prh1 mediates arf7-lbd dependent auxin signaling to regulate lateral … · 1 1 prh1 mediates...

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1 PRH1 mediates ARF7-LBD dependent auxin signaling to regulate lateral root

2 development in Arabidopsis thaliana

3 Feng Zhang1, Wenqing Tao1, Ruiqi Sun1, Junxia Wang1, Cuiling Li1, Xiangpei Kong1,

4 Huiyu Tian1 and Zhaojun Ding1,*

5 1The Key Laboratory of the Plant Cell Engineering and Germplasm Innovation,

6 Ministry of Education, School of Life Sciences, Shandong University, Qingdao

7 266237, Shandong, China

8

9 *Corresponding author:

10 Zhaojun Ding

11 Address: The Key Laboratory of Plant Cell Engineering and Germplasm Innovation,

12 Ministry of Education, College of Life Science, Shandong University, Qingdao

13 266237, Shandong, People’s Republic of China

14 E-mail: [email protected]

15 Tel: (+86)0532 58630889

16

17 Abstract

18 The development of lateral roots in Arabidopsis thaliana is strongly dependent on

19 signaling directed by the AUXIN RESPONSE FACTOR7 (ARF7), which in turn

20 activates LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factors

21 (LBD16, 18, 29 and 33). Here, the product of PRH1, a PR-1 homolog annotated

22 previously as encoding a pathogen-responsive protein, was identified as a target of

23 ARF7-mediated auxin signaling and also as participating in the development of lateral

24 roots. PRH1 was shown to be strongly induced by auxin treatment, and plants lacking

25 a functional copy of PRH1 formed fewer lateral roots. The transcription of PRH1 was

26 controlled by the binding of both ARF7 and LBDs to its promoter region. An

27 interaction was detected between PRH1 and GATA23, a protein which regulates cell

28 identity in lateral root founder cells.

29 Key Words: Lateral Root, Auxin, ARF7, LBD, PRH1

.CC-BY 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted February 22, 2019. . https://doi.org/10.1101/558387doi: bioRxiv preprint

https://doi.org/10.1101/558387

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30

31 Author Summary

32 In Arabidopsis thaliana AUXIN RESPONSE FACTOR7 (ARF7)-mediated auxin

33 signaling plays a key role in lateral roots (LRs) development. The LATERAL ORGAN

34 BOUNDARIES DOMAIN (LBD) transcription factors (LBD16, 18, 29 and 33) act

35 downstream of ARF7-mediated auxin signaling to control LRs formation. Here, the

36 PR-1 homolog PRH1 was identified as a novel target of both ARF7 and LBDs

37 (especially the LBD29) during auxin induced LRs formation, as both ARF7 and

38 LBDs were able to bind to the PRH1 promoter. More interestingly, PRH1 has a

39 physical interaction with GATA23, which has been also reported to be up-regulated

40 by auxin and influences LR formation through its regulation of LR founder cell

41 identity. Whether the interaction between GATA23 and PRH1 affects the stability

42 and/or the activity of either (or both) of these proteins remains an issue to be explored.

43 This study provides improves new insights about how auxin regulates lateral root

44 development.


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45 Introduction

46 The architecture of the root system depends on the density of lateral roots (LRs)

47 formed along with the extent of root branching. LRs are initiated from mature

48 pericycle cells lying adjacent to the xylem pole, referred to in Arabidopsis thaliana as

49 the xylem pole pericycle [1, 2]. A subset of these cells, namely the founder cells,

50 undergo a series of highly organized divisions to form an LR primordium, which

51 eventually develops into an LR. The entire process of LR development, from its

52 initiation to its emergence, is regulated by auxin [1, 3, 4]. The accumulation of auxin

53 in protoxylem cells results in the priming of neighboring pericycle cells to gain

54 founder cell identity, forming an LR pre-branch site [5, 6]. This auxin signaling is

55 mediated by the proteins ARF7 and ARF19, which act in the IAA28-dependent

56 pathway [7, 8]. LR initiation depends on the transcriptional activation, through the

57 involvement of the transcription factor LBD16 and LBD18, of either E2Fa or CDK

58 A1;1 and CYC B1;1 [9, 10]. Other downstream targets such as EXP14 and EXP17 and

59 the products of certain cell wall loosening-related genes are also either directly or

60 indirectly regulated by LBD18 to promote the emergence of an LR [10, 11, 12].

61 LBD18 has been also found to control LR formation through its interaction with GIP1,

62 which results in the transcriptional activation of EXP14 [13].

63 LBD transcription factors are among the most well studied downstream targets of

64 ARF7 and ARF19 [14]. ARF7 has also been found to control LR emergence through

65 its regulation of IDA and genes which encode leucine-rich repeat receptor-like kinases

66 such as HAE and HSL2 [15]. These interactions take place in the overlaying tissues,

67 thereby influencing cell wall remodeling and cell wall degradation during LR

68 emergence [15]. The product of PRH1 (At2g19990), a homolog of PR-1, is currently

69 annotated as encoding a pathogen responsive protein [16]. Here, this protein has been

70 identified as a novel target of ARF7-mediated auxin signaling, and evidence is

71 provided of its involvement in LR development.

