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Proteomic Analysis of SUMO1-Sumoylome Changes During Defense 4 Elicitation in Arabidopsis

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Proteomic Analysis of SUMO1-Sumoylome Changes During Defense 4 Elicitation in Arabidopsis bioRxiv preprint doi: https://doi.org/10.1101/2020.08.02.233544; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Running Title: SUMO1-substrate identification in plant immunity 2 3 Proteomic analysis of SUMO1-SUMOylome changes during defense 4 elicitation in Arabidopsis 5 6 Kishor D. Ingole1,2 Shraddha K. Dahale1 and Saikat Bhattacharjee1* 7 8 1Laboratory of Signal Transduction and Plant Resistance, UNESCO-Regional Centre for Biotechnology 9 (RCB), NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad- 121 10 001, Haryana, India. 11 12 2Kalinga Institute of Industrial Technology (KIIT) University, Bhubaneswar- 751 024, Odisha, India. 13 14 Author for Correspondence: 15 Saikat Bhattacharjee 16 Tel: (+91) 0129-2848837 17 email: [email protected] 18 19 ORCID 20 Saikat Bhattacharjee: https://orcid.org/0000-0003-1369-7700 21 Kishor D. Ingole: https://orcid.org/0000-0003-1946-6639 22 Shraddha K. Dahale: https://orcid.org/0000-0001-8458-3984 23 24 25 26 27 28 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.02.233544; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 29 Abstract 30 Rapid adaptation of plants to developmental or physiological cues is facilitated by specific receptors 31 that transduce the signals mostly via post-translational modification (PTM) cascades of downstream 32 partners. Reversible covalent attachment of SMALL UBIQUITIN-LIKE MODIFIER (SUMO), a 33 process termed as SUMOylation, influence growth, development and adaptation of plants to various 34 stresses. Strong regulatory mechanisms maintain the steady-state SUMOylome and mutants with 35 SUMOylation disturbances display mis-primed immunity often with growth consequences. Identity of 36 the SUMO-substrates undergoing SUMOylation changes during defences however remain largely 37 unknown. Here we exploit either the auto-immune property of an Arabidopsis mutant or defense 38 responses induced in wild-type plants against Pseudomonas syringae pv tomato (PstDC3000) to enrich 39 and identify SUMO1-substrates. Our results demonstrate massive enhancement of SUMO1-conjugates 40 due to increased SUMOylation efficiencies during defense responses. Of the 261 proteins we identify, 41 29 have been previously implicated in immune-associated processes. Role of others expand to diverse 42 cellular roles indicating massive readjustments the SUMOylome alterations may cause during induction 43 of immunity. Overall, our study highlights the complexities of a plant immune network and identifies 44 multiple SUMO-substrates that may orchestrate the signalling. 45 46 Keywords: Post-translational modification (PTM), SUMOylation, autoimmunity, PstDC3000, LC- 47 MS/MS, plant-pathogen interaction, plant defense response. 48 49 50 51 52 53 54 55 56 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.02.233544; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 57 INTRODUCTION 58 Post-translational modifications (PTMs) by reversible attachment of the SMALL UBIQUITIN- 59 LIKE MODIFIER (SUMO), through a process termed as SUMOylation, dynamically and reversibly 60 modulate target protein fate, localization, or function (reviewed in Morrell and Sadanandom, 2019). In 61 plants, abiotic or biotic cues cause massive changes on global SUMOylome (Bailey et al. 2015; Miller et 62 al. 2010; Colignon et al. 2017a). Though primarily utilized to regulate nuclear functions such as DNA 63 replication, chromatin remodeling, transcription in eukaryotes, role of SUMOylation extend to other 64 organellar processes (Colignon et al. 2017a; Miller et al. 2010). Nevertheless, the vast majority of nuclear 65 proteins as SUMO-substrates is indicative of the rapid mechanism to modulate transcriptome in response 66 to stimulus. 