Effects of Endobacterium (Stenotrophomonas maltophilia) on Pathogenesis-Related Gene Expression of Pine Wood Nematode (Bursaphelenchus xylophilus) and Pine Wilt Disease

Abstract

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is responsible for devastating epidemics in pine trees in Asia and Europe. Recent studies showed that bacteria carried by the PWN might be involved in PWD. However, the molecular mechanism of the interaction between bacteria and the PWN remained unclear. Now that the whole genome of B. xylophilus (Bursaphelenchus xylophilus) is published, transcriptome analysis is a unique method to study the role played by bacteria in PWN. In this study, the transcriptome of aseptic B. xylophilus, B. xylophilus treated with endobacterium (Stenotrophomonas maltophilia NSPmBx03) and fungus B. xylophilus were sequenced. We found that 61 genes were up-regulated and 830 were down-regulated in B. xylophilus after treatment with the endobacterium; 178 genes were up-regulated and 1122 were down-regulated in fungus B. xylophilus compared with aseptic B. xylophilus. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were used to study the significantly changed biological functions and pathways for these differentially expressed genes. Many pathogenesis-related genes, including glutathinone S-transferase, pectate lyase, ATP-binding cassette transporter and cytochrome P450, were up-regulated after B. xylophilus were treated with the endobacterium. In addition, we found that bacteria enhanced the virulence of PWN. These findings indicate that endobacteria might play an important role in the development and virulence of PWN and will improve our understanding of the regulatory mechanisms involved in the interaction between bacteria and the PWN.

Keywords: Bursaphelenchus xylophilus; bacteria; differentially expressed genes; pine; transcriptome.

Figure 1

Gene Ontology (GO) terms for the transcriptome sequences derived from differently treated B. xylophilus (p < 0.05). Gray bars indicate significantly enriched GO terms in endobacterium-treated PWN samples (Bx_b) compared to aseptic PWNs (Bx_a); black bars indicated significantly enriched GO terms in fungus PWNs (Bx_fungus) compared to aseptic PWNs (Bx_a).

Figure 2

Putative cell wall hydrolase genes in B. xylophilus treated with endobacterium and fungus B. xylophilus (more than two-fold change in B. xylophilus treated with endobacterium or wild-type B. xylophilus). Bx_b: B. xylophilus treated with endobacterium, Bx_fungus: fungus B. xylophilus.

Figure 3

Putative detoxification-related genes most differentially expressed, with higher levels in B. xylophilus treated with S. maltophilia NSPmBx03 and fungus B. xylophilus. Bx_a: aseptic B. xylophilus, Bx_b: B. xylophilus treated with S. maltophilia NSPmBx03, Bx_fungus: fungus B. xylophilus. The left of the vertical axis indicates down-regulated genes, and the right indicates up-regulated genes.

Figure 4

Putative reproduction-related genes in B. xylophilus treated with endobacterium and fungus B. xylophilus (fold-change > 2). Bx_a: aseptic B. xylophilus, Bx_b: B. xylophilus treated with S. maltophilia NSPmBx03, Bx_fungus: fungus B. xylophilus. The left of the vertical axis indicates down-regulated genes, and the right indicates up-regulated genes.

Figure 5

Relative pathways significantly enriched in differentially expressed genes (DEGs) of PWNs treated with endobacterium and fungus PWNs (p < 0.005). DEGs were enriched in detoxification pathways and turpentine-related metabolism pathway. Bx_b: B. xylophilus treated with S. maltophilia NSPmBx03, Bx_fungus: fungus B. xylophilus.

Figure 6

Relative gene expression. (A) Expression levels of 16 genes in sequencing results. RPKM, reads per kilobase per million reads; (B) qRT-PCR results of 16 differentially expressed genes under sterile, endobacterium treatment, and fungus conditions. White bars indicate expression levels of the genes in control samples (Bx_a); gray bars indicate expression levels of genes in endobacterium-treated PWN samples (Bx_b); black bars indicate expression levels of genes in fungus PWN samples (Bx_fungus). Data represent the means ± SD (n = 3). SD, standard deviations.

Figure 7

Symptoms of P. massoniana inoculated with different PWN treatments. (A) Symptoms after 0 day of inoculation; (B) Symptoms after 21 days; (C) Symptoms after 26 days; (D) Symptoms after 30 days. Pines inoculated with: L1, fungus B. xylophilus; L2, B. xylophilus treated with S. maltophilia NS PmBx 03; L3, aseptic B. xylophilus; L4, S. maltophilia NSPmBx 03; and L5, sterile water.