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. 2023 Jul 13;24(9):1033–1046. doi: 10.1111/mpp.13334

The Bursaphelenchus xylophilus candidate effector BxLip‐3 targets the class I chitinases to suppress immunity in pine

Yi‐Jun Qiu 1,2, Xiao‐Qin Wu 1,2,✉, Tong‐Yue Wen 1,2, Long‐Jiao Hu 1,2,3, Lin Rui 1,2, Yan Zhang 1,2, Jian‐Ren Ye 1,2,✉
PMCID: PMC10423331  PMID: 37448165

Abstract

Lipase is involved in lipid hydrolysis, which is related to nematodes' energy reserves and stress resistance. However, the role of lipases in Bursaphelenchus xylophilus, a notorious plant‐parasitic nematode responsible for severe damage to pine forest ecosystems, remains largely obscure. Here, we characterized a class III lipase as a candidate effector and named it BxLip‐3. It was transcriptionally up‐regulated in the parasitic stages of B. xylophilus and specifically expressed in the oesophageal gland cells and the intestine. In addition, BxLip‐3 suppressed cell death triggered by the pathogen‐associated molecular patterns PsXEG1 and BxCDP1 in Nicotiana benthamiana, and its Lipase‐3 domain is essential for immunosuppression. Silencing of the BxLip‐3 gene resulted in a delay in disease onset and increased the activity of antioxidant enzymes and the expression of pathogenesis‐related (PR) genes. Plant chitinases are thought to be PR proteins involved in the defence system against pathogen attack. Using yeast two‐hybrid and co‐immunoprecipitation assays, we identified two class I chitinases in Pinus thunbergii, PtChia1‐3 and PtChia1‐4, as targets of BxLip‐3. The expression of these two chitinases was up‐regulated during B. xylophilus inoculation and inhibited by BxLip‐3. Overall, this study illustrated that BxLip‐3 is a crucial virulence factor that plays a critical role in the interaction between B. xylophilus and host pine.

Keywords: Bursaphelenchus xylophilus, class III lipase, class I chitinase, effector, Pinus thunbergii

The Bursaphelenchus xylophilus candidate effector BxLip‐3 is a crucial virulence factor that interacts with two class I chitinases, PtChia1‐3 and PtChia1‐4, in Pinus thunbergii to attenuate host defences.

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1. INTRODUCTION

Plants have evolved a network of immune responses against pathogen infection. Plant basal immunity relies on the activation of plant pattern recognition receptors by pathogen‐associated molecular patterns (PAMPs) to trigger PAMP‐triggered immunity (PTI) to suppress pathogen proliferation, including reactive oxygen species (ROS) burst, callose deposition, and induction of defence‐related gene expression (Hammond‐Kosack & Jones, 1996; Jones & Dangl, 2006; Lee & Hwang, 2005; Spoel & Dong, 2012). In turn, host‐adapted pathogens are equipped with diverse virulence effectors to modify host physiology and manipulate host immunity, thus enhancing pathogen proliferation. For example, the Sclerotinia sclerotiorum effector SsPINE1 targets the plant polygalacturonase‐inhibiting protein (PGIP) to enhance the degradation of pectin, a component of plant cell walls, resulting in the enhancement of fungal parasitism (Wei et al., 2022); the Phytophthora capsici effector PcAvh103 specifically interacts with the lipase domain of Enhanced Disease Susceptibility 1 (EDS1) to disrupt the EDS1–Phytoalexin Deficient 4 (PAD4) immune signalling pathway, suppressing plant immunity (Li, Wang, et al., 2020); and the Meloidogyne javanica effector Mj2G02 interferes with jasmonic acid (JA) signalling by manipulating several JA‐related genes to suppress cell death and promote parasitism in the host (Song et al., 2021). Therefore, pathogens need to evolve unceasingly to diversify effectors for further parasitism.

Pine trees, which are widely used in windbreak planting, sand erosion prevention, and soil and water conservation, are of great significance to the carbon cycle and the ecological environment (Shinya et al., 2013). Additionally, as fast‐growing tree crops in the Northern Hemisphere, they are important sources of forest products, contributing to forest construction and the economy (Yu et al., 2022). However, pine trees now face a catastrophic situation because of the pine wood nematode (PWN) Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD). PWNs can cause death in most Pinus species on a massive scale, resulting in severe environmental destruction and enormous economic losses in eastern Asia and Europe, especially in China, Japan, and Korea (Futai, 2013; Mamiya & Kiyohara, 1972; Mota et al., 1999; Robertson et al., 2011). They are migratory endoparasitic nematodes, which have a complex life cycle that includes phytophagous and mycophagous stages (Futai, 2013). At the phytophagous stage, the nematode feeds on pine parenchyma cells, leading to cell death. After the death of the host, the nematode feeds on fungi. The spread of nematodes depends on the insect vector Monochamus spp. (Kobayashi et al., 2003). Every year, mature beetles lay their eggs in dead trees. The following year, the beetles emerge from the dead pine trees, carrying PWNs in their respiratory system, and feed on the young branches of healthy pine trees (Futai, 2013). These feeding wounds provide entry portals for the nematode. Once inside the pine cortex, the nematode feeds on host parenchyma cells and spreads to the whole trunk through the tree's vascular system and resin canals (Feng et al., 2022; Mamiya, 2012). To defend against nematode infection in the early stages, the tree shows a strong defence response and releases ROS, polyphenolic compounds, terpenoids, and lipid peroxides (Fukuda, 1997). Like other pathogenic microorganisms, B. xylophilus secretes various effectors to overcome plant immunity for successful host colonization. Several effectors from B. xylophilus are capable of suppressing plant defence responses. They can suppress programmed cell death (PCD) triggered by different elicitors in Nicotiana benthamiana and regulate the expression of pathogenesis‐related (PR) genes in Pinus thunbergii (Hu, Wu, Wen, Qiu, et al., 2022; Li, Hu, et al., 2020; Wen et al., 2021; Zhang et al., 2022). Nevertheless, many important aspects of the other immunosuppression mechanisms of the nematode are yet unknown. Identifying more effector proteins in B. xylophilus and exploring the mechanisms of defence suppression by these effectors could be an effective way to control this destructive pest.