72 Results

73 PRH1 is a target of ARF7-mediated auxin signaling

74 To identify additional targets involved in ARF7-mediated auxin signaling and LR


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75 development (Fig 1A and 1B), a comparison was made between the root

76 transcriptomes of eight-day-old wild type (WT) or arf7 mutant seedlings exposed to

77 auxin treatment respectively. A total of 74 genes was classified as DEGs (P < 0.0001,

78 log2 fold change >1) in WT but not in arf7 mutant, and these were then compared to

79 the arf7 and WT transcriptome after auxin treatment (Fig 1C and S1A Fig).

80 Twenty-three DEGs (P < 0.0001, log2 fold change >1.5) were selected as the

81 candidate target genes induced by auxin, which were also dependent on ARF7 (S1B

82 Fig). We then compared the expression patterns of these 23 candidate genes in lateral

83 root initiation using Arabidopsis eFP Browser

84 (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). As a result of this analysis, eight

85 genes were found to be involved in LR development. The most strongly differentially

86 transcribed of these genes was PRH1 (At2g19990) (S1C Fig).

87 PRH1, a homolog of PR-1 which has been annotated as encoding a

88 pathogen-responsive protein [16]. Though PRH1 was highly induced by the auxin

89 treatment in WT, it was completely non-responsive to auxin treatment in arf7

90 according to our qPCR assays (Fig 1D). The GUS expression analysis in 8-day-old

91 seedlings carrying a PRH1pro::GUS transgene implied that PRH1 is mainly

92 expressed in the site of LR initiated, which was enhanced by exogenous auxin

93 treatment (S2 Fig). The GUS signal was detected only at the LR primordia in WT

94 background (Fig 2A), and the level of GUS activity was greatly enhanced following

95 exposure of the plants to auxin (Fig 2B). When the PRH1pro::GUS transgene was

96 expressed in the arf7 mutant background, the PRH1pro::GUS expression was

97 strongly suppressed, and auxin treatment only slightly induced the expression of

98 PRH1pro::GUS (Fig 2C and 2D). The conclusion was that the auxin-responsive gene

99 PRH1 is expressed in the LR primordia and its expression is dependent on

100 ARF7-mediated auxin signaling.

101 PRH1 acts downstream of ARF7 to regulate LR development

102 To address if auxin-induced PRH1 expression is involved into LR development, we

103 examined the LR phenotypes in both prh1 mutant and the PRH1 over-expression lines

104 (S3A-3C Fig). The expression level of PRH1 in its T-DNA insertion mutants and the


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105 over-expression lines were verified (S3D Fig). Examination of LR development in

106 prh1-1 mutant plant showed that the absence of a functional copy of PRH1 inhibited

107 the formation of both emerged LRs and total LRs (Fig 3A and 3B). Though LR

108 development is not greatly affected by the over-expression of PRH1 in a WT

109 background, over-expression of PRH1 could significantly increase the LR numbers of

110 arf7 mutant plants (Fig 3A and 3B). In summary, PRH1 acts downstream of ARF7 to

111 regulate LR development.

112

113 ARF7 directly regulates PRH1 transcription

114 The observation that the abundance of PRH1 transcript was reduced in the arf7

115 mutant by auxin treatment (Fig 1D) suggested that the gene was transcriptionally

116 regulated by the auxin response factor ARF7. The gene’s promoter sequence harbors

117 the known auxin response elements (AuxREs) TGTC (Fig 4A). A transient expression

118 assay carried out in A. thaliana leaf protoplasts showed that ARF7 was able to

119 activate a construct comprising the PRH1 promoter fused with LUC. The

120 co-expression of PRH1pro::LUC and 35S::ARF7 had a large positive effect on the

121 intensity of the luminescence signal (Fig 4B), consistent with the idea that ARF7 is

122 able to activate the PRH1pro::LUC construct. A ChIP-qPCR assay confirmed the

123 association of ARF7 with the AuxRE elements present on the PRH1 promoter (Fig

124 4A and 4C), and a yeast one hybrid assay showed that ARF7 was able to bind to the

125 PRH1 promoter (Fig 4D). The conclusion was that the effect of ARF7 binding to the

126 PRH1 promoter was regulating the gene’s transcription, thereby influencing LR

127 development.

