The key molecular pattern BxCDP1 of Bursaphelenchus xylophilus induces plant immunity and enhances plant defense response via two small peptide regions

Abstract

The migratory plant-parasitic nematode Bursaphelenchus xylophilus is the pathogen of the pine wilt disease (PWD), causing serious damage to pine forests in China. During the process of plant resistance to multiple pathogens, plant immunity plays a key role. In this current study, the pathogen-associated molecular pattern (PAMP) BxCDP1 in B. xylophilus has been identified, but the host target protein of BxCDP1 and its key amino acid region inducing the plant immunity have yet to be elucidated. We found that BxCDP1 could trigger superoxide production, H₂O₂ production, and callose deposits. A RING-H2 finger protein 1 (RHF1) of Pinus thunbergii was screened and characterized as a target protein of BxCDP1 by yeast two-hybrid and co-immunoprecipitation (Co-IP). Moreover, two peptides (namely M9 and M16) proved to be key regions of BxCDP1 to induce PAMP-triggered immunity (PTI) in Nicotiana benthamiana, which also induced the expression of pathogenesis-related (PR) genes (PtPR-3, PtPR-4, and PtPR-5) in P. thunbergii and enhanced the resistance of the host to B. xylophilus. These results indicate that BxCDP1 plays a critical role in the interaction between B. xylophilus and P. thunbergii, and both peptides M9 and M16 have the potential to be developed and utilized as immune inducers of pines against B. xylophilus in future.

Keywords: Bursaphelenchus xylophilus; Pinus thunbergii; immunity induction; key peptides; protein interaction.

FIGURE 1

BxCDP1 induces the accumulation of superoxide and hydrogen peroxide and callose deposits in Nicotiana benthamiana. Representative N. benthamiana leaves stained using sodium azide after 3 h inoculation with purified GFP protein (A) and BxCDP1 protein (B). Representative N. benthamiana leaves stained using 3,3’-diaminobenzidine (DAB) after 3 h inoculation with purified GFP protein (C) and BxCDP1 protein (D). Representative N. benthamiana leaves stained using aniline blue after 3 h inoculation with purified GFP protein (E) and BxCDP1 protein (F). These infiltration assays were, respectively, performed three times, and three different plants with three inoculated leaves were used in each assay. (G) Quantification using ImageJ software. The relative staining area of DAB or NBT by purified BxCDP1 protein treated in N. benthamiana was calculated. The staining area of DAB or NBT by GFP treated was set as 1. (H) The number of callose deposits per microscopic field was quantitated using ImageJ software. The relative number of callose deposits by purified BxCDP1 protein treated in N. benthamiana was counted. The number of callose deposits by GFP treated was set as 1. Data are the means, and the error bars represent ± SD from three biological replicates. Different letters on top of the bars indicate statistically significant differences (p < 0.05, t-test) as measured by Duncan’s multiple range test.

FIGURE 2

BxCDP1 interacts with Pinus thunbergii protein PtRHF1. (A) BxCDP1 interacts with PtRHF1 in yeast. Yeast strain Y2H Gold co-carrying pGBKT7-BxCDP1 and pGADT7-PtRHF1 was grown on SD/-Trp/-Leu and the selective medium SD/-Trp/-Leu/-Ade/-His+X-α-gal+AbA+1mM 3-AT. (B) BxCDP1 interacts with PtRHF1 in vivo. Co-immunoprecipitation (Co-IP) was performed on extracts of Nicotiana benthamiana leaves expressing both BxCDP1-HA and PtRHF1-GFP. BxCDP1-HA and PtRHF1-GFP were detected using anti-HA and anti-GFP antibodies by Western blot. The immune complexes were pulled down using anti-HA agarose beads.

