A Bursaphelenchus xylophilus effector BxICD1 inducing plant cell death, concurrently contributes to nematode virulence and migration

Abstract

The migratory endoparasitic phytonematodes Bursaphelenchus xylophilus is the causal agent of pine wilt disease and causes significant economic damage to pine forests in China. Effectors play a key role in the successful parasitism of plants by phytonematodes. In this study, 210 genes obtained by transcriptomics analyses were found to be upregulated in B. xylophilus infecting Pinus massoniana that were not functionally annotated nor reported previously in B. xylophilus infecting P. thunbergii. Among these differentially expressed genes, a novel effector, BxICD1, that could induce cell death in the extracellular space of Nicotiana benthamiana was identified. BxICD1 was upregulated in the early stages of infection, as shown by RT-qPCR analyses. In situ hybridization analysis showed that BxICD1 was expressed in the esophageal gland of nematodes. The yeast signal sequence trap system indicated that BxICD1 possessed an N-terminal signal peptide with secretion functionality. Using an Agrobacterium-mediated transient expression system, it was demonstrated that the cell death-inducing activity of BxICD1 was dependent on N. benthamiana brassinosteroid-insensitive 1-associated kinase 1 (NbBAK1). Finally, BxICD1 contributed to B. xylophilus virulence and migration in host pine trees, as demonstrated by RNAi silencing assays. These findings indicate that BxICD1 both induces plant cell death and also contributes to nematode virulence and migration in P. massonian.

Keywords: Bursaphelenchus xylophilus; Pinus massoniana; effector; plant cell death activation; transcriptome.

Figure 1

Differentially expressed genes at the mycetophagous and phytophagous stages of Bursaphelenchus xylophilus. (A) Heatmap of differentially expressed genes at the mycetophagous and phytophagous stages. (B) Validation of differentially expressed genes (DEGs). Validation of seven DEGs identified in the RNA-seq dataset by quantitative reverse transcription PCR. The relative expression levels were calculated using the 2^−ΔΔCt method; data represent shown as the mean ± standard deviation (SD) of three replicated; the experiments were performed twice with similar results. Bx_Pe1, Bx_Pe2, and Bx_Pe3 correspond to the three B. xylophilus samples from the mycetophagous stage; Bx_Pm1, Bx_Pm2, and Bx_Pm3 correspond to the three B. xylophilus samples from the phytophagous stage.

Figure 2

Screening of candidate effectors from Bursaphelenchus xylophilus capable of inducing plant cell death. (A) Schematic diagram of the location of Agrobacterium tumefaciens-injected tobacco leaves. INF1 is Phytophthora infestans elicitin, a known apoplastic protein that induces cell death in Nicotiana benthamiana, used as a positive control. eGFP, an enhanced green fluorescent protein that cannot induce cell death, was used as a negative control. “Effector” represents candidate effectors with the native signal peptide; “effector^Δsp” represents candidate effectors without the native signal peptide. (B) Ten candidate effectors were expressed in N. benthamiana leaves. The infiltration assay was performed thrice, and five different plants with two inoculated leaves were used in each assay. Similar results were obtained from all experiments.

Figure 3

Localization of BxICD1 mRNA and secretion function analysis of BxICD1 signal peptide. (A) Localization of BxICD1 mRNA in esophageal gland cells of Bursaphelenchus xylophilus by in-situ hybridization. Fixed nematodes were hybridized with digoxigenin-labeled antisense (left) and sense (right) cDNA probes from BxICD1. S, stylet; M, median bulb; GC, esophageal gland. Scale bars = 50 µm (B) Secretion function analysis of the BxICD1 signal peptide. The predicted signal peptide of BxICD1 was cloned into the yeast vector pSUC2 to generate pSUC2:SP^BxICD1-invertase constructs, and pSUC2: SP^Avr1b-Invertase was used as a positive control. YTK12 can grow on YPDA plates. CMD-W media was used to ensure the expression of pSUC2-derived plasmids. Invertase can reduce 2,3,5-Triphenyltetrazolium Chloride (TTC) to insoluble red-colored 1,3,5-Triphenylformazan (TPF).

Figure 4

Apoplastic localization of BxICD1 is required for the induction of cell death in Nicotiana benthamiana. (A) Agrobacterium strain GV3101 carrying the fusion constructs BxICD1:eGFP, BxICD1^Δsp:eGFP and the eGFP control were infiltrated in Nicotiana benthamiana leaves for transient expression analysis. (B) N. benthamiana leaves expressing BxICD1:eGFP, BxICD1:eGFP^Δsp and eGFP were treated with 30% glycerol for plasmolysis. GFP signals were observed at 3 days after infiltration. The red asterisks indicate the apoplast region. Scale bars=50 µm. (C) BxICD1-triggered cell death in N. benthamiana. Agrobacterium strain GV3101 carrying the constructs BxICD1:eGFP, BxICD1^Δsp:eGFP, eGFP, and INF1 were infiltrated in N. benthamiana leaves for transient expression analysis. The leaf phenotypes were observed after 3 days. (D) Quantification of cell death by measuring electrolyte leakage in N. benthamiana leaves at 3 days post-infiltration with constructs encoding the indicated proteins. Data represent the mean of three repeats ± SD. Three independent experiments were performed with similar results, with three technical replicates for each reaction. (E) Western blot analysis was performed to confirmed the expression of proteins in N. benthamiana. Ponceau S staining of RuBisCO was used as indicate the protein loading control.

Figure 5

BxICD1-triggered cell death depends on NbBAK1. (A) NbBAK1, but not NbSOBIR1, is required for BxICD1-triggered cell death in Nicotiana benthamiana. N. benthamiana plants were subjected to VIGS by inoculation with TRV constructs (TRV:eGFP, TRV : NbBAK1, or TRV : NbSOBIR1). Three weeks after inoculation, BxICD1, INF1, the empty vector (EV), and eGFP were transiently expressed in the gene-silenced leaves and photographed after 3 d. The infiltration experiment was performed thrice, with three different plants with three inoculated leaves were used in each assay. All experiments were performed with similar results. (B) Western blot analysis confirmed the expression of BxICD1 protein in gene-silenced plants. Ponceau S staining of RuBisCO was used as indicate the protein loading control. (C, D) The relative expression levels of NbBAK1 and NbSOBIR1 in gene-silenced plant were determined by RT-qPCR. Data represent the mean of three replicates ± SD. Three independent experiments were performed with similar results. Asterisks indicate significant differences (P<0.05).

Figure 6

BxICD1 contributes to the virulence and migration of Bursaphelenchus xylophilus. (A) BxICD1 silencing efficiency after treatment with BxICD1 dsRNA in B. xylophilus. Asterisks indicate significant differences (P<0.05). ns indicates no significant differences (P≥0.05). (B) Representative photographs of Pinus massoniana seedlings at 12 days and 20 days post-inoculation. dseGFP, dsBxICD1 and CK correspond to nematodes inoculated in eGFP-, BxICD1- and non-dsRNA solution, respectively. (C) The morbidity of Pinus massoniana seedlings inoculated with dseGFP-, dsBxICD1-, and non-dsRNA solution -treated nematodes. (D) The disease severity index of P. massoniana seedlings inoculated with dseGFP-, dsBxICD1-, and non-dsRNA solution-treated nematodes. (E) Schematic depiction of nematode inoculation and sampling. (F) The number of nematodes in Area A, Area B, and Area C wood sections under three different treatments. “ns” indicates no sigificant difference (p≥0.05).