128

129 Auxin-induced PRH1 expression is regulated by LBD29

130 Members of the LBD family are known to function downstream of ARF7 to control

131 LR initiation and emergence [14, 17, 18, 19]. When the transcription level of PRH1

132 was investigated in the three single lbd mutants lbd16(Salk_040739), lbd18

133 (Salk_038125) and lbd29 (Salk_071133) combined with or without exogenous auxin

134 treatment, the observation was that the gene was strongly repressed in the single


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135 mutants. Though the auxin induced PRH1 expression was normal in lbd16 or lbd18,

136 the induction was highly reduced in lbd29, indicating LBD29 plays a crucial role in

137 auxin induced PRH1 expression (S4A Fig). Consistently, the over-expression of

138 LBD29 strongly up-regulates PRH1 expression (S4B Fig). In addition, we also

139 observed the up-regulation of PRH1 expression in LBD16 or LBD18 over-expression

140 lines (S4B Fig) These results suggest that LBDs regulate the transcription of PRH1,

141 auxin induced expression of RPH1 is highly dependent on LBD29.

142

143 LBDs regulate PRH1 by binding to its promoter

144 The transcriptional response of PRH1 to the loss-of-function and over-expression of

145 certain LBDs implied that these transcription factors can also regulate PRH1. Though

146 the PRH1 promoter has the typical LBD protein binding elements including CGGCG

147 and CACGTG (S5A Fig). Although the yeast one hybrid assay produced no direct

148 evidence for the binding of LBD proteins to the PRH1 promoter (S5B Fig), but the

149 ChIP-qPCR assay did support the association of LBD16, LBD18 and LBD29 with the

150 PRH1 promoter (Fig 5A). Thus a transient expression assay in A. thaliana leaf

151 protoplasts was used to investigate whether LBD16, LBD18 and/or LBD29 was able

152 to influence the expression of the PRH1pro::LUC transgene. The outcome of

153 co-expressing this construct with each of 35S::LBD16, 35S::LBD18 or 35S::LBD29

154 was to enhance the expression of PRH1pro::LUC (Fig 5B). When a pair of effector

155 transgenes (either 35S::LBD16 plus 35S::LBD18, 35S::LBD18 plus 35S::LBD29 or

156 35S::LBD16 plus 35S::LBD29) was introduced, the PRH1pro::LUC expression was

157 even more strongly activated (Fig 5B). The overall conclusion was that the LBDs

158 regulate PRH1 expression by directly binding to its promoter.

159

160 LBD regulated LR development is partially dependent on PRH1

161 To further study if LBDs regulated PRH1 transcription is involved into

162 LBD-mediated LR development, we crossed 35S::PRH1 with lbd16, lbd18 or lbd29

163 mutants and analyzed LR phenotypes of the generated double mutants. Compared to

164 lbd16, lbd18, lbd29 single mutant plants, the generated 35S::PRH1/lbd16, 35S::PRH1


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165 lbd18 or 35S::PRH1/lbd29 double mutants have significantly increased LR numbers

166 (Fig 6), indicating that PRH1 is involved into LBD-mediated LR development.

167

168 PRH1 interacts with GATA23

169 The subcellular localization of PRH1 is in the nuclei of N. benthamiana leaf cells (S6

170 Fig). To explore the candidate genes interacting with PRH1 in the process of

171 regulating LR development, a yeast two hybrid screen of an A. thaliana root cDNA

172 library was carried out, using PRH1 cDNA as the bait. One of the positive clones

173 isolated was found to include the coding sequence of GATA23, a gene which encodes

174 a transcription factor known to control LR founder cell identity (Fig 7A) [8]. The

175 interaction between PRH1 and GATA23 was confirmed using a Co-IP assay, in which

176 C-terminal Myc-tagged full length PRH1 cDNA (PRH1-Myc) and C-terminal

177 GFP-tagged full length GATA23 cDNA (GATA23-GFP) were transiently

178 co-expressed in A. thaliana leaf protoplasts (Fig 7B). When the immunoprecipitated

179 proteins were probed with an anti-GFP antibody, it was found that PRH1-Myc

180 co-immunoprecipitated with GATA23-GFP. The suggestion was therefore that PRH1

181 interacts with GATA23 in planta. Further evidence for the interaction between PRH1

182 and GATA23 was obtained using a BiFC assay, in which a strong YFP signal was

183 observed in the nuclei of N. benthamiana epidermal cells co-expressing PRH1 fused

184 to the N-terminal half of YFP and GATA23 fused to its C-terminal half (Fig 7C). The

185 interpretation of this result was that the auxin inducible protein PRH1 interacts with

186 GATA23. Meanwhile the transcriptional level of GATA23 was significantly

187 down-regulated in arf7, lbd16, lbd18 and lbd29 single mutant (S7 Fig). Together,

188 auxin induced PRH1 interacts with GATA23, which has been also reported to be

189 up-regulated by auxin [20].

190 According to this study together with the previous reports, a proposed model was

191 given in Fig 8. PRH1 is involved in ARF7-LBD dependent auxin signaling pathway

192 regulating lateral root development. PRH1 acts as a downstream gene of both ARF7

193 and LBDs by the direct transcriptional regulation [14]. Nuclear localized PRH1 can

194 interact with another transcription factor GATA23, known to act downstream of


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195 ARF7 and the LBDs, to mediate LR formation [8, 20].

