ORIGINAL PAPER

Design of artificial miRNA for redundant silencing of BrassicaSHP1 and SHP2: transient assay-based validation of transcriptcleavage from polyploid Brassicas

Priyanka Dhakate • S. M. Shivaraj •

Anandita Singh

Received: 24 October 2013 / Revised: 10 February 2014 / Accepted: 10 May 2014

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2014

Abstract Asynchronous pod-shatter results in significant

yield losses in canola. SHATTERPROOF1 (SHP1) and

SHATTERPROOF2 (SHP2) regulate pod shatter in

Brassicaceae. This study describes the potential of an

artificial RNA (amiRNA)-based approach for redundant

silencing of SHP1/SHP2 homologs in Brassicas for intro-

ducing shatter resistance. After surveying the natural var-

iation in Brassica SHP homologs, amiR-bnashp1 and

amiR-bnashp2 were designed and processed from miR319a

precursor in N. benthamiana. Analysis of thermodynamic

interaction between amiRNAs and SHP1/SHP2 homologs

predicted high binding energy for amiR-bnashp2 [total free

energy of binding (TFEB): -23.54 kcal/mol] and not for

amiR-bnashp1 (TFEB: 6.01 kcal/mol). As predicted, agro-

co-infiltration assay validated amiR-bnashp2-mediated

cleavage of SHP1 and SHP2 homologs from B. napus,

B. juncea and A. thaliana, while amiR-bnashp1 failed to

direct cleavage of these homologs. In conclusion, for trait

manipulation in polyploid genomes deployment of amiR-

NAs is suggested to silence redundant genes. Furthermore,

an a priori knowledge on transcriptome potential of gen-

ome concerned is a prerequisite for predicting efficiency

and specificity of target selection by amiRNA(s).

Keywords miRNA � Natural variation � N. benthamiana

assay � Pod-shatter resistance � Polyploid genome � Trait

manipulation

Introduction

Synchronized seed dispersal is an important agronomic trait.

In crops like rapeseed, asynchronous pod-shatter results in

tremendous yield losses (MacLeod 1981). Genetic modifi-

cations permit introduction of shatter resistance in crops to

curb such losses. In Arabidopsis, SHATTERPROOF1

(SHP1) and SHATTERPROOF2 (SHP2) are key genes

regulating pod shatter. These genes redundantly govern

lignification of valve margin cells and define dehiscence

zone (Liljegren et al. 2000). Additionally, AGAMOUS (AG)

(Savidge et al. 1995; Flanagan et al. 1996), SEEDSTICK

(STK) (Favaro et al. 2003; Pinyopich et al. 2003), INDE-

HISCENT (IND) (Liljegren et al. 2004) and ALCATRAZ

(ALC) (Rajani and Sundaresan 2001) control valve margin

formation. SHP orthologs have been reported from plant

species including; Solanum esculentum (Vrebalov et al.

2009), Malus domestica (Van der Linden et al. 2002), Pru-

nus persica (Tani et al. 2007), Phaseolus vulgaris (Nanni

et al. 2011), Taihangia rupestris (Lu et al. 2007), Citrus

sinensis (Araujo et al. 2013), Vitis vinifera (Boss et al. 2001)

and Brassica rapa and B. napus (Spence et al. 1996).

Translation of genomic information from Arabidopsis to

crop Brassicas for trait manipulation is challenging as

polyploidy-induced gene amplification and fractionation

events have resulted in variable number of homologs

(Lagercrantz 1998; Lan et al. 2000; Babula et al. 2003;

Osborn 2004; Lysak et al. 2005; Parkin et al. 2005; Sankoff

et al. 2012). Paralogs in polyploid genomes accumulate

mutations owing to relaxed selection pressure on individual

Communicated by E. Schleiff.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11738-014-1589-6) contains supplementarymaterial, which is available to authorized users.

P. Dhakate � S. M. Shivaraj � A. Singh (&)

Department of Biotechnology, TERI University,

10 Institutional Area, Vasant Kunj, New Delhi 110070, India

e-mail: asingh@teri.res.in

123

Acta Physiol Plant

DOI 10.1007/s11738-014-1589-6

copies and are known to have different evolutionary fates

(Adams 2007). Such a parallel may also be speculated for

SHP1 and SHP2 in polyploid Brassicas.

With this background, we present artificial miRNA

(amiRNA)-based approach for down-regulation of SHP1/

SHP2, for achieving pod-shatter resistance. Between

popularly employed RNAi approaches, amiRNAs are

preferred since siRNAs-based intron-hairpin constructs

are associated with widespread off-target silencing (Sab-

lok et al. 2011). In amiRNA technology, principles gov-

erning natural miRNA-based target selection are used by

graphical interfaces such as Web MicroRNA Designer 3

(WMD3) to predict candidate amiRNAs (Ossowski et al.

personal communications). Optimal candidate(s) are

engineered in a native miRNA precursor backbone (pre-

amiRNAs) that processes a mature amiRNA in vivo. The

latter directs cleavage of intended target(s) resulting in

desirable phenotype(s). amiRNAs hence promise efficient

silencing of target genes that are not regulated by natural

miRNAs.

Materials and methods

Plant growth conditions and nucleic acid isolation

B. juncea var. Varuna and B. napus var. GSL1 were grown

under field conditions while Arabidopsis thaliana and

Nicotiana benthamiana were grown in a growth chamber

under continuous light at 22 �C and 65–70 % relative

humidity. Siliques from Brassica and Arabidopsis were

harvested 20 days after pollination (DAP) and flash frozen

in liquid nitrogen. Total RNA from Arabidopsis siliques

was isolated using method described by Meng and Feldman

(2010), while total RNA from Brassica siliques and N.

benthamiana leaves was isolated using TRIzol reagent

(Invitrogen) as per manufacturer’s instructions.

Primer design for retrieval of Brassica orthologs

Arabidopsis SHP1 (AT3G58780) and SHP2 (AT2G42830)

were employed to retrieve Brassica SHP1/SHP2 homologs

from GenBank (http://blast.ncbi.nlm.nih.gov) using BLASTN

with default parameters. B. napus orthologs of SHP1 (Gen-

Bank ID: AY036062) and SHP2 (GenBank ID: EU424342,

EU424343) were employed for designing primers to isolate

orthologs from Indian Brassicas. The oligonucleotide

sequences used were, BnaSHP1_Fwd: 50-ATGGATGAAGG

TGGGAGTAGT-30 and BnaSHP1_Rev: 50-TTAAACAAGT

TGAAGAGGAGGTTG-30; BnaSHP2_Fwd: 50-ATGGAGG

GTGGTGCGAGT-30 and BnaSHP2_Rev: 50-TTAAACAAG

TTGGAGAGGTGGTTG-30. All oligonucleotides were syn-

thesized at MWG Biotech, Munich, Germany.

