Subcellular localization, gene expression analysis, dual-luciferase reporter assay, electrophoretic mobility shift assay, and transgenic Arabidopsis experiments were performed to elucidate the molecular mechanism of AbMYB11 in regulating disease resistance.

Results: MeJA application greatly improved mushroom resistance to P. tolaasii infection, and suppression of the phenylpropanoid pathway significantly weakened this effect. MeJA treatment stimulated the accumulation of phenylpropanoid metabolites, which was accompanied by increased the activities of biosynthetic enzymes and the expression of phenylpropanoid pathway-related genes (AbPAL1, Ab4CL1, AbC4H1) and an AbPR-like gene, further confirming the critical role of the phenylpropanoid pathway in MeJA-induced responses to P. tolaasii. Importantly, AbMYB11, localized in the nucleus, was rapidly induced by MeJA treatment under P. tolaasii infection; it transcriptionally activated the phenylpropanoid pathway-related and AbPR-like genes, and AbMYB11 overexpression in Arabidopsis significantly increased the transcription of phenylpropanoid-related genes, the accumulation of total phenolics and flavonoids, and improved resistance to P. tolaasii.

Conclusion: This study clarified the pivotal role of AbMYB11 as a regulator in disease resistance by modulating the phenylpropanoid pathway, providing a novel idea for the breeding of highly disease-resistant edible mushrooms and plants.

Keywords: Brown spot disease; Button mushroom; Methyl jasmonate; Phenylpropanoid metabolism; Pseudomonas tolaasii.

Graphical abstract

Fig. 1

Effects of pretreatments with MeJA alone or combined with different inhibitors on the resistance of A. bisporus to bacterial brown spot disease. (A), The development of bacterial brown spot disease was recorded at storage times of 36, 48, 60, and 72 h. (B), The disease-related indicators, the antioxidant enzyme activities, and the phenylpropanoid pathway-related indexes. The disease-related indicators include the incidence rate, disease index, PPO activity, and MDA content; the antioxidant enzymes include POD, SOD, and CAT; the phenylpropanoid pathway-related indexes include PAL, C4H, 4CL, and CAD activity and the content of total phenolics, total flavonoids, and lignin. Each square represents normalized values (shown a color scale), which is the relative level of each indicator. Values are the mean of three replicates. MeJA, methyl jasmonate; AOA, aminooxy acetic acid, a competitive inhibitor of PAL; PIP, piperonylic acid, an irreversible inhibitor of C4H; MDCA, 3,4-(methylenedioxy) cinnamic acid, a competitive inhibitor of 4CL.

Fig. 2

Expression patterns of phenylpropanoid pathway- and pathogenesis-related genes. Effects of MeJA pretreatment on the expression of five genes related to the phenylpropanoid pathway and a pathogenesis-related gene in the fruiting bodies of A. bisporus involved in resistance to P. tolaasii. Data are the mean of three replicates ± standard errors. Different lowercase letters above the bars denote significant differences between two groups at P<0.05.

Fig. 3

Identification, characterization, subcellular localization, and expression patterns of AbMYB11. (A), Multiple alignments of the MYB domains of AbMYB11 with PoMYB1 (AXB87541.1) and PoMYB2 (AXB87528.1) from Pleurotus ostreatus; GsMYB1 (PRJNA476322) and GsMYB2 (PRJNA71455) from Ganoderma sinense; NtMYB1 (NP_001312530.1), NtMYB2 (NP_001312096.1) and NtMYB3 (BAB70512.1) from Nicotiana tabacum; AtMYB1 (NP_194999), AtMYB2 (AAF25950) and AtMYB3 (NP_001330337) from Arabidopsis thaliana; OsMYB (BAD81765.1) from Oryza sativa; Hs_c-Myb (NP_001123644), Hs_A-Myb (CAA31656) and Hs_B-Myb (CAA31655) from Homo sapiens; DmMYB (CAA29373) from Drosophila melanogaster; and GgMYB (Q03237) from Gallus gallus. The blue cylinders above the sequences show the locations of the imperfect tandem repeats of R1, R2, and R3 within the MYB domain. α1-α9 indicate alpha-helices. Amino acids with 100 % identity are shaded in red, and those with 80 % to 100 % identity are shaded in yellow. The black triangles below the sequence denote eleven DNA-binding amino acid residues. (B), Tertiary structure of the SANT domains of AbMYB11 predicted by I-TASSER. Putative amino residues in R3 that could bind to DNA are shown as stick models. (C), Subcellular localization of AbMYB11 via the transient expression of the fusion protein (AbMYB11-GFP) and 35S-GFP in tobacco leaves. GFP and mCherry signals were captured using laser confocal microscopy after 2–3 d of infiltration. Bars = 20 μM. (D), Effect of MeJA pretreatment on the relative expression levels of AbMYB11 during the process of AbMYB11 under P. tolaasii infection.

Fig. 4

AbMYB11 activates the expression of AbPAL1, AbC4H1, Ab4CL1, and AbPR-like by binding to specific cis-acting elements in the promoter. (A), Construction diagrams of effector and reporter plasmids for dual-luciferase reporter (DLR) experiments. (B), The regulation of AbMYB11 on each promoter was defined according to the relative LUC/REN ratio. The LUC/REN ratios of the empty plasmid plus promoter were set to 1 to calibrate relative values. Each value represents the mean ± SE of six independent biological replicates. Different letters above the bars indicate significant differences (P<0.05). (C-F), EMSA results show the binding ability of AbMYB11 to the promoters of AbPAL1, AbC4H1, Ab4CL1, and AbPR-like, respectively. The promoter DNA sequence of each target gene containing MYB-binding motifs (AACTG or ACCTACC, red letters) was used as a wild probe. Empty MBP proteins were incubated with the biotin-labeled probe and served as a negative control; 50- and 100-fold excesses of unlabeled (mutated) probes were used for competition. MBP-AbMYB11 proteins were incubated with mutated probes assess the binding specificity of AbMYB11 to the binding motif. Mu means mutated probes in which the AACTG and ACCTACC motifs were replaced by TTTTT and TTTTTTT, respectively. “+” and “–” represent presence and absence, respectively. Arrows indicate the shifted bands or free probe bands.

Fig. 5

AbMYB11 overexpression improves the resistance of Arabidopsis plants to P. tolaasii and activates the phenylpropanoid pathway. (A), qPCR analysis of AbMYB11 expression. (B), Semiquantitative RT-PCR analysis of AbMYB11 expression. (C), The disease symptoms of Arabidopsis leaves. (D), Trypan blue staining of Arabidopsis leaves. (E), Total content of phenolics and flavonoids. (F), qPCR analysis of AtPAL1, AtC4H, At4CL1, and AtPR1 expression. Data in (A)-(F) were all collected from 6-week-old wild-type (Col-0) and transgenic Arabidopsis leaves 24 h after inoculation (dpi) with P. tolaasii. Relative expression levels of target genes in (A) and (F) were normalized using the reference gene (AtActin2) and shown as the mean ± SE.

Fig. 6

Proposed function of the 3R type MYB TF AbMYB11 in the resistance of A. bisporus to bacterial brown spot disease. Exogenous MeJA and inoculation with P. tolaasii could up-regulate the expression of AbMYB11. AbMYB11 is involved in MeJA-induced resistance to P. tolaasii by the activation of the phenylpropanoid pathway-related and AbPR-like genes. Question marks indicate that AbMYB11 possibly has additional target genes. Dashed arrows indicate mechanisms that have not yet been proven.