196

197 Discussion

198 The product of the auxin-induced gene PRH1 controls LR formation

199 The role of auxin in LR development has been extensively investigated. During the

200 initiation of LR, auxin signals are transduced through the two distinct

201 AUX/IAA-ARFs modules IAA14-ARF7/ARF19 and IAA12-ARF5 [21-25]. This

202 process establishes LR founder cell identity, which is the initial step towards the

203 formation of a LR. Auxin is also involved in LR emergence, since it controls the

204 activation of IDA, HAE and HSL2 which encode cell wall remodeling enzymes and of

205 genes encoding various expansins in the endodermal cells which lie above the LR

206 primordia [12, 26, 27]. Members of the LBD gene family encode transcription factors,

207 characterized by their LOB domain [28], which act downstream of ARF7-mediated

208 auxin signaling to control LR formation [13, 17, 18, 29].

209 Here, the PR-1 homolog gene PRH1 was identified as a novel target of

210 ARF7-mediated auxin signaling. Disruption of PRH1 function compromised both the

211 initiation and emergence of LR, as demonstrated by the reduction in the number of

212 LR developed by the PRH1 loss-of-function mutant (Fig 3). Although the

213 over-expression of PRH1 in a WT background was not associated with any LR

214 phenotype, its over-expression in arf7, lbd16, 1bd18 or lbd29 mutant backgrounds led

215 to a partial rescue of the compromised LR phenotype (Figs 3 and 6). The result

216 implied that the regulation of LR formation carried out by PRH1 likely requires a

217 degree of coordination with other components. The PRH1 promoter directs its product

218 primarily to the LR primordia (as shown by the site of GUS expression in plants

219 expressing a PRH1pro::GUS transgene), which is fully consistent with the proposed

220 role of PRH1 in LR formation. Auxin treatment strongly induced the expression of the

221 PRH1pro::GUS transgene in a WT (Fig 2A and 2B), but not in an arf7 background

222 (Fig 2C and 2D), which implied that the auxin-induced expression of PRH1 must

223 depend on the presence of a functional copy of ARF7. LBD proteins, especially

224 LBD29, are also involved in the induction by auxin of PRH1. Experimentally, it was


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225 shown that both ARF7 and LBDs were able to bind to the PRH1 promoter (Figs 4 and

226 5). It will be of future interest to investigate how ARF7 coordinates with LBDs to

227 mediate auxin signaling and therefore to control the expression of PRH1.

228 PRH1 interacts with GATA23 to control LR formation

229 The homo- and heterodimerization of LBD proteins are thought to be an important

230 determinant of LR formation [29, 30]. The AtbZIP59 transcription factor has been

231 reported to form complexes with LBDs to direct auxin-induced callus formation and

232 LR development [31]. All these results suggest that homodimerization and

233 heterodimerization of signaling components are important in LR formation. Here, it

234 was shown that GATA23, a protein which influences LR formation through its

235 regulation of LR founder cell identity [8], interacts with PRH1 (Fig 7). According to

236 the previous study together with our results, both GATA23 and PRH1 are auxin

237 induced, a process which is dependent on auxin response factor ARF7 (Figs 1 and 2)

238 [8]. Whether the interaction between GATA23 and PRH1 affects the stability and/or

239 the activity of either (or both) of these proteins remains an issue to be explored.

240


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241 Materials and Methods

242 Plant materials and growing conditions

243 A. thaliana ecotype Col-0 was used as the wild type (WT), and all mutants and

244 transgenes were constructed in a Col-0 background. The mutants comprised a set of

245 T-DNA insert lines obtained from the Arabidopsis Biological Resource Center

246 (ABRC, Columbus, OH, USA), namely prh1-1 (GK_626H08), prh1-2 (Salk_014249),

247 lbd16 (Salk_040739), lbd18 (Salk_038125) [32] and lbd29 (Salk_071133) [33], along

248 with arf7 (Salk_040394) [22]; the transgenic lines were Super::LBD29-GFP [34],

249 35S::MYC-LBD16, 35S::MYC-LBD18, 35S::PRH1, 35S::GFP-PRH1 and

250 PRH1pro::GUS. Seeds were surface-sterilized by fumigation with chlorine gas, and

251 then plated on solidified half strength Murashige and Skoog (MS) medium. All of the

252 seeds were held for two days at 4℃ and finally grown in a greenhouse delivering a 16

253 h photoperiod with a constant temperature of 22℃.