Chitinase is a glycosyl hydrolase enzyme that can hydrolyse the β‐1,4‐glycosidic bonds of chitin randomly. Chitin is widely distributed in nature, occurring in plants, animals, fungi, and bacteria (Hou et al., 2019; Shih et al., 2001). Chitinases are classified into seven biochemical classes (I–VII) based on the presence or absence of conserved chitin‐binding (CBD) and chitinolytic domains and the variable hinge domain (Grover, 2012; Peery et al., 2021). Most plant chitinases belong to class I, II, and IV chitinases, which have homologous catalytic domains and correspond to glycosyl hydrolase family 19 (Han et al., 2016; Shakhbazov & Kartel, 2008). These chitinases are thought to be involved in the defence system against pathogen attack as PR proteins in higher plants (Gomez et al., 2002). Evidence is mounting that chitinases enhance resistance to pathogens in pines (Davis et al., 2002; Liu et al., 2011). Furthermore, chitinases extracted from Lysobacter capsici decrease hatching and cause damage to eggshells of Meloidogyne spp. (Lee et al., 2014). A study of the Trichoderma harzianum chitinase demonstrated that enhancement of fungal chitinase activity promoted fungal infection of root‐knot nematode eggs (Sharon et al., 2001). However, little is known about pine chitinases. To the best of our knowledge, there are no reports on chitinase in P. thunbergii, nor on the role of chitinase in the interaction between pine and B. xylophilus.

In a previous transcriptome analysis, multiple B. xylophilus candidate effectors were identified, one of which is a lipase (Hu et al., 2021). Recent research indicated that lipase may serve as a virulence biomarker, as its levels were significantly increased in the secretome of a virulent isolate compared to an avirulent isolate (Cardoso et al., 2022). However, its precise function remains to be elucidated. Here, we focused on this candidate effector and named it BxLip‐3. It was notably up‐regulated upon infection and could suppress PCD triggered by PsXEG1 and BxCDP1. In particular, the immunosuppression function of BxLip‐3 relies on its Lipase‐3 domain. Using RNA interference (RNAi) assays, we verified the significant role of BxLip‐3 in B. xylophilus infection. BxLip‐3 represses the activity of antioxidant enzymes and the relative expression of PR genes to suppress plant immunity. Moreover, we report for the first time that BxLip‐3 interacts with two class I chitinases, namely PtChia1‐3 and PtChia1‐4, and regulates their expression. Collectively, we show that BxLip‐3 is a vital effector that is required for the virulence of B. xylophilus.

2. RESULTS

2.1. Identification of the B. xylophilus candidate effector BxLip‐3

The BxLip‐3 (BXY_0824600) gene sequence was identified from the B. xylophilus transcriptome (accession number PRJNA397001) at the early stages of infection (Hu et al., 2021). The BxLip‐3 cDNA encodes a 285‐amino‐acid sequence with a 15‐amino‐acid N‐terminal signal peptide (SP) without transmembrane domains and with a class III lipase domain. There are many B. xylophilus isolates, some with strong virulence and some with weak virulence. To explore BxLip‐3 sequence polymorphisms, we sequenced the BxLip‐3 coding region from three highly virulent B. xylophilus isolates (AMA3, AA3, and ZL1) and three weakly virulent isolates (USA, HE2, and YW4) (Ding et al., 2016). The results display that BxLip‐3 has no polymorphism among the six strains, demonstrating that BxLip‐3 is highly conserved among different isolates (Figure S1).

2.2. BxLip‐3 is up‐regulated during parasitic stages and predominantly expressed in the dorsal gland and intestine

The transcript levels of BxLip‐3 were determined by reverse transcription–quantitative PCR (RT‐qPCR) at nine time points after nematode inoculation. The mycophagous stage was set as 0 h, and the BxLip‐3 transcript level at this stage was set as 1 as a reference to calculate the relative expression in other stages. The results showed that BxLip‐3 was up‐regulated after nematode inoculation and reached a peak at 5 days postinoculation (dpi) (Figure 1a). Then, the expression of BxLip‐3 decreased, and it remained relatively low even though pine seedlings began to show wilting symptoms and chlorosis of the needles (15 dpi) or PWD was severe (20 dpi) (Figure S2). These findings confirm that BxLip‐3 plays a vital role in the nematode parasitic stages.

FIGURE 1.

FIGURE 1

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Expression pattern of BxLip‐3. (a) The relative expression level of BxLip‐3 at nine time points after Bursaphelenchus xylophilus inoculation, as determined by reverse transcription–quantitative PCR (RT‐qPCR) analysis. Nematodes were collected from the whole Pinus thunbergii seedlings inoculated with B. xylophilus (isolate AMA3) at different time points. The relative expression level of BxLip‐3 was calculated by the comparative threshold method. The RT‐qPCR values were normalized to the transcript level of Actin. Values represent the mean ± standard deviation of three independent biological samples. Different letters indicate statistically significant differences as determined using Duncan's multiple range test (p < 0.05). (b) Expression of BxLip‐3 in the dorsal gland (DG) and intestine as determined by in situ hybridization. Nematodes were collected from whole seedlings 12 h after nematode inoculation. Fixed nematodes were hybridized with digoxigenin (DIG)‐labelled BxLip‐3 sense and antisense cDNA probes. M, median bulb. Scale bars = 20 μm.

To confirm the tissue‐specific expression of BxLip‐3 in B. xylophilus, we collected PWNs from whole seedlings using the Baermann funnel technique 12 h after nematode inoculation into pine stems and used in situ hybridization assays. Using digoxigenin (DIG)‐labelled antisense cDNA probes, we found that BxLip‐3 was specifically expressed in the dorsal gland and intestine of B. xylophilus (Figure 1b). These findings suggest that BxLip‐3 can be secreted into plant tissues and it is vital in the parasitic stages of B. xylophilus.

2.3. BxLip‐3 suppresses PCD triggered by PsXEG1 and BxCDP1 in Nicotiana benthamiana

To verify the immunosuppressive ability of BxLip‐3, we infiltrated Agrobacterium tumefaciens strains carrying BxLip‐3 without the SP (BxLip‐3ΔSP) into N. benthamiana leaves 16 h before infiltration with Agrobacterium strains carrying the Phytophthora sojae PAMP PsXEG1 and the B. xylophilus novel molecular pattern BxCDP1. Green fluorescent protein (GFP) was used as a negative control. We found that full‐length BxLip‐3 could not suppress cell death, while BxLip‐3ΔSP could suppress PCD triggered by PsXEG1 and BxCDP1 in N. benthamiana (Figure 2a). GFP did not suppress PsXEG1‐ and BxCDP1‐triggered necrosis‐like symptoms in N. benthamiana. Cell death is generally accompanied by electrolyte leakage, and therefore electrolyte leakage is often measured to quantify the degree of cell necrosis (Mittler et al., 1999). Electrolyte leakage in N. benthamiana triggered by PsXEG1 and BxCDP1 was markedly reduced in the presence of BxLip‐3ΔSP, further indicating that BxLip‐3ΔSP suppresses PCD (Figure 2b). The expression of these proteins (GFP, BxLip‐3, BxLip‐3ΔSP, PsXEG1, and BxCDP1) in N. benthamiana leaves was validated by western blot analysis (Figure 2c). Moreover, a previous study showed that N. benthamiana PTI marker genes (NbAcre31, NbPTI5, and NbCyp71D20) were up‐regulated by treatment with BxCDP1 in response to biotic stresses (Hu et al., 2020). To assess whether BxLip‐3 (without the SP) suppresses immune responses other than cell death, we measured whether PTI marker genes triggered by BxCDP1 could be suppressed in N. benthamiana by transiently expressing BxLip‐3. The BxLip‐3rec protein was produced in Escherichia coli, and a peptide (His‐tag) encoded by the empty vector (EV) was used as a negative control. The purified recombinant proteins were assessed by western blotting to confirm successful purification (Figure S3a). RT‐qPCR analysis showed that BxLip‐3 reduced the relative expression level of NbAcre31 and NbCyp71D20 (Figure S3b). These results show that BxLip‐3 can suppress PCD in N. benthamiana.

