Mutation of a Conserved Motif of PP2C.D Phosphatases Confers SAUR Immunity … · SAUR gene family...

Mutation of a Conserved Motif of PP2C.D PhosphatasesConfers SAUR Immunity and Constitutive Activity1[OPEN]

Jeh Haur Wong,2 Angela K. Spartz,2 Mee Yeon Park, Minmin Du, and William M. Gray3,4

Department of Plant and Microbial Biology, University of Minnesota, St. Paul, Minnesota 55108

ORCID IDs: 0000-0002-3697-6051 (J.H.W.); 0000-0001-8247-3925 (M.Y.P.); 0000-0003-2858-8582 (M.D.); 0000-0002-1320-290X (W.M.G.).

The phytohormone auxin promotes the growth of plant shoots by stimulating cell expansion via plasma membrane (PM) H1-ATPase activation, which facilitates cell wall loosening and solute uptake. Mechanistic insight was recently obtained bydemonstrating that auxin-induced SMALL AUXIN UP RNA (SAUR) proteins inhibit D-CLADE TYPE 2C PROTEINPHOSPHATASE (PP2C.D) activity, thereby trapping PM H1-ATPases in the phosphorylated, activated state, but howSAURs bind PP2C.D proteins and inhibit their activity is unknown. Here, we identified a highly conserved motif near theC-terminal region of the PP2C.D catalytic domain that is required for SAUR binding in Arabidopsis (Arabidopsis thaliana).Missense mutations in this motif abolished SAUR binding but had no apparent effect on catalytic activity. Consequently,mutant PP2C.D proteins that could not bind SAURs exhibited constitutive activity, as they were immune to SAURinhibition. In planta expression of SAUR-immune pp2c.d2 or pp2c.d5 derivatives conferred severe cell expansion defects andcorresponding constitutively low levels of PM H1-ATPase phosphorylation. These growth defects were not alleviated by eitherauxin treatment or 35S:StrepII-SAUR19 coexpression. In contrast, a PM H1-ATPase gain-of-function mutation that results in aconstitutively active H1 pump partially suppressed SAUR-immune pp2c.d5 phenotypes, demonstrating that impaired PM H1-ATPase function is largely responsible for the reduced growth of the SAUR-immune pp2c.d5 mutant. Together, these findingsprovide crucial genetic support for SAUR-PP2C.D regulation of cell expansion via modulation of PM H1-ATPase activity.Furthermore, SAUR-immune pp2c.d derivatives provide new genetic tools for elucidating SAUR and PP2C.D functions andmanipulating plant organ growth.

The phytohormone auxin coordinates plant growthand development by regulating the fundamental pro-cesses of cell division, expansion, and differentiation(Strader and Zhao, 2016; Zhao, 2018). Plants perceivethe auxin signal through a coreceptor complex com-prising TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX PROTEINS (TIR1/AFBs)and AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA)transcriptional repressors in the nucleus, which uponauxin binding leads to AUX/IAA ubiquitylation bythe SCFTIR1/AFB complex and subsequent degradationby the 26S proteasome (Gray et al., 2001; Dharmasiriet al., 2005; Kepinski and Leyser, 2005). Aux/IAA

degradation results in the derepression of AUXINRESPONSE FACTOR transcription factors, leading tochanges in auxin-responsive gene expression that sub-sequently direct auxin-induced physiological and de-velopmental responses (Lavy and Estelle, 2016). Earlyauxin-responsive genes are induced within minutes,including AUX/IAAs, GRETCHEN HAGEN3s (GH3s),and SMALL AUXIN UP RNAs (SAURs; Hagen andGuilfoyle, 2002). While induction of AUX/IAA andGH3 expression attenuates auxin signaling throughfeedback regulation (Lavy and Estelle, 2016), up-regulation of SAUR expression has been proposed topromote cell expansion leading to organ growth (Spartzet al., 2012, 2014).In Arabidopsis (Arabidopsis thaliana), the plant-specific

SAUR gene family consists of 81 members (includingtwo pseudogenes), which share high sequence similar-ity. Most, but not all, SAUR genes are auxin inducible(Ren andGray, 2015).SAUR expression is also controlledthrough mRNA instability and posttranslational controlthrough the ubiquitin-26S proteasome pathway (Giland Green, 1996; Knauss et al., 2003; Chae et al., 2012;Spartz et al., 2012; Vi et al., 2013), although the reg-ulation of these aspects of SAUR expression is poorlyunderstood. In many cases, the rapid degradationof SAUR proteins can be overcome with the additionof GFP or epitope tags, which partially stabilizethe proteins (Chae et al., 2012; Spartz et al., 2012).This strategy has enabled gain-of-function geneticapproaches to elucidate SAUR function. Arabidopsis

1This work was supported by the National Science Foundation(MCB-1613809 to W.M.G.) and the National Institutes of Health(GM067203 to W.M.G.).

2These authors contributed equally to the article.3Author for contact: [email protected] author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:William M. Gray ([email protected]).

W.M.G., J.H.W., and A.K.S. designed the research; J.H.W., A.K.S.,M.Y.P., andM.D. performed the experiments; W.M.G. supervised theexperiments; J.H.W. and W.M.G. wrote the article with contributionsfrom all authors.

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plants expressing stabilized SAUR19 or SAUR63 fu-sion proteins display increased cell expansion pheno-types, suggesting that these SAURs promote cellexpansion. For example, 35S:GFP-SAUR19 plants dis-play increased hypocotyl length and leaf size, whilePSAUR63:SAUR63:GFP plants exhibit long hypocotyl,petal, and stamen filament phenotypes (Franklin et al.,2011; Chae et al., 2012; Spartz et al., 2012).

Recent findings suggest that SAURs activate plasmamembrane (PM) H1-ATPases to promote cell expan-sion via an acid growth mechanism. Based on physio-logical studies correlating auxin-induced expansionwith apoplastic acidification, the acid growth theorywas first proposed in 1970 (Rayle and Cleland, 1970,1980, 1992; Hager, 2003). H1 pump activation reducesapoplastic pH, which activates expansins and cellwall modification enzymes to enable cell expansion(McQueen-Mason et al., 1992; Cosgrove, 2016). Addi-tionally, increased PM H1-ATPase activity hyperpo-larizes the PM, leading to solute and water uptake andincreased turgor that drives cell expansion. Takahashiet al. (2012) provided mechanistic insight into thisprocess by demonstrating auxin-induced phosphoryl-ation of the penultimate Thr within the autoinhibitorydomain of PM H1-ATPases. Phosphorylation of thisresidue (corresponding to Thr-947 of ARABIDOPSISH1 ATPASE [AHA2]) promotes 14-3-3 protein bind-ing and H1 pump activation (Fuglsang et al., 1999;Kinoshita and Shimazaki, 1999; Jelich-Ottmann et al.,2001). In addition to increased cell expansion pheno-types, 35S:GFP-SAUR19 seedlings also exhibit increasedPM H1-ATPase activity and Thr-947 phosphorylation(Spartz et al., 2014). SAUR proteins promote PMH1-ATPase activation by inhibiting D-CLADETYPE 2C PROTEIN PHOSPHATASES (PP2C.Ds),which can directly dephosphorylate Thr-947 of PMH1-ATPases (Spartz et al., 2014, 2017). This suggests amodel whereby auxin induction of SAUR expressionleads to a reduction in PP2C.D activity, thus leading toincreased PM H1-ATPase phosphorylation, whichdrives cell expansion. Consistent with this model,plants constitutively overexpressing GFP-SAUR19exhibit reduced apoplastic pH and auxin-independentelongation growth (Spartz et al., 2014, 2017; Fendrychet al., 2016; Barbez et al., 2017).

