Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this bottleneck by developing a workflow combining cell-free DNA template generation, cell-free protein synthesis, and binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to evaluate 135 previously published antibodies targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including all 8 antibodies previously granted emergency use authorization for coronavirus disease 2019 (COVID-19), and demonstrate identification of the most potent antibodies. We also evaluate 119 anti-SARS-CoV-2 antibodies from a mouse immunized with the SARS-CoV-2 spike protein and identify neutralizing antibody candidates, including the antibody SC2-3, which binds the SARS-CoV-2 spike protein of all tested variants of concern. We expect that our cell-free workflow will accelerate the discovery and characterization of antibodies for future pandemics and for research, diagnostic, and therapeutic applications more broadly. Antibody discovery is bottlenecked by the individual expression and evaluation of antigen specific hits. Here, the authors build an antibody screening workflow leveraging cell-free protein synthesis that enables expression and evaluation of hundreds of antibody fragments in less than 24 h. Antibodies have also recently garnered attention as potential countermeasures for emerging pathogens, having been used as both prophylaxis and therapy against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and coronavirus disease 2019 (COVID-19)6.Antibodies are widely used in diagnostics and as drugs. They are the critical component in immunoassays enabling rapid diagnostics8 sequences to a pool of 103 to 104 candidates targeting the desired antigen. However, once this pool of candidates is generated, state-of-the-art workflows rely on labor-intensive procedures , etc.) to evaluate and identify the best antibody candidates8. These procedures take weeks to months and represent a major speed and throughput bottleneck in antibody discovery.Contemporary workflows for antibody discovery commonly utilize synthetic selections or the isolation of single B cell clones from convalescent patients or animals to go from >1010 or non-overlapping epitopes12 to resist viral escape and increase the ability to neutralize viral variants13; both of which require intensive screening campaigns. A further challenge is that existing antibody discovery processes frequently have low efficiency, with few of the screened candidates having potent neutralizing activity, as has been the case for SARS-CoV-2 (Supplementary Table The effort to identify antibodies against emerging threats like SARS-CoV-2 during the coronavirus disease 2019 (COVID-19) pandemic has highlighted the importance of (i) rapid and high-throughput antibody discovery platforms and (ii) identifying high-affinity antibodies targeting conserved15, the manufacture of proteins without living cells using crude extracts or purified components, is an attractive tool to overcome these limitations. A variety of CFPS systems for antibody expression have been developed22; however, few of these studies have focused on the functional screening of antibodies, and most methods rely on techniques that are not suitable for high-throughput screening like the use of purified plasmids or labor-intensive ELISAs23.Cell-free protein synthesis (CFPS)24, and (iv) acoustic liquid handling that enables a highly parallel and miniaturized workflow. This integrated workflow enables a single researcher to express and profile the antigen-specific binding of hundreds of antibodies in less than 24 h. As a model, we apply our workflow to profile a diverse set of 135 previously published antibodies targeting the SARS-CoV-2 spike glycoprotein, and show that our workflow identifies all 8 neutralizing antibodies that had been granted emergency use authorization (EUA) by the United States Food and Drug Administration for the treatment of COVID-19. In addition, we screen 119 antibodies derived from mice immunized against the SARS-CoV-2 spike glycoprotein and identify several candidate neutralizing antibodies.Here, we describe a CFPS-based integrated pipeline for antibody expression and evaluation to address screening limitations in current antibody discovery pipelines. The workflow leverages four key developments Fig. : (i) DNA25. The method consists of a Gibson assembly step, followed by PCR amplification of the linear expression template (LET) using the unpurified Gibson assembly product as a template. The key idea was to create a versatile approach for construction of DNA templates without the requirement of cell culture, allowing for DNA assembly and amplification in less than 3 h entirely in 384-well plates.We first implemented a method for cell-free DNA assembly and amplification by adapting and optimizing recently reported protocols for rapid construction of DNA templates for CFPS26. These sequences were subsequently amplified by PCR to generate LETs  strain, which contains mutations in the E. coli reductase genes trxB and gor to enable the formation of disulfide bonds in the cytoplasm27. By pretreating the extract with the reductase-inhibitor iodoacetamide (IAM) to further stabilize the redox environment30 and supplementing the reaction with purified E. coli disulfide bond isomerase DsbC and prolyl isomerase FkpA32, we successfully expressed and assembled full-length trastuzumab 31. Consistent assembly under the same experimental conditions is important for screening since individual conditions cannot be optimized for hundreds or thousands of antibodies. We thus opted to use the synthetically dimerized antigen-binding fragment  format and, like Ojima-Kato et al.18, found that the assembly of sdFabs were more uniform than their corresponding Fabs in CFPS for a panel of antibodies  for binding of the SARS-CoV-2 Receptor Binding Domain (RBD). Our determined rank order of IC50s Fig. .Furthermore, we utilized AlphaLISA to develop a sdFab assembly screen to monitor antibody fragment expression and assembly in CFPS, a step that traditionally requires SDS-PAGE. The measurement immobilizes the heavy and light chains of the sdFab to the AlphaLISA beads, resulting in signal when the two chains are assembled 50. We used these assays as a rapid screen for S6P binding and ACE2 competition at a single unknown concentration of CFPS-derived antibody.Using the developed workflow, we next evaluated a set of 115 SARS-CoV-2 targeted antibodies that were selected based on the availability of sequence, structural, binding, and neutralization data, with 84 being drawn from Brouwer et al.FPS Fig. . MeasureTo determine the robustness of the workflow, antibody fragments were expressed and evaluated in triplicate. We observed that independent replicates were consistent with one another and exhibited average coefficients of variation (standard deviation divided by the mean) in the range of 0.15–0.22 , r = 0.41 , and r = 0.40  for Figs. While there is only a weak correlation between our AlphaLISA data and the corresponding Brouwer et al. S6P binding ELISA data, the RBD binding ELISA data, and the pseudovirus neutralization data 67. For all 8 EUA antibody fragments, we observed S6P binding, RBD binding, ACE2 competition, and assembly  by the US Food and Drug Administration for prophylaxis or treatment of COVID-19 67. Notably, we observed that S2H97 exhibited ACE2 competition, which like S309, is reported to bind an epitope adjacent to the receptor binding motif and not compete with ACE2 for binding10. The antibody 54042-4 exhibited weak binding to the RBD but showed low assembly signal, indicative of poor expression or assembly , we observed binding and competition for 10 of 11 antibody fragments tested, with the results largely consistent with published literatureata Fig. . Based o68. For these antibodies, the normalized S6P binding AlphaLISA measurement correlates well with loss in neutralization potency ). Also consistent with literature, S309 exhibited binding to the SARS-CoV spike protein, whereas no other EUA Ab is reported to bind SARS-CoV59.We further profiled the binding of this set of high interest antibodies against SARS-CoV-2 variants of concern (VOC) including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron  as well as several other human coronaviruses including SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E Fig. . Our dat64. S2K146 did not exhibit binding to the SARS-CoV spike, despite being reported to neutralize SARS-CoV65. S2P6 additionally exhibited binding to MERS-CoV and HCoV-OC43 consistent with the literature on this antibody61. For S2P6, we observed heterogeneity in binding signal to different S6P variants, possibly a result of the target epitope of S2P6 being near the C-terminus of S6P and near to the C-terminal avi tag (a site-specific biotinylation), which may impede immobilization on the AlphaLISA bead. Neutralization data against all VOC for the tested bnAbs are not available, but the binding profiles for those characterized are generally consistent with literature. Against Omicron BA.1, S2K146 exhibits strong binding whereas S2X259, S2H97, and SARS2-38 all exhibit reduced binding70. Similarly, S2K146 exhibits strong binding to all Omicron variants except BA.4/5, whereas S2X259 and S2H97 exhibit reduced binding to the other Omicron sub-lineages72. Taken together, our results indicate that the CFPS-derived antibody fragment binding patterns are consistent with those reported in literature.For the set of bnAbs, we observed cross-reactivity with the SARS-CoV spike protein for S2P6, S2X259, DH1047, C118, and S2H97 all of which are reported to bind to this antigen73 and isolated spike-positive activated B cells 10 days later using fluorescence-activated cell sorting. The pooled sorted B cells were sequenced to identify paired heavy and light chains, which were codon optimized and ordered commercially as synthetic DNA .No statistical method was used to predetermine sample size. Previous experience with the measurement techniques and their dynamic ranges was used to determine sample sizes. Large-scale experiments . The Benjamini and Hochberg False Discovery Rate procedure50 was used to correct for multiple testing. Statistical analyses were performed in python. Two-sided t-tests  were performed using the scipy package and the FDR procedure was performed using the statsmodels package with a family-wise error rate of 5%. For antibody screening, the following samples were used as a measurement of background, and the combined data were used in the t-test. Assembly: No DNA and beads only controls. S6P binding: No DNA and beads only controls. RBD binding: No DNA and beads only controls. ACE2 competition: No DNA and αHER2.Samples were considered different from background if they exhibited Pearson correlation coefficients were calculated using GraphPad Prism 9.Single-cell BCR sequences were analyzed with Cell Ranger v3.1.0. AlphaLISA data were analyzed with Prism 9.5.1. Images were processed using ImageJ2 v2.9.0/1.53t. Plate reader data were processed using