PpMYC2 and PpJAM2/3 antagonistically regulate lignin synthesis to cope with the disease in peach fruit

Abstract

Transcription factors MYC2 and JAMs play a crucial role in regulating disease resistance in model plants. However, their regulatory mechanisms of disease resistance in non-model plants, particularly in postharvest fruits, remain largely unknown. In this study, we observed that PpMYC2 expression was up-regulated in peach fruit following infection by Monilinia fructicola, while the expressions of PpJAM2/3 were down-regulated. Furthermore, we found that PpMYC2 positively regulated the resistance against M. fructicola, whereas PpJAM2/3 negatively regulated it. Through a combined DNA affinity purification and RNA sequencing analysis for PpMYC2, we identified lignin synthesis genes (PpPAL1, PpC4H, Pp4CL1, PpCSE and PpCCoAOMT1) as candidate target genes. Subsequent assays, including dual-luciferase reporter assay, transient overexpression and silencing assays, electrophoretic mobility shift assay and yeast one-hybrid assay demonstrated that PpMYC2 activated the transcription of these five genes by binding to their promoters, promoting lignin accumulation. Conversely, PpJAM2 inhibited the transcription of PpC4H and PpCSE, while PpJAM3 inhibited Pp4CL1 and PpCCoAOMT1. Additionally, PpJAM2 or PpJAM3 interfered with PpMYC2's activation of their common target genes by competitively binding to the promoters. In conclusion, when peach fruit is infected with M. fructicola, up-regulation of PpMYC2 promotes lignin synthesis, while down-regulation of PpJAM2/3 reduces their inhibitory effects, ultimately resulting in lignin accumulation to combat the disease infection. Our study provides new insights into the molecular mechanisms of disease response in postharvest peach fruit.

Keywords: Monilinia fructicola; Prunus persica; competitive binding; lignin synthesis; transcription factor.

Figure 1

Disease resistance of peach fruit after transient overexpression and silencing of PpMYC2 and PpJAM2/3. (a‐c) Expression patterns of PpMYC2 and PpJAM2/3 at different time points in peach fruit after inoculation with sterile water (CK) or M. fructicola. Data are presented as mean ± SE (n = 3). (d‐f) The transcription levels of PpMYC2 and PpJAM2/3 in the overexpression peach fruit. The empty pART‐CAM vector was used as the control. Data are presented as mean ± SE (n = 6). (g‐h) The phenotype and lesion diameter of the overexpression peach fruit at 72 h after M. fructicola inoculation. Peach fruit was inoculated with M. fructicola at 48 h after transient transformation with overexpression vectors. Data are presented as mean ± SE (n = 3). (i‐k) The transcription levels of pathogenesis‐related protein (PR) genes in peach fruit following transient overexpression of PpMYC2 and PpJAM2/3. Data are presented as mean ± SE (n = 6). (l‐n) The transcription levels of PpMYC2 and PpJAM2/3 in the silencing peach fruit. The empty pTRV2 vector was used as the control. Data are presented as mean ± SE (n = 6). (o, p) The phenotype and lesion diameter of the silencing peach fruit at 72 h after M. fructicola inoculation. Peach fruit was inoculated with M. fructicola at 10 days after transient transformation with silencing vectors. Data are presented as mean ± SE (n = 3). (q‐s) The transcription levels of PR genes in peach fruit following transient silencing of PpMYC2 and PpJAM2/3. Data are presented as mean ± SE (n = 6). Asterisks indicate significant differences (**, P < 0.01; *, P < 0.05; Student's t‐test).

Figure 2

Combination analysis of DAP‐seq and RNA‐seq identified the potential target genes of PpMYC2. (a) Overlapping peaks and distance from the centre of the binding site to transcription start site (TSS) for all peaks. (b) Distribution of PpMYC2‐binding peaks on gene functional elements. (c) The top three conserved motifs enriched within the recognition interval of PpMYC2. (d) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of PpMYC2‐binding genes. The gene ratio represents the number of peak‐associated genes in each pathway as a percentage of the number of all peak‐associated genes. (e) KEGG pathway enrichment analysis of differentially expressed genes (DEGs) between the control and PpMYC2‐overexpressed peach fruit. The gene ratio represents the number of DEGs in each pathway as a percentage of the number of all DEGs. (f) Venn diagram of intersection DEGs by DAP‐seq and RNA‐seq. The genes on promoters bound by PpMYC2 were used for intersection analysis with the DEGs of RNA‐seq. (g) The heatmap of DEGs (PpPAL1, PpC4H, Pp4CL1, PpCSE and PpCCoAOMT1) between the control and PpMYC2‐overexpressed peach fruit. The empty pART‐CAM vector was used as the control. Blue indicates low expression and red indicates high expression. (h) The motifs in the promoters of PpPAL1, PpC4H, Pp4CL1, PpCSE and PpCCoAOMT1. The promoter length and the motifs are indicated with black lines and red diamonds, respectively. (i‐n) Expression patterns of PpPAL1, PpC4H, Pp4CL1, PpCSE and PpCCoAOMT1 and lignin content at different time points in peach fruit inoculated with sterile water or M. fructicola. Data are presented as mean ± SE (n = 3). Asterisks indicate significant differences (**, P < 0.01; *, P < 0.05; Student's t‐test).