The Arabidopsis genome encodes nine PP2C.Dmembers, which belong to the Mg21/Mn21-dependentPP2C family of protein phosphatases (Fuchs et al.,2013). Individual members display unique subcellularlocalizations, however, suggesting functional specific-ity (Tovar-Mendez et al., 2014; Ren et al., 2018). Nota-bly, like SAUR19 and PM H1-ATPases, PP2C.D2,PP2C.D5, and PP2C.D6 localize exclusively to the PM.We recently demonstrated that the pp2c.d2/5/6 triplemutant phenocopies 35S:GFP-SAUR19 gain-of-func-tion plants in a variety of growth assays and in bio-chemical assays examining PM H1-ATPase Thr-947phosphorylation status (Ren et al., 2018). Togetherwith our biochemical findings that SAURs inhibitPP2C.D enzymatic activity, these findings provide

strong evidence supporting the hypothesis that SAURand PP2C.D proteins function antagonistically to reg-ulate PM H1-ATPase activity and expansion growth.Missing from this model, however, is strong geneticsupport derived from saur loss-of-function mutants.Recently, saur16/50 double mutants were shown to ex-hibit modest organ-specific cell expansion defects (Sunet al., 2016). However, these mutant phenotypes weredramatically weaker than those conferred by PP2C.Doverexpression (Spartz et al., 2014; Ren et al., 2018).While this difference could be due to ectopic PP2C.Dexpression, given the large number of SAUR genes inplant genomes, it seems likely that extensive functionalredundancy exists within the large SAUR gene family.Given the plethora of SAUR genes in Arabidopsis andother plants, we investigated the possibility that theSAUR binding and phosphatase activities of PP2C.Dproteins were genetically separable. If so, pp2c.d mu-tants that cannot bind SAURs yet retain phosphataseactivity may be able to serve as genetic proxies for saurloss-of-function plants. Here, we identify a highlyconserved, unique motif near the C terminus of thecatalytic domain of PP2C.D proteins that is essentialfor SAUR binding. We find that single missense mu-tations within this motif abolish SAUR binding andinhibition, resulting in phosphatases with constitu-tive enzymatic activity. Our findings demonstratethat SAUR-immune pp2c.d2 and pp2c.d5 derivativesconstitutively dephosphorylate and inhibit PMH1-ATPases, thus restricting plant cell elongation andorgan growth.

RESULTS

PP2C.D1 Deletion Analysis

To begin to investigate the SAUR-binding determi-nants of PP2C.D phosphatases, we conducted a dele-tion analysis of PP2C.D1 and assessed binding activityin yeast two-hybrid assays. We chose PP2C.D1 as itexhibits the most robust interaction with SAUR19 inthis system. PP2C.D phosphatases are composed pri-marily of the core PP2C catalytic domain (amino acids41–342 of PP2C.D1) with short N- and C-terminalextensions. PP2C.D1 containing a deletion of theC-terminal 26 amino acids (D345-370) retained theability to interact with SAUR19. Longer C-terminaldeletions (D332-370 and D292-370), however, abol-ished SAUR binding (Supplemental Fig. S1A). Ex-pression of these truncated proteins was confirmedby immunoblot analysis (Supplemental Fig. S1B).Likewise, a short N-terminal deletion (D1-36) retainedSAUR-binding activity, but the longer D1-116 mutantderivative did not. In this case however, we were un-able to detect expression of the D1-116 mutant deriv-ative, so it remains unclear whether this region isinvolved in SAUR binding. We also tested a deletionwithin the catalytic domain (D227-255) that had noeffect on SAUR-binding activity (Supplemental Fig.S1, A and C).

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Identification of PP2C.D Missense Mutations That PreventSAUR Binding

Our deletion studies indicated that the C-terminalregion of PP2C.D1 was required for SAUR binding.However, since we could not conclusively eliminateother regions, we generated a library of pp2c.d1 mis-sense mutants using error-prone PCR and tested in-teractions with SAUR19 in a reverse yeast two-hybridscreen. Upon screening ;1,500 library clones, 56 can-didates were identified that abolished interactionwith SAUR19 and that also passed retesting follow-ing plasmid rescue and retransformation and immu-noblot analysis to verify expression. Sequence analysisrevealed that 19 of the 56 candidates contained singlemissense mutations within the PP2C.D1 coding se-quence (Fig. 1A). The remaining candidates containedtwo ormoremutations or rearrangements andwere notanalyzed further.Strikingly, over half of the missense mutations

identified localized to the very C-terminal end of thecatalytic domain (amino acids 317–336; Fig. 1, A and B).Multiple sequence alignment of the ArabidopsisPP2C.D proteins revealed that this region was nearlyperfectly conserved among all nine ArabidopsisPP2C.D members, as well as PP2C.D orthologs from avariety of angiosperms as well as bryophytes (Fig. 1C).Furthermore, this sequence is unique to D-clade phos-phatases and represents an insertion that is missing inall other PP2C families, with the exception of a looselyrelated sequence in C-clade PP2Cs (Supplemental Fig.S2). To further test the importance of thismotif in SAURbinding, select pp2c.d1missense mutants were tested inin vitro pull-down assays with SAUR19. Both thepp2c.d1M317K and pp2c.d1L322Q mutations completelyabolished SAUR19 binding, whereas the pp2c.d1D321Gmutation modestly reduced binding in this assay(Fig. 1D).Given the high degree of sequence conservation of

this motif among PP2C.D family members, we exam-ined the possibility that mutations analogous to thoseidentified in PP2C.D1 could also disrupt SAUR bindingwhen introduced into other PP2C.D family members.Since the pp2c.d1M317K mutation completely abolishedSAUR binding in both two-hybrid and pull-downassays, we chose this residue to test this possibilityand generated pp2c.d2M331K, pp2c.d3M330K, pp2c.d4M331K,and pp2c.d5M328K mutant derivatives. Indeed, yeasttwo-hybrid assays revealed that these analogousmutations conferred the same effect on these PP2C.Dfamily members by preventing interaction withSAUR19 (Fig. 1E).The above findings indicate that a highly conserved

motif near the C terminus of the PP2C.D catalytic do-main is essential for SAUR-binding activity. To inves-tigate whether this domain is sufficient to mediateSAUR interaction, we replaced the C-terminal aminoacids (407–434) of the A-clade PP2C, ABA INSENSI-TIVE1 (ABI1), with the C terminus (amino acids313–370) of PP2C.D1. In yeast two-hybrid assays,

however, SAUR19 did not interact with this ABI1-PP2C.D1 fusion protein (Supplemental Fig. S3A). Ex-pression of the hybrid protein was verified by westernblotting (Supplemental Fig. S3B). Together, our find-ings suggest that the C terminus of the PP2C.D catalyticdomain is essential, but not sufficient, for SAURbinding.

