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

A secreted Botrytis cinerea stage-specific effector promotes virulence by targeting the plant ROS-generating machinery | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A secreted Botrytis cinerea stage-specific effector promotes virulence by targeting the plant ROS-generating machinery Amir Sharon, kai Bi, Ziyao Wang, Xiaofei Nie, Yong Liang, Wenjun Zhu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4918366/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction 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 perception 3 . 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 interaction 9 , while the Tin2 effector is utilized following host penetration, within plants cells 10 . 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 leaves 11 . 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 pathogens 14 . Unlike the diverse range of effectors used by biotrophic pathogens, necrotrophic pathogens primarily employ effectors that induce host cell death 4 . 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 death 15 . 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 spread 22 . 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 agents 31 . 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 virulence 32 , whereas plants expressing of anti-apoptotic genes can block RCD and prevent B. cinerea infection 33 . 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 lycopersici 34 . 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. Materials and Methods 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 1.63 37 . 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. benthamiana 38 . 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-Δ bccelp1 ; 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 cassette 39 ; 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 MgCl 2 , 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 Na 3 VO 4 , 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 |log 2 Fold 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.4.2.0 42 . 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 protocols 43 . 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 described 44 . Pathogenicity assays were performed on leaves of French bean ( P. vulgaris ) and N. benthamiana as described previously 24 . For inoculation of bean leaves, 7.5 µl of conidia suspensions (2 × 10 5 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 × 10 4 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 previously 24 . 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 mm 3 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 described 45 . 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. Results 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. 1 a, b). A BLASTp search of the predicted Bcin09g02160 protein against the NCBI database showed homology to the CELP0028-effector-like protein from Blumeria graminis 34 . 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 secretome 23 . 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. 1 a, 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. 1 a, 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. 1 c). Hence, BcCELP1 must enter the plant cell to induce cell death, similar to the previously characterized cytoplasmic CDIP BcCrh1 24 . 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. 1 a, 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. 1 a, 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. 2 a), callose deposition (Fig. 2 b), and elevated transcription of defense-related marker genes (Fig. 2 c). These responses were similar to those observed following treatment with the cytoplasmic CDIP BcCrh1 24 . 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. 2 d). 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 Δ bccelp1 deletion strain was unaffected in terms of hyphal growth rate (Fig. 3 a), spore germination, and infection cushion formation (Supplementary Fig. 6), but showed defects in sporulation and sclerotia formation (Fig. 3 b, 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. 3 d, 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. 4 a). 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. 4 b, 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 D bccelp1 deletion strain (Fig. 3 d, 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 D bccelp1 mutant with the CELP0028 domain under the control of the bccelp1 promoter completely restored the pathogenicity defect of the D bccelp1 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. 6 a). 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. 6 a). Similarly, the S. sclerotiorum SsCELP1 protein was found to physically associate with NbRACK1 (Fig. 6 a). Finally, we verified the interaction between BcCELP1 and NbRACK1 in planta using Co-IP and BiFC assays (Fig. 6 b, 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 attack 46 . BcCELP1 promotes RACK1-RBOHB interaction As RACK1A is localized inside the plant cell 47 , 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. 6 d). RACK1 is known to associate with the plant ROS-generating enzyme RBOHB, which is localized at the cell membrane 48 . 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. 6 e), 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. 7 a). Transient co-expression of NbRACK1 and NbRBOHB in N. benthamiana increased the plant’s sensitivity to B. cinerea (Fig. 7 b), while A. thaliana rack1 and rbohb T-DNA insertion mutant lines exhibited less sensitivity to infection by the fungus (Fig. 7 c). A recent study showed that the secreted peroxidase BcCcp1 removes plant-derived H 2 O 2 and is necessary for ROS detoxification and pathogen invasion 49 . Importantly, the secretion of BcCcp1 is specifically enhanced during the early infection stage at 24 hpi, but decreases at 36 hpi 49 . 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 Δ bcccp1 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. Discussion B. cinerea infection is a multi-layered process governed by the exchange of a wide range of factors that collectively determine disease development and severity 18 . In addition to PCWDEs and toxins, B. cinerea utilizes a cocktail of CDIPs to facilitate the rapid killing of host cells 15 . 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 host 19 . 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 pathogens 55 . 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 invasion 58 . 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 pathways 59 . In plants, RACK1 is involved in diverse biological processes, including growth, development, phytohormone responses, protein translation, micro-RNA biogenesis and multiple environmental stimuli responses 60 . 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. sclerotiorum 67 . 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 H 2 O 2 68 . 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. 6 a), and B. cinerea infection downregulates the catalases expression of N. benthamiana and P. vulgaris (Fig. 7 a), 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 interactions 69 . 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 infection 70 . A recent study provides evidence that Botrytis employs unique spatio-temporal penetration mechanics, depending on the actin skeleton 71 . 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 colonization 70 , 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 infection 23 . 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 Δ bccelp1 , 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 H 2 O 2 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, H 2 O 2 began to accumulate in the mesophyll layer as early as 24 hpi and was associated with the spread of cell death 58 , 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. 