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Background Insect resistance in crops represents a main challenge for agriculture. Transgenic approaches based on proteins displaying insect resistance properties are widely used as efficient breeding strategies. To extend the spectrum of targeted pathogens and overtake the development of resistance, molecular evolution strategies have been used on genes encoding these proteins to generate thousands of variants with new or improved functions. The cotton boll weevil (Anthonomus grandis) is one of the major pests of cotton in the Americas. An α-amylase inhibitor (α-AIC3) variant previously developed via molecular evolution strategy showed inhibitory activity against A. grandis α-amylase (AGA). Results We produced in a few days considerable amounts of α-AIC3 using an optimised transient heterologous expression system in Nicotiana benthamiana. This high α-AIC3 accumulation allowed its structural and functional characterizations. We demonstrated via MALDI-TOF MS/MS technique that the protein was processed as expected. It could inhibit up to 100% of AGA biological activity whereas it did not act on α-amylase of two non-pathogenic insects. These data confirmed that N. benthamiana is a suitable and simple system for high-level production of biologically active α-AIC3. Based on other benefits such as economic, health and environmental that need to be considerate, our data suggested that α-AIC3 could be a very promising candidate for the production of transgenic crops resistant to cotton boll weevil without lethal effect on at least two non-pathogenic insects. Conclusions We propose this expression system can be complementary to molecular evolution strategies to identify the most promising variants before starting long-lasting stable transgenic programs.
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R E S E A R C H A R T I C L E Open Access
Nicotiana benthamiana is a suitable
transient system for high-level expression
of an active inhibitor of cotton boll weevil
Guilherme Souza Prado
, Pingdwende Kader Aziz Bamogo
, Joel Antônio Cordeiro de Abreu
François-Xavier Gillet
, Vanessa Olinto dos Santos
, Maria Cristina Mattar Silva
, Jean-Paul Brizard
Marcelo Porto Bemquerer
, Martine Bangratz
, Christophe Brugidou
, Drissa Sérémé
Maria Fatima Grossi-de-Sa
and Séverine Lacombe
Background: Insect resistance in crops represents a main challenge for agriculture. Transgenic approaches based
on proteins displaying insect resistance properties are widely used as efficient breeding strategies. To extend the
spectrum of targeted pathogens and overtake the development of resistance, molecular evolution strategies have
been used on genes encoding these proteins to generate thousands of variants with new or improved functions.
The cotton boll weevil (Anthonomus grandis) is one of the major pests of cotton in the Americas. An α-amylase
inhibitor (α-AIC3) variant previously developed via molecular evolution strategy showed inhibitory activity against
A. grandis α-amylase (AGA).
Results: We produced in a few days considerable amounts of α-AIC3 using an optimised transient heterologous
expression system in Nicotiana benthamiana. This high α-AIC3 accumulation allowed its structural and functional
characterizations. We demonstrated via MALDI-TOF MS/MS technique that the protein was processed as expected.
It could inhibit up to 100% of AGA biological activity whereas it did not act on α-amylase of two non-pathogenic
insects. These data confirmed that N. benthamiana is a suitable and simple system for high-level production of
biologically active α-AIC3. Based on other benefits such as economic, health and environmental that need to be
considerate, our data suggested that α-AIC3 could be a very promising candidate for the production of transgenic
crops resistant to cotton boll weevil without lethal effect on at least two non-pathogenic insects.
Conclusions: We propose this expression system can be complementary to molecular evolution strategies to
identify the most promising variants before starting long-lasting stable transgenic programs.
Keywords: Transient protein expression, α-amylase inhibitors, Gene silencing suppressors, Cotton boll weevil
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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* Correspondence:
Maria Fatima Grossi-de-Sa and Séverine Lacombe are contributed equally to
this work.
IRD, CIRAD, Université Montpellier, Interactions Plantes Microorganismes et
Environnement (IPME), Montpellier, France
INERA/LMI Patho-Bios, Institut de LEnvironnement et de Recherches
Agricoles (INERA), Laboratoire de Virologie et de Biotechnologies Végétales,
Ouagadougou, Burkina Faso
Full list of author information is available at the end of the article
Prado et al. BMC Biotechnology (2019) 19:15
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Biotic stresses such as insect pests induce dramatic dam-
ages in crops throughout the world, leading to signifi-
cant losses for growers. To defend against these stresses,
chemical treatments are largely used. However, due to
health, environmental and cost concerns, for years atten-
tion has focused on genetic resistance, both in terms of
conventional and transgenic applications [1,2].
The most common transgenic plants displaying insect
resistance (IR) carry genes encoding crystal toxins (Cry)
from the soil bacterium Bacillus thuringiensis (Bt). Cry
proteins solubilize in the insect midgut, where they
become active and lead to cell lysis and insect death. Cry
proteins are toxic to insects but not to humans or other
vertebrates [3]. Despite a quite narrow range of control
pathogens and low accumulation levels in plants, Bt IR
crop plants represent one of the most successful achieve-
ments in plant transgene technology [2]. Currently, several
Bt plants, including corn, cotton and soybean, grow under
field conditions worldwide [4]. However, lack of high dose
cry expression in plants still can lead to the selection of
insect varieties that acquire resistance against the toxic
effects of the Cry molecules via adaptation [5].