SAUR-Immune PP2C.Ds Exhibit ConstitutivePhosphatase Activity

We next asked if mutations in the C-terminalmotif affect PP2C.D phosphatase activity. Wild-typePP2C.D1 and the M317K, R318K, D321G, andL322Q mutant derivatives were purified from Esche-richia coli as GST fusion proteins and tested in in vitrophosphatase assays with the chromogenic substrate,p-nitrophenylphosphate (pNPP). All mutant deriva-tives exhibited phosphatase activity comparable towild-type PP2C.D1, indicating that the mutations donot have major effects on catalytic activity (Fig. 2A).Since our previous work demonstrated strong

SAUR9 inhibition of several Arabidopsis PP2C.Dphosphatases (Spartz et al., 2014), we used SAUR9 toexamine the ability of SAURs to inhibit the pp2c.d1M317Kmutant as well as the analogous mutant derivatives ofPP2C.D2, PP2C.D4, and PP2C.D5. The addition of re-combinant SAUR9 protein to pNPP phosphatase assaysstrongly inhibited wild-type PP2C.D1, PP2C.D2, andPP2C.D5 and, to a somewhat lesser extent, PP2C.D4(Fig. 2B). In contrast, phosphatase activities of theM→Kpp2c.d mutant derivatives were not inhibited whatso-ever by SAUR9 addition. Together with our proteininteraction data, these findings demonstrate that SAURbinding activity is required for SAUR inhibition ofPP2C.D activity. pp2c.d mutations that disrupt SAURbinding result in constitutively active phosphatasesthat are immune to SAUR inhibition.Our previous findings demonstrated that several

PP2C.Ds can interact with AHA2 and dephosphorylatethe C-terminal Thr-947 residue (Spartz et al., 2014; Renet al., 2018). To investigate whether SAUR-immunePP2C.D proteins could still bind AHA2, we per-formed bimolecular fluorescence complementation(BiFC) assays where AHA2 was coexpressed in Nicoti-ana benthamiana leaves with either wild-type PP2C.D1,PP2C.D2, PP2C.D5, and PP2C.D6 or the M→K mutantpp2c.d derivatives. Strong PM-localized yellow fluo-rescent protein (YFP) signal was observed in all cases(Fig. 2C; Supplemental Fig. S4), demonstrating thatthe M→Kmutants retain the ability to interact with PMH1-ATPases.To determine if SAUR-immune PP2C.Ds could still

dephosphorylate the Thr-947 residue of AHA2, weconducted in vitro AHA2 dephosphorylation assaysas previously described (Spartz et al., 2014). Thisassay used yeast-expressed AHA2 that is phosphory-lated on Thr-947. Both wild-type and SAUR-immunePP2C.Ds could dephosphorylate AHA2, as shown by

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Analysis of SAUR-Immune PP2C.D Phosphatases

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GST-14-3-3 far-western gel-blotting assays, which arewidely employed for assessing Thr-947 phosphoryla-tion status (Fuglsang et al., 1999; Kinoshita andShimazaki, 1999; Hayashi et al., 2010). Consistent

with our pNPP assay findings, SAUR9 stronglyinhibited wild-type PP2C.D proteins from dephos-phorylating AHA2 but had no effect on the SAUR-immune PP2C.D mutant derivatives (Fig. 2D). These

Figure 1. Screening of SAUR interaction mutants of PP2C.D1. A, Diagram of the PP2C.D1 protein with point mutations thatabolish SAUR19 binding indicated. B, Reverse yeast two-hybrid assay of wild-type PP2C.D1 or mutant pp2c.d1 derivatives withSAUR19. Growth on SC-Leu,Trp,His (-L-T-H) 1 3-amino-19, -29, -49 triazole (3-AT) medium is indicative of PP2C.D1-SAUR19interaction. C, Multiple sequence alignment of partial PP2C.D amino acid sequences from a variety of plants. Not all tomato(Solanum lycopersicum) and rice (Oryza sativa) members are included. PP2C.D orthologs from other species were retrieved fromhttps://phytozome.jgi.doe.gov/pz/portal.html. Red asterisks indicate the positions of PP2C.D1 residues identified in the reversetwo-hybrid screen. The red arrowhead indicates the conserved Met residue, which was mutated to Lys in other PP2C.D familymembers. D, In vitro pull-down assay of SAUR19 bywild-type PP2C.D1 or mutated pp2c.d1 derivatives. Immunoblots are shownin the top row, whereas Ponceau S-stained membranes are shown in the bottom row. All samples were run on the same gel andblotted together. Extraneous lanes were digitally spliced out of the blot as indicated. E, Yeast two-hybrid assay of wild-typePP2C.D or mutant pp2c.d isoforms with SAUR19. In B and E, images are composites where individual images were cropped anddigitally extracted for comparison.


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findings clearly demonstrate that SAUR-immunePP2C.Ds are specifically defective in SAUR bindingand retain both phosphatase activity and the ability tointeract with PM H1-ATPases.

SAUR-Immune pp2c.d2 and pp2c.d5 RepressCell Expansion

Our prior genetic analysis of the PP2C.D familyrevealed that PP2C.D2, PP2C.D5, and PP2C.D6 are theprimary family members involved in controlling AHAphosphorylation status in planta to regulate cell elon-gation (Ren et al., 2018). To examine the physiologicaleffects of constitutively active SAUR-immune PP2C.Denzymes, we generated native promoter:pp2c.d2M331K-GFP and native promoter:pp2c.d5M328K-GFP constructsand introduced them into the respective pp2c.d2or pp2c.d5 mutant backgrounds. Wild-type native

promoter:PP2C.D2-GFP and native promoter:PP2C.D5-GFP lines were included as controls. Both the wild-typeand SAUR-immune pp2c.d2/d5-GFP fusions decoratedthe PM, indicating that the M→K mutations did notcompromise localization of the SAUR-immune PP2C.Dsin either cotyledon pavement cells or root tips(Supplemental Fig. S5, A and B). Notably, however,the SAUR-immune pp2c.d2- and pp2c.d5-expressingplants exhibited pavement cells significantly smallerthan wild-type PP2C.D2-GFP and PP2C.D5-GFP con-trols (Supplemental Fig. S5, C and D).In our previous analysis of PPP2C.D:PP2C.D2/5/6-HA/

GFP transgenic lines, we found that plant growth ishighly sensitive to PP2C.D dosage (Ren et al., 2018). Inthat prior study, the majority of transgenic linesexpressed low levels of PP2C.D-HA/GFP and com-plemented (or slightly overcomplemented) pp2c.d mu-tant phenotypes. Due to position effects, however, asmall subset of transgenic lines expressed high levels of

Figure 2. Analogous mutations in other PP2C.D members also confer SAUR immunity. A, In vitro phosphatase assay of pp2c.d1missense mutant proteins. Values represent means (n 5 3) 6 SD. B, Phosphatase activities of wild-type PP2C.D (WT) or pp2c.dSAUR-immune (SI) M→Kmissensemutant proteins in the absence or presence of SAUR9. Values represent means (n5 3)6 SD. C,BiFC assay demonstrating protein-protein interaction of wild-type PP2C.D5 or pp2c.d5M328K with AHA2 after transient ex-pression in N. benthamiana leaves. Images are composites where individual images were cropped and digitally extracted forcomparison. Bars5 50mm. D, In vitro phosphatase assay showing Thr-947 dephosphorylation of yeast-expressed AHA2 bywild-type PP2C.Ds or SAUR-immune pp2c.d isoforms.Where indicated, 6xHis-SAUR9was added to a threefoldmolar excess relativeto PP2C.D. The slight mobility difference between wild-type PP2C.D1 and pp2c.d1M317K is due to a second 6xHis tag in themutant construct.



