1 Plant Physiology Manuscript Running head: A set of LIC vectors ...

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Plant Physiology Manuscript

Running head: A set of LIC vectors for functional studies in plants

Name: Dolf Weijers

Address: Dreijenlaan 3, 6703HA Wageningen, The Netherlands

Telephone number: +31 (0) 317-482866

E-mail: [email protected]

Journal research area: Breakthrough Technologies

Plant Physiology Preview. Published on May 11, 2011, as DOI:10.1104/pp.111.177337

Copyright 2011 by the American Society of Plant Biologists

www.plantphysiol.orgon February 7, 2018 - Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

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A versatile set of Ligation-Independent Cloning vectors for functional studies in plants

Bert De Rybel1, Willy van den Berg1, Annemarie Lokerse, Che-Yang Liao2, Hilda van

Mourik, Barbara Möller, Cristina Llavata Peris and Dolf Weijers*

Wageningen University, Laboratory of Biochemistry, Dreijenlaan 3, 6703 HA Wageningen,

The Netherlands



3

This work was supported by a long-term FEBS fellowship and a Marie Curie long term FP7

Intra-European Fellowship (IEF-2009-252503) to B.D.R. and by funding from the

Netherlands Organization for Scientific Research (NWO; ALW-816.02.014 and ALW-VIDI-

864.06.012) and from the European Commission 7th Framework Program (Initial Training

Network “SIREN”; Contract no. 214788) to D.W.

1 These authors contributed equally to this work 2 Present address: Institute for Biology III, Freiburg University, D-79104 Freiburg, Germany * To whom correspondence should be addressed: [email protected]



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Abstract

With plant molecular biology entering the omics era, there is a need for simple cloning

strategies that allow high throughput to systematically study the expression and function of

large numbers of genes. Such strategies would facilitate the analysis of gene (sub-) families

and/or sets of co-expressed genes identified by transcriptomics. Here, we provide a set of 34

Ligation-Independent Cloning (LIC) binary vectors for expression analysis, protein

localization studies and misexpression that will be made freely available. This set of pPLV or

Plant LIC Vectors offers a fast alternative to standard cloning strategies involving ligase or

recombination enzyme technology. We demonstrate the use of this strategy and our new

vectors by analyzing the expression domains of genes belonging to two subclades of the basic

helix-loop-helix (bHLH) transcription factor family. We show that neither the closest

homologs of TARGET OF MONOPTEROS 7 (TMO7/ATBS1) nor the members of the ATBS1

INTERACTING FACTOR (AIF) subclade of putative TMO7 interactors are expressed in the

embryo and that there is very limited co-expression in the primary root meristem. This

suggests that these bHLH transcription factors are most likely not involved in TMO7-

dependent root meristem initiation.



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Introduction

Whole genome analysis is becoming a standard analysis tool in reverse genetics plant

research. Furthermore, there is often the need to study large gene-families in Arabidopsis due

to redundancy. For these and other reasons, there is an increasing need in plant research for

fast cloning strategies. Besides speed, these methods have to be characterized by easy

handling in order to, for example, verify protein localizations with moderately high

throughput. Unfortunately, most currently available cloning methods are not able to combine

these characteristics. Current cloning procedures are either laborious and slow (such as

classical cloning) or quick but expensive (such as the Gateway technology (Curtis and

Grossniklaus, 2003; Karimi et al., 2007)). Other, more recent advances such as BAC

recombineering (Zhou et al., 2011), whilst allowing precision cloning, have a clear

disadvantage in that they introduce not only a gene of interest, but a complete genomic

region. An emerging single step method that is very suitable for moderate high throughput

cloning is ligation-independent cloning (LIC) (Li and Elledge, 2007; Eschenfeldt et al.,

2009). The LIC cloning system is characterized by a few simple steps, including linearization

of the vector, amplification of the fragment of interest, the creation of sticky ends on the

vector and insert by the 3’-5’ exonuclease activity of T4 DNA polymerase and subsequent

annealing of the fragment into the vector (Figure 1). A related type of LIC cloning has been

described, facilitating the assembly of multiple fragments in one reaction, called SLIC

(Sequence and Ligation Independent Cloning), using in vitro homologous recombination and

single-strand annealing (Li and Elledge, 2007). For most projects however, single purpose

LIC cloning is sufficient.

