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