provided relevant materials and resources; W.C., W.W. and H.P. Natl. Biomass was measured by genomic DNA quantitative PCR. 2010 - MAN TGM 4x4. Xiang, W. et al. http://dx.doi.org/10.1038/protex.2012.046 (2012), Watson, M. et al. Google Scholar. When amplification is complete, the RT-PCR products can be analyzed with gel electrophoresis.[48][49]. 185, 27592773 (2003), Suwa, Y. et al. Disease lesion size caused by the wildtype strain was also significantly reduced by application of AtPGIP1, although production of SsPINE1 allowed the wildtype strain to initiate infection even in the presence of excess AtPGIP1 (Fig. An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens. Nature 421, 756760 (2003). (S2G and S2H), An increased colocalization of endogenous or inducible HER2 in Golgi marker (arrowed) was observed upon extended treatment of cGAMP in HER2-driven tumor line BT474 (S2G) or HER2 Tet-On DLD1 cells (S2H). nov. and its phylogenetic relationship. Proc. The disadvantage of the two-step approach is susceptibility to contamination due to more frequent sample handling. 16, 30553071 (2014), Hovanec, T. A., Taylor, L. T., Blakis, A. To date, how fungal pathogens specifically overcome PGIP inhibition is unknown. 11, 6570 (2006). Unless specified, n=3 independent experiments. Cell 56, 723737 (2014). Biol. Kerppola, T. K. Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Microbiol. are named as inventors of a patent that is based on the presented findings. with n=3 replicates, Treatment means are significantly different from that of the wildtype Col-0 at P<0.05 (*), 0.01 (**) or 0.001 (***), respectively, in two-tailed t tests. SsPINE1 contributes to S. sclerotiorums exceptional ability to macerate host tissue by inactivating PGIPs and protecting PG activity. Plant pathogens degrade cell wall through secreted polygalacturonases (PGs) during infection. 6), suggesting that ectopic expression of SsPINE1 in the host can adequately restore virulence of the SsPINE1 mutant beyond the wild-type level. Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR). Juge, N. Plant protein inhibitors of cell wall degrading enzymes. 42, D222D230 (2014), Benson, D. A. et al. The experiment was performed three times independently with similar results obtained. Drivers Cab with AC, Cruise control and Parking heater. Prodigal: prokaryotic gene recognition and translation initiation site identification. Expression of SsPINE1-GFP in A. thaliana was confirmed by Western blot and fluorescence microscopy (Supplementary Fig. Nat Commun 13, 2213 (2022). Phosphorylated arginine residues (pArg) serve as a degradation signal that is recognized by the ClpC:ClpP (ClpCP) protease, a quasi-proteasomal particle critical for microbial protein quality control, stress tolerance, and pathogenicity (. RNA isolation of cells cultured within 3-D collagen gel will be performed in a manner similar to that of RNA isolation from tissues.. 1. ITC experiments with BacPROTAC-4 and BacPROTAC-4a were performed in a buffer containing 50mM Tris pH7.5, 300mM NaCl, 0.5mM TCEP. Supplementary Figure 7 HER2 protects cancer cells from STING-mediated antitumor immunity. 10c), another notorious broad host range necrotrophic plant pathogen8. Ueki, I. F. et al. nviron. Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Biochem. Australas. Microbiol. Anti-tuberculosis lead molecules from natural products targeting Mycobacterium tuberculosis ClpC1. Growth rate of SsPG1-knockout mutants was also compared with that of the wildtype on medium with polygalacturonic acid (2%) as the carbon source. Bio-Rad CFX96 Real-Time PCR Detection System; Bio-Rad DNA Engine Opticon and Opticon 2 Real Time PCR Detection Systems; GoTaq qPCR Master Mix Technical Manual PDF (442 KB) English. Yang, G. et al. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira-contigs in new metagenomes from engineered systems. Supplementary Figure 8 Unprocessed images of blots. Homokaryotic BcPINE1 replacement mutants were verified using PCR with primers BoE15-L/BoE13-R (Supplementary Table2) specific for the BcPINE1 (BC1G_04506) locus. 