FIGURE 2.

FIGURE 2

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BxLip‐3 suppresses PsXEG1‐ and BxCDP1‐triggered cell death in Nicotiana benthamiana. (a) Co‐expression of BxLip‐3 (without signal peptide) suppressed PsXEG1‐ and BxCDP1‐triggered cell death in N. benthamiana. Pictures were taken at 5 days postinoculation. The experiments were repeated at least three times with similar results. (b) Quantification of suppression of cell death by measuring electrolyte leakage in N. benthamiana. GFP, green fluorescent protein. The data shown are from three independent experiments. Different letters indicate statistically significant differences as determined using Duncan's multiple range test (p < 0.05). (c) Immunoblot analysis of proteins from N. benthamiana leaves transiently expressing target proteins. Protein loading is indicated by Ponceau S staining of RuBisCO.

2.4. Lipase‐3 domain of BxLip‐3 is sufficient for immunosuppression function in N. benthamiana

To determine whether small peptide regions in BxLip‐3 inhibit PsXEG1‐induced cell death, we transiently expressed eight deletion mutants of BxLip‐3 16 h before infiltrating PsXEG1 into N. benthamiana. The results showed that mutants M1–M6 retained their PCD suppression activity (Figure 3a). In particular, the peptide region BxLip‐384–200 (BxLip‐3‐M6) is a relatively small peptide region to suppress cell death. Further truncation of peptide regions showed that mutants M7 and M8 had completely lost their immunosuppressive ability. We found that the observed cell death symptoms were highly correlated with the degree of electrolyte leakage in N. benthamiana leaves. The peptide region BxLip‐384–220 (BxLip‐3‐M5) is the Lipase‐3 domain of BxLip‐3. Interestingly, the electrolyte leakage of N. benthamiana leaves coexpressing PsXEG1 and BxLip‐3‐M5 was significantly lower than that of other leaves, indicating that region M5 has an even stronger ability to suppress PsXEG1‐induced cell death than the smaller mutant M6 (Figure 3b). Our findings suggest that the Lipase‐3 domain of BxLip‐3 is crucial to the suppression function.

FIGURE 3.

FIGURE 3

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The Lipase‐3 domain of BxLip‐3 is required to retain the immunosuppression ability in Nicotiana benthamiana. (a) Structural analysis of the region of BxLip‐3 required for suppression. The BxLip‐3 protein and mutants are shown as horizontal blue bars, with the signal peptide (SP) in yellow and the Lipase‐3 domain in green. Agrobacterium‐mediated transient expression of full‐length BxLip‐3, BxLip‐3ΔSP, and BxLip‐3 mutants (M1–M8) was performed 16 h before infiltration of the elicitor PsXEG1 into N. benthamiana leaves. Photographs were taken 5 days after inoculation. (b) Quantification of suppression of cell death by measuring electrolyte leakage in N. benthamiana. BxLip‐3 and its mutants were infiltrated into N. benthamiana leaves 16 h before infiltration of the elicitor PsXEG1. Electrolyte leakage from leaf discs was measured at 5 days postinfiltration. Coexpression of green fluorescent protein (GFP) was used as a negative control. The data are shown are the mean ± standard deviation from three independent experiments. Different letters indicate statistically significant differences (p < 0.05), as determined by Duncan's multiple range test.

2.5. BxLip‐3 contributes to B. xylophilus virulence

To assess the role of BxLip‐3 in the virulence of B. xylophilus, we used RNAi methods to silence the BxLip‐3 gene in the AMA3 isolate. We inoculated pines with treated nematodes 48 h after soaking them in double‐stranded RNA (dsRNA) solution. RT‐qPCR analysis confirmed that BxLip‐3 was successfully silenced, with transcript levels decreased by approximately 80% (Figure S4a). Most needles in the control groups of pines inoculated with dsGFP‐treated PWNs and wild‐type (WT) PWNs showed needle leaf yellowing, with the morbidity reaching 70% at 15 dpi in both groups (Figure 4a,b). Notably, at the same time, symptoms were significantly reduced in the pines inoculated with dsBxLip‐3‐treated nematodes, and the morbidity of this group was only 40%. The disease severity index also proved that silencing of BxLip‐3 could delay disease onset (Figure 4c). The Botrytis cinerea mycelium on the potato dextrose agar (PDA) plates used for culturing the PWNs was observed, and the PWNs on the B. cinerea plates and in P. thunbergii seedlings were counted. The results demonstrated that all treated PWNs showed similar reproduction and feeding rates (Figure S4b–d). These data indicate that BxLip‐3 is required for the virulence of B. xylophilus. We further evaluated whether BxLip‐3 silencing in B. xylophilus influences the pine defence response. We found that the activity of the antioxidant enzymes peroxidase (POD) and catalase (CAT) in pine increased when BxLip‐3 was silenced (Figure 4d,e). Likewise, we analysed the relative expression of PR genes 12 h after PWN inoculation using RT‐qPCR (Figure 4f,g). PtPR‐3 (encoding a class I chitinase) and PtPR‐6 were markedly up‐regulated in P. thunbergii when BxLip‐3 was silenced. These results imply that BxLip‐3 plays a significant role in suppressing immune responses in P. thunbergii.

FIGURE 4.

FIGURE 4

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BxLip‐3 contributes to Bursaphelenchus xylophilus virulence. (a) Inoculation assay of pine seedlings. Based on the colour of the needles, the morbidity degree of the Pinus thunbergii seedlings was different. At 15 days postinoculation (dpi), eight seedlings inoculated with wild‐type (WT) or dsGFP‐treated B. xylophilus turned yellow or brown, and five seedlings inoculated with dsBxLip‐3‐treated B. xylophilus turned yellow. (b, c) The infection rates and the disease severity index of P. thunbergii seedlings under three different treatments. The experiment was performed three times independently with similar results, including 10 seedlings/group one time and six seedlings/group the other two times. (d, e) The activity of catalase (CAT) and peroxidase (POD) in P. thunbergii infected with BxLip‐3 dsRNA‐treated nematodes was increased compared with controls. (f, g) The relative transcript levels of pathogenesis‐related genes (PtPR‐3 and PtPR‐6) in P. thunbergii infected with dsBxLip‐3‐treated nematodes were up‐regulated compared with controls. In (d)–(g), stems of approximately 1 cm in length were selected to measure enzyme activity or to extract RNA at 12 h postinoculation. The data are shown as the mean ± standard deviation from three independent experiments. Different letters indicate statistically significant differences (p < 0.05), as determined by Duncan's multiple range test.