On the other hand, plants are equipped with natural
defence systems against pests such as insects. These
defences mainly involve antimetabolite proteins that in-
duce alterations to the digestive system of insect pests.
The transfer of proteinase inhibitor genes from one plant
to another has been widely used to develop insect-resist-
ant plants [68]. For example, when expressed in
Nicotiana benthamiana, a beetroot gene encoding a pro-
teinase inhibitor induces resistance to lepidopteran insect
pests [9]. Lectins are plant carbohydrate-binding proteins
that present a high toxicity to phytophagous insects
[10]. Lectins have been used in genetic transformation
to provide resistance against spider mite in papaya
[11]. Chitinases are also plant-expressed proteins that
text [12,13].
Alpha-amylase inhibitors (α-AI) produced in common
bean (Phaseolus vulgaris) and other Phaseolus species
act on α-amylase present in insect guts by inhibiting the
processing of complex sugars and, consequently, the
growth of insect larvae [14]. They exist as two isoforms,
α-AI1 and α-AI2, that undergo proteolytic cleavage from
a preprotein to two polypeptides: α- and β-subunits [15].
In addition, amino acid hydrolysis occurs at the
C-terminal ends of both α- and β-subunits, giving rise to
10 and 15 kDa chains, respectively [16]. Even if the
unprocessed and processed forms accumulated in plants,
it has been shown that proteolysis is required for inhibi-
tory activity [15]. Despite a relatively high similarity,
α-AI1 and α-AI2 act on specific and distinct spectra of
insect α-amylases [14]. Transgenic processes to express
bean α-AI have been widely used on several plant
species for the improvement of IR [1720].
Despite the efficiency of these IR strategies, the
spectrum of insects controlled by any given protein is
quite narrow. Moreover, whatever the controlling strat-
egy is, it must face the development of resistant insects.
Hence, to extend the spectrum of target pathogens and
to overtake the development of insect resistance,
molecular evolution strategies have been used on ori-
ginal IR proteins to generate thousands of variants
with potentially new or improved functions [21,22].
New resistances have been identified from these li-
braries for the cotton boll weevil (A. grandis), sugar-
cane giant borer (Telchin licus licus)andmustard
aphid (Acyrthosiphon pisum)[2326]. These findings
highlight the importance of the variant libraries to
create new IR to harmful insect pests that act on
major crops worldwide. However, even with this im-
portant agricultural interest, a deep characterization
of these proteins is required to demonstrate their
economic interest and safety impact such as allergenic
issues [27].
Systems allowing low-cost and rapid screening of these
libraries are necessary to identify the most promising
variants before starting long-term and costly transgenic
programmes. Cry and trypsin inhibitor variants are
expressed in phage systems before in vitro screening of
inhibitory activity [2325]. However, this phage-based
system is not suitable for plant variants requiring post-
translational modifications for their activities, such as
α-AI. Moreover, the final goal is to express these variants
in plants, implying that they would be processed by the
plant cell machinery. Consequently, plant-based systems
could be more convenient than phage- or prokaryote-
based systems to screen these variants and select the
most promising ones. The model plant Arabidopsis
thaliana has been used to stably express α-AI variants.
This system allowed the identification of a very promis-
ing variant, α-AIC3 that was able to inhibit 77% of the
α-amylases from the insect A. grandis, whereas the
original α-AI forms were ineffective. This variant differs
from the original sequence by several amino acid
changes induced by the molecular evolution strategy
performed [26]. This outcome represents an important
finding for the cotton culture in the Americas, where A.
grandis is among the major insect pests. Consequently a
deep characterization of this variant should be done
before starting a promising transgenic cotton program.
However, A. thaliana transgenic-based screenings may
not be suitable for evaluating potentially interesting
proteins from thousands of variant libraries. Therefore,
in order to characterize accurately such protein variants,
it is crucial to establish an alternative and robust
plant-based expression system that allows the expression
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of recombinant proteins at high yield and with accuracy
in terms of post-translational modifications.
In recent years, advances in biotechnology have led to
the emergence of plants as bioreactors for the produc-
tion of proteins of interest not only in stable transgenic
systems but also in transient systems [28]. The first
crucial advance was the use of transient expression sys-
tems relying on Agrobacterium as a vector to deliver
DNA encoding proteins of interest directly into leaf cells
by syringe infiltration so-called agroinfiltration [29].
Moreover, protein production can be increased by the
co-expression of viral proteins displaying suppression of
gene silencing activity. Indeed, the presence of such viral
proteins in transient expression systems allows overcom-
ing the gene silencing triggered by the plant defence
machinery to specifically degrade foreign nucleic acids.
Consequently, the yield of the protein of interest is dra-
matically increased by 50 fold or more [30,31].
Here, we describe a high-yielding, easier, quicker and
cheaper system compared to the stable transformation
of A. thaliana. This well-known system is based on the
transient expression of the protein of interest in N.
benthamiana leaves (see for review [32]). As previously
described, a combination of three viral suppressors of
gene silencing are used to improve the expression in
terms of accumulation levels [31]. We focused on an
α-AIC3 variant that was previously demonstrated to act
on one of the most damaging insects to cotton culture
in the Americas the cotton boll weevil (A. grandis)
[26]. We showed that these proteins that were transiently
expressed in N. benthamiana leaves, accumulated at high
levels and exhibited their expected post-translation matur-
ation and in vitro function on the target insect enzyme.