14, 213219 (2006), Article Microbiol. Each sample was tagged with an excess of the respective TMT labelling reagent (1:20, peptide:TMT label) and incubated at room temperature for one hour. ADS were made on agar plates containing 0.5% polygalacturonic acid and 0.8% agarose in 100mM sodium acetate buffer (pH 4.6). 1a), which demonstrates that SsPINE1 is a critical component for S. sclerotiorum virulence and SsPINE1-GFP is functional. PLoS One 8, e80835 (2013), Article About 500bps each of the 5 and 3 flanking sequences of SsPINE1 or SsPG1 open reading frames (ORF) were amplified from genomic DNA of the wildtype strain WMA1 with appropriate primer pairs (Supplementary Table2). 75, 195204 (2011), Martiny, A. C., Albrechtsen, H. J., Arvin, E. & Molin, S. Identification of bacteria in biofilm and bulk water samples from a nonchlorinated model drinking water distribution system: detection of a large nitrite-oxidizing population associated with Nitrospira spp. AtPGIP1, P1, P2 and P3 were cloned into plasmid pPIK9K that includes the poly-histidine tag. PubMed B. 43, D30D35 (2015), Markowitz, V. M. et al. The video shows the map of the tetramer of ClpC hexamers (10) and the map obtained refining a single ClpC hexamer (3.7). Total RNA samples of fungi and plants were isolated using RNeasy Mini Kit (QIAGEN) according to the manufacturers protocols. The experiment was performed three times with similar results. 08 August 2022, The ISME Journal 4c). Plant Pathol. Metagenomic bins are numbered as in Supplementary Table 8. & Wang, Y. 3922 wiseman blvd ste 304. most comfortable dining chairs Kroemer, G., Senovilla, L., Galluzzi, L., Andre, F. & Zitvogel, L. Natural and therapy-induced immunosurveillance in breast cancer. Typical results from the NEBNext Library Quant Kit on a Bio-Rad CFX96 Touch (A) and an Applied Biosystems 7500 Fast real-time qPCR instrument (B). Based on your Freezer Program type, you are trying to add a product to your cart that is either not allowed or not allowed with the existing contents of your cart. 5) were applied to Sclerotinia sclerotiorum inoculum of wildtype strain WMA1 and SsPINE1-deletion mutant KoSsPINE1-7 in detached pea leaf bioassays with PBS buffer as a control for comparison. Arch. 2022, Received in revised form: Front. Dou, Z. et al. Acad. Department of Plant Pathology, Washington State University, Pullman, WA, 99164, USA, Wei Wei,Kiwamu Tanaka,George Vandemark&Weidong Chen, State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwestern A&F University, Yangling, 712100, Shaanxi, China, Department of Crop & Soil Sciences, Washington State University, Pullman, WA, 99164, USA, Hao Peng,Karen A. Sanguinet&Weidong Chen, School of Life Science and Technology, Wuhan Polytechnic University, Wuhan, Hubei, 430023, China, Molecular Plant Science Program, Washington State University, Pullman, WA, 99164, USA, Kiwamu Tanaka,Karen A. Sanguinet&Weidong Chen, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China, USDA Agricultural Research Service, Grain Legume Genetics and Physiology Research Unit, Pullman, WA, 99164, USA, You can also search for this author in (S4F), Cotransfection of AKT1, but not AKT2 or AKT3, resulted in the effective TBK1 modification. 9, 29933007 (2007), Chain, P. et al. Columns show the mean normalized spectral abundance factor (NSAF), error bars show 1s.d. Unless specified, n=3 independent experiments. and F.Z. Wei, W., Xu, L., Peng, H. et al. N-glycosylation shields Phytophthora sojae apoplastic effector PsXEG1 from a specific host aspartic protease. Kishi, S. et al. Science 300, 11481151 (2003). Microbiol. Li, R. et al. Therefore, SsPG1 was selected for further study. Until now, this two-step reaction was thought to involve two organisms in a cross-feeding interaction. Targeted protein degradation: expanding the toolbox. Natl. The cDNA coding regions of AtPGIP1 and SsPINE1 were amplified by PCR with primers containing restriction sites (Supplementary Table4), and the amplified fragments were inserted into pGADT7 and pGBKT7, respectively. Agar discs (4mm diam.) Commun. The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant-pathogen interactions. Zhang, Q. et al. Curr. Clinical diganostics company Novacyt Google Scholar. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. J. Bacteriol. ADS The research was supported in part by the USDA National Sclerotinia Initiative to W.C. and by the National Natural Science Foundation of China grant number 31701737 to W.W. The emerging colonies on the DDO plates were subjected to three rounds of higher stringency selection on SD/- Ade/-His/-Leu/-Trp/X--Gal/Aureobasidin A (QDO/X/A) agar plates to obtain pure blue colonies. Previous studies showed that purified Sclerotinia PGs were very sensitive to inhibition by plant PGIPs19,44, but such sensitivity is lost in bioassays since S. sclerotiorum can readily infect and macerate host tissue, even in PGIP-overexpressing plants19,44, which may be at least partially due to the PGIP inactivation by endogenous SsPINE1. The products of RT-PCR can be analyzed with gel electrophoresis. The complementation vectors were also used to transform the wildtype strain to generate over-expression mutants for testing secretion of SsPINE1 and SsPG1. N. inopinata amoC to other amoC and pmoC genes (81 taxa, 993 alignment positions). 6a, b). Immunity 41, 843852 (2014). a Deletion of BcPINE1 reduced virulence of B. cinerea on Arabidopsis lines. & Barber, G. N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Microbiol. Overexpression of AtPGIP1-3xFlag in the transgenic plants was verified by Western blot with anti-Flag antibody and RT-qPCR and normalized using UBQ5 gene expression. PubMed Central Mol. The expression vectors were linearized with endonuclease SacI and used to transform the yeast P. pastorisi GS115 using Frozen-EZ Yeast Transformation II kit (Zymo Research, Irvine, CA 92614, U.S.A). However, how S. sclerotiorum specifically overcomes PG inhibition imposed by plant PGIPs remains unknown. P.X. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Microbiol. The mutation variants were fused with a His-tag and expressed in yeast. Pie charts indicate statistical support of branches based on maximum likelihood (ML; 1,000 bootstrap iterations) and Bayesian inference (BI; posterior probability, 4 independent chains). The natural product cyclomarin kills Mycobacterium tuberculosis by targeting the ClpC1 subunit of the caseinolytic protease. Nat. Environ. CAS Phthalimide conjugation as a strategy for invivo target protein degradation. P.X. carried out most of the experiments. Protac-induced protein degradation in drug discovery: breaking the rules or just making new ones?. Nitrosomonas Nm143-like ammonia oxidizers and Nitrospira marina-like nitrite oxidizers dominate the nitrifier community in a marine aquaculture biofilm. F.E.M., S.K., J.L., A.M., M.K., and T.C. Environ. and D.H. performed the cryo-EM analysis. Mol. 9, 593618 (2017). Natl Acad. performed proteomics measurements and data analysis; T.R. (S3A and S3B), No HER2-mediated TBK1 tyrosine phosphorylation was observed (S3A), where LCK-mediated tyrosine phosphorylation of TBK1 was set as a positive control. Annu. 7b, c). Kabbage, M., Williams, B. Sharma, S. et al. Guido), both SsPINE1 mutants caused significantly (39 to 45%) smaller disease lesions than the wildtype strain (Fig. Support values indicate the consensus probability from 5 independent chains. The fusion gene SsPINE1-GFP without signal peptide was amplified with primers YEST-E1ECOR1-L and YESTE1GFPNOT-R) from the CO-IP plasmid pCNG-SsPINE1 and cloned into the EcoRI and NotI sites of the plasmid pPIC9K, resulting in the expression vector pPIC-SsPINE1 (Supplementary Table3). Microbiol. HER2 overcomes PTEN (loss)-induced senescence to cause aggressive prostate cancer. 21, 15521560 (2011), Gregor, I., Drge, J., Schirmer, M., Quince, C. & McHardy, A. C. PhyloPythiaS+: A self-training method for the rapid reconstruction of low-ranking taxonomic bins from metagenomes. ISME J. Tu, D. et al. Microbiol. Sci. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. Sci. https://www.r-project.org/ (R Foundation for Statistical Computing, 2013). PubMed Central b Lesion area caused by WMA1, KOSsPINE1-7 and KOSsPG1-3 on Col-0, 35S::SsPINE1 and 35S::AtPGIP1 Arabidopsis lines at 48 hpi. Total synthesis of cyclomarin A, a marine cycloheptapeptide with anti-tuberculosis and anti-malaria activity. Biol. This is achieved by monitoring the amplification Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. For example, Sclerotinia sclerotiorum SsPG6 activity was severely inhibited by Brassica napus BnPGIP119. Rep. 50, 911 (2003). Sci. 3a, lane indicated by an arrow), indicating that SsPINE1 is more readily associated with AtPGIP1 than does SsPG1. Environ. Sella, L., Tomassini, A., DOvidio, R., Favaron, F. & Camillo de Lellis, S. Expression of two Sclerotinia sclerotiorum endo-PG genes correlates with endo-polygalacturonase activity during Glycine max colonization. Download PDF. Biol. (D)Cryo-EM density of the tyrosine-bearing pore loops overlaid with the final model. SsPINE1 targets a plant PGIP which is highly conserved; thus, SsPINE1 contributes to broad spectrum of virulence, consistent with the wide host range of S. sclerotiorum. The authors are grateful for using the analytical facilities of the Centre for Chemical Microscopy (ProVIS) (Helmholtz Centre for Environmental Research), which is headed by H. Richnow (Department of Isotope Biochemistry) and supported by European Regional Development Funds (EFREEurope funds Saxony) and the Helmholtz Association. Tanner, N.A., Zhang, Y. and Evans, T.C. 1a). Nature 528, 504509 (2015). PubMed CAS Reasonable aliquots of the compounds are available until stocks run out from the, Cell debris was removed by centrifugation. absence of the wildtype allele in the genome by detection with RT-PCR and Southern blot analysis). Construction of targeted mycobacterial mutants by homologous recombination. Plant Pathol. Image, Download Hi-res 1a, b). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Biotechnol. c, Phylogenetic relationship of Ca. Can I use dUTP and UDG for carryover contamination prevention with the WarmStart LAMP Master Mix? Gennari, R. et al. Since both SsPINE1 and SsPG1 interact with AtPGIP1, they may compete for binding. SsPG1 was tested in various concentrations to show quantitative nature of the enzyme assay. Microbiol. Basic local alignment search tool. Hippo signalling governs cytosolic nucleic acid sensing through YAP/TAZ-mediated TBK1 blockade. J . Sistigu, A. et al. FEMS Microbiol. This Expedition Truck has an extra 2 person fixed bunk bed for instant accessible sleeping space. Similarly, SsPG1-GFP without signal peptide was amplified using primers YEST-PG1ECOR1-L/ YEST-PG1NOT-R from pCNG-SsPG1, and AtPGIP1 without signal peptide using primers YEST-AD1RCOR1-L/ YEST-AD1NOT1-R from pCHNF3-AtPGIP1 and cloned into plasmid pPIK9K, resulting in plasmids pPIC-SsPG1 and pPIC-AtPGIP1, respectively (Supplementary Table3). 1c).Such porous structure provides large available culture surface on MCs (S3D), Cytosolic sensing of DNA analogs also induced the activation of HER2, along with the phosphorylation of AKT1, in HER2 Tet-On DLD1 cells. Google Scholar. Scale bars=20 m. [47] Additionally, planning and design of quantification studies can be technically challenging due to the existence of numerous sources of variation including template concentration and amplification efficiency. Together these data indicate that both sCym-1 and dCymM moieties can be incorporated into the chemical adaptor, tolerating distinct linker attachment points to yield potent and selective degraders. Phytopathology 108, 11281140 (2018). J. Please sign back in to continue your session. Appl. Sci. Representative images are shown. [23], The measurement approaches of end-point RT-PCR requires the detection of gene expression levels by the use of fluorescent dyes like ethidium bromide,[24][25] P32 labeling of PCR products using phosphorimager,[26] or by scintillation counting. The yeast expression system (Invitrogen, Carlsbad, CA) using the methylotrophic yeast Pichia pastoris GS115 was used to express AtPGIP1 and the fusion proteins of SsPINE1-GFP and SsPG1-GFP. For example, the polyphagous necrotrophic pathogen S. sclerotiorum has a genome that encodes at least five endo-polygalacturonases that are expressed at different infection stages and conditions. Sign up for the Nature Briefing: Cancer newsletter what matters in cancer research, free to your inbox weekly. 