Python 3.8.8. The Python code to analyze plate reader data was not central to the research and was not deposited.18. Example plasmid maps of the aHER2 heavy and light chain sdFabs can be found in the Source data. Antibody sequences were collected from literature and their light chains were classified as either kappa or lambda via the terminal residue of the J-segment in the VL domain. The VH and VL domains were subsequently fused to their corresponding human constant heavy (Uniprot P0DOX5) or human constant light (kappa CL Uniprot P01834 or lambda 1 CL Uniprot P0CG04) chains. At the N-terminus of the VH and VL domains, we chose to include a modified expression tag based on the first 5-residues of the E. coli chloramphenicol acetyltransferase gene followed by a Tobacco Etch Virus (TEV) protease cleavage site 79 as opposed to the previously published SKIK tag80. The heavy chain was fused to the LZA heterodimer subunit (AQLEKELQALEKENAQLEWELQALEKELAQK) and a strep II tag or super FLAG (sFLAG) tag81. The light chain was fused to the LZB heterodimer subunit (AQLKKKLQALKKKNAQLKWKLQALKKKLAQK). Antibodies in the screen in Fig. sdFabs were assembled based on a modified version of previously published protocolssdFab heavy chain constant strepII tagged:[MEKKIENLYFQS][VH_Sequence][ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC]GGGGS[AQLEKELQALEKENAQLEWELQALEKELAQK]GSSA[WSHPQFEK].sdFab heavy chain constant super FLAG (sFLAG) tagged:[MEKKIENLYFQS][VH_Sequence][ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC]GGGGS[AQLEKELQALEKENAQLEWELQALEKELAQK]GSSA[DYKDEDLL].sdFab light chain kappa:[MEKKIENLYFQS][VL_Sequence][RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]GGGGS[AQLKKKLQALKKKNAQLKWKLQALKKKLAQK].sdFab light chain lambda 1:[MEKKIENLYFQS][VL_Sequence][GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS]GGGGS[AQLKKKLQALKKKNAQLKWKLQALKKKLAQK].Proteins to be manufactured via CFPS were codon optimized using the IDT codon optimization tool and ordered as double-stranded linear DNA containing the desired Gibson assembly overhangs from IDT or GenScript. sfGFP was ordered containing the two pJL1 Gibson assembly overhangs. Antibody VH DNA was ordered with the pJL1 5′ and the human IgG1 heavy chain constant 5′ Gibson overhangs. Antibody VL DNA was ordered with the pJL1 5′ and the human Ig light chain kappa or lambda 1 Gibson assembly overhangs. DNA was resuspended at a concentration of 50 ng/μL and used without amplification.Additional linear DNA components for Gibson assembly  were ordered as gblocks from IDT. These components were amplified using PCR using Q5 Hot Start DNA polymerase  following manufacturer instructions. Amplified DNA was purified using the DNA Clean and Concentrate Kit  and diluted to a concentration of 50 ng/μL. Sequences of the utilized components are listed below, with Gibson assembly sequences being denoted by underlined lowercase text and primers for a given amplicon being listed below the DNA sequence.Gibson assembly overhangs:tttgtttaactttaagaaggagatatacat.pJL1 5′ Gibson: gtcgaccggctgctaacaaagcccgaaagg.pJL1 3′ Gibson: gcgtcaacaaaaggtccttcagttttcccattagcccct.Human IgG1 heavy chain constant 5′ Gibson: cgcacggtcgcggcgccgtctgtctttatttttcctcct.Human Ig light chain kappa 5′ Gibson: ggccaacccaaagcaaacccaactgtcactttgttcccg.Human Ig light chain lambda 5′ Gibson: Linear pJL1 plasmid backbone (Addgene plasmid # 69496):gtcgaccggctgctaacaaagcccgaaaggAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGCCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCTTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATtttgtttaactttaagaaggagatatacat.pJL1_F: gtcgaccggctgcta.pJL1_R: atgtatatctccttcttaaagttaaacaaaattatttcta.Linear sdFab heavy chain constant strepII tagged:gcgtcaacaaaaggtccttcagttttcccattagcccctTCTTCTAAGTCAACTAGTGGCGGTACTGCCGCTCTTGGGTGTTTGGTTAAAGATTACTTCCCAGAACCGGTTACGGTCTCGTGGAACTCTGGTGCACTGACATCGGGCGTACATACATTTCCCGCAGTTTTGCAGTCTTCGGGACTGTATTCTCTTTCATCGGTGGTTACAGTCCCTAGCTCTTCCCTGGGTACACAGACCTACATTTGTAATGTTAATCATAAGCCGAGTAATACTAAGGTGGATAAAAAGGTGGAACCGAAGTCTTGTGGTGGTGGCGGGTCAGCTCAACTGGAGAAGGAGTTACAGGCACTGGAAAAAGAGAATGCTCAACTTGAGTGGGAATTACAGGCGTTAGAAAAAGAACTGGCCCAGAAGGGTTCTAGCGCATGGTCACATCCCCAGTTCGAAAAATAAgtcgaccggctgctaacaaagcccgaaagg.Linear sdFab heavy chain constant super FLAG tagged:gcgtcaacaaaaggtccttcagttttcccattagcccctTCTTCTAAGTCAACTAGTGGCGGTACTGCCGCTCTTGGGTGTTTGGTTAAAGATTACTTCCCAGAACCGGTTACGGTCTCGTGGAACTCTGGTGCACTGACATCGGGCGTACATACATTTCCCGCAGTTTTGCAGTCTTCGGGACTGTATTCTCTTTCATCGGTGGTTACAGTCCCTAGCTCTTCCCTGGGTACACAGACCTACATTTGTAATGTTAATCATAAGCCGAGTAATACTAAGGTGGATAAAAAGGTGGAACCGAAGTCTTGTGGTGGTGGCGGGTCAGCTCAACTGGAGAAGGAGTTACAGGCACTGGAAAAAGAGAATGCTCAACTTGAGTGGGAATTACAGGCGTTAGAAAAAGAACTGGCCCAGAAGGGTGGAGCCAGTCCAGCAGCTCCTGCGCCTGGCGGGGACTACAAAGATGAAGACCTTCTTTAAgtcgaccggctgctaacaaagcccgaaagg.IgGC_F: GCGTCAACAAAAGGTCCTTCAGTTTTC.pJL1_3′Gib_R: CCTTTCGGGCTTTGTTAGCAGC.Linear sdFab light chain kappa constant:cgcacggtcgcggcgccgtctgtctttatttttcctcctTCTGATGAACAGCTTAAATCTGGGACAGCTTCTGTTGTATGTTTATTAAACAACTTTTACCCGCGTGAGGCAAAAGTTCAATGGAAGGTAGACAACGCACTGCAAAGCGGAAATTCGCAGGAGTCAGTTACCGAACAGGATTCCAAGGATAGTACCTACTCCTTAAGTTCAACATTAACCCTGTCAAAGGCGGACTATGAAAAACATAAGGTATATGCCTGCGAAGTAACTCATCAGGGCTTATCATCCCCAGTTACAAAATCTTTCAACCGTGGAGAATGCGGCGGCGGAGGTAGCGCGCAGCTTAAGAAAAAATTGCAAGCCCTTAAAAAAAAAAATGCCCAACTTAAATGGAAGCTGCAAGCCTTAAAAAAGAAATTGGCGCAGAAGTAAgtcgaccggctgctaacaaagcccgaaagg.kLC_F: TCGCGGCGCCGTCTG.pJL1_3′Gib_R: CCTTTCGGGCTTTGTTAGCAGC.Linear sdFab light chain lambda 1 constant:ggccaacccaaagcaaacccaactgtcactttgttcccgCCCTCAAGCGAGGAACTTCAGGCTAATAAGGCCACGCTTGTTTGCCTGATCTCAGACTTTTATCCCGGTGCCGTAACAGTGGCTTGGAAGGCAGATGGTTCGCCGGTCAAAGCGGGCGTGGAAACTACAAAGCCATCGAAACAGTCAAACAATAAATATGCGGCATCAAGTTACTTGAGCCTTACCCCAGAACAGTGGAAGTCACACCGCTCGTACAGTTGTCAAGTTACACACGAGGGAAGTACAGTTGAAAAGACCGTTGCCCCAACTGAATGTTCAGGCGGTGGTGGCTCAGCGCAGTTAAAGAAAAAACTGCAGGCTTTGAAGAAAAAGAATGCTCAATTAAAGTGGAAATTGCAGGCGTTGAAGAAGAAACTTGCGCAGAAGTAAgtcgaccggctgctaacaaagcccgaaagg.lLC_F: GGCCAACCCAAAGCAAACC.pJL1_3′Gib_R: CCTTTCGGGCTTTGTTAGCAGC.82.Linear sfGFP (same DNA sequence as Addgene Plasmid #102634). Note that the sequence of sfGFP is heavily modified and contains mutations from Bundy et al.tttgtttaactttaagaaggagatatacatATGAGCAAAGGTGAAGAACTGTTTACCGGCGTTGTGCCGATTCTGGTGGAACTGGATGGCGATGTGAACGGTCACAAATTCAGCGTGCGTGGTGAAGGTGAAGGCGATGCCACGATTGGCAAACTGACGCTGAAATTTATCTGCACCACCGGCAAACTGCCGGTGCCGTGGCCGACGCTGGTGACCACCCTGACCTATGGCGTTCAGTGTTTTAGTCGCTATCCGGATCACATGAAACGTCACGATTTCTTTAAATCTGCAATGCCGGAAGGCTATGTGCAGGAACGTACGATTAGCTTTAAAGATGATGGCAAATATAAAACGCGCGCCGTTGTGAAATTTGAAGGCGATACCCTGGTGAACCGCATTGAACTGAAAGGCACGGATTTTAAAGAAGATGGCAATATCCTGGGCCATAAACTGGAATACAACTTTAATAGCCATAATGTTTATATTACGGCGGATAAACAGAAAAATGGCATCAAAGCGAATTTTACCGTTCGCCATAACGTTGAAGATGGCAGTGTGCAGCTGGCAGATCATTATCAGCAGAATACCCCGATTGGTGATGGTCCGGTGCTGCTGCCGGATAATCATTATCTGAGCACGCAGACCGTTCTGTCTAAAGATCCGAACGAAAAAGGCACGCGGGACCACATGGTTCTGCACGAATATGTGAATGCGGCAGGTATTACGTGGAGCCATCCGCAGTTCGAAAAATAAgtcgaccggctgctaacaaagcccgaaagg.84. 20 ng of purified, linear pJL1 backbone, 20 ng of purified, linear sdFab VH or VL constant DNA, and 20 ng of the protein open reading frame insert were combined in 2 μL Gibson assembly reactions and incubated at 50 °C for 30 min. The unpurified assembly reactions were diluted in 40 μL of nuclease-free water  and 1 μL of the diluted reaction was used as the template for a PCR to generate linear expression templates (LETs) for CFPS. Linear expression templates were amplified via PCR using the pJL1_LET_F (ctgagatacctacagcgtgagc) and pJL1_LET_R (cgtcactcatggtgatttctcacttg) primers in a 50 μL PCR reaction using the Q5 Hot Start DNA polymerase  following manufacturer instructions.Gibson assembly was used to assemble protein open reading frame DNA with the pJL1 backbone following the published protocol with the addition of 3.125 μg/mL of ET SSB P. pyralis luciferase containing a c-terminal strepII tag  used as a negative control is below and was cloned into the pJL1 vector.The DNA sequence of the atggaagacgctaagaacattaagaagggacctgctccattctaccccctcgaagacggcactgcaggtgagcagcttcataaagcgatgaagcgttatgcgttagttcctggcacgatcgccttcactgacgcgcacatcgaagtcaatatcacctacgctgaatactttgagatgagtgtgcgtctggcggaagccatgaagcgttatggccttaacacgaaccaccgcatcgttgtttgtagcgagaattccttacaattcttcatgcccgtccttggcgcgctgtttattggtgtggccgttgcaccagccaatgacatctataatgagcgcgagttgttgaactccatgaacatttctcaaccaacagtggtgttcgtttcaaagaaaggcttacagaaaatcttaaacgttcaaaagaaactgccgattatccagaagatcatcattatggatagtaagactgactaccagggcttccagtcaatgtatacattcgtgacgagtcacctgcccccgggttttaacgagtacgactttgtcccagagagctttgatcgcgacaagaccatcgccctcattatgaatagcagtggttcgacgggtagcccaaagggagtggccctgccccatcgtaccgcgtgcgtccgtttctcccatgcccgcgacccaattttcggcaatcaaatcatccccgacacggcaatcttgtcggtcgtcccgtttcaccatggctttggaatgtttacgacactcggttacctcatctgcggtttccgcgtcgttctgatgtatcgcttcgaggaagagttgttcttacgttcgcttcaggactacaagattcaatccgcccttctggtccccactttgttcagtttctttgctaagagcaccttaattgataagtatgacctctccaacttacacgagattgcgagcggtggtgctcccctcagcaaagaggttggagaggcggttgctaagcgttttcatctgcccggtatccgtcaaggttacggcctcaccgaaaccacttctgccattcttatcactccggaaggtgacgataagcctggggcagtgggtaaagttgtacccttcttcgaggctaaggttgtggatttagatacggggaagaccttaggtgtgaaccagcgcggtgaactgtgcgttcgcggtccgatgattatgtcgggttatgttaatgaccccgaggctacgaacgcgcttatcgataaggacggttggcttcattccggcgacatcgcttactgggatgaggatgagcacttcttcatcgttgaccgtctgaagagtctcatcaagtataagggatgtcaagtcgctccggcagagttagagagcatcttactccagcaccctaatatcttcgatgctggggttgccgggctcccaggcgacgatgccggcgagctgccggcggcggtagttgttttagagcatggcaagaccatgaccgaaaaggagattgtagactacgtcgcgagtcaagtaaccacagcgaagaagctccgcggtggagtggtctttgttgacgaggtgcctaaaggcctgacgggcaaacttgacgcgcgtaagatccgtgagatcctcatcaaagcgaagaagggtgggaagagtaagctggggagttcaggttggtcccacccgcaatttgagaagtga.E. coli OrigamiTM B(DE3)  extracts were prepared using a modified version of established protocols86. Briefly, a 150 mL OrigamiTM B(DE3) starter culture was inoculated in LB from a glycerol stock and cultured in a 250 mL baffled flask at 37 °C for 16 h. The 2xYTP was prepared without glucose in 75% of the final volume and sterilized using an autoclave. A 4x glucose solution was prepared and autoclaved separately, then added to the medium immediately before use. The starter cultures were used to inoculate 1 L of 2xYTPG media  in a 2.5 L Full-Baffle Tunair shake flask at an initial OD600 of 0.08. Cells were cultured at 37 °C at 220 RPM in a shaking incubator. Cultures were grown until OD600 0.4-0.6, at which point the expression of T7 RNA polymerase was induced by the addition of IPTG to a final concentration of 0.5 mM. Cells were harvested at an OD600 of 2.5 via centrifugation at 12,000 × g for 1 min at 4 °C. Cell pellets were washed three times with 25 mL S30 buffer per 50 mL culture . Pellets were resuspended in 1 mL S30 buffer per gram of cell mass. Cell suspensions were lysed using a single pass on an Avestin EmulsiFlex-B15 Homogenizer at a lysis pressure of 24,000 PSI. Cell debris was separated via centrifugation at 18,000 × g for 20 min, and the clarified lysate was collected, flash-frozen in liquid nitrogen, and stored at −80 °C.E. coli MRE600 tRNA (Roche 10109541001), 100 mM NAD, 50 mM CoA, 4 mM oxalic acid, 1 mM putrescine, 1 mM spermidine, 57 