A secreted Botrytis cinerea stage-specific effector promotes virulence by targeting the plant ROS-generating machinery

doi:10.21203/rs.3.rs-4918366/v1

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A secreted Botrytis cinerea stage-specific effector promotes virulence by targeting the plant ROS-generating machinery

https://doi.org/10.21203/rs.3.rs-4918366/v1

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Necrotrophic plant pathogens are assumed to exploit the plant hypersensitive response (HR), but the molecular mechanism underlying this exploitation remains largely unclear. Here, we report the discovery and characterization of BcCELP1, an early infection-specific, cell death-inducing effector required for plant colonization by the phytopathogenic fungus Botrytis cinerea. We demonstrate that BcCELP1 is necessary during the initial stage of plant colonization, and that it interacts with the host scaffold protein NbRACK1, promoting NbRACK1’s interaction with the reduced nicotinamide adenine dinucleotide phosphate oxidase NbRBOHB, and thereby contributing to excessive ROS production. We further show that BcCELP1 is produced and specifically leveraged during plant invasion to facilitate the formation of necrotic tissue patches, which serve as foci for subsequent fungal spread. Misregulation of bccelp1 disrupts pathogen development, resulting in reduced disease symptoms. Collectively, these findings reveal an unsuspected sophisticated strategy employed by a necrotrophic pathogen, whereby a fungal effector activates the host ROS-generating machinery in a stage-specific manner to promote effective invasion.

Biological sciences/Plant sciences

Biological sciences/Microbiology/Fungi/Fungal pathogenesis

The co-evolutionary ‘arms race’ between host plants and pathogens over millions of years has culminated in a two-tiered innate immune system in plants, consisting of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI)^1,2. To successfully invade and cause disease in plants, pathogens deploy a large arsenal of effectors that facilitate host colonization by e.g., weakening physical barriers, and suppressing or evading immune perception³. Tight regulation of the timing, level and spatial expression of these effectors is critical for their proper function.

Plant pathogens are typically classified into three categories: biotrophs, which infect living plants and depend on robust immune suppression; necrotrophs, which feed on dead tissue and seek to activate cell death; and hemibiotrophs, which exhibit an initial biotrophic phase followed by a necrotrophic phase^4–6. Recent advances have deepened our understanding of the sophisticated spatial and temporal regulation of effectors, depending on the stage of infection. In the biotrophic smut fungi, Ustilago maydis, which infects maize, the effector Pep1 is secreted during the initial penetration of maize tissues ^7,8. As the later infection progresses, effector Pit2 is deployed to maintain a biotrophic interaction⁹, while the Tin2 effector is utilized following host penetration, within plants cells¹⁰.

The expression of effector coding genes in U. maydis is not only infection stage-specific, but also organ-specific. For example, effector SEE1, which is implicated in proliferation of leaf cells but not immature tassels, plays a role in tumor progression in maize leaves¹¹. Hemibiotrophic pathogens, Colletotrichum higginsianum and Phytophthora sojae, both exhibit waves of effector expression during infection, expressing cell death–suppressing effectors during the biotrophic phase, and cell death–inducing effectors following the switch to necrotrophy^12,13.

While there is clear evidence that biotrophs and hemibiotrophs tailor their effector ‘cocktail’ to specific infection stages and plant organs, few studies have investigated how effectors are transcriptionally regulated over time in necrotrophic pathogens¹⁴. Unlike the diverse range of effectors used by biotrophic pathogens, necrotrophic pathogens primarily employ effectors that induce host cell death⁴. Previously, necrotrophic fungal plant pathogens were thought to cause host cell death through relatively simple mechanisms involving the secretion of phytotoxic molecules and the degradation of plant cell walls. However, accumulating evidence suggests that necrotrophic pathogens interact with their hosts in a more nuanced and intricate manner, extending beyond mere induction of cell death¹⁵.

Botrytis cinerea is one of the most notorious and ubiquitous necrotrophic fungal phytopathogens, known for its broad host range. Capable of infecting hundreds of plant species, B. cinerea causes economic losses worldwide, affecting both pre- and postharvest agriculturally important crops^16,17. Currently, there is no complete genetic resistance to B. cinerea in host cultivars^18,19 and control of the disease primarily relies on chemical fungicides, which not only pose an environmental hazard, but are also increasingly compromised by the development of resistance^20,21. The main driver of B. cinerea infection has long been considered to be the massive secretion of plant cell wall degrading enzymes (PCWDEs) and phytotoxins. However, more recent studies have uncovered a more complex infection process: B. cinerea early stage infection, during which local necrotic lesions are formed, and later stage infection characterized by fast-spreading lesions. Research into the disease dynamics further identified an intermediate stage bridging the transition from local infection to lesion spread²².

As a typical necrotrophic filamentous fungus, B. cinerea secretes various cell death-inducing proteins (CDIPs), many of which have been identified^15,23–30. A proposed model suggests that B. cinerea utilizes these CDIPs to establish initial local infection sites while avoiding early detection by plant defense agents³¹. In addition to these agents, it has also been hypothesized that B. cinerea manipulates the plant’s regulated cell death (RCD) machinery to promote both local and spreading host cell death. This hypothesis is supported by evidence showing that the hypersensitive response (HR), a plant defense against biotrophic pathogens, is necessary for B. cinerea infection^15,32. Moreover, B. cinerea mutants that overexpress HR-inducing protein 1 (Hip1) display enhanced virulence³², whereas plants expressing of anti-apoptotic genes can block RCD and prevent B. cinerea infection³³. Despite these insights, the precise mechanisms by which putative pathogen-derived activators target host plant HR remain to be disclosed.

