Validation of Monilinia fructicola Putative Effector Genes in Different Host Peach (Prunus persica) Cultivars and Defense Response Investigation

Abstract

Monilinia fructicola is the most common and destructive brown rot agent on peaches. Knowledge of gene expression mediating host-pathogen interaction is essential to manage fungal plant diseases. M. fructicola putative virulence factors have been predicted by genome investigations. The pathogen interaction with the host was validated. Five M. fructicola isolates were inoculated on two cultivars (cv.s) of peach (Prunus persica (L.) Batsch) 'Royal Summer' and 'Messapia' with intermediate and late ripening periods, respectively. The expression pattern of 17 candidate effector genes of M. fructicola with functions linked to host invasion and fungal life, and seven peach genes involved in the immune defense system were monitored at 0, 2, 6, 10, and 24 h-post inoculation (hpi). All fungal isolates induced similar brown rot lesions on both cv.s whereas the modulation of effector genes was regulated mainly at 2, 6, and 10 hpi, when disease symptoms appeared on the fruit surface, confirming the involvement of effector genes in the early infection stage. Although differences were observed among the fungal isolates, the principal component investigation identified the main differences linked to the host genotype. The salicylic acid and jasmonate/ethylene signaling pathways were differently modulated in the host independent from the fungal isolate used for inoculation. On plants susceptible to brown rot, the pathogen may have adapted to the host's physiology by modulating its effectors as weapons.

Keywords: brown rot; defense genes; fungal effector proteins; gene expression; stone fruit.

Figure 1

Disease assessment on wounded peach fruit cv. ‘Royal Summer’ (a–e) and cv. ‘Messapia’ (f–j) 24 h post inoculation (hpi) with 30 μL conidia suspension (10⁹ conidia/mL) of AN7 (a,f), AN26 (b,g), AN12 (c,h) AN13 (d,i), and AN14 (e,j) M. fructicola isolates. Brown rot severity (k) caused by M. fructicola isolates at 24 hpi after inoculation on peach fruit cv. ‘Royal Summer’ and cv. ‘Messapia’. Data represent the mean of two experiments with four inoculations per fruit and three fruits per isolate and experiment (n = 24). Data were analyzed by analysis of variance. Means and standard deviation are not significantly different (p ≤ 0.05) according to the Tukey HSD post hoc test.

Figure 2

Expression profiles of 17 putative effector genes assayed (Table 1) by RT-qPCR. RNA was isolated from five M. fructicola isolates (AN7, AN26, AN12, AN13, and AN14) after inoculation on peach cv. ‘Royal Summer’ at 0, 2, 6, 10, and 24 hpi. The data were represented as fold-change compared to 0 hpi (h0*), which is given a value of 1. Means were determined with data from two biological replicates with three technical replicates each (n = 6). For each gene of each fungal isolate, columns with different letters are significantly different (p ≤ 0.05; Tukey HSD post hoc test).

Figure 3

Heatmap showing hierarchical cluster analysis of 17 M. fructicola putative effector genes (Table 1) expressed across all samples (different fungal isolates AN7, AN26, AN12, AN13, and AN14 at 0, 2, 6, 10, and 24 hpi) on the two cv.s ‘Royal Summer’ (a) and ‘Messapia’ (b). The genes (rows) and samples (columns) are clustered using the Pearson Correlation distance and complete linkage hierarchical clustering. The gradient scale represents expression levels, with red showing the highest expression to blue with the lowest expression level.

Figure 4

Expression profiles of 17 putative effector genes assayed (Table 1) by RT-qPCR. RNA was isolated from five M. fructicola isolates (AN7, AN26, AN12, AN13, and AN14) after inoculation on cv. ‘Messapia’ peach at 0, 2, 6, 10, and 24 hpi. The data were represented as fold-change compared to 0 hpi (h0*), which is given a value of 1. Means were determined with data from two biological replicates with three technical replicates each (n = 6). For each gene of each fungal isolate, columns with different letters are significantly different (p ≤ 0.05; Tukey HSD post hoc test).

Figure 5

Expression profiles of seven key peach defense genes (Table 1) assayed by RT-qPCR detected in ‘Royal Summer’ after inoculation with AN7, AN26, AN12, AN13, and AN14 M. fructicola fungal isolates at 0, 2, 6, 10, and 24 hpi. The data were represented as fold-change compared to 0 hpi (h0*) which is given a value of 1. Means were determined with data from two biological replicates with three technical replicates each (n = 6). For each gene of each fungal isolate, columns with different letters are significantly different (p ≤ 0.05; Tukey HSD post hoc test).

Figure 6

Heatmap showing hierarchical cluster analysis of seven genes (Table 1) expressed in peaches across all samples on the ‘Royal Summer’ cv. (a) and ‘Messapia’ cv. (b) after inoculation with AN7, AN26, AN12, AN13, and AN14 M. fructicola fungal isolates at 0, 2, 6, 10, and 24 hpi. The scale represents expression levels, with red showing the highest expression to blue with the lowest expression.

Figure 7

Expression profiles of seven key peach defense genes (Table 1) assayed by RT-qPCR detected in ‘Messapia’ after inoculation with M. fructicola fungal isolates AN7, AN26, AN12, AN13, and AN14 at 0, 2, 6, 10, and 24 hpi. The data were represented as fold-change compared to 0 hpi (h0*) which is given a value of 1. Means were determined with data from two biological replicates with three technical replicates each (n = 6). For each gene of each fungal isolate, columns with different letters are significantly different (p ≤ 0.05; Tukey HSD post hoc test).

Figure 8

Principal component analysis (PCA) of gene expression dataset. PCA biplot (score plot) visualizing projections onto the first two principal components. The biplot graph represents the relationship among the expression of 17 putative effector genes (Table 1) and 7 peach expression genes highlighted by a red circle (see Table 1 for details) used as vectors and the peach cv.s ‘Messapia’ and ‘Royal Summer’ used as groups. The two main components (Dim1 and Dim2) explained 24.3% and 14.8% of the variance, respectively. Different colors and symbols represent the different cv.s: (• blue), ‘Messapia’ and (▲ yellow), ‘Royal Summer’. Ellipses indicate a confidence interval of 50%. The direction and length of the arrows illustrate how each effector (vector) contributes to the first two components in the PCA. Aligned vectors indicate a strong positive correlation between the two effectors (<30%). Vectors at right angles/opposites (>90%) indicate no correlation/negative correlation, respectively.