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Müller, T. et al. Plant infection by the necrotrophic fungus Botrytis requires actin-dependent generation of high invasive turgor pressure. bioRxiv (2023). Additional Declarations There is NO Competing Interest. Supplementary Files 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 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4918366","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":345453490,"identity":"16b96d0f-3481-485d-b722-67e4ad4e857c","order_by":0,"name":"Amir Sharon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIie2PsQrCMBCGLwi6WLKeg/oKEdfisyQE6lLQURA0IGR1E8GncHMsFHQRXQtdKkInB910M4IOiqSODvnILUc+/v8AHI6/hCjz/I8l7xcqAT6/vhRWGBR/KGBR6CzWl9tqP6JzuUZy7QhViTPILAqmYrLwtiliEkgGXApVDZi9mFFKRKcISdjKgJeEgtB+S9Mo5KZ32Ex65wj4WCh6sivMKODpCFkSEpMSC4UFKa1HMU/L2nKbtxkPNm2NOYtsSiPtHk2xDm1sZI5nf1ifUnk4XAeW89/gZspmol8Fh8PhcHznDieGTztz+ZPxAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-6045-9169","institution":"Institute for Cereal Crops Improvement","correspondingAuthor":true,"prefix":"","firstName":"Amir","middleName":"","lastName":"Sharon","suffix":""},{"id":345453491,"identity":"db9ccf50-d59d-4789-9690-39291e5cb5f6","order_by":1,"name":"kai Bi","email":"","orcid":"","institution":"WHPU","correspondingAuthor":false,"prefix":"","firstName":"kai","middleName":"","lastName":"Bi","suffix":""},{"id":345453492,"identity":"48754585-cf83-4ba1-af70-a793eb20907d","order_by":2,"name":"Ziyao Wang","email":"","orcid":"","institution":"College of Life Science and Technology, Wuhan Polytechnic University","correspondingAuthor":false,"prefix":"","firstName":"Ziyao","middleName":"","lastName":"Wang","suffix":""},{"id":345453493,"identity":"4919d06b-2a25-4914-8a8f-076eaec5f4cc","order_by":3,"name":"Xiaofei Nie","email":"","orcid":"","institution":"College of Life Science and Technology, Wuhan Polytechnic University","correspondingAuthor":false,"prefix":"","firstName":"Xiaofei","middleName":"","lastName":"Nie","suffix":""},{"id":345453494,"identity":"aa4643f1-2671-4474-915e-8388e8402b0e","order_by":4,"name":"Yong Liang","email":"","orcid":"","institution":"Institute for Cereal Crops Research, School of Plant Sciences and Food Security, Tel Aviv University, Tel Aviv, Israel","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Liang","suffix":""},{"id":345453495,"identity":"4899990b-2526-4382-af59-48b9acf1edce","order_by":5,"name":"Wenjun Zhu","email":"","orcid":"https://orcid.org/0000-0002-9026-7221","institution":"College of Life Science and Technology, Wuhan Polytechnic University","correspondingAuthor":false,"prefix":"","firstName":"Wenjun","middleName":"","lastName":"Zhu","suffix":""}],"badges":[],"createdAt":"2024-08-15 09:20:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4918366/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4918366/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63450816,"identity":"cc4db63e-918f-4063-8ec1-d6f1083fde42","added_by":"auto","created_at":"2024-08-28 09:20:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":857480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe CELP0028 domain is necessary and sufficient for induction of cell death by\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eBcCELP1\u003c/strong\u003e. \u003cstrong\u003ea, \u003c/strong\u003eSchematic presentation of predicted signal peptide and the BcCELP1 domains: SP - green, uncharacterized region - grey, CELP0028 domain - blue. \u003cstrong\u003eb, \u003c/strong\u003eCell death induction assay. \u003cem\u003eN. benthamiana \u003c/em\u003eleaves were infiltrated with Agrobacterium strains transformed with the indicated fragments. Leaves were photographed five days after agroinfiltration. \u003cstrong\u003ec, \u003c/strong\u003eSubcellular localization of GFP-fusion proteins transiently expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Samples were harvested two days after agroinfiltration, submerged for 20 min in 0.8 M mannitol to induce plasmolysis, and then scanned with a confocal microscope. ‘Merged’ represents an overlap of fluorescence and bright field images. White asterisks mark apoplastic space between the cell wall (blue arrow) and plasma membrane (red arrow) in plasmolysed plant cells.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4918366/v1/a30cd331d7e22786e4d2629b.png"},{"id":63450817,"identity":"76309c1d-19f9-4118-948b-edfaa1506a38","added_by":"auto","created_at":"2024-08-28 09:20:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":581964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBcCELP1 triggers plant immunity responses and enhances plant resistance to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. cinerea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eLeaves of \u003cem\u003eN. benthamiana \u003c/em\u003ewere infiltrated with Agrobacterium strains that harbored a construct encoding GFP (35S:GFP, negative control), BcCELP1 (35S: BcCELP1), or the CELP0028 domain (35S:CELP domain). \u003cstrong\u003ea, \u003c/strong\u003eROS accumulation. Two days after agroinfiltration, representative \u003cem\u003eN. benthamiana\u003c/em\u003e leaves were taken and stained with DAB, then decolorized with ethanol and staining intensity was quantified with ImageJ. All data from three independent biological replicates are plotted as black dots. \u003cstrong\u003eb, \u003c/strong\u003eCalose deposition. Two days after agroinfiltration, leaves were harvested and stained with aniline blue, cleared with ethanol, and images were captured under a fluorescent microscope. Callose deposition was quantified as the number of individual depositions per square millimetre using ImageJ. All data from three independent experiments are presented as black dots. Bar = 100 μm. \u003cstrong\u003ec, \u003c/strong\u003eRelative expression of \u003cem\u003eN. benthamiana\u003c/em\u003edefense-related marker genes. Samples were harvested two days after agroinfiltration, then gene expression levels were determined by qRT-PCR and normalized with \u003cem\u003eN. benthamiana\u003c/em\u003e \u003cem\u003eEF-1α\u003c/em\u003e gene. Expression in GFP treatment plants was set as 1 and was compared with expression levels in plants agroinfiltrated with a construct for expression of the \u003cem\u003eB. cinerea \u003c/em\u003eCDIP \u003cem\u003ebccrh1 \u003c/em\u003egene (35S:BcCrh1). Values represent mean ± SD (n = 9) from three independent biological replications and three technical replications. Asterisks represent statistical differences between all other treatments and GFP treatment control (p \u0026lt; 0.01, unpaired two-tailed Student's \u003cem\u003et\u003c/em\u003e tests). \u003cstrong\u003ed, \u003c/strong\u003eInfection assay. Two days after agroinfiltration, leaves were inoculated with \u003cem\u003eB. cinerea\u003c/em\u003e mycelia plugs, the inoculated plants were incubated for an additional 72 h in a moist chamber and then symptoms were photographed and recorded. Graph shows data from three independent biological replications.. Whiskers of the boxplots in (\u003cstrong\u003ea\u003c/strong\u003e), (\u003cstrong\u003eb\u003c/strong\u003e), and (\u003cstrong\u003ed\u003c/strong\u003e) show the minimum and maximum values; center lines of boxplots display the median values; box limits indicate the 25th and 75th percentiles. Different letters indicate statistical differences at \u003cem\u003eP\u003c/em\u003e ≤ 0.01 using one-way ANOVA.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4918366/v1/d8305ee4387487b327daed57.png"},{"id":63451558,"identity":"e4855a73-1d5e-44ee-a2da-628cf76880da","added_by":"auto","created_at":"2024-08-28 09:28:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1542266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBcCELP1 is necessary for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. cinerea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e pathogenicty. a, \u003c/strong\u003eColony morphology, \u003cstrong\u003eb\u003c/strong\u003e, sporulation, and \u003cstrong\u003ec\u003c/strong\u003e, sclerotia formation of \u003cem\u003eB. cinerea \u003c/em\u003ewild type (wt), \u003cem\u003ebccelp1 \u003c/em\u003edeletion (Δ\u003cem\u003ebccelp1\u003c/em\u003e), and the Δ\u003cem\u003ebccelp1\u003c/em\u003e complemented with \u003cem\u003ebccelp1 \u003c/em\u003eunder the native (Com-\u003cem\u003ebccelp1\u003c/em\u003e) and the constitiive H\u003csub\u003e2\u003c/sub\u003eB (OE-\u003cem\u003ebccelp1\u003c/em\u003e) promoters. For morphology (a) and sporulation (b) fungi were cultured on GB5+Glucose medium at 22°C with continuous fluorescent light). Colony diameter (\u003cstrong\u003ea\u003c/strong\u003e) was measured every day for three days and growth rate (mm/d) was calculated. Data show mean ± SD (n = 6) from three independent biological replications. For sporulation (\u003cstrong\u003eb\u003c/strong\u003e), pictures were taken after eight days of incubation and spores were collected and counted. Data represent mean ± SD (n = 7) from three independent biological replications. For sclerotia formation (\u003cstrong\u003ec\u003c/strong\u003e), fungi were cultured on PDA at 22°C under continuous darkness. Pictures were taken after 15 days and sclerotia were counted. Values represent mean ± SD (n = 8) from three independent biological replications. \u003cstrong\u003ed, \u003c/strong\u003eInfection assay. Bean leaves were inoculated with spore suspensions of different \u003cem\u003eB. cinerea \u003c/em\u003estrains, including wt, \u003cem\u003ebccelp1\u003c/em\u003e knock out strain (Δ\u003cem\u003ebccelp1\u003c/em\u003e), \u003cem\u003ebccelp1\u003c/em\u003e complementation strain under the native \u003cem\u003ebccelp1 \u003c/em\u003epromoter (Com-\u003cem\u003e bccelp1\u003c/em\u003e), and \u003cem\u003ebccelp1\u003c/em\u003e overexpression strain (OE-\u003cem\u003ebccelp1\u003c/em\u003e), bccelp1 complementation strain under the \u003cem\u003ebcspl1\u003c/em\u003e promoter (Com-\u003cem\u003ePro\u003c/em\u003e\u003csup\u003e\u003cem\u003ebcspl1\u003c/em\u003e\u003c/sup\u003e-\u003cem\u003ebccelp1\u003c/em\u003e)). Pictures were taken and lesion size recorded at 48 hpi\u0026nbsp; and 72 hpi. Box limits in the graph show the 25th and 75th percentiles. The center lines of boxplots indicate the median values; whiskers extend to minimum and maximum values from the 25th and 75th percentiles; Individual data points are indicated as black dots. At least 16 sample points from three independent biological replications were used for statistical analysis. Different letters indicate statistical differences at \u003cem\u003eP\u003c/em\u003e ≤ 0.01 using one-way ANOVA.