Effector-like proteins with CELP0023 domains are widely distributed in fungi, including Blumeria graminis, Exserohilum turcicum, Stemphylium lycopersici³⁴. However, the roles of these CELP0023 domain-containing proteins in fungal interactions with host plants are largely unknown. Here we identify BcCELP1, a B. cinerea cell death-inducing effector containing a CELP0023 domain and crucial for plant invasion. We show that BcCELP1 interacts with the RACK1-RBOHB protein complex, manipulating ROS production by the host plant to facilitate infection. Collectively, our results demonstrate the orchestrated expression of a B. cinerea effector that specifically mediates plant invasion by modulating the plant ROS production system.

Strains and the growth of plants

Botrytis cinerea B05.10 and derived transgenic strains were routinely cultured on potato dextrose agar (PDA, Acumedia) and maintained at 22°C under continuous fluorescent light, supplemented with near UV (black) light. For transgenic strains, the medium was modified by adding 100 µg/ml hygromycin B (Calbiochem) and/or 100 µg/ml Nourseothricin (Sigma-Aldrich). Escherichia coli strains DH5α and BL21 (DE3) were used for plasmid construction and protein expression, respectively. Agrobacterium tumefaciens strain GV3101 was used for A. tumefaciens-mediated transient expression of proteins in N. benthamiana leaves. All bacteria were grown on LB agar plates or in LB liquid medium supplemented with 100 µg/ml ampicillin, 50 µg/ml kanamycin, and 50 µg/ml rifampicin at 37°C for E. coli strains (DH5α and BL21) and 28°C for A. tumefaciens (GV3101). French bean (Phaseolus vulgaris L. genotype N9059), N. benthamiana, A. thaliana (ecotype Columbia-0), tomato (Solanum lycopersicum) cv. Hawaii 7998, and maize (Zea mays) cv. silver queen were grown in a climate-controlled growth chamber at 20°C for A. thaliana and 25°C for all other plant species, with a 16-h/8-h light/dark cycle.

Bioinformatics analysis

The B. cinerea genes sequences were retrieved from the B. cinerea genomic sequence database (https://mycocosm.jgi.doe.gov/Botci1/Botci1.home.html). The presence of N-terminal signal peptides was predicted using the SignalP 5.0 server (http://www.cbs.dtu.dk/services/SignalP/)³⁵, while TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/)³⁶ was used for the prediction of transmembrane helices in proteins. Homologous sequences of BcCELP1 in other species were identified by querying the protein sequence against the NCBI database from the genome of the corresponding species, using BLASTp search programs (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with default settings. Multiple Sequence Alignment (MSA) analysis was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/), and the results were visualized with MView Version 1.63³⁷.

Plasmid construction

The primers used in this study are listed in Supplementary Table S1. All plasmids were sequence-verified prior to further transformations. All amplicons were then cloned into linearized vectors using an E. coli DH5α-mediated DNA assembly method. Binary plasmids based on the 2 × 35S-MCS‐eGFP (pCNG) vector were used for transient gene expression in N. benthamiana³⁸. Full-length target genes were cloned from B. cinerea cDNA using gene-specific primers and Phusion High-Fidelity DNA Polymerases (NEB). Truncated mutant variants were generated and site-directed PCR mutagenesis was conducted on the targeted sequences according to specific requirements. To generate B. cinerea mutant strains, several plasmids were constructed: 1) To delete the bccelp1 gene, the 5’- and 3’-homologous flanks (500 bp) of the targeted gene were amplified and fused to each side of a hygromycin-resistance cassette, producing the bccelp1 deletion plasmid, pTZ-Δbccelp1; 2) To construct the bccelp1 complementation plasmids, a fused fragment containing the 1,000 bp upstream regulatory region and the coding region of bccelp1 was amplified by PCR and cloned into vector pNAN-OGG, which contains a nourseothricin resistance cassette³⁹; 3) To generate fungal strains that express bccelp1 under the control of the bcspl1 promoter, the bccelp1 clone was introduced into pNAN-OGG, between the bcspl1 promoter and B. cinerea Tgluc terminator; 4) To generate the bccelp1 over-expression vector, the full-length ORF of bccelp1 was placed under the control of the H2B promoter in the pH2G vector; 5) To express and purify the protein, the coding sequence excluding the SP (Signal peptide) was amplified and cloned into pET-14b (+) (Novagen), generating the expression vector pET14b-6xHis-bccelp1.

DNA and RNA manipulation

Genomic DNA and total RNA extraction was performed using Extract-N-Amp™ Tissue PCR Kits (Sigma/aldrich) and TRIzol reagent (Sigma/Invitrogen). For cDNA synthesis, total RNA was digested with DNase I (Thermo Scientific) and first-strand cDNA was synthesized from 1 µg of DNA-free RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). This was followed by quantitative reverse transcription-PCR (qRT-PCR) with SYBR™ Green PCR Master Mix using StepOne (Applied Biosystems) Real-time PCR instruments. Relative expression levels were determined using the 2^− ΔΔCT method with three independent biological replicates. The relative fold change in mRNA levels was normalized to B. cinerea bcgpdh (BC1G_05277) for the B.cinerea samples and NbActin for N. benthamiana samples.