56, 311338 (2018). Larabi, A. et al. The pellet was resuspended in lysis buffer, loaded on a HiLoad 26/10 Q Sepharose High Performance column (GE Healthcare) equilibrated with lysis buffer and eluted in a gradient to 1M KCl. The genome of this chemolithoautotrophic organism encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. 8c). Structure and mechanism of the hexameric MecA-ClpC molecular machine. 03 June 2022, Nature Communications Given the advantages of bi-functional degraders, such as their modular design and possibility to repurpose diverse protein ligands, BacPROTACs could open new horizons to develop antimicrobials with high selectivity and species specificity. The S. sclerotiorum genome contains a single copy of SsPINE1 (Ss1g_08128) (https://www.broadinstitute.org/fungal-genome-initiative/sclerotinia-sclerotiorum-genome-project). Such a clear enhanced resistance by PGIP-overexpression to the SsPINE1 mutant but only subtle difference to the wild-type strain further demonstrates the critical role of SsPINE1 in Sclerotinia virulence. Sci. Plant Microbe Interact. Among the five interacting proteins, A. thaliana polygalacturonase-inhibitor 1 (AtPGIP1) is the only protein that has a known function35. PGs from S. sclerotiorum and other fungi play important roles in inducing necrosis and degrading pectin of plant cell walls in macerating host tissue and releasing nutrients for fungal growth10,11,46. Department of Microbiology and Ecosystem Science, Division of Microbial Ecology, University of Vienna, Althanstrasse 14, Vienna, 1090, Austria, Holger Daims,Petra Pjevac,Ping Han,Craig Herbold,Marton Palatinszky,Julia Vierheilig&Michael Wagner, Winogradsky Institute of Microbiology, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, bld. 2c). You are using a browser version with limited support for CSS. Previous research showed that PGIP binding affinity is influenced by amino acid residues on the -sheet-formed concave surface, which determines the specificity for PGs40,41. Rev. Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex, A family of conserved bacterial virulence factors dampens interferon responses by blocking calcium signaling, Development of BacPROTACs that reprogram ClpCP, Model substrates and bi-functional compounds used to address BacPROTAC-1-induced activity of, BacPROTAC binding induces ClpC reassembly and activation, BacPROTAC-1 induces formation of an active ClpC oligomer, BacPROTAC-1 induces formation of an active ClpC oligomer, related to, Cryo-EM data collection, refinement, and validation statistics, Cryo-EM structure of the activated ClpC hexamer in complex with a BacPROTAC-tethered substrate, Cryo-EM structure of the activated ClpC hexamer in complex with a BacPROTAC-tethered substrate, related to, Extending the BacPROTAC approach to mycobacteria, Characterization of compounds and BacPROTACs binding ClpC1, BacPROTACs can reprogram the mycobacterial ClpC1P1P2, BacPROTACs induce protein degradation in mycobacteria, BacPROTACs mediate selective POI degradation in mycobacteria, Induced degradation of target proteins in mycobacteria, BacPROTAC-induced degradation (BID) approach in mycobacteria, BacPROTAC-induced degradation (BID) approach in mycobacteria, related to, Analytical size exclusion chromatography (SEC), Negative staining EM sample preparation, data collection and processing, Cryo-EM sample preparation and data collection, Cryo-EM analysis of the ternary complex between ClpC, BacPROTAC-1 and mSA: the tetramer of hexamers, Cryo-EM analysis of the ternary complex between ClpC, BacPROTAC-1 and mSA: the hexameric building block, Cultivation and Susceptibility Tests Using, Sample preparation for quantitative mass spectrometry analysis, High pH chromatography and LC-MS/MS analysis, https://doi.org/10.1016/j.cell.2022.05.009, BacPROTACs mediate targeted protein degradation in bacteria, View Large The qRTPCR reaction (20 L) consisted of 10 L of iTaq Universal SYBR Green Supermix (Bio-Rad, CA, USA), 1 L of primers containing 900 nM of forward and reverse primers, and 2 L of template cDNA. Plant Sci. The experiment was performed three times with similar results. This research was sponsored by the National Science Foundation for Distinguished Young Scholars (grant no. VAT will be added later in the checkout.Tax calculation will be finalised during checkout. 