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PP2C.D-HA/GFP, resulting in reductions in cellexpansion and organ growth due to overexpression.Therefore, to assess the functional consequences ofSAUR-immune pp2c.d mutants and avoid the compli-cation of overexpression, we intentionally selectedpp2c.d2M331K-GFP and pp2c.d5M328K-GFP lines thatexpressed lower levels of the fusion protein than thecorresponding PP2C.D2-GFP and PP2C.D5-GFP wild-type controls (Fig. 3B, bottom; Supplemental Fig. S6B,bottom).

Plants expressing the M→K SAUR-immune pp2c.d2/d5-GFP derivatives exhibited severe dwarf phenotypeswith dramatic reductions in leaf size and petiole length(Fig. 3, A and C; Supplemental Fig. S6, A and C).In contrast, PP2C.D2-GFP and PP2C.D5-GFP plantslargely resembled wild-type controls, although mi-nor reductions in organ size were observed. Over-expression of PP2C.D1 or PP2C.D5 was previouslyreported to confer severe dwarf phenotypes (Spartz

et al., 2014; Ren et al., 2018). However, the dwarfismresulting from expression of the SAUR-immunepp2c.d2/d5-GFP constructs is clearly not the result ofoverexpression, as mutant protein levels were lowerthan those of the corresponding wild-type PP2C.D2/D5-GFP proteins (Fig. 3B; Supplemental Fig. S6B).Rather, we hypothesize that the severe phenotypesresulting from pp2c.d2M331K and pp2c.d5M328K expres-sion are the consequence of the constitutively activephosphatase activity of these mutant derivatives due tothe loss of SAUR binding.

To extend our in vitro findings that SAUR-immunephosphatases exhibit constitutive activity, we ex-amined AHA Thr-947 phosphorylation status inpp2c.d2M331K-GFP and pp2c.d5M328K-GFP seedlings.Despite the fact that the SAUR-immune derivativeswere expressed at lower levels than their wild-typecounterparts, a dramatic reduction in Thr-947 phos-phorylation was observed in the mutants compared

Figure 3. Expression of pp2c.d5M328K in Arabidopsis confers dramatic cell expansion defects. A, Twenty-four-day-old plants.Three independent pp2c.d5M328K-GFP lines are shown. Bar5 1 cm. B, Examination of AHAThr-947 phosphorylation in 7-d-oldlight-grown seedlings by GST-14-3-3 far-western assay. PP2C.D5-GFP and pp2c.d5M328K-GFP protein expression levels areshown in the bottom image. C, Leaf blade area of 24-d-old plants. Values represent means (n5 30, three leaf blades per plant)6SD. D, Seven-day-old light-grown seedlings. Bar5 1mm. E, Hypocotyl length of 7-d-old light-grown seedlings. Error bars indicateSD (n5 20). F, Mean hypocotyl epidermal cell length of 7-d-old light-grown seedlings. The apical-most five cells from 10 seedlingsweremeasured (n5 50). Error bars indicate SD. G, Five-day-old etiolated seedlings. Bar5 2mm.H, Seven-week-old plants. Bar55 cm. I, Plant height of 7-week-old plants. Error bars indicate SD (n 5 10). Different letters above bars indicate significant dif-ferences (P , 0.05) when analyzed by one-way ANOVA with Tukey’s honestly significant difference (HSD) test. In D and G,images are composites where individual images were cropped and digitally extracted for comparison.


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with the PP2C.D2-GFP and PP2C.D5-GFP control lines(Fig. 3B; Supplemental Fig. S6B). This finding stronglysuggests that, as per our in vitro findings, the SAUR-immunity mutations result in constitutively activephosphatases and supports our prior genetic and bio-chemical findings that SAURs negatively regulatePP2C.D activity to activate PM H1-ATPases (Spartzet al., 2014; Ren et al., 2018).In young seedlings, hypocotyl lengths of the SAUR-

immune pp2c.d2 and pp2c.d5mutants were significantlyreduced in both light- and dark-grown seedlings (Fig. 3,D, E, and G; Supplemental Fig. S6, D, E, G, and H). Thereduction of hypocotyl length was the result of de-creased cell elongation, as hypocotyl epidermal cells inthe SAUR-immune mutants were approximately 30%to 40% shorter than in wild-type Columbia-0 (Col-0;Fig. 3F; Supplemental Fig. S6F). In addition, the inflo-rescences of both SAUR-immune pp2c.d2 and pp2c.d5mutants were approximately 70% shorter than those ofcontrol plants, giving them very short stature (Fig. 3, Hand I; Supplemental Fig. S6, I and J). This dwarf phe-notype contrasts with the increased organ growthphenotypes resulting from overexpression of stabilizedSAUR19 (Spartz et al., 2012). The SAUR-immunepp2c.d2/d5 plants also produced small flowers withshort carpels and stamens (Supplemental Fig. S7, A andB). Stamen filaments of pp2c.d5M328K flowers wereshorter than those of pp2c.d2M331K, consistent with thehigher expression of PP2C.D5 in stamen filaments aspreviously reported (Ren et al., 2018). The short fila-ments resulted in a mechanical defect where self-fertilization could not occur, and hence led to unde-veloped siliques bearing no seeds in the case ofpp2c.d5M328K or reduced seed yields in pp2c.d2M331Kplants (Supplemental Fig. S7, C and D). We observedrare cases of pp2c.d5M328K flowers producing short si-liques with a few seeds, but manual pollination wasgenerally needed to obtain seeds. Similar findings werereported for PP2C.D5 overexpression lines (Ren et al.,2018).