254 Transgene constructs and the generation of transgenic lines

255 The 1,692 nt region upstream of the PRH1 start cordon was used as the PRH1

256 promoter, and was inserted, along with the PRH1 coding sequence into a

257 pENTRTM/SD/D-TOPOTM plasmid (ThermoFisher). The construct was subsequently

258 recombined with several Gateway destination vectors, namely PK7WG2 [35] for

259 over-expression without any tags, pEXT06/G (BIOGLE) for over-expression with an

260 N-GFP tag, PGWB18 for over-expression with an N-Myc tag, PBI221 for

261 over-expression, PKGWFS7.1 [35] for promoter analysis with C-GFP/GUS,

262 pX-nYFP (35S::C-nYFP)/pX-cYFP (35S::C-cYFP) for the bimolecular fluorescence

263 complementation (BiFC) assay, and pGADT7 (AD)/pGBKT7 (BD) (Clontech,

264 Mountain view, CA, USA) for the yeast hybrid assays. Transgenesis was effected

265 using the floral dip method [36], employing transgene constructs harbored by

266 Agrobacterium tumefaciens strain GV3101.

267 Microscopy

268 Sub-cellular structures and GFP activity generated by transgenes were imaged using

269 laser-scanning confocal microscopy. LR numbers and the GUS activity generated by


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270 transgenes were imaged by light microscopy. For the examination of LR,

271 eight-day-old seedlings were treated following the procedure given by (Malamy and

272 Benfey, 1997). The length of primary roots was derived from digital images using

273 Image J software (imagej.nih.gov/ij/). The GUS staining assay followed Liu et al.

274 [37].

275 RNA isolation and quantitative real time PCR (qPCR) analysis

276 RNA was extracted from the roots of eight-day-old seedlings (some of which had

277 been treated with 10 μM naphthalene acetic acid (NAA), obtained from Sigma (St.

278 Louis, MO, USA)) using a TIANGEN Biotech (Beijing) Co. RNA simple Total RNA

279 kit (www.tiangen.com), following the manufacturer’s protocol. A 2 μg aliquot of the

280 RNA was used as the template for synthesizing the first cDNA strand, using a Fast

281 Quant RT Kit (TIANGEN Biotech (Beijing) Co.). Subsequent qPCRs were processed

282 on an MyiQ Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA), using

283 the Super Real PreMix Plus SYBR Green reagent (TIANGEN Biotech (Beijing) Co.).

284 Each sample was represented by three biological replicates, and each biological

285 replicate by three technical replicates. The AtACTIN2 (At3g18780) sequence was used

286 as the reference. Details of the relevant primer sequences are given in S1 Table .

287 RNA-Seq analysis

288 RNA submitted for RNA-seq analysis was isolated from the roots of eight-day-old

289 WT and arf7 seedlings (both NAA-treated and non-treated samples), using a RNeasy

290 Mini Kit (Qiagen, Hilden, Germany). The RNA-seq analysis were processed

291 following the methods given by [38].

292 Chromatin immunoprecipitation coupled to quantitative PCR (ChIP-qPCR) analysis

293 DNA harvested from eight-day-old seedlings of WT and transgenic lines harboring

294 either 35S::MYC-ARF7, Super::LBD29-GFP, 35S::MYC-LBD16 or

295 35S::MYC-LBD18 was cross-linked in a solution containing 1% v/v formaldehyde.

296 The ChIP procedure followed that given by [39], and employed antibodies

297 recognizing MYC and GFP (Abcam, UK). The immune-precipitated DNA and total

298 input DNA were analyzed by qPCR. Details of the relevant primers are given in S1

299 Table.


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300 Yeast one-hybrid (Y1H) and yeast two-hybrid (Y2H) assays

301 Y1H assays were carried out using the Matchmaker Gold yeast one hybrid Library

302 Screening System (Biosciences Clontech, Palo Alto, CA, USA). The PRH1 promoter

303 was inserted into the pAbAi vector, and the bait vector (PRH1pro-pAbAi) was

304 linearized, restricted by BbsI, and introduced into the Y1H Goldeast strain along with

305 the prey vector AD-ARF7. Transformants were grown on SD/-Leu-Ura dropout plates

306 containing different concentrations of aureobasidin A (CAT#630499, Clontech, USA).

307 The primers used for generating the various constructs are listed in S1Table. Y2H

308 assays were performed as recommended in the BD Matchmaker Library Construction

309 and Screening kit (Clontech, USA). The complete coding sequence of PRH1 was

310 fused to the GAL4 DNA binding domain (BD) (pGBKT7), and that of GATA23 to the

311 GAL4 activation domain (AD) (pGADT7), and the two constructs were introduced

312 into yeast strain Y2HGold. Interactions were detected following the manufacturer’s

313 protocol (Clontech, USA). The primers used for generating the various constructs are

314 listed in S1 Table.

315 Dual-luciferase transient expression assay in A. thaliana protoplasts

316 Protoplasts were isolated from three week old WT leaves following Yoo et al. [40].

317 The ligation of the PRH1 promoter to pGreen0800-LUC was realized by enzyme

318 digestion and ligation. The primers used for this procedure are listed in S1 Table [41].

