RESUMEN

For insect-borne pathogens, phoretic ability is important not only to spread more widely and efficiently but also to evolve virulence. Bursaphelenchus xylophilus, the causal agent of pine wilt disease, is transmitted by the cerambycid beetle Monochamus alternatus, which is associated with pine tree host. Their specific phoretic ability to appropriate vectors depending on their life cycle is critical for efficient transfer to the correct host and is expected to enhance virulence. We evaluated how B. xylophilus acquired a specific relationship with M. alternatus with a focus on Bursaphelenchus okinawaensis, a close relative of B. xylophilus that has evolved a relationship with a cerambycid beetle vector. Bursaphelenchus okinawaensis has a single dispersal stage (dauer) larva (third-stage dispersal [DIII] larva), whereas B. xylophilus has two distinct dispersal stages (DIII and fourth-stage dispersal [DIV] larva). Also, the dauer formation in B. okinawaensis is not completely dependent on its beetle vector, whereas DIV larvae of B. xylophilus are induced by volatile from the beetle vector. We investigated the induction conditions of dauer larvae in B. okinawaensis and compared to with B. xylophilus. The dauer percentages of B. okinawaensis significantly increased when the nematode population on the plate increased or when we propagated the nematodes with a crude extract of cultured nematodes, which likely contained dauer-inducing pheromones. In addition, dauer formation tended to be enhanced by the crude extract at high temperatures. Furthermore, when we propagated the nematodes with M. alternatus pupae until the beetles eclosed, B. okinawaensis significantly developed into dauer larvae. However, only 1.3% of dauer larvae were successfully transferred to M. alternatus, the rate lower than that of B. xylophilus. DIII and DIV of B. xylophilus were induced by increasing the nematode population and the presence of the beetle vector, respectively. These results suggest that B. okinawaensis has acquired specificity for the cerambycid beetle through dauer formation, which is efficiently induced in the presence of the beetle, and the DIV larval stage, exclusive to the xylophilus group, may be crucial for high transfer ability to the beetle vector.

RESUMEN

Pine wilt disease (PWD), which is caused by the pine wood nematode Bursaphelenchus xylophilus, is among the most serious tree diseases worldwide. PWD is thought to be initiated by sequential excessive hypersensitive responses to B. xylophilus. Previous studies have reported candidate pathogenic molecules inducing hypersensitive responses in pine trees susceptible to B. xylophilus. The functions of some of these molecules have been analyzed in model plants using transient overexpression; however, whether they can induce hypersensitive responses in natural host pines remains unclear due to the lack of a suitable functional analysis method. In this study, we established a novel functional analysis method for susceptible black pine (Pinus thunbergii) seed embryos using transient overexpression by the Apple latent spherical virus vector and investigated five secreted proteins of B. xylophilus causing cell death in tobacco to determine whether they induce hypersensitive responses in pine. We found that three of five molecules induced significantly higher expression in pathogenesis-related genes ( p < 0.05), indicating hypersensitive response in pine seed embryos compared with mock and green fluorescence protein controls. This result suggests that tobacco-based screening may detect false positives. This study is the first to analyze the function of pathogenic candidate molecules of B. xylophilus in natural host pines using exogenous gene expression, which is anticipated to be a powerful tool for investigating the PWD mechanism.

RESUMEN

Pine wilt disease, caused by the pinewood nematode, Bursaphelenchus xylophilus, is one of the world's most serious tree diseases. Although the B. xylophilus whole-genome sequence and comprehensive secretome profile have been determined over the past decade, it remains unclear what molecules are critical in pine wilt disease and govern B. xylophilus virulence in host pine trees. Here, a comparative secretome analysis among four isolates of B. xylophilus with distinct virulence levels was performed to identify virulence determinants. The four candidate virulence determinants of B. xylophilus highly secreted in virulent isolates included lipase (Bx-lip1), glycoside hydrolase family 30 (Bx-GH30), and two C1A family cysteine peptidases (Bx-CAT1 and Bx-CAT2). To validate the quantitative differences in the four potential virulence determinants among virulence groups at the protein level, we used real-time reverse-transcription polymerase chain reaction analysis to investigate these determinants at the transcript level at three time points: pre-inoculation, 3 days after inoculation (dai), and 7 dai into pine seedlings. The transcript levels of Bx-CAT1, Bx-CAT2, and Bx-GH30 were significantly higher in virulent isolates than in avirulent isolates at pre-inoculation and 3 dai. A subsequent leaf-disk assay based on transient overexpression in Nicotiana benthamiana revealed that the GH30 candidate virulent factor caused cell death in the plant. Furthermore, we demonstrated that Bx-CAT2 was involved in nutrient uptake for fungal feeding via soaking-mediated RNA interference. These findings indicate that the secreted proteins Bx-GH30 and Bx-CAT2 contribute to B. xylophilus virulence in host pine trees and may be involved in pine wilt disease.

RESUMEN

Pine wilt disease (PWD) is an infectious disease of pines that typically kills affected trees. The causal pathogen of PWD is the pine wood nematode (PWN), Bursaphelenchus xylophilus. Understanding of the disease has advanced in recent years through the use of a highly sensitive proteomics procedure and whole genome sequence analysis; in combination, these approaches have enabled identification of proteins secreted by PWNs. However, the roles of these proteins during the onset of parasitism have not yet been elucidated. In this study, we used a leaf-disk assay based on transient overexpression in Nicotiana benthamiana to allow functional screening of 10 candidate pathogenic proteins secreted by PWNs. These proteins were selected based on previous secretome and RNA-seq analyses. We found that five molecules induced significant cell death in tobacco plants relative to a GFP-only control. Three of these proteins (Bx-TH1, Bx-TH2, and Bx-CPI) may have a role in molecular mimicry and likely make important contributions to inducing hypersensitive responses in host plants.

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RESUMEN

Phenotypic plasticity is one of the most important strategies used by organisms with low mobility to survive in fluctuating environments. Phenotypic plasticity plays a vital role in nematodes because they have small bodies and lack wings or legs and thus, cannot move far by themselves. Bursaphelenchus xylophilus, the pathogenic nematode species that causes pine wilt disease, experiences fluctuating conditions throughout their life history; i.e., in both the phytophagous and mycetophagous phases. However, whether the functional morphology changes between the life phases of B. xylophilus remains unknown. Our study revealed differences in the ultrastructure of B. xylophilus between the two phases. Well-developed lateral alae and atrophied intestinal microvilli were observed in the phytophagous phase compared with the mycetophagous phase. The ultrastructure in the phytophagous phase was morphologically similar to that at the dauer stage, which enables the larvae to survive in harsh environments. It suggests that the living tree represents a harsh environment for B. xylophilus, and ultrastructural phenotypic plasticity is a key strategy for B. xylophilus to survive in a living tree. In addition, ultrastructural observations of obligate plant-parasitic species closely related to B. xylophilus revealed that B. xylophilus may be in the process of adapting to feed on plant cells.

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