We proposed this system to be complementary to mo-
lecular evolution strategies to allow easy selection and
characterization (within a few days) of the most promising
variants from molecular evolution libraries before starting
stable transgenic programs.
α-AIC3 expression in N. benthamiana leaves
To optimize the accumulation of α-AIC3 in N. benthami-
ana leaves, the aic3 gene was transiently co-expressed in
4-week-old wild-type N. benthamiana plants together
with genes encoding three viral gene silencing suppres-
sors. It has been previously demonstrated that these
suppressors act synergistically by inhibiting three different
steps of the gene silencing defence mechanism [31].
Agroinfiltrated leaf regions were collected at 5 dpi and
weighted, after which protein was extracted. A total of
40 μg of soluble proteins representing approximately 10
mg of fresh leaves was separated by 15% (m/v) SDS-PAGE
and blotted onto a nitrocellulose membrane. The
Coomassie Blue-stained gel (Fig. 1a) showed additional
bands of lower molecular mass for samples 2 (pBI-
N61:α-AIC3) compared to samples 1 (pBIN61), suggesting
that this difference was due to the aic3 gene expression
and protein accumulation in the leaves. This result was
confirmed by Western blot (Fig. 1b) using a specific
anti-α-AIC3 primary antibody; samples 1 did not show
any visible band or signal, but samples 2 presented a
pattern composed of three intense bands. The lower bands
were very intense and referred to the processed α-AIC3
forms, which may correspond to α-andβ-subunits of 12
kDa and 15 kDa, respectively. Moreover, bands of higher
molecular weight also appeared that were approximately
28 kDa, strongly suggesting that they correspond to the
unprocessed forms of α-AIC3; these bands had a less
intense signal than the bands attributed to the processed
subunits. The results here indicate that α-AIC3 was
successfully expressed and mostly processed according to
the expected proteolytic processing. Furthermore, the
generated bands were not linear but dispersed. These
patterns suggest several isoforms that could result from
the expected post-translational maturation processes for
these inhibitors including amino acid hydrolysis at the
C-terminus ends of both subunits and glycosylation
[16]. However, despite their accurate size, we cannot
exclude that the observed bands were due to protein
aggregation or degradation. The following structural
and functional characterization were performed to
exclude this possibility.
α-AIC3 yield and expression level analysis and protein
The yield of the protein of interest in the dialyzed
samples was measured by indirect ELISA using a specific
anti-α-AIC3 primary antibody. The expression level of
α-AIC3 was estimated for the pBIN61:α-AIC3 samples,
considering pBIN61 samples as negative controls. In the
first experiment, 13.6 ng of α-AIC3 out of 40 ng of total
protein were detected, indicating a yield of 34% of Total
Soluble Proteins (TSP) for the heterologous protein.
Based on the percentage of the specific expression of
α-AIC3, this amount corresponded to a yield of 0.1 mg/g
fresh weight (FW) tissue or 100 mg/kg FW. In another
experiment, 70.4 ng of α-AIC3 of 160 ng of total protein
was detected, indicating a yield of 44% TSP for the
heterologous protein and corresponding to 0.15 mg/g
FW or 150 mg/kg FW. For the protein purification, a
dialyzed extract was used, and proteins were loaded on a
gel-filtration column for performing size exclusion
chromatography (SEC). A total of 90 fractions consisting
of 2 mL each were obtained. The chromatograms
showed different peaks for fractions 1014, 1620 and
3058 (Fig. 2a). Hence, some fractions (12, 17, 18, 19,
26, 37, 40, and 42) from each peak were selected to per-
form electrophoresis and separate samples to further
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Fig. 1 Detection of α-AIC3 expression in presence of gene silencing suppressor combination (P0, P1 and P19). a- Coomassie Blue-stained 15%
SDS-PAGE consisting of 40 μg of total protein from crude extracts of pBIN61 samples (1) and pBIN61:α-AIC3 samples (2) from N. benthamiana
leaves co-expressing these vectors with the three gene silencing suppressors . b- Western blot of corresponding Coomassie Blue-stained gel
using a specific primary anti-α-AIC3 antibody. Expected bands for whole and unprocessed α-AIC3 (27 kDa), as well as for its subunits (α-subunit,
12 kDa, and β-subunit, 15 kDa), are shown. M: Molecular marker
Fig. 2 α-AIC3 purification through size exclusion chromatography. a- Chromatogram generated from molecular size exclusion chromatography of
α-AIC3-expressing N. benthamiana extracts after dialysis against water. The indicated peaks comprise fractions 1014, 1620 and 3058. A total of
180 mL of eluted volume was obtained, distributed in 90 fractions of 2 mL each. Software: UNICORN6.4 (GE Healthcare). b- Silver-stained 15%
SDS-PAGE of selected SEC fractions (15 μL). CE: crude extract; W: washing; numbers: selected SEC fractions. c- Western blot of 15% SDS-PAGE gel
using a specific primary anti-α-AIC3 antibody. Sample analysed consists of combined fractions 17, 18 and 19 of purified and concentrated α-AIC3.