SAUR-Immune pp2c.d2 and pp2c.d5 Block Auxin-MediatedHypocotyl Elongation

Together, the above findings suggest that SAUR-immune pp2c.d2 and pp2c.d5 mutants exhibit constitu-tive phosphatase activity via loss of SAUR inhibition,resulting in continual PM H1-ATPase dephosphoryla-tion that prevents proper cell expansion for plantgrowth. Therefore, if auxin normally promotes cell ex-pansion by inducing SAUR expression to inhibitPP2C.D activity and activate H1 pumps, the SAUR-immune pp2c.d2/d5 mutants should not exhibit auxin-mediated organ growth. To test this possibility, weexamined hypocotyl lengths of SAUR-immune pp2c.d2/d5-GFP seedlings and the respective PP2C.D2-GFP andPP2C.D5-GFP controls following treatment with piclo-ram. This synthetic auxin promotes robust hypocotylelongation and has been shown to rapidly induce the

expression of many SAURs in hypocotyls (Chapmanet al., 2012). Treatment with 2 mM picloram increasedwild-type Col-0 hypocotyl length by approximately1.7-fold (Fig. 4, A–D). Likewise, the PP2C.D2-GFP andPP2C.D5-GFP lines exhibited significantly longer hy-pocotyls on picloram compared with control medium.The slightly less robust increase in hypocotyl lengthsof these transgenics compared with wild-type controlsis presumably the result of weak overexpression (Renet al., 2018). Importantly, however, unlike Col-0 and thewild-type PP2C.D2/D5-GFP seedlings, lines expressingthe SAUR-immune pp2c.d2/d5-GFP constructs dis-played no significant increase in hypocotyl length fol-lowing picloram treatment (Fig. 4, A–D). Cell lengthmeasurements confirmed that picloram promoted anincrease in hypocotyl cell length of the control geno-types but not the SAUR-immune lines (Fig. 4E). Similarresults were obtained using 602 proauxin (Savaldi-Goldstein et al., 2008), with 602 treatment having nosignificant effect on SAUR-immune pp2c.d2 hypocotyllength (Fig. 4F). SAUR-immune pp2c.d5 seedlingsexhibited a slight response to 602, but this was dra-matically reduced compared with the wild-type con-trols. In contrast to the auxin-resistant hypocotylphenotype conferred by the expression of SAUR-immune pp2c.d2/d5-GFP constructs, these lines exhibi-ted wild-type-like sensitivity to auxin in root growthinhibition assays (Supplemental Fig. S8), demonstrat-ing that SAUR-immune pp2c.d seedlings can still re-spond to auxin.The above findings suggest that auxin-induced ex-

pression of SAURs (or any other auxin-regulated genes)cannot overcome the inhibitory effects of the SAUR-immune PP2C.D derivatives on cell expansion. To ex-amine this possibility more directly, we crossed thepp2c.d2M331K-GFP and pp2c.d5M328K-GFP lines withplants expressing a 35S:StrepII-SAUR19 overexpressionconstruct (Spartz et al., 2012). We previously demon-strated that overexpression of SAUR19 could suppressthe growth defects conferred by overexpression ofwild-type PP2C.D5 (Ren et al., 2018). While SAUR19overexpression promoted increases in the hypocotyllengths and leaf sizes of Col-0, PP2C.D2-GFP, andPP2C.D5-GFP control plants, no effect whatsoever wasapparent when SAUR19 was overexpressed in thepp2c.d2M331K-GFP and pp2c.d5M328K-GFP plants (Fig. 4,G–L; Supplemental Fig. S9). These findings furthersupport our conclusion that these mutations conferSAUR immunity and constitutive phosphatase activityregardless of the SAUR protein dosage.

SAUR-Immune PP2C.D Phenotypes Are PartiallySuppressed by Constitutive PM H1-ATPase Activity

There are 11 genes in Arabidopsis encoding PM H1-ATPases. AHA1 and AHA2 are the major contributors,together constituting 70% to 80% of the total H1-ATPase activity in most plant organs (Haruta et al.,2010). The open stomata2 (ost2-2) mutant contains two



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Figure 4. SAUR immunity confers resistance to auxin and SAUR19 overexpression. A, Three-day-old light-grown Col-0, pp2c.d5,PP2C.D5-GFP, and pp2c.d5M328K-GFP seedlings were transferred to Arabidopsis with Suc (ATS) medium 6 2 mM picloram andgrown an additional 4 d. Bar5 1 mm. B, Mean hypocotyl length of seedlings grown as described in A. Error bars indicate SD (n520). C andD, pp2c.d2M331K-GFP and control seedlings grown as described in A. Error bars indicate SD (n5 20). E, Mean hypocotylepidermal cell length of seedlings in A and C. Error bars indicate SD (n5 50). Asterisks indicate statistically significant differences(P , 0.001) when analyzed by two-tailed Student’s t test. n.s., Not significant (P . 0.05). F, Mean hypocotyl length of seedlingstreated with or without 2 mM 602 proauxin according to the procedure in A. Error bars indicate SD (n5 17). G to L, The effects of35S:StrepII-SAUR19 (S19-OX) expression on the growth of control and pp2c.d5M328K-GFP plants. G, Seven-day-old light-grownseedlings grown on unsupplemented ATS medium. Bar5 1 mm. H, Hypocotyl length of 7-d-old light-grown seedlings. Error barsindicate SD (n5 20). I, Five-day-old etiolated seedlings. Bar5 2 mm. J, Hypocotyl length of 5-d-old etiolated seedlings. Error bars


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point mutations (L169F and G867S) in AHA1 that resultin constitutive H1 pump activity (Merlot et al., 2007).Although not yet confirmed, this has been proposed tobe the consequence of the mutations abolishing intra-molecular interactions between the autoinhibitoryC-terminal domain and cytosolic regions elsewhere inthe protein, thus trapping the H1 pump in the activatedstate. The resulting gain-of-function ost2-2 phenotypespartially phenocopy those resulting from SAUR19overexpression or pp2c.d2/5/6 mutation (Spartz et al.,2014; Ren et al., 2018). Furthermore, ost2-2 mutantphenotypes are enhanced when the mutation is com-bined with the aha2-5 null mutation (Fendrych et al.,2016).To investigate whether constitutive PM H1-ATPase

activity could suppress the dwarf phenotype conferredby SAUR-immune phosphatases, we introduced thePPP2C.D5:pp2c.d5M328K-GFP construct into the ost2-2aha2-5 genetic background. Analysis of two indepen-dent lines expressing the SAUR-immune pp2c.d5 con-struct revealed reduced AHA Thr-947 phosphorylationstatus compared with ost2-2 aha2-5 controls (Fig. 5, Aand B). Despite this reduction in AHA phosphoryla-tion, pp2c.d5M328K-GFP expression in the ost2-2 aha2-5plants did not result in severe dwarfism (Fig. 5, C andD). However, when these lines were put through abackcross to remove the ost2-2 and aha2-5 mutations,the pp2c.d5M328K-GFP transgene once again conferred adramatic dwarf phenotype (Fig. 5C, right), indicatingthat the SAUR-immune pp2c.d5 growth defects arePMH1-ATPase dependent. Likewise, the reduction inhypocotyl lengths resulting from pp2c.d5M328K-GFPexpression was partially suppressed in the ost2-2 aha2-5 background (Fig. 5, E–H). That said, ost2-2 aha2-5[pp2c.d5M328K-GFP] hypocotyls were still signifi-cantly shorter than those of the ost2-2 aha2-5 parentalline. This modest reduction could be due to decreasedphosphorylation status of the remaining AHA pro-teins, or this finding may indicate that PP2C.D5regulates additional proteins that contribute toelongation growth. Lastly, several adult phenotypes ofpp2c.d5M328K expressionwere also largely suppressed inthe ost2-2 aha2-5 background, including the severe re-ductions in stature, stamen filament length, and fertility(Fig. 5, I–M). These findings suggest that the constitu-tively active ost2-2 mutant protein functions indepen-dently of Thr-947 phosphorylation status and hence isless responsive to constitutively active SAUR-immunepp2c.d5. Importantly, these findings support our con-tention that reduced PM H1-ATPase activity is a majorcontributor to the cell expansion phenotypes conferredby SAUR-immune PP2C.D phosphatases.