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4918366/v1/781c84b0020348f36de2e8ff.png"},{"id":63450828,"identity":"c0115816-1c0d-4f6e-9167-5c46bf0da710","added_by":"auto","created_at":"2024-08-28 09:20:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":847929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSecretion of BcCELP1 during plant infection. \u003c/strong\u003eOnion epidermis was inoculated with droplets of spore suspensions of \u003cem\u003eB. cinerea\u003c/em\u003e strains that express free GFP (OE-GFP) and BcCELP1-GFP fusion protein (OE-BcCELP1-GFP). Samples were collected at 36 hpi and scanned with a confocal microscope. \u003cstrong\u003ea, \u003c/strong\u003eThe GFP signal in the OE-GFP strain is localized in hyphae, \u003cstrong\u003eb, \u003c/strong\u003eThe GFP signal in the BcCELP1-GFP fusion protein is localized in the onion cell near hyphal tips and diffused into spaces surrounding the infection sites.\u003cstrong\u003e \u003c/strong\u003eThe bottom images show enlargement of the boxed area in the top images. Bar = 50 μm in \u003cstrong\u003ea \u003c/strong\u003eand\u003cstrong\u003e b\u003c/strong\u003e. Bar = 20 μm in \u003cstrong\u003ec\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4918366/v1/c8b87fc2881144f7d97800f6.png"},{"id":63450822,"identity":"96ed93ed-5e5d-4f44-8c38-5dca45fe4042","added_by":"auto","created_at":"2024-08-28 09:20:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":529220,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe CELP0028 domain of BcCELP1 is essential and sufficient for its full pathogenicity. \u003c/strong\u003eBean leaves were inoculated with spore suspensions of \u003cem\u003eB. cinerea\u003c/em\u003e wild type (wt), native CELP0028 domain complementation strain (Com-CELP domain), CELP0028 domain with mutation of C234, and C244 complementation strain (Com-CELP\u003csup\u003eC234AC244A\u003c/sup\u003e domain). All the complementation strains were generated from Δ\u003cem\u003ebccelp1\u003c/em\u003e. Pictures were taken and lesion size recorded at 48 hpi (top) and 72 hpi (bottom). At least 16 sample points from three independent biological replications were used for statistical analysis. In box plots, center lines represent the median values, box edges show the 25th and 75th percentiles, whiskers extend to minimum and maximum values from the 25th and 75th percentiles. Individual data points are indicated as black dots. Different letters indicate statistical differences at \u003cem\u003eP\u003c/em\u003e ≤ 0.01 using one-way ANOVA.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4918366/v1/251d3b34b5687d623cbd55bd.png"},{"id":63451557,"identity":"dc0b8a48-83ea-4b84-8197-92a7bcbf372d","added_by":"auto","created_at":"2024-08-28 09:28:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":376131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction of BcCELP1 with the scaffold protein NbRACK1. a, \u003c/strong\u003eYeast two-hybrid assay to identify the interaction of BcCELP1 and NbRACK1. SD/-Trp-Leu medium (SD-LW) was used to confirm the designed transformation events. SD/-Trp-Leu-His-Ade medium (SD-LWHA) containing AbA and X-α-gal was used to screen yeast strains with the positive protein-protein interaction.\u003cstrong\u003e b, \u003c/strong\u003eCo-immunoprecipitation (Co-IP) assay. BcCELP1-GFP or empty-GFP was co-expressed with the NbRACK1-Flag in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. The immune complexes were immunoprecipitated with an α-GFP antibody (α-GFP IP), and the bound protein was detected by immunoblotting with α-FALG antibody. BcCELP1-GFP, but not empty-GFP, co-precipitated with NbRACK1-Flag. \u003cstrong\u003ec, \u003c/strong\u003eBiFC (Bimolecular fluorescence complementation) assay. \u003cem\u003eN. benthamiana\u003c/em\u003e leaves were agroinfiltrated with a fresh mixture of \u003cem\u003eA. tumefaciens\u003c/em\u003e harboring the constructs BcCELP1-YFPn+NbRACK1-YFPc, or the negative controls BcCELP1-YFPn+YFPc and YFPn+NbRACK1-YFPc. The fluorescence of YFP was monitored at 3 d post-agroinfiltration using a confocal microscope. Bars = 20 μm. \u003cstrong\u003ed, \u003c/strong\u003eSubcellular localization of BcCELP1 and NbRACK1 following transient expression in \u003cem\u003eN. benthamiana \u003c/em\u003eepidermal cells. Samples were scanned with a confocal microscope and pictures were taken to monitor the fluorescence of GFP and mCherry at 3 d post-agroinfiltration. Bars = 20 μm. \u003cstrong\u003ee, \u003c/strong\u003eEffect of co-expression of BcCELP1 on abundance of NbRBOHB-NbRACK1interaction. NbRBOHB-Flag and NbRACK1-HA were co-expressed with/without BcCELP1-GFP in\u003cem\u003e N. benthamiana \u003c/em\u003ecells. IP was performed using anti-HA affinity gel, followed by western blot analysis using GFP, HA or Flag antibody. The relative NbRBOHB-Flag band intensities were quantified with ImageJ. Ponceau S staining was used as sample loading control.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4918366/v1/cd9a29c210d6379b770f1173.png"},{"id":63450827,"identity":"b08af51a-8b25-4fbb-9985-a39d9a37e715","added_by":"auto","created_at":"2024-08-28 09:20:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":354554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHost ROS-generating complex RACK1-RBOHB facilitates \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. cinerea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e host invasion. a, \u003c/strong\u003eExpression patterns of \u003cem\u003eRACK1\u003c/em\u003e, \u003cem\u003eRBOHB\u003c/em\u003e and \u003cem\u003eCAT1 \u003c/em\u003efollowing \u003cem\u003eB. cinerea \u003c/em\u003einfection of \u003cem\u003eN. benthamiana \u003c/em\u003eand\u003cem\u003e P. vulgaris\u003c/em\u003e. \u003cem\u003eN. benthamiana \u003c/em\u003eand\u003cem\u003e P. vulgaris \u003c/em\u003eleaves were inoculated with \u003cem\u003eB. cinerea\u003c/em\u003e spore suspension. The inoculated plants were incubated in a moist chamber, samples were harvested two days after inoculation and expression levels of \u003cem\u003eRACK1\u003c/em\u003e, \u003cem\u003eRBOHB\u003c/em\u003e and \u003cem\u003eCAT1\u003c/em\u003e genes were determined by qRT-PCR using the \u003cem\u003eEF-1α\u003c/em\u003e gene as a normalizer. Values represent mean ± SD (n = 9) from three independent biological replications and three technical replicatios. Asterisks represent statistical differences of \u003cem\u003eB. cinerea\u003c/em\u003e inoculated treatment (BC) compared with control treatment plants (Ctr) (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, unpaired two-tailed Student's t tests). \u003cstrong\u003eb, \u003c/strong\u003eInfection assay of \u003cem\u003eB. cinerea \u003c/em\u003eon \u003cem\u003eN. benthamiana \u003c/em\u003eleaves over-expressing the \u003cem\u003eNbRACK1\u003c/em\u003eand \u003cem\u003eNbRBOHB\u003c/em\u003e genes\u003cem\u003e. N. benthamiana \u003c/em\u003eleaves were infiltrated with\u003cem\u003eAgrobacterium \u003c/em\u003estrains that were transformed with the indicated genes. Two days after treatment the leaves were inoculated with \u003cem\u003eB. cinerea\u003c/em\u003e, the inoculated plants were kept for 72 h in a moist chamber, and then symptoms were recorded. The graph shows data from three independent biological replications. \u003cstrong\u003ec, \u003c/strong\u003eInfection assay of \u003cem\u003eA. thaliana \u003c/em\u003eT-DNA insertion mutant lines (\u003cem\u003erack1\u003c/em\u003eand \u003cem\u003erbohb\u003c/em\u003e). \u003cem\u003eA. thaliana \u003c/em\u003eleaves were inoculated with droplets of \u003cem\u003eB. cinerea \u003c/em\u003espore suspension, the plants were incubated in a moist chamber and symptoms were recorded 72 hpi. Images show the representative inoculated leaves and individual plants. Graph shows data from three independent biological replications (n = 22). Whiskers of the boxplots in (\u003cstrong\u003eb\u003c/strong\u003e) and (\u003cstrong\u003ec\u003c/strong\u003e) show the minimum and maximum values; center lines of boxplots display the median values; box limits indicate the 25th and 75th percentiles. T-DNA insertions were confirmed by PCR using a specific primer of the T-DNA border (LBb1.3 primer）and gene-specific primers (LP and RP Supplementary Table 2, LP1/RP1 for SALK_009406.56.00.x (\u003cem\u003erack1\u003c/em\u003e); LP2/RP2 for for SALK_099459.48.45.x (\u003cem\u003erbohb\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4918366/v1/2c53bbc6255cc3022683b26c.png"},{"id":67167563,"identity":"56320a09-333c-4f31-b7bf-e0e476edb86b","added_by":"auto","created_at":"2024-10-22 02:08:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7862126,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4918366/v1/8c428188-1286-455d-9420-e7ad498e8fca.pdf"},{"id":63451559,"identity":"e028f4ee-fa48-4612-ae7b-6c2586c84e01","added_by":"auto","created_at":"2024-08-28 09:28:16","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16578,"visible":true,"origin":"","legend":"Supplementary Table 1. List of PCR primerss used in this study","description":"","filename":"SupplementaryTable1.ListofPCRprimerssusedinthisstudy.