2.6. BxLip‐3 physically interacts with the class I chitinases in pine

Because BxLip‐3 is a secreted virulence protein, it was used as bait in a yeast two‐hybrid (Y2H) assay to screen a P. thunbergii cDNA library for potential host targets. We preliminarily identified two primary targets, class I chitinases that were captured more than five times in two independent screens, and selected them for further investigation. We acquired the two whole coding sequences from P. thunbergii according to the specific primer of Pinus taeda, and named them PtChia1‐3 and PtChia1‐4. Targeted Y2H assays (Figure 5a) and co‐immunoprecipitation (Co‐IP) assays (Figure 5b,c) confirmed BxLip‐3 interacted with the two chitinases. Both the Y2H and Co‐IP results also demonstrate that the Lipase‐3 domain of BxLip‐3 (BxLip‐3‐M5) interacts with PtChia1‐3 and PtChia1‐4 in P. thunbergii.

FIGURE 5.

FIGURE 5

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BxLip‐3 interacts with PtChia1‐3 and PtChia1‐4. (a) Yeast two‐hybrid interaction between BxLip‐3 and two chitinases. Co‐transformants grown on medium lacking tryptophan and leucine (SD/−Trp /−Leu) demonstrate that both bait and prey plasmids are present in yeast; only yeast cells containing the BxLip‐3 bait or the BxLip‐3‐M5 bait plus the PtChia1‐3 or PtChia1‐4 prey, or the positive control interaction of 53 bait plus T prey, grew or turned blue on the selective medium lacking tryptophan, leucine, and histidine containing X‐α‐Gal and aureobasidin A (Aba) (SD/−Trp/−Leu/−His+X‐α‐Gal+Aba). (b, c) BxLip‐3 (b) and BxLip‐3‐M5 (c) interact with PtChia1‐3 and PtChia1‐4, as determined by a co‐immunoprecipitation (Co‐IP) assay. Western blot analysis confirmed the expression of the input proteins: BxLip‐3‐HA (anti‐HA antibody), BxLip‐3‐M5‐RFP (anti‐RFP antibody), and PtChia1‐3‐GFP, PtChia1‐4‐GFP, and GFP (anti‐GFP antibody). The immune complexes were pulled down using anti‐HA or anti‐GFP agarose beads. PtChia1‐3‐GFP and PtChia1‐4‐GFP were detected after Co‐IP with the samples expressing BxLip‐3 (anti‐GFP antibody), but GFP was not detected. BxLip‐3‐M5‐RFP was detected after Co‐IP with the samples expressing PtChia1‐3‐GFP or PtChia1‐4‐GFP (anti‐RFP antibody), but BxLip‐3‐M5‐RFP was not detected with samples expressing GFP. Protein loading is indicated by Ponceau S staining of RuBisCO.

2.7. BxLip‐3 and two class I chitinases colocalize in the nucleus and the cytoplasm in N. benthamiana

To determine whether BxLip‐3 and its targets colocalize in plant cells, we examined the subcellular localization of these proteins. Red fluorescent protein (RFP), RFP‐BxLip‐3, and different mutant fusion proteins were coexpressed with GFP‐PtChia1‐3 or GFP‐PtChia1‐4 by agroinfiltration in N. benthamiana leaves. Confocal imaging showed that PtChia1‐3 and PtChia1‐4 colocalized with RFP‐BxLip‐3 in both the nucleus and the cytoplasm when coexpressed in N. benthamiana (Figure 6a). The expression of these proteins was confirmed by western blotting (Figure 6b). These findings further confirm that BxLip‐3 interacts with PtChia1‐3 and PtChia1‐4.

FIGURE 6.

FIGURE 6

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BxLip‐3 colocalizes with PtChia1‐3 and PtChia1‐4 in the nucleus and the cytoplasm. Proteins were expressed in Nicotiana benthamiana through agroinfiltration. (a) Confocal microscopy imaging of N. benthamiana leaves with transient expression of red fluorescent protein (RFP)‐tagged BxLip‐3, green fluorescent protein (GFP)‐tagged PtChia1‐3 and PtChia1‐4 shows that BxLip‐3 colocalizes with PtChia1‐3 and PtChia1‐4 in both the nucleus and the cytoplasm. Pictures taken 36 h postinfiltration show cells cotransformed with BxLip‐3 (red channel; left panel) and PtChia1‐3 and PtChia1‐4 (green channel; middle left panel). Bright field images (middle right panel) and the overlay (right panel) are also shown. N, nucleus; C, cytoplasm. Scale bars, 10 μm. (b) Expression of RFP‐BxLip‐3 together with GFP‐PtChia1‐3 and GFP‐PtChia1‐4 was confirmed by western blotting using anti‐GFP and anti‐RFP. Protein loading is indicated by Ponceau S staining of RuBisCO.

2.8. BxLip‐3 inhibits the expression of PtChia1‐3 and PtChia1‐4 in P. thunbergii

To elucidate whether PtChia1‐3 and PtChia1‐4 react to nematode inoculation, we measured their expression levels by RT‐qPCR at nine different time points in P. thunbergii inoculated with nematode suspension (in sterile water). To reduce the effects of wounds on chitinase expression levels, another group of seedlings was treated with sterile water at similar inoculation sites. The results demonstrated that the relative expression levels of PtChia1‐3 and PtChia1‐4 followed a similar trend (Figure 7a,b). As expected, the chitinases were up‐regulated when P. thunbergii faced wounding or nematode attack. However, upon wounding, chitinase expression was up‐regulated at very early stages and then remained low. In contrast, the chitinase expression pattern was more complex in pines inoculated with nematodes. First, their expression levels were up‐regulated during the early stages of nematode–pine interaction, reaching the first peak at 12 h postinoculation (hpi). Then, their expression levels gradually decreased. Interestingly, their expression levels increased dramatically at 10 dpi, when seedlings had not yet developed symptoms (Figure S2a). At 15 dpi, pine seedlings began to show symptoms, and a few needles turned yellow (Figure S2b). Chitinase expression levels remained high at this time point, but declined at 20 dpi, when most needles turned yellow and the pines wilted (Figure S2c). This expression pattern supports the idea that PtChia1‐3 and PtChia1‐4 are involved in the defence response of pine to B. xylophilus infection. Interestingly, we found that the expression of chitinases induced by wounding was much higher than that induced by PWNs at 6 hpi, indicating that PWNs might secrete some substance to inhibit their expression. Furthermore, our results described above showed that silencing of BxLip‐3 up‐regulated the expression of PtPR‐3 (encoding a class I chitinase) (Figure 4f). Therefore, we quantified the expression of PtChia1‐3 and PtChia1‐4 in P. thunbergii after BxLip‐3 silencing. RT‐qPCR assays showed that their expression levels increased compared with control groups when BxLip‐3 was silenced (Figure 7c). In addition, we tested whether BxLip‐3 could suppress the expression of PtChia1‐3 and PtChia1‐4. We injected purified recombinant proteins or sterile water into the stem of P. thunbergii 3 h before nematode inoculation, and collected stems at 6 hpi. Sterile water and a peptide (His‐tag) encoded by the empty vector were used as negative controls. The results showed that the expression levels of PtChia1‐3 and PtChia1‐4 decreased in BxLip‐3rec‐treated pines compared with negative control groups (Figure 7d). Thus, we infer that BxLip‐3 could inhibit the expression of PtChia1‐3 and PtChia1‐4 in P. thunbergii.