Cloning and sequence characterization of amiRNA

and SHP1/SHP2 orthologs

For cDNA synthesis, 1 lg of DNaseI (Fermentas)-treated

total RNA was reverse transcribed using RevertAidTM H

Minus first strand cDNA synthesis kit (Fermentas). PCR was

carried out in a 20 ll reaction using 1 ll of template cDNA

with final concentration of 1X reaction buffer, 2.5 mM

MgSO4, 0.2 mM of dNTPs, 0.5 lM of each primer and

1U Pfu DNA polymerase (Fermentas). Thermo-cycling

parameters for amplification of homologs were programmed

as initial denaturation at 95 �C for 2 min followed by 35

cycles of 95 �C for 30 s, 55 �C for 30 s, 72 �C for 60 s fol-

lowed by a final extension at 72 �C for 5 min. The amplicons

were gel-purified, A-tailed, and subsequently ligated

into pGEMR-T Easy vector (Promega). Ligation mixture was

transformed into Escherichia coli DH5a strain. Positive

clones were sequence characterized. Brassica SHP1/SHP2

homologs isolated from B. napus, B. juncea, and A. thaliana

prefixed with a ‘‘Bna’’, ‘‘Bju’’, and ‘‘Ath’’, respectively.

Phylogenetic analysis

Sequences were aligned using Clustal X ver. 2.0

(Larkin et al. 2007). Pairwise alignment scores were cal-

culated using BioEdit ver. 7.0.5.3 (Hall 1999). Phyloge-

netic relationships were analyzed using Bayesian statistical

framework implemented in BEAST (Drummond and

Rambaut 2007). The alignment file in.nex format was used

to generate.xml file through BEAUti ver. 1.6.2 with default

parameters (Hasegawa, Kishino and Yano model of DNA

substitution; Strict clock with rate = 1.0; Tree prior =

Coalescent Tree; Constant Size; Markov chain Monte

Carlo (MCMC) Chain length = 100 00 000). The.xml file

was used as input file in BEAST ver. 1.6.2 to permute trees

which were analyzed through Tree Annotator ver. 1.6.2

(Drummond and Rambaut 2007) and visualized using

FigTree ver. 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/).

Prediction of amiRNAs and pre-amiRNAs design

amiRNAs targeting SHP1/SHP2 homologs were designed

using MicroRNA designer (WMD3) tool (Schwab et al.

2006) that predicts best amiRNA after scanning the

sequence of intended target transcripts (http://wmd3.wei

gelworld.org/cgi-bin/webapp.cgi; Ossowski et al. personal

communications). This tool was originally launched for

A. thaliana but now accommodates [ 30 additional plant

species for which genomes have been fully annotated or

extensive EST information is available. For input of

sequences in WMD3 for amiRNA design, SHP1 and SHP2

homologs isolated from B. napus var. GSL1 were used

individually as input sequences in WMD3 designer keeping

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minimum number of included targets as one, off-target

selection as two and selecting B. napus PUTv173a (PGDB)

genome library. Top ranking candidates, amiR-bnashp1

and amiR-bnashp2, were selected from discrete outputs of

predicted amiRNAs specific to BnaSHP1 and BnaSHP2,

respectively. To replace the native miR319a sequence in

the precursor backbone with amiRNAs, an independent set

of overlapping PCRs were carried out following the pro-

tocol prescribed by WMD3 designer tool (Ossowski et al.

personal communications). To design oligonucleotides for

site-directed mutagenesis, the putative amiRNA sequences

were independently used as inputs in the ‘‘oilgo’’ tool of

WMD. Four oligonucleotide sequences (oligo I–oligo IV)

were designed by WMD3 for each amiRNA. These oli-

gonucleotides were based on amiRNA and amiRNA*

sequences and were engineered into pre-miR319a precur-

sor backbone present in plasmid pRS300. For designing

pre-amiR-bnashp1, following set of oligonucleotides were

used: oligo I: 50-GATATTACACCCGATCCATGCTGTC

TCTCTTTTGTATTCC-30; oligo II: 50-GACAGCATGGA

TCGGGTGTAATATCAAAGAGAATCAATGA-30; oligo

III: 50-GACAACATGGATCGGCTGTAATTTCACAGGT

CGTGATATG-30: oligo IV: 50-GAAATTACAGCCGAT

CCATGTTGTCTACATATATATTCCT-30. For designing

pre-amiR-bnashp2, following oligonucleotides were used:

oligo I: 50-GATTAGTATTGAGTATTAGCTTCTCTCTC

TTTTGTATTCC-30; oligo II: 50-GAGAAGCTAATACTC

AATACTAATCAAAGAGAATCAATGA-30; oligo III:

50-GAGACGCTAATACTCTATACTATTCACAGGTCG

TGATATG-30; oligo IV: 50-GAATAGTATAGAGTAT

TAGCGTCTCTACATATATATTCCT-30. Additionally,

primer A: 50-CTGCAAGGCGATTAAGTTGGGTAAC-30

and primer B: 50-GCGGATAACAATTTCACACAGGAA

ACA G-30 designed on pRS300 plasmid backbone were also

used. The template used for site-directed mutagenesis was

pRS300 that harbors a native pre-miR319a. To replace 21 nt

miR319a and miR319a* with predicted amiRNA and ami-

RNA*, independent PCRs were set up using specific primer

combinations. Briefly, three amplicons were amplified, viz.

‘‘a’’ (using oligos A and IV), ‘‘b’’ (using oligos III and II)

and ‘‘c’’ (using oligos I and B). An overlapping PCR was

employed to fuse amplicons ‘‘a’’ to ‘‘c’’ using oligos A and

B. The resulting amplicon ‘‘d’’ corresponded to amiRNA

and amiRNA* lodged in backbone of pre-miR319a. To

amplify fragments ‘‘a’’ to ‘‘c’’, PCR was carried out in

individual 50 ll reactions using 1 ll of template plasmid

DNA diluted to 1:100 with final concentration of 1X reac-

tion buffer, 0.2 mM of dNTPs, 0.4 lM of each primer and

1U Pfu DNA polymerase (Fermentas). Thermo-cycling

parameters were programmed as initial denaturation at

95 �C for 2 min followed by 24 cycles of 95 �C for 30 s,

50 �C for 30 s for B and 55 �C for ‘‘a’’ and ‘‘c’’ and 72 �C

for 40 s, followed by a final extension at 72 �C for 1 min.