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4918366/v1/2ab6fd1bf3c91d35aecd3acf.xlsx"},{"id":63450819,"identity":"e97dcdd3-e3da-464f-99c6-c9954b1982f7","added_by":"auto","created_at":"2024-08-28 09:20:17","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":46929,"visible":true,"origin":"","legend":"Supplementary Table 2. Summary of IP-LC-MS-MS data","description":"","filename":"SupplementaryTable2.SummaryofIPLCMSMSdata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4918366/v1/d4bf8542cb1379985c82da08.xlsx"},{"id":63451560,"identity":"426fc1e1-2e80-4643-83c7-fcc003c61f4a","added_by":"auto","created_at":"2024-08-28 09:28:16","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9773,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 3. List of B. cinerea strains used in this study\u003c/p\u003e","description":"","filename":"SupplementaryTable3.ListofB.cinereastrainsusedinthisstudy.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4918366/v1/879757e248d7fba0eff48240.xlsx"},{"id":63450825,"identity":"d247c902-7137-42e9-8e2d-dac8c12e62b0","added_by":"auto","created_at":"2024-08-28 09:20:17","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":6644527,"visible":true,"origin":"","legend":"","description":"","filename":"BcCELP1MSNCSupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4918366/v1/2efe670fed6ff42b92818d88.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A secreted Botrytis cinerea stage-specific effector promotes virulence by targeting the plant ROS-generating machinery","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe co-evolutionary \u0026lsquo;arms race\u0026rsquo; 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)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. 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 perception\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Tight regulation of the timing, level and spatial expression of these effectors is critical for their proper function.\u003c/p\u003e \u003cp\u003ePlant 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\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. 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, \u003cem\u003eUstilago maydis\u003c/em\u003e, which infects maize, the effector Pep1 is secreted during the initial penetration of maize tissues \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. As the later infection progresses, effector Pit2 is deployed to maintain a biotrophic interaction\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, while the Tin2 effector is utilized following host penetration, within plants cells\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe expression of effector coding genes in \u003cem\u003eU. maydis\u003c/em\u003e 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 leaves\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Hemibiotrophic pathogens, \u003cem\u003eColletotrichum higginsianum\u003c/em\u003e and \u003cem\u003ePhytophthora sojae\u003c/em\u003e, both exhibit waves of effector expression during infection, expressing cell death\u0026ndash;suppressing effectors during the biotrophic phase, and cell death\u0026ndash;inducing effectors following the switch to necrotrophy\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile there is clear evidence that biotrophs and hemibiotrophs tailor their effector \u0026lsquo;cocktail\u0026rsquo; to specific infection stages and plant organs, few studies have investigated how effectors are transcriptionally regulated over time in necrotrophic pathogens\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Unlike the diverse range of effectors used by biotrophic pathogens, necrotrophic pathogens primarily employ effectors that induce host cell death\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. 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 death\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBotrytis cinerea\u003c/em\u003e is one of the most notorious and ubiquitous necrotrophic fungal phytopathogens, known for its broad host range. Capable of infecting hundreds of plant species, \u003cem\u003eB. cinerea\u003c/em\u003e causes economic losses worldwide, affecting both pre- and postharvest agriculturally important crops\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Currently, there is no complete genetic resistance to \u003cem\u003eB. cinerea\u003c/em\u003e in host cultivars\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e 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\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The main driver of \u003cem\u003eB. cinerea\u003c/em\u003e 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: \u003cem\u003eB. cinerea\u003c/em\u003e 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 spread\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs a typical necrotrophic filamentous fungus, \u003cem\u003eB. cinerea\u003c/em\u003e secretes various cell death-inducing proteins (CDIPs), many of which have been identified\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan additionalcitationids=\"CR24 CR25 CR26 CR27 CR28 CR29\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. A proposed model suggests that \u003cem\u003eB. cinerea\u003c/em\u003e utilizes these CDIPs to establish initial local infection sites while avoiding early detection by plant defense agents\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In addition to these agents, it has also been hypothesized that \u003cem\u003eB. cinerea\u003c/em\u003e manipulates the plant\u0026rsquo;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 \u003cem\u003eB. cinerea\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Moreover, \u003cem\u003eB. cinerea\u003c/em\u003e mutants that overexpress HR-inducing protein 1 (Hip1) display enhanced virulence\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, whereas plants expressing of anti-apoptotic genes can block RCD and prevent \u003cem\u003eB. cinerea\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Despite these insights, the precise mechanisms by which putative pathogen-derived activators target host plant HR remain to be disclosed.\u003c/p\u003e \u003cp\u003eEffector-like proteins with CELP0023 domains are widely distributed in fungi, including \u003cem\u003eBlumeria graminis\u003c/em\u003e, \u003cem\u003eExserohilum turcicum\u003c/em\u003e, \u003cem\u003eStemphylium lycopersici\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, the roles of these CELP0023 domain-containing proteins in fungal interactions with host plants are largely unknown. Here we identify BcCELP1, a \u003cem\u003eB. cinerea\u003c/em\u003e 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 \u003cem\u003eB. cinerea\u003c/em\u003e effector that specifically mediates plant invasion by modulating the plant ROS production system.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains and the growth of plants\u003c/h2\u003e \u003cp\u003e \u003cem\u003eBotrytis cinerea\u003c/em\u003e B05.10 and derived transgenic strains were routinely cultured on potato dextrose agar (PDA, Acumedia) and maintained at 22\u0026deg;C under continuous fluorescent light, supplemented with near UV (black) light. For transgenic strains, the medium was modified by adding 100 \u0026micro;g/ml hygromycin B (Calbiochem) and/or 100 \u0026micro;g/ml Nourseothricin (Sigma-Aldrich). \u003cem\u003eEscherichia coli\u003c/em\u003e strains DH5α and BL21 (DE3) were used for plasmid construction and protein expression, respectively. \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 was used for \u003cem\u003eA. tumefaciens\u003c/em\u003e-mediated transient expression of proteins in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. All bacteria were grown on LB agar plates or in LB liquid medium supplemented with 100 \u0026micro;g/ml ampicillin, 50 \u0026micro;g/ml kanamycin, and 50 \u0026micro;g/ml rifampicin at 37\u0026deg;C for \u003cem\u003eE. coli\u003c/em\u003e strains (DH5α and BL21) and 28\u0026deg;C for \u003cem\u003eA. tumefaciens\u003c/em\u003e (GV3101). French bean (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e L. genotype N9059), \u003cem\u003eN. benthamiana\u003c/em\u003e, \u003cem\u003eA. thaliana\u003c/em\u003e (ecotype Columbia-0), tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) cv. Hawaii 7998, and maize (Zea mays) cv. silver queen were grown in a climate-controlled growth chamber at 20\u0026deg;C for \u003cem\u003eA. thaliana\u003c/em\u003e and 25\u0026deg;C for all other plant species, with a 16-h/8-h light/dark cycle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eB. cinerea\u003c/em\u003e genes sequences were retrieved from the \u003cem\u003eB. cinerea\u003c/em\u003e genomic sequence database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mycocosm.jgi.doe.gov/Botci1/Botci1.home.html\u003c/span\u003e\u003cspan address=\"https://mycocosm.jgi.doe.gov/Botci1/Botci1.home.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The presence of N-terminal signal peptides was predicted using the SignalP 5.0 server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbs.dtu.dk/services/SignalP/\u003c/span\u003e\u003cspan address=\"http://www.cbs.dtu.dk/services/SignalP/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e35\u003c/sup\u003e, while TMHMM Server v. 