FIGURE 7.

FIGURE 7

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The expression pattern of PtChia1‐3 and PtChia1‐4 in Pinus thunbergii inoculated with Bursaphelenchus xylophilus. (a) The relative expression of PtChia1‐3 in P. thunbergii. (b) The relative expression of PtChia1‐4 in P. thunbergii. Pine stem samples were collected at different time points after being treated with sterile water or B. xylophilus (isolate AMA3) suspension to analyse the changes in the expression of PtChia1‐3 and PtChia1‐4. (c) The relative expression of PtChia1‐3 and PtChia1‐4 in P. thunbergii under three different treatments. In total, 3000 dsBxLip‐3‐treated nematodes, dsGFP‐treated nematodes, or wild‐type (WT) nematodes were inoculated into 3‐year‐old P. thunbergii seedlings. After 6 h, stem samples were collected to analyse the changes in the expression of PtChia1‐3 and PtChia1‐4. (d) The relative expression of PtChia1‐3 and PtChia1‐4 in P. thunbergii treated with sterile water, a peptide (His‐tag) encoded by the empty vector (EV), or BxLip‐3rec protein. Three‐year‐old seedlings were inoculated with 1 mL of a purified recombinant protein solution (50 μg/mL) or 1 mL sterile water. Nematode inoculation was performed 3 h later and stems were collected at 6 h postinoculation. Reverse transcription–quantitative PCR analysis was conducted to determine the relative gene expression levels of the two chitinases. The data are shown as the mean ± standard deviation from three independent experiments. Different letters indicate statistically significant differences (p < 0.05), as determined by Duncan's multiple range test.

3. DISCUSSION

During nematode–plant interactions, PWNs secrete diverse effectors into the host tissues, which play central roles in modulating host immunity to facilitate plant parasitism. In a previously reported transcriptome analysis, BxLip‐3, a class III lipase from B. xylophilus, was identified as a candidate effector (Hu et al., 2021). RT‐qPCR analysis showed that BxLip‐3 expression was up‐regulated at the early parasitic stage of B. xylophilus, suggesting a potential role in early nematode–plant interactions (Figure 1a). PWNs can complete a generation in 4–5 days at 26°C or in 3 days at 30°C (Futai, 2013). With the spread and propagation of nematodes in pine, the expression of BxLip‐3 reached a peak at 5 dpi. These findings indicate that BxLip‐3 produced a marked effect mainly through the initial establishment of the plant–nematode interaction at the early stages of nematode parasitism. Genes expressed exclusively in the nematode oesophageal gland cells and secreted through the stylet may play critical roles during nematode parasitism (Mitchum et al., 2013). In this study, we performed in situ hybridization analysis and found that BxLip‐3 was expressed in oesophageal gland cells and the intestine in PWNs, indicating the possibility of BxLip‐3 secretion via the nematode stylet. The expression pattern and the localization of BxLip‐3 during infection support the hypothesis that the gene encodes an effector.

Similar to other pathogens, B. xylophilus secretes different effectors at different parasitic stages, and these effectors function in different ways, for example by suppressing plant defence responses. B. xylophilus effectors BxSCD1, Bx‐FAR‐1, and BxSCD3 have been found to be up‐regulated during the early parasitic stages (2.5, 12, and 24 h), and they can suppress PAMP‐triggered cell death (Hu, Wu, Wen, Qiu, et al., 2022; Li, Hu, et al., 2020; Wen et al., 2021). Additionally, BxCDP1 from B. xylophilus has been characterized as a PAMP that could induce cell death and PTI in the host (Hu et al., 2020). Here, BxLip‐3 was identified to suppress PCD triggered by PsXEG1 and BxCDP1 and to inhibit the expression of PTI marker genes (NbCyp71D20 and NbAcre31) induced by BxCDP1 (Figures 2 and S3). Unlike the above effectors, BxLip‐3 is not only expressed at the earlier parasitic stages but also at the later stages. These results suggest that B. xylophilus, like other pathogens, secretes various effectors that coordinate to inhibit plant immunity.

Lipids are the main constituents of cell membranes and are known to influence pathogenesis and resistance mechanisms associated with plant–microbe interactions (Cardoso et al., 2022; Shah, 2005). Lipases can hydrolyse long‐chain acyl‐triglycerides into di‐ and monoglycerides, glycerol, and free fatty acids in many important biological processes (Pascoal et al., 2018). Caenorhabditis elegans can maintain whole‐body energy homeostasis by promoting lipid hydrolysis in response to adverse stresses (Hu, 2007; Lapierre et al., 2013). Here, we first reported that the Lipase‐3 domain is essential to the immunosuppressive ability of BxLip‐3 (Figure 3), highlighting the roles that the domain plays in the effector function.

RNAi is a valuable technique for studying gene function on account of its sequence‐specific degradation of mRNA via homologous dsRNA, which inhibits gene function posttranscriptionally (Hammond et al., 2001). To validate the role of BxLip‐3 in B. xylophilus virulence in pines and the development of PWD, BxLip‐3 was silenced, which resulted in a delay in the appearance of symptoms of P. thunbergii. POD and CAT are antioxidant enzymes responsible for clearing and decomposing toxic ROS and free radicals in plants, helping plants resist disease. BxLip‐3 decreased the contents of these antioxidant enzymes, resulting in suppression of host immunity, which could facilitate nematode infection. PR proteins are closely related to plant disease resistance. Our results showed that the expression of PtPR‐3 (encoding a class I chitinase) and PtPR‐6 (a JA/ethylene‐responsive gene) was up‐regulated when P. thunbergii was infected by BxLip‐3 dsRNA‐treated nematodes, indicating that BxLip‐3 might interfere with host signalling pathways and immune responses. In a previous study, BxLip‐3 was identified as a potential virulence biomarker, which was particularly abundant in the secretome of the virulent PWN isolate (Cardoso et al., 2022). These findings demonstrated that BxLip‐3 might be a pathogenicity protein contributing to B. xylophilus virulence.

