Pathophysiology and transcriptomic analysis of Picea koraiensis inoculated by bark beetle-vectored fungus Ophiostoma bicolor

Abstract

Ophiostomatoid fungi exhibit a complex relationship with bark beetles; exhausting of host tree defenses is traditionally regarded as one of the key benefits provided to beetle vectors. Ophiostoma bicolor is one of the dominant species of the mycobiota associated with Ips genus bark beetles which infect the spruce trees across the Eurasian continent. Host spruce trees resist fungal invasion through structural and inducible defenses, but the underlying mechanisms at the molecular level, particularly with respect to the interaction between bark beetle-associated fungi and host trees, remain unclear. The aim of this study was to observe the pathological physiology and molecular changes in Picea koraiensis seedlings after artificial inoculation with O. bicolor strains (TS, BH, QH, MX, and LWQ). This study showed that O. bicolor was a weakly virulent pathogen of spruce, and that the virulent of the five O. bicolor strains showed differentiation. All O. bicolor strains could induce monoterpenoid release. A positive correlation between fungal virulence and release of monoterpenoids was observed. Furthermore, the release rate of monoterpenoids peaked at 4 days post-inoculation (dpi) and then decreased from 4 to 90 dpi. Transcriptomic analysis at 4 dpi showed that many plant-pathogen interaction processes and mitogen-activated protein kinase (MAPK) metabolic processes were activated. The expression of monoterpenoid precursor synthesis genes and diterpenoid synthesis genes was upregulated, indicating that gene expression regulated the release rate of monoterpenoids at 4 dpi. The enriched pathways may reveal the immune response mechanism of spruce to ophiostomatoid fungi. The dominant O. bicolor possibly induces the host defense rather than defense depletion, which is likely the pattern conducted by the pioneers of beetle-associated mycobiota, such as Endoconidiophora spp.. Overall, these results facilitate a better understanding of the interaction mechanism between the dominant association of beetles and the host at the molecular level.

Keywords: Ips; monoterpenoids; ophiostomatoid fungi; spruce; transcriptome.

FIGURE 1

Disease development of P. koraiensis stems infected by O. bicolor at 30 and 90 dpi.

FIGURE 2

Release rates of six monoterpenes in different treatments (μg/h, mean ± SE, N = 3).

FIGURE 3

Global evaluation of transcriptome sequencing data of spruce. (A) Hierarchical clustering analysis of gene expression shown the correlation among samples. (B) Venn diagram of DEGs in compared between TS, LWQ, and control treatments. DEGs were selected using fold change ≥ 4, FDR correction < 0.01. (C) Volcano plot of all detected genes. Red represents upregulation; green represents downregulation; black represents non-differentially expression. (D) TFs differentially expressed under O. bicolor. X-axis represents the number of DEGs, and Y-axis represents the number names of transcription factor family.

FIGURE 4

Top 20 KEGG pathway analysis of DEGs.

FIGURE 5

Transcriptional profiling of differentially expressed genes (DEGs) associated with Terpenoid backbone biosynthesis, flavonoid biosynthesis and phenylpropanoid biosynthesis pathway. The log₂FC (fold change) values for the DEGs were used for each treatment (TS vs. Control and LWQ vs. Control). The progression of the color scale from blue to red represents an increase in the log₂FC values. Sequences of all new genes were in Supplementary Table 7. (A) The DEGs involved in terpenoid backbone biosynthesis pathway. (B) The DEGs involved in flavonoid biosynthesis pathway. (C) The DEGs involved in phenylpropanoid biosynthesis pathway.

FIGURE 6

Validation of RNA-seq data by RT-qPCR. R was the correlation coefficient between the FPKM value of RNA-seq and the relative expression level of RT-qPCR, and p was the significance between the FPKM value of RNA-seq and the relative expression level of RT-qPCR. p < 0.01 means a significant correlation at the 0.01 level.