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbs.dtu.dk/services/TMHMM/\u003c/span\u003e\u003cspan address=\"http://www.cbs.dtu.dk/services/TMHMM/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e36\u003c/sup\u003e 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 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with default settings. Multiple Sequence Alignment (MSA) analysis was performed using Clustal Omega (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/Tools/msa/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/Tools/msa/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the results were visualized with MView Version 1.63\u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction\u003c/h2\u003e \u003cp\u003eThe primers used in this study are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. All plasmids were sequence-verified prior to further transformations. All amplicons were then cloned into linearized vectors using an \u003cem\u003eE. coli\u003c/em\u003e DH5α-mediated DNA assembly method. Binary plasmids based on the 2 \u0026times; 35S-MCS‐eGFP (pCNG) vector were used for transient gene expression in \u003cem\u003eN. benthamiana\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Full-length target genes were cloned from \u003cem\u003eB. cinerea\u003c/em\u003e 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 \u003cem\u003eB. cinerea\u003c/em\u003e mutant strains, several plasmids were constructed: 1) To delete the \u003cem\u003ebccelp1\u003c/em\u003e gene, the 5\u0026rsquo;- and 3\u0026rsquo;-homologous flanks (500 bp) of the targeted gene were amplified and fused to each side of a hygromycin-resistance cassette, producing the \u003cem\u003ebccelp1\u003c/em\u003e deletion plasmid, pTZ-Δ\u003cem\u003ebccelp1\u003c/em\u003e; 2) To construct the \u003cem\u003ebccelp1\u003c/em\u003e complementation plasmids, a fused fragment containing the 1,000 bp upstream regulatory region and the coding region of \u003cem\u003ebccelp1\u003c/em\u003e was amplified by PCR and cloned into vector pNAN-OGG, which contains a nourseothricin resistance cassette\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e; 3) To generate fungal strains that express \u003cem\u003ebccelp1\u003c/em\u003e under the control of the \u003cem\u003ebcspl1\u003c/em\u003e promoter, the \u003cem\u003ebccelp1\u003c/em\u003e clone was introduced into pNAN-OGG, between the \u003cem\u003ebcspl1\u003c/em\u003e promoter and \u003cem\u003eB. cinerea\u003c/em\u003e Tgluc terminator; 4) To generate the \u003cem\u003ebccelp1\u003c/em\u003e over-expression vector, the full-length ORF of \u003cem\u003ebccelp1\u003c/em\u003e 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-\u003cem\u003ebccelp1\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDNA and RNA manipulation\u003c/h2\u003e \u003cp\u003eGenomic DNA and total RNA extraction was performed using Extract-N-Amp\u0026trade; 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 \u0026micro;g 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\u0026trade; Green PCR Master Mix using StepOne (Applied Biosystems) Real-time PCR instruments. Relative expression levels were determined using the 2\u003csup\u003e\u0026minus;\u0026thinsp;ΔΔCT\u003c/sup\u003e method with three independent biological replicates. The relative fold change in mRNA levels was normalized to \u003cem\u003eB. cinerea bcgpdh\u003c/em\u003e (\u003cem\u003eBC1G_05277\u003c/em\u003e) for the \u003cem\u003eB.cinerea\u003c/em\u003e samples and \u003cem\u003eNbActin\u003c/em\u003e for \u003cem\u003eN. benthamiana\u003c/em\u003e samples.\u003c/p\u003e \u003cp\u003e \u003cb\u003eA. tumefaciens\u003c/b\u003e \u003cb\u003e-mediated transient expression\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe constructs were introduced into \u003cem\u003eA. tumefaciens\u003c/em\u003e strain GV3101 through electroporation. After selection via selective antibiotics (50 \u0026micro;g/ml of kanamycin and 50 \u0026micro;g/ml of rifampin), individual colonies verified by PCR were cultured in LB liquid medium at 28\u0026deg;C in a shaking incubator at 220 rpm for 24 h. The bacteria were harvested by centrifugation (2,000 \u0026times; g for 5 min) and resuspended in MES buffer (10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM MES, 200 \u0026micro;M acetosyringone, pH 5.7) at room temperature (RT) for 3 h before infiltration. \u003cem\u003eN. benthamiana\u003c/em\u003e plants, grown for 4\u0026ndash;5 weeks in the greenhouse, were infiltrated using a needleless syringe with \u003cem\u003eA. tumefaciens\u003c/em\u003e 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\u0026ndash;3 d after agroinfiltration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSDS PAGE and Western blotting\u003c/h2\u003e \u003cp\u003eFor 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 Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, 1 mM dithiothreitol (DTT), 0.5% Triton X-100, 10% glycerol and 1 \u0026times; protease inhibitor cocktail]. The extract was centrifuged at 12,500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 20 min at 4\u0026deg;C and the supernatant was transferred to a new tube. The samples were then boiled for 15 min at 95\u0026deg;C with 4 \u0026times; SDS protein loading buffer, loaded onto a gel for SDS polyacrylamide gel electrophoresis, and protein was detected by immunoblotting with the indicated antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePlant defense response assay\u003c/h2\u003e \u003cp\u003eTo assess the plant defense-inducing activity of BcCELP1, \u003cem\u003eN. benthamiana\u003c/em\u003e 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, \u003cem\u003eN. benthamiana\u003c/em\u003e leaves were sampled 48 h post-agroinfiltration with analysis conducted as previously described \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Total RNA was extracted from the leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e 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 \u003cem\u003eB. cinerea\u003c/em\u003e mycelia plugs at 48 h after agroinfiltration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRNA preparation and RNA-Seq analysis\u003c/h2\u003e \u003cp\u003eTranscriptome analysis was conducted on \u003cem\u003eN. benthamiana\u003c/em\u003e plants transiently expressing BcCELP1 as follows: Total RNA was extracted from \u003cem\u003eN. benthamiana\u003c/em\u003e 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\u0026rsquo;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 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://mycocosm.jgi.doe.gov/Botci1/Botci1.home.html\" target=\"_blank\"\u003ewww.genereadtech.com/\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.genereadtech.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). 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 \u003cem\u003eN. benthamiana\u003c/em\u003e reference genome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioweb01.qut.edu.au/benthTPM/download.html\u003c/span\u003e\u003cspan address=\"https://bioweb01.qut.edu.au/benthTPM/download.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) 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\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, with a cutoff criteria of |log\u003csub\u003e2\u003c/sub\u003eFold Change| \u0026ge; 1 and FDR (false discovery rate)\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The resulted DEGs were subjected to GO terms and KEGG pathways GSEA (Gene Set Enrichment Analysis) using clusterProfile v.4.2.0\u003csup\u003e42\u003c/sup\u003e. 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eExpression and purification of proteins\u003c/h2\u003e \u003cp\u003eRecombinant proteins were produced in \u003cem\u003eE. coli\u003c/em\u003e strain BL21 (DE3) cells containing the pET-14b (+) expressing vector. BL21 cells were grown in LB medium at 37\u0026deg;C until OD600 reached 0.5\u0026ndash;0.8, then cultures were induced with 0.2 mM isopropyl-1-thio-b-D-galactopyranoside (IPTG) and incubated at 16\u0026deg;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 \u0026times; g for 30 min at 4\u0026deg;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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLeaf infiltration assay with purified proteins\u003c/h2\u003e \u003cp\u003eTo test the cell death-inducing activity of the recombinant proteins, an infiltration experiment was carried out using leaves from \u003cem\u003eN. benthamiana\u003c/em\u003e, \u003cem\u003eS. lycopersicum\u003c/em\u003e, and \u003cem\u003eZ. mays.\u003c/em\u003e Purified proteins were infiltrated into the leaves using a 25 \u0026micro;M protein solution. Infiltrated leaves were photographed 2\u0026ndash;3 days after treatment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration of transgenic mutant strains of\u003c/b\u003e \u003cb\u003eB. cinerea\u003c/b\u003e \u003cb\u003eand phenotypic assays of the transformants\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePEG-mediated protoplast transformation of \u003cem\u003eB. cinerea\u003c/em\u003e was performed according to published protocols\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. 