91, fiu031 (2015), Article Sci. P values are indicated, by ANOVA test and Bonferroni correction in statistics. Article Nat. The interaction between SsPINE1 and AtPGIP1 was further confirmed by an in vivo co-immunoprecipitation (Co-IP) assay (Fig. PubMed The extreme sensitivity of the technique can be a double edged sword since even the slightest DNA contamination can lead to undesirable results. USA 108, 1639216397 (2011). A RNA target (HMBS2) was amplified from Jurkat total RNA using the WarmStart LAMP Kit and OptiGene Master Mix (ISO-001). What is the optimal LAMP/RT-LAMP amplification temperature? were added to 0.5g SsPG1 in 225-l volume (0.5% polygalacturonic acid in 100mM sodium acetate buffer). Albersheim and Anderson15 predicted that potential PGIP inhibitors should have higher affinity with PGIPs and/or be present at higher abundance to be functionally effective. Nitrogen cycling and microbial cooperation in the terrestrial subsurface, A nitrite-oxidising bacterium constitutively consumes atmospheric hydrogen, Disturbance-based management of ecosystem services and disservices in partial nitritation-anammox biofilms, https://img.jgi.doe.gov/cgi-bin/m/main.cgi, http://dx.doi.org/10.1038/protex.2012.046, Extended Data Figure 1 Photomicrographs and cell diagram of. Google Scholar. You are using a browser version with limited support for CSS. Interestingly, in the SsPG1-deletion mutants, no obvious compensation by other SsPGs was observed (Fig. CAS Cancer cell-autonomous contribution of type I interferon signaling to the efficacy of chemotherapy. Highly accurate protein structure prediction with AlphaFold. Ou, Y. H. et al. (S6B and S6C), The effect of HER2 on alleviating HU-induced cellular senescence was entirely dependent of the presence of STING, as revealed by -Gal staining (S6B) and the SASP (S6C). 6, 847862 (2012), Angel, R. Total nucleic acid extraction from soil. However, in S. sclerotiorum, the importance of PGs was shown through enzyme analysis and gene expression patterns11,38. Immunol. Plants produce polygalacuturonase-inhibiting proteins (PGIPs) to counteract cell wall degradation by pathogenic microbes. N. inopinata genome. 74, 10181027 (2010), Article In conclusion, our data demonstrate that BacPROTACs represent a versatile molecular tool that can be applied to various protein substrates and allow integration of diverse head groups and chemical linkers. 56, 919 (2020). Plant Microbe Interact. 5a, b). Phytopathology 87, 161163 (1997). Since PG-PGIP binding is reversible39, we tested if the addition of a competing protein could dissociate the SsPINE1/AtPGIP1 or SsPG1/AtPGIP1 complexes formed in vivo. What are the advantages of using standard LAMP (NEB #E1700, M1708, E1708) over pH-based colorimetric LAMP (NEB #M1800, M1804)? A slight decrease in the ammonium concentration was observed in these experiments and also in an abiotic control incubation containing only medium and 1mM ammonium, but no cells (data points for this control show means of two technical replicates). The purified proteins were analyzed by immunoblotting with anti-6X His EPITOPE TAG (RABBIT) Antibody (ROCKLAND Cat No.666-401-382S). Similar to the situation with S. sclerotiorum, overexpression of AtPGIP1 enhanced resistance to B. cinerea, particularly to the BcPINE1-knockout mutant KoBcPINE1-6. 2. After sequencing the 130 positive clones, 108 prey proteins were identified (Supplementary Table1). For Extended Data Fig. Peptides were labelled for quantification in a multiplexed setup with TMT isobaric mass tags (TMTpro 16plex). FEMS Microbiol. 72, 62996315 (2006), Radajewski, S. et al. Microbiol. Microbiol. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities. Sci. 175, 438456 (2017). 41, D636D647 (2013), Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Med. 5c), demonstrating the role of SsPG1 in securing nutrients from pectin-like substances.

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