Botrytis cinerea B05.10 was used as wild-type strain and control strain in this study. All B. cinerea strains, including two independent BcXyl1 knockout mutants and two complementary transformants, were routinely maintained in 15% glycerol at -80°C and grown on PDA at 22°C, respectively. Agrobacterium tumefaciens AGL-1 were grown on LB (Kan and Rif) medium at 28°C. To obtain conidia, B. cinerea grown on tomato-PDA plates (39 g of potato dextrose agar plus 250 g of homogenized tomato fruits per liter) as explained previously (Benito et al., 1998). N. benthamiana and tomato (Solanum lycopersicum) plants were grown at 27 °C in a greenhouse with a day/night period of 14/10 h and 60% relative humidity (RH).

The open reading frame of BcXyl1 (amplified with primers BcXyl1 F/BcXyl1 R; Supplementary Table S2) and C^130-155 were amplified by PCR from cDNA of the wild-type strain B05.10 and the fragment fused with a myc tag and a 6xHis tag at the C terminus was cloned into the pPICZαA vector at the BamHI and EcoRI sites. The recombinant plasmid pPICZαA-BcXyl1 and pPICZαA-C^130-155 were linearized with PmeI and transformed into Pichia pastoris KM71H for expression. The transformed yeasts were grown and induced in BMGY (buffered glycerol complex medium) and BMMY (buffered methanol complex medium), respectively (Easy Select Pichia expression kit; Invitrogen). Then, the supernatant was collected (3000 g for 10 min at 4°C) and purified using nickel affinity chromatography. The purified C^130-155, BcXyl1^rec, or BcXyl1 were kept in protein buffer (20 mM Tris, pH 8.0) and further detected via SDS-PAGE and Western blotting. The concentration of the purified protein was measured using Easy II Protein Quantitative Kit (BCA) and the protein was then stored at -80°C.

To transiently express truncated mutants of the BcXyl1 protein in leaves, DNA sequences encoding different fragments (BcXyl1, N⁸⁰, N¹³⁰, N¹⁵⁵ C⁸⁰, C¹³⁰, C¹⁵⁵, and C^130-155) were amplified by PCR from cDNA of the wild-type strain B05.10 and inserted into pYBA1132 vector at the XbaI and BamHI sites and then transformed into the A. tumefaciens strain GV3101. Agroinfiltration assays were performed on N. benthamiana plants. Agrobacterium-mediated transient expression was performed as described (Ma et al., 2015). Leaves were scored and photographed 6 days after initial inoculation. Each assay was performed on six leaves from three individual plants, and repeated at least three times.

To determine the relationship between the enzymatic activity and cell death-inducing activity of BcXyl1, we constructed BcXyl1^rec mutant, which abolished the enzymatic activity. According to multiple sequence alignment, two potentially highly conserved catalytic residues (E104 and E157) were the critical catalytic sites of BcXyl1. Next, two glutamic acid residues were substituted by Gln using the Quick Change^TM Site-Directed Mutagenesis Kit (Stratagene, United States). BcXyl1^rec was expressed in P. pastoris and carried out the enzyme assay.

The xylanase activity was assayed via the method as described previously (Biely et al., 1988). The purified BcXyl1^rec or BcXyl1 (500 ng) and substrate (1% beechwood xylan) were co-incubated in citrate phosphate McIlvaine buffer, pH 5, at 35°C for 10 min (total volume: 125 μl). All samples were incubated at 100°C for 10 min to end the assays reactions. The amount of reducing sugars released from xylan was quantified using a standard calibration curve obtained with the dinitrosalicylic acid procedure. The experiment was replicated three times.

To confirm whether BcXyl1 was secreted into the apoplast and the relationship between enzymatic activity of BcXyl1 and cell death-inducing activity, transient expression in N. benthamiana was performed. Three sequences (BcXyl1, BcXyl1^-ΔSP, and BcXyl1^rec) were cloned into the pYBA1132 vector which contained a C-terminal GFP tag at the XbaI and BamHI sites, and then transformed into the A. tumefaciens strain GV3101. All primers are listed in Supplementary Table S2. Plant total protein extractions and immunoblots were assessed as previously described (Yu et al., 2012). All proteins were analyzed by immunoblots using anti-GFP-tag primary monoclonal antibody. The blots were visualized using the Odyssey^® LI-COR Imaging System. Rubisco was used to confirm the equal protein loading.