A. tumefaciens -mediated transient expression

The constructs were introduced into A. tumefaciens strain GV3101 through electroporation. After selection via selective antibiotics (50 µg/ml of kanamycin and 50 µg/ml of rifampin), individual colonies verified by PCR were cultured in LB liquid medium at 28°C in a shaking incubator at 220 rpm for 24 h. The bacteria were harvested by centrifugation (2,000 × g for 5 min) and resuspended in MES buffer (10 mM MgCl₂, 10 mM MES, 200 µM acetosyringone, pH 5.7) at room temperature (RT) for 3 h before infiltration. N. benthamiana plants, grown for 4–5 weeks in the greenhouse, were infiltrated using a needleless syringe with A. tumefaciens cell suspension adjusted to a final OD600 of 0.5. Infiltrated leaves were photographed five days after infiltration. Expression of all proteins was verified by western blot analysis 2–3 d after agroinfiltration.

SDS PAGE and Western blotting

For protein detection following agroinfiltration, protein samples from agroinfiltrated plants were ground in lysis buffer [20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM NaF, 2 mM Na₃VO₄, 1 mM dithiothreitol (DTT), 0.5% Triton X-100, 10% glycerol and 1 × protease inhibitor cocktail]. The extract was centrifuged at 12,500 × g for 20 min at 4°C and the supernatant was transferred to a new tube. The samples were then boiled for 15 min at 95°C with 4 × SDS protein loading buffer, loaded onto a gel for SDS polyacrylamide gel electrophoresis, and protein was detected by immunoblotting with the indicated antibodies.

Plant defense response assay

To assess the plant defense-inducing activity of BcCELP1, N. benthamiana plants were agroinfiltrated with constructs, including pCNG-BcCELP1 (35S: BcCELP1), pCNG-CELP domain (35S: CELP domain), pCNG-BcCrh1 (35S: BcCrh1) and pCNG-EV (35S: GFP). For quantitative analysis of reactive oxygen species (ROS) accumulation and callose deposition, N. benthamiana leaves were sampled 48 h post-agroinfiltration with analysis conducted as previously described ²⁴. Total RNA was extracted from the leaves of N. benthamiana at 48 h after agroinfiltration, and qRT-PCR analysis was performed to measure plant defense-related marker gene expression levels. Meanwhile, the agroinfiltrated area was inoculated with B. cinerea mycelia plugs at 48 h after agroinfiltration.

RNA preparation and RNA-Seq analysis

Transcriptome analysis was conducted on N. benthamiana plants transiently expressing BcCELP1 as follows: Total RNA was extracted from N. benthamiana leaves 3 days post-agroinfiltration from both the transient GFP expression group and the transient BcCELP1 expression group using the Spin Column Plant total RNA Purification Kit, according to the manufacturer’s protocol (Sangon Biotech, Shanghai, China). cDNA libraries were constructed and sequenced on the Illumina HiSeq platform (Illumina Inc., San Diego, CA, USA) by Wuhan Gene Read Biotechnology Co. Ltd, Wuhan, China (www.genereadtech.com/). fastp (v.0.23.2) was used to remove low-quality reads and adaptors from the raw RNA-Seq sequences. The trimmed sequenced reads were then mapped to the N. benthamiana reference genome (https://bioweb01.qut.edu.au/benthTPM/download.html) using HISAT2 v.2.2.1, and the expression level was calculated using featureCounts v.2.0.1. The gene expression level was determined according to the FPKM. Differentially expressed genes (DEGs) analysis between transient GFP and BcCELP1 expression groups was performed using the DESeq2 v.1.20 package^40,41, with a cutoff criteria of |log₂Fold Change| ≥ 1 and FDR (false discovery rate) < 0.05. The resulted DEGs were subjected to GO terms and KEGG pathways GSEA (Gene Set Enrichment Analysis) using clusterProfile v.4.2.0⁴². Three biological repeats were used for the transient GFP expression group and two biological repeats were used for the transient BcCELP1 expression group in this transcriptome analysis.

Expression and purification of proteins

Recombinant proteins were produced in E. coli strain BL21 (DE3) cells containing the pET-14b (+) expressing vector. BL21 cells were grown in LB medium at 37°C until OD600 reached 0.5–0.8, then cultures were induced with 0.2 mM isopropyl-1-thio-b-D-galactopyranoside (IPTG) and incubated at 16°C for 18 h. Cell pellets were collected by centrifugation at 5000 g for 15 min, then resuspended in 50mM Tris-HCl buffer (pH 8.0) containing 0.5M NaCl, 20 mM imidazole and protease inhibitors (complete EDTA-free tablets, one tablet per 50 ml, Roche), followed by sonication and centrifugation at 23,000 × g for 30 min at 4°C for 30 min. Histidine-tagged proteins were purified from cleared lysate using Ni-NTA resin (Thermo Scientific, Waltham, MA, USA). The protein was further cleaned and concentrated using a 10-kDa molecular weight cut-off Amicon Ultra centrifugal filter (Millipore).

Leaf infiltration assay with purified proteins

To test the cell death-inducing activity of the recombinant proteins, an infiltration experiment was carried out using leaves from N. benthamiana, S. lycopersicum, and Z. mays. Purified proteins were infiltrated into the leaves using a 25 µM protein solution. Infiltrated leaves were photographed 2–3 days after treatment.