319 The PRH1pro::LUC reporter plasmid was transferred into Arabidopsis protoplast

320 cells with one or more effector plasmids (ARF7, LBD16, LBD18, LBD29 – PBI221);

321 the empty vector PBI221 was used as the negative control. The dual-luciferase assay

322 kit (Promega, USA) allows for the quantification of both firefly and renilla luciferase

323 activity. Signals were detected with a Synergy 2 multimode micro-plate (Centro

324 LB960, Berthold, Germany).

325 Bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation

326 (Co-IP) assays

327 For the BiFC assays, Ag. tumefaciens (strain GV3101) transformants carrying either

328 PRH1-YFPN or GATA23-YFPC were co-infiltrated into the leaves of N. benthamiana,

329 or with an empty vector. After incubation, the plants were kept in the dark for 24 h,


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330 then moved to a lit greenhouse for 72 h. The leaves were monitored by laser scanning

331 microscopy in order to detect fluorescent signals generated by YFP. For the Co-IP

332 assay the target genes were constructed to the over-expression vectors [42], and the

333 plasmids of 35S::PRH1-GFP and 35S::GATA23-MYC were co-transformed into A.

334 thaliana protoplasts using the method described above, then the protoplasts were held

335 for 12-16 hours in darkness with the temperature of 22℃. The protoplasts were

336 collected and the subsequent work was operated following the protocol for

337 immunoprecipitation of Myc-fusion proteins using Myc-Trap®_MA (Chromotek,

338 Germany). Both the input and the IP samples were separated by SDS-polyacrylamide

339 gel electrophoresis, then transferred on to a membrane and probed with the relevant

340 antibody.

341 Statistical analysis

342 Tests were applied to the data to check for normality and homogeneity before

343 attempting an analysis of the variation. The data were analyzed using routines

344 implemented in Prism 5 software (GraphPad Software). The Students’ t-test was

345 applied to compare pairs of means. Values have been presented in the form mean±SE;

346 the significance thresholds were 0.05 (*), 0.01 (**) and 0.001 (***). Comparisons

347 between multiple means were made using a one-way analysis of variance and Tukey’s

348 test.

349 Accession numbers.

350 Sequence data referred to in this paper have been deposited in both the Arabidopsis

351 Genome Initiative and the GenBank/EMBL databases under the accession numbers:

352 ARF7 (At5g20730, NP_851046), PRH1 (At2g19990, NP_179589), LBD16

353 (At2g42430, NP_565973), LBD18 (At2g45420, NP_850436), LBD29 (At3g58190,

354 NP_191378), GATA23 (At5g26930, NP_198045), The RNA-seq data are available in

355 the Gene Expression Omnibus database under accession number GSE122355.

356 Acknowledgments

357 We thank Prof. Yuxin Hu and Prof. Eva Benkova for sharing published materials.

358


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519

520

521 Supplemental data

522 S1 Fig. Transcriptomic analysis of WT and arf7 roots before and after auxin

523 treatment.

524 (A) Numbers of DEGs in arf7 roots compared to WT with auxin treatment or not. (B)

525 Selected candidate genes from both the up-regulated DEGs in WT by auxin and

526 down-regulated genes in arf7 (the bigger purple circle), and the 8 targets involved in

527 lateral root development further refined by Arabidopsis eFP Browser from the 23

528 selected candidates (the smaller yellow circle). (C) The expression pattern and change

529 folds of 8 targets at the site of lateral roots initiation in WT and slr-1 under the

530 condition of auxin treatment or not.

531 S2 Fig. Expression pattern of PRH1 in intact seedlings before (left) and after

532 (right) a 3 h exposure to 10 μM NAA.

533 GUS signals appear blue. GUS: β-glucuronidase, NAA, naphthalene acetic acid. Bar:

534 0.5 cm.

535 S3 Fig. LR density and the transcription level of PRH1 in wild type (WT), prh1

536 mutants and its over-expression lines.

537 (A) The number of LR primordia per cm along the primary root. (B) LRE density per

538 cm along the primary root. (C) LRT density per cm along the primary root in WT,


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539 prh1-1, prh1-2 and plants harboring 35S::PRH1. LRP: LR primordia, LRP: LR

540 primordia, LRE: emerged LRs, LRT: total LRs including the LRP and LRE. Values

541 shown as mean±SE, three biological replicates in the experiment, 20 plant seedlings

542 for each repeat. *: means differ significantly (P<0.05) from the WT control. (D)

543 PRH1 transcription level in the roots of WT, prh1 mutants and plants over-expressing

544 PRH1. Data represent means±SE, three biological replicates in the experiment. **,

545 ***: means differ significantly (P<0.01, P<0.001) from the WT control.

546 S4 Fig. The transcription of PRH1 is regulated by LBD16, LBD18 and LBD29.

547 (A) Transcription level of PRH1 in the roots of WT, lbd16, lbd18 and lbd29 seedlings

548 exposed to 10 μM auxin for 4 hours (orange) or not treated (brown) before extracting

549 total RNA. Different letters atop the columns indicate significant (P<0.05) differences

550 in abundance. (B) Transcription level of PRH1 is enhanced in each of the LBD16, 18

551 and 29 over-expression lines. Data represent means±SE, three biological replicates in

552 the experiment **, ***: means differ significantly (P<0.01, P<0.001) from the WT

553 control.

