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Discovery of Novel Defense Regulated WD40-Repeat Proteins DRW1/2 and Their Roles In bioRxiv preprint doi: https://doi.org/10.1101/786848; this version posted September 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Discovery of novel Defense Regulated WD40-repeat proteins DRW1/2 and their roles in 2 plant immunity * † *† 3 Authors: Jimi C. Miller , Brenden Barco , & Nicole K. Clay 4 Current Address: 5 *Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510, 6 USA. 7 †Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, 8 CT 06510, USA. 9 10 Author Contributions: B.B. performed the phylogenetic analysis. J.C.M. performed the homology 11 modeling, split-luciferase complementation, co-localization, MAPK assays, and Pto DC3000 12 infections. N.K.C. generated the agb1 drw1-1, agb1 drw2-1, and drw1 drw2 mutants, performed 13 the A. brassicicola fungal infections and contributed to Pto DC3000 infections. J.C.M. interpreted 14 the results and wrote the manuscript. 15 bioRxiv preprint doi: https://doi.org/10.1101/786848; this version posted September 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 16 ABSTRACT 17 Plant heterotrimeric G proteins transduce extracellular signals that activate plant immunity. Plants 18 encode canonical and non-canonical Gα and Gγ subunits, but only a single canonical Gβ subunit 19 is known. The existence of only one Gβ subunit limits the number of heterotrimeric G protein 20 combinations able to transduce different signals. It remains unknown whether non-canonical Gβ 21 subunits exist. Here, we identify two WD40-repeat genes that negatively regulate plant immunity. 22 The proteins encoded by these two genes, DEFENSE REGULATED WD40-REPEAT 1 and 2 23 (DRW1/2), are structurally similar to AGB1. DRW2 localizes to the plasma membrane and 24 interacts with the canonical Gα and Gγ subunits. Reduced levels of DRW in the drw1 and drw2 25 single mutants resulted in greater MAPK activation in response to flagellin treatment and conferred 26 increased resistance to the bacterial pathogen Pseudomonas syringae. Furthermore, the drw1 drw2 27 double-mutant also displayed increased MAPK activation upon flagellin treatment and broad- 28 spectrum resistance against bacterial and fungal pathogen infection. The function of DRW1 and 29 DRW2 is opposite of AGB1, which promotes immune signaling, suggesting that the function of 30 these potential non-canonical Gβ subunits are not conserved with the canonical Gβ subunit. Our 31 study identifies additional heterotrimeric G protein components, greatly increasing the number of 32 heterotrimeric G protein complexes that participate in signal transduction. 33 bioRxiv preprint doi: https://doi.org/10.1101/786848; this version posted September 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 34 INTRODUCTION 35 Plants respond to and survive the many different environmental stresses they are subjected to, 36 including pathogen infection. Plants perceive pathogens through the utilization of cell surface 37 pattern recognition receptors (PRRs). PRRs bind to conserved pathogen-associated molecular 38 patterns (PAMPs) such as flagellin from bacteria or chitin from fungal cell walls (Boller and Felix, 39 2009). The PRR FLAGELLIN-SENSITIVE 2 (FLS2) binds flagellin and initiates immune 40 signaling, which in turn leads to the activation of defense responses such as the production of 41 reactive oxygen species (ROS), activation of the mitogen-activated protein kinase (MAPK) 42 cascade, transcriptional upregulation of pathogen-induced genes, and production of defense 43 metabolites (Asai et al., 2002; Zipfel et al., 2004; Nürnberger and Lipka, 2005; Chinchilla et al., 44 2007; Boller and Felix, 2009). However, little is known about the signal transduction pathways 45 between the receptor complex and the nucleus. 46 Immune signaling from FLS2 is transduced by a membrane-localized heterotrimeric G protein 47 complex that activates downstream effectors. The heterotrimeric G protein complex is composed 48 of a Gα, Gβ, and Gγ subunit (Temple and Jones, 2007; Oldham and Hamm, 2008). The inactive 49 GDP-bound Gα subunit associates with the PRR and the obligate Gβγ heterodimer. When the PRR 50 is activated, this causes a conformational change in the Gα to exchange GDP for GTP, causing the 51 active GTP-bound Gα subunit to dissociate from the PRR complex and the Gβγ heterodimer 52 (Temple and Jones, 2007). The Gβγ heterodimer becomes activated after dissociating from the 53 GTP-bound Gα subunit and subsequently activates downstream effectors (Temple and Jones, 54 2007). 