To test the induction of cell death or PTI responses, BcXyl1 and C^130-155 were dissolved in PBS and infiltrated into the leaves of N. benthamiana and tomato plants using a syringe. Plants were grown in a greenhouse with a day/night period of 14/10 h. The cell-death response was investigated after 48 h treated with BcXyl1, C^130-155, or PEVC (P. pastoris culture supernatant from an empty vector control strain, purified in the same way as BcXyl1). To further investigate cell death, trypan blue staining was performed by boiling leaf tissues in a mixture of phenol, lactic acid, glycerol, and distilled water containing 1 mg/ml trypan blue (1:1:1:1) for 1 min. The samples were then soaked in 2.5 mg/ml chloral hydrate overnight. The accumulation of ROS in plant leaves was stained by 3′3-diaminobenzidine (DAB) and Nitroblue Tetrazolium (NBT) solution as described previously (Bindschedler et al., 2006). To visualize callose deposition, 4-week-old N. benthamiana leaves were infiltrated with 1 μM recombinant proteins and stained with aniline blue at 24 h post-treatment, as described previously (Chen et al., 2012). To assay electrolyte leakage, the N. benthamiana leaves treated with proteins were harvested at different time points and submerged in sterile water at 4°C. Ion conductivity was measured using a conductivity meter. To test whether BcXyl1 could confer plants disease resistance, the purified BcXyl1, C^130-155, or PEVC was individually syringe-infiltrated into 4-week-old N. benthamiana and tomato leaves. Five microliters of 2 × 10⁶ conidia/ml B. cinerea and TMV-GFP were placed on the systemic leaves, respectively. The inoculated plants were placed in a greenhouse at 25°C with a day/night period of 14/10 h. Lesion diameter of B. cinerea and the number of TMV-GFP lesions on N. benthamiana leaves were evaluated at 2 and 4 days post-inoculation, respectively. All experiments were performed three times.

To test whether BcXyl1 functioned as a virulence factor of B. cinerea, the wild-type strain and derived mutants, including the BcXyl1 deletion (ΔBcXyl1-1 and ΔBcXyl1-2) and complementary mutants (ΔBcXyl1-1-C and ΔBcXyl1-2-C) were used in this study. Four-week-old N. benthamiana leaves were inoculated with 5 μL of 2 × 10⁶ conidia/ml B. cinerea. The inoculated plants were placed in a greenhouse with a day/night period of 14/10 h. The lesion development of B. cinerea on the N. benthamiana leaves was evaluated at 2 days post-inoculation by determining the average lesion diameter. Tomato, grape, and apple fruits (commercially obtained) were washed under running tap water and surface sterilized by immersion for 5 min in ethanol. After air drying, fruits were inoculated with 5 μL of 2 × 10⁶ conidia/ml B. cinerea. Fruits were incubated at 25°C under conditions of high humidity on water-soaked filter paper in closed containers. The lesion development of B. cinerea on the fruits was evaluated at 3 days post-inoculation by determining the average lesion diameter. All the experiments were performed three times.

To determine whether BAK1 or SOBIR1 participate in induction of cell death by BcXyl1, VIGS was performed. NbBAK1 or NbSOBIR1 gene was silenced using VIGS, as described previously (Kettles et al., 2016). A. tumefaciens strain harboring constructs (pTRV1, pTR::BAK1 or pTRV1, pTRV2::SOBIR1) were infiltrated into the N. benthamiana leaves. pTRV2::GFP was used as the control and the expression levels of BAK1 and SOBIR1 were determined by qRT-PCR. Agroinfiltration assays were performed on N. benthamiana plants using Bcl-2-associated X protein (BAX) as positive controls. Phenotypes were photographed 6 days after infiltration. All the experiments were performed three times.

To measure the expression of BcXyl1 during infection, 4-week-old N. benthamiana or 4-week-old tomato plants were inoculated with B. cinerea 2 × 10⁶ conidia/ml. We selected 10 indicated time points during different stages of post-inoculation to determine expression patterns of BcXyl1 by qPCR. All samples were stored at -80°C. Total RNA of B. cinerea was extracted with the E.Z.N.A.^® Total RNA Kit I according to the manufacturer’s instructions and stored at -80°C. For the measurement of defense-related genes expression, leaves of 4-week-old N. benthamiana plants were treated with 1 μM purified BcXyl1, C^130-155, or PEVC. The leaves were obtained at the indicated time points, immediately frozen in liquid nitrogen, and stored at -80°C. The EasyPure Plant RNA Kit (TransGen Biotech) was used to extract total RNA. After isolation of total RNA, qPCR was performed using a TransStart Green qPCR SuperMix UDG according to the manufacturer’s instructions (TransGen Biotech). qRT-PCR was performed under the following conditions: an initial 95°C denaturation step for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. N. benthamiana EF-1a (P43643.1) and B. cinerea Bcgpdh gene (BC1G_05277) were used as endogenous plant controls and used to quantify fungal colonization, respectively. qPCR assays were repeated at least twice, each repetition with three independent replicates (Livak and Schmittgen, 2001). All primers are listed in Supplementary Table S2. The relative transcript levels among various samples were determined using the 2^-ΔΔCT method with three independent determinations (Livak and Schmittgen, 2001).