DISCUSSION

Cell elongation is a crucial process that affects organgrowth and development to give shape and form to theplant. Our previous studies demonstrated that auxin-induced cell elongation in hypocotyls is mediated bySAUR and PP2C.D proteins, which function in an an-tagonistic manner to control phosphorylation and ac-tivity of PM H1-ATPases and perhaps other proteinsubstrates (Spartz et al., 2014, 2017; Ren et al., 2018).Among the nine D-clade PP2C phosphatases, PP2C.D2,PP2C.D5, and PP2C.D6 act in a largely redundantmanner to inhibit cell expansion in hypocotyls andseveral other organs, while the remaining familymembers do not appear to contribute to these growthprocesses (Ren et al., 2018). While analysis of pp2c.dloss-of-function mutants by Ren et al. (2018) clearlyimplicated PP2C.D2, PP2C.D5, and PP2C.D6 as nega-tive regulators of cell expansion, due to the plethora ofSAUR genes present in plant genomes, the evidencethat auxin-induced SAUR proteins antagonize thesephosphatases has been limited to gain-of-functionSAUR overexpression and in vitro biochemicalapproaches. In this study, we provide an approach topotentially circumvent the complications of SAUR ge-netic redundancy by identifying mutant PP2C.D de-rivatives that retain phosphatase activity but areincapable of interacting with SAUR proteins, thus ren-dering the phosphatases immune to SAUR inhibition.Our random mutagenesis of PP2C.D1 identified a

motif near the C terminus of the catalytic domain that isessential for SAUR binding. This motif is extremelyhighly conserved in all D-clade family members fromArabidopsis as well other species representing bothvascular and nonvascular plants. Compared with otherPP2Cs, this motif is present as a unique insertion, withthe lone exception of the C-clade phosphatases, whichcontain a related but loosely conserved sequence. Theseven member C-clade PP2Cs include POLTERGEISTand the related POLTERGEIST-LIKE (PLL) phospha-tases (Yu et al., 2003), which regulate stem cell initiationand maintenance through CLAVATA3/WUSCHEL-RELATED HOMEOBOX signaling pathways (Gagneand Clark, 2010). Since SAUR19 overexpression con-fers a variety of cell expansion phenotypes but no ob-vious meristematic defects, it seems unlikely thatSAURs regulate C-clade phosphatase activity. Fur-thermore, we examined potential interactions betweenSAUR19 and PLL4 in both yeast two-hybrid and BiFCinteraction assays but obtained negative results usingboth approaches (Supplemental Fig. S10).While mutations in the C-terminal SAUR-interaction

motif abolish SAUR binding, these mutations do not

Figure 4. (Continued.)indicate SD (n5 20). K, Twenty-five-day-old plants. Bar5 1 cm. L, Leaf blade area of 25-d-old plants. Values representmeans (n530, three leaf blades per plant)6 SD. Different letters above bars indicate significant differences (P, 0.05) when analyzed by one-way ANOVAwith Tukey’s HSD test. In A, C, G, and I, images are composites where individual images were cropped and digitallyextracted for comparison.



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Figure 5. pp2c.d5M328K is partially suppressed in ost2-2 aha2-5. A, Examination of AHA Thr-947 phosphorylation in 7-d-oldlight-grown seedlings byGST-14-3-3 far-western gel assay. B, Immunoblot examining pp2c.d5M328K-GFP protein expression levelin 7-d-old light-grown seedlings. SEC12 is shown as a loading control. C, Twenty-four-day-old plants. The left image shows acomparison between ost2-2 aha2-5 with or without the pp2c.d5M328K-GFP transgene and pp2c.d5M328K-GFP in the pp2c.d5mutant background as a reference. The right image shows two independent lines of ost2-2 aha2-5[pp2c.d5M328K-GFP] afterbackcrossing the transgene into the pp2c.d5 AHA1 AHA2 background. Bar5 1 cm. D, Leaf blade area of 25-d-old plants. Valuesrepresent means (n5 30, three leaf blades per plant)6 SD. E, Seven-day-old light-grown seedlings. Bar5 1 mm. F, Five-day-oldetiolated seedlings. Bar5 2 mm. G, Mean hypocotyl length of 7-d-old light-grown seedlings. Error bars indicate SD (n 5 20). H,


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appear to affect catalytic activity of PP2C.D enzymes.Consequently, PP2C.D proteins containing mutationsin this motif are immune to SAUR inhibition and dis-play constitutive phosphatase activity both in vitroand in planta. For example, absolutely no SAUR inhi-bition was detected in our phosphatase assays ofmutant derivatives of pp2c.d1, pp2c.d2, pp2c.d4, orpp2c.d5 in biochemical assays employing either thechemical substrate, pNPP, or a native substrate, AHA2.Furthermore, when expressed in Arabidopsis fromtheir respective native promoters, the pp2c.d2 orpp2c.d5 SAUR-immune derivatives conferred constitu-tive low Thr-947 phosphorylation status of PM H1-ATPases, severe dwarfism, and several other cellexpansion phenotypes. While overexpression of wild-type PP2C.D5 confers very similar phenotypes, co-overexpression of SAUR19 suppresses all of thegrowth defects resulting from PP2C.D5 overexpression(Ren et al., 2018). In marked contrast, however, thegrowth defects of the SAUR-immune pp2c.d2M331K andpp2c.d5M328K mutants were not restored whatsoever byoverexpression of StrepII-SAUR19. Also consistentwith this premise, auxin treatment did not promotehypocotyl elongation in these SAUR-immune pp2c.dmutants. Together, these findings provide a strongcomplement to our previous analysis of pp2c.d2 andpp2c.d5 loss-of-function mutants (Ren et al., 2018),clearly demonstrating that these phosphatases playvital roles in regulating PMH1-ATPase activity and cellexpansion.Our findings also provide additional support for the

long-standing acid growth hypothesis. It has previ-ously been established that auxin promotes PM H1-ATPase phosphorylation and activation (Takahashiet al., 2012) and that this process involves SAUR-PP2C.D2/5/6 regulatory modules (Spartz et al., 2014;Fendrych et al., 2016; Ren et al., 2018). We foundthat introduction of the dominant ost2-2 mutation en-coding a constitutively activated mutant isoform of theAHA1 PM H1-ATPase into the pp2c.d5M328K back-ground significantly suppressed many of the cell ex-pansion defects of this SAUR-immune mutant. Thisfinding strongly suggests that the growth defects con-ferred by SAUR-immune pp2c.d5 are largely the resultof the inability to activate PM H1-ATPases. Interest-ingly, this suppression occurred despite the fact thatThr-947 phosphorylation was dramatically lower inthe pp2c.d5M328K background. We hypothesize that thisindicates that the mutant ost2-2 isoform does not re-quire Thr-947 phosphorylation for activation. Rather,as previously suggested, the mutations may disrupt

intramolecular contacts with the C-terminal auto-inhibitory domain (Merlot et al., 2007), therebybypassing the normal requirement of phosphorylationfor pump activation.Apart from the PM-localized PP2C.D2, PP2C.D5, and