Generation of transgenic mutant strains of B. cinerea and phenotypic assays of the transformants

PEG-mediated protoplast transformation of B. cinerea was performed according to published protocols⁴³. Details of the transgenic strains are provided in Supplementary Table S3. For each mutant strain, at least three independent single spore isolates were obtained from independent colonies. The mutants were identified by diagnostic PCR using specific primer pairs detailed in Supplementary Table S1. The characterization of the transformants, including mycelial growth rates, conidial germination, and spore and sclerotium development was conducted as previously described⁴⁴. Pathogenicity assays were performed on leaves of French bean (P. vulgaris) and N. benthamiana as described previously²⁴. For inoculation of bean leaves, 7.5 µl of conidia suspensions (2 × 10⁵ spores ml^− 1 in GB5 minimal medium with 2% glucose) were placed on the first two primary leaves of 10-day-old plants and the plants were incubated in a closed box with 100% humidity at 22°C. Lesion diameter was measured at 48 hpi and 72 dpi.

Fluorescence and confocal microscopy

To localize GFP-labeled BcCELP1 during infection, conidia were suspended in PDB medium, and 10 µl droplets of the spore suspension, containing 1 × 10⁴ conidia ml^− 1, were placed on onion epidermal cells and incubated at 20°C under moist conditions. Samples were prepared for microscopy observation at designated time points. Confocal microscopy was performed with a Zeiss LSM780 confocal microscope system and ZEISS ZEN 3.0 (blue edition) imaging software as described previously²⁴. Briefly, eGFP, mCherry and YFP fluorescence were collected using excitation laser wavelengths of 488 nm, 561 nm and 510 nm, respectively. eGFP, mCherry and YFP emissions were collected selectively in the range of 493–535 nm, 560–615 nm and 530 nm. Imaging for the detection of aniline blue-stained callose was performed on a Zeiss Axio imager M1 microscope and Carl Zeiss AxioVision Rel. 4.8 Software. A UV laser (405 nm and 430–550 nm excitation and emission, respectively) was used for visualization of Aniline blue-stained callose.

Co-immunoprecipitation (Co-IP) and LC-MS/MS analysis

Total proteins were extracted from N. benthamiana leaves using a cell lysis buffer for IP (Beyotime, Haimen, China, P0013) 2 days after agroinfiltration. Samples were incubated on ice for 30 min, centrifuged at 13,000 g for 15 min (4°C), and the supernatant was collected and filtered through a 0.45 µm filter. 10 µl of anti-GFP agarose beads (chromotek) were incubated with 5 mL of the filtered supernatant at 4°C on a rotary shaker overnight (12 h). The samples were centrifuged at 1,000 g for 5 min at 4°C, and the beads were collected and washed 5 times, each with 1 ml of cold IP buffer. To elute the bound proteins from the beads, the samples were boiled with 1 × SDS loading buffer at 100°C for 10 min and the proteins were separated by SDS-PAGE (12% separating gel, 80 V for 15 min, then 120 V for 15 min). The gel was stained with Coomassie brilliant blue dye, and cut into 1 mm³ pieces. The gel cubes were then decolorized by incubation in 50 mM ammonium bicarbonate/acetonitrile (1:1, v/v) solution, and the proteins were digested with trypsin in preparation for Nano LC-MS/MS Analysis. The raw MS data were analyzed and searched against the target protein database using Maxquant (1.6.2.10). Only peptides identified with high confidence (with enzyme specificity set to trypsin; maximum missed cleavages set to 2; precursor ion mass tolerance set to 20 ppm, and MS/MS tolerance 20 ppm) were chosen for downstream protein identification analysis. To confirm the interaction between BcCELP1 and NbRACK1, a Co-IP assay was conducted as follows: pCNG-BcCELP1, pCNG-EV and pCNF3-NbRACK1 constructs were transiently co-expressed by agroinfiltration of 4–5 week-old N. benthamiana leaves. Total proteins were extracted from leaves using a cell lysis buffer, and subjected to anti-GFP agarose beads IP (chromotek) as previously described⁴⁵. Eluted proteins were subjected to immunoblot analysis using anti-GFP antibodies and anti-FALG antibodies. Total proteins were loaded as an input control.

Yeast two-hybrid analyses

To verify protein interaction, bait (pGBKT7) and prey (pGADT7) plasmids were co-transformed into the yeast strain AH109 using the LiAc/SS carrier DNA/PEG method, following the manufacturer’s instructions. Selective dropout (SD)/‐Trp‐Leu (DDO) medium was used to screen the desired plasmids (pGBKT7 and pGADT7) harboring yeast transformants. Yeast cells were further validated on SD/‐Trp‐Leu‐His‐Ade medium supplemented with 20 µg /ml X‐α‐galactosidase (X‐α‐gal) and 100 ng/ml aureobasidin A (AbA) (QDO/X/A). Yeast plates were kept in 28°C growth chambers for 4 days before imaging.

BcCELP1 is a novel cell death-inducing protein

Bcin09g02160 was identified as a cell death-inducing protein (CDIP) within the secretome of B. cinerea at an early infection stage (28hpi)²³ (Fig. 1a, b). A BLASTp search of the predicted Bcin09g02160 protein against the NCBI database showed homology to the CELP0028-effector-like protein from Blumeria graminis³⁴. Accordingly, we named the putative protein BcCELP1. Multiple Sequence Alignment (MSA) revealed the presence of a conserved fungal CELP0023 domain between amino acids 84–251 (Supplementary Fig. 1a). A phylogenetic tree constructed using the MSA result showed that CELP0023 domain-containing proteins are widely distributed across fungi, with many found in plant pathogens (Supplementary Fig. 1b). Further bioinformatics analysis using SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP/) indicated the presence of an N-terminal secretion signal peptide (SP) within amino acids 1–19 (Supplementary Fig. 1c). The functionality of this SP was confirmed by a yeast signal sequence trap assay, which demonstrated that the SP of BcCELP1 was sufficient for the secretion of invertase in a yeast system (Supplementary Fig. 1d). This finding is consistent with the detection of the BcCELP1 within the fungal secretome²³.