BcXyl1 gene and 500 bp flanking sequences of the target gene were amplified from B. cinerea B05.10 wild-type strain genomic DNA. Two flanking sequences of the target gene and hygromycin resistance cassette were constructed into a fusion fragment using a nested PCR reaction, which is subsequently introduced into the binary vector pGKO2 gateway. To generated complementary transformants, the donor vector pCT-HN containing BcXyl1 gene was integrated into the mutant transformants using a previously described Agrobacterium-mediated transformation method (Liu et al., 2013). All mutants were identified using PCR with the corresponding primers. All primers are listed in Supplementary Table S2.

All the experiments and data presented here were performed at least three repeats. The data are presented as the means and standard deviations. Statistical Analysis System (SAS) software was used to perform the statistical analysis via Student’s t-test.

BcXyl1 was identified by searching the B. cinerea genome sequence. The open reading frame of BcXyl1 (GenBank: ATZ53308.1) is 987 bp encoding a 329 aa protein with a predicted N-terminal signal peptide (1–20 aa), and no transmembrane helices of BcXyl1 were found, suggesting that it may be secreted into extracellular space. The bioinformatics analysis suggested that BcXyl1 belongs to SGNH hydrolase subfamily and has a highly strong similarity to fungal endo-β-1,4-xylanases.

Previous studies showed that xylanases in pathogenic microorganisms were implicated in the pathogenicity. In order to assess the role of BcXyl1 to B. cinerea virulence, we first analyzed the expression patterns of BcXyl1 during different stages of post-inoculation. qRT-PCR results suggested that when the spore suspension of B. cinerea was inoculated onto leaves of N. benthamiana and tomato, transcript level of BcXyl1 increased rapidly and reached a maximum of about 26-fold to 28-fold at 2 days post-inoculation, and then rapidly declined and maintained a level that was slightly higher than the initial level during later stages (Figure 1).

To more directly explore the biological roles of BcXyl1 during infection, we constructed two BcXyl1 deletion mutants in B. cinerea (ΔBcXyl1-1 and ΔBcXyl1-2) and two rescued strains (ΔBcXyl1-1-C and ΔBcXyl1-2-C), and the ability of the resulting mutants to infect various plant organisms was evaluated. All mutants showed no significant differences with the wild-type stain in growth rate and colony morphology on PDA plates (Supplementary Figure S2). N. benthamiana leaves were inoculated with spore suspension of the wild type and mutants, and lesion size was measured 48 h after inoculation. Interestingly, the deletion of BcXyl1 displayed significantly reduced virulence and produced much smaller lesions on leaves of N. benthamiana than the WT strain at 48 hpi (Figures 2A,B). The rescued strains recovered the high virulence phenotypes. And two BcXyl1 deletion mutants displayed much weaker disease symptoms and lesion diameter than the wild-type strain and the complement strains (ΔBcXyl1-1-C and ΔBcXyl1-2-C) on grape, tomato, and apple fruits 72 h post-inoculation (Figures 2A,B). These results indicated that BcXyl1 functioned as a virulence factor that contributes to B. cinerea virulence on host plants.

To further confirm whether BcXyl1 could induce cell death in N. benthamiana, we expressed BcXyl1 in the yeast P. pastoris using the pPICZαA vector (pPICZαA: BcXyl1). Moreover, the recombinant protein BcXyl1, with a size of 35 kDa, was infiltrated into the mesophyll of N. benthamiana leaves with different concentrations (Supplementary Figure S3). The area of necrosis occurred and increased with increasing concentrations of BcXyl1 from 800 nM to 2 μM after infiltration 3 days, whereas no cell death activity was detected in the leaves treated with PEVC (P. pastoris culture supernatant from an empty vector control strain, purified in the same way as BcXyl1) (Figures 3A,B).