PP2C.D6 family members negatively regulating PMH1-ATPase activity to control cell expansion, thefunctions of the remaining D-clade phosphatases areless well understood. PP2C.D1 has been implicated inapical hook development (Sentandreu et al., 2011;Spartz et al., 2014) and as a negative regulator of leafsenescence, the latter of which may be mediatedthrough PP2C.D1 control of SENESCENCE-ASSOCI-ATED RECEPTOR-LIKE KINASE activity (Xiao et al.,2015). PP2C.D3 (also known as PP2C38) was reportedto negatively regulate the Arabidopsis cytoplasmicBOTRYTIS-INDUCED KINASE1 (BIK1) to suppressBIK1-mediated plant immunity (Couto et al., 2016).Whether SAURs are involved in regulating PP2C.D1and PP2C.D3 in these processes remains to be deter-mined. That said, SAUR36 was found to promote leafsenescence (Hou et al., 2013), suggesting that SAUR36and PP2C.D1 may in fact function antagonistically toregulate senescence. Given the extreme conservationamong D-clade members of the C-terminal SAUR in-teraction motif, and that we have demonstrated thatmutations in this motif confer immunity to SAUR in-hibition of PP2C.D1, PP2C.D2, PP2C.D4, and PP2C.D5,we hypothesize that analogous mutations in otherfamily members will have similar effects. Unfortu-nately, we have been unable to test this possibility di-rectly, as we have thus far not been able to developin vitro phosphatase assays with recombinantlyexpressed PP2C.D3, PP2C.D6, PP2C.D7, PP2C.D8, orPP2C.D9. If so, however, expression of SAUR-immunederivatives of these additional D-clade members fromtheir respective native promoters may provide uniquegain-of-function genetic tools to assign functions tothese phosphatases.Lastly, we suggest that SAUR-immune derivatives of

PP2C.D2 and PP2C.D5may provide novel genetic toolsfor investigating the roles of PM H1-ATPasesthroughout plant development. Currently, such geneticstudies are hampered by the fact that aha1 and aha2single mutants only exhibit weak conditional pheno-types and the double mutant is embryo lethal (Harutaet al., 2010, 2015). Since SAUR-immune pp2c.d2and pp2c.d5 repress H1-ATPase activity via constitu-tive Thr-947 dephosphorylation, expression of thesemutant derivatives from inducible, tissue-specific, andcell type-specific promoters would provide a unique

Figure 5. (Continued.)Mean hypocotyl length of 5-d-old etiolated seedlings. Error bars indicate SD (n5 20). I, Flowers. The bottom images show flowerswith sepals and petals removed to expose carpels and stamens. Bars 5 1 mm. J, Seven-week-old plants. Bar 5 5 cm. K, Plantheight of 7-week-old plants. Error bars indicate SD (n 5 10). L, Inflorescence stems showing siliques. White asterisks indicateundeveloped siliques in ost2-2 aha2-5 double mutant and ost2-2 aha2-5 [pp2c.d5M328K-GFP] independent lines. Bar5 5 cm. M,Percentage of developed and undeveloped siliques. Error bars indicate SD (n5 200, 20 siliques per plant). Different letters abovebars indicate significant difference (P, 0.05) when analyzed by one-way ANOVAwith Tukey’s HSD test. In C, E, F, and I, imagesare composites where individual images were cropped and digitally extracted for comparison.



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approach for down-regulating PM H1-ATPase activityin a temporal and spatially controlled fashion to probeH1-ATPase functions in specific processes, cells, ororgans.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

All Arabidopsis (Arabidopsis thaliana) lines used in this studywere in the Col-0 ecotype. Seedlings were grown on ATS medium (Lincoln et al., 1990) at 22°Cunder long-day conditions (60–80 mE m22 s21) unless specified otherwise.Transgenic plants were generated through the floral dip method using Agro-bacterium tumefaciens strain GV3101 (helper plasmid pMP90) harboring thebinary vector with the desired target genes (Clough and Bent, 1998). TheStrepII-SAUR19 and PP2C.D5-HA transgenic plants were described previ-ously (Spartz et al., 2012; Ren et al., 2018).

For auxin treatment, seedlings were grown on ATS plates for 3 d at 22°Cbefore being transferred to plates containing 2 mM picloram (4-amino-3,5,6-trichloro-picolinic acid), 2 mM 602 proauxin, or 1 mM IAA. Seedlingswere grown under long-day conditions as described above. The seedlingswere grown for 4 d before hypocotyls and roots were imaged and mea-sured at described below.

Gene Cloning

The QuikChange XL site-directed mutagenesis kit (Agilent Technologies)was used to perform site-directed mutagenesis on pENTR/D-TOPO clonesharboring the full-length cDNA or genomic DNA of PP2C.D phosphatasesusing the primer pairs listed in Supplemental Table S1. The PP2C.D genomicDNA clones have been previously described (Ren et al., 2018). The mutatedPP2C.D cDNAs were recombined into pET32a-GW or pDEST15-GW for bac-terial expression using Gateway LR clonase II enzyme mix (Life Technologies).Mutated PP2C.D genomic DNAs were recombined into pGWB204 for stableexpression in Arabidopsis using the floral dip method. For ABI1-D1 fusionconstruction, overlap-extension PCR was performed. The ABI1-F and SKLA-Rprimerswere used to amplify the N terminus of ABI1 coding sequence, whereasSKLA-F and D1-R primers were used to amplify the C terminus of PP2C.D1coding sequence as listed in Supplemental Table S1. The resulting PCR productswere thenmixed and used as template to amplify the ABI1-D1 fusion sequence,which was subsequently cloned into pENTR/D-TOPO entry clone.

Plant Growth Measurement

A SPOT Insight camera mounted on an Olympus SZX12 stereomicroscopewas used to photograph seedling hypocotyls for length measurements. For celllengthmeasurements, images of propidium iodide-stainedhypocotyl epidermalcells were acquired using a Nikon Ti2 A1si Confocal system (Nikon USA). Allimage measurements were performed using ImageJ software. The ImageJplugin PaCeQuant was used to automatically quantify cotyledon pavement cellarea and perimeter, together with manual validation to exclude inaccuratelysegmented pavement cells for each measurement (Möller et al., 2017).