The amplified products were eluted from 2 % agarose gels.

For amplicon fragment ‘‘d,’’ PCR was carried out in indi-

vidual 50 ll reactions using 0.5 ll of amplicons ‘‘a’’, ‘‘b’’

and ‘‘c’’ with final concentration of 1X reaction buffer,

0.2 mM of dNTPs, 0.4 lM of each primer and 1U Pfu DNA

polymerase (Fermentas). Thermo-cycling parameters were

programmed as initial denaturation at 95 �C for 2 min fol-

lowed by 24 cycles of 95 �C for 30 s, 55 �C for 30 s and

72 �C for 90 s, followed by a final extension at 72 �C for

7 min. The amplicons thus obtained were gel-eluted, cloned

in pGEMR-T Easy vector system (Promega) and sequence

characterized (Macrogen, Korea). amiR-bnashp1 and

amiR-bnashp2, and homologs BnaSHP1a, BnaSHP2a,

BnaSHP2b, BjuSHP2a, AthSHP2 and AthSHP1 were sub-

cloned in pCHF3 vector under the control of 35S CaMV

promoter and transformed in A. tumifaciens strain GV3101.

Thermodynamic analyses

Minimum free energy (MFE) that determines stability of

secondary structure of precursor miRNAs was evaluated

for pre-amiRNAs using RNAfold web server (http://rna.

tbi.univie.ac.at/cgi-bin/RNAfold.cgi). Total free energy of

binding (TFEB) defines the interaction between 80-bp

sequence contexts of target site within transcripts with

miRNAs. It was calculated as sum total of energy gained

from duplex formation (EDF), energy lost as opening

energy for the longer sequence (OELS) and opening

energy for the shorter sequence (OESS) using RNAup

(Muckstein et al. 2006) keeping default parameters.

Tobacco transient assays in N. benthamiana

Primary cultures of constructs overexpressing BnaSHP1a,

BnaSHP2a, BnaSHP2b, BjuSHP2a, AthSHP2, AthSHP1,

amiR-bnashp1 and amiR-bnashp2 were set up. Agro-co-

infiltration assays as described by De Felippes and Weigel

(2010) were carried out on young leaves of 4-week-old N.

benthamiana plants. For negative controls, co-infiltrations

were performed by mixing Agrobacterium suspensions over-

expressing intended targets with empty vectors instead

amiRNAs, and Agrobacterium suspensions overexpressing

amiRNAs with empty vector instead of intended targets in a

ratio of 1:1. The suspensions were individually injected into

the abaxial surface of leaves. Leaf samples were harvested

after 72 h of infiltration and stored at -70 �C until further use.

Analysis of amiRNA biogenesis and target cleavage

Total RNA was extracted from leaf samples and used for

cDNA synthesis to simultaneously monitor accumulation

of mature amiRNA and cleaved target. One lg of DNaseI

(Fermentas)-treated total RNA was incubated with 0.5 lM

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of the stem-loop primer at 65 �C for 5 min. For detection

of amiRNA expression, stem-loop primers were designed

as explained by Chen et al. (2005) for amiR-bnashp1

(50-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCA

CTGGATACGACCAGCAT-30), and amiR-bnashp2 (50-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG

GATACGACGAAGCT-30). Further, to validate amiR-

bnashp1 and amiR-bnashp2 expression, second strand

synthesis was carried out using forward primers 50-GCGG

CGGTATTACACCCGATCC-30 and 50-GCGGCGGTTAG

TATTGAGTATT-30, respectively.

Stem-loop-based primers were also adapted for detection

of cleaved product. The amiRNA-binding site and pre-

sumptive cleavage site were in silico mapped on target

transcripts. For designing stem-loop primers for reverse

transcription, the region upstream to the presumptive cleav-

age site was surveyed. These stem-loop primers have typical

44-bp stem-loop backbone (Chen et al. 2005) with 6-bp

sequence at the 30 end that is complementary to the 30 end of

the cleaved transcript. Hence, to reverse transcribe cleaved

transcripts of SHP1/SHP2, stem-loop primers 50-GTCGTA

TCCAGTGCAGGGTCCGAGGTATTCGCACTGGATAC

GACGTATTA-30 and 50-GTCGTATCCAGTGCAGGGTCC

GAGGTATTCGCACTGGATACGACATCCAT-30, respec-

tively, were used. The reaction mixture comprised of 0.5

lM stem-loop primers,1 X RT buffer, 0.25 mM dNTPs, and

200 U of H minus M-MuLV Reverse Transcriptase (Re-

vertAidTM H Minus first strand cDNA synthesis kit, Fer-

mentas). For detection of amiRNA biogenesis and target

cleavage product, reverse transcription was carried out for

30 min at 50 and 45 �C, respectively, followed by incuba-

tion at 16 �C for 30 min. Further, to detect cleaved targets,

forward primers were designed upstream to the cleavage

site on the transcripts, while a universal reverse primer was

used that was designed on the stem-loop. To detect cleaved

SHP1 and SHP2 transcripts by respective amiRNAs (amiR-

bnashp1 and amiR-bnashp2), forward primers 50-ATTCA

GAATTCGAACAGGCA-30 and 50-TGTCTTGTGTGACG

CTGAGG-30 were used, respectively. However, to detect

SHP1 cleaved products by amiR-bnashp2, forward primer

50-ATGGATGAAGGTGGGAGTAGT-30 was employed.

The universal reverse primer designed on the backbone of

the stem-loop 50-GTGCAGGGTCCGAGGT-30 was com-

mon in all the reactions.

Results

Sequence characterization of Brassica SHP1/SHP2

orthologs

Four SHP2 orthologs (BjuSHP2a, BjuSHP2b, BjuSHP2c

and BjuSHP2d) from B. juncea var. Varuna and two

orthologs (BnaSHP2a and BnaSHP2b) from B. napus

var. GSL1 were isolated. In addition, two SHP1 ortho-

logs, BnaSHP1a and BnaSHP1b, from B. napus var.

