Transcriptomic Response of Resistant (PI613981- Malus sieversii) and Susceptible ("Royal Gala") Genotypes of Apple to Blue Mold (Penicillium expansum) Infection

Abstract

Malus sieversii from Central Asia is a progenitor of the modern domesticated apple (Malus × domestica). Several accessions of M. sieversii are highly resistant to the postharvest pathogen Penicillium expansum. A previous study identified the qM-Pe3.1 QTL on LG3 for resistance to P. expansum in the mapping population GMAL4593, developed using the resistant accession, M. sieversii -PI613981, and the susceptible cultivar "Royal Gala" (RG) (M. domestica), as parents. The goal of the present study was to characterize the transcriptomic response of susceptible RG and resistant PI613981 apple fruit to wounding and inoculation with P. expansum using RNA-Seq. Transcriptomic analyses 0-48 h post inoculation suggest a higher basal level of resistance and a more rapid and intense defense response to wounding and wounding plus inoculation with P. expansum in M. sieversii -PI613981 than in RG. Functional analysis showed that ethylene-related genes and genes involved in "jasmonate" and "MYB-domain transcription factor family" were over-represented in the resistant genotype. It is suggested that the more rapid response in the resistant genotype (Malus sieversii-PI613981) plays a major role in the resistance response. At least twenty DEGs were mapped to the qM-Pe3.1 QTL (M × d v.1: 26,848,396-28,424,055) on LG3, and represent potential candidate genes responsible for the observed resistance QTL in M. sieversii-PI613981. RT-qPCR of several of these genes was used to validate the RNA-Seq data and to confirm their higher expression in MS0.

Keywords: Penicillium expansum; malus domestica; necrotrophic pathogens; postharvest pathogens; qualitative trait loci; wound healing.

Figure 1

Principal Coordinates Analysis (PCoA) of Malus sieversii–PI613981 (denoted as MS) vs. “Royal Gala” (denoted as RG) parental lines. (A) PCoA plot at time 0; (B) PCoA plot of wounded (denoted as W) vs. Penicillium expansum inoculated (denoted as P) RG apple fruits at 6, 24, and 48 h; (C) PCoA plot of W vs. P MS apple fruits at 6, 24, and 48 h; and, (D) PCoA plot of P. expansum inoculated (P) RG vs. MS apple fruits at 6, 24, and 48 hpi.

Figure 2

Number of up– and down–regulated genes [FDR p ≤ 0.01 and log₂(ratio)≤-1 or log₂(ratio)≥1] in MS (solid bars) and RG (striped bars) parental lines at 6, 24, and 48 h after wounding or after P. expansum inoculation compared to non–treated apples (Time 0). Numbers between parentheses indicate the number of DEGs located within the qM–Pe3.1 QTL on LG3.

Figure 3

Venn diagrams showing the numbers of common and specific DE genes (FDR p value≤0.01) at 6 (A), 24 (B), and 48 (C) h after wounding (W) or P. expansum inoculation (P) in the parental lines Malus sieversii–PI613981 (MS) and Malus x domestica “Royal Gala” (RG).

Figure 4

Hierarchical cluster (HCL) analysis and heatmaps of differentially expressed genes (two–factor ANOVA) based on genotype (A), time course of P. expansum infection (B), and the interaction (C). Heatmap of genes included in the significant biological processes “response to biotic stimulus” (D) and “lipid metabolic process (E) based on the list of DEGs during the time course (D) or the interaction (E). Abbreviations: Malus sieversii–PI613981 resistant apple (MS) and Malus x domestica “Royal Gala” susceptible apple (RG) at time 0 and inoculated with Penicillium expansum (P) at 6, 24, and 48 hpi. Data are expressed as log₂(ratios).

Figure 5

Overview of metabolic pathways (A,C) and of genes related to biotic stress (B,D) for each DEGs upon P. expansum infection after 6 hpi in the resistant MS parent (A,B) or in the susceptible RG parent (B,D) vs. Time 0. The scale bar displays changes in gene expression as log₂ (ratio MS6P/MS0) or log₂(ratio RG6P/RG0) that were significant (FDR p value≤0.01). Genes induced due to the P. expansum infection are highlighted in red and repressed genes are highlighted in blue. CHO, carbohydrates; OPP, oxidative pentose phosphate; TCA: tricarboxylic acid cycle.

Figure 6

Parametric analysis of gene–set enrichment (PAGE) using AgriGo to identify enriched GO terms onto the biological term inclusive of gene expression levels. The colored blocks represent the level of up/downregulation of each term at a certain time–point. The yellow–to–red, cyan–to–blue, and grayscale represent the term is upregulated, downregulated, or no–significant change, respectively. The adjusted p-value of the term determines the degree of color saturation of the corresponding box. MSW/MS0 = comparison of M. sieversii at 6, 12, and 48 h after wounding, relative to Time 0. RGW/RG0 = comparison of “Royal Gala” at 6, 12, and 48 h after wounding, relative to Time 0. MSPE/MS0 = comparison of M. sieversii at 6, 12, and 48 h post-inoculation with P. expansum, relative to Time 0. RGPE/RG0 = comparison of “Royal Gala” at 6, 12, and 48 h post–inoculation with P. expansum, relative to Time 0.