RESEARCH ARTICLE

Complex structures of transgene rearrangement implicate novelmechanisms of RNA-directed DNA methylationand convergent transcription

Zheng Liu • Shuangcheng Gao • Shumin Zhang •

Shangjun Yang • Ning Sun

Received: 13 July 2013 / Accepted: 4 September 2013 / Published online: 26 September 2013

� The Genetics Society of Korea 2013

Abstract An RNAi construct for silencing FtsZ gene

functioning on plastid division in higher plants was trans-

formed into tobacco genome. Sequencing of the flanking

sequences of the T-DNA insertion site in a positive trans-

genic plant 2-3, which showed wildtype phenotype,

revealed that the construct had rearranged, resulting in

convergent transcription of nptII gene by 35S promoter and

nos promoter with both transcripts expressing at low level.

Although it is possible that the sense and antisense tran-

scripts can form dsRNA, they did not trigger silencing of

nptII gene derived from a FtsZ silenced plant 2-12 in the

crossed plant 2-392-12. Furthermore, although small

interfering RNA can trigger DNA methylation of the

silencing locus, our investigation revealed that the crossed

plant 2-392-12 showed partial methylation of the non-

silencing transgene locus but full methylation of the non-

rearranged silencing locus, suggesting that the efficiency of

RNA-directed DNA methylation is associated with its

original DNA locus.

Keywords Convergent transcription � RNA-

directed DNA methylation � RNA interference �Transgene rearrangement

Introduction

RNA interference (RNAi), also called posttranscriptional

gene silencing (PTGS), is an evolutionarily conserved

mechanism for silencing gene expression. It is a universal,

highly conserved pathway found in a large variety of

eukaryotic organisms such as plants (Napoli et al. 1990;

Smith et al. 1990; van der Krol et al. 1990), animals (Fire

et al. 1998; Zamore et al. 2000; Elbashir et al. 2001) and

fungi (Cogoni et al. 1996). Initiation of RNAi requires

production of primary double-stranded RNAs (dsRNAs).

There are potentially three different sources in plants: First,

dsRNA may be produced through convergent transcription

or transcription of inverted repeat DNA. Second, a target

sequence may not be transcribed at all, but dsRNA origi-

nates from an unlinked homologous transcribed locus or an

exogenous source. Third, dsRNA may be produced by an

RNA dependent RNA polymerase (RDR) utilizing a single-

stranded RNA template, using either endogenous plant

RDR or viral RDR for replication. These long dsRNAs are

cleaved into 21–25 nucleotide (nt) primary small interfer-

ing RNAs (siRNAs) by the enzyme Dicer (Hannon 2002).

The antisense strand of siRNAs is then incorporated into

the RNA induced silencing complex (RISC) to guide the

mRNA cleavage (Liu et al. 2004; Rivas et al. 2005). These

cleaved transcripts are used as templates by RDR to syn-

thesize long dsRNA which is subsequently diced into

secondary siRNAs. Secondary siRNAs with RISC then

perform amplification of the target mRNA degradation

(Ghildiyal and Zamore 2009). Although high conservation

of RNAi between animals and plants occur, some differ-

ences in siRNA induced silencing have been reported. For

example, only a *21 nt siRNA class is produced in

mammalian cells and in Drosophila embryos, whereas two

distinct classes, i.e. *21 and *24 nt siRNAs are observed

Z. Liu (&) � S. Zhang � S. Yang

College of Life Sciences, Hebei University, Baoding 071002,

Hebei, China

e-mail: liuzhengxp@yeah.net

S. Gao

College of Agronomy, Henan University of Science and

Technology, Luoyang 471003, Henan, China

N. Sun

The Affiliated School of Hebei Baoding Normal,

Baoding 071000, Hebei, China

123

Genes Genom (2014) 36:95–103

DOI 10.1007/s13258-013-0147-8

in plants; secondary siRNAs spread only towards 50

direction in C. elegans, while both 50 and 30 spreading

directions exist in plants (Lu et al. 2004); efficiency of

mRNA cleavage is greatly reduced when mismatches

happened between siRNA and the target mRNA in Dro-

sophila. In contrast, a small number of mismatches can be

allowed in plants (Kusaba 2004).