GSL1 were isolated. Higher polymorphism was observed

in SHP1 compared to SHP2 at both cDNA and protein

level. Pairwise nucleotide sequence identity ranged from

91.4 to 95.7 % and 95.6 to 99.7 %, within Brassica

SHP1 and SHP2, respectively (ESM_1.pdf). However,

between Brassica SHP1 and SHP2, pairwise identity

ranged from 78.2 to 79.6 % (ESM_1.pdf). Table 1

summarizes details of isolated sequences and their pre-

dicted proteins. Two size variants, BnaSHP1a (750 bp)

and BnaSHP1b (747 bp), were obtained sharing 93.2 %

nucleotide identity (ESM_1.pdf), while the deduced

proteins, BnaSHP1a (248aa) and BnaSHP1b (249aa),

share 94.3 % identity (ESM_2.pdf). In contrast to Bras-

sica SHP1, no length variation was observed within

Brassica SHP2 sequences. Deduced Brassica SHP2 (244

aa) shared identity ranging between 98.3 and 99.5 %

(ESM_2.pdf). Comparison of isolated SHP1/SHP2

sequences with reported ones presented conserved

MADS domain and K-box (ESM_3.pdf).

The phylogram revealed close relationship among iso-

lated and reported SHP1/SHP2 sequences with ortholog-

specific grouping of Brassica SHP1 and SHP2 corre-

sponding to clades I and II, respectively (Fig. 1).

Features of designed artificial miRNAs

Several optimal amiRNAs were predicted for BnaSHP2,

whereas none were identified for BnaSHP1. Stem-loop

structures of native miR319a precursor along with amiR-

bnashp1 and amiR-bnashp2 lodged in the miR319a pre-

cursor backbone were analyzed using RNAfold (Zuker and

Stiegler 1981). Modified and native precursors of miRNA

319a were found to share similar fold-back structures and

free energy (DG) implying comparable stabilities. The

MFE of amiR-bnashp1, amiR-bnashp2 and native miRNA

319a were found to be -107.96 kcal/mol, -105.57 kcal/

mol and -112.64 kcal/mol, respectively, (Fig. 2a). The

designed amiRNA precursors were engineered under the

control of 35S CaMV promoter in pCHF3 binary vector

(Fig. 2b).

Hybridization kinetics of amiRNAs

The designed amiRNAs were analyzed for their ability to

redundantly silence SHP1/SHP2 homologs from Brassica

spp. To this effect, binding sites for amiR-bnashp1 and

amiR-bnashp2 were mapped to Brassica SHP1/SHP2

homologs (Fig. 3). amiR-bnashp1 target site was located

at position 586 nt to 606 nt, downstream to K box in all

Brassica SHP1 homologs. Only BnaSHP1a target site

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bore a perfect complimentarity to amiR-bnashp1, while

all other targets harbored mismatches varying from 1 to 5

nt. On the other hand, target site for amiR-bnashp2

mapped to position 294 nt to 314 nt corresponding to

K-Box and was largely conserved. One mismatch was

observed at 7th position of amiR-bnashp2 at its binding

site in all SHP1/SHP2 homologs but BnaSHP2a,

BjuSHP2b and BjuSHP2c. The predicted cleavage site

located between the 11th and 12th position of the target

site was a perfect match across all homologs. Analysis of

variation in the 80-bp sequence context around amiR-

bnashp2-binding site (ESM_3.pdf) in Brassica SHP1/

SHP2 homologs presented a genetic variation ranging

between 6.3–1.3 and 3.8–1.3 % for SHP1 and SHP2

homologs, respectively (Table 2).

To identify potential amiRNA-binding sites in tran-

scripts of un-intended targets of B. rapa and B. napus

background and rule out the possibility of off-target

silencing, mature amiRNA sequences were used as a query

for launching a BLASTn search against the available B.

rapa genome (BRAD database) and B. napus ESTs (Gen-

Bank). No off-targets were detected having less than five

mismatches to the designed amiRNAs (data not shown)

implying that designed amiRNAs were specific to SHP1/

SHP2 homologs of Brassica. The summary of thermody-

namics of interaction between amiRNAs and Brassica

SHP1/SHP2 targets is provided in Tables 3 and 4. TFEB,

determining amiRNA-binding site accessibility of Brassica

SHP1/SHP2 targets, was maximally influenced by EDF.

Energy requirements of amiR-bnashp2 were lower (aver-

age EDF: -29.67 kcal/mol) for binding to its respective

target site in all isolated SHP1/SHP2 homologs as com-

pared to amiR-bnashp1 (average EDF: -8.94 kcal/mol).

Additionally, Brassica SHP1/SHP2 homologs were

predicted to interact more effectively with amiR-bnashp2

(average TFEB: -23.54 kcal/mol) than with amiR-

bnashp1 (average TFEB: -6.01 kcal/mol). This result

suggests an efficient in vivo cleavage of transcripts medi-

ated by amiR-bnashp2 than amiR-bnashp1.

Agro-co-infiltration assays for amiRNA biogenesis

and target cleavage

The efficacy of amiR-bnashp1 and amiR-bnashp2 in

directing cleavage of BnaSHP2a, BnaSHP2b, BjuSHP2a,

AthSHP2, BnaSHP1a and AthSHP1 was empirically vali-

dated though tobacco transient assays. Mature amiRNAs

were detected from all the experimental samples wherein

constructs overexpressing amiR-bnashp1 and amiR-

bnashp2 were infiltrated (Fig. 4a, (ESM_4.pdf) indicating

efficient biogenesis and processing. Amplicons corre-

sponding to cleaved transcripts of expected sizes were

detected when Agrobacterium overexpressing amiR-

bnashp2 was co-infiltrated with Agrobacterium over-

expressing BnaSHP1a, BnaSHP2, BjuSHP2a, AthSHP2,

BnaSHP1a and AthSHP1 (Fig. 4b). No amplicons were

detected when Agrobacterium overexpressing amiR-

bnashp1 was co-infiltrated with Agrobacterium over-

expressing any of the Brassica SHP1/SHP2 homologs (data

not shown). Additionally, no amplicons were obtained in

controls wherein constructs overexpressing either target or

amiRNA were infiltrated with Agrobacterium over-

expressing vector implying efficient detection of only

intended cleaved products (Fig. 4a, c).

Amplicons corresponding to cleaved transcripts of

SHP1/SHP2 were cloned and sequence confirmed. These

aligned with their respective full-length cDNA sequences

with 100 % identity (data not shown).

Table 1 Description of isolated

and reported SHP1/SHP2

orthologs from B. juncea var.