Despite its potential to become a good alternative for current cloning strategies, LIC cloning

has not been readily used in plant research so far, perhaps due to the absence of a

comprehensive set of vectors. Over the past years, only a small number of LIC-based vectors

have been made available for protein production and purification (Doyle et al., 2005;

Bardóczy et al., 2008), in planta expression (Oh et al., 2010) and construction of hairpin

constructs (Xu et al., 2010; Hauge et al., 2009). Although these vectors are very useful for

these purposes, a comprehensive collection of LIC-based vectors in plant research was

missing.

Here, we describe the creation of a multipurpose set of 34 LIC-compatible Plant LIC Vector

(pPLV) vectors (Figure 3, Table 1) for expression analysis, protein localization studies and

various misexpression analyses in Arabidopsis thaliana and other plant species.



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Generating a series of vectors for Ligation-Independent Cloning

We generated a versatile set of LIC vectors for general use in plant molecular biology. These

include vectors for expression analysis of promoter fragments, protein localization studies,

misexpression (using several different promoters) as well as standard empty vectors to

generate custom LIC vectors for other purposes (Table 1, Figure 1). A range of fluorescent

proteins, triple GFP, sYFP, sCFP and tandemTomato were selected and used due to their

higher quantum yields compared to the original fluorescent proteins (Kremers et al., 2006;

Shaner et al., 2004). Furthermore, the pGIIK/B/H-LIC-sYFP-NOSt and pGIIK/B/H-LIC-

sCFP-NOSt vectors can be used for FRET-FLIM analyses to detect protein-protein

interactions in plants. Almost all of the vectors are available with different antibiotic

resistances, increasing the versatility of this set of vectors through combinatorial use of

multiple transgenes. All constructed vectors are based on a binary pGreenII (pGII) vector

backbone with kanamycin (K), phosphinothricin/basta (B) or hygromycin (H) resistance

(Hellens et al., 2000); in which a custom LIC-site was introduced using EcoRI and BamHI

restriction sites (see Figure 1).

The pGIIK/B/H-LIC-NOSt vectors served as the base for the LIC vectors that were

generated; except for the pPLV04 vector, which was modified from a previously described

pGIIB-SV40-3GFP-NOSt vector (Takada and Jurgens, 2007). The custom LIC site that was

introduced contains a unique HpaI restriction site, which is used for linearizing vectors (see

Figure 1). The vectors used for expression analysis and protein localization were created by

introducing the respective fluorescent protein or GUS fragment in the BamHI restriction site

at the 3’ flank of the LIC site. In protein localization vectors, the resulting linker between the

genomic fragment (without stop codon) and the fluorescent protein is illustrated in Figure 2.

The forward primer used to amplify the fluorescent proteins also created a SpeI restriction

site, which was used to introduce the SV40 nuclear localization signal in the vectors used for

expression analysis of promoter fragments. To allow cloning of the tandemTomato vectors, a

remnant 424 basepair Lac promoter fragment, which caused a reverse reading frame in

combination with the tandem Tomato, was removed. This Lac promoter fragment at the 3’

end of the construct was removed by cutting with NotI and StuI restriction enzymes en

reintroducing an MluI restriction site. Standard analyses of protein localization are done using

the endogenous promoter by cloning the full genomic fragment in the protein localization

vectors. This has the advantage of capturing all potential regulatory sequences both in the



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promoter region and in intronic regions. Nevertheless, these vectors could also be easily

adapted to drive expression from different promoters by inserting these into the multiple

cloning site upstream of the LIC site by standard cloning strategies or by cloning a chimeric

construct (created using standard overlap extension PCR) into the existing vectors. For the

misexpression vectors, the p35S and pRPS5a (Odell et al., 1985; Weijers et al., 2001)

promoters were introduced using KpnI and ApaI restriction enzymes. The pRPS5a promoter

fragment contained an HpaI restriction site, which had to be mutated in order to enable

linearization of the vector. For this, we mutated the GTTAAC sequence of the HpaI site into

GTAAAC using site directed mutagenesis (Sawano and Miyawaki, 2000) without affecting

the promoter activity. The pMP promoter (Schlereth et al., 2010) was introduced using KpnI

and XhoI restriction enzymes and the GAL4-dependent pUAS (upstream activating sequence;

(West et al., 1984; Weijers et al., 2003)) fragment was cloned using PstI and BamHI

restriction sites. An overview of the set of LIC vectors is given in Figure 3 and Table 1.