The four bands analysed by mass spectrometry are indicated. M: Molecular marker
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identify presence of α-AIC3 subunits. Silver staining
demonstrated that fractions 17, 18 and 19 (Fig. 2b)
presented expected bands for α-AIC3. Indeed, these
patterns were very similar to the one previously revealed
via Western blotting using a specific anti-α-AIC3 pri-
mary antibody (Fig. 1b). Based on these results, these
fractions were pooled concentrated and separated again
for Coomassie Blue staining and western blotting. Four
main bands were clearly detected by western blotting at
the expected size for unprocessed (two bands around 28
kDa), β(15 kDa) and αsubunits (12 kDa) (Fig. 2c).
Corresponding bands visualized on Coomassie staining
gel were excised to structurally characterize the proteins
and confirm identity with the protein of interest.
Structural characterization
Spots were excised from the four bands and prepared
for MALDI-TOF MS/MS analysis. Spectra of the
generated peptides were fragmented, some of which are
shown in Fig. 3with respect to both α-AIC3 subunits.
For the α-subunit, one of the four possible tryptic
peptides was detected and confirmed after sequencing:
AFYSAPIQIR. This finding indicates a coverage of 10 of
73 amino acid residues for the α-subunit, resulting in 14%
coverage. For the β-subunit, five of the twelve possible
tryptic peptides were detected and confirmed after sequen-
This finding indicates a coverage of 65 of 135 residues for
the β-subunit, resulting in 48% coverage. In total, six
peptides were detected, sequenced and confirmed, indicat-
ing a coverage of 75 of 221 residues for α-AIC3 or 34%
coverage (Fig. 4). The peptide of the α-subunit was found
in all samples corresponding to bands at 28 kDa, 25 kDa
and 12 kDa. The five peptides of the β-subunit were found
in the samples related to bands 28 kDa, 25 kDa and15 kDa.
This finding strongly supports that bands at 15 kDa and
12 kDa represent the β-andα-subunits, respectively, and
Fig. 3 MALDI-TOF MS/MS spectra of fragmented peptides from α-AIC3. Above: parent ion corresponding to an α-subun it peptide [ M + H]
= 1165.7
Da; predicted sequence: AFYSAPIQIR. Below: parent ion corresponding to a β-subunit peptide [M + H]
= 1986.7 Da; predicted sequence:
SVPWDVHDYDGQNAEVR. Software: FlexAnalysis 3.3 (Bruker Daltonics)
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that bands 28 kDa and 25 kDa represent the whole unpro-
cessed protein, since it contains sequences of both sub-
units. However, peptides detected do not cover both N-
and C-terminus ends of each subunit. Consequently, we
cannot exclude that subunits were not intact.
Functional characterization
Based on the DNS method, the α-AIC3-containing
samples of N. benthamiana showed an average inhib-
ition of 98.5% in A.grandis α-amylase (AGA) activity
when using 1 unit of enzyme and 100 μg of soluble pro-
tein. This inhibition level was validated based on the in-
hibition obtained using the same amount of A. thaliana
α-AIC3-containing samples, which completely inhibited
the AGA activity. The experiments were repeated for
extracts from three different agroinfiltrations. These re-
sults showed that the AGA activity inhibition level var-
ied from 96.7 to 100% (Fig. 5). The same extracts of the
third agroinfiltration were simultaneously used to assess
the inhibition level for AMA and SFA, and did not show
any significant inhibition activity (Fig. 5), since absor-
bances were the same for reactions with or without
α-AIC3 and containing active Apis mellifera amylase
(AMA) and Spodoptera frugiperda amylase (SFA) en-
zymes. Hence, regardless of any assays using AMA and
SFA specific inhibitor controls because of their currently
Fig. 4 Total sequenced peptides from the α-AIC3 chain. α-AIC3 whole sequence, showing amino acid residues of the α- and β-subunits; the
respective peptides were identified, fragmented and sequenced via MALDI-TOF MS/MS and are highlighted inside the rectangles. In total, six
peptides were sequenced, one for the α-subunit and five for the β-subunit. C-terminal end peptides, which were cleaved off to yield the mature
subunits, for both subunits are underlined in the figure
Fig. 5 α-AIC3 inhibitory level against target (AGA Anthonomus grandis amylase) and non-target (AMA Apis mellifera and SFA Spodoptera
frugiperda) enzymes. The inhibition levels presented here are based on 100 μg of total soluble protein. The assay results were generated based on
three independent experiments. Error bars represent the standard deviation
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unavailability, these data suggested that α-AIC3 produced
in N. benthamiana was unable to inhibit α-amylase from
A. mellifera and S. frugiperda. Altogether, these results
showed that the protein of interest exhibits its expected
activity on AGA but exhibits no inhibitory activity against
the amylases of these non-target insect species.
The work presented here demonstrates that N. benthami-
ana coupled with the use of a cocktail of gene silencing
suppressors is a suitable system for quickly and easily
producing a quite high level of an α-AI variant, α-AIC3.
The yield was estimated in the range of 100 to 150mg/kg
FW. Plant biosystem yield for proteins of interest is quite
variable, reaching up to 2 g/kg of FW in the case of
optimised viral based technology [33]. Here, we are in the
upper range for non- viral based technology. Moreover,
we demonstrated that all the expected α-subunit,
β-subunit and unprocessed forms accumulated, mostly
consisting of the processed α-andβ-subunits. Finally, the
expected protein functionality as an inhibitor of AGA was
demonstrated, and α-AIC3 did not inhibit the α-amylases
of two non-target insects tested (A. mellifera and S.
frugiperda), preliminarily suggesting that α-AIC3 is
biologically and environmentally safe. However, it is
recommended to perform in vivo studies of α-AIC3,
giving rise to an even more realistic result regarding
the protein yield and safety,furnishing data concerning
the feasibility to produce genetically modified cotton
plants that could be resistant to A. grandis without
triggering biosafety traits such as environmental im-
balances or allergenicity.