Varuna, B. napus var. GSL1 and

A. thaliana

a Percentage identity was

estimated by aligning SHP1

orthologs from Brassicas and A.

thalianab Percentage identity was

estimated by aligning SHP2

orthologs from Brassicas and A.

thaliana

Gene Homologs (GenBank ID) CDS Protein % Identitya % Identityb

Length (bp) Length (aa) CDS Protein CDS Protein

SHP1 BnaSHP1 (AY036062) 750 249 91.0 92.3 81.9 80.7

BnaSHP1a (JQ973084) 750 249 91.2 90.7 79.6 79.1

BnaSHP1b (JQ973085) 747 248 92.6 93.1 79.2 78.7

AthSHP1 (JQ973091) 747 248 100 100 81.1 83.1

SHP2 BnaSHP2a (EU424342) 735 244 81.0 82.7 90.7 92.3

BnaSHP2b (EU424343) 735 244 80.8 82.7 90.5 92.3

BnaSHP2a (JQ973082) 735 244 80.7 82.7 88.6 92.3

BnaSHP2b (JQ973083) 735 244 80.7 82.7 88.7 92.3

BjuSHP2a (JQ973089) 735 244 80.8 82.7 88.6 92.3

BjuSHP2b (JQ973088) 735 244 80.8 82.3 88.0 91.9

BjuSHP2c (JQ973087) 735 244 80.4 82.7 88.3 92.3

BjuSHP2d (JQ973090) 735 244 78.4 82.7 87.6 92.3

AthSHP2 (JQ973092) 741 246 81.1 83.1 100 100

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Discussion

The present study describes a potential of an artificial

miRNA-based strategy to achieve redundant silencing of

SHP1/SHP2 homologs in polyploid genomes of Brassicas.

Herein, two amiRNAs, viz. amiR-bnashp1 and amiR-

bnashp2 were designed and tested for efficient biogenesis

and cleavage of targets. Based on predictions of thermo-

dynamics and in vivo interactions between amiRNAs and

multiple SHP1/SHP2 targets, we prescribe amiR-bnashp2

as a candidate to engineer pod-shatter resistance in crop

Brassicas.

As a first step, diversity in homologs of SHP1/SHP2 in

Indian cultivars of B. napus and B. juncea was analyzed

to predict efficacy of amiRNA(s)-based silencing in

respective genetic backgrounds. Variation was uncovered

in SHP1/SHP2 homologs isolated from B. juncea and B.

napus. Observed sequence variation within SHP1/SHP2

homologs implicated their origin from multiple genomic

loci. Nevertheless, conservation in the domain structure of

SHP1/SHP2 homologs coupled with overall sequence

identity indicated that these redundantly control pod shat-

ter. Increased sequence variation in SHP1 compared to

SHP2 suggested purifying selection on Brassica SHP2.

Such differential selection pressures on paralogs leading to

multiple evolutionary fates have been reported in polyploid

genomes by several workers (Veitia 2005; Otto and

Whitton 2000). Phylogenetic analysis of SHP1/SHP2

homologs revealed an ortholog-specific clustering sug-

gesting that speciation preceded gene duplication. The

BnaSHP1 (AY036062)

BnaSHP1a (JQ973084)

BnaSHP1b (JQ973085)

BnaSHP2a (EU424342)

BnaSHP2b (EU424343)

BnaSHP2a (JQ973082)

BnaSHP2b (JQ973083)

BjuSHP2a (JQ973089)

BjuSHP2b (JQ973088)

BjuSHP2c (JQ973087)

BjuSHP2d (JQ973090)

AthSHP1 (JQ973091)

AthSHP2 (JQ973092)

0.02

Fig. 1 The phylogram generated using Bayesian methods depicts ortholog-specific grouping of SHP1 and SHP2 from B. juncea, B. napus, and

A. thaliana. Homologs from A. thaliana form distinct out-groups

Acta Physiol Plant

123

subclades, however, depicted clustering of homologs

derived from homeologous blocks of the genome. The

Brassica and Arabidopsis ancestral split (Koch et al. 2001;

Lysak et al. 2005; Ziolkowski et al. 2006) is reflected by

Brassica and Arabidopsis homologs forming distinct

clusters.

CAAACACACGCUC

GGA

CGC

AUA

UU

AC

ACAUG

UUC

AU A

CAC

U UA

AU

AC

U CG

CU

GU

UU

UG A A U

UG

AU

GU

UU

UA

GGAAUAUAU

AUG

U AGACA

ACAUGGAUCGG

CUGUAAU

UUCA

CAGGU

CGUGA

U AUGAUUCAAUU

AG

CU

UCCG

AC

UCAUUCAU

CCA

AA

UA

CCGAGUCG

CCAAA

A U U CAAAC

UAG

ACUCGU

UA

AA

UG

AAUGAAUGA

UG

CGGU

AGA

CA

AAUUGGAUCAU U

GAU

UCU

CUU

UGAU

AUUACA

CCCGAUCCAUG

CUGUCU C

UCU

UUU

GUAUUCCA

A U UUUC

UUG A U U A AUC

U U UCC

UG C A C AAA

AACAUGCUUGAUCC

AC

UA

AG

UGACAU

AU

AUG

C UGC

CUUCG

UA

UA

UA

UAG

UUCUG

GUAAA

AUUAA

CAU

UUUGG

GUUUAUCU

UUA

UUU

AA

GG

CA

UC

GCCAUG

5’

3’

*A

NRi

ma

5’

3’

AN

Rima

CAAACACACGCUC

GGA

CGC

AUA

UU

AC

ACAUG

UUC

AU A

CAC

U UA

AU

AC

U CG

CU

GU

UU

UG A A U

UG

AU

GU

UU

UA

GGAAUAUAU

AUG

U AGAG

AGAGCUUCCUU

GAGUCCA

UUCA

CAGGU

CGUGA

U AUGAUUCAAUU

AG

CU

UCCG

AC

UCAUUCAU

CCA

AA

UA

CCGAGUCG

CCAAA

A U U CAAAC

UAG

ACUCGU

UA

AA

UG

AAUGAAUGA

UG

CGGU

AGA

CA

AAUUGGAUCAU U

GAU

UCU

CUU

UGAU

UGGACUG

AAGGGAGCUCC

CUCU C

UCU

UUU

GUAUUCCA

A U UUUC

UUG A U U A AUC

U U UCC

UG C A C AAA

AACAUGCUUGAUCC

AC

UA

AG

UGA

CAU

AU

AUG

C UGC

CUUCG

UA

UA

UA

UAG

UUCUG

GUAAA

AUUAA

CAU

UUUGG

GUUUAUCU

UUA

UUU

AA

GG

CA

UC

GCCAUG

5’

3’

*A

NRi

ma

5’

3’