The overall efficiency of the LIC-based vector cloning described here is very high, although

it is important to note that, in our hands, LIC cloning works best when high concentrations

(about 1.5 μg for one T4 treatment) of very pure vector and PCR fragment are used for the T4

treatment. Although there can be a large variability in the number of colonies after

transformation, the rate of positive colonies is usually high (between 60 and 100%). Due to

the reduced stability of the triple GFP construct in E. coli, cloning with the pPLV04 vector as

well as cloning larger fragments (> 5kb) into any of the vectors can be less efficient.

Nonetheless, we routinely clone fragments of up to 5kb in several of these LIC vectors.

Examples of constructs generated by LIC

To provide an example of the speed and efficiency of our LIC-based vectors, we analyzed the

promoter expression domains of TARGET OF MONOPTEROS 7 (TMO7/ATBS1) (Wang et

al., 2009; Schlereth et al., 2010) and its three closest homologs, namely PRE1/BNQ1,

PRE2/BNQ2 and PRE4/BNQ3 (Lee et al., 2006; Mara et al., 2010).

TMO7 was first identified as Activation-Tagged Bri1-Suppressor1 (ATBS1) in a screen for

genes that could rescue the dwarfed bri1 phenotype when overexpressed (Wang et al., 2009).

More recently, TMO7/ATBS1 (from now on referred to as TMO7) was found to be a direct

target of MP and is required during embryogenesis (Schlereth et al., 2010). Specifically,

TMO7 was shown to move from the proembryo towards the uppermost suspensor cell to

specify this cell as hypophysis, which is required for establishing the primary root meristem



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(Schlereth et al., 2010). Although RNAi suppression of TMO7 led to embryo defects and a

low rate of rootless seedlings (Schlereth et al., 2010), it is still possible that other, closely

related genes act redundantly with TMO7. As a first step in addressing this issue, we tested

the expression of TMO7 and its 4 closest homologs by fusing their promoters to SV40-

3xGFP in the pPLV4 vector (Figure 3). None of the TMO7 homologs showed any expression

during embryo development (data not shown), therefore, we analyzed the expression patterns

in the post-embryonic root. Consistent with its role in the establishment of the primary root

meristem, TMO7 was strongly expressed in the quiescent centre (QC) and surrounding

proximal stem cells, but absent from the columella root cap cells (Figure 4). The TMO7

homolog bHLH161/PRE4/BNQ3 only expressed in the lateral root cap, while bHLH166,

bHLH136/PRE1/BNQ1 and bHLH134/PRE2/BNQ2 are weakly expressed in the root cap and

columella cells (Figure 4). Hence it appears that none of the TMO7-relatives shows a strong

co-expression with TMO7 during embryogenesis or in the primary root meristem. In the

mature root however, there is an overlap in expression domains for TMO7, bHLH161 and

bHLH166; but not for bHLH136 or bHLH134 (Supplemental Figure 1).

In a yeast two-hybrid screen, four closely related bHLH transciption factors were shown to be

able to interact with TMO7 and were named ATBS1 interacting factors (AIF1-4) (Wang et

al., 2009). Although TMO7 was shown to interact with AIF1-4 in vitro and in vivo (in

seedlings overexpressing both AIF1 and TMO7), it is not clear whether these genes are

actually expressed in the same tissues and thus if their interaction is biologically meaningful.

To address this question, we analyzed the expression of the four AIF1-4 genes as well as that

of the related bHLH151/UPB (Tsukagoshi et al.), bHLH158 and bHLH159 genes. Again,

none of these genes appears to be expressed during embryo development (data not shown). In

the primary root meristem, bHLH150/AIF1 expression cannot be detected, while

bHLH148/AIF2, bHLH147/AIF3 and bHLH149/AIF4 are all expressed in the root cap and

lower columella cells (Figure 4). A similar expression pattern was observed for bHLH158,

while bHLH159 is expressed in the lateral root cap and vascular tissues. Similar to published

data (Tsukagoshi et al., 2010), bHLH151/UPB is expressed in the lateral root cap and in the

vascular tissues (Figure 4). In the mature root, bHLH149/AIF4, bHLH150/AIF1,

bHLH151/UPB and bHLH159 are expressed in all cell types, while bHLH148/AIF2 and

bHLH147/AIF3 appear to be more specific for vascular tissues (Supplemental Figure 1).