Concerning the inhibitory assays, we could achieve up
to 100% inhibition of AGA using 100 μgofN. benthami-
ana extracts, indicating that up to 44 μgofα-AIC3 were
used to inhibit 1 U of AGA, while the complete inhib-
ition of AGA was also achieved using the same amount
of total protein from A. thaliana seeds. Silva et al. [26]
demonstrated that the inhibitory activity was 77% when
using 85 μg of total protein from A. thaliana leaves ex-
pressing this inhibitor at an expression level of 0.2% of
TSP, which is very low compared to the level in transient
expression obtained here. This means that, from the
total protein, approximately 170 ng of α-AIC3 are neces-
sary to inhibit 77% of the AGA activity, and in our study,
we can estimate that approximately 200 ng of α-AIC3
are enough to inhibit 1 U of AGA if considering similar
expression levels of α-AIC3 in seeds as in leaves for
transgenic Arabidopsis as reported in the case of 35S
transgenic constructs [34]. These reproducible Arabi-
dopsis values validate the functional assay used here.
For N. benthamiana extracts, the amount of inhibitor
used to inhibit completely the AGA activity was much
higher, since 100 μg of soluble proteins contain up to
44 μgofα-AIC3 (considering an expression level of 44%
TSP). As the kinetic parameters of this enzymatic assay
were not known, we cannot exclude that this high
amount of inhibitor present in N. benthamiana extract
saturated the assay. Thus, fewer protein amounts could
have triggered similar inhibition levels. Furthermore, we
must also consider that the amount of useful α-AIC3,
i.e., the amount that effectively participates in the
enzyme inhibition was considerably lower than 44 μg.
Indeed, based on the western (Fig. 1b), part of total
α-AIC3 is composed of unprocessed chains, unable to
inhibit the AGA activity as the post-translational
processing is imperative for the acquisition of biological
activity in α-AI proteins [15]. Moreover, from results
from MALDI-TOF MS/MS analysis, we cannot deter-
mine the amount of α- and β-subunits produced that are
fully active.
Regardless of this, the transient expression system
remains a suitable alternative to stable expression sys-
tems because former exhibits practicality: it is simple as
it does not require complex materials nor techniques
and provides considerable amounts of protein without
the need for regenerating plants and selecting transfor-
mants. Dias et al. [35] also used tobacco-based expres-
sion to produce an α-amylase inhibitor (αBIII) of Secale
cereale in Nicotiana tabacum seeds via stable expres-
sion. This system also yielded low protein levels (0.1
0.29% TSP) that were similar to those in A. thaliana
[26] and achieved a maximum inhibition of only 41% of
AGA activity when using 250 mg of crude protein
Altogether, these data suggest that this N. benthami-
ana transient expression system may be suitable for the
rapid, easy and efficient production of α-AI variants
obtained from molecular evolution strategies for prelim-
inary functional screening and biosafety studies. The
α-AIC3 variant analysed here was identified from a li-
brary that consisted of more than 8000 variants [26].
With such transient system, this library could be
efficiently exploited to identify variants with new or
improved IR functions against major pests as describe
above for the potato gene encoding a disease resistant
protein against a virus [36,37].
Nicotiana-based transient expression systems have
been widely used to express proteins of interest, such as
those for vaccines and biopharmaceuticals [28]. Fewer
examples have been described for proteins of agricul-
tural interest. Farnham and Baulcombe [36] produced a
variant library using random mutagenesis from a potato
gene encoding a disease resistant protein (Rx) against a
subset of potato virus X (PVX). Those authors used
transient expression in N. tabacum to screen 1920
variants. Thirteen of those variants induced a cell-death
response in the presence of the PVX coat protein,
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indicative of disease resistance [36]. The same potato
gene encoding Rx resistant protein was also used to
generate a library of 1500 mutants that were transiently
expressed in N. benthamiana together with the gene
encoding for the Poplar mosaic virus (PopMV) coat
protein. This phenomenon allowed the identification of
four variants inducing a cell-death response related to
resistance to this new virus [37]. Similar to the results
reported here, these studies indicate the interest in this
dual variant library/N. benthamiana transient expression
strategy for its easy, rapid, low-cost ability to identify
new or improved disease resistance genes from
thousands of variants.
Other proteins are known to be associated with IR and
have been used in genetic transformation to bring new
IR to important crops worldwide [2]. Molecular evolu-
tion libraries consisting of thousands of variants have
been developed for Cry proteins and proteinase inhibi-
tors. These variants have been screened using phage-
based assays to identify variants with new IR functions
not present in the original forms [23,24]. Because the
ultimate goal was to exploit these variants in transgenic
plants, the N. benthamiana transient expression system
presented here would be more accurate for the heterol-
ogous expression of the variants with the goal of
performing functional tests in order to preview the re-
sponses of the transformed plant as a definite host. As
such, functional tests must serve as a filtering step, as it
is more difficult to regenerate several plants displaying a
wide selection of candidate variants for additional sort-
ing of promising proteins and events. Therefore, heterol-
ogous expression could save time and material and
could reduce the complexity of the process for obtaining
transformed and commercially feasible events. For this,
it is important to gradually characterize the candidates,
as was done in this study. An in vitro stage of
characterization is needed to validate the proposed activ-
ity against the target. However, it is suitable to proceed
with an in vivo and complementary stage of assays in
which the insects are grown in the presence of the mole-
cules, checking systemic effects in the insect. Once the
biological activity is confirmed following this stepwise
study, investigations on genetic transformation will be
much more reliable since regenerated plants will display
the same in vivo observed activity.