FIGURE 3

Relative transcript levels of PtRHF1 in Pinus thunbergii. (A) The PtRHF1 was upregulated at the early stages of Bursaphelenchus xylophilus infection. The P. thunbergii inoculated without B. xylophilus was used as a control. (B) The relative expression of PtRHF1 was induced when purified BxCDP1 protein was inoculated into P. thunbergii. The P. thunbergii inoculated with purified GFP protein was used as a control. Data are the means, and the error bars represent ± SD from three biological replicates. Different letters on top of the bars indicate statistically significant differences (p < 0.05, t-test) as measured by Duncan’s multiple range test.

FIGURE 4

Analysis of domain by constructing deletion mutants of BxCDP1. The representative Nicotiana benthamiana leaves at 7 days after agroinfiltration carrying BxCDP1 and its 19 deletion mutants. M9, M15, and M16 were the shortest regions that could trigger cell death in N. benthamiana. The infiltration assay was performed three times and three different N. benthamiana with three inoculated leaves were used in each assay.

FIGURE 5

Western blot analysis of proteins from Nicotiana benthamiana leaves transiently expressing target proteins fused with anti-red fluorescent protein (anti-RFP) tags. Protein loading is shown by Ponceau S staining of RuBisCO.

FIGURE 6

Detection of the relative expression of PAMP-triggered immunity (PTI) marker gene after injecting with three key peptides of BxCDP1 in Nicotiana benthamiana. (A–C) Transcriptional upregulation of N. benthamiana PTI marker genes triggered by 300 nM synthesized peptides M9, M15, and M16 in N. benthamiana. The N. benthamiana leaves infiltrated with synthesized peptide M14 were used as a control. The assay was conducted three times, and three different plants with three inoculated leaves were used in each assay. Data are the means, and the error bars represent ± SD from three biological replicates. Different letters on top of the bars indicate statistically significant differences (p < 0.05, t-test) as measured by Duncan’s multiple range test.

FIGURE 7

Relative transcript levels of pathogenesis-related (PR) genes in Pinus thunbergii inoculated with three synthesized peptides and Bursaphelenchus xylophilus. (A) The relative expression of PtPR-3 was detected by RT-qPCR. (B) The relative expression of PtPR-4 was detected by RT-qPCR. (C) The relative expression of PtPR-5 was detected by RT-qPCR. The P. thunbergii seedlings were treated with three synthesized peptides M9, M15, and M16 for 4 h and then inoculated with B. xylophilus. After 6 h, 2 cm stems in the length of P. thunbergii seedlings were used for RNA extraction, and the relative expressions of PtPR-3, PtPR-4, and PtPR-5 were detected. The P. thunbergii seedlings inoculated with B. xylophilus after inoculating with synthesized peptide M14 were used as the control. The inoculation assay was repeated three times, and in each assay, three different seedlings for each treatment were used. Data are the means, and the error bars represent ± SD from three independent experiments. Different letters on top of the bars indicate statistically significant differences (p < 0.05, t-test) as measured by Duncan’s multiple range test.

FIGURE 8

Peptides M9 and M16 of BxCDP1 can promote the resistance of Pinus thunbergii to Bursaphelenchus xylophilus. (A) The infection rates of P. thunbergii seedlings under four different treatments. (B) The disease severity index of P. thunbergii seedlings under four different treatments. The P. thunbergii seedlings were treated with three synthesized peptides M9, M15, and M16 for 4 h and then inoculated with 1500 B. xylophilus. The P. thunbergii seedlings inoculated with 1500 B. xylophilus after treating with synthesized peptide M14 for 4 h were used as control. Data are the means of three independent inoculation experiments and error bars (±SD) from three independent inoculation experiments. Different capital letters above the bars indicate significant differences between inoculation times in the same treatments (p < 0.05, t-test), as measured by Duncan’s multiple range test; different lowercase letters above the bars indicate significant differences between different treatments at the same inoculation time (p < 0.05, t-test), as measured by Duncan’s multiple range test. The infection experiment was repeated three times with three replicate P. thunbergii seedlings per treatment. (C) Representative photographs of P. thunbergii at 1 day, 12 days, and 15 days post-inoculation. The B. xylophilus infection assay was conducted three times.