mM HEPES pH 7.2, 2 mM of each amino acid, 33.3 mM PEP, 20% v/v E. coli extract, varying concentrations of DNA template, and the remainder water. The preparation of these reagents has been described in detail elsewhere87. For DNA templates, plasmids were used at a concentration of 8 nM, and unpurified linear PCR products were used at 6.66% (v/v). For the expression of antibodies, each template was added to a final concentration of 6.66% (v/v). For antibody and sdFab expression 4 mM oxidized glutathione, 1 mM reduced glutathione, 14 μM of purified DsbC, and 50 μM FkpA were also supplemented to the reactions. In addition, for oxidizing CFPS reactions, cell-extracts were treated with 500 μM iodoacetamide (IAM) at room temperature for 30 min before use in CFPS88. All reaction components were assembled on ice and were either run as 12 μL reactions in 1.5 mL microtubes or 2 μL reactions in 384-well plates . For 2 μL reactions, components were transferred to the plate using an Echo 525 acoustic liquid handler. A mix containing all the CFPS components except for the DNA was dispensed from 384PP Plus plates  using the BP setting. The DNA (unpurified PCR products) was dispensed from a 384LDV Plus plate  using the GP setting. Reactions were allowed to proceed at 30 °C for 20 h.CFPS reactions were composed of the following reagents: 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 1.2 mM ATP, 0.5 mM of each CTP, GTP, and UTP. 0.03 mg/mL folinic acid, 0.17 mg/mL 86. Radioactive leucine was added to CFPS at a final concentration of 10 μM of L-[14C(U)]-leucine , followed by precipitation of the expressed proteins and scintillation counting89. To quantify sfGFP fluorescence, 2 μL of a CFPS reaction was diluted in 48 μL of water in a Black Costar 96 Well Half Area Plate. Fluorescence was measured using a BioTek SynergyTM H1 plate reader with excitation and emission wavelengths of 485 and 528, respectively. Scintillation counts and fluorescence were fit to determine a standard curve for use with non-radioactive samples.To quantify sfGFP fluorescence, a standard curve was prepared using previously reported methodsTM . FluoroTectTM was included in the CFPS reaction at 3.33%v/v. After protein synthesis, RNAseA  was added to 0.1 mg/mL and the sample was incubated at 37 °C for 10 min. 3 μL of the CFPS and RNAseA mixture were combined with 4x loading buffer  and the samples were subsequently denatured at 70 °C for 3 min, then separated via SDS-PAGE and imaged using a LI-COR Odyssey Fc imager on the 600 channel. Densitometry was performed using the ImageJ software.To visualize antibody assembly, proteins were labeled during CFPS with FluoroTect77. DsbC  and FkpA  were ordered as gBlocks from IDT containing a c-terminal, TEV cleavable his tag (GSENLYFQSGSHHHHHHHHHH) and cloned into pET28a. Plasmid maps of both DsbC and FkpA are available in the Source Data. Plasmids were transformed into BL21 StarTM DE3, plated on LB agar, and cultured overnight at 37 °C. 1 L of Overnight Express TB  was inoculated by scraping all colonies on a transformation plate and cultured at 37 °C in 2.5 L tunair flasks  at 220 rpm overnight. Cells were harvested, resuspended at a ratio of 1 g cell mass to 4 mL resuspension buffer , 1 mg/mL lysozyme, 62.5 U/mL cell suspension of benzonase ) and lysed using an Avestin B15 homogenizer at 24,000 PSI. The lysate was spun down 14,000 × g for 10 min and the clarified supernatant was incubated with Ni-NTA Agarose  for 60 min on an end-over-end shaker. The resin was spun down 2500l × g for 2 min, the supernatant removed, resuspended in wash buffer , loaded on a gravity flow column, and subsequently washed with 20X resin volumes of wash buffer. Protein was eluted using elution buffer  and exchanged into 50 mM HEPES pH 7.4, 150 mM NaCl using PD-10 desalting columns  according to manufacturer instructions.Protein expression, purification, and his tag removal were performed similarly to previously reportedHis tags were removed via cleavage by ProTEV Plus . Before cleavage, 10% v/v glycerol was added to the protein. ProTEV Plus was added to a concentration of 0.5 U/μg purified protein and DTT was added to a concentration of 1 mM. Cleavage reactions were carried out at 30 °C for 4 h. Free His tag and ProTEV Plus were removed by incubating with Ni-NTA Agarose for 1 h at 4 °C and collecting the supernatant. Proteins were subsequently concentrated to >1 mg/mL . His tag removal was validated via SDS-PAGE and the AlphaScreen Histidine (Nickel Chelate) Detection Kit .90.AlphaLISA reactions were carried out in 50 mM HEPES pH 7.4, 150 mM NaCl, 1 mg/mL BSA, and 0.00015 v/v TritonX-100 (hereafter referred to as Alpha buffer). All components were dispensed using an Echo 525 liquid handler from a 384-Well Polypropylene 2.0 Plus microplate  using the 384PP_Plus_GPSA fluid type. All components of the AlphaLISA reactions were prepared as 4x stocks and added as 0.5 μL to the final 2 μL reaction to achieve the desired concentration. All AlphaLISA reactions were performed with CFPS reactions diluted to a final concentration of 0.025 v/v. AlphaLISA beads were combined to prepare a 4X stock in Alpha buffer immediately before use and added to the proteins to yield a concentration of 0.08 mg/mL donor beads and 0.02 mg/mL acceptor beads in the final reaction. All reactions were incubated with AlphaLISA beads for at least 1 h before measurement. AlphaLISA measurements were taken on a Tecan Infinite M1000 Pro plate reader using the AlphaLISA filter with an excitation time of 100 ms, an integration time of 300 ms, and a settling time of 20 ms. Before measurement, plates were allowed to equilibrate inside the instrument for 10 min. For measurements involving sdFabs, protein A AlphaLISA beads were avoided due to the ability of protein A to bind human subgroup VH3 FabsThe impact of CFPS reagents on AlphaLISA was determined by serially diluting the specified reagents in Alpha buffer and combining them with the specified AlphaLISA conditions. The TrueHits kit  was used to assess the impact of the CFPS reagents on the Alpha detection chemistry. CFPS reagents were mixed with the donor and acceptor beads and incubated for 2 h before measurement. His tagged RBD  and human FC tagged human ACE2  were used to evaluate the impact of CFPS reagents on capture chemistries. RBD and ACE2 were diluted in Alpha buffer, mixed at a final reaction concentration of 10 nM each, combined with the CFPS reagents, and allowed to incubate for 1 h. Donor and acceptor beads were subsequently added and allowed to incubate for a further hour before measurement. Protein A Alpha donor beads , Ni-Chelate AlphaLISA acceptor beads , and anti-6xhis AlphaLISA acceptor beads  were utilized for detection.50 values were recorded from the product page at the time of purchase and converted to μg/mL assuming a MW of 150,000 Da if reported in M. Antibodies were serially diluted in Alpha buffer and mixed with SARS-CoV-2 RBD  at a concentration of 10 nM in the final reaction and incubated for 1 h. Mouse FC tagged human ACE2  was subsequently added and incubated for 1 h, followed by simultaneous addition of the acceptor and donor beads. AlphaLISA detection was performed using Anti-Mouse IgG Alpha Donor beads  and Strep-Tactin AlphaLISA Acceptor beads . IC50 values were calculated using Prism 9 by fitting the normalized data to [Inhibitor] vs. response–Variable slope (four parameters) fit with the max constrained to a value of 1.The commercial neutralizing antibody ACE2 competition experiment was performed with the following antibodies: nAb1 , nAb2 , nAb3 , nAb4 . ELISA ICFor all antibody screening experiments, the different reagents and AlphaLISA conditions used are described in Supplementary Table Assembly AlphaLISA reactions consisted of sdFab expressing CFPS and either Rabbit Anti-Human kappa light chain antibody  or Rabbit Anti-Human lambda light chain . CFPS reaction containing the expressed sdFab of interest was mixed with the appropriate anti-light chain antibody and allowed to equilibrate for two hours before the simultaneous addition of the acceptor and donor beads.SARS-CoV-2 S6P binding AlphaLISA reactions consisted of sdFab expressing CFPS and SARS-CoV-2 S6P CFPS reaction containing the expressed sdFab of interest was mixed with the S6P and allowed to equilibrate for two hours before the simultaneous addition of the acceptor and donor beads.SARS-CoV-2 RBD binding AlphaLISA reactions consisted of sdFab expressing CFPS and SARS-CoV-2 RBD. CFPS reaction containing the expressed sdFab of interest was mixed with the RBD and allowed to equilibrate for two hours before the simultaneous addition of the acceptor and donor beads.ACE2 and RBD competition AlphaLISA reactions consisted of sdFab expressing CFPS, human ACE2, and SARS-CoV-2 S6P. CFPS reaction containing the expressed sdFab of interest was first mixed with S6P and allowed to incubate for 1 h. Subsequently, ACE2 was added and allowed to equilibrate for a further 1 h before the simultaneous addition of the acceptor and donor beads.77), SARS-CoV-2 S6P Beta/B.1.351 , SARS-CoV-2 S6P Gamma/P.1 , SARS-CoV-2 S6P Delta/B.1.617.2 , SARS-CoV-2 S6P Omicron/BA.1 , SARS-CoV-2 S6P Omicron/BA.2 , SARS-CoV-2 S6P Omicron/BA.2.12.1 , SARS-CoV-2 S6P Omicron/BA.4/5 , SARS-CoV S2P , MERS-CoV S2P , HCoV-HKU1 S , HCoV-OC43 S , HCoV-NL63 S , and HCoV-229E S .For SARS-CoV-2 variant and other non-SARS-CoV-2 coronavirus binding experiments, AlphaLISA measurements were carried out in the same manner as described for SARS-CoV-2 S6P. The following Hisx6-tagged proteins were used. SARS-CoV-2 S6P , SARS-CoV-2 S6P Alpha/ B.1.1.7  of human ACE2. All three components were incubated for an additional hour prior to simultaneous addition of AlphaLISA beads. Reactions were incubated for 2 h prior to measurement.For RBD and ACE2 bridging experiments SARS-CoV-2 RBD, human ACE2, and the specified dilution of CFPS  were incubated for 1 h prior to the simultaneous addition of the AlphaLISA beads. Reactions were incubated for 2 h prior to measurement.10 viral particles (vp) of ChAd-SARS-CoV-2-S73 in 50 µl of PBS via intramuscular injection in the hind leg. Draining inguinal lymph nodes were collected 10 days later and processed into a single-cell suspension. Cells were stained with biotinylated recombinant SARS-CoV-2 spike (S2P) for 30 min at 4 °C then washed twice with FACS buffer followed by staining with anti-CD19 BV421 (BioLegend # 115537), anti-CD4 FITC (BioLegend # 100405), anti-IgD-PE-Cy7 (BioLegend # 405719), Streptavidin APC (BioLegend # 405207), aqua cell viability dye (Invitrogen L34957), and anti-mouse CD16/CD32 Fc block (BioLegend # 156607). Spike-positive activated B cells (live singlet CD4- CD19+ IgDlo Streptavidin+) were bulk sorted on BD FACSAriaII sorter.Female C57BL/6 (Strain: 000664) were purchased from The Jackson Laboratory. Six-week-old animals were immunized with 10The following 10x Genomics kits were used for libraries preparation: Chromium Single Cell 5′ Library and Gel Bead Kit v2 (PN-1000006), Chromium Single Cell A Chip Kit (PN-1000152), Chromium Single Cell V(D)J Enrichment Kit, Mouse B cell (96rxns) (PN-1000072), and Single Index Kit T (PN-1000213). The GEM generation and barcoding was followed by cDNA preparation then GEM RT reaction and bead cleanup steps. Purified cDNA was amplified for 10–14 cycles then cleaned up using SPRIselect beads. cDNA concentration was determined by running samples on a Bioanalyzer. BCR target enrichments were done on the full-length cDNA followed by BCR libraries preparation as recommended by 10x Genomics Chromium Single Cell V(D)J Reagent Kits (v1 Chemistry) user guide. The cDNA Libraries were sequenced on Novaseq S4 (Illumina), targeting a median sequencing depth of 5000 read pairs per cell.Cell Ranger v3.1.0 for alignment against the GRCm38 mouse reference v3.1.0 , generating 3760 assembled high-confidence BCR sequences for 4420 cells. Sequences for screening were selected randomly from the top clonal groups with >10 members in the clonal group. Cellranger vdj output was then parsed using Change-O v0.4.6 within the immcantation suite. Additional quality control included examining sequences to be productively rearranged and have valid V and J gene annotations. Furthermore, only cells with exactly one heavy chain sequence paired with at least one light chain sequence were kept.Demultiplexed pair-end FASTQ reads from 10x Genomics single-cell V(D)J profiling were preprocessed using the “cellranger vdj” command from Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