Plant breeding has been recently revolutionized with
the advent of genome editing technologies allowing
precise modifications in genomic sequences with the
so-called genome engineering [38,39]. Several econom-
ically important species, such as cotton, are suitable tar-
gets for these technologies [40,41], especially concerning
agronomic traits. These technologies have been success-
fully used in maize, soybean and rice to induce exact
mutations in specific genes, leading to herbicide tolerance
[4244]. Resistance development against biotic stresses
can also benefit of these technologies as shown by the
development of a genome-edited tomato displaying pow-
dery mildew resistance [45] and an engineered cucumber
showing broad virus resistance [46]. Based on that,
we can speculate that the dual strategy variant li-
brary/N. benthamiana transient expression allowing
identification of variants of interest could be followed by
genome editing technologies to precisely induce modifica-
tions in the genome of crops. Results presented here sug-
gest that α-AIC3 would be an ideal candidate to evaluate
this hypothesis, as well as producing genome-edited plants
displaying new or improved IR through α-AI specific
modifications. Moreover, whatever the gene of interest, N.
benthamiana system presented here could be a useful tool
to rapidly and easily identify variants that could be
integrated in plant genomes through genome editing
In this study, we reported successful transient expression
of α-amylase inhibitors using N. benthamiana-based
system with a recent established combination of gene
silencing suppressors. We showed that this system is
highly suitable for producing variants of mutant inhibi-
tors, which were expressed not only at a very high yield
but also with the correct, albeit incomplete, processing,
preserving the expected biological function.
Expression vectors and silencing suppressors
The experiments were performed using Agrobacterium
tumefaciens C58C1 strain harbouring pBIN61:α-AIC3
expression vector for producing the protein of interest
or empty pBIN61 vector for negative control. Based on
our previous work demonstrating the positive effect of
the simultaneous expression of gene silencing suppres-
sors on the accumulation of candidate protein by
blocking the gene silencing defence mechanism [31],
these additional gene silencing suppressor vectors were
used for the co-expression with pBIN61 vectors. They
encoded for P0 from Beet western yellow virus
(pBIN61:P0 vector) [47], P1 from Rice yellow mottle virus
(pCambia1300:P1Tz3 vector) [48] and P19 from Cymbid-
ium ringspot virus (pBIN61:P19 vector) [49]. Each of them
was cloned into expression vectors and transformed in
Agrobacterium tumefaciens C58C1 strain.
Gene design, synthesis and cloning
The nucleotide sequence for the gene (aic3) encoding
the α-AIC3 variant was obtained in silico via reverse
translation and codon optimization of the α-AIC3 pro-
tein sequence [GenBank:AGB50990.1], as reported by
Silva et al. [26]. Codon optimization was performed with
Prado et al. BMC Biotechnology (2019) 19:15 Page 8 of 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Gene Designer 2.0 software [50] based on the codon
usage table for N. benthamiana species, available at
Kazusa Codon Usage Database. The nucleotide se-
quence for the corresponding native signal peptide
(MASSNLLSLALFLVLLTHANS) was also retrieved
and codon-optimized. The final insert sequence was
flanked by 5-XbaIand3-BamHI restriction sites,
and a Kozak consensus sequence (GCCACC) was
inserted immediately upstream of the start codon. No
restriction sites for XbaIandBamHI were detected
within the CDS. SignalP 4.1 Server was used for sig-
nal peptide detection and validation. The sequence
was synthesized de novo by Epoch Life Science® and
cloned into the XbaI-BamHI cloning sites of the
pUC18 vector to generate pUC18:α-AIC3. The aic3
gene was then excised from pUC18:α-AIC3 and cloned
into the XbaIandBamHI sites of the pBIN61 binary
expression vector, which was previously described by
Bendahmane et al. [51] under the control of the constitu-
tive CaMV 35S promoter and terminator to generate pBI-
N61:α-AIC3 that was used to transform A. tumefaciens
strain C58C1 via electroporation. The cloning into the
pBIN61 vector was confirmed by sequencing using the
M13 forward primer and carried out by Beckman Coulter
Genomics®. Cells were also transformed with empty
vectors; these cells served as negative controls.
Agroinfiltration, plant material and experimental
Strains harbouring empty pBIN61, pBIN61:α-AIC3,
pBIN61:P0, pCambia1300:P1Tz3 and pBIN61:P19 vec-
tors were separately grown overnight from precultures
at 28 °C and 200 rpm in an orbital shaker using LB
medium containing rifampicin (100 μg/mL) and kanamy-
cin (50 μg/mL). The cultures were pelleted by centrifu-
gation for 10 min at 4000 g, after which the pellets were
resuspended in 10 mM MgCl
to a final OD600 of 0.5.