AN

Rima

CAAACACACGCUC

GGA

CGC

AUA

UU

AC

ACAUG

UUC

AU A

CAC

U UA

AU

AC

U CG

CU

GU

UU

UG A A U

UG

AU

GU

UU

UA

GGAAUAUAU

AUG

U AGAGA

CGCUAAUACUC

UAUACUA

UUCA

CAGGU

CGUGA

U AUGAUUCAAUU

AG

CU

UCCG

AC

UCAUUCAU

CCA

AA

UA

CCGAGUCG

CCAAA

A U U CAAAC

UAG

ACUCG

UUA

AA

UG

AAUGAAUGA

UG

CGG

UAGA

CA

AAUUGGAUCAU U

GAU

UCU

CU

UUGAU

UAGUAU

UGAGUAUUAGCU

UCUCU C

UCU

UUU

GUAUUCCA

A U UUUC

UUG A U U A AUC

U U UCC

UG C A C AAA

AACAUGCUUGAUCC

AC

UA

AG

UGA

CAU

AU

AUG

C UGC

CUUCG

UA

UA

UA

UAG

UUCUG

GUAAA

AUUAA

CAU

UUUGG

GUUUAUCU

UUA

UUU

AA

GG

CA

UC

GCCAUG

5’

3’

AN

Rima

5’

3’

*A

NRi

mamiR319a

ΔG = -112.64 kcal/mol ΔG = -107.96 kcal/molamiR-bnashp1 amiR-bnashp2

ΔG = -105.57 kcal/mol

ter

BamHI

35S

CamV

KpnI

5’-UUAGUAUUGAGUAUUAGCUUC-3’

3’-UAUCAUAUCUCAUAAUCGCAG-5’ amiR-bnashp2*amiR-bnashp2

pre-amiR-bnashp2

a

b

5’-UAUUACACCCGAUCCAUGCUG-3’

3’-UUAAUGUCGGCUAGGUACAAC-5’ amiR-bnashp1*

amiR-bnashp1

ter

BamHI

35S

CamV

KpnI

pre-amiR-bnashp1

Fig. 2 Fold-back structures of amiR-bnashp1 and amiR-bnashp2

precursors. a Minimum free energy (MFE)-based secondary struc-

tures of native miR319a, amiR-bnashp1 and amiR-bnashp2. The

complementary regions formed between amiRNA/amiRNA* on

precursor are highlighted. b Scheme illustrating engineering of

amiR-bnashp1 and amiR-bnashp2 overexpression constructs. amiR-

NAs were cloned under a 35S CaMV promoter between KpnI and

BamHI sites of pCHF3

MADS superfamily

1 125 250 375 500 625 736

DNA binding site K-box superfamily

314294 586 606

5’-GAAGCUAAUACUCAAUACUA-3’

5’-GAAGCUAAUACUCAGUACUA-3’

5’-GAAGCUAAUACUCAAUACUA-3’

5’-GAAGCUAAUACUCAGUACUA-3’

5’-GAAGCUAAUACUCAAUACUA-3’

5’-GAAGCUAAUACUCAAUACUA-3’

5’-GAAGCUAAUACUCAGUACUA-3’

Predicted site for amiR-bnashp2

BnaSHP2a

BnaSHP2b

BjuSHP2a

BjuSHP2b

BjunSHP2c

AthSHP2

BjuSHP2d 5’-GAAGCUAAUACUCAGUACUA-3’

5’-GAAGCUAAUACUCAGUACUA-3’BnaSHP1a

BnaSHP1b 5’-GAAGCUAAUACUCAGUACUA-3’

AthSHP1 5’-GAAGCUAAUACUCAGUACUA-3’

Predicted amiR-shp2 3’-CUUCGAUUAUGAGUUAUGAUU-5’

Predicted site for amiR-bnashp1 5’-CAGCAUGGAUCGGGUGUAAU-3’

BnaSHP1a 5’-CAGCAUGGAUCGGGUGUAAU-3’

5’-CAGCAGGAAUCGAGUGUGAU-3’AthSHP1

Predicted amiR-shp1 3’-GUCGUACCUAGCCCACAUUAU-5’

BnaSHP1b 5’-CAGCAGGAUUCGAGUGUAAU-3’

BnaSHP2a

BnaSHP2bBjuSHP2a

BjuSHP2b

BjunSHP2c

BjunSHP2d

5’-CAGCAGGAAGCGAGUGUGAU-3’

5’-CAGCAGGAAGCGAGUGUGAU-3’5’-CAGCAGGAAGCGAGUGUGAU-3’

5’-CAGCAGGAAGCGAGUGUGAU-3’

5’-CAGCAGGAACCGAGUGUGAU-3’

5’-CAGCAGGAACCGAGUGUGAU-3’

AthSHP2 5’-CAACAAGAAUCGAGUGUGAU-3’

Fig. 3 Mapping of amiRNA- binding sites on SHP1/SHP2 homologs.

amiR-bnashp1 and amiR-bnashp2 target sites, indicated with gray

boxes, were mapped on SHP1/SHP2 homologs. MADS and K-box

domains are indicated with black boxes. Target sites were aligned to

their respective amiRNAs. A single mismatch was observed between

mature amiR-bnashp2 and its binding site in SHP1/SHP2 homologs,

indicated as boxed region

Acta Physiol Plant

123

To achieve pod-shatter resistance in polyploid Brassica

genomes, the designed amiRNA(s) should effectively tar-

get diverse transcripts from multiple loci of SHP1/SHP2

paralogs. With this background, amiR-bnashp1 and amiR-

bnashp2 were designed while keeping off-target selection

parameter at two, to ensure redundant silencing of multiple

SHP transcripts. Although several optimal candidate

amiRNAs were retrieved for BnaSHP2, only sub-optimal

amiRNAs were predicted for BnaSHP1 since the latter

lacked an appropriate sequence context satisfying numer-

ous criteria required for binding of mature amiRNA

(Schwab et al. 2006). Of all the optimal amiRNAs pre-

dicted against BnaSHP2, amiR-bnashp2 was chosen since

its target site was found conserved in SHP1 and SHP2

homologs from B. juncea, B. napus and Arabidopsis SHP2

and promised redundant silencing in these genetic back-

grounds. amiR-bnashp1 was also designed and tested in

spite of being predicted sub-optimal, since true in planta

response can only be assessed empirically. Even WMD3

tool prescribes in vivo testing of sub-optimal amiRNAs.

Stability of pre-amiRNAs (DG), ensuring efficient bio-

genesis of mature amiRNA was inferred by analysis of

secondary structures of precursor backbone harboring

amiRNAs vis-a-vis natural miRNA. Equivalent MFE val-

ues of pre-amiR-bnashp1, pre-amiR-bnashp2 and native

miR319a precursor structures suggested equivalent effi-

ciency of amiRNA biogenesis. This was validated by

detection of robust expression of mature amiRNAs across

all experiments conducted in the present study.