Shigella flexneri to selectively internalize into glioblastoma (GBM) brain tumor cells as an initial step to generating a bacterial-based drug delivery system.The use of microorganisms as drug delivery systems to treat cancer has expanded recently, including FDA approval of certain viruses as oncolytics. Microorganisms have several unique benefits compared to traditional pharmacologic agents including dose independence, the ability to produce therapeutic proteins locally within the tumor, and simplicity of administration. However, current microbial delivery systems such as AAV9 and herpes virus have limited cassette sizes, minimal cancer cell selectivity, and low innate cytotoxicity. To address these issues, we sought to generate a strain of S. flexneri that selectively internalize into GBM cells using iterative co-cultured assays.We generated After 50 rounds of co-culture, the new strain infected 95 percent of GBM cells in 2 hours. GBM-infecting Shigella demonstrate a 124-fold preference for internalizing in nine different GBM cell lines compared to Normal Astrocytes (NA) controls. Additionally, we developed an in-cell western to identify GBM-infecting Shigella clones that preferentially internalize in patient samples without iterative co-culture. Finally, we demonstrate internalization into GBM cells is mediated via a factor modified by myristoylation.In conclusion, here we present a novel bacterial platform that preferentially internalizes in brain tumor cells. This system provides numerous potential benefits over current interventions and other microbial strategies for treating brain tumors. Microorganism-based drug delivery systems are emerging as a promising approach to treating solid tumors –4. Most Glioblastoma (GBM) is the most common malignant, primary brain tumor observed in adults, and patients diagnosed with GBM demonstrate a median survival of only 15 months . The staShigella flexneri, a gram-negative intracellular bacterium similar to the viruses discussed above. Importantly, Shigella has a type 3 secretion system capable of administering toxic proteins into the host cell cytosol  (30 g/L) + agar (15 g/L), and Congo Red (100 mg/L) sterile plates and incubated overnight at 30°C or 37°C. Individual colonies were expanded in sterile Tryptic Soy Broth (30 g/L) using a shaking incubator (250 rpm) at 30°C.Shigella was streaked out from the frozen stock onto TSB (30 g/L) + Congo Red (0.01% w/v) plates and incubated overnight at 37°C. Colonies were picked and grown for approximately 2 hours (OD650 = 0.6) (® EHEC (Cat#: 751630). Band intensity was measured using ImageJ.At 24 hours prior to testing, 0 = 0.6) . SamplesShigella colony was picked using a pipet tip and resuspended in 20 μL nuclease-free water. A colony was incubated for 10 min at 95°C and then centrifuged at 1,000 rcf for 1 min to remove membrane components.A single Supernatant at a volume of 5 μL was used as the template for a PCR assay to quantify the presence of DNA encoding for Shiga toxin(s). Potential DNA regions were amplified with the Phusion taq polymerase kit  using previously described primers that are specific for Shiga toxin 1 (Stx1 – primer pair: ATGTCAGAGGGATAGATCCA and TATAGCTACTGTCACCAGACAAT), Shiga toxin 2 (Stx2 – primer pair: AGTTCTGCGTTTTGTCACTGTC and CGGAAGCACATTGCTGATT), or positive control virulence factor F (virF – primer pair: AGCTCAGGCAATGAAACTTTGAC and TGGGCTTGATATTCCGATAAGTC) . Bands wS. flexneri was streaked onto TSB agar plates with 0.01% Congo Red and grown at 37°C overnight. The next day, 96 red colonies of Shigella were picked using a filtered p200 pipet tip. Each colony was added into 400 μL of TSB in a deep-well block plate and incubated at 37°C, 250 rpm, until an OD650 of 0.6 was reached (approximately 2.5 to 3 hours). Shigella suspensions were combined and pelleted into a 50-mL conical tube. After washing three times with 25 mL of 10% glycerol, Shigella was resuspended in 5 mL of 10% glycerol. Shigella was electroporated with 200 ng of pRedTKI plasmid DNA (Addgene Plasmid #51628) and recovered in TSB for 1 hour at 30°C, followed by plating on TSB + 50 μg/mL Kan agar plates with 0.01% Congo Red overnight. The pRedTKI plasmid introduces the Lambda red genes necessary for homologous recombination along with conferring Kan resistance.Genes were deleted as previously described using standard Lambda red recombineering methods with minor modifications , 27. S. Shigella + pRedTKI colonies were picked and incubated at 30°C, 250 rpm, until an OD650 of 0.6 was reached (approximately 3 hours); during the last 30 min of incubation, l-arabinose was added at 10 mmol/L to induce the Lambda red recombination genes along with 50 μg/mL kanamycin to maintain transmission of the pRedTKI plasmid. Bacteria were pelleted in a 50-mL conical tube by centrifugation at 4,000 × g, transferred to a 1.5-mL Eppendorf tube, washed three times with 500 μL of 10% glycerol, and resuspended in 50 μL of 10% glycerol. Using an electroporation cuvette with a 2 mm gap, Shigella were transfected via electroporation with 200 ng of I-SceI-flanking resistance cassette with 70-bp homology adjacent to the gene to be deleted. The cassette for MsbB1 was as follows: TGGTGCGGGGCAAGTTGTGCCGCTACACTATCACCAGATTGATTTTTGCCTTATCCGAAACTGGAAAAGCTAGGGATAACAGGGTAATATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTACGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATAGGGATAACAGGGTATAAAAGCCTCTCGCGAGGAGAGGCCTTCGCCTGATGATAAGTTCAAGTTTGCTTCAGAATATTCGAAATCT.The cassette for MsbB2 was as follows: AATTAAGGTTAGATGTATTCTCTGAATAAAATATTAATGATGATTATGGTAGGGGCATTCGCACTAAATATAGGGATAACAGGGTAATATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTACGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATAGGGATAACAGGGTAATAATTATAAAGTACAGGTATTTCCACTAGTTGTTTCTTACAGGTTACCAATCGAAACACATCCCCCTTCCG.Shigella was allowed to recover in Super Optimal broth with Catabolite repression (SOC) for 3 hours at 30°C and plated on TSB + 50 μg/mL Kan + 12.5 μg/mL chloramphenicol (Cam) agar plates with 0.01% Congo Red overnight.The primer pair for identifying MsbB1 was TTGAACTTATCATCAGGCGAAGGCCTC and CGGCTTTTTTTATTTGGTGCGGGG, and that for MsbB2 was CTGCTATCCGCTCTTTGGATGCA and CTACACAGTCCTCCGTGCCAA. Electroporated Shigella + pRedTKI + Cam resistance cassette was incubated at 30°C, 250 rpm, until an OD650 of 0.6 (approximately 3 hours) in 3 mL TSB + 50 μg/mL Kan + 20 mmol/L isopropyl β-d-1-thiogalactopyranoside (IPTG) to induce I-SceI expression to excise the Cam resistance gene. A sample of liquid culture was seeded onto TSB + 50 μg/mL Kan + agar plates with 0.01% Congo Red and incubated overnight. Modifications were verified by PCR.To complete the gene deletion protocol, a single colony of l-cysteine, NaOH, CaCl2, DNAse I, EDTA, and papain for 35 min, and then inactivated in serum media. Cells then underwent trituration, were strained through a 100-μm cell strainer, and were centrifuged for 5 min at 200 × g, 4°C. The supernatant was aspirated, and cells were counted and replated at 750,000 cells/mL. Neurobasal media at a volume of 2 mL with B-27 supplement, GlutaMAX, and 1.25% FBS was added to a culture plate. Thirty percent of the media was replaced twice per week. After 3 days of culture, 5-fluoro-2′-deoxyuridine (FUDR) was added to remove dividing cells. All mammalian cells were cultured at 37°C, 5% CO2.GBM cell lines were obtained from Dr. John Kuo  or ATCC . Normal astrocytes were purchased from Lonza (Cat#: CC-2565) and grown according to the manufacturer’s protocol without antibiotics. Mammalian cells were grown in tissue culture-treated T25 or T75 flasks in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), GlutaMAX, and 1% sodium pyruvate (without pen/strep) –30. CellMammalian cells were maintained in culture as described above. At 24 hours prior to co-culture assays, the media was changed without removing cells from the tissue culture flask. Cells grown to 80% confluency in a T25 flask were used for each round of co-culture.S. flexneri was streaked on TSB agar plates with 0.01% Congo Red and grown at 37°C overnight. The next day, 96 red colonies of Shigella were picked using a filtered p200 pipet tip and added to individual wells of a deep-well block plate that contained 400 μL of TSB. The deep-well block plate was incubated at 37°C, 250 rpm, until an OD650 of 0.6 was reached (approximately 2.5 to 3 hours). A control well was used to estimate the Shigella growth rate. After reaching the mid-log phase (0.6 OD650), individual wells were pooled, and the Shigella was concentrated to 2 × 108 cfu/mL (OD650 of 1.0 = 8.0 × 108). Next, 250 μL of Shigella concentrate was added to ~5 mL of GBM, normal astrocyte (NA), or rNeuron media. Shigella-containing media at a volume of 5 mL was added to the T25 cell culture flask containing mammalian cells. The T25 culture flask was centrifuged at 200 × g for 10 min and then incubated at 37°C, 5% CO2, for 30 min. After 30 min, culture media was removed by aspiration. The T25 flask was washed four times using 5 mL of phosphate-buffered saline (PBS) for 1 min with gentle agitation. Next, 5 mL of mammalian culture media containing 20 μg/mL of gentamicin was added to the T25 flask. The T25 flask was incubated at 37°C, 5% CO2, for an additional 90 min. After 90 min, the media was aspirated, and cells were washed four times with PBS, 1 min per wash, with gentle agitation.2, until GBM, NA, or rNeuron cells detached from the plate. Mammalian cell membranes were mechanically disrupted by pulling the cell suspension through a 27Ga needle 10 times. The mammalian cell lysate was centrifuged at 1,000 × g for 2 min to pellet any membrane-associated  Shigella. Mammalian cell lysate at a volume of 20 μL was plated onto a TSB + 0.01% Congo Red agar plate and grown at 37°C overnight. Individual colonies were expanded and frozen in 25% glycerol or used directly for the next round of co-culture assays.After the final wash, 1 mL of Accutase  was added to the T25 flask. The T25 flask was incubated at 37°C, 5% COd-Lysine, No. 1.5 glass-bottom dishes .At 24 hours prior to internalization, GBM cells were grown to ~80% confluence using the protocol described above in the mammalian cell culture section on Poly-Shigella colony was picked, grown to the mid-log phase, and co-cultured with mammalian cells in the microscopy dish using the protocol described above in the co-culture assay section. After the final wash, cells were fixed with 4% paraformaldehyde (PFA)  for 10 min and then washed three times with PBS.An individual PBS staining solution at a volume of 1 mL containing wheat germ agglutinin-fluorescein  and Hoechst 33342  was added to the fixed cells and incubated for 15 min. Cells were washed three times with PBS. After the final wash, 1 mL of PBS was added to the dish so cells did not dry out during imaging. Cells were imaged using a 60× objective on a Nikon Eclipse Ti2 confocal microscope. Images were processed and analyzed using the publicly available ImageJ software package.Shigella antibody  in 1 mL PBS + 30 mg/mL BSA + 0.1% Triton X-100 for 90 min at room temperature. After incubation, cells were washed three times with PBS. Next, cells were incubated with anti-rabbit Alexa Fluor 488 secondary  in PBS + 30 mg/mL BSA + 5% donkey serum + 0.1% Triton X-100  for 60 min. Cells were washed three times with PBS. Next, cells were incubated with Phalloidin Dye 594  and Hoechst 33342  in PBS  for 15 min. Cells were imaged using a 60× objective on a Nikon Eclipse Ti2 confocal microscope. Images were processed and analyzed using the publicly available ImageJ software package.Cells were permeabilized by adding 1 mL of 0.1% Triton X-100 in PBS for 5 min. Blocking buffer at a volume of 1 mL  + 5% donkey serum + 0.1% Triton X-100) was added to each dish, and the cells were incubated at 4°C overnight. The next day, cells were washed three times with PBS. Next, cells were incubated with an anti-At 24 hours prior to internalization, GBM or NA cells were grown to 80% confluence as described in the mammalian tissue culture section above on a tissue culture-treated, black well, clear bottom, 96-well tissue culture plate.Shigella colonies were picked and expanded as described above in a deep block 96-well plate. Shigella culture at a volume of 40 μL from a single well was added to 200 μL of GBM or NA media previously plated into each well of the clear-bottom, black, 96-well plate. The 96-well plate was centrifuged at 200 × g for 10 min and then incubated at 37°C, 5% CO2, for 30 min. Culture media was removed after 30 min by dumping the supernatant into a sterile glass dish containing bleach. The plate was washed four times with PBS using a similar method to remove the supernatant. Next, 200 μL of mammalian cell media containing 20 μg/mL gentamicin was added to each well. The plate was incubated at 37°C, 5% CO2, for 90 min. After incubation, the plate was washed four times with PBS. Cells were fixed by adding 200 μL of 4% PFA to each well and incubating for 10 min. The plate was washed three times with PBS. Cells were permeabilized by adding 200 μL of PBS containing 0.1% Triton X-100 to each well for 5 min. Blocking buffer containing 1% BSA and 1% donkey serum was added to each well, and the plate was incubated at 4°C overnight. The next day, the plate was washed three times with PBS. The plate was incubated with anti-Shigella antibody  in PBS + 30 mg/mL BSA + 0.1% Triton X-100 (100 μL per well) for 90 min. The plate was washed with PBS three times. Finally, each well was incubated with IR800CW anti-rabbit secondary  in PBS + 30 mg/mL BSA + 5% donkey serum + 0.1% Triton X-100 for 60 min. The plate was washed three times with PBS and imaged on a LI-COR Fc scanner using a 2-min medium resolution scan. A standard curve of Shigella was used on each plate to convert the IR800 signal to the number of bacteria per well. It was important to ensure that the moles of anti-Shigella antibody greatly exceeded the moles of Shigella to ensure the saturated binding assumption was valid in order to quantify the number of bacteria per well.Individual Shigella internalization in cell lines were determined using ANOVA with a 1% false discovery rate used as the threshold for significance. For microscopy assays, ANOVA was used to determine differences between groups with a 5% false discovery rate used as the threshold for significance.Data are generally presented as means with standard deviation. All validation experiments were conducted with a minimum of two independent replicates. For microscopy experiments, a minimum of three fields were quantified from at least two independent experiments. Significant differences between Shigella clones that preferentially internalize into brain tumors using iterative co-culture assays. Briefly, S. flexneri, an intracellular bacterium, was streaked onto agar plates. Clones containing the virulence factor needed to survive inside mammalian cells were expanded and incubated with a brain tumor cell line, U-251 GBM. Bacteria that internalized into GBM cells were harvested and used for additional rounds of co-culture.As described in Shigella strain that is safe to use as a therapeutic platform, we identified a strain that did not contain Shiga toxins. We probed S. flexneri strain 2475 serotype 2a for DNA regions encoding Shiga toxins using previously described primers. As expected, we did not observe a band at the expected size for the PCR product  when stained with a DNA intercalating dye Hoechst 33342 (rendered blue). The number of GBM cells demonstrating an internalized bacterium increased after each round of Shigella co-culture, starting with 5% ± 2.5% in round 10 and enriching to 95% ± 1% by round 50 of co-culture. The percentage of GBM cells with an internalized bacterium was calculated by taking three randomly selected fields and dividing the total number of infected cells by the total number of U-251 cells present , phalloidin-AF594, and Hoechst 33342 then imaged via confocal microscopy. The DNA rod-like structures observed inside GBM cells throughout this study co-localized with an anti-Shigella antibody  lines were incubated with GBM-infecting Shigella. Internalized Shigella was harvested and plated to count the number of internalized bacteria per group. GSC 115 showed the highest internalization at 123,800 bacteria/5 × 105 GBM cells. To demonstrate the specificity of the platform, GBM-infecting Shigella were incubated with normal astrocytes and internalized bacteria counted. Approximately 383 bacteria internalized/~300 NA cells (two separate patient cell lines) were observed. Next, GBM-infecting Shigella and compared the data to those of the parental strain. We identified 177 mutations in 46 genes . Removal of both MsbB1 and MsbB2 completely abrogated internalization to an average of <1 bacterium recovered. Confocal imaging of GBM cells treated with either R50, MsbB1 KO, or MsbB1 and MsbB2 KO demonstrate a significant decrease in cells internalizing Shigella (Shigella-positive cells dropped from 95% to 25% (p < 0.05 via ANOVA). Quantifying the fluorescent Shigella signal indicated that MsbB1 removal reduced the mean signal to 0.425 rfu compared to 6.16 rfu for R50 . Removal of both MsbB1 and MsbB2 resulted in 0% of cells with a Shigella internalized and a fluorescent signal of 0.009 rfu (~684-fold decrease). GBM cells that did demonstrate Shigella internalization after removal of myristoylation factor MsbB1 had similar numbers of internalized Shigella compared to R50. The growth curves of R50, MsbB1 KO, and MsbB1 and MsbB2 KO Shigella are presented in We removed MsbB1 and MsbB2 myristoylation factors from the round 50 GBM-infecting ineering –37, remoShigella Figure 5S. flexneri clones that selectively internalize into GBM cell lines. We first selected a strain of Shigella that does not express Shiga toxins to improve the safety profile of the platform  , doxycycline, or polymyxins, can serve as master suicide switch(es) to immediately halt GBM-infecting Shigella activity in the case of adverse events such as fever or general infection  , 41. A rblastoma . Additioe genome , 27. Thun system , 20. In n system , 24, 27.nfection . It is iShigella to internalize into GBM cells as a foundation for a drug delivery system. We envision this platform serving as a tool for neurosurgeons to clean the margins of a GBM post-resection. GBM-infecting Shigella could be administered into the surgical cavity before closing to infect and eradicate cancerous cells in the invasive margin that cause disease recurrence. This approach bypasses the need for systemic administration and reduces the potential for internalization into off-target cells in the body. However, many steps are required before GBM-infecting Shigella are suitable for a drug delivery platform including stabilizing genetic material, controlling the bacterial population, ensuring intracellular replication, modulating immunogenicity, and weaponizing the system with cytotoxic proteins such as ribosome toxins (gelonin), cytokines (IL-2 and TNF), and autophagy-inducing proteins (caspases)  –46. AddiShigella into GBM cells. Removal of MsbB1 and MsbB2 myristoylation enzymes completely abrogates the internalization of GBM-infecting Shigella in GBM cells. Previous reports indicate that removing MsbB genes does not impair the internalization of Shigella in intestinal cells, the typical target of wild-type Shigella or Salmonella Typhimurium invasion into tumor cells  is essential for GBM-infecting Shigella to internalize into GBM cells. However, our data do not indicate what that factor is. Whole genome sequencing did not identify obvious mutations in MsbB genes or a known myristoylated protein; however, future RNA-sequencing, epigenetic, and/or proteomic studies of Shigella membrane-bound proteins could identify factors with differential copy number, alterations in promoters, and/or de-novo localization to bacterial membranes that are driving the observed changes in Shigella internalization in GBM cells. These studies can be narrowed bioinformatically by eliminating proteins of interest that do not contain an N-terminal glycine  that is required for modification with myristic acid  can be found in the article/Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. Ethical approval was not required for the studies on animals in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.AS designed assays and wrote the manuscript, GF designed assays and edited the manuscript, BD conceptualized the study and edited the manuscript, and BU conceptualized the study, designed assays, and wrote the manuscript. All authors contributed to the article and approved the submitted version.