Additionally, we analyzed the protein localization (pPLV16 or pGIIB-LIC-sYFP-tNOS

vector) for some of these bHLH transcription factors to support the expression domains found

using the pPLV04 (pGIIK-LIC-SV40-3GFP-tNOS) vector (Supplemental Figure 2). All



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analyzed protein localization domains fully overlapped with the promoter expression

domains and with available data for bHLH151/UPB (Tsukagoshi et al., 2010), supporting the

validity of this set of vectors.

Discussion

We have created a set of LIC-compatible vectors for diverse purposes in plant molecular

biology research, including expression analysis of promoter fragments, protein localization

studies and misexpression. LIC cloning allows for a single-step way to clone in a moderately

high throughput fashion. Despite the obvious advantages of LIC cloning, there are some

drawbacks using this system. The most obvious downside is that every fragment needs to be

sequenced when used in different vectors, which is not the case for Gateway cloning. This

highlights the need for a high quality proofreading polymerase enzyme for amplification. In

our hands however, we only very rarely encounter erroneous base pairs (e.g. 1 mistake in 10-

20 kb sequenced; in line with the error-rate of the proofreading polymerase used). Another

disadvantage is that cloned fragments cannot be recombined as with Gateway cloning.

However, in our experience this is not a problem for most purposes since the majority of

cloning reactions are single-purpose projects.

The set of vectors presented here has allowed us to misexpress all members of a gene family

of over 20 members from 4 different promoters and to determine/validate transcription

patterns for up to 150 genes identified in microarray experiments (data will be published

elsewhere). Here, as an example, we show the use of one of our LIC-compatible vectors to

examine the promoter expression domains of the TMO7 subclade of bHLH transcription

factors and its putative AIF interactors. Interestingly, none of the examined homologs

showed expression or overlap in expression with TMO7 in the embryo or in the hypophysis

descendants in the primary root meristem; further supporting the proposed single gene

function for TMO7 in root meristem establishment (Schlereth et al., 2010). Furthermore,

there is no expression of the putative AIF1-4 interactors in the embryo, nor is there a co-

expression with the TMO7 expression domain in the primary root meristem. Notably,

bHLH150/AIF1 does not appear to be expressed in the embryo nor the root meristem,

precluding the possibility of an interaction with TMO7 in these tissues. In conclusion, our

analysis has shown that none of the TMO7 homologs nor its putative interactors are likely to

be involved in the process of TMO7-dependent root meristem initiation.



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In the primary root however, several of the analyzed bHLH transcription factors have

overlapping expression domains with TMO7, allowing potential interactions in these tissues.

Furthermore, as the TMO7 protein was shown to move from the proembryo to the future

hypophysis during embryonic root development (Schlereth et al., 2010), we cannot exclude a

similar movement to the columella cells in the primary root meristem. Therefore, an overlap

in the protein expression domains (and potential for interaction) between TMO7 and

bHLH148/AIF2, bHLH147/AIF3 and bHLH149/AIF4 in the columella region remains

possible. In any case, further research is required to investigate the biological significance of

these interactions. Furthermore, it is important to note that the expression domains of

previously published genes, such as bHLH151/UPB (Tsukagoshi et al., 2010), are identical to

what we have found using our LIC-compatible vectors, supporting the quality of our set of

vectors.

In conclusion, we believe that this set of LIC-compatible vectors will provide a useful

resource for researchers in plant biology that depend highly on cloning of large numbers of

constructs. Therefore, the availability of this quick and versatile cloning system may aid

progress in the current omics era.



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Materials and Methods

The pPLV vectors (for Plant LIC Vectors) are all available from the NASC stock center for

distribution. The sequences of all vectors have been added to the manuscript as a

supplemental text file (Supplemental Dataset), and are accessible at Genbank

(http://www.ncbi.nlm.nih.gov/genbank/). Accession numbers are provided in Figure 3.

To introduce a fragment of interest into a LIC-based vector, extensions are added to the

fragment during amplification by PCR using primers with LIC adapter sites (see Table 1).