The sequence context of amiRNA-binding site has been

reported to influence its accessibility to amiRNAs (Kertesz

et al. 2007). In our study, analysis of thermodynamic

interactions revealed differential accessibility of amiRNAs

to respective binding sites on target transcripts. This was

contributed mainly by the sequence of target site and to a

lesser degree by the sequence context. Total energy of

binding (TFEB), indicative of target site accessibility to

respective amiRNAs, is defined as a sum total of energy

gained due to binding of the mature 21 nt i.e., energy of

duplex formation (EDF), and energy losses incurred due to

opening of short sequences (OESS) and opening of long

sequences (OELS) as described by Kertesz et al. (2007).

amiR-bnashp1 and amiR-bnashp2 were found to have

widely contrasting TFEB. Lower TFEB of amiR-bnashp2

suggested strong hybridization with respective target sites

across Brassica homologs of SHP1/SHP2. On the other

hand, high TFEB of amiR-bnashp1 with all SHP1/SHP2

homologs suggested its inefficiency in directing transcript

cleavage. This finding was in corroboration with WMD3-

based predictions that similarly berated amiR-bnashp1 as

sub-optimal.

In order to test the veracity of RNAup-based predictions,

representative homologs (BnaSHP2a, BnaSHP2b,Ta

ble

2P

erce

nta

ge

sim

ilar

ity

shar

edin

80

-bp

seq

uen

ceco

nte

xt

surr

ou

nd

ing

amiR

-bn

ash

p2

targ

etsi

teac

ross

SH

P1/S

HP

2o

rth

olo

gs

Bn

aS

HP

1(A

Y0

36

06

2)

Bn

aS

HP

1a

Bn

aSH

P1

bA

thS

HP

1B

na

SH

P2

aB

na

SH

P2

bB

na

SH

P2

b(E

U4

24

34

3)

Bn

aS

HP

2a

(EU

424

34

2)

Bju

SH

P2a

Bju

SH

P2

bB

juS

HP

2c

Bju

SH

P2d

Ath

SH

P2

Bn

aS

HP

1(A

Y0

36

06

2)

10

0

Bn

aS

HP

1a

95

.01

00

Bn

aS

HP

1b

93

.79

8.7

10

0

Ath

SH

P1

91

.29

6.2

97

.51

00

Bn

aS

HP

2a

83

.78

8.7

90

.09

0.0

10

0

Bn

aS

HP

2b

81

.28

6.2

87

.58

7.5

96

.21

00

Bn

aS

HP

2b

(EU

424

34

3)

81

.28

6.2

87

.58

7.5

96

.21

00

10

0

Bn

aS

HP

2a

(EU

424

34

2)

83

.78

8.7

90

.09

0.0

1.0

96

.29

6.2

10

0

Bju

SH

P2a

82

.58

5.0

86

.28

6.2

95

.09

8.7

98

.79

5.0

10

0

Bju

SH

P2b

82

.58

5.0

86

.28

6.2

95

.09

8.7

98

.79

5.0

10

01

00

Bju

SH

P2c

83

.78

8.7

90

.09

0.0

98

.79

7.5

98

.79

8.7

96

.29

6.2

10

0

Bju

SH

P2d

83

.78

8.7

90

.09

0.0

98

.79

7.5

97

.59

8.7

96

.29

6.2

10

01

00

Ath

SH

P2

83

.78

8.7

87

.58

7.5

88

.78

7.5

87

.58

8.7

86

.28

6.2

87

.58

7.5

10

0

Acta Physiol Plant

123

BjuSHP2a, AthSHP2, BnaSHP1a and AthSHP1) were

chosen for functional validation of amiRNAs through

detection of target cleavage product. The transcripts

differed from each other at three levels, viz. target site

per se, the 80-bp sequence context of the amiRNA-binding

site and genetic backgrounds from which these were

isolated.

For functional validation, stem-loop primers-based

reverse transcription was also adapted to detect cleaved

products using co-infiltration assays. We optimized strin-

gent temperature conditions for reverse transcription such

that considerable spatial constraints are imposed on stem-

loop primers. Reverse transcription was thus allowed only

from a cleaved target transcripts and not intact ones. In our

opinion, the cleaved transcripts presented an interface at 30

end on to which the stem-loop primers attach specifi-

cally. Further, base stacking of the stem enhanced the

thermal stability of the RNA–DNA heteroduplex (Chen

et al. 2005). Co-infiltration of Agrobacterium strains

overexpressing BnaSHP2a, BnaSHP2b, BjuSHP2a, AthS-

HP2, BnaSHP1a and AthSHP1 individually with Agro-

bacterium strains overexpressing amiR-bnashp2, therefore,

resulted in detection of cleavage products for all tested

transcripts. Absence of amplicons in stem-loop primed

reverse transcription on tissue samples wherein Agrobac-

teria overexpressing target transcripts were co-infiltrated

with empty vector presented a scenario wherein no artifi-

cial miRNA was expressed while an intact target transcript

had accumulated. Our results validated the specificity of

stem-loop primers in selectively reverse transcribing the

cleaved transcripts and not the intact ones. Our data also

suggests that cleavage product was obtained only when

hybridization energy values between the target transcripts

and amiRNAs were optimum. More specifically, TFEB

values ranging from -22.38 kcal/mol to -25.45 kcal/mol

were found to be necessary and sufficient to direct cleavage

of the target(s). Based on our finding, we prescribe TFEB-

based analysis to predict the efficacy of a designed

amiRNA.

In vivo studies revealed that a single mismatch in

sequences corresponding to amiR-bnashp2 seed region of

the transcripts (BnaSHP2b, BjuSHP2a, AthSHP2,

BnaSHP1a and AthSHP1) was tolerated and resulted in

efficient target cleavage. Hence, amiR-bnashp2 is antici-

pated to direct cleavage of other SHP1/SHP2 transcripts,

such as BjuHSP2b and BjuSHP2c, that harbor identical

binding sites. Furthermore, amiR-bnashp2-mediated

cleavage was observed even in SHP1 and SHP2 homologs

with nucleotide sequence variability of 8.8 and 13.8 %,

across the entire sequence and in 80-bp sequence context

around its target site, respectively. We may herein con-

clude that amiR-bnashp2 is capable of directing cleavage

of multiple transcripts corresponding to SHP1/SHP2 loci if

sequence identities are maintained in target site and its

flanking regions.

Absence of amiR-bnashp1-mediated cleavage of

BnaSHP1a and AthSHP1 was anticipated since the average

TFEB was found to be high (-6.01 kcal/mol) which may

be attributed to up to five mismatches observed between

amiR-bnashp1 and their binding sites in isolated homologs.

Hence, amiR-bnashp1 was taken as an efficient experi-

mental control for this study.