To examined the cell death-inducing activity of BcXyl1 in plants other than N. benthamiana, we infiltrated BcXyl1 (1 μM) into the leaves of several plants, including tomato, soybean, and cotton. BcXyl1 could induce significant cell death in these plants while PEVC did not (Figure 3C). So, BcXyl1 has ability to induce cell death in several plant species.

BcXyl1 has a signal peptide with 20 amino acids and no transmembrane helices, implying that BcXyl1 might be a secreted protein. In order to check if, as previously hypothesized, BcXyl1 was secreted into the apoplast to induce cell death response, we transiently expressed the full length BcXyl1 and BcXyl1^-ΔSP (lacking the signal peptide) in N. benthamiana by agroinfiltration. The results showed that BcXyl1 containing signal peptide induced cell death in N. benthamiana, whereas BcXyl1^-ΔSP lacking signal peptide abolished the ability to trigger cell death at 5 days after agroinfiltration (Supplementary Figure S4A). The protein expression level of BcXyl1 and BcXyl1^-ΔSP in N. benthamiana were detected by immunoblot (Supplementary Figure S4B). So, all results showed BcXyl1 was delivered into the apoplast to induce cell death in several plant species.

Previous reports showed that xylanases from fungi had ability to degrade xylan (Brutus et al., 2005). Interestingly, purified BcXyl1 had a xylanase activity using low viscosity xylane (LVX) as substrate (Supplementary Table S1). The sequence alignment results showed that BcXyl1 included two potentially highly conserved catalytic residues (E104 and E157), which are essential for the xylanase activity (Supplementary Figure S1). In addition, the enzymatic activity of CWDEs was required for cell death activity, and in a few cases, the cell death-inducing activity was found to be independent of the enzymatic activity. To determine the relationship between the enzymatic activity and cell death-inducing activity of BcXyl1, we generated a site-directed mutant (BcXyl1^rec) that two glutamic acid residues were substituted by Gln using site-directed mutagenesis and expressed the mutant protein in P. pastoris (Figure 4A). Enzymatic assays with purified BcXyl1^rec showed the xylan-degrading xylanase activity was abolished (Supplementary Table S1). Surprisingly, although BcXyl1^rec absent the ability of xylanase activity, retained the same cell death-inducing activity as the wild-type (BcXyl1) (Figure 4B). Further, A. tumefaciens infiltration assays showed that BcXyl1^rec and BcXyl1 induced similar visible cell death symptoms in N. benthamiana leaves 4 days post-inoculation (Figure 4B). Western blot assays showed that the accumulation of BcXyl1 and BcXyl1^rec was similar (Figure 4C). These results confirmed that BcXyl1 did not need to xylanase activity to induce cell death in N. benthamiana.

Some cell death-inducing proteins are recognized by plant immune system and activate host PTI responses, bring a series of typical characteristics such as accumulation of ROS, leakage of ion electrolytes, expression of defense genes, and callose deposition (Frías et al., 2012; Zhang et al., 2014, 2015). To examine whether BcXyl1 could induce typical PTI responses, the leaves of N. benthamiana and tomato plants were infiltrated with 1 μM BcXyl1. The ability of BcXyl1 to induce the accumulation of ROS in the infiltrated leaves was studied. The hydrogen peroxide (H₂O₂) and superoxide anion (O_2-) production levels were assayed using DAB and NBT, respectively. A clear brown and blue precipitate was observed in leaves treated with BcXyl1, whereas the leaves treated with PEVC showed opposite patterns of DAB and NBT signal (Figure 5A). Meanwhile, BcXyl1 also induced electrolyte leakage and displayed an increase in conductivity, while PEVC exhibited barely change at the same concentration (Figure 5B). BcXyl1 was shown to cause significantly upregulation of seven genes associated with PTI and defense response in N. benthamiana leaves 12 h after treatment with BcXyl1; these genes included PR-1a and PR-5, which are involved in the SA-dependent defense pathway, PAL (phenylalanine ammonia lyase), NPR1 (the non-expressor of pathogenesis related 1), HSR203J and HIN1, which are two HR marker genes in tobacco, and COI1 (CORONATINE INSENSITIVE 1), which is JA responsive (Figure 5C). We finally examined callose deposition in leaves treated with BcXyl1, PEVC, or flg22. Furthermore, N. benthamiana leaves infiltrated with BcXyl1 or flg22 exhibited strong callose deposition compared with those infiltrated with PEVC, which exhibited undetectable levels of callose deposition (Figure 5D). These data indicated that BcXyl1 could induce typical PTI responses.