This LIC-compatible fragment is next treated with T4 DNA polymerase and an excess of

dGTP. The 3’-5’ exonuclease activity of the T4 DNA polymerase creates 15 base pair (bp)

single strand overhangs (Figure 1). LIC-vectors are prepared by linearizing using a unique

restriction site in the LIC-site of the vector. Subsequent T4 DNA polymerase treatment with

excess of dCTP then creates 15 bp single strand overhangs, complementary to those available

on the T4-treated fragment (Figure 1). Vector and insert are then combined, allowed to

anneal and transformed into a bacterial host, which will repair the introduced nicks. The

sequence of the LIC-site itself ensures correct orientation of the inserted fragment. A detailed

protocol can be found below in this section and a quick lab protocol in the Supplemental Data

online.

Preparation of vectors

For a standard preparation, 2 to 4 μg of vector is cut with 1 μl HpaI fast cut restriction

enzyme (Fermentas) in duplicate for 2 hours at 37°C. Linearized vector is next purified from

agarose gel using QIAEXII gel extraction kit (Qiagen) and duplicates are pooled. Linearized

vectors are then precipitated overnight (or minimum 2 hours) using 0.5 volumes ammonium

acetate (7.5M) and 2.5 volumes of 100 % ethanol at -20°C. The precipitated vector is pelleted

by centrifugation for 30 minutes at maximum speed. The supernatant is removed and the

pellet is washed with 100 μl of 70 % ethanol followed by a 100 % ethanol wash. The pellet is

next dried and resuspended in 50 μl water (at 50°C for 5 to 10 minutes). For T4 treatment

(New England Biolabs), 200-400 ng of linearized vector, 4 μl 10X T4 buffer, 4 μl 100mM

dCTP, 2 μl 100mM dithiothreitol, 0.4 μl bovine serum albumin, 0.8 μl T4 DNA polymerase

(New England Biolabs) and H2O to 40 μl total volume is mixed. The mixture is centrifuged at

maximum speed for 1 minute, incubated at 22°C for at least 30 minutes (up to 2 hours),

inactivated at 75°C for 20 minutes and centrifuged again at maximum speed for 1 minute. T4



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treated vectors can be stored at 4°C until further use.

Preparation of fragments

The DNA fragment of choice is first amplified by PCR using primers with respective LIC-

adapter sites (dependent on destination vector, see Table 1). PCR is performed in 50 μl

volume in duplicate using Phusion Flash (Finnzymes) polymerase (or another high quality

polymerase enzyme with proof reading) using the amplification protocol provided by the

supplier. Fragments are next purified from agarose gel using QIAEXII gel extraction kit

(Qiagen) and duplicates are pooled. For T4 treatment (New England Biolabs), 200-400 ng of

purified fragment, 2 μl 10X T4 buffer, 2 μl 100mM dGTP, 1 μl 100mM dithiothreitol, 0.2 μl

bovine serum albumin, 0.4 μl T4 DNA polymerase (New England Biolabs) and H2O to 20 μl

total volume is mixed. The mixture is centrifuged at maximum speed for 1 minute, incubated

at 22°C for at least 30 minutes (up to 2 hours), inactivated at 75°C for 20 minutes and

centrifuged again at maximum speed for 1 minute. T4 treated fragments can be stored at 4°C

until further use.

Annealing, transformation in E. coli and sequence verification

To anneal the linearized, T4 treated vector and the T4 treated PCR fragment, 10-40 ng vector

and insert are combined in a 1:3 molar ratio for 30 minutes to 2 hours at 22°C (usually about

1 to 3 μl each) or overnight at 4°C. The whole mixture is then transformed into

electrocompetent DH5α E. coli cells (transformation efficiency > 107 cfu/μg) and plated on

LB-agar plates with 25 mg/l kanamycin as antibiotic (see Table 1) and incubated at 37°C

overnight. A T4 treated vector without added insert can be used to analyze the amount of

background colonies. The next day, colonies are verified for inserts using colony PCR and

positives grown overnight in 6 ml LB with 25 mg/l kanamycin. Plasmids are extracted

(GeneJET Plasmid Mini Prep Kit from Fermentas), checked by restriction digest before

sequencing. Because of the proofreading DNA polymerase, point mutations are very

uncommon.