Extensive support has been gathered indicating that an

RNA-directed DNA methylation (RdDM) mechanism in

plants plays a key role in silencing associated with DNA

methylation (Zhang and Zhu 2012). In Arabidopsis, a

silencer locus generates two classes of small interfering

RNAs, i.e. 21-nt and 24-nt siRNAs respectively. 21-nt

siRNAs are produced by dicer (Dicer-Like 4)-mediated

cleavage of dsRNAs which are synthesized by RDR6 and

are involved in mRNA degradation and translation inhibi-

tion (Parent et al. 2012). In contrast, 24-nt siRNAs which

are generated by Dicer-Like 3 in an RDR2-dependent

pathway are responsible for methylation of the transcribed

region during PTGS (Daxinger et al. 2009). Cytosine

methylation in the symmetric context 50CG30 is observed at

the highest frequency within methylated regions of the

plant genome with the next to 50CHG30. Even methylation

of cytosines in other asymmetric contexts, such as CHH,

can also be found (Law and Jacobsen 2010). However the

mechanism of partial methylation remains unclear.

In eukaryotic genomes, cis-natural sense antisense

transcripts (cis-NATs) are usually produced at the same

genome locus by complementary strands of DNA. It is

estimated that 4–26 % of total protein-coding (PC) genes

are transcribed as cis-NATs in higher eukaryotes (Solda

et al. 2008). For example, in 26,939 annotated genes of

Arabidopsis, 3,027 NATs are co-expressed with their sense

transcripts and both sense and antisense transcripts of

another 7,598 genes are found to be accumulated in spe-

cific tissues (Yamada et al. 2003). There are usually three

types of cis-NATs. In type I, sense and antisense transcripts

are complementary in their 30 ends. In type II, these two

transcripts are complementary in their 50 ends. In type III,

one transcript is entirely homologous to the second tran-

script (Zhan and Lukens 2013). Analysis of cis-NATs in

Arabidopsis showed that they are co-expressed at high

frequency and remarkably broad transcript levels and nat-

ural antisense small RNA (nat-siRNA) production from

cis-NATs is limited, indicating that small RNAs play

limited role in cis-NAT regulation (Zhan and Lukens

2013). It was estimated that cis-NATs exhibit diverse

mechanisms: firstly, some NATs can favor the formation of

dsRNAs, which are then degraded to nat-siRNAs that can

initiate silencing of sense or antisense transcripts; secondly,

convergent transcription of sense and antisense transcripts

can cause transcriptional interference based on the sup-

pressive influence of both transcriptional processes; thirdly,

the local chromatin environment affects the transcriptional

competence of cis-NAT genes, resulting in antagonistic

expression patterns of the two convergently transcribed

genes (Kunova et al. 2012). So, why do some NATs enter

into RNAi pathway, while others do not? We may question

whether co-existence of natural sense and antisense tran-

scripts is related to their special secondary structure, e.g.

lacking stem loop structure, as such structure can induce

silencing efficiently?

Here, we take advantage of a rearranged filamenting

temperature-sensitive mutant Z (FtsZ) transgenic tobacco

(Nicotiana tobacum) plant 2-3, which did not show

silencing phenomena, to study RdDM and convergent

transcription. In this transgenic plant, FtsZ transgenes

contain a unique XhoI site which is sensitive to DNA

methylation. When crossed with a silenced FtsZ transgenic

plant 2-12 to produce the plant 2-392-12, siRNA did not

trigger full methylation of non-silencing transgene locus,

but full methylation of the non-rearranged silencing locus

in the crossed plant 2-392-12. Also, in the rearranged plant

2-3, the Neomycin Phosphotransferase II (nptII) gene was

convergently transcribed by the 35S promoter and nos

promoter respectively. Sense and antisense nptII transcripts

derived from the plant 2-3 co-exist in the crossed plant

2-392-12 and did not induce silencing of nptII transcript

by secondary siRNAs of silencing spread mechanism

derived from the silenced plant 2-12. These results indi-

cated that firstly, there would exist a novel mechanism in

which the aboriginality of the DNA locus can be identified

by RdDM; secondly, stem loop structure would play a key

role in the efficiency of transgene induced silencing.