Chlamydomonas reinhardtii delta tubulin (uni3) and epsilon tubulin (bld2) genes through an unknown mechanism. Our studies revealed that intact PCR products for both these genes could be obtained upon PCR amplification from plasmid templates carrying these genes. However, interestingly, purification of these PCR products led to their cleavage through an unidentified mechanism. This cleavage persisted despite using different PCR purification kits. Deleting a synthetic intron within the delta tubulin gene also did not have any effect on this cleavage.In this study, we report an unusual phenomenon of the self-cleavage of purified PCR products of codon-optimized Centrosomes consist of orthogonally-paired cylindrical centrioles enveloped by the protein-dense pericentriolar material .Chlamydomonas reinhardtii delta and epsilon tubulin proteins that are coded by theuni3 andbld2 genes respectively, play a crucial role in maintaining the proper triplet microtubule architecture of centrioles . To obtain further insights into the functions of these genes in regulating centriole architecture, we wanted to generate purified PCR products ofChlamydomonas reinhardtiidelta and epsilon tubulin cDNAs carrying synthetic introns that were codon-optimized for expression inC. elegans. These purified PCR products would enable us to perform downstream functional assays to further characterize the functions of these genes. Although we were successful in obtaining full length PCR products of codon-optimizedChlamydomonas reinhardtiidelta and epsilon tubulin genes, these PCR products exhibited an unexpected cleavage upon their purification using either the Invitrogen Purelink™ Quick PCR purification kit or the Qiaquick® PCR purification kit.As shown in, both the delta and epsilon tubulin PCR products but not a non-relevant control PCR product (dyn-1) exhibited cleavage after their purification using the Invitrogen Purelink™ Quick PCR purification kit or the Qiaquick® PCR purification kit. Purification of the delta and epsilon tubulin PCR products using a non-kit-based isopropanol precipitation method as well as upon their gel extraction using a Qiaquick Gel Extraction kit , both yielded this similar pattern of cleavage. To confirm that the cleaved fragments indeed correspond to the PCR products of the delta and epsilon tubulin genes, DNA sequencing of these lower sized fragments was performed after excising these separated bands from the agarose gel. Our sequencing results confirmed that the lower sized fragments that were observed between 650 bp and 1000 bp upon agarose gel electrophoresis of the purified delta and epsilon tubulin PCR productscontained DNA sequences from these respective genes. Our sequencing analysis identified a synthetic intron close to the genomic region where the delta and epsilon tubulin PCR products were being cleaved. Since the sequence of this synthetic intron was identical between the delta and epsilon tubulin genes, we questioned whether the presence of this synthetic intron was responsible for the cleavage of the purified PCR products of these genes. To address this, site-directed mutagenesis was performed to remove this synthetic intron from the sequence of the delta tubulin gene and the PCR purification procedure was repeated. As shown indeletion of the synthetic intron close to the cleavage region within the delta tubulin gene did not eliminate cleavage of the purified PCR product. These data indicate that this synthetic intron does not contribute to this observed cleavage phenomenon. In the future, studies should be directed to further characterize the molecular mechanisms mediating the cleavage of theChlamydomonas reinhardtii delta and epsilon tubulin PCR products upon purification. Since we have invested a considerable amount of time and resources on this project, we would like to share these findings with the scientific community and with other researchers working on these genes to prevent a further loss of their valuable time and resources.Centrosomes are the major microtubule organizing centers of most animal cells. They contribute to a variety of cellular functions including spindle assembly, cell polarity, cell shape and cilia assembly (Reviewed in NiggI) PCR conditions, analysis, and purificationPCR was performed using a C1000™ Thermal Cycler . 35 cycles of amplification were used for 50 µL PCR reaction volume consisting of:uni3,bld2,dyn-1), 1 µL - 10 mM dNTPs, 2 µL- 50 mM MgSO4, 5 µL - 10X Hi-Fi Buffer, 0.2 µL - Platinum®TaqDNA Polymerase Hi -Fi enzyme, and molecular biology water was used to bring the final volume to 50 µL.1 µL- 10 µM forward primer, 1 µL- 10 µM reverse primer, 35 ng- plasmid template  was obtained from a synthesized plasmid construct that contains the coding sequence for blue fluorescent protein with homology arms to theC. elegans dyn-1 gene.The control PCR product (dyn-1) PCR:PCR conditions for the control  Site-directed mutagenesisuni3 gene except that the Phusion® High-Fidelity DNA Polymerase was used for PCR amplification.For performing the site-directed mutagenesis, the PCR reaction mixture comprised of all the same components used in PCR amplification of thePCR conditions for delta tubulin intron deletion using site-directed mutagenesis:Initial denaturation: 98°C - 30 sDenaturation: 98°C - 10 sAnnealing: 68.6°C - 30 sExtension: 72°C - 6 minFinal Extension: 72°C - 10 minSite-directed mutagenesis was followed by digestion with 5 units of Dpn1 for 2 hours, followed by bacterial transformation and plasmid purification using the QIAprep® Spin Miniprep kit. The isolated plasmid samples were sent for whole plasmid sequencing . Upon analyzing the sequencing results, the plasmid with successful intron deletion was selected and used for further analysis.Primers and sequences used for studyI) Primers for PCR amplification of Epsilon tubulinForward primer: 5’- ATT CGA ATA TAT ATT GTC AGT TG -3’Reverse primer: 5’- ATA CGA GGA TTA TGG TAC AAG -3’II) Primers for PCR amplification of Delta tubulinForward primer: 5’- TTG TTT CTT TCT TTT AAT GTT AAA TAT TTC CAG AAC TAT GCC ATG -3’Reverse primer: 5’- GAT AAA ATA ATT ATT CGG GCA GTA ATA AAA CAG GGA TCT ATC ACT TC -3’III) Primers for site-directed mutagenesis for Delta tubulin with one intron deletedForward primer: 5’- TCG AGG AGG CCG GAC TCA AGG GAC AAT CCT CCG GAC CAG G -3’Reverse primer: 5’- CCT GGT CCG GAG GAT TGT CCC TTG AGT CCG GCC TCC TCG A -3’(dyn-1):IV) Primers for amplification of controlForward primer: 5’- AAA ATC GAT TTT CAG GTA GTT CAG C -3’Reverse primer: 5’- TTG ATC ACA GGG ATC AAC GCC -3’Chlamydomonas reinhardtii delta tubulin (uni3) cDNA codon optimized for expression inC. elegans (contains synthetic introns (lowercase + underlined) and homology arms for CRISPR genome editing (only lowercase)):V) Sequence ofgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGTCGTCGCCGGAGCCCGTTCCGCCGCCGCCGCCTCCGGATCCTGGTGGCGTTACCCATCCTCCGGATACCTCGTCATGCAATCCGGATCCGGAAACAACTGGGCCCAAGGATTCCACGGATACGGACCACAAGTCCACGAGGACGCCCTCGACCTCGTCCGTAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGAGGTCGAGCACGCCGACTCCCTCACCGGATTCCTCCTCCTCCAATCCATGGCCGGAGGAACCGGAGCCGGACTCGGAACCTACGTCGCCGAGGCCCTCCGTGACGAGTACCACTCCGCCTTCGTCGCCAACTGCTGCGTCTGGCCATACGAGTCCGGAGAGGTCATCGTCCAACCATACAACACCCTCCTCACCCTCTCCCACCTCGCCGACGTCTCCGACGGACTCGTCCTCCTCGAGAACGAGGCCCTCCACCGTACCGCCGCCAAGCTCTACGGAATCGCCCGTCCATCCTTCGGAGTCCGTGGACGTGTCCTCGGACGTGCCGGAGAGTCCCGTGTCGAGGAGGCCGGACTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagGGACAATCCTCCGGACCAGGAGGATGGGGAGTCTGCACCGCCCCACTCGCCGAGCTCGTCACCCGTCTCTGCGGACACCCAGCCTACCGTCTCCTCACCCTCCGTTCCGTCCCACAACTCCCACCAGCCAACATCGACTTCACCACCTTCACCTGGCCAGCCCTCACCAAGCGTCTCCGTCAAATGCTCGTCACCGGATCCGTCCTCGAGGAGGGACTCGACTGGTCCATCACCCCACAATCCCCAGGAGCCGCCGCCGCCCTCGGAGCCGGACTCGCCGGACCAACCGTCAACCGTGCCCTCGCCTCCTGGCTCATCCTCCGTGGACAAGGAGCCGCCGAGGCCGACGTCGGAGAGTTCGCCGACCCAGCCCTCTCCGCCGCCTGGTCCCCAGAGCCACTCTCCGTCTCCTACTCCACCGGACGTTTCGGACGTTGCGCCATGTCCGCCTGCCTCCTCTCCAACGACCGTCACTGCGTCGGACCAATCCAACGTATGCAAGAGCACGCCTACGGAATGCTCGAGTCCCGTGCCTTCGTCCACCAATACGAGAAGTACGGACTCTCCGTCGCCGAGTTCCAAGACTGCTTCGCCCGTATCGAGGACATCGCCCAACGTTACGCCCGTCTCGACTACAAAGACCATGACGGTGATTATAAAGATCATGATATCGATTACAAGGATGACGATGACAAGATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGtgatagatccctgttttattactgcccgaataattattttatc-3’5’-ttgtttctttcttttaatgttaaatatttccagaactATGCCATGCATCACCCTCCAACTCGGACAATGCGGAAACCAACTCGGATGCTCCCTCTTCAACACCCTCGCCACCGAGTTCTCCTCCCACGACTACGGAACCGACGCCGTCCACGAGTACTTCCGTCCATCCGCCGACCCAAACCTCTACACCGCCCGTTCCGTCCTCATCGACATGGAGCCAAAGChlamydomonas reinhardtii epsilon tubulin (bld2) cDNA codon optimized for expression inC. elegans (contains introns (lowercase + underlined) and homology arms for CRISPR genome editing (only lower case)):VI) Sequence ofgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagATCCGTCTCACCGCCGAGGACTGCGACTCCCTCCAATCCTTCATGGTCCTCCACTCCCTCGGAGGAGGAACCGGATCCGGAGTCGGAACCTACATCGTCCGTATGCTCGCCGACGAGTTCCCAGGAGTCTTCCGTTTCACCGGATCCGTCTTCCCATCCGAGGACGACGACGTCGTCACCTCCCCATACAACGCCATGCTCGCCCTCGGACAACTCGTCGAGCACGCCGACTGCGTCCTCCCAATCGAGAACCAAGCCCTCATCGACATCGTCAACCGTACCGAGGCCGCCCGTGACCGTGCCGCCGCCGCCGACGCCGCCGCCTCCGCCGTCTCCGGACTCAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGGATCCGGAGGAGGATCCAAGCCATTCGACTCCATGAACGGAGTCGCCGCCTCCCTCCTCCTCCACCTCACCGCCTCCGTCCGTTTCGAGGGACCACTCAACGTCGACCTCAACGACATCACCATGAACCTCGTCCCATACCCACGTATGCACTTCCTCCTCTCCTCCATGTCCCCACTCCAACCACCACCAAAGGACAAGGACCCACGTACCCTCGACCAAGTCCGTGTCTTCGGAGACGTCTTCTCCCGTGAGCACCAACTCATCCGTGCCGACCCACGTGCCGCCACCTACCTCGCCTGCGGACTCATCGCCCGTGGACCAACCGCCACCATGGCCGACATCAACCGTAACGTCGCCCGTCTCCGTCCACAACTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagATGGTCCACTGGAACTCCGAGGGATTCAAGCTCGGAATCTGCTCCACCCCACCAGTCGGATGCCCATTCGGACTCCTCTGCCTCGCCAACAACACCGCCATCGCCCACACCTTCACCACCATGCGTGAGCGTTTCGACAAGCTCTACAAGCGTCGTTTCTACACCCACCACTACGAGCAATACATGGACCCAGGAGGATTCACCTCCGCCATGGAGGTCGTCGGAGACCTCACCGCCCAATACCGTGCCCTCGAGGGAGCCACCCAAGCCCCACCACTCACCCGTCTCCGTCCACGTGGACTCTCCTTCCTCCCAGGAGGTTCCGGTGGTTCTGGTGGATCCGGTAAGCCTATCCCAAATCCTTTGTTGGGTCTGGACTCCACGATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGtgataatattcatttaatccaacttgtaccataatcctcgtat-3’5’- attcgaatatatattgtcagttgttctgtttgtcgtcgtgATGCCACGTGAGCTCGTCACCATCCAAGTCGGACAATGCGGAAACCAAGTCGGATGCCGTTTCTGGGAGCTCGCCCTCCGTGAGCACGCCGCCTACAACACCAAGGGAGTCTACGACGAGGCCCTCTCCTCCTTCTTCCGTAACGTCGACACCCGTGTCGAGCCACCACGTAACCTCCCAGTCGGAGAGGGACGTGGAGCCATCCGTACCCTCAAGGCCCGTTCCGTCATCGTCGACATGGAGTGCGGAGTCATCAACGAGATGCTCAAGGGACCACTCGGAGAGGTCCTCGACACCCGTCAACTCGTCTCCGACGTCTCCGGAGCCGGAAACAACTGGGCCCACGGACACCACGAGTACGGACCACGTTACCACGACGCCATCCTCGACAAG(dyn-1):VI) Sequence of the positive control used in this study5’- AAAATCGATTTTCAGGTAGTTCAGCGTATAACCACCAGGATCAGCGATGGTTTCGGAATTGATTAAAGAAAATATGCACATGAAGCTCTACATGGAGGGAACCGTCGACAACCACCACTTTAAATGTACCTCCGAGGGAGAGGGAAAGCCATACGAGGGAACCCAAACCATGCGTATCAAGGTCGTCGAGGGTGGTCCGCTCCCATTCGCCTTTGATATCCTCGCCACCTCCTTCCTCTATGGTTCCAAGGTAAGTTTAAACATATATATACTAACTAACCCTGATTATTTAAATTTTCAGACCTTCATCAACCACACCCAAGGAATCCCAGACTTTTTTAAACAATCCTTCCCAGAGGGATTCACCTGGGAGCGTGTCACCACCTACGAGGACGGAGGAGTCCTCACCGCCACCCAAGACACCTCCCTCCAAGACGGATGCCTCATCTACAACGTCAAGGTAAGTTTAAACAGTTCGGTACTAACTAACCATACATATTTAAATTTTCAGATCCGTGGAGTCAACTTCACCTCCAACGGACCAGTCATGCAAAAGAAGACCCTCGGATGGGAGGCCTTCACCGAGACCCTCTACCCAGCCGACGGAGGACTCGAGGGACGTAACGACATGGCCCTCAAGCTCGTCGGAGGATCCCACCTCATCGCCAACGCCAAGGTAAGTTTAAACATGATTTTACTAACTAACTAATCTGATTTAAATTTTCAGACCACCTACCGTTCCAAGAAGCCAGCCAAGAACCTCAAGATGCCAGGAGTCTACTACGTCGACTACCGTCTCGAGCGTATCAAGGAGGCCAACAACGAGACCTACGTCGAGCAACACGAGGTCGCCGTCGCCCGTTACTGCGACCTCCCATCCAAGCTCGGACACAAGCTCAACTACCCATATGATGTTCCAGATTACGCTGGAGGATCTGGAGGCGGTTCTGGCGGAGGTTCTGGTATGTCGTGGCAAAACCAGGGAATGCAGGCGTTGATCCCTGTGATCAA – 3’