55 The Gβ and Gγ subunits form a Gβγ heterodimer through a coiled-coil interaction between the N- 56 terminal α-helices on the Gβ and Gγ subunits (Sondek et al., 1996). This heterodimer is obligatory bioRxiv preprint doi: https://doi.org/10.1101/786848; this version posted September 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 57 as the Gβ subunit will not fold properly in the absence of the Gγ subunit (Higgens and Casey, 58 1994). Interaction with the Gγ subunit promotes plasma membrane localization of the Gβ subunit 59 through the C-terminal prenylation site on the Gγ subunit that attaches it the plasma membrane 60 (Yasuda et al., 1996). However, there are reports in which the Gβ subunit is able to localize to the 61 plasma membrane independently of the Gγ subunit (Ullah et al., 2008; Navarro-Olmos et al., 2010; 62 Hackenberg et al., 2013). 63 Unlike animals, plants encode two different classes of heterotrimeric G proteins, canonical and 64 non-canonical. The canonical heterotrimeric G proteins include the Gα subunit GUANINE 65 NUCLEOTIDE-BINDING PROTEIN ALPHA-1 SUBUNIT (GPA1), the Gβ subunit 66 ARABIDOPSIS GTP BINDING PROTEIN BETA 1 SUBUNIT (AGB1), and the Gγ subunits 67 ARABIDOPSIS G PROTEIN GAMMA-SUBUNIT 1 and 2 (AGG1 and AGG2). The canonical 68 heterotrimeric G proteins were discovered through homology to the animal heterotrimeric G 69 proteins (Ma et al., 1990; Weiss et al., 1994; Mason & Botella, 2000, 2001). The non-canonical 70 heterotrimeric G proteins include the Gα subunits EXTRA-LARGE GUANINE NUCLEOTIDE- 71 BINDING PROTEIN 1/2/3 (XLG1/2/3), and the Gγ subunit ARABIDOPSIS G PROTEIN 72 GAMMA-SUBUNIT 3 (AGG3). The non-canonical heterotrimeric G proteins were identified by 73 searching for conserved domains between the non-canonical and canonical heterotrimeric G 74 proteins, such as the Ras domain of the Gα subunit or the isoprenylation motif at the C-terminus 75 of the Gγ subunit (Lee & Assmann, 1999; Assmann, 2002; Chakravorty et al., 2011). Despite these 76 conserved domains, the non-canonical heterotrimeric G proteins share low homology (less than 77 20% protein sequence identity) to the plant canonical heterotrimeric G proteins and animal 78 heterotrimeric G proteins (Lee & Assmann, 1999; Assmann, 2002; Chakravorty et al., 2011). bioRxiv preprint doi: https://doi.org/10.1101/786848; this version posted September 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 79 The Gβ subunit only contains two domains: an N-terminal α-helix and a seven tandem WD40- 80 repeat domain that adopts an asymmetrical seven-bladed β-propeller-like structure (Lambright et 81 al., 1996; Smith et al., 1999; Ullah et al., 2008; Adams et al., 2011; Ruiz et al., 2012). These two 82 domains are not unique to Gβ subunits as there are many other WD40 repeat proteins that share a 83 similar structure to the animal and plant Gβ subunits (Neer et al., 1994; Fulop et al., 1999; van 84 Nocker and Ludwig, 2003; Stirnimann et al., 2010; Xu and Min, 2011). This poses a challenge in 85 identifying novel non-canonical Gβ subunits in plants. 86 Both the canonical and non-canonical plant heterotrimeric G proteins are involved in plant 87 development and immunity (Zhang et al., 2018; Liu et al., 2013; Trusov et al., 2006; Trusov et al., 88 2007; Xu et al., 2015; Zhang et al., 2008). Loss of either XLG2 or XLG3 results in increased 89 susceptibility to the bacterial pathogen Pseudomonas syringae and the fungal pathogen Fusarium 90 oxysporum (Maruta et al., 2015). Interestingly, only XLG2 seems to be involved in resistance 91 toward the fungal pathogen Alternaria brassicicola, even though loss of both XLG2 and XLG3 92 causes severe susceptibility to all three pathogens (Maruta et al., 2015). XLG2 and XLG3 interact 93 with the Gβγ heterodimer as well as the PRR FLS2 (Maruta et al., 2015; Liang et al., 2016). 94 Specifically, XLG2 forms a heterotrimeric complex with the AGB1-AGG1/2 heterodimer that 95 binds to inactive FLS2 at the plasma membrane (Liang et al., 2016). Upon flagellin binding, FLS2 96 initiates dissociation of the heterotrimeric G protein complex, causing phosphorylation of XLG2 97 to enhance the production of reactive oxygen species (ROS) (Liang et al., 2016). The Gβγ 98 heterodimer is critical for proper immune signaling as loss of either the Gβ subunit AGB1 or both 99 redundant Gγ subunits AGG1 and AGG2 results in increased susceptibility to P. syringae and the 100 fungal pathogens F. oxysporum, Botrytis cinerea, and A. brassicicola (Liu et al., 2013; Trusov et bioRxiv preprint doi: https://doi.org/10.1101/786848; this version posted September 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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