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 \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The characterization of the transformants, including mycelial growth rates, conidial germination, and spore and sclerotium development was conducted as previously described\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Pathogenicity assays were performed on leaves of French bean (\u003cem\u003eP. vulgaris\u003c/em\u003e) and \u003cem\u003eN. benthamiana\u003c/em\u003e as described previously\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. For inoculation of bean leaves, 7.5 \u0026micro;l of conidia suspensions (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e spores ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 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\u0026deg;C. Lesion diameter was measured at 48 hpi and 72 dpi.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence and confocal microscopy\u003c/h2\u003e \u003cp\u003eTo localize GFP-labeled BcCELP1 during infection, conidia were suspended in PDB medium, and 10 \u0026micro;l droplets of the spore suspension, containing 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e conidia ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, were placed on onion epidermal cells and incubated at 20\u0026deg;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 previously\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. 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\u0026ndash;535 nm, 560\u0026ndash;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\u0026ndash;550 nm excitation and emission, respectively) was used for visualization of Aniline blue-stained callose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation (Co-IP) and LC-MS/MS analysis\u003c/h2\u003e \u003cp\u003eTotal proteins were extracted from \u003cem\u003eN. benthamiana\u003c/em\u003e 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 \u003cem\u003eg\u003c/em\u003e for 15 min (4\u0026deg;C), and the supernatant was collected and filtered through a 0.45 \u0026micro;m filter. 10 \u0026micro;l of anti-GFP agarose beads (chromotek) were incubated with 5 mL of the filtered supernatant at 4\u0026deg;C on a rotary shaker overnight (12 h). The samples were centrifuged at 1,000 \u003cem\u003eg\u003c/em\u003e for 5 min at 4\u0026deg;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 \u0026times; SDS loading buffer at 100\u0026deg;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 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e 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\u0026ndash;5 week-old \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Total proteins were extracted from leaves using a cell lysis buffer, and subjected to anti-GFP agarose beads IP (chromotek) as previously described\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Eluted proteins were subjected to immunoblot analysis using anti-GFP antibodies and anti-FALG antibodies. Total proteins were loaded as an input control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eYeast two-hybrid analyses\u003c/h2\u003e \u003cp\u003eTo 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\u0026rsquo;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 \u0026micro;g /ml X‐α‐galactosidase (X‐α‐gal) and 100 ng/ml aureobasidin A (AbA) (QDO/X/A). Yeast plates were kept in 28\u0026deg;C growth chambers for 4 days before imaging.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBcCELP1 is a novel cell death-inducing protein\u003c/h2\u003e \u003cp\u003eBcin09g02160 was identified as a cell death-inducing protein (CDIP) within the secretome of \u003cem\u003eB. cinerea\u003c/em\u003e at an early infection stage (28hpi)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). A BLASTp search of the predicted Bcin09g02160 protein against the NCBI database showed homology to the CELP0028-effector-like protein from \u003cem\u003eBlumeria graminis\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Accordingly, we named the putative protein BcCELP1. Multiple Sequence Alignment (MSA) revealed the presence of a conserved fungal CELP0023 domain between amino acids 84\u0026ndash;251 (Supplementary Fig.\u0026nbsp;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.\u0026nbsp;1b). Further bioinformatics analysis using SignalP 5.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbs.dtu.dk/services/SignalP/\u003c/span\u003e\u003cspan address=\"http://www.cbs.dtu.dk/services/SignalP/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) indicated the presence of an N-terminal secretion signal peptide (SP) within amino acids 1\u0026ndash;19 (Supplementary Fig.\u0026nbsp;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.\u0026nbsp;1d). This finding is consistent with the detection of the BcCELP1 within the fungal secretome\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCytoplasmic localized BcCELP1 triggers plant cell death\u003c/h2\u003e \u003cp\u003eAgroinfiltration of \u003cem\u003eN. benthamiana\u003c/em\u003e leaves with 35S: BcCELP1 triggered local cell death within five days post inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). Infiltration of \u003cem\u003eN. benthamiana\u003c/em\u003e leaves with agrobacterium expressing the \u003cem\u003ebccelp1\u003c/em\u003e without the secretion signal (BcCELP1\u003csup\u003e20\u0026ndash;251\u003c/sup\u003e) 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Hence, BcCELP1 must enter the plant cell to induce cell death, similar to the previously characterized cytoplasmic CDIP BcCrh1\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe BcCELP1 CELP0028 domain is sufficient for induction of plant cell death\u003c/h2\u003e \u003cp\u003eTo 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 \u003cem\u003eN. benthamiana\u003c/em\u003e. The CELP0028 domain (residues 84\u0026ndash;251) proved to be sufficient for full cell death-inducing activity, and deletion of this domain resulted in a complete loss of activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). MSA revealed the presence of four conserved cysteine residues within the CELP0028 domain at positions 91, 201, 234, and 244 (Supplementary Fig.\u0026nbsp;1a). ConSurf and alphafold2 analyses predicted functional roles for the cysteine residues at positions 234 and 244 (Supplementary Fig.\u0026nbsp;2a, b). Consistent with these predictions, substitutions of both cysteine residues with alanine (C234A/C244A) abolished the protein\u0026rsquo;s cell death-inducing activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). Immunoblot analysis of protein extracts from agroinfiltrated \u003cem\u003eN. benthamiana\u003c/em\u003e leaves showed degradation of the full length BcCELP1 but not of the CELP0028 domain alone (Supplementary Fig.\u0026nbsp;3), suggesting that the protein is degraded in planta and that this degradation is mediated by the N\u0026rsquo; variable region of the protein.\u003c/p\u003e \u003cp\u003eTo determine whether the other diverse CELP0023 domain-containing fungal proteins can also trigger cell death in \u003cem\u003eN. benthamiana\u003c/em\u003e, we tested \u003cem\u003eSlerotinia sclerotiorum\u003c/em\u003e SsCELP1, \u003cem\u003eColletotrichum higginsianum\u003c/em\u003e ChCELP1 and \u003cem\u003eFusarium grminearum\u003c/em\u003e FgCELP1 by agroinfiltration. Among these proteins, only SsCELP1 triggered cell death symptoms similar to those caused by BcCELP1 (Supplementary Fig.\u0026nbsp;4). A purified protein infiltration assay showed that both BcCELP1 and the CELP0028 domain alone caused comparable cell death in \u003cem\u003eN. benthamiana\u003c/em\u003e and tomato leaves. Consistent with findings reported for other \u003cem\u003eB. cinerea\u003c/em\u003e CDIPs, BcCELP1 did not promote necrosis in monocots (Supplementary Fig.\u0026nbsp;5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBcCELP1 triggers plant immunity responses\u003c/h2\u003e \u003cp\u003eInfiltration of \u003cem\u003eN. benthamiana\u003c/em\u003e leaves with agrobacterium expressing \u003cem\u003ebccelp1\u003c/em\u003e resulted in the accumulation of ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), callose deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), and elevated transcription of defense-related marker genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). These responses were similar to those observed following treatment with the cytoplasmic CDIP BcCrh1\u003csup\u003e24\u003c/sup\u003e. Furthermore, agroinfiltration of \u003cem\u003eN. benthamiana\u003c/em\u003e leaves with either the full length \u003cem\u003ebcclep1\u003c/em\u003e or the CELP domain fragment reduced disease symptoms compared to control leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Thus, similar to the majority of other \u003cem\u003eB. cinerea\u003c/em\u003e CDIPs, BcCELP1 not only induces plant cell death, but also triggers plant defense responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBcCELP1 is required for\u003c/b\u003e \u003cb\u003eB. cinerea\u003c/b\u003e \u003cb\u003epathogenicity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine whether BcCELP1 is necessary for fungal pathogenic development, we generated \u003cem\u003ebccelp1\u003c/em\u003e deletion mutants and tested their pathogenicity. The Δ\u003cem\u003ebccelp1\u003c/em\u003e deletion strain was unaffected in terms of hyphal growth rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), spore germination, and infection cushion formation (Supplementary Fig.