Acetosyringone (4-hydroxy-3,5-dimethoxyacetophenone)
was added to each suspension to a final concentration of
100 μM for virulence induction, and the suspensions
were incubated at 24 °C for 3 h. Agroinfiltration cocktails
were prepared by combining cultures for co-infiltration:
for the negative control, the pBIN61 culture was
combined with the cultures of silencing suppressors
(pBIN61:P0:P1:P19, 3:1:1:1, v/v:v/v), and the same
procedure was employed for protein expression, in
which the pBIN61:α-AIC3 culture was combined with
the cultures of silencing suppressors (pBIN61-α-
AIC3:P0:P1:P19, 3:1:1:1, v/v/v/v). Cocktails were infil-
trated into the leaves of 4 weeks old wild-type N.
benthamiana plants using syringes without needle. Four
plants were used for the negative control per experi-
ment, while twelve plants were used for α-AIC3 expres-
sion. The plants were placed in a growth chamber and
cultivated for 5 days before harvesting (12 h of light per
day, 24 °C, 60% relative humidity). Three independent
experiments were performed to generate three biological
replicates for subsequent molecular analysis.
Protein extraction, dialysis and concentration
Infiltrated leaf tissues were harvested from the plants at
5 days post-infiltration (dpi). The fresh leaves were com-
bined in their respective groups (negative control and
α-AIC3 expression), weighted, frozen in liquid nitrogen
and then ground using a mortar and pestle. Protein ex-
traction was performed by adding 700 μL of extraction
buffer (20 mM Tris-Cl, 100 mM NaCl, 10 mM Na
O, 25 mM D-glucose, 0.1% Triton X-100, 5 mM
EGTA, 5% (v/v) glycerol, 5 mM dithiotreitol, and 1 mM
phenylmethanesulfonyl fluoride, pH 7.4) to 300 mg of
tissue powder. Crude extracts were incubated on ice for
20 min, strongly shaken for 20 min at 4 °C using a vortex
and centrifuged at 14000 gfor 30 min at 4 °C. The total
soluble proteins (TSP) were recovered from the superna-
tants and dialyzed against water (1 mL of extract per
200 mL of distilled water) using Slide-A-LyzerG2
Dialysis Cassettes (ThermoFisher Scientific) that had a
10 kDa molecular weight cut-off (MWCO). The dialyzed
samples were clarified by centrifugation at 14000 gfor
10 min and quantified by a Bio-Rad® Bradford protein
assay [52] based on a bovine serum albumin (BSA)
(Sigma Aldrich) standard curve.
SDS-PAGE and Western blot
A total of 40 μg of protein for each extract was subjected
to low-pressure drying, resuspended in 15 μL of pure
water and then diluted in protein loading buffer [53]
with 2-mercaptoethanol. The samples were incubated at
95 °C for 5 min, loaded and then separated by 15% (m/v)
SDS-PAGE. A mirror gel was also made for protein de-
tection via immunoblotting. Proteins were stained with
Coomassie Brilliant Blue G-250 or blotted onto a nitro-
cellulose membrane at 5 V for 20 min in a Trans-Blot®
SD semi-dry system (Bio-Rad) after the membrane and
gel were treated with blotting buffer (20 mM Tris base,
150 mM glycine, 20% methanol, pH 8.3) for 10 min.
Western blot analysis proceeded by blocking the mem-
brane with a 3% (m/v) solution of skimmed milk powder
in TBS-T buffer (20 mM Tris base, 150 mM NaCl, 0.1%
Tween 20, pH 7.5) for 2 h under shaking. The protein
was probed by adding a primary specific anti-α-AIC3
rabbit IgG (GenScript) to the TBS-T buffer (1:2500 of
antibody:buffer, or at 0.4 μg.mL
), after which the
membrane was incubated for 2 h under shaking. After
six five-minute rinses with TBS-T buffer, the bound anti-
bodies were probed by adding an AP-conjugated second-
ary goat anti-rabbit IgG (Sigma Aldrich) to the TBS-T
buffer (13,000 of antibody:buffer, or 0.3 μg.mL
), after
Prado et al. BMC Biotechnology (2019) 19:15 Page 9 of 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
which the membrane was incubated again for 1 h under
shaking. Subsequent washing followed as described, and
the proteins were detected using a colorimetric AP
substrate reagent kit (Bio-Rad) according to the manu-
facturers instructions.
ELISA: α-AIC3 quantification
Dialyzed samples were used to estimate the expression
level of α-AIC3 in the total protein by indirect ELISAs.
Assays were performed in triplicate by coating 96-well
microplates with 40 ng or 160 ng of total protein. A
standard curve of protein amount (R
= 0.9948) was
constructed based on a gradient from 0.2 ng to 200 ng,
in a total of 11 dilutions, of both the bacterial and puri-
fied β-subunits of α-AIC3 previously produced and
kindly provided by Dr. Leonardo Macedo (Embrapa
Genetic Resources and Biotechnology, Brasília, Brazil).