Materials and methods

Plasmid construction and plant transformation

A pair of primers (TFtsZ for CACCATGGCTACTTGTAC

ATCAGCTG and TFtsZrev CGCCTGCTTGCTTTCGTTG

GCAG) was synthesized to amplify the 50 end (560 bp) of

tobacco FtsZ cDNA according to its sequence from a

database in Gene Bank (AJ311847). CACC was added at

the 50 end of TFtsZfor primers. PCR fragment of FtsZ gene

was directly cloned into pENTR/D-TOPO vector (invitro-

gen) and then cloned to the RNAi construct pK7GW1WG2

by the gateway method to produce the transformation

vector pK7GW1WG2FtsZ (Fig. 1a).

Leaf pieces of sterile tobacco as explants and the

Agrobacterium tumefaciens strain LBA4404, carrying this

destination vector were used for plant transformation. The

overnight transformed A. tumefaciens cell culture was

centrifuged and the cell pellet was evenly resuspended in

MSR3 media (4.2 g l-1 MS salt (Duchefa)/30 g l-1

96 Genes Genom (2014) 36:95–103

123

Sucrose/1 ml l-1 R3 vitamins, pH 5.9) (R3 Vitamins:

1 mg ml-1 Thiamine/0.5 mg ml-1 Nicotinic Acid/0.5 mg

ml-1 Pyridoxine) to A600 \ 0.2. Leaf pieces (1 9 1 cm2)

were transformed by placing into the cell suspension and

agitated gently for 15 min. The explants were put on M1

medium (MSR3 medium with 7 g l-1 Agar/0.9 mg l-1

indole-3-acetic acid (IAA)/1.75 mg l-1 Zeatin) and incu-

bated under white fluorescent light at a photon flux of

27 lE m-2 s-1 with a 16 h light/8 h dark period at

25 ± 2 �C. After 1 day, the leaf pieces were transferred

onto selective M13 plates containing antibiotics (M1

medium with 400 lg ml-1 Amoxycillin Na/K Clavulanate

(Duchefa) and 50 mg l-1 Kanamycin sulphate), and incu-

bated as above. The explants were transferred to fresh M13

media plates every 3 weeks until calli and shoot developed.

Individual shoots from calli were cut and placed in MSR3

pots containing 7 g l-1 Agar/50 lg ml-1 kanamycin sul-

phate/400 lg ml-1 Amoxycillin Na/K Clavulanate to

induce growth of roots. Selected rooting shoots were taken

and grown in compost in a glasshouse.

Southern analysis

Genomic DNA was extracted from young leaves of T2

transgenic homozygous lines using the GenEluteTM Plant

Genomic DNA Miniprep Kit (Sigma). 20 lg DNA was

digested with EcoRI and XhoI overnight at 37 �C and

separated in 0.8 % agarose gels. The DNA was transferred

to Genescreen hybridization membrane (Perkin Elmer Life

Science) using a standard neutral transfer procedure

(Sambrook et al. 1989). Prehybridization was carried out at

65 �C for 1 h in 59 SSPE/59 Denhardt’s solution/1 %

sodium dodecyl sulphate (SDS)/100 lg ml-1 sheared and

denatured salmon sperm DNA. Hybridization by adding

DNA probe was carried out at 65 �C overnight, followed

by washes with 29 SSC/0.1 % SDS and 0.29 SSC/0.1 %

SDS at 65 �C. Signals were detected using autoradio-

graphic film (KODAK X-OMAT AR).