Cytoplasmic localized BcCELP1 triggers plant cell death

Agroinfiltration of N. benthamiana leaves with 35S: BcCELP1 triggered local cell death within five days post inoculation (Fig. 1a, b). Infiltration of N. benthamiana leaves with agrobacterium expressing the bccelp1 without the secretion signal (BcCELP1^20–251) induced cell death symptoms comparable to those induced by the full length BcCELP1, suggesting that BcCELP1 is targeted to the cytoplasmic space, where it induces cell death (Fig. 1a, b). In line with these results, BcCELP1-GFP fusion protein accumulated inside plant cells in these leaves. By contrast, in control leaves treated with agrobacterium expressing GFP fused to the PR3 signal peptide, the fluorescent signal was observed in the apoplastic space (Fig. 1c). Hence, BcCELP1 must enter the plant cell to induce cell death, similar to the previously characterized cytoplasmic CDIP BcCrh1²⁴.

The BcCELP1 CELP0028 domain is sufficient for induction of plant cell death

To identify the specific epitope responsible for cell death-inducing activity, we generated truncated mutant variants of BcCELP1 and tested their ability to induce cell death through agroinfiltration of N. benthamiana. The CELP0028 domain (residues 84–251) proved to be sufficient for full cell death-inducing activity, and deletion of this domain resulted in a complete loss of activity (Fig. 1a, b). MSA revealed the presence of four conserved cysteine residues within the CELP0028 domain at positions 91, 201, 234, and 244 (Supplementary Fig. 1a). ConSurf and alphafold2 analyses predicted functional roles for the cysteine residues at positions 234 and 244 (Supplementary Fig. 2a, b). Consistent with these predictions, substitutions of both cysteine residues with alanine (C234A/C244A) abolished the protein’s cell death-inducing activity (Fig. 1a, b). Immunoblot analysis of protein extracts from agroinfiltrated N. benthamiana leaves showed degradation of the full length BcCELP1 but not of the CELP0028 domain alone (Supplementary Fig. 3), suggesting that the protein is degraded in planta and that this degradation is mediated by the N’ variable region of the protein.

To determine whether the other diverse CELP0023 domain-containing fungal proteins can also trigger cell death in N. benthamiana, we tested Slerotinia sclerotiorum SsCELP1, Colletotrichum higginsianum ChCELP1 and Fusarium grminearum FgCELP1 by agroinfiltration. Among these proteins, only SsCELP1 triggered cell death symptoms similar to those caused by BcCELP1 (Supplementary Fig. 4). A purified protein infiltration assay showed that both BcCELP1 and the CELP0028 domain alone caused comparable cell death in N. benthamiana and tomato leaves. Consistent with findings reported for other B. cinerea CDIPs, BcCELP1 did not promote necrosis in monocots (Supplementary Fig. 5).

BcCELP1 triggers plant immunity responses

Infiltration of N. benthamiana leaves with agrobacterium expressing bccelp1 resulted in the accumulation of ROS (Fig. 2a), callose deposition (Fig. 2b), and elevated transcription of defense-related marker genes (Fig. 2c). These responses were similar to those observed following treatment with the cytoplasmic CDIP BcCrh1²⁴. Furthermore, agroinfiltration of N. benthamiana leaves with either the full length bcclep1 or the CELP domain fragment reduced disease symptoms compared to control leaves (Fig. 2d). Thus, similar to the majority of other B. cinerea CDIPs, BcCELP1 not only induces plant cell death, but also triggers plant defense responses.

BcCELP1 is required for B. cinerea pathogenicity

To determine whether BcCELP1 is necessary for fungal pathogenic development, we generated bccelp1 deletion mutants and tested their pathogenicity. The Δbccelp1 deletion strain was unaffected in terms of hyphal growth rate (Fig. 3a), spore germination, and infection cushion formation (Supplementary Fig. 6), but showed defects in sporulation and sclerotia formation (Fig. 3b, c). The mutant was slightly less pathogenic than the wild type strain, as indicated by statistically significant reductions in lesion size at 48 hpi and 72 hpi (Fig. 3d, e). Complementation of the deletion mutant with bccelp1 under its native promoter, as well as overexpression of bccelp1, recovered the wild-type phenotype (Fig. 3). The mutant OE-BcCELP1-GFP restored the virulence defect of the deletion mutant, confirming the normal biological function of the BcCELP1-GFP fusion protein. An onion epidermis infection assay with the OE-GFP overexpression strain demonstrated that the free GFP protein was localized to the cytoplasm in the fungus (Fig. 4a). By contrast, during tissue colonization, the BcCELP1-GFP protein was secreted from hyphal tips and diffused into the apoplastic space surrounding the infection site (Fig. 4b, c). Collectively, these results show that BcCELP1 is necessary for full virulence of the fungus and that the protein is specifically secreted into the apoplastic space during plant invasion.

The timing of transcriptional activation of bccelp1 is indispensable for its virulence function

Consistent with the presence of the BcCELP1 in the early secretome, expression levels of the bccelp1 gene increased almost 90-fold at 24 hpi and 20-fold at 36 hpi, before sharply decreasing to basal expression levels (Supplementary Fig. 7). To determine whether the temporal activation of bccelp1 is required for its role in pathogenicity, we generated a strain that expresses the BcCELP1 protein under the control of the bcspl1 promoter, which is specifically upregulated during the late infection stage (Supplementary Fig. 8). The bccelp1 deletion strain complemented with bccelp1 expressed under the bcspl1 promoter remained hypovirulent, like the parental Dbccelp1 deletion strain (Fig. 3d, e). This finding indicates that BcCELP1 is specifically required for pathogenicity at the early infection stage.

