Dual RNA-Sequencing Analysis of Resistant (Pinus pinea) and Susceptible (Pinus radiata) Hosts during Fusarium circinatum Challenge

Abstract

Fusarium circinatum causes one of the most important diseases of conifers worldwide, the pine pitch canker (PPC). However, no effective field intervention measures aiming to control or eradicate PPC are available. Due to the variation in host genetic resistance, the development of resistant varieties is postulated as a viable and promising strategy. By using an integrated approach, this study aimed to identify differences in the molecular responses and physiological traits of the highly susceptible Pinus radiata and the highly resistant Pinus pinea to F. circinatum at an early stage of infection. Dual RNA-Seq analysis also allowed to evaluate pathogen behavior when infecting each pine species. No significant changes in the physiological analysis were found upon pathogen infection, although transcriptional reprogramming was observed mainly in the resistant species. The transcriptome profiling of P. pinea revealed an early perception of the pathogen infection together with a strong and coordinated defense activation through the reinforcement and lignification of the cell wall, the antioxidant activity, the induction of PR genes, and the biosynthesis of defense hormones. On the contrary, P. radiata had a weaker response, possibly due to impaired perception of the fungal infection that led to a reduced downstream defense signaling. Fusarium circinatum showed a different transcriptomic profile depending on the pine species being infected. While in P. pinea, the pathogen focused on the degradation of plant cell walls, active uptake of the plant nutrients was showed in P. radiata. These findings present useful knowledge for the development of breeding programs to manage PPC.

Keywords: Fusarium circinatum; Pinus pinea; Pinus radiata; conifer defense; disease differential susceptibility; dual RNA-Seq; host-pathogen interaction.

Figure 1

Survival probability plot determined using the Kaplan–Meier estimate of the survival function for P. pinea and P. radiata seedlings inoculated with F. circinatum. Different letters indicate significant p-values.

Figure 2

Needle gas exchange-related parameters of P. pinea and P. radiata inoculated with F. circinatum and controls at 4 dpi. (A) Stomatal conductance. (B) Transpiration rate. (C) Net CO₂ assimilation rate. (D) Sub-stomatal CO₂ concentration. Error bars show the standard deviation. Different letters above the bars indicate significant differences (ANOVA, p < 0.05).

Figure 3

Two-dimensional scatterplot of the principal component analyses (PCA) for (A) Pinus radiata and P. pinea different treatments and (B) Fusarium circinatum infecting every host species. The distances approximate the typical log₂ fold changes between the samples.

Figure 4

Venn diagram showing the number of P. pinea and P. radiata genes up-regulated (A) and down-regulated (B).

Figure 5

Representation of the most significantly (p < 0.05) enriched GO terms (biological processes) of the up-regulated genes of P. pinea (blue bars) and P. radiata (green bars) infected by F. circinatum.

Figure 6

Differentially expressed genes (DEGs) with a role in signal perception. Numbers represent the log₂ fold change value based on the comparison of the transcript levels between the pine seedlings infected by F. circinatum and mock-inoculated control. Groups of genes are abbreviated as follows: RLKs, receptor-like kinases; LecRLKs, lectin domain-containing receptor kinase; STKs, serine/threonine-protein kinases; LRR-RL ST, receptor-like serine/threonine-protein kinase with leucine-rich repeat domain; STPK HT1-like, serine threonine-protein kinase HT1-like; WAKL, wall-associated receptor kinase-like; LRR-RLKs, leucine-rich repeat receptor-like protein kinases; CRKs, cysteine-rich receptor-like protein kinase; MAPKK, mitogen-activated protein kinase kinase; MAPKKK, mitogen-activated protein kinase kinase kinase; PP2A, serine/threonine phosphatases type 2A.

Figure 7

Simplified scheme of the flavonoid and lignin biosynthetic pathway. Some critical up-regulated (green) or down-regulated (red) enzymes of P. pinea are indicated and abbreviated as follows: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate: CoA ligase; CHS, chalcone synthase; CHI, flavanone isomerase; F3H, Flavanone 3-hydroxylase; DFR, dihydroflavonol 4-reductase; LDOX, leucoanthocyanidin dioxygenase; OMT, O-methyltransferase; UFGT, UDP-glucose:flavonoid 3-O-glucosyl transferase; RT, putative rhamnosyltransferase; CCoAOMT, caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase; FLS, flavonol synthase; LAR, leucanthocyanidin reductase. Individual differentially expressed genes (DEGs) with annotated functions are listed along the Y-axis for P. pinea and P. radiata. The bars in the X-axis represent the level of log₂ fold change value based on the comparison of the transcript levels between the pine seedlings infected by F. circinatum and mock-inoculated control. Red bars denote the level of down-regulation, and green bars indicate the level of up-regulation.

Figure 8

Differentially expressed genes (DEGs) encoding cell wall modifying family proteins. Individual genes are listed along the Y-axis. In X-axis is represented the log₂ fold change value based on the comparison of the transcript levels between F. circinatum infected Pinus species and mock-inoculated plants.

Figure 9

Differentially expressed genes (DEGs) encoding enzymes of defense hormone biosynthesis. Individual genes are listed along the Y-axis. In X-axis is represented the log₂ fold change value based on the comparison of the transcript levels between F. circinatum infected Pinus species and mock-inoculated plants.

Figure 10

Representation of the 10 most significantly (p < 0.05) enriched GO terms (biological processes) of the up-regulated (green bars) and down-regulated (red bars) genes of F. circinatum infecting P. pinea compared with P. radiata.