\u0026nbsp;6), but showed defects in sporulation and sclerotia formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, e). Complementation of the deletion mutant with \u003cem\u003ebccelp1\u003c/em\u003e under its native promoter, as well as overexpression of \u003cem\u003ebccelp1\u003c/em\u003e, recovered the wild-type phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe timing of transcriptional activation of\u003c/b\u003e \u003cb\u003ebccelp1\u003c/b\u003e \u003cb\u003eis indispensable for its virulence function\u003c/b\u003e\u003c/p\u003e \u003cp\u003eConsistent with the presence of the BcCELP1 in the early secretome, expression levels of the \u003cem\u003ebccelp1\u003c/em\u003e gene increased almost 90-fold at 24 hpi and 20-fold at 36 hpi, before sharply decreasing to basal expression levels (Supplementary Fig.\u0026nbsp;7). To determine whether the temporal activation of \u003cem\u003ebccelp1\u003c/em\u003e is required for its role in pathogenicity, we generated a strain that expresses the BcCELP1 protein under the control of the \u003cem\u003ebcspl1\u003c/em\u003e promoter, which is specifically upregulated during the late infection stage (Supplementary Fig.\u0026nbsp;8). The \u003cem\u003ebccelp1\u003c/em\u003e deletion strain complemented with \u003cem\u003ebccelp1\u003c/em\u003e expressed under the \u003cem\u003ebcspl1\u003c/em\u003e promoter remained hypovirulent, like the parental D\u003cem\u003ebccelp1\u003c/em\u003e deletion strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, e). This finding indicates that BcCELP1 is specifically required for pathogenicity at the early infection stage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eThe CELP0028 domain is sufficient for pathogenicity\u003c/h2\u003e \u003cp\u003eComplementation of the D\u003cem\u003ebccelp1\u003c/em\u003e mutant with the CELP0028 domain under the control of the \u003cem\u003ebccelp1\u003c/em\u003e promoter completely restored the pathogenicity defect of the D\u003cem\u003ebccelp1\u003c/em\u003e mutant, unlike complementation with the mutated CELP0028\u003csup\u003eC234AC244A\u003c/sup\u003e, which failed to restore this defect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results show that the functional CELP0028 domain is both sufficient and essential for the role of BcCELP1 in the pathogenicity of \u003cem\u003eB. cinerea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eBcCELP1 interacts with the scaffold protein NbRACK1\u003c/h2\u003e \u003cp\u003eTo 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 \u003cem\u003eN. benthamiana\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The conserved cysteine residues of BcCELP1 proved not to be required for interaction with NbRACK1, as interaction was still observed between the BcCELP1\u003csup\u003eC234AC244A\u003c/sup\u003e mutant and NbRACK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Similarly, the \u003cem\u003eS. sclerotiorum\u003c/em\u003e SsCELP1 protein was found to physically associate with NbRACK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Finally, we verified the interaction between BcCELP1 and NbRACK1 in \u003cem\u003eplanta\u003c/em\u003e using Co-IP and BiFC assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNbRACK1 is required for BcCELP1 cell death-inducing activity.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe transiently expressed BcCELP1 in an \u003cem\u003eN. benthamiana\u003c/em\u003e plant in which \u003cem\u003eNbRACK1\u003c/em\u003e was silenced using VIGS and monitored the extent of BcCELP1-triggered cell death. The cell death-inducing activity of BcCELP1 was significantly compromised in \u003cem\u003eNbRACK1\u003c/em\u003e-silenced plants, indicating that NbRACK1 is functionally necessary for BcCELP1-triggered cell death in \u003cem\u003eN. benthamiana\u003c/em\u003e (Supplementary Fig.\u0026nbsp;9).\u003c/p\u003e \u003cp\u003eTo further explore the virulence mechanism of BcCELP1, we transiently expressed BcCELP1 in \u003cem\u003eN. benthamiana\u003c/em\u003e 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.\u0026nbsp;10a). We validated the RNA-Seq results by qPCR analysis of six DEGs (Supplementary Fig.\u0026nbsp;10b).\u003c/p\u003e \u003cp\u003eGO GSEA (Gene Set Enrichment Analysis) showed enrichment of activated genes, including GO terms \u0026lsquo;GO: 0042542 - response to hydrogen peroxide\u0026rsquo;, \u0026lsquo;GO: 0000302 - response to reactive oxygen species\u0026rsquo; and \u0026lsquo;GO: 0006979 - response to oxidative stress\u0026rsquo;. The down-regulated genes were enriched in GO terms \u0026lsquo;GO: 0006270 - DNA replication initiation\u0026rsquo;, \u0026lsquo;GO: 0000079 - regulation of cyclin-dependent protein serine/threonine kinase activity\u0026rsquo;, and \u0026lsquo;GO: 0000280 - nuclear division\u0026rsquo; (Supplementary Fig.\u0026nbsp;10c).\u003c/p\u003e \u003cp\u003eKEGG-GSEA (Gene Set Enrichment Analysis) also showed that the activated genes were enriched in KEGG terms \u0026lsquo;ko03050 - Proteasome\u0026rsquo;, \u0026lsquo;ko04136 - Autophagy - other\u0026rsquo;, \u0026lsquo;ko04016 - MAPK signaling pathway \u0026ndash; plant\u0026rsquo;, and \u0026lsquo;ko04626 - Plant-pathogen interaction\u0026rsquo;. Down-regulated genes were enriched in KEGG terms \u0026lsquo;ko00195 - Photosynthesis\u0026rsquo;, \u0026lsquo;ko00710 - Carbon fixation in photosynthetic organisms\u0026rsquo;, and \u0026lsquo;ko03030 - DNA replication\u0026rsquo; in the transient BcCELP1 expression lines (Supplementary Fig.\u0026nbsp;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 attack\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eBcCELP1 promotes RACK1-RBOHB interaction\u003c/h2\u003e \u003cp\u003eAs RACK1A is localized inside the plant cell\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, 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 \u003cem\u003ein planta\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eRACK1 is known to associate with the plant ROS-generating enzyme RBOHB, which is localized at the cell membrane\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. 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 \u003cem\u003eN. benthamiana\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee), suggesting that BcCELP1 promotes NbRACK1-NbRBOHB interaction in the ROS-generating complex.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHost ROS-generating pathway facilitates\u003c/b\u003e \u003cb\u003eB. cinerea\u003c/b\u003e \u003cb\u003eplant invasion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe 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 \u003cem\u003eN. benthamiana\u003c/em\u003e and \u003cem\u003eP. vulgaris RACK1\u003c/em\u003e and \u003cem\u003eRBOHB\u003c/em\u003e genes were both upregulated following \u003cem\u003eB. cinerea\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Transient co-expression of \u003cem\u003eNbRACK1\u003c/em\u003e and \u003cem\u003eNbRBOHB\u003c/em\u003e in \u003cem\u003eN. benthamiana\u003c/em\u003e increased the plant\u0026rsquo;s sensitivity to \u003cem\u003eB. cinerea\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), while \u003cem\u003eA. thaliana rack1\u003c/em\u003e and \u003cem\u003erbohb\u003c/em\u003e T-DNA insertion mutant lines exhibited less sensitivity to infection by the fungus (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA recent study showed that the secreted peroxidase BcCcp1 removes plant-derived H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and is necessary for ROS detoxification and pathogen invasion\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Importantly, the secretion of BcCcp1 is specifically enhanced during the early infection stage at 24 hpi, but decreases at 36 hpi\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. To investigate whether the ROS detoxification mechanism deployed by BcCcp1 contributes to \u003cem\u003eB. cinerea\u003c/em\u003e virulence specifically during the early infection stage, we carried out a pathogenicity assay on the mutants with constitutive expression of \u003cem\u003ebcccp1\u003c/em\u003e. Similar to the Δ\u003cem\u003ebcccp1\u003c/em\u003e strain, the OE-\u003cem\u003ebcccp1\u003c/em\u003e strains also resulted in smaller lesions than the wild type at 72 hpi (Supplementary Fig.\u0026nbsp;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 \u003cem\u003eB. cinerea\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003eCollectively, 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.