The CELP0028 domain is sufficient for pathogenicity

Complementation of the Dbccelp1 mutant with the CELP0028 domain under the control of the bccelp1 promoter completely restored the pathogenicity defect of the Dbccelp1 mutant, unlike complementation with the mutated CELP0028^C234AC244A, which failed to restore this defect (Fig. 5). These results show that the functional CELP0028 domain is both sufficient and essential for the role of BcCELP1 in the pathogenicity of B. cinerea.

BcCELP1 interacts with the scaffold protein NbRACK1

To gain insight into the plant processes affected by BcCELP1, we performed immunoprecipitation (IP) combined with LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) to identify potential plant targets of BcCELP1. The IP-LC-MS/MS results indicated that the N. benthamiana scaffold protein NbRACK1 interacts with BcCELP1 (Supplementary Table S2). Mapping the interaction domains between BcCELP1 and NbRACK1 using a yeast two-hybrid interaction assay revealed that both the full-length BcCELP1 and the truncated CELP1 domain were able to interact with NbRACK1 and a truncated NbRACK1-WD12 (Fig. 6a). The conserved cysteine residues of BcCELP1 proved not to be required for interaction with NbRACK1, as interaction was still observed between the BcCELP1^C234AC244A mutant and NbRACK1 (Fig. 6a). Similarly, the S. sclerotiorum SsCELP1 protein was found to physically associate with NbRACK1 (Fig. 6a). Finally, we verified the interaction between BcCELP1 and NbRACK1 in planta using Co-IP and BiFC assays (Fig. 6b, c).

NbRACK1 is required for BcCELP1 cell death-inducing activity.

We transiently expressed BcCELP1 in an N. benthamiana plant in which NbRACK1 was silenced using VIGS and monitored the extent of BcCELP1-triggered cell death. The cell death-inducing activity of BcCELP1 was significantly compromised in NbRACK1-silenced plants, indicating that NbRACK1 is functionally necessary for BcCELP1-triggered cell death in N. benthamiana (Supplementary Fig. 9).

To further explore the virulence mechanism of BcCELP1, we transiently expressed BcCELP1 in N. benthamiana and performed RNA sequencing (RNA-Seq) on GFP- (control) and BcCELP1-treated plants. Analysis of the RNA-Seq data identified 8,840 DEGs between the BcCELP1- and GFP- (control) treated leaves. Of these, 5,393 DEGs were downregulated and 3,447 upregulated (Supplementary Fig. 10a). We validated the RNA-Seq results by qPCR analysis of six DEGs (Supplementary Fig. 10b).

GO GSEA (Gene Set Enrichment Analysis) showed enrichment of activated genes, including GO terms ‘GO: 0042542 - response to hydrogen peroxide’, ‘GO: 0000302 - response to reactive oxygen species’ and ‘GO: 0006979 - response to oxidative stress’. The down-regulated genes were enriched in GO terms ‘GO: 0006270 - DNA replication initiation’, ‘GO: 0000079 - regulation of cyclin-dependent protein serine/threonine kinase activity’, and ‘GO: 0000280 - nuclear division’ (Supplementary Fig. 10c).

KEGG-GSEA (Gene Set Enrichment Analysis) also showed that the activated genes were enriched in KEGG terms ‘ko03050 - Proteasome’, ‘ko04136 - Autophagy - other’, ‘ko04016 - MAPK signaling pathway – plant’, and ‘ko04626 - Plant-pathogen interaction’. Down-regulated genes were enriched in KEGG terms ‘ko00195 - Photosynthesis’, ‘ko00710 - Carbon fixation in photosynthetic organisms’, and ‘ko03030 - DNA replication’ in the transient BcCELP1 expression lines (Supplementary Fig. 10d). These results indicate that BcCELP1 activates the plant defense, in particularly the ROS-generating pathway(s), and simultaneously causing growth arrest, a response typical for stress-response situations, including during defense against pathogen attack⁴⁶.

BcCELP1 promotes RACK1-RBOHB interaction

As RACK1A is localized inside the plant cell⁴⁷, the interaction of BcCELP1 with NbRACK1A indicates that, following secretion of BcCELP1 to the apoplast, the protein is internalized by the plant cells. To investigate this further, we examined the subcellular localization of BcCELP1-GFP when co-expressed in planta with NbRACK1-RFP using confocal microscopy. The GFP signal of BcCELP1-GFP overlapped with the RFP signal of NbRACK1-RFP in the cytoplasm and cell membrane. Notably, NbRACK1 predominantly co-localized with BcCELP1 at the cell membrane, while in the free GFP control group, NbRACK1 was primarily observed in the cytoplasm, (Fig. 6d).

RACK1 is known to associate with the plant ROS-generating enzyme RBOHB, which is localized at the cell membrane⁴⁸. To test the biological significance of the BcCELP1-NbRACK1 interaction, we examined the impact of BcCELP1 on the stability of the RACK1-RBOHB complex. To this end, we co-expressed NbRACK1-HA and NbRBOHB-FLAG with either BcCELP1-GFP or GFP in N. benthamiana leaves, and analyzed the quantities of the different proteins using immunoblotting. Co-expression of BcCELP1 with NbRACK1 and NbRBOHB resulted in increased abundance of the NbRBOHB-NbRACK1 interaction, and the level of NbRBOHB significantly increased in the presence of BcCELP1 (Fig. 6e), suggesting that BcCELP1 promotes NbRACK1-NbRBOHB interaction in the ROS-generating complex.