Identifying PWN‐secreted effectors' interactors could assist in the functional characterization of the effectors (Chen et al., 2018). Our previous results showed that BxLip‐3 can inhibit the expression of PtPR‐3 (encoding a class I chitinase). Through Y2H, Co‐IP, and subcellular localization assays, we identified two class I chitinases as targets of BxLip‐3. Plant chitinases are thought to be closely related to plant defence against phytopathogens, and so are pine chitinases. The induction of chitinase expression upon pathogen attack and wounding has been demonstrated in Pinus species (Davis et al., 2002; Kolosova et al., 2014; Liu et al., 2005). Likewise, our findings demonstrate that wounding and nematode inoculation can induce chitinase expression. Unlike the short‐term expression of chitinase induced by wounding, the expression of chitinases induced by nematode infection lasts longer and is more intense at the later stages of nematode inoculation. In contrast, the expression level of BxLip‐3 reached its peak at 5 dpi, earlier than that of chitinases, inferring that the pine response might lag behind nematodes in the later stage of the pine–nematode interaction. Surprisingly, the expression of these chitinases induced by wounding was much higher than that induced by nematode inoculation at 6 h, indicating that B. xylophilus might secrete some substances to repress the expression of chitinases (Figure 7a,b). Correspondingly, silencing of BxLip‐3 up‐regulated the expression of PtChia1‐3 and PtChia1‐4 in P. thunbergii, while the addition of purified BxLip‐3rec protein had the opposite effect. Therefore, we deduce that BxLip‐3 might suppress the expression of PtChia1‐3 and PtChia1‐4 to interfere with the ability of pine to resist B. xylophilus infection.

Chitin, which is present in the eggshell, cuticle, pharynx, and microfilariae sheath of nematodes, can be hydrolysed by chitinase (Adam et al., 1996; Bird & McClure, 1976; Fanelli et al., 2005; Veronico et al., 2001; Zhang et al., 2005). Studies have shown that chitinase secreted by bacteria and fungi can antagonize nematodes (Castaneda‐Alvarez & Aballay, 2016; Lee et al., 2014; Sharon et al., 2001). We speculate that PtChia1‐3 and PtChia1‐4 may hydrolyse chitin and harm nematodes. It has been reported that after inoculation with Heterobasidion annosum, the Norway spruce chitinase is quickly up‐regulated to reduce or prevent pathogen colonization (Hietala et al., 2004). This up‐regulation also occurs in the class I chitinase PcChia1‐1 from Pinus contorta upon Grossmannia clavigera infection (Kolosova et al., 2014). Whether PtChia1‐3 and PtChia1‐4 contribute to P. thunbergii resistance to pathogens is an attractive question that needs to be explored in the future. Given that chitin exists in pathogens but not in plants, chitinases may serve as resistance biomarkers. Thus, a more thorough understanding of chitinases is warranted. This was demonstrated in recent studies showing that class I chitinases play key roles in PTI, which can be triggered by the effective PAMP chitin (Buendia et al., 2018; Mengiste, 2012). Nevertheless, the presence of BxLip‐3 reduced the expression of PtChia1‐3 and PtChia1‐4. According to the above results, BxLip‐3 also suppresses PCD triggered by the PAMPs PsXEG1 and BxCDP1 in N. benthamiana, indicating that BxLip‐3 can inhibit PTI through multiple pathways. Together, we inferred that BxLip‐3 suppresses plant immunity to help protect nematodes and promote parasitism.

In this study, we characterized a B. xylophilus candidate effector, BxLip‐3, that suppresses plant immunity in N. benthamiana and contributes to B. xylophilus virulence. The class I chitinases PtChia1‐3 and PtChia1‐4 were found to be targets of BxLip‐3 in P. thunbergii. On the one hand, our study provides significant evidence to further explore the pine–nematode interaction. On the other hand, the identification of effector targets conduces to the exploration of the resistance of pines and supports breeding programmes to generate plants that are resistant to this pest.

4. EXPERIMENTAL PROCEDURES

4.1. PWN culture and plant material

B. xylophilus strains used in this study included AMA3 (Anhui province, China), AA3 (Anhui province, China), ZL1 (Zhejiang province, China), USA (United States), HE2 (Hubei province, China), and YW4 (Yunnan province, China). All strains were maintained in the Forest Protection Laboratory, Nanjing Forestry University and cultured on B. cinerea grown on PDA at 25°C in the dark. N. benthamiana was maintained in the greenhouse at 25°C with a photoperiod of 16 h light/8 h darkness, and leaves of 4–5‐week‐old N. benthamiana plants were used in the infiltration assay. Three‐year‐old P. thunbergii seedlings were cultivated at temperatures ranging from 28°C to 32°C with the relative humidity ranging from 65% to 75%.

4.2. Inoculation and sampling method

We used a sterile blade to cut the bark on the stem at a height of approximately 10 cm in the aboveground part of the plant to reach the xylem, and sterile cotton balls were inserted with forceps. Then, nematode suspension containing 5000 nematodes was dropped onto the cotton on each plant. The inoculated pine seedlings were placed in the incubation room after inoculation to ensure that the nematode solution did not leak out. After inoculating P. thunbergii with PWNs for 6 h, 12 h, 24 h, 2 days, 5 days, 10 days, 15 days, and 20 days, we selected stems of approximately 1 cm in length below the wound to extract plant RNA. All the rest of the seedlings were cut into small segments, which were split into halves. The nematodes were extracted from these segments via the Baermann funnel method over 3 h. The Baermann funnel method was performed as previously described (Hooper et al., 2005; Son & Moon, 2013; Staniland, 1954).

4.3. Total RNA extraction and cDNA synthesis

The total RNA from each N. benthamiana or P. thunbergii sample was extracted using the RNAprep Pure Plant Plus Kit (Polysaccharides & Polyphenolics‐rich) (TIANGEN). Total RNA from the nematodes was extracted using TRIzol reagent (Invitrogen). First‐strand cDNA for RT‐qPCR was synthesized from 1 μg of total RNA using HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme) according to the manufacturer's protocol.

4.4. Real‐time PCR

Real‐time qPCR was performed in a reaction volume of 20 μL that included 20 ng of cDNA, 0.2 μΜ gene‐specific primers, 0.2 μΜ ROX reference dye, 10 μL of 2×SYBR Green Pro Taq HS Premix (Accurate Biology), and RNase‐free water according to the manufacturer's instructions. The relative expression values were quantified using Actin of B. xylophilus and PtEF1α of P. thunbergii as reference genes.

4.5. Plasmid construction

BxLip‐3 and its eight mutant variants were cloned using cDNA from B. xylophilus (AMA3). The amplified fragments were ligated into pBINRFP (pCAM1300‐RFP) or PVX (pGR107) using the appropriate restriction enzymes and a ClonExpress II One Step Cloning Kit (Vazyme). The genes encoding PtChia1‐3 and PtChia1‐4 were cloned using P. thunbergii cDNA. These amplified fragments of PtChia1‐3 and PtChia1‐4 were also ligated into pBINGFP (a plasmid encoding GFP). The primers we used are listed in Table S1.