554 S5 Fig. The interaction between LBDs and PRH1 promoter in yeast one-hybrid.

555 (A) Structure of the PRH1 promoter, showing the putative binding motifs of LBD18

556 (red square) and LBD29 (yellow square). (B) Yeast one-hybrid binding assay

557 containing the interaction between LBD16, LBD18, LBD29 and PRH1 promoter.

558 S6 Fig. PRH1 is deposited in the nuclei of N. benthamiana leaf cells. A

559 35S::GFP-PRH1 transgene was infiltrated into N. benthamiana leaves, and epidermal

560 cells were scanned for the expression of GFP. GFP: green fluorescent protein. Scale

561 bar: 20 μm.

562 S7 Fig. The transcription level of GATA23 is repressed in the roots of the

563 mutants arf7, lbd16, lbd18 and lbd29.

564 Data represent means±SE, three biological replicates in the experiment. *, **: means

565 differ significantly (P<0.05, P<0.01) from the WT control.

566 S1 Table. Primers used in this study.


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567 Figure legends

568 Fig 1. PRH1 is a downstream target of ARF7-mediated auxin signaling.

569 (A) Eight-day-old seedlings of WT and arf7. Scale bar: 1 cm. (B) The ratio between

570 the number of LRs and the length of the primary root of WT and arf7 seedlings. Data

571 shown as mean±SE, three biological replicates in the experiment, 20 plant seedlings

572 for each repeat. ***: the mutant’s performance differs significantly (P<0.001) from

573 that of WT. (C) RNA-seq analysis: RNA was extracted from the roots of

574 eight-day-old seedlings exposed or not exposed to auxin. DEGs: differentially

575 expressed genes, NAA: naphthalene acetic acid. (D) Transcription level of PRH1 in

576 the roots of eight-day-old WT and arf7 seedlings exposed (green) or not exposed

577 (orange) to 10 μmol NAA for 4 hours. Transcript abundances assayed by qPCR.

578 Values represent averages of three biological replicates in the experiment, and the

579 total RNA was extracted from about 100 seedlings for each repeat. Error bars

580 represent SE, ***: the mutant’s performance differs significantly (P<0.001) from that

581 of WT.

582 Fig 2. Histological localization and expression of PRH1pro::GUS transgene

583 during LR formation.

584 (A) and (B) PRH1 is expressed in the LR primordia in WT seedlings not exposed to

585 auxin(A), and the intensity of its expression is strongly enhanced when the plants

586 were treated with auxin (B). (C) and (D) GUS activity is suppressed in non-treated

587 arf7 seedlings (C), and even more so in treated ones (D). NAA: naphthalene acetic

588 acid. Before GUS staining the seedlings were soaked for 4 hours in liquid half

589 strength Murashige and Skoog (MS) medium, which containing 10 μmol NAA or not.

590 The developmental stages (I through VIII) of the LRs are indicated in the bottom left

591 corner. GUS (β-glucuronidase) signals are shown in blue. Scale bars: 100 μm.

592 Fig 3. PRH1 acts downstream of ARF7-mediated LR development.

593 (A) Eight-day-old seedlings of WT, arf7, prh1-1 mutants and transgenic lines

594 harboring either 35S::PRH1 or 35S::PRH1/arf7. Bar: 0.5 cm. (B) The ratio between

595 the number of LRs and the length of the primary root. LRP: LR primordia. LRE:

596 emerged LRs, LRT: all the LRs including the LRP and LRE. Data shown as mean±SE,


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597 three biological replicates in the experiment, 20 plant seedlings for each repeat. The

598 sterisks indicate means which differ significantly (P<0.05) from one another.

599 Fig 4. PRH1 is regulated by ARF7 at the transcriptional level.

600 (A) Structure of PRH1 promoter and the fragments used in the CHIP-qPCR assay.

601 AuxREs are indicated by red squares, and black lines show the promoter regions

602 containing the AuxREs used in this assay. NC: negative control. (B) ARF7

603 transactivates the PRH1 promoter in A. thaliana leaf protoplasts. The left hand panel

604 is a schematic of the effector (35S::ARF7) and reporter (PRH1pro::LUC) constructs.