Host ROS-generating pathway facilitates B. cinerea plant invasion

The HR is highly effective in blocking biotrophic and hemibiotrophic pathogens, but is less effective, and may even be counterproductive, against necrotrophic pathogens. Interestingly, the expression levels of N. benthamiana and P. vulgaris RACK1 and RBOHB genes were both upregulated following B. cinerea infection (Fig. 7a). Transient co-expression of NbRACK1 and NbRBOHB in N. benthamiana increased the plant’s sensitivity to B. cinerea (Fig. 7b), while A. thaliana rack1 and rbohb T-DNA insertion mutant lines exhibited less sensitivity to infection by the fungus (Fig. 7c).

A recent study showed that the secreted peroxidase BcCcp1 removes plant-derived H₂O₂ and is necessary for ROS detoxification and pathogen invasion⁴⁹. Importantly, the secretion of BcCcp1 is specifically enhanced during the early infection stage at 24 hpi, but decreases at 36 hpi⁴⁹. To investigate whether the ROS detoxification mechanism deployed by BcCcp1 contributes to B. cinerea virulence specifically during the early infection stage, we carried out a pathogenicity assay on the mutants with constitutive expression of bcccp1. Similar to the Δbcccp1 strain, the OE-bcccp1 strains also resulted in smaller lesions than the wild type at 72 hpi (Supplementary Fig. 11). These results further confirm that ROS detoxification by BcCcp1 promotes pathogen invasion in the early infection stage at 24 hpi, while the host ROS-generating process is necessary for following stages of B. cinerea infection.

Collectively, our results show that, unlike the roles of host-derived ROS in defense against biotropic pathogens, necrotrophic pathogens utilize ROS-generating process to destroy host tissues, which facilitates disease progression in an invasion stage-specific manner.

B. cinerea infection is a multi-layered process governed by the exchange of a wide range of factors that collectively determine disease development and severity¹⁸. In addition to PCWDEs and toxins, B. cinerea utilizes a cocktail of CDIPs to facilitate the rapid killing of host cells¹⁵. A previous study on S. sclerotiorum proposes that it is not cell death itself but rather the type of cell death -- whether driven by the host (autophagy) or the pathogen (apoptosis) -- which plays a decisive role in the outcome of a given plant-pathogen interaction^50,51. A similar scenario has been proposed for B. cinerea, based on evidence that this fungus manipulates the plant towards committing death by targeting host HR and PCD machinery, rather than indiscriminately killing its host¹⁹. It is assumed that B. cinerea promotes oxidative bursts and hypersensitive cell death in host plants to facilitate host colonization^52,53. Based on this model, B. cinerea may manipulate the plant regulated cell death (RCD) machinery to facilitate the formation of local lesions^15,17,54, and effectors that target the plant RCD machinery are likely to be involved in disease progression.

In this study, we have identified a novel virulence effector, BcCELP1, specific to the early infection stage of B. cinerea. BcCELP1 promotes early invasion by targeting the RACK1-RBOHB protein complex and manipulating the RCD machinery of the host. The HR, which involves the generation of ROS and activation of RCD processes, is considered to be one of the most crucial factors in hindering invasion of biotrophic and hemibiotrophic pathogens⁵⁵. However, HR does not protect hosts against infection by necrotrophic pathogens. Indeed, aggressive necrotrophic microbes, such as B. cinerea and S. sclerotiorum, probably utilize the host HR for rapid colonization. Notably, the level of generation and accumulation of ROS during HR are correlated positively with the growth and spread of B. cinerea^56,57. However, there is also evidence that ROS-mediated HR-like cell death can block B. cinerea at very early stages of infection (4 hpi). It has therefore been proposed that the timing, localization and function of ROS accumulation are critical factors in determining its role in the development of B. cinerea invasion⁵⁸.

As a member of the tryptophan–aspartate repeat (WD repeat) domain-containing proteins, RACK1 (receptor for activated C kinase 1) is strictly conserved across eukaryotes, and acts as a versatile scaffold protein involved in various signaling pathways⁵⁹. In plants, RACK1 is involved in diverse biological processes, including growth, development, phytohormone responses, protein translation, micro-RNA biogenesis and multiple environmental stimuli responses⁶⁰. Emerging evidence indicates that RACK1 also plays key roles in plant innate immunity against biotrophic and hemibiotrophic pathogens. For example, the OsRACK1A–OsRBOHB immune complex and its mediated ROS production are required for immunity of rice plants against Prycularia oryzae and Ustilaginoidea virens^47,48. In this work, we reveal a new pathogenic mechanism of necrotrophs, whereby B. cinerea secretes BcCELP1 into host cells to target NbRACK1A, promoting the NbRACK1A–NbRBOHB module interaction and triggering a ROS burst that increases plant susceptibility to infection. These findings illustrate that, while biotrophic and necrotrophic pathogens have evolved distinct virulence strategies, both types have converged on virulence factors that manipulate a host ROS generation-associated protein complex to promote infection.