RNA extraction and northern analysis

RNA was extracted from leaves using methods similar to

the description by Han and Grierson (2002). About 3–5 g

of leaf tissue was frozen in liquid nitrogen and ground to a

fine powder in a mortar. RNA extraction buffer (50 mM

Tris ± HCl, pH 8.3, 1 % triisopropylnaphthalene sul-

phonic acid (Na? salt), 6 % 4-aminosalicylic acid, 5 %

phenol mix (mixture of 50 g phenol, 7 ml m-creosol and

0.05 g 8-hydroxyquinoline in 15 ml water) was mixed with

the powder on ice at a ratio of 1 ml to 1 g FW. And then

the frozen slurry was extracted twice with phenol/chloro-

form. The nucleic acids were precipitated by addition of 1

volume isoproponal and 0.1 volume 3 M sodium acetate

(pH 5) and refrigeration at -20 �C overnight. After cen-

trifugation at 13,0009g for 30 min the pellet was washed

with 70 % ethanol and redissolved in 1 ml of water. An

equal volume of 8 M LiCl were added and mixed. After

precipitation at -20 �C for 3 h followed by centrifugation

(21,0009g, 4 �C) for 30 min, the pellet was dissolved in

water and treated with RNAase-free DNAase. They were

extracted with phenol/chloroform once, precipitated and

redissolved in 50 ll water. RNA was separated on a

Pnos attB1

P35S RBFtsZa

XhoI

LB

Tnos

nptII T35S

attB1

FtsZb

attB2

intronXhoI

LB

Tnos part

P35S

attB1

FtsZa

attB2

intron

Tnos part

nptII

XhoI

Pnos

T35S

attB1

FtsZb

XhoI

A

B

Fig. 1 RNAi construct containing 50 FtsZ transgene for transforma-

tion and its rearranged form in line 2-3 plant. a In RNAi construct

pK7GW1WG2FtsZ, there is only one single XhoI site in each FtsZ

transgene, which can give 1.3 kb band when XhoI cut this construct if

there is no DNA methylation happens. EcoRI did not cut this

construct. The first FtsZ transgene was put in antisense orientation

and the second one in sense orientation when they are transcribed by

35S promoter, therefore to form double stranded RNA to enter RNAi

pathway. b The RNAi construct pK7GW1WG2FtsZ was rearranged

when it was transformed to line 2-3 plant. When 35S promoter

transcribes the new construct, it will give two antisense FtsZ

transcripts and one antisense nptII. Nos promoter transcribes one

sense NptII. When XhoI cut this rearranged construct, it gives 2.8 kb

band

Genes Genom (2014) 36:95–103 97

123

25 mM sodium phosphate (pH 6.5)/3.7 % formaldehyde/

1.0 % agarose gel and blotted to Genescreen hybridization

membrane as described by Hamilton et al. (1998). Prehy-

bridization was carried out at 42 �C in 50 % formamide/

1 % SDS/1 M NaCl/10 % dextran sulphate/100 lg ml-1

sheared and denatured salmon sperm DNA. Hybridization

was carried out at the same temperature by using DNA

probes. The filters were washed in 29 SSC/0.1 % SDS and

0.19 SSC/0.1 % SDS at 65 �C.

Extraction and detection of small RNAs

Small RNAs were extracted and transferred to Hybond-Nx

membrane (Amersham Pharmacia Biotech) as described

previously (Hamilton and Baulcombe 1999). The initial

steps for small RNA extraction were the same as those

described above for total RNA extraction. After the first

ethanol precipitation, the pellet was re-dissolved in 2 ml

water. High molecular weight nucleic acids were removed

by precipitation with an equal volume of 20 % PEG8000/

1 M NaCl and small RNAs were enriched by ion-exchange

chromatography using a Qiagen-tip 20 (Qiagen). The small

RNAs were precipitated with 3 volumes of 100 % ethanol

and the resulting pellet was redissolved in formamide.

Small RNAs were separated through 15 % polyacrylamide/

7 M urea/0.59 tris–borate EDTA gel, transferred onto

Hybond Nx filters by electrophoretic transfer at 250 mA for

30 min using Bio-Rad Trans-BlotTM (Bio-Rad) and cross-

linked by UV (1.2 9 105 J) using a Stratalinker� (Strata-

gene). Prehybridization was performed in 40 % formam-

ide/7 % SDS/0.3 M NaCl/0.05 M Na2HPO4–NaH2PO4

(pH 7)/19 Denhardt’s solution/100 lg ml-1 sheared and

denatured salmon sperm DNA for 30 min at 30 �C.