Figure 3

Effects of PpMYC2 and PpJAM2/3 on the transcription activities of lignin synthesis genes. (a) Schematic diagram of the effector and reporter constructs for dual‐luciferase reporter (DLR) assay. (b–d) Effects of PpMYC2 and PpJAM2/3 on transcriptional activities of lignin synthesis genes in tobacco leaves. The ratio of LUC/REN of the empty vector (62SK) plus promoter was used as a calibrator (set as 1). Data are presented as mean ± SE (n = 6). (e–h) The transcription levels of lignin synthesis genes and lignin content in peach fruit with transient overexpression of PpMYC2 and PpJAM2/3. The data are expressed as mean ± SE (n = 6). (i–l) The expression levels of lignin synthesis genes and lignin content in peach fruit with transient silencing of PpMYC2 and PpJAM2/3. Data are expressed as mean ± SE (n = 6). Asterisks indicate significant differences (**, P < 0.01; *, P < 0.05; Student's t‐test).

Figure 4

The interaction of PpMYC2 and PpJAM2/3 with the promoters of the target genes in vitro and vivo. (a) Schematic diagram of the bait and the prey constructs for yeast one‐hybrid (Y1H) assay. (b‐j) Y1H assay showing the binding of PpMYC2 and PpJAM2/3 to the promoters of the target genes. AD‐Empty was used as the negative control. (k‐s) Electrophoretic mobility shift assay (EMSA) showing the binding of PpMYC2 and PpJAM2/3 to the promoters of the target genes. The unlabelled probes served as competitors. The symbols ‘‐’, ‘+’ and ‘+ +’ indicate the absence, presence and increasing amounts, respectively.

Figure 5

Disease resistance of Arabidopsis from mutants, wild type (WT) and overexpression lines. (a) Identification of transgenic Arabidopsis. (b) The phenotype of Arabidopsis leaves from different lines at 2 days after infection with M. fructicola. (c) DAB staining of Arabidopsis leaves from different lines at 2 days after infection with M. fructicola. (d) Lesion diameter of Arabidopsis leaves from different lines at 2 days after infection with M. fructicola. (e) Lignin content in mutants, WT and overexpression lines. The leaves of 4‐week‐old Arabidopsis were used to determine lignin content. (f‐h) Expression levels of lignin synthesis genes in mutants, WT and overexpression lines. The leaves of 4‐week‐old Arabidopsis were used to determine lignin content. Data are presented as mean ± SE (n = 3). Distinct letters are assigned to show significant differences (P < 0.05; Tukey's HSD test).

Figure 6

PpJAM2/3 interfere with the transcription of target genes by competitively binding to the promoters with PpMYC2. (a‐d) Electrophoretic mobility shift assay (EMSA) showing the competitive binding of PpJAM2/3 and PpMYC2 to the promoters of common target genes. His protein served as a negative control. (e‐h) PpJAM2/3 interfered with PpMYC2 in activating the transcription of common target genes. The symbols ‘‐’ and ‘+’ indicate the absence and presence, respectively. Data are presented as mean ± SE (n = 6). Distinct letters are assigned to show significant differences (P < 0.05; Tukey's HSD test).

Figure 7

Possible molecular mechanism of PpMYC2 and PpJAM2/3 synergistically regulating disease resistance in peach fruit. The symbols ‘−’ and ‘+’ indicate the decrease and the increase, respectively. The red arrow indicates an activation effect, while the grey flat end indicates an inhibition effect. The greater the binding proportion of PpMYC2 and PpJAM2/3 to the cis element, the stronger the competition for binding to the promoter. After M. fructicola infection, the expression of PpMYC2 in peach fruit was up‐regulated, whereas the expressions of PpJAM2 and PpJAM3 were down‐regulated. The up‐regulation of PpMYC2 enhanced the transcriptional activation of PpPAL1, PpC4H, PpCSE, Pp4CL1 and PpCCoAOMT1, thereby promoting lignin accumulation. Simultaneously, the down‐regulation of PpJAM2 decreased the transcription of PpC4H and PpCSE, and the down‐regulation of PpJAM3 reduced the transcription of Pp4CL1 and PpCCoAOMT1, collectively alleviating the inhibition of lignin synthesis. These two regulation ways ultimately resulted in an increase in lignin synthesis serving as a protective response against infection by M. fructicola.