4.6. A. tumefaciens infiltration assays

The A. tumefaciens infiltration assays were performed as previously described (Zhang et al., 2022). Agrobacterium cells carrying BxLip‐3 were infiltrated into leaves using a needleless syringe. The same infiltration site was challenged 16 h later with Agrobacterium carrying an elicitor. Each experiment was performed on six leaves from three individual plants and repeated at least three times.

4.7. Electrolyte leakage assay

Electrolyte leakage in the inoculated N. benthamiana leaves was measured as described previously (Mittler et al., 1999; Yu, Tang, et al., 2012).

4.8. Total protein extraction and western blot analysis

Total protein extraction and western blot analysis were carried out as described previously (Wen et al., 2021).

4.9. Confocal microscopy

For fluorescence observations, patches of N. benthamiana leaves were collected at 36 hpi and used for confocal laser scanning microscopy with an LSM710 microscope (Zeiss) with a 40× objective lens. Fluorescence of GFP was elicited at 514 nm and detected at 520–540 nm. Fluorescence of RFP was elicited at 561 nm and detected at 590–630 nm.

4.10. In situ hybridization

An 810‐bp PCR product was amplified from B. xylophilus AMA3 cDNA. Antisense and sense DIG‐labelled probes were prepared separately by unidirectional PCR with these templates. The primers we used are listed in Table S1. In situ hybridization was performed as described previously (de Boer et al., 1998) using a DIG High Prime RNA Labeling and Detection Starter Kit I (Roche). Finally, the samples were observed under an Axio Image M2 microscope.

4.11. RNAi design and treatment for inoculation assays

The dsRNA corresponding to BxLip‐3 and the negative control GFP were synthesized using the MEGAscript RNAi kit (ThermoFisher) according to the manufacturer's instructions. Afterward, the nematodes (a mixture of juveniles and adults) were soaked in BxLip‐3 dsRNA, GFP dsRNA, and non‐dsRNA solutions and then incubated at 20°C in a shaking incubator at 180 rpm for 48 h. We measured the silencing efficiency of BxLip‐3 by RT‐qPCR with specific primers.

In the infection assay, each 3‐year‐old P. thunbergii seedling was inoculated with 3000 dsBxLip‐3‐treated PWNs, dsGFP‐treated PWNs, or WT PWNs. The seedlings inoculated only with WT nematodes were regarded as a blank control. The disease severity index is a composite indicator of seedling morbidity and disease severity. Based on the colour of the needles, the disease severity index of the P. thunbergii seedlings was classified into five different grades (Rempel & Hall, 1996; Yu, Wu, et al., 2012): 0, all needles are green; I, a few needles have turned yellow; II, approximately half of the needles have turned yellow or brown; III, most of the needles have turned brown; and IV, the entire seedling has withered. The morbidity of seedlings was calculated using as A/C and the disease severity index was calculated as ∑(A × B)/(C × D) × 100%, where A is the number of sick seedlings per grade, B is the corresponding grade of the sick seedlings, C is the number of seedlings inoculated in each treatment, and D is the most serious grade (Rempel & Hall, 1996).

4.12. Expression of recombinant proteins

The recombinant pET‐32a‐BxLip‐3 expression plasmid carrying a thioredoxin‐6×His‐tag was transformed into chemically competent E. coli Transetta(DE3) cells (TransGen Biotech). The competent cells were grown in liquid Luria‐Bertani medium with ampicillin at 37°C for 12 h to a final OD at 600 nm of 0.4–0.6. Then, we added isopropyl β‐d‐1‐thiogalactopyranoside (final concentration, 0.1 mM) to the medium and continued to culture the cells at 16°C at 150 rpm for 16 h. The cultures were harvested using centrifugation at 10,000 g for 20 min at 4℃ and were resuspended in 20 mL phosphate‐buffered saline (pH 7.4) Finally, we sonicated the suspension and centrifuged it at 10,000 g for 20 min at 4℃ to collect the supernatant. Purification of the recombinant protein from the supernatant was performed using Ni‐NTA 6FF (prepacked gravity column) (Sangon Biotech). The fusion protein encoded by just the pET‐32a(+) vector was used as a control.

4.13. Enzyme activity assay

Samples of homogenized stems (100 mg) approximately 1 cm in length were mixed with 1 mL lysis solution on ice. CAT and POD activities in P. thunbergii inoculated with nematodes with different treatments were determined by detection kits (Comin) according to the manufacturer's instructions.

4.14. Y2H assay

The yeast library was constructed as previously described (Hu, Wu, Wen, Ye, et al., 2022). The coding sequences of BxLip‐3 (without SP), PtChia1‐3, and PtChia1‐4 were cloned into pGBKT7 or pGADT7 to generate bait and prey vectors. Synthetic defined (SD) bases included a yeast nitrogen base, ammonium sulphate, and glucose as a carbon source. Amino acids were not added so dropout supplements must be added separately to the SD base to make SD medium lacking the specified nutrients, allowing for selection of transformed yeast clones. 5‐Bromo‐4‐chloro‐3‐indolyl‐α‐d‐galactopyranoside (X‐α‐Gal) is used to rapidly detect protein interactions, and aureobasidin A (AbA) is used for screening with very low background signals. The indicated construct pairs were co‐transformed into Y2H Gold yeast cells and transformed colonies were selected on SD medium lacking Leu and Trp (SD/−Trp/−Leu). The transformants were transferred to selective medium lacking Leu, Trp, and His containing X‐α‐Gal and Aba (SD/−Trp/−Leu/−His+X‐α‐Gal+AbA) for interaction analysis.

4.15. Co‐IP assay

For the Co‐IP assay, the BxLip‐3 (without SP), PtChia1‐3, and PtChia1‐4 sequences were inserted into the PVX or pBINGFP vector and introduced into A. tumefaciens GV3101, which was then used to transform N. benthamiana leaves. A. tumefaciens strains containing the PVX‐BxLip‐3 and pBINGFP‐PtChia1‐3 constructs were co‐infiltrated in N. benthamiana. Likewise, A. tumefaciens strains containing the PVX‐BxLip‐3 and pBINGFP‐PtChia1‐4 constructs were used in agroinfiltration. Co‐IP was performed with leaf lysate from the co‐infiltrated leaves of N. benthamiana 48 h after co‐infiltration. Immunoblotting was performed as previously described (Yang et al., 2018).

CONFLICT OF INTEREST STATEMENT

The authors declare no competing interests.

Supporting information

Figure S1 The amino acid sequence of BxLip‐3 in the six Bursaphelenchus xylophilus isolates.