67 SUMOylation cascade recruits processed SUMOs to covalently attach via an isopeptide bond to ε- 68 amino group of lysine residues on target proteins. The modified lysine often is a part of a partially conserved 69 motif, ψ-K-X-D/E (ψ = hydrophobic amino acid, X= any amino acid, D/E= aspartate/glutamate). A 70 conjugatable SUMO, subsequently forms a thiol-ester bond with the SUMO E1 ACTIVATING ENZYME 71 (SAE). A trans-esterification reaction further shuttles SUMO to the SUMO E2 CONJUGATING ENZYME 72 1 (SCE1) and then to target lysine on SUMOylation substrates. SUMO1 can conjugate to itself and form 73 poly-SUMO1 chains (Miller et al. 2010). SUMO-substrate specificities are regulated by SUMO E3 74 LIGASES such as HIGH PLOIDY2/METHYL METHANE SULFONATE21 (HPY2/MMS21) and SAP 75 and MIZ1 (SIZ1) (Morell and Sadanandom, 2019). Fate of covalently-attached SUMOs may either include 76 de-conjugation and recycling by SUMO proteases such as EARLY IN SHORT DAYS 4 (ESD4), its closest 77 homolog ESD4-LIKE SUMO PROTEASE 1 (ELS1), or targeted proteolysis of the substrate through 78 ubiquitin-mediated pathway. SUMO-interactions are also non-covalent in nature facilitated by hydrophobic 79 amino acid-rich SUMO-interaction motifs (SIMs) present in the recipient. SIMs populate several 80 SUMOylation-associated proteins implying strong auto-regulatory mechanisms (Wang et al. 2010). Unlike 81 fruit fly, worm, or yeast, humans and plants express multiple SUMO isoforms (Flotho and Melchior, 2013). 82 In Arabidopsis thaliana, 4 SUMO isoforms are expressed namely SUMO1, SUMO2, SUMO3 and SUMO5 83 suggestive of substrate specificities and/or SUMO crosstalks (van den Burg et al. 2010). 84 Distinctions between self and non-self in plants is facilitated by the robust two-layered immune 85 system (Jones and Dangl, 2006). Primary defense signalling routes responds to recognition via surface- 86 localized pattern-recognition receptors (PRRs) for conserved molecular signatures called pathogen- or 87 microbe-associated molecular patterns (PAMPs/MAPMs) present on pathogens, to elicit PAMP-triggered 88 immunity (PTI). Manipulative pathogens secrete effectors to suppress PTI which in turn are detected by 89 intracellular resistance (R) proteins to activate a more amplified effector-triggered immunity (ETI). ETI in 90 many cases lead to a programmed cell death called hypersensitive response (HR) (Jones and Dangl, 2006). 91 The hormone salicylic acid (SA) plays a central role in plant immunity and its accumulation drives 92 intricately interconnected networks causing increased expression of PATHOGENESIS RELATED bioRxiv preprint doi: https://doi.org/10.1101/2020.08.02.233544; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 93 PROTEIN 1/2 (PR1/2) and other defense-associated genes (Loake and Grant, 2007). A key mediator of SA- 94 signaling is ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) the null-mutant of which dramatically 95 decreases plant combat abilities (Wiermer et al. 2005). Preventing mis-priming of immunity and regulating 96 of its amplitudes especially in the context of activation of R proteins is performed by negative regulators 97 such as SUPPRESSOR OF rps4-RLD1 (SRFR1) (Kim et al. 2010). A srfr1-4 mutant is growth retarded, 98 has elevated SA-based defences, and is enhanced resistant to the bacterial pathogen Pseudomonas syringae 99 pv tomato DC3000 (PstDC3000). SRFR1 encodes a tetratricopeptide (TPR) protein with versatile 100 interactors such as multiple R proteins, EDS1, co-chaperone SUPPRESSOR OF THE G2 ALLELE OF 101 skp1 (SGT1), and members of the TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription 102 factors (Bhattacharjee et al. 2011; Kim et al. 2014, 2010; Li et al. 2010). Loss of EDS1 abolishes growth 103 retardation and increased immunity of srfr1-4 implicating constitutive SA-signaling sectors for these 104 defects. These features make srfr1-4 an excellent system for studying defense signaling processes. 105 Host SUMOylome readjustments influence immune outcomes and been comprehensively 106 highlighted in several excellent reviews (Augustine and Vierstra, 2018; van den Burg and Takken, 2010). 107 Arabidopsis siz1-2, SUMO protease mutants OVERLY TOLERANT TO SALT 1/2 (OTS1/2), or esd4-1 plants 108 accumulate elevated SA, constitutively express PR proteins and are increased resistant to PstDC3000 (Lee 109 et al. 2006; Bailey et al. 2015; Villajuana-Bonequi et al. 2014). Plants null for SUM1 and expressing 110 microRNA-silenced SUM2 (sum1-1 amiR-SUM2) also have heightened immunity (van den Burg et al. 111 2010). Constitutive activation of SA-dependent defenses occur in SUM1/2/3-overexpressing transgenic 112 plants (van den Burg et al. 2010). From these reports, it is increasingly evident that unregulated increases 113 (eds4-1, ots1/2 or over-expressing SUM1/2) or decreases (in siz1-2 or sum1-1 amiR-SUM2) in global 114 SUMOylome bear immune consequences. It is also not surprising
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