A simplified plant transformation procedure allowing moderate throughput

Plasmids are transformed into electrocompetent Agrobacterium tumefaciens GV3101

containing the pGreen helper plasmid pSOUP (Hellens et al., 2000) using standard protocols

and plated on LB plates with the appropriate antibiotics (Table 1). Following two days of

growth at 28°C, a smear of multiple colonies is inoculated into 20 ml liquid LB medium with



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the appropriate antibiotics and grown overnight at 28°C in a shaker. The next day, the

volume of the culture is increased to 50 ml LB with antibiotics and grown, again at 28°C, to

an OD600 of around 0.7 (0.5 to 0.9 is acceptable). If the optical density is too high, the

cultures can be diluted to the correct OD600 using LB. Next, 2.5 g sucrose and 10-20 μl Silwet

is added to 50 ml culture and shaken until the sucrose is dissolved. Five to ten plants are then

floral dipped in this mixture, placed in a box and covered with cling film for one day before

growing them in the growth room until seeds can be harvested.

Plant growth and selection

Plants (Col-0) ecotype were grown under standard conditions at 23 degrees Celsius in a 16

hours light / 8 hours dark cycle. Selection for transgenes was performed on solid MS medium

supplemented with 25 mg/L kanamycin (pPLV04) or 15 mg/L phosphinotricin (pPLV16).

Microscopy

Gene expression or protein accumulation was analyzed in roots of homozygous T3 lines

carrying a single T-DNA insert as determined by segregation of kanamycin or PPT

resistance. Four- to five-day old vertically grown seedlings were incubated in water

containing 1 μM FM4-64 (Invitrogen) for 1 minute, and subsequently imaged on a Zeiss

LSM510 confocal laser scanning microscope.

Accession numbers

All vector sequences have been deposited at Genbank, and can be found using the following

accession numbers: JF909454 (pPLV01), JF909455 (pPLV02), JF909456 (pPLV03),

JF909457 (pPLV04), JF909458 (pPLV05), JF909459 (pPLV06), JF909460 (pPLV07),







JF909485 (pPLV32), JF909486 (pPLV33), JF909487 (pPLV34).



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Figure and Table legends

Figure 1. LIC cloning procedure with modified LIC site. Vectors are first digested with

HpaI restriction enzyme and fragments are amplified by PCR with primers containing the

LIC adapter sites. Overhangs are made by the 3’-5’ exonuclease activity of T4 DNA

polymerase in excess of dCTP or dGTP for the vector and fragment respectively. The sticky

end overhangs that are created allow for easy annealing of vector and insert.

Figure 2. The linker introduced by LIC cloning. As an example, the linker is shown

between a genomic fusion without stop codon and a fluorescent protein of choice (sYFP) by

LIC cloning.

Figure 3. Overview of LIC vectors. Indicated are left and right border (LB and RB), NOS

promoter and terminator (pNOS and NOSt), resistance genes (B/K/H), nuclear localization

signal (SV40), the LIC site (LIC), specific promoters for misexpression (p35S, pRPS5a, pMP

and upstream activating sequence or UAS) and respective fluorescent proteins (GFP, sCFP,

sYFP, tandemTomato).

Figure 4. Overview of promoter expression patterns of a subclade of TMO7 related

bHLH transcription factors using the pGIIK-LIC-VP40-3GFP-NOSt vector. The

phylogenetic tree shows a subclade of the bHLH transcription factors based on full-length

protein sequences. Branch lengths indicate phylogenetic distances (see scale bar: fraction of

deviations). Confocal images of primary root meristems were counter stained using FM4-64

dye (red).



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Table 1. Overview of LIC-compatible vectors. Respective use, names, antibiotic resistances, remarks, size in base pairs and required LIC-

adapter sites for forward and reverse primers used to amplify the required fragment are indicated.

Use pPLV Vector Antibiotic resistance adapter forward primer 5'-3' adapter reverse primer 5'-3'

basic vector for custom use pPLV01 pGIIB-LIC-NOSt Basta/ppt - -

pPLV02 pGIIK-LIC-NOSt Kanamycin - -

pPLV03 pGIIH-LIC-NOSt Hygromycin - -

promoter analysis pPLV04 pGIIK-LIC-SV40-3xGFP-NOSt Kanamycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAA-

pPLV05 pGIIB-LIC-SV40-sYFP-NOSt Basta/ppt TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAA-

pPLV06 pGIIK-LIC-SV40-sYFP-NOSt Kanamycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAA-

pPLV07 pGIIB-LIC-SV40-sCFP-NOSt Basta/ppt TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAA-

pPLV08 pGIIK-LIC-SV40-sCFP-NOSt Kanamycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAA-

pPLV09 pGIIH-LIC-SV40-sCFP-NOSt Hygromycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAA-