Hybridization was in the same solution for 16 h at 30 �C

and the filters were washed with 29 SSC/0.2 % SDS at

50 �C for 3 9 10 min.

Hybridization probes

All probes were cloned, fully sequenced and gel purified

prior to use. DNA probes were random prime labelled with

[a-32P] dCTP using a RediprimeTM II (Amersham, UK)

kit. For southern analysis, the DNA probes corresponding

to 35S promoter and intron respectively were derived from

the plasmid pK7GW1WG2. The primers (50-TTGCTTTGA

AGACGTGGTTG-30 and 50-CGATTCAAGGCTTGCTTC

AT-30) were used to amplify 35S promoter as probe and the

primers (50-TCAAGCTGACCTGCAAACAC-30 and 50-TG

CCTCTTCTTACGGCTTTC-30) were used for intron

probe. The DNA probe used for the detection of endoge-

nous FtsZ transcript expression was made from the 30 half

of the FtsZ cDNA and did not overlap with the FtsZ

fragment used for RNAi purpose. The primers TFtsZ2f (50-

AAGGGATTGCTGCTTTGAGA-30) and TFtsZ2r (50-TG

AACCACCTTCCAAAAAGG-30) were used for this pur-

pose. The DNA probe used for detection of nptII transcript

expression was made from the nptII gene of pK7GW1WG2

plasmid by PCR using primers 50-TCAAGCTGACCTGCA

AACAC-30 and 50-TGCCTCTTCTTACGGCTTTC-30. An

antisense-specific 32P-UTP-labelled riboprobe correspond-

ing to the 50 end (560 bp) of tobacco FtsZ cDNA for small

RNA detection was generated using an in vitro transcrip-

tion system (Promega) and as described by Smith et al.

(1988). The riboprobe was treated by RNase-free DNase

(Promega) to remove the DNA template. To hydrolyse the

riboprobe to an average size of 50 nt, 200 ml of alkaline

buffer (120 mM Na2CO3, 80 mM NaHCO3) was added to

10 ml riboprobe and incubated at 60 �C for 3 h. The

primers TFtsZf, TFtsZr, TFtsZ2f, TFtsZ2r mentioned above

were used as markers to estimate the sizes of small RNA

molecules. The size of TFtszf oligonucleotide is 26 nt.

TAIL-PCR amplification

TAIL-PCR was conducted as described by Singer and

Burke (2003) to identify to flanking sequences of the

T-DNA insertion site in the non-silenced transgenic plant

2-3. 6 AD primers, for which sequences are the same as

they described, were used for three rounds of PCR as

described in this paper. PCR cycles were also set as they

described.

Results and discussion

Identification of a rearranged but non silenced FtsZ

transgene construct in tobacco

FtsZ RNAi construct (Fig. 1a) was used for A. tumefaciens

mediated tobacco transformation. Southern analysis of 16

putative FtsZ transgenic lines showed that although the

plant 2-3 is transgenic (Fig. 2a, b), this plant showed a

normal chloroplast phenotype (Fig. 3b) as that in wild type

plant (Fig. 3a), rather than one big single chloroplast as

seen in silenced plants (Fig. 3c, d). Northern hybridization

by using 30 FtsZ cDNA as a probe also showed expression

of the endogenous FtsZ transcript in the plant 2-3 (Fig. 2c).

In order to know why silencing did not happen in this plant,

even though it is transgenic, TAIL-PCR was conducted to

check the T-DNA insertion site and its flanking sequences.

The work showed that the fragment containing the 35S

promoter, one FtsZ transgene and intron was inserted into

the nptII terminator by an unknown mechanism (Fig. 1b).

When the 35S promoter transcribes the construct, it gives a

transcript containing two antisense FtsZs and one antisense

98 Genes Genom (2014) 36:95–103

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nptII. The Nos promoter transcribes from the opposite

orientation to give one sense nptII. Southern analysis

(Fig. 2a, b) showed that the plant 2-12 is transgenic with

single copy insertion. This line was a FtsZ silenced plant

with one big chloroplast per cell (Fig. 3c) and no endog-

enous FtsZ RNAs were detected (Fig. 2c). The plant 2-3

and the plant 2-12 were crossed to study RdDM and con-

vergent transcription. This crossed plant 2-392-12 also

showed one big chloroplast per cell (Fig. 3d) as in line

2-12, indicating that FtsZ gene was silenced.