Recent reports showed that fungi CWDEs could confer plants disease resistance (Gui Y. et al., 2017; Gui Y.-J. et al., 2017; Zhu et al., 2017). To further confirm the role of BcXyl1 in conferring resistance to plant diseases, the N. benthamiana leaves were treated with 1 μM BcXyl1or PEVC, and after 2 days, the systemic leaves were inoculated with TMV-GFP and B. cinerea spore suspension. BcXyl1-treated tobacco plants enhanced disease resistance against TMV, and the number of TMV-GFP lesions of BcXyl1-treated leaves was significantly decreased than that of the leaves treated with PEVC (Figure 6A). Meanwhile, BcXyl1 led to more resistance to the B. cinerea infection in N. benthamiana, as significantly lower lesions size on leaves compared with the leaves treated with PEVC controls (Figure 6B). Furthermore, in tomato plants that were pre-infiltrated with BcXyl1, lesion size on the B. cinerea-infected leaves was significant smaller compared with lesions size on leaves in plants that were pre-infiltrated with PEVC (Figure 6C). Together, these results strongly suggested that BcXyl1 conferred plants disease resistance.

The plant receptors often recognize specific small protein epitopes of PAMP to induce plant immunity (Rotblat et al., 2002). To delineate the elicitor active peptide of BcXyl1, we generated N-terminal and C-terminal truncated mutants and detected the ability to induce cell death by agroinfiltration in N. benthamiana leaves (Figure 7A). We found that the N-terminal truncated mutant (N¹⁵⁵) maintained the ability of cell death-inducing, whereas expression of N⁸⁰ and N¹³⁰ did not trigger cell death. The C-terminal truncated mutants (C⁸⁰ and C¹³⁰) induced cell death, but C¹⁵⁵ resulted in the loss of cell death-inducing activity in N. benthamiana. Further, C^130-155 induced the same cell death symptom compared with full-length BcXyl1 in N. benthamiana (Figure 7B). Hence, C^130-155 was identified as the functional peptide of BcXyl1 to induce cell death in N. benthamiana. To probe whether C^130-155 induced plant immune responses, purified C^130-155 was used to infiltrate plants leaves. We found that like BcXyl1, C^130-155 could induce typical PTI responses, including accumulation of ROS, leakage of ion electrolytes, expression of defense genes, and callose deposition (Figure 5). Meanwhile, C^130-155 could also enhance resistance to B. cinerea and TMV in plants (Figure 6). These results suggested that a small peptide of BcXyl1 was sufficient for elicitor function.

The plant PRRs, such as the LRR RLKs BAK1 and SOBIR1, were employed to participate in multiple PRR pathways, including cell death induction (Monaghan and Zipfel, 2012; Liebrand et al., 2014; Gravino et al., 2016). For example, BAK1 was required for cell death inducing of GH12 members (Ma et al., 2015; Zhu et al., 2017). As demonstrated above, BcXyl1 was secreted into the apoplast to induce cell death. To determine whether BAK1 and SOBIR1 participated in induction of cell death by BcXyl1, we used virus-induced gene silencing (VIGS) to induce the gene silencing of BAK1 or SOBIR1 in N. benthamiana leaves. Three weeks after viral inoculation to silence BAK1, transient expression of BcXyl1 in N. benthamiana did not result in cell death after agroinfiltration with BcXyl1 expression constructs. Treatment of BAK1-silenced plants with Bcl-2-associated protein X (BAX) was used as a control, which resulted in cell death induction (Figure 8A). The results of SOBIR1-silenced plants were in accordance with BAK1-silenced plants, BcXyl1 did not trigger cell death, while BAX was still capable of inducing cell death (Figure 8A). Immunoblotting confirmed that BcXyl1 were successfully expressed at the expected size in N. benthamiana plants inoculated with TRV::BAK, TRV::SOBIR1, or TRV::GFP (Figure 8B). qPCR analysis confirmed that the expression of BAK1 or SOBIR1 expression was markedly reduced upon inoculation with the TRV::BAK or TRV::SOBIR1, with an expression level about 20% in comparison with inoculation with TRV::GFP (Figure 8C). From these results, we inferred that BAK1 and SOBIR1 (a LRR-RLP/SOBIR1/BAK1 complex) were required for BcXyl1-triggered cell death in N. benthamiana.