pPLV10 pGIIB-LIC-SV40-tdTomato-NOSt Basta/ppt TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAA-

pPLV11 pGIIK-LIC-SV40-tdTomato-NOSt Kanamycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAA-

pPLV12 pGIIH-LIC-SV40-tdTomato-NOSt Hygromycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAA-

pPLV13 pGIIB-LIC-GUS-NOSt Basta/ppt TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAA-

pPLV14 pGIIK-LIC-GUS-NOSt Kanamycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAA-

pPLV15 pGIIH-LIC-GUS-NOSt Hygromycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAA-

protein localization pPLV16 pGIIB-LIC-sYFP-NOSt Basta/ppt TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

pPLV17 pGIIK-LIC-sYFP-NOSt Kanamycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

pPLV18 pGIIH-LIC-sYFP-NOSt Hygromycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

pPLV19 pGIIB-LIC-sCFP-NOSt Basta/ppt TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

pPLV20 pGIIK-LIC-sCFP-NOSt Kanamycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

pPLV21 pGIIH-LIC-sCFP-NOSt Hygromycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

pPLV22 pGIIB-LIC-tdTomato-NOSt Basta/ppt TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

pPLV23 pGIIK-LIC-tdTomato-NOSt Kanamycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

pPLV24 pGIIH-LIC-tdTomato-NOSt Hygromycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

pPLV13 pGIIB-LIC-GUS-NOSt Basta/ppt TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

pPLV14 pGIIK-LIC-GUS-NOSt Kanamycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

pPLV15 pGIIH-LIC-GUS-NOSt Hygromycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

misexpression pPLV25 pGIIB-p35S-LIC-NOSt Basta/ppt TAGTTGGAATAGGTTC- AGTATGGAGTTGGGTTC-

pPLV26 pGIIK-p35S-LIC-NOSt Kanamycin TAGTTGGAATAGGTTC- AGTATGGAGTTGGGTTC-

pPLV27 pGIIH-p35S-LIC-NOSt Hygromycin TAGTTGGAATAGGTTC- AGTATGGAGTTGGGTTC-

pPLV28 pGIIB-pRPS5a-LIC-NOSt Basta/ppt TAGTTGGAATAGGTTC- AGTATGGAGTTGGGTTC-

pPLV29 pGIIB-pMP-LIC-NOSt Basta/ppt TAGTTGGAATAGGTTC- AGTATGGAGTTGGGTTC-

pPLV30 pGIIK-pMP-LIC-NOSt Kanamycin TAGTTGGAATAGGTTC- AGTATGGAGTTGGGTTC-

pPLV31 pGIIH-pMP-LIC-NOSt Hygromycin TAGTTGGAATAGGTTC- AGTATGGAGTTGGGTTC-

pPLV32 pGIIB-UAS-LIC-NOSt Basta/ppt TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

pPLV33 pGIIK-UAS-LIC-NOSt Kanamycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

pPLV34 pGIIH-UAS-LIC-NOSt Hygromycin TAGTTGGAATGGGTTCGAA- TTATGGAGTTGGGTTCGAAC-

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pGIIK/B/H-LIC-NOSt4750 bp

K/B/Hresistance

NOSt

NOSt

pNOS

ori

LB

RB

LIC

5’GAATTCTAGTTGGAATGGGTTAACCCAACTCCATAAGGATCC 3’3’CTTAAGATCAACCTTACCCAATTGGGTTGAGGTATTCCTAGG 5’

EcoRI BamHIHpaI

cut with HpaI

T4 treatment + dCTP

5’GAATTCTAGTTGGAATGGGTT AACCCAACTCCATAAGGATCC 3’3’CTTAAGATCAACCTTACCCAA TTGGGTTGAGGTATTCCTAGG 5’

5’GAATTC AACCCAACTCCATAAGGATCC 3’3’CTTAAGATCAACCTTACCCAA CCTAGG 5’

PCR fragment:

5’ TAGTTGGAATGGGTTCGAA------TTCGAACCCAACTCCATAA 3’3’ ATCAACCTTACCCAAGCTT------AAGCTTGGGTTGAGGTATT 5’

5’ TAGTTGGAATGGGTTCGAA------TTCG 3’3’ GCTT------AAGCTTGGGTTGAGGTATT 5’

T4 treatment + dGTP

fragment of interestLIC adapter LIC adapter

Vector:

Figure 1. LIC cloning procedure with modified LIC site. Vectors are first digested with HpaI restriction enzyme and fragments are amplified by PCR with primers containing the LIC adapter sites. Overhangs are made by the 3’-5’ exonuclease activity of T4 DNA polymerase in excess of dCTP or dGTP for the vector and fragment respectively. The sticky end overhangs that are created allow for easy annealing of vector and insert.



genomic fragment (-STOP) gtt cga acc caa ctc cat aag gat ccc ATG-sYFP

... Val Arg Thr Gln Leu His Lys Asp Pro Met ...