Rearrangement of introduced DNA often occurs when

performing plant transformation. It was thought that

homologous recombination, synthesis-dependent mecha-

nism(s) and concerted action of several DNA break-repair

mechanisms were involved in transgene locus rearrange-

ment (Svitashev et al. 2002). Although the patterns of the

rearrangement were diverse, the change from sense to

antisense transgene was usually observed after transfor-

mation. For example, rearrangement of sense transgene to

antisense orientation resulted in the production of aberrant

Fig. 2 Southern, northern blotting and small RNAs detection of FtsZ

transgenic plants. a and b Southern blotting by using 35S promoter

and intron respectively derived from RNAi construct pK7GW1WG2

as probes. Line 2-3 and line 2-12 showed one single positive band

which means single insertion of the construct in plant genome.

c Northern blotting by using 30 FtsZ cDNA as probe to detect

endogenous FtsZ expression. Line 2-3 showed expression of endog-

enous FtsZ transcript, however, line 2-12 did not show expression of

endogenous FtsZ transcript. d Small RNAs detection. Small RNAs

can not be detected in line 2-3, indicating that RNAi did not happen,

whereas, small RNAs can be detected in line 2-12, indicating that

RNAi occurred. The primers TFtsZf, TFtsZr, TFtsZ2f, TFtsZ2r were

used as markers to estimate the sizes of small RNA molecules.

Because the probe for small RNAs detection was chosen from the 50

region of FtsZ cDNA in an antisense orientation, only 26 nt TFtsZf

oligonucleotides can be hybridized

Genes Genom (2014) 36:95–103 99

123

RNAs which triggered RNA silencing (Morino et al. 2004).

In this study, our plant 2-3 also contained the rearrange-

ment of the 35S promoter and one transgene FtsZ which

resulted in the ntpII gene being transcribed in a convergent

fashion to give a sense transcript from the Nos promoter

and an antisense transcript from the 35S promoter

(Fig. 1b). The rearrangement of the transgene FtsZ moti-

vated us with RdDM study and the convergent transcription

of the nptII gene provided us with an opportunity for fur-

ther understanding on silencing related phenomena.

The rearranged FtsZ construct was only partially

methylated in the crossed plant 2-392-12

Plant total DNA was digested by EcoRI and XhoI. EcoRI

does not cut within the construct inserted into the plant

genome, whereas XhoI can cut inside the FtsZ transgene

fragment to give a 1.3 kb fragment. Due to the rearrange-

ment that transferring the fragment of the 35S promoter,

the FtsZ transgene and intron to the site inside the nptII

terminator, a 2.8 kb fragment should result when DNA of

the plant 2-3 is cut by XhoI (Fig. 4).

DNA methylation is associated with RNA silencing.

Methylation of promoter regions interferes with the tran-

scription of genes, resulting in transcriptional silencing,

whereas methylation of the coding region does not stop

transcription. siRNAs play a key function in silencing

related DNA methylation (Zhang and Zhu 2012). Here we

have found direct evidence that RNAi caused by transgene

dsRNAs is directly associated with the transgene 50CG30

methylation in the higher plant tobacco. The XhoI site

(CTCGAG) is sensitive to DNA methylation and there is

one single XhoI site within the FtsZ transgene region

(Fig. 1). In the line 2-3, the transgene DNA can be cut by

XhoI to get a 2.8 kb band as expected, indicating that no

DNA methylation happened in this construct (Fig. 4).