Blumeria graminis forma specialis tritici (B.g. tritici) is the airborne fungal pathogen that causes powdery mildew disease on hexaploid bread wheat. Calmodulin-binding transcription activators (CAMTAs) regulate plant responses to environments, but their potential functions in the regulation of wheat–B.g. tritici interaction remain unknown. In this study, the wheat CAMTA transcription factors TaCAMTA2 and TaCAMTA3 were identified as suppressors of wheat post-penetration resistance against powdery mildew. Transient overexpression of TaCAMTA2 and TaCAMTA3 enhanced the post-penetration susceptibility of wheat to B.g. tritici, while knockdown of TaCAMTA2 and TaCAMTA3 expression using transient- or virus-induced gene silencing compromised wheat post-penetration susceptibility to B.g. tritici. In addition, TaSARD1 and TaEDS1 were characterized as positive regulators of wheat post-penetration resistance against powdery mildew. Overexpressing TaSARD1 and TaEDS1 confers wheat post-penetration resistance against B.g. tritici, while silencing TaSARD1 and TaEDS1 enhances wheat post-penetration susceptibility to B.g. tritici. Importantly, we showed that expressions of TaSARD1 and TaEDS1 were potentiated by silencing of TaCAMTA2 and TaCAMTA3. Collectively, these results implicated that the Susceptibility genes TaCAMTA2 and TaCAMTA3 contribute to the wheat–B.g. tritici compatibility might via negative regulation of TaSARD1 and TaEDS1 expression. Triticum aestivum L.) has served as a major staple food for thousands of years and provided about 20% of the calories consumed by humans [Blumeria graminis forma specialis tritici (B.g. tritici), leading to 5–50% yield losses [B.g. tritici-resistant wheat cultivars [B.g. tritici and identify key regulators of wheat resistance against powdery mildew disease.As one of the most widely grown small-grain cereal crops, bread wheat  transcription factors, calmodulin-binding transcription activators (CAMTAs) play important roles in regulating plant growth, development, and responses to environmental stresses [CAMTA genes differentially respond to environmental cues like drought, salinity, and extreme temperatures in the model plant Arabidopsis thaliana [Arabidopsis mutant camta1 exhibited hypersensitivity to cold and drought stress, and AtCAMTA1 was shown to regulate the expression of cold and drought-responsive genes like AtRD26, AtERD7, AtCBF2, and AtRAB18 [Arabidopsis mutant camta6 exhibited hypersensitivity to NaCl treatment, and AtCAMTA6 was demonstrated to regulate expression of salt resilience-related genes, including + TRANSPORTER1HIGH-AFFINITY K, SALT OVERLY SENSITIVE1, and +/H+ ANTIPORTERNa [Arabidopsis AtCAMTA3 was shown to function in concert with AtCAMTA1 and AtCAMTA2 in suppressing plant defense responses [B.g. tritici remains largely unknown.As Castresses ,20,21. Fthaliana ,24,25,26 AtRAB18 ,24,25,26TIPORTER . In addiesponses ,30,31,32TaCAMTA2 and TaCAMTA3, were characterized as Susceptibility (S) genes contributing to wheat–B.g. tritici compatibility. Transient overexpression of TaCAMTA2 and TaCAMTA3 resulted in enhanced wheat post-penetration susceptibility to B.g. tritici, while transient silencing of TaCAMTA2 and TaCAMTA3 led to attenuated wheat post-penetration susceptibility to B.g. tritici. Furthermore, overexpressing TaSARD1 and TaEDS1 could confer wheat post-penetration resistance against powdery mildew, while silencing TaSARD1 and TaEDS1 enhanced wheat post-penetration susceptibility to B.g. tritici. Moreover, TaCAMTA2 and TaCAMTA3 were demonstrated to negatively regulate the expression of the defense genes TaSARD1 and TaEDS1. These results strongly support that S genes TaCAMTA2 and TaCAMTA3 partially redundantly suppress wheat post-penetration resistance against B.g. tritici presumably via the negative regulation of expressions of defense genes TaSARD1 and TaEDS1.In this research, two CAMTA transcription factor genes, Arabidopsis CAMTA transcription factor AtCAMTA3 plays a vital role in the regulation of plant immunity [B.g. tritici interaction. To this end, we first searched the reference genome of the hexaploid bread wheat by using the amino acid sequence of Arabidopsis AtCAMTA3 (At2g22300) as a query and obtained TaCAMAT2 and TaCAMTA3, the most closely related homologs of AtCAMTA3, in bread wheat. Three highly homologous sequences of TaCAMAT2 genes separately located on chromosomes 4A, 4B, and 4D were obtained from the genome sequence of the hexaploid wheat and designated as TaCAMTA2-4A (TraesCS4A02G407100), TaCAMTA2-4B (TraesCS4B02G306300), and TaCAMTA2-4D (TraesCS4D02G304500). Similarly, three highly homologous sequences of TaCAMAT3 genes separately located on chromosomes 2A, 2B, and 2D were obtained from the genome sequence of the hexaploid wheat and designated as TaCAMTA3-2A (TraesCS2A02G163000), TaCAMTA3-2B (TraesCS2B02G188800), and TaCAMTA3-2D (TraesCS2D02G169900).Previous studies revealed that the immunity ,30,31,32Arabidopsis AtCAMTA3. In addition, TaCAMTA2-4A, TaCAMTA2-4B, TaCAMTA2-4D, TaCAMTA3-2A, TaCAMTA3-2B, and TaCAMTA3-2D proteins all contain a conserved CG-1 DNA-binding domain at their N-terminal parts, a transcription factor immunoglobulin-like (TIG) DNA-binding domain, several ankyrin repeats (ANK) in the middle parts, as well as two IQ CaMB motifs (IQXXXRGXXXR) at their C-termini  increased from 56% for the empty vector (OE-EV) control to above 70% on wheat cells overexpressing TaCAMTA2 or TaCAMTA3 genes. These results suggested that overexpression of TaCAMAT2 and TaCAMTA3 could significantly enhance wheat post-penetration susceptibility to B.g. tritici.To study the function of TaCAMAT2 and TaCAMTA3 in the regulation of wheat–B.g. tritici interaction, we employed transiently induced gene silencing (TIGS) assays to silence all endogenous TaCAMAT2 or TaCAMTA3 genes in the epidermal cell of the B.g. tritici-susceptible wheat cultivar Yannong 999. After inoculation of conidia from the virulent B.g. tritici isolate E09, the frequency of fungal haustorium formation in the transformed plant cells was scored. As shown in TaCAMAT2 or TaCAMTA3 genes resulted in a marked HI% decrease to about 27%, compared to 33% for empty vector (EV) controls. Significantly, simultaneous silencing of TaCAMAT2 and TaCAMTA3 could lead to a further decrease in HI% to approximately 13%, suggesting that TaCAMTA2 and TaCAMTA3 might partially redundantly suppress post-penetration resistance of wheat to B.g. tritici.To further verify the function of TaCAMAT2 or TaCAMTA3 genes in the leaves of the B.g. tritici-susceptible wheat cultivar Yannong 999. qRT-PCR showed that the endogenous transcript level of TaCAMAT2 or TaCAMTA3 was substantially reduced in the indicated VIGS plants  declined to approximate 40% on BSMV-TaCAMTA2as plants and 47% on BSMV-TaCAMTA3as plants, compared with 55% for the BSMV-γ plants -induced gene silencing (BSMV-VIGS) to silence all endogenous S plants C. Thereaγ plants D. NotablSYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (AtSARD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (AtEDS1) in A. thaliana [Arabidopsis AtSARD1 (At1g73805) and AtEDS1 (At3g48090) as a query and obtained TaSARD1 and TaEDS1, the most closely related homologs of AtSARD1 and AtEDS1, in bread wheat. Five highly homologous sequences of TaSARD1 genes separately located on chromosomes 6A, 6B, and 6D were obtained from the genome sequence of the hexaploid wheat and designated as TaSARD1.1-6A (TraesCS6A02G091700), TaSARD1.1-6B (TraesCS6B02G119900), TaSARD1.1-6D (TraesCS6D02G080500), TaSARD1.2-6A (TraesCS6A02G296600), and TaSARD1.2-6D (TraesCS6D02G276800). Similarly, three highly homologous sequences of TaEDS1 genes separately located on chromosomes 5A, 5B, and 5D were obtained from the genome sequence of the hexaploid wheat and designated as TaEDS1-5A, TaEDS1-5B, and TaEDS1-5D [Previous studies revealed that AtCAMTA3 could regulate the expression of defense genes thaliana ,30,31,32aEDS1-5D .Arabidopsis AtSARD1. In addition, TaSARD1.1-6A, TaSARD1.1-6B, TaSARD1.1-6D, TaSARD1.2-6A, and TaSARD1.2-6D proteins all contain a CBP60-conserved domain  resistance genes and quantitative trait loci (QTL) contributed to wheat resistance to B.g. tritici and have been employed in wheat breeding for powdery mildew resistance [B.g. tritici underlies wheat’s susceptibility to powdery mildew. A plethora of wheat S genes have been identified to facilitate compatibility by inducing B.g. tritici (pre)penetration, suppressing wheat immunity, and supporting the sustenance of B.g. tritici [S genes TaWIN1, TaKCS6, and TaECR were revealed to facilitate the conidial germination of B.g. tritici by promoting the biosynthesis of wheat cuticular wax, whereas wheat S gene TaSTP13 encodes a sugar transporter facilitating wheat hexose accumulation for B.g. tritici acquisition [TaMLO, TaEDR1, and TaPOD70 genes contribute to wheat susceptibility to powdery mildew by suppressing plant defense responses [B.g. tritici by suppressing defense-related transcriptional reprogramming in bread wheat [Powdery mildew, caused by the adapted fungal pathogen oduction ,4. To imeraction ,4. Powdesistance ,4. Compa tritici ,35. For uisition ,39,40,41esponses ,45,46,47ad wheat ,51,52,53TaCAMAT2 and TaCAMTA3 were identified as the most closely related homologs of AtCAMTA3, which is consistent with the reported phylogenetic analysis of the CAMTA homologs in different species [TaCAMAT2 and TaCAMTA3 are characterized as wheat S genes contributing to the wheat post-penetration susceptibility to B.g. tritici in this study. Overexpression