9 AA linker introduced by LIC

Figure 2. The linker introduced by LIC cloning. As an example, the linker is shown between a genomic fusion without stop codon and a fluorescent protein of choice (sYFP) by LIC cloning.



B/K/H NOSt

LIC

pNOS

RBLB

NOSt

B

LIC RBLB

pRPS5aNOSt NOStpNOS

LIC RBLB

sYFPNOSt NOStpNOSB/K/H

LIC RBLB

sCFPNOSt NOStpNOSB/K/H

LIC RBLB

NOSt NOStpNOS tdTomatoB/K/H

LIC RBLB

p35SNOSt NOStpNOSB/K/H

B/K/H

LIC RBLB

pMPNOSt NOStpNOS

LIC RBLB

UASNOSt NOStpNOSB/K/H

B/K

LIC RBLB

SV40NOSt NOStpNOS sYFP

LIC RBLB

SV40NOSt pNOS NOStsCFPB/K/H

B/K/H

LIC RBLB

SV40NOSt NOStpNOS tdTomato

K

LIC RBLB

SV40NOSt pNOS NOStGFP GFP GFP

mis

expr

essi

onpr

otei

n lo

calis

atio

nex

pres

sion

ana

lysi

s

LIC RBLB

NOSt NOStpNOSB/K/H GUS

1 (B)2 (K)3 (H)

4 (K)

5 (B)6 (K)

7 (B)8 (K)9 (H)

10 (B)11 (K)12 (H)

13 (B)14 (K)15 (H)

16 (B)17 (K)18 (H)

19 (B)20 (K)21 (H)

22 (B)23 (K)24 (H)

25 (B)26 (K)27 (H)

28 (B)

29 (B)30 (K)31 (H)

32 (B)33 (K)34 (H)

pPLV

Figure 3. Overview of LIC vectors. Indicated are left and right border (LB and RB), NOS promoter and terminator (pNOS and NOSt), resistance genes (B, Basta; K, Kanamycin; H, Hygromycin), nuclear localization signal (SV40), the LIC site (LIC), specific promoters for misexpression (p35S, pRPS5a, pMP and upstream activating sequence or UAS) and fluorescent proteins (GFP, sCFP, sYFP, tdTomato). pPLV numbers are indicated for each vector,as well as Genbank accession numbers.

GenBank

JF909457

JF909458JF909459

JF909460JF909461JF909462

JF909463JF909464JF909465

JF909466JF909467JF909468

JF909485JF909486JF909487

JF909469JF909470JF909471

JF909472JF909473JF909474

JF909475JF909476JF909477

JF909478JF909479JF909480

JF909481

JF909482JF909483JF909484

JF909454JF909455JF909456



bHLH148 bHLH149

bHLH150 bHLH151 bHLH158

bHLH134

bHLH136bHLH161 bHLH166

bHLH147

bHLH159

AT1G74500/bHLH135/TMO7 AT3G47710/bHLH161

AT3G28857/bHLH166 AT5G39860/bHLH136

AT5G15160/bHLH134

AT3G06590/bHLH148

AT3G17100/bHLH147

AT1G09250/bHLH149

AT3G05800/bHLH150

AT2G47270/bHLH151

AT2G43060/bHLH158

AT4G30410/bHLH159

0.1

AIF-clade

TMO7-clade

TMO7 - bHLH135

Figure 4. Overview of promoter expression patterns of a subclade of TMO7 related bHLH transcription factors using the pGIIK-LIC-VP40-3GFP-NOSt vector. The phylogenetic tree shows a subclade of the bHLHtranscription factors based on full-length protein sequences. Branch lengths indicate phylogenetic distances (see scale bar: fraction of deviations). Confocal images of primary root meristems were counter stained using FM4-64 dye (red).



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