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eB. cinerea\u003c/em\u003e infection is a multi-layered process governed by the exchange of a wide range of factors that collectively determine disease development and severity\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In addition to PCWDEs and toxins, \u003cem\u003eB. cinerea\u003c/em\u003e utilizes a cocktail of CDIPs to facilitate the rapid killing of host cells\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. A previous study on \u003cem\u003eS. sclerotiorum\u003c/em\u003e 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\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. A similar scenario has been proposed for \u003cem\u003eB. cinerea\u003c/em\u003e, based on evidence that this fungus manipulates the plant towards committing death by targeting host HR and PCD machinery, rather than indiscriminately killing its host\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. It is assumed that \u003cem\u003eB. cinerea\u003c/em\u003e promotes oxidative bursts and hypersensitive cell death in host plants to facilitate host colonization\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Based on this model, \u003cem\u003eB. cinerea\u003c/em\u003e may manipulate the plant regulated cell death (RCD) machinery to facilitate the formation of local lesions\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, and effectors that target the plant RCD machinery are likely to be involved in disease progression.\u003c/p\u003e \u003cp\u003eIn this study, we have identified a novel virulence effector, BcCELP1, specific to the early infection stage of \u003cem\u003eB. cinerea\u003c/em\u003e. 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 pathogens\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. However, HR does not protect hosts against infection by necrotrophic pathogens. Indeed, aggressive necrotrophic microbes, such as \u003cem\u003eB. cinerea\u003c/em\u003e and \u003cem\u003eS. sclerotiorum\u003c/em\u003e, 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 \u003cem\u003eB. cinerea\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. However, there is also evidence that ROS-mediated HR-like cell death can block \u003cem\u003eB. cinerea\u003c/em\u003e 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 \u003cem\u003eB. cinerea\u003c/em\u003e invasion\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs a member of the tryptophan\u0026ndash;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 pathways\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. In plants, RACK1 is involved in diverse biological processes, including growth, development, phytohormone responses, protein translation, micro-RNA biogenesis and multiple environmental stimuli responses\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Emerging evidence indicates that RACK1 also plays key roles in plant innate immunity against biotrophic and hemibiotrophic pathogens. For example, the OsRACK1A\u0026ndash;OsRBOHB immune complex and its mediated ROS production are required for immunity of rice plants against \u003cem\u003ePrycularia oryzae\u003c/em\u003e and \u003cem\u003eUstilaginoidea virens\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. In this work, we reveal a new pathogenic mechanism of necrotrophs, whereby \u003cem\u003eB. cinerea\u003c/em\u003e secretes BcCELP1 into host cells to target NbRACK1A, promoting the NbRACK1A\u0026ndash;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.\u003c/p\u003e \u003cp\u003eOne 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\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. 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)\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. When uncontrolled, this activation of the HR is followed by so-called runaway cell death that facilitates the spreading of host plant cell death\u003csup\u003e\u003cspan additionalcitationids=\"CR64 CR65\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. In this case, cell death induced by HR keeps propagating, which contributes to the spreading invasion of necrotrophic pathogens, such as \u003cem\u003eB. cinerea\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eS. sclerotiorum\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. 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 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e68\u003c/sup\u003e. The substantial enhancement in mulberry's resistance to \u003cem\u003eB. cinerea\u003c/em\u003e was accompanied by increased catalase (CAT) activity. Interestingly, we found that BcCELP1 also interacts with NbCAT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), and \u003cem\u003eB. cinerea\u003c/em\u003e infection downregulates the catalases expression of \u003cem\u003eN. benthamiana\u003c/em\u003e and \u003cem\u003eP. vulgaris\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea), underscoring the potentially important role of host ROS-generating pathway manipulation in the invasion strategy of \u003cem\u003eB. cinerea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eNotably, 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\u0026ndash;pathogen interactions\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent 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\u0026ndash;promoting effectors during later stages of infection\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. A recent study provides evidence that \u003cem\u003eBotrytis\u003c/em\u003e employs unique spatio-temporal penetration mechanics, depending on the actin skeleton\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. 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 colonization\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e, 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.\u003c/p\u003e \u003cp\u003eDuring the initial infection stage, \u003cem\u003eB. cinerea\u003c/em\u003e primarily relies on inducing local necrosis to establish infection foci for the subsequent infection\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. 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 \u003cem\u003ebcelp1\u003c/em\u003e, that limited to activated at later stage, failed to restore the phenotypic defect of Δ\u003cem\u003ebccelp1\u003c/em\u003e, highlighting the necessity of appropriate timing of \u003cem\u003ebccelp1\u003c/em\u003e expression to mediate different infection stages. Interestingly, the mutant strain with overexpression of \u003cem\u003ebccelp1\u003c/em\u003e retains normal pathogenicity, suggesting as long as timely activation of \u003cem\u003ebccelp1\u003c/em\u003e during the early phase is sufficient to conserve its function. Previous research suggested that an H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e burst in the very early stage of infection (4 hpi) in \u003cem\u003esitiens\u003c/em\u003e, the abscisic acid-deficient tomato mutant, contributes to its resistance to \u003cem\u003eB. cinerea\u003c/em\u003e. Instead, in the susceptible wild type, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e began to accumulate in the mesophyll layer as early as 24 hpi and was associated with the spread of cell death\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, which is in accordance with the expression levels of the \u003cem\u003ebccelp1\u003c/em\u003e 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis 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.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eYuan, M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Pattern-recognition receptors are required for NLR-mediated plant immunity. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e592\u003c/strong\u003e, 105-109 (2021).\u003c/li\u003e\n \u003cli\u003eNgou, B. P. M., Ahn, H.-K., Ding, P. \u0026amp; Jones, J. D. 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Plant-pathogen effectors: cellular probes interfering with plant defenses in spatial and temporal manners. \u003cem\u003eAnnual review of phytopathology\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 419-441 (2016).\u003c/li\u003e\n \u003cli\u003eM\u0026uuml;ller, T.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Plant infection by the necrotrophic fungus Botrytis requires actin-dependent generation of high invasive turgor pressure. \u003cem\u003ebioRxiv\u003c/em\u003e (2023).\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4918366/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4918366/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNecrotrophic 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 \u003cem\u003eBotrytis cinerea\u003c/em\u003e. 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\u0026rsquo;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 \u003cem\u003ebccelp1\u003c/em\u003e 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.\u003c/p\u003e","manuscriptTitle":"A secreted Botrytis cinerea stage-specific effector promotes virulence by targeting the plant ROS-generating machinery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-28 09:20:12","doi":"10.21203/rs.3.rs-4918366/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b12fbc15-a73d-4971-8a30-51bdb9498574","owner":[],"postedDate":"August 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":36618120,"name":"Biological sciences/Plant sciences"},{"id":36618121,"name":"Biological sciences/Microbiology/Fungi/Fungal pathogenesis"}],"tags":[],"updatedAt":"2024-10-22T02:00:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-28 09:20:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4918366","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4918366","identity":"rs-4918366","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}