of TaCAMTA2 and TaCAMTA3 in the leaf epidermal cell by transient gene expression assays led to enhanced wheat susceptibility to B.g. tritici, while knockdown of TaCAMTA2 and TaCAMTA3 expression using transient- or virus-induced gene silencing resulted in compromised wheat post-penetration susceptibility to B.g. tritici. Interestingly, a gain-of-function mutation in SIGNAL RESPONSIVE1 (SR1), which encodes the Arabidopsis homologs of wheat TaCAMTA2 and TaCAMTA3, could suppress the edr2-associated powdery mildew resistance [sr1-4D single mutant is more susceptible to Arabidopsis powdery mildew (Golovinomyces cichoracearum), whereas the sr1-1 null mutant plants displayed enhanced post-penetration resistance against G. cichoracearum [Arabidopsis AtCAMTA1 was revealed to function partially redundantly with AtCAMTA2 and AtCAMTA3 in suppressing plant immunity [TaCAMAT2 and TaCAMTA3 could lead to a further decrease in the HI% and MI% compared with single silencing of TaCAMAT2 or TaCAMTA3, supporting the fact that TaCAMTA3 functions partially redundantly with TaCAMAT2 in suppressing wheat post-penetration resistance against B.g. tritici. In Arabidopsis, CAMTA transcription factors AtCAMTA1, AtCAMTA2, and AtCAMTA3 partially redundantly suppress the biosynthesis of salicylic acid (SA) and N-hydroxypipecolic acid (NHP), a metabolite duo essential for systemic acquired resistance (SAR) [S genes TaCAMAT2 and TaCAMTA3 in the regulation of SA and NHP biosynthesis, as well as SAR establishment, in bread wheat in future research.Through homology-based searching,  species . TaCAMATsistance . The sr1racearum . In addiimmunity ,31,32. Ice (SAR) ,31,32. TTaSARD1 and TaEDS1 are identified as positive regulators of wheat resistance against B.g. tritici in this study. Overexpression of TaSARD1 or TaEDS1 in the leaf epidermal cell by transient gene expression assays led to enhanced wheat post-penetration resistance to B.g. tritici, while knockdown of TaSARD1 or TaEDS1 expression using transient- or virus-induced gene silencing resulted in increased wheat post-penetration susceptibility to B.g. tritici. In Arabidopsis, transcription factor AtSARD1 functions in concert with AtCBP60g to activate the expression of SID2 (SA INDUCTION DEFICIENT 2), which encodes isochorismate synthase 1 (ICS1), essential for pathogen-induced SA biosynthesis [Arabidopsis AtEDS1 was shown to heterodimerize with its partners, phytoalexin deficient 4 (PAD4) or senescence-associated gene 101 (SAG101), to play signaling roles in ETI as well as SA-dependent and SA-independent PTI pathways [TaPR1, TaPR2, and TaPR5 induced by B.g. tritici infection were attenuated by silencing of TaSARD1 or TaEDS1, suggesting that the SARD1-EDS1-SA defense axis might be partially conserved between model plant Arabidopsis and crop plant bread wheat. Therefore, it is intriguing to examine the potential regulation of wheat SA biosynthesis and signaling by TaSARD1 and TaEDS1 in future research.ynthesis ,55,56. Apathways ,62,63,64TaSARD1 and TaEDS1 were significantly enhanced by silencing TaCAMTA2 and TaCAMTA3. Notably, simultaneous silencing TaCAMAT2 and TaCAMTA3 could lead to a further increase in the expression levels of TaSARD1 and TaEDS1 compared with single silencing TaCAMAT2 or TaCAMTA3, indicating that TaCAMTA2 and TaCAMTA3 partially redundantly suppress expressions of TaSARD1 and TaEDS1. In Arabidopsis, AtCAMTA3 could bind to the promoter region of AtEDS1 by recognizing the CGCG box, thereby directly repressing the expression of AtEDS1 [AtSARD1 was demonstrated to be negatively regulated by partially redundant AtCAMTA1, AtCAMTA2, and AtCAMTA3, presumably via an indirect effect [SARD1 and EDS1 by partially redundant CAMTA3 and its homologs might be partly conserved between the model plant Arabidopsis and the important crop bread wheat. Indeed, the expressions of SA defense marker genes TaPR1, TaPR2, and TaPR5 induced by B.g. tritici infection were found to be potentiated by silencing TaCAMAT2 or TaCAMTA3 in this study. However, binding sites for TaCAMAT2 and TaCAMTA3 in the promoter regions of TaSARD1 and TaEDS1 genes remain to be identified.In this study, expression levels of f AtEDS1 ,29,30,31t effect ,29,30,31TaCAMAT2 and TaCAMTA3 are identified as wheat S genes partially redundantly suppressing post-penetration resistance against powdery mildew, presumably via negative regulation of the expressions of defense genes TaSARD1 and TaEDS1. Genetic manipulation of S genes TaMLO and TaEDR1 via targeting induced local lesions in genomes (TILLING) and genome editing techniques like transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) 9 systems compromised wheat compatibility with B.g. tritici and conferred wheat resistance against powdery mildew [TaCAMAT2 and TaCAMTA3 in wheat breeding for powdery mildew resistance in future research.Herein, y mildew ,71,72,73B.g. tritici strain E09 was maintained on the leaves of Jing411 plants. Conidia of B.g. tritici strain E09 were used for the inoculation of Jing411 leaves in the study of wheat–powdery mildew interaction. Arabidopsis thaliana used in this study was grown in the greenhouse under a 16 h/8 h light period at 23 ± 1 °C with 70% relative humidity.The seedlings of bread wheat cultivar Yannong999 used in this study were grown in a growth chamber under a 16-h/8-h, 20 °C/18 °C day/night cycle with 70% relative humidity. The GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (TaGAPDH) was set as the internal control and expressions of TaGAPDH, TaCAMTA2, TaCAMTA3, TaSARD1, TaEDS1, TaPR1, TaPR2 and TaPR5 were analyzed using the primers 5′-TTAGACTTGCGAAGCCAGCA-3′/5′-AAATGCCCTTGAGGTTTCCC-3′, 5′-TACAGAAGTTGCAACAG-3′/5′-ATCTCCGTCGACTCCTCA-3′, 5′-CCTGACAAACAACTTGA-3′/5′-CGCCAGCTGCA TCGCTT-3′, 5′-GCGAGTAATGAAAGCAT-3′/5′-TTAATCAACTTGATCCC-3′, 5′-TGAAAGACAGGGTGGGT-3′/5′-CGAAGGCACAAGTCTCG-3′, 5′-GAGAATGCAGACGCCCAAGC-3′/5′-CTGGAGCTTGCAGTCGTTGATC-3′, 5′-AGGATGTTGCTTCCATGTTTGCCG-3′/5′-AAGTAGATGCGCATGCCGTTGATG-3′, and 5′-CTTCTACATCAAGA ACAACTG-3′/5′-CAGTCGCCGGTCTGGCAG-3′.Total RNA was extracted from the wheat leaves using the EasyPure Plant RNA kit  and 2 μg of RNA was used to synthesize the cDNA template using the TransScript one-step gDNA removal and cDNA synthesis supermix  according to the manufacturer’s instructions. The real-time PCR assay was performed using the ABI real-time PCR system with the qPCR Master Mix . The expression of traditional housekeeping gene TaCAMTA2, TaCAMTA3, TaSARD1, and TaEDS1 was cloned into the pCa-γbLIC vector to create the BSMV-TaCAMTA2as, BSMV-TaCAMTA3as, BSMV-TaSARD1as, and BSMV-TaEDS1as constructs using the primer pair 5′-AAGGAAGTTTATACCATCATTAGCACTTGG-3′/5′-AACCACCACCACCGTCACTTTTGGAATTACATTC-3′, 5′-AAGGAAGTTTACATTATGCACCTGCGAGGA-3′/5′-AACCACCACCACCGTTCAGTGCACTTTGGTGAGC-3′, 5′-AAGGAAGTTTATGGTTCTAGTATCTATAAG-3′/5′-AACCACCACCACCGTGTTTGGAACCAGTTATTCG-3′, and 5′-AAGGAAGTTTAAGCGAATTCCCAACAGGTG-3′/5′-AACCACCACCACCGTAGACGGGGAAGTGTCAATC-3′. The BSMV-mediated gene silencing in wheat leaves was performed as described by Zhi et al. (2020) [B.g. tritici strain E09 conidia. About 72 h post-B.g. tritici inoculation, leaf segments were fixed with ethanol: acetic acid solution  and kept in the destaining solution . Before mounting for microscopy, B.g. tritici-infected leaves were stained with 0.1% (w/v) Coomassie Brilliant Blue R250 to visualize the fungal epiphytic structure, as reported previously [The antisense fragment of . (2020) . About 1eviously .TaCAMTA2, TaCAMTA3, TaSARD1, and TaEDS1 were, respectively, amplified using the primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTACCATCATTAGCACTTGG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCACTTTTGGAATTACATTC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCATTATGCACCTGCGAGGA-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGTGCACTTTGGTGAGC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTGGTTCTAGTATCTATAAG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCGTTTGGAACCAGTTATTCG-3′, and 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAGCGAATTCCCAACAGGTG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCAGACGGGGAAGTGTCAATC-3′, and cloned into the pIPKb007 vector using a Gateway cloning system to create the TIGS-TaCAMTA2, TIGS-TaCAMTA3, TIGS-TaSARD1, and TIGS-TaEDS1 constructs. The coding regions of TaCAMTA2-4A, TaCAMTA2-4B, TaCAMTA2-4D, TaCAMTA3-2A, TaCAMTA3-2B, TaCAMTA3-2D, TaSARD1.1-6A, TaSARD1.1-6B, TaSARD1.1-6D, TaSARD1.2-6A, TaSARD1.2-6B, TaSARD1.2-6D, TaEDS1-5A, TaEDS1-5B, and TaEDS1-5D were, respectively, amplified using the primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGAGGGCCGGCGCTAC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGAAATAGCCCGGCAACG-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGAGGGCCGGCGCTAC-3′/5′-GGGGACCACTTTGTACAAGAAAGCT GGGTCCTAGAAATAGCCAGGCAACG-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGAGGGCCGGCGCTAC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGAAATAGCCCGGCAACG-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGAGATGCACAAGTAC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACAAAATATTGGACATCG-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGAGATGCACAAGTAC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACAAAACAGTGGACATCG-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGAGATGCACAAGTAC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACAAAATAGTGGACATCG-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGTGCGAAGGCCGCG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGTGCGAAGGCCGCG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGTGCGAAGGCCGCG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCGGTGCGAAGGCCCCG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCGGTGCGAAGGCCACG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGATGGACACCCCGCC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACGAAGGCACAAGTCTCGC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGATGGACACCCCGCC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACGAAGGCACAAGTCTCGC-3′, and 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGATGGACACCCCGCC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACGAAGGCACAAGTCTCGC-3′, and cloned into the pIPKb001 vector. The single-cell transient gene silencing and expression were conducted essentially as described  [B.g. tritici strain E09 conidia, the leaf segments were stained for GUS activity 48 h post-B.g. tritici inoculation. Before mounting for microscopic analysis, the leaves were stained with 0.1% (w/v) Coomassie Brilliant Blue R250 to visualize the fungal epiphytic structure.Antisense fragments of ., 2020) . Briefly