One of the most efficient mechanisms employed by plants to combat attacks by biotrophic pathogen is the generation of an oxidative burst that can trigger hypersensitive cell death^61,62. However, the role of host-derived ROS in the interaction of necrotrophic pathogens and plants are complex: ROS can induce host local cell death to block pathogen colonization, acting as signaling molecules to activate the expression of defense-related genes; but they can also elicit the hypersensitive response (HR)⁴⁹. When uncontrolled, this activation of the HR is followed by so-called runaway cell death that facilitates the spreading of host plant cell death^63–66. In this case, cell death induced by HR keeps propagating, which contributes to the spreading invasion of necrotrophic pathogens, such as B. cinerea^15,56 and S. sclerotiorum⁶⁷. A recent study demonstrated that maize catalases played key roles in sugarcane mosaic virus (SCMV) multiplication and infection by catalyzing the decomposition of excess cellular H₂O₂⁶⁸. The substantial enhancement in mulberry's resistance to B. cinerea was accompanied by increased catalase (CAT) activity. Interestingly, we found that BcCELP1 also interacts with NbCAT1 (Fig. 6a), and B. cinerea infection downregulates the catalases expression of N. benthamiana and P. vulgaris (Fig. 7a), underscoring the potentially important role of host ROS-generating pathway manipulation in the invasion strategy of B. cinerea.

Notably, both host plants and their pathogens produce the same types of ROS in the course of their interaction. However, the details underlying the goals and uses of host and pathogen ROS in mediating these interactions remain largely elusive. Previous studies have typically focused on either the plant or the pathogen, without simultaneously considering both organisms' ROS mechanisms. An integrated approach, in which both sides of the coin are taken into account, is expected to advance our understanding of how ROS affect host–pathogen interactions⁶⁹.

Recent evidence has greatly enhanced our understanding of how plant pathogens deploy effectors in a spatial and temporal manner, depending on the stage of infection. In general, obligate biotrophs secrete effectors to suppress plant immune recognition and ensure host cell survival, while hemibiotrophs initially secrete effectors promoting cell survival, but switch to secreting cell death–promoting effectors during later stages of infection⁷⁰. A recent study provides evidence that Botrytis employs unique spatio-temporal penetration mechanics, depending on the actin skeleton⁷¹. However, relatively little detail is available in terms of how the necrotrophic pathogens manipulate effectors during different infection stages in an ingenious spatial and temporal manner. It is hypothesized that necrotrophic pathogens can also deploy stage-specific secreted effectors to manipulate their hosts and facilitate colonization⁷⁰, though the mechanistic details are not fully understood. This hypothesis is supported by some previously identified CDIPs, such as BcXYG1 and BcCrh1, which exhibit specific high expression pattern and local necrosis-inducing activity at the early infection stage.

During the initial infection stage, B. cinerea primarily relies on inducing local necrosis to establish infection foci for the subsequent infection²³. We have uncovered a sophisticated temporal regulation mechanism in which the novel stage- specific CDIP effector BcCELP1 is deployed during the early invasion stage to modulate ROS production in a spatial and temporal manner. Misexpression of bcelp1, that limited to activated at later stage, failed to restore the phenotypic defect of Δbccelp1, highlighting the necessity of appropriate timing of bccelp1 expression to mediate different infection stages. Interestingly, the mutant strain with overexpression of bccelp1 retains normal pathogenicity, suggesting as long as timely activation of bccelp1 during the early phase is sufficient to conserve its function. Previous research suggested that an H₂O₂ burst in the very early stage of infection (4 hpi) in sitiens, the abscisic acid-deficient tomato mutant, contributes to its resistance to B. cinerea. Instead, in the susceptible wild type, H₂O₂ began to accumulate in the mesophyll layer as early as 24 hpi and was associated with the spread of cell death⁵⁸, which is in accordance with the expression levels of the bccelp1 gene increased from 24 hpi. Our results also hint that coordinated expression of specific effector(s) during various stages of invasion is critical, and this regulation mechanism is conserved across a wide range of plant pathogens, from biotrophs to necrotrophs. Nonetheless, the regulatory network controlling the expression of effector genes and secretion of necrotrophic pathogens is yet to be elucidated.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (Grant No. 32372514), Research and Innovation Initiatives of WHPU (Grant No. 2024J02) to K.B. and BARD grant #5261-20C to A.S.

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There is NO Competing Interest.

SupplementaryTable1.ListofPCRprimerssusedinthisstudy.xlsx
Supplementary Table 1. List of PCR primerss used in this study
SupplementaryTable2.SummaryofIPLCMSMSdata.xlsx
Supplementary Table 2. Summary of IP-LC-MS-MS data
SupplementaryTable3.ListofB.cinereastrainsusedinthisstudy.xlsx
Supplementary Table 3. List of B. cinerea strains used in this study
BcCELP1MSNCSupplementaryInformation.docx

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A secreted Botrytis cinerea stage-specific effector promotes virulence by targeting the plant ROS-generating machinery

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Abstract

Figures

Introduction

Materials and Methods

Strains and the growth of plants

Bioinformatics analysis

Plasmid construction

DNA and RNA manipulation

SDS PAGE and Western blotting

Plant defense response assay

RNA preparation and RNA-Seq analysis

Expression and purification of proteins

Leaf infiltration assay with purified proteins

Fluorescence and confocal microscopy

Co-immunoprecipitation (Co-IP) and LC-MS/MS analysis

Yeast two-hybrid analyses

Results

BcCELP1 is a novel cell death-inducing protein

Cytoplasmic localized BcCELP1 triggers plant cell death

The BcCELP1 CELP0028 domain is sufficient for induction of plant cell death

BcCELP1 triggers plant immunity responses

The CELP0028 domain is sufficient for pathogenicity

BcCELP1 interacts with the scaffold protein NbRACK1

BcCELP1 promotes RACK1-RBOHB interaction

Discussion

Declarations

Acknowledgements

References

Additional Declarations

Supplementary Files

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