Click here for additional data file. (4.9MB, tif)

Figure S2 Inoculation assay of pine seedlings for reverse transcription–quantitative PCR analysing the expression of BxLip‐3, PtChia1‐3, and PtChia1‐4. Bursaphelenchus xylophilus (the AMA3 isolate) was inoculated into 3‐year‐old Pinus thunbergii seedlings. At different stages of disease development, we selected seedling stems of approximately 1 cm in length to extract RNA. Meanwhile, we collected nematodes from the remaining seedling stems using the Baermann funnel technique. (a) At 10 days postinoculation (dpi), the needles of P. thunbergii seedlings stayed green and the whole tree showed no disease symptoms. (b) At 15 dpi, a few needles began to turn yellow. (c) At 20 dpi, most of the needles were yellow, and the pine tree wilted obviously. Black arrow, inoculation site.

Click here for additional data file. (13.5MB, tif)

Figure S3 BxLip‐3 suppressed the expression of PTI marker genes triggered by BxCDP1 in Nicotiana benthamiana. (a) Western blot analysis of fusion protein expression. The fusion protein of the recombinant pET‐32a(+) vector carrying BxLip‐3 and the protein encoded by the pET‐32a(+) vector alone were expressed in Escherichia coli Transetta(DE3). After purification on a Ni‐NTA column, the proteins were analysed by western blotting using an anti‐His antibody. (b) The transcript levels of NbCyp71D20 and NbAcre31 induced by BxCDP1 in N. benthamiana leaf tissues expressing the fusion proteins. We infiltrated the BxCDP1rec protein into N. benthamiana leaves 16 h after expressing the BxLip‐3rec protein or a peptide (His‐tag) encoded by the empty vector alone (EV). After 6 h, the leaf samples were collected to analyse the transcript levels of NbCyp71D20 and NbAcre31 by reverse transcription–quantitative PCR (RT‐qPCR). The RT‐qPCR values were normalized to the transcript level of NbEF1α. The data are presented as the mean ± standard deviation from three independent experiments. Different letters indicate statistically significant differences (p < 0.05), as determined by Duncan’s multiple range test.

Click here for additional data file. (451.1KB, tif)

Figure S4 The effect of BxLip‐3 silencing on the reproduction and feeding rate of Bursaphelenchus xylophilus. (a) The silencing efficiency of BxLip‐3 in B. xylophilus. (b) The number of nematodes on Botrytis cinerea over 6 days. Each dish of B. cinerea was inoculated with 100 nematodes. (c) The number of nematodes in Pinus thunbergii inoculated with dsRNA‐treated nematodes. In total, 3000 nematodes were inoculated per pine tree and sampled at 16 days postinoculation. (d) The propagating quantity of B. xylophilus cultured on B. cinerea.

Click here for additional data file. (4.2MB, tif)

Table S1 List of primers used in this study.

Click here for additional data file. (17.5KB, docx)

ACKNOWLEDGEMENTS

This work was supported by the National Key Research and Development Program of China (2021YFD1400903), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX20_0901).

Qiu, Y.‐J. , Wu, X.‐Q. , Wen, T.‐Y. , Hu, L.‐J. , Rui, L. , Zhang, Y. et al. (2023) The Bursaphelenchus xylophilus candidate effector BxLip‐3 targets the class I chitinases to suppress immunity in pine. Molecular Plant Pathology, 24, 1033–1046. Available from: 10.1111/mpp.13334

Contributor Information

Xiao‐Qin Wu, Email: xqwu@njfu.edu.cn.

Jian‐Ren Ye, Email: jrye@njfu.edu.cn, Email: njfu_jrye@163.com.

DATA AVAILABILITY STATEMENT

All relevant data can be found within the manuscript and its supporting information. The nucleotide sequences of PtChia1‐3 and PtChia1‐4 are available in GenBank at https://www.ncbi.nlm.nih.gov/genbank/ under accession numbers OP574696 and OP574697, respectively.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 The amino acid sequence of BxLip‐3 in the six Bursaphelenchus xylophilus isolates.

Click here for additional data file. (4.9MB, tif)

Figure S2 Inoculation assay of pine seedlings for reverse transcription–quantitative PCR analysing the expression of BxLip‐3, PtChia1‐3, and PtChia1‐4. Bursaphelenchus xylophilus (the AMA3 isolate) was inoculated into 3‐year‐old Pinus thunbergii seedlings. At different stages of disease development, we selected seedling stems of approximately 1 cm in length to extract RNA. Meanwhile, we collected nematodes from the remaining seedling stems using the Baermann funnel technique. (a) At 10 days postinoculation (dpi), the needles of P. thunbergii seedlings stayed green and the whole tree showed no disease symptoms. (b) At 15 dpi, a few needles began to turn yellow. (c) At 20 dpi, most of the needles were yellow, and the pine tree wilted obviously. Black arrow, inoculation site.

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Figure S3 BxLip‐3 suppressed the expression of PTI marker genes triggered by BxCDP1 in Nicotiana benthamiana. (a) Western blot analysis of fusion protein expression. The fusion protein of the recombinant pET‐32a(+) vector carrying BxLip‐3 and the protein encoded by the pET‐32a(+) vector alone were expressed in Escherichia coli Transetta(DE3). After purification on a Ni‐NTA column, the proteins were analysed by western blotting using an anti‐His antibody. (b) The transcript levels of NbCyp71D20 and NbAcre31 induced by BxCDP1 in N. benthamiana leaf tissues expressing the fusion proteins. We infiltrated the BxCDP1rec protein into N. benthamiana leaves 16 h after expressing the BxLip‐3rec protein or a peptide (His‐tag) encoded by the empty vector alone (EV). After 6 h, the leaf samples were collected to analyse the transcript levels of NbCyp71D20 and NbAcre31 by reverse transcription–quantitative PCR (RT‐qPCR). The RT‐qPCR values were normalized to the transcript level of NbEF1α. The data are presented as the mean ± standard deviation from three independent experiments. Different letters indicate statistically significant differences (p < 0.05), as determined by Duncan’s multiple range test.

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Figure S4 The effect of BxLip‐3 silencing on the reproduction and feeding rate of Bursaphelenchus xylophilus. (a) The silencing efficiency of BxLip‐3 in B. xylophilus. (b) The number of nematodes on Botrytis cinerea over 6 days. Each dish of B. cinerea was inoculated with 100 nematodes. (c) The number of nematodes in Pinus thunbergii inoculated with dsRNA‐treated nematodes. In total, 3000 nematodes were inoculated per pine tree and sampled at 16 days postinoculation. (d) The propagating quantity of B. xylophilus cultured on B. cinerea.

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Table S1 List of primers used in this study.

Click here for additional data file. (17.5KB, docx)

Data Availability Statement

All relevant data can be found within the manuscript and its supporting information. The nucleotide sequences of PtChia1‐3 and PtChia1‐4 are available in GenBank at https://www.ncbi.nlm.nih.gov/genbank/ under accession numbers OP574696 and OP574697, respectively.

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