605 The empty vector pBI221 was used as a negative control; the right hand panel shows

606 the ratio of ARF7 drived LUC and the empty vector (negative control) to 35S

607 promoter drived REN respectively. LUC: firefly luciferase activity, REN: renilla

608 luciferase activity. Values shown as mean±SE, three biological replicates in the

609 experiment. *: means differ significantly (P<0.05) from the negative control. (C)

610 ARF7 is associated with the PRH1 promoter according to a CHIP-qPCR assay.

611 Chromatin isolated from a plant harboring 35S::MYC-ARF7 and a WT mock control

612 was immunoprecipitated with anti-MYC antibody followed the amplification of

613 regions P1, P2 and P3. The coding region segment NC was used as the negative

614 control. The ChIP signal represents the ratio of bound promoter fragments (P1-P3) to

615 total input after immuno-precipitation. Values shown as mean±SE, three biological

616 replicates in the experiment. **, ***: means differ significantly (P<0.01, P<0.001)

617 from the WT control. (D) Physical interaction of ARF7 with the PRH1 promoter

618 according to a Y1H assay. The plasmid pGADT7-ARF7 was introduced into Y1H

619 Gold cells harboring the reporter gene PRH1pro::AbAr and the cells were grown on

620 SD/-Ura-Leu medium in the presence of 30 or 50 ng/mL aureobasidin A (AbA). The

621 empty vector pGADT7 (AD) was used as a negative control.

622 Fig 5. The transcription of PRH1 is activated by LBD binding to its promoter.

623 (A) LBDs associated with the promoter of PRH1 according to a CHIP-qPCR assay.

624 The upper panel shows the structure of the PRH1 promoter, and five uniformly

625 distributed sites (black lines) were selected for the CHIP-qPCR assay. Sites II and III

626 include the LBD29 and LBD18 binding motif (See S5 Fig). NC: negative control. The


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627 lower panel demonstrates the ratio of bound promoter fragments (Ⅰ-Ⅴ) to total input,

628 derived from experiments involving WT seedlings (negative control) and those

629 harboring one of the transgenes 35S::Myc-LBD16 (Myc-LBD16), 35S::Myc-LBD18

630 (Myc-LBD18), Super::LBD29-GFP (LBD29-GFP). Values shown as mean±SE, three

631 biological replicates in the experiment. *, **: means differ significantly (P<0.05,

632 P<0.01) from the WT control. (B) LBDs transactivate the PRH1 promoter in A.

633 thaliana leaf protoplasts as shown by a transient dual-luciferase assay. The reporter

634 gene (PRH1 promoter driving LUC) was co-transformed with one or two of the

635 constructs 35S::LBD16, 35S::LBD18 and 35S::LBD29. The empty vector pBI221 was

636 used as negative control. Values shown as mean±SD, three biological replicates in the

637 experiment. *, **: means differ significantly (P<0.05, P<0.01) from the WT control.

638 Fig 6. The over-expression of PRH1 partially alleviates the defective development

639 of LRs in the lbd mutant.

640 (A) Eight-day-old seedlings of WT, the mutants lbd16, lbd18 and lbd29, and the

641 transgenic lines harboring 35S::PRH1. Scale bar: 5 mm. (B) Density of LRP (LR

642 primordia), LRE (emerged LRs) and LRT (Total LRs containing LRP and LRE)

643 formed in eight-day-old seedlings. Data shown as means±SE, three biological

644 replicates in the experiment, 20 plant seedlings for each repeat. *: means differ

645 significantly (P<0.05) as a result of over-expressing PRH1 in the mutant

646 backgrounds.

647 Fig 7. PRH1 physically interacts with GATA23.

648 (A) PRH1 interacts with GATA23 in a yeast two hybrid assay. Yeasts were grown on

649 both Synthetic Complete-Leu-Trp (SD-L-T) and Synthetic Complete-Leu-Trp-His

650 (SD-L-T-H) medium. The interaction of pGADT7-GATA23 (AD-GATA23) with

651 pGBKT7-PRH1 (BD-PRH1) was assayed by contrasting the growth of the positive

652 and negative controls on SD-L-T-H. (B) The interaction of PRH1 with GATA23 was

653 assayed by Co-IP. The protoplasts transiently expressing PRH1-MYC and/or

654 GATA23-GFP were prepared from A. thaliana leaves, the tagged proteins were

655 immunoprecipitated using an agarose-conjugated anti-MYC matrix, and the blots

656 were probed with anti-GFP and/or anti-MYC antibody. (C) Bimolecular fluorescence


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657 complementation (BiFC) was used to assay the interaction of PRH1 with GATA23.

658 PRH1 was fused with the N-terminal fragment of YFP (YFPN) and GATA23 with the

659 C-terminal fragment of YFP (YFPC); the transgenes were infiltrated into N.

660 benthamiana and the leaves were assayed for YFP fluorescence. Empty vectors were

661 used as the negative control. Scale bar: 40 μm.

662 Fig 8. A working model of the regulation of LR development by PRH1.

663 PRH1 acts downstream of ARF7 and LBDs. Auxin-induced PRH1 transcription is

664 mediated by both ARF7 and LBDs via their binding to its promoter. The transcription

665 factor GATA23, known to act downstream of ARF7 and the LBDs, mediates auxin

666 signaling during LR formation and interacts with PRH1.


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