Assays for small RNAs also indicated that no siRNA, the

trigger of RdDM, is produced in this plant (Fig. 2d). On the

other hand, in the plant 2-12, transgene DNA could not be

cut by XhoI, showing a [20 kb band (Fig. 4) and this is

accompanied by detectable siRNAs (Fig. 2d). More inter-

estingly, when the siRNAs were introduced into the non-

silenced plant 2-3 by crossing with the silenced 2-12 plant

(the crossed plant 2-392-12), southern hybridization

Fig. 3 Phenotypes of tobacco

FtsZ non-silenced and silenced

transgenic plants. Under

microscopy, the wildtype

(a) and line 2-3 plant (b)showed the a lot of small

chloroplasts in a cell, whereas

line 2-12 (c) and crossed line

2-392-12 (d) showed a single

big chloroplast phenotype in a

single cell. Scale bars are

10 lm

100 Genes Genom (2014) 36:95–103

123

(Fig. 4a) showed one band of the same size as line 2-12 and

three additional bands bigger than that of plant 2-3. The

biggest band in the crossed plant 2-392-12 should be the

one from the plant 2-12. The other three additional bands

indicate partial methylation of the 2-3 transgene construct;

this is also indicated by the fact that in the crossed plant

2-392-12, the 2.8 kb band (Fig. 4b) disappeared. Of these

three bands, the biggest band is likely to be a result of the

full methylation of both XhoI sites located within the two

FtsZ transgenes in the rearranged construct, whereas the

other two bands indicate that only one XhoI site is meth-

ylated, leaving another XhoI site unmethylated (Fig. 4a).

This indicated that the efficiency of DNA methylation in

the plant 2-3 was lower than that of the plant 2-12 DNA. It

seems that siRNA would have an affect on the pattern or

level of DNA methylation. siRNAs can induce DNA

methylation of their original DNA efficiently. However,

this efficiency drops when they target an exogenous DNA

locus derived from the plant 2-3, although the DNA

sequence is the same. It was concluded that splicing related

proteins, e.g. exon junction complex, played a key role on

translation, surveillance and localization of the spliced

mRNA. These proteins first deposited on mRNA during

splicing to provide a position-specific memory of the

splicing event, and then transported into the cytoplasm to

regulate mRNA expression at post-transcriptional level

(Chorev and Carmel 2012). Therefore, it is reasonable to

hypothesize that both the original DNA hairpin locus and

the subsequent siRNA produced were deposited by some

specific proteins for memory during RNAi. When siRNA

entered into the nucleus, the proteins it bonded with could

specifically recognize the proteins which deposited on the

original DNA locus. Thus, an accurate feedback loop

between the siRNA and its original DNA locus were

formed for efficient RdDM. However, a different mecha-

nism would exist when the siRNA targeted to the other

DNA locus with the same DNA sequence as that of the

original one, exhibiting low efficiency of RdDM.

Convergent transcription of nptII gene in the rearranged

FtsZ transgenic plant did not reach silencing threshold

In order to test if convergent transcription without a stem

loop structure could induce silencing efficiently, 30 lg of

total RNA from wt, the plants 2-3 and 2-12, and the crossed

plant 2-392-12 was used for Northern hybridization using

the nptII gene as a probe (Fig. 5). The non-silenced plant

2-3 showed a faint band of about 3 kb, which is the tran-

script containing one antisense FtsZ RNA, intron, antisense

nptII, and another antisense FtsZ RNA presumably result-

ing from transcription by the 35S promoter. Compared with

the plant 2-12 and crossed plant 2-392-12, which have

sense nptII bands at \1 kb, the sense nptII RNA in the

plant 2-3 showed a larger but fainter band (about 1.5 kb).

This may be due to the construct rearrangement site in the

plant 2-3 being within nptII nos terminator. When tran-

scribing the sense nptII gene, RNA polymerase could read

through it and go further beyond due to the separation of

nos terminator into two parts resulting in inefficient tran-

scriptional termination. Also, because the 35S promoter

and nos promoter read nptII gene in a convergent orien-

tation, RNA polymerases may block or inhibit each other

by the mechanism of RNAi (Shearwin et al. 2005). Such

inhibition could be the reason that the expression levels of

both genes were significantly lower than that in the plant

2-12. We also found that although in the crossed plant

2-392-12, 3 kb band (containing two antisense FtsZ and

one antisense nptII RNA) and 1.5 kb band (containing one

sense FtsZa and one sense nptII RNA) were degraded by

the siRNAs of FtsZ derived from the plant 2-12, the tran-

script of the sense nptII gene (\1.0 kb) derived from the

Fig. 4 FtsZ plants southern hybridization cut by EcoRI and XhoI.