Reverse Two-Hybrid Screen

Yeast parent vectors pBI880 and pBI771 and Saccharomyces cerevisiae strainYPB2 were described by Gray et al. (1999). pBI880 containing full-lengthSAUR19 and pBI771 containing full-length PP2C.D1 have been previouslydescribed (Spartz et al., 2014). To facilitate mutant screening, the PP2C.D1coding sequence was divided into N-terminal (encoding amino acids 1–107),central (encoding amino acids 105–272), and C-terminal (encoding amino acids260–370) thirds. Each third was subjected to identical error-prone PCR usingpBI771[PP2C.D1] template DNA and employing Sigma RedExtract-n-AMPready mix supplemented with 0.1 mM MnCl2 and an additional 2 mM dTTP anddCTP. To generate libraries of mutant PP2C.D1 clones, EcoRI and SpeI restric-tion sites were introduced into the wild-type pBI771[PP2C.D1] vector at nu-cleotide positions 316 and 780 of the PP2C.D1 cDNA using site-directedmutagenesis. This vector was digested with NotI and EcoRI, EcoRI and SpeI, orSpeI and SalI to generate gapped plasmids corresponding to the above three

PP2C.D1 regions. Each gapped vector was then mixed with the correspondingmutagenized PCR products, and the mixture was used to transform YPB2 cellscarrying the pBI880[SAUR19] bait vector. Homologous recombination betweenthe ends of the PCR products (corresponding to the amplification primers) andthe gapped vector generated libraries of pBI771[PP2C.D1] mutant derivatives.Transformants were selected on SC(-L-T), and then colonies were picked andstreaked onto both SC(-L-T) and SC(-L-T-H) 1 5 mM 3-AT for screening. Col-onies that grew on SC(-L-T) but not SC(-L-T-H)1 3-ATmediumwere identifiedas potential SAUR19 noninteractors and characterized further. This includedimmunoblot screening with purified a-PP2C.D1 antibody to ensure expressionand eliminate stop codonmutants, sequencing, retransformation of the plasmidinto yeast, and retesting for yeast two-hybrid interaction with SAUR19. Ap-proximately 500 colonies were tested for each of the three PP2C.D1 intervals.

Yeast Two-Hybrid Assays

Additional protein interaction assays of SAUR19with PP2C.D phosphataseswere carried out in both GAL4 (pBI880/pBI771; Kohalmi et al., 1998) and lex-A-based (pBTM116/pACT2; Weber et al., 2005) yeast two-hybrid systems. Themutated PP2C.D inserts in pENTR/D-TOPO vectors were cloned into the preyvector pACT2 through Gateway LR recombination as described above. Yeaststrain L40ccU3 [MATa, his3-200, trp1-901, leu2-3, 112ade2 LYS:(lexAop)4-HIS3,URA:(lexAop)8-lacZ, GAL4, gal80] was cotransformed with pBTM116-SAUR19and pACT2-PP2C.D vectors.

Protein Expression and Phosphatase Assays

GST- or 6xHis-tagged SAUR or PP2C.D proteins were purified from Esch-erichia coli cultures as previously described (Spartz et al., 2014). For phosphataseassays, 0.15mMPP2C.D protein was preincubatedwith 1.2mM SAURproteins oran equivalent amount of elution buffer for 10 min at room temperature. Proteinmixtures were then added into assay buffer (75 mM Tris, pH 7.6, 10 mM MnCl2,100 mM NaCl, 0.5 mM EDTA, pH 8, and 5 mM pNPP), and A405 was recordedusing a Powerwave 340 plate reader (Biotek Instruments) every 1 min for20 min.

For in vitro AHA2 dephosphorylation assays, yeast PMs were purified fromRS-72 cells expressing AHA2 protein as previously described (Panaretou andPiper, 2006; Spartz et al., 2014). The isolated membranes were resuspended inbuffer with 5 mM potassium phosphate, pH 7.8, 3 mM KCl, 0.1 mM EDTA, pH 8,330 mM Suc, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride. The 6xHis-SAUR9 (or buffer control) was preincubated with 6xHis-PP2C.D on ice for10 min prior to addition to the assay reaction. The protein mixture was thenadded into reaction buffer containing 2 mg of yeast PM proteins and 1.8 mMMnCl2 with final volume of 15 mL. The reaction mixture was incubated at 25°Cfor 5 min before stopping with SDS-PAGE sample buffer. GST-14-3-3 far-western gel blotting was performed to assess AHA2 Thr-947 phosphorylationstatus as previously described (Hayashi et al., 2010; Spartz et al., 2014).

In Vitro Pull-Down Assays

GST-PP2C.D1 and 6xHis-SAUR19 constructs and purification methodswere described previously (Spartz et al., 2014). Mutant GST-PP2C.D1 con-structs were made by QuikChange site-directed mutagenesis with the primerslisted in Supplemental Table S1. Following expression and purification, ap-proximately 1 mg each of GST-PP2C.D1 and SAUR19 was incubated in 250 mLof buffer C (Gray et al., 1999) for 1 h at 4°C, and beads werewashed two times inbuffer C with a final wash in buffer C1 0.1% (v/v) Nonidet P-40. Immunoblotsof pulled down SAUR19 protein were performed with affinity-purifieda-SAUR19 (Spartz et al., 2012).

BiFC Assay

pENTR/D-TOPO vectors containing full-length cDNA sequences withoutstop codons ofwild-type ormutated PP2C.Dwere recombined into the pSPYCEdestination vector for BiFC expression constructs (Walter et al., 2004). The ex-pression constructs with PP2C.D1, PP2C.D2, PP2C.D5, PP2C.D6, AHA2, andAUX1 inserts were reported previously (Spartz et al., 2014; Ren et al., 2018). TheBiFC assays were performed transiently in approximately 5-week-old Nicotianabenthamiana leaves as previously described (Schütze et al., 2009). YFP fluores-cence signals were observed 3 d postinfiltration. The images were acquiredusing a Nikon Ti2 A1si Confocal system (Nikon USA).


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Statistical Analyses

JMP Pro 13.1 software suite (SAS Institute) was used to performANOVA forall statistical analyses. Results were grouped by letters, with different lettersindicating significant differences (P , 0.05) based on Tukey’s HSD test.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under the following accession numbers: SAUR19 (At5g18010),SAUR9 (At4g36110), PP2C.D1 (At5g02760), PP2C.D2 (At3g17090), PP2C.D3(At3g12620), PP2C.D4 (At3g55050), PP2C.D5 (At4g38520), PP2C.D6 (At3g51370),PP2C.D7 (At5g66080), PP2C.D8 (At4g33920), PP2C.D9 (At5g06750), AHA2(At4g30190), and AUX1 (At2g38120).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. PP2C.D1 deletion analysis.

Supplemental Figure S2. Multiple sequence alignment of ArabidopsisPP2C phosphatases.

Supplemental Figure S3. Yeast two-hybrid assay testing protein-proteininteractions between an ABI1-D1 fusion protein and SAUR19.

Supplemental Figure S4. BiFC assay of PP2C.D and SAUR-immune de-rivatives with AHA2.

Supplemental Figure S5. Subcellular localization of pp2c.d2M331K-GFP andpp2c.d5M328K-GFP.

Supplemental Figure S6. Expression of pp2c.d2M331K in Arabidopsis con-fers dramatic cell expansion defects.

Supplemental Figure S7. pp2c.d2M331K and pp2c.d5M328K plants havesmaller floral organs.

Supplemental Figure S8. Auxin inhibition of root growth.

Supplemental Figure S9. pp2c.d2M331K is insensitive to SAUR19overexpression.

Supplemental Figure S10. Yeast two-hybrid and BiFC assays testingprotein-protein interaction between PLL4 and SAUR19.

Supplemental Table S1. List of oligonucleotides.

ACKNOWLEDGMENTS

We thank the College of Biological Sciences Imaging Center for assistancewith confocal microscopy and Dr. Anton Sanderfoot (University of Wiscon-sin-La Crosse) for providing SEC12 antisera.

Received April 23, 2019; accepted June 21, 2019; published July 16, 2019.

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Mutation of a Conserved Motif of PP2C.D Phosphatases Confers SAUR Immunity … · SAUR gene family...

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