Virulence Biomarkers of Bursaphelenchus xylophilus: A Proteomic Approach

Abstract

The pinewood nematode (PWN), Bursaphelenchus xylophilus, one of the most serious forest pests worldwide, is considered the causal agent of the pine wilt disease (PWD). The main host species belong to the genus Pinus, and a variation in the susceptibility of several pine species to PWN infection is well-known. It is also recognized that there is variation in the virulence among B. xylophilus isolates. In the present study, we applied a quantitative mass spectrometry-based proteomics approach to perform a deep characterization of proteomic changes across two B. xylophilus isolates with different virulence from different hosts and geographical origins. A total of 1,456 proteins were quantified and compared in the two isolates secretomes, and a total of 2,741 proteins were quantified and compared in the nematode proteomes in pine tree extract and fungus stimuli conditions. From the proteomic analyses, a group of proteins was selected and identified as potential virulence biomarkers and shed light on putative most pathogenic proteins of this plant-parasitic nematode. Proteomic data are available via ProteomeXchange with identifier PXD029377.

Keywords: pine trees; pine wilt disease; pinewood nematode; plant–nematode interactions; proteomics; secretome; virulence.

FIGURE 1

Quantitative analysis of secreted proteins of Bursaphelenchus xylophilus isolates. Volcano plot (A) and pie chart (B) reflecting the results from the statistical analysis of the 1,456 proteins quantified among the secretomes of B. xylophilus virulent (Sec_BxV) and avirulent (Sec_BxAv) isolates. Statistical analysis was performed by the Mann-Whitney U test, and statistical significance was considered for p < 0.05.

FIGURE 2

Gene ontology (GO) enrichment analysis of the 656 increased proteins in Bursaphelenchus xylophilus avirulent secretome (Sec_BxAv). An enriched bar chart reflecting GO enrichment analysis of the 656 proteins increased in Sec_BxAv performed against all the 1,456 quantified proteins in the secretomes using the statistical Fisher’s Exact Test with p < 0.05.

FIGURE 3

Number of differentially expressed proteins from the 2,741 proteins quantified among the proteomes of virulent Bursaphelenchus xylophilus isolate (BxV) and avirulent isolate (BxAv) under fungal (fungus) or under pine extract (PE) stimulus. Statistical analysis was performed by a Kruskal–Wallis test followed by the Dunn’s test of multiple comparisons, and statistical significance was considered for an adjusted p < 0.05.

FIGURE 4

Quantitative analysis of whole nematodes proteins and gene ontology enrichment analysis of differentially expressed proteins in Bursaphelenchus xylophilus isolates under fungal stimulus. Volcano plot (A) and pie chart (B) reflecting the results from the statistical analysis of the proteomes of B. xylophilus virulent isolate (BxV_Fungus) and avirulent isolate (BxAv_Fungus) with statistical analysis of the 1,892 proteins quantified and differentially expressed (p < 0.05), considering all conditions, performed by the Kruskal–Wallis test, followed by the Dunn’s test for multiple comparisons. An enriched bar chart reflecting GO enrichment analysis of the 959 proteins increased in BxAv_Fungus (C) and 532 proteins increased in BxV_Fungus (D) against all the 2,741 quantified proteins in the proteomes using the statistical Fisher’s Exact Test (p < 0.05).

FIGURE 5

Quantitative analysis of whole nematodes proteins and gene ontology enrichment analysis of differentially expressed proteins in Bursaphelenchus xylophilus isolates under pine extract (PE) stimulus. Volcano plot (A) and pie chart (B) reflecting the results from the statistical analysis of the proteomes of B. xylophilus virulent isolate (BxV_PE) and avirulent isolate (BxAv_PE) with statistical analysis of the 1,892 proteins quantified and differentially expressed (p < 0.05), considering all conditions, performed by the Kruskal–Wallis test followed by the Dunn’s test for multiple comparisons. An enriched bar chart reflecting GO enrichment analysis of the 571 proteins increased in BxAv_PE (C) and 473 proteins increased in BxV_PE (D) against all the 2,741 quantified proteins in the proteomes using the statistical Fisher’s Exact Test (p < 0.05).

FIGURE 6

Increased proteins between each pair of conditions. A Venn diagram showing the number of unique and overlapping increased proteins between the proteomes of Bursaphelenchus xylophilus virulent isolate (BxV) and avirulent isolate (BxAv) under fungus (BxV_Fungus/BxAv_Fungus) and BxV and BxAv under pine extract (PE) (BxV_PE/BxAv_PE).

FIGURE 7

Quantitative analysis of selected 146 nematode proteins and gene ontology (GO) enrichment analysis of 30 differentially expressed proteins in Bursaphelenchus xylophilus isolates under pine extract (PE) stimulus. Heatmap of a normalized protein level in the proteomes of B. xylophilus in the four conditions: avirulent isolate under fungal stimulus (BxAv_Fungus) and under PE stimulus (BxAv_PE) and virulent isolate under fungal stimulus (BxV_Fungus) and under PE stimulus (BxV_PE) (A). Volcano plot reflecting the results from the statistical analysis of the proteomes of BxV_PE and BxV_Fungus performed by the Kruskal–Wallis test followed by the Dunn’s test for multiple comparisons and with statistical significance considered for a p < 0.05 (B). Gene Ontology distribution of the 30 selected proteins increased in virulent proteome (BxV) based on functional annotation with biological process (BP), molecular function (MF), and cellular component (CC) GO terms (C).