a 20 lg total genome DNAs quantified by Nanodrop Spectropho-

tometer were completely digested by EcoRI and XhoI and were

loaded on 0.8 % agarose gel. Southern analysis using the intron probe

showed three additional bigger bands in the crossed 2-392-12 plant

compared with that in the 2-3 plant in which there was only one

2.8 kb band. However, in the 2-12 plant it gave[20 kb band instead

of hypothesized 1.3 kb band. This demonstrated that full methylation

happened in the 2-12 plant, whereas in the crossed plant only partial

methylation can happen when transferring RNAi pathway into it by

crossing between the not silenced plant 2-3 and the silenced plant

2-12. b Hypothesized southern picture, in that 2-12 plant should give

1.3 kb band by XhoI digestion and 2-392-12 crossed plant should

give 1.3 and 2.8 kb bands if no methylation happened on XhoI site

located within FtsZ transgene

Genes Genom (2014) 36:95–103 101

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plant 2-12 was not degraded by RNAi mechanism (Fig. 5).

One possibility is that it was reported by crossing and

grafting assays that silencing can spread along the target

gene to up- or down-stream regions (Garcia-Perez et al.

2004). This means that siRNAs of nptII derived from the 3

and 1.5 kb RNAs, which contain antisense and sense nptII,

could be produced in the crossed plant by this mechanism.

However in our assays, such nptII silencing signals did not

induce, or not efficiently induce, silencing of the sense

nptII RNA derived from the plant 2-12 (a strong \1 kb

band existed in the crossed plant 2-392-12, Fig. 5). As

transcription of the 3 and 1.5 kb RNAs derived from the

plant 2-3 was low, there would be insufficient siRNAs of

nptII produced by the silencing spread mechanism to

trigger silencing of the sense nptII genes derived from the

plant 2-12. In other words, siRNAs of nptII derived from

the plant 2-3 were produced at a concentration lower than

the threshold and as a result, silencing of sense nptII genes

of the plant 2-12 was not initiated. Another possibility is

that convergent transcription may produce sense and anti-

sense transcripts, which would form double stranded RNAs

for silencing (Lechtenberg et al. 2003). As intron splicing

could facilitate the formation of dsRNAs and silencing

(Smith et al. 2000), it is possible that without a loop

structure, the sense and antisense nptII did not form stable

dsRNAs. Thus siRNAs of nptII would not be produced at

all or they were produced in much less degree leading to

inefficient RNA silencing. By combining these data, we

have demonstrated that the convergent transcription nptII

gene without a loop structure did not reach the threshold of

RNA silencing. It should be noted that not all cis-NATs in

plants generate small RNAs. For examples, only 179

among 994 cis-NATs in soybean were known to produce

small RNAs (Hu et al. 2013); in Arabidopsis, although

some natural antisense transcript microRNAs (nat-miR-

NAs) overlop the 30UTR of other protein coding genes,

they did not target them for cleavage (Fahlgren et al. 2007;

Rajagopalan et al. 2006; Sunkar and Zhu 2004). The

mechanism explaining why they did not enter RNAi

pathway and their biological functions remain elusive. Is it

possible that the lack of a stem-loop structure was a shared

character among these NATs? As a prerequisite to answer

this question, it is necessary to analyze statistically the ratio

of cis-NATs without a loop to all NATs and investigate the

secondary structures of those cis-NATs which did not

trigger cleavage of their target mRNAs.

In conclusion, by analyzing a rearranged FtsZ transgene

construct, we have found that efficiency of RdDM is

associated with its original DNA locus, and convergent

transcription can not induce silencing efficiently, probably

due to low expression of both genes or lack of a stem loop

structure.

Acknowledgments We thank the Scientific Research Foundation

for the Returned Overseas Chinese Scholars by State Education

Ministry of China (No. 2011-1139), the Talent Introduction Project of

Hebei University (No. 2010-185) for funding.

Conflict of interest The authors declare no conflict of interest.

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