In the absence of complete representation of SHP1/

SHP2 transcript sequences from B. napus and B. juncea,

prediction of true in planta response is not feasible.

However, in genomes where whole genome sequence is

available, our predictions based on thermodynamic inter-

actions between amiRNA and target allow quick assess-

ment on actual in planta response. We suggest that

energetics of interactions be routinely assessed for short-

Table 3 Hybridization energy (TFEB) of amiR-bnashp1 and SHP1/

SHP2 target pairs indicating high free energy requirement for its

binding site (RNAup)

EDF

(kcal/mol)

OELS

(kcal/mol)

OESS

(kcal/mol)

TFEB

(kcal/mol)

BnaSHP2a -9.42 4.55 0.03 -4.85

BnaSHP2b -6.30 1.81 0.02 -4.48

BjuSHP2a -8.12 5.08 0.03 -3.02

BjuSHP2b -6.30 3.31 0.02 -2.97

BjuSHP2c -6.30 2.10 0.02 -4.18

BjuSHP2d -8.12 5.08 0.03 -3.02

BnaSHP1a -6.30 2.10 0.02 -4.18

BnaSHP1b -6.30 1.99 0.02 -4.29

AthSHP2 -18.49 9.67 0.03 -8.79

AthSHP1 -23.34 6.01 0.03 -17.31

Table 4 Hybridization energy (TFEB) of amiR-bnashp2 and SHP1/

SHP2 target pairs indicating low free energy requirement for its

binding site (RNAup)

EDF

(kcal/mol)

OELS

(kcal/mol)

OESS

(kcal/mol)

TFEB

(kcal/mol)

BnaSHP2a -29.71 4.44 1.56 -23.72

BnaSHP2b -29.71 2.70 1.56 -25.45

BjuSHP2a -29.71 5.78 1.56 -22.38

BjuSHP2b -29.43 4.33 1.56 -23.54

BjuSHP2c -29.43 2.91 1.56 -24.96

BjuSHP2d -29.71 5.78 1.56 -22.38

BnaSHP1a -29.71 5.03 1.56 -23.12

BnaSHP1b -29.71 3.16 1.56 -24.99

AthSHP2 -29.71 4.48 1.56 -23.67

AthSHP1 -29.71 5.31 1.56 -22.84

Acta Physiol Plant

123

listing candidate amiRNAs when knockdown mutants are

to be analyzed in a high-throughput mode. In conclusion,

we have successfully designed an efficient amiRNA (amiR-

bnashp2) capable of redundantly silencing of the SHP1/

SHP2 homologs from Brassica and Arabidopsis, as evi-

denced by detection of cleavage product of SHP1 and

SHP2 transcripts from, B. napus, B. juncea and Arabi-

dopsis. Our method circumvents the requirement for

engineering customized amiRNAs specifically targeting

individual members of redundant gene families followed

by tedious gene-stacking efforts. We recommend adoption

of this strategy in related Brassica genomes to tackle yield

losses owing to pod shatter.

Author contribution A.S. conceived the study. P.D. and

A.S. designed the experiments, analyzed the data and

prepared the manuscript. P.D. and S.M. performed the

experiments. All authors have seen the manuscript and

agree to the same.

Acknowledgments This work was supported by grants (BT/

PR10071/AGR/36/31/2007 and BT/PR628/AGR/36/674/2011)

received from Department of Biotechnology, Govt. of India. The

authors thank Dr. Sandip Das for offering valuable suggestions.

Plasmid pRS300 was received as a kind gift from Prof. Detlef Weigel.

Financial assistance as SRF to Priyanka Dhakate from Council of

Scientific and Industrial Research and to S.M. Shivaraj from

Department of Biotechnology, Govt. of India is gratefully

200 bp

400 bp

SHP1SHP1/SHP2SHP2 cleaved productscleaved products

M

305 bp

152 bp

Bna

SH

P2a

+am

iR-b

ansh

p2

Bna

SH

P2b

+am

iR-b

ansh

p2

Bju

SH

P2a

+am

iR-b

ansh

p2

Ath

SH

P2

+am

iR-b

ansh

p2

Bna

SH

P1a

+am

iR-b

ansh

p2

Ath

SH

P1

+am

iR-b

ansh

p2

100 bp

M

400 bp

amiR-bnashp2 amiR-bnashp2

amiR

-ban

shp2

+A

thS

HP

1

amiR

-ban

shp2

+B

naS

HP

2a

amiR

-ban

shp2

+B

naS

HP

2b

amiR

-ban

shp2

+B

juS

HP

2a

amiR

-ban

shp2

+A

thS

HP

2

amiR

-ban

shp2

+B

naS

HP

1

amiR

-ban

shp2

+E

mpt

y ve

ctor

Neg

etai

vete

mpl

ate

cont

rol

Neg

etai

vete

mpl

ate

cont

rol

100 bp

400 bp

Experimental controlsExperimental controls

a

b

M Bna

SH

P2a

+E

mpt

y ve

ctor

Bna

SH

P2b

+E

mpt

y ve

ctor

Bju

SH

P2a

+E

mpt

y ve

ctor

Ath

SH

P2

+E

mpt

y ve

ctor

Bna

SH

P1a

+E

mpt

y ve

ctor

Ath

SH

P1

+E

mpt

y ve

ctor

Neg

etai

vete

mpl

ate

cont

rol

C

Fig. 4 Detection of amiR-bnashp2 and cleaved products of SHP1/

SHP2 homologs in tobacco transient assays. a Gel picture depicts

biogenesis of mature amiR-bnashp2 in Nicotiana leaf samples co-

infiltrated with Agrobacterium suspensions overexpressing amiR-

bnashp2 and transcripts BnaSHP2a, BnaSHP2b, BjuSHP2a, AthS-

HP2, BnaSHP1a and AthSHP1 (lane 1–6). Lane 7 corresponds to

stem-loop PCR product for amiR-bnashp2 in empty vector control.

Lane 8 represents negative control with template absent. b Gel picture

depicts PCR products corresponding to cleaved transcripts of

respective Nicotiana leaf samples. Lane 7 represents negative control

with template absent. The difference in length of the detected

fragments is due to different primer sets used for detecting cleaved

SHP2 (lane 1–4, 152 bp) and SHP1 transcripts (lane 5–6, 305 bp).

c Gel picture depicts negative controls for panel B. Absence of

amplicons confirms failure of stem-loop mediated reverse transcrip-

tion from intact transcripts

Acta Physiol Plant

123

acknowledged. Infrastructural support from TERI and TERI Univer-

sity are duly acknowledged.

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