The samples were diluted in coating buffer (50 mM
sodium bicarbonate/carbonate, pH 9.6), and the coated
plates were incubated at 4 °C for 18 h. Samples were in-
cubated at 37 °C for 1 h and washed thrice with 200 μL
of PBS-T buffer (136 mM NaCl, 3 mM KCl, 10 mM
, 2 mM KH
, and 0.05% Tween 20, pH
7.4). The membrane was blocked by using 3% (m/v)
gelatin in PBS-T buffer for 2 h at 37 °C. The samples
were discarded, washed and incubated together with
100 μL of a primary anti-α-AIC3 antibody (GenScript)
diluted in PBS-T buffer with 1% gelatin (1:1000 of anti-
body:buffer, v/v, or at 1 μg.mL
) for 2.5 h, at 37 °C. The
samples were then washed and incubated together with
100 μL of an HRP-conjugated secondary goat anti-rabbit
IgG H + L (Bio-Rad) in PBS-T buffer with 1% (m/v)
gelatin (1:3000 antibody:buffer, v/v, or at 0.3 μg.mL
for 1 h at 37 °C. The samples were detected with 100 μL
of a revealing solution as peroxidase substrate consisting
of 10 mL of phosphate-citrate buffer (24.3 mM citric
acid, 51.4 mM Na
, and 0.06% H
, pH 5.0) and
1 mg of 3,3,5,5-tetramethylbenzidine (TMB) (Sigma
Aldrich). The colour reaction was stopped after 15 min
at room temperature with 100 μL of stop solution (3 M
). The absorbance values were read at 450 nm
using a SpectraMax 190 microplate reader (Molecular
Devices), and the samples were analysed according to
the appropriate calculations using Excel 2007 software
Protein purification
Dialyzed samples of the expressed α-amylase inhibitor
were also used for purification via size exclusion chro-
matography (SEC) using a HiLoad 16/600 Superdex 75
pg (GE Healthcare) 120 mL column. As such, 15 mL of
extract was completely dried under reduced pressure
and resuspended in 1 mL of equilibration buffer (PBS
1X, 1 mM EDTA, 1 mM EGTA, and 1 mM dithiotreitol,
pH 7.4). Afterward, the column was washed with 120 mL
of distilled water at a flow rate of 1 min/mL and then
equilibrated with 240 mL of equilibration buffer at the
same flow rate. The protein solution was loaded on the
column, and 180 mL of equilibration buffer was injected
at a continuous flow rate of 1 min/mL for elution;
fractions were collected every 2 min. Chromatography
was performed using an ÄKTAprime plus protein purifi-
cation system (GE Healthcare), and chromatogram peaks
at 280 nm were generated and analysed by UNICORN
6.4 software (GE Healthcare). Ninety fractions (2 mL
each) were collected, and 15 μL of each fraction of the
different peaks were separated by 15% (m/v) SDS-PAGE
for silver staining according to the methods of
Switzer et al. [54]. Fractions corresponding to the
α-AIC3 peak were combined, lyophilized, resuspended
in ultrapure water and quantified. Aliquots of 20 μg
of proteins were separated by electrophoresis using
15% (m/v) SDS-PAGE.
In-gel digestion and mass spectrometry (MALDI-TOF)
Spots of bands were excised from purified α-AIC3 corre-
sponding bands, i.e., processed and unprocessed forms,
and prepared for trypsin-based in-gel digestion. The
samples were destained three times with 30% (v/v) etha-
nol under vigorous shaking for 20 min. Afterward,
samples were dehydrated with a solution of 50% (v/v)
acetonitrile (ACN) and 25 mM NH
for 15 min,
after which 200 μL of 100% (v/v) ACN was added to the
recovered gel pieces, which were then shook for 10 min.
The supernatant was discarded, the pieces were dried at
room temperature and 15 μL of activated trypsin
(Promega), which was prepared in digestion buffer ac-
cording to the manufacturers instructions, was added.
The mixture was then incubated on ice for 30 min. Di-
gestion proceeded by adding 25 μLof50mMNH
to the samples, which were then incubated at 37 °C for
18 h. The hydrolysis products were collected, desalted,
concentrated and purified using C18 resin ZipTip®
pipette tips (Merck Millipore) according to the manufac-
turers instructions, although peptides were eluted with
80% (v/v) aqueous ACN. The resulting peptides were
dried under reduced pressure and resuspended in 10 μL
of ultrapure and sterile water. Molecular mass analyses
of α-AIC3 and its fragments were performed by
MALDI-TOF MS/MS. A saturated α-cyano-4-hydroxy-
cinnamic acid (CHCA, Sigma Aldrich) solution at 10
mg/mL was prepared in a 1:1 (v/v) aqueous acetonitrile
solution containing 0.3% TFA. The solution of the
hydrolysis products was mixed with CHCA solution
(CHCA:sample, 3:1, v/v), spotted onto a MALDI target
plate, and completely dried for crystallization at room
temperature before analysis. Desorption/ionization, analysis
Prado et al. BMC Biotechnology (2019) 19:15 Page 10 of 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
and detection of peptides were performed using an Auto-
flexSpeed mass spectrometer (Bruker Daltonics), and
ionization was carried out in positive reflection mode.
Spectra were acquired based on external calibration using
Protein Calibration Standard II (Bruker Daltonics) in ac-
cordance with the manufacturers instructions. Peptide
fragmentations were performed by using the LIFTmethod
[55]. MS/MS spectra were manually interpreted, and the
corresponding peptides were sequenced from the b/y series
using FlexAnalysis 3.3 software (Bruker Daltonics). The
peptide sequences were compared to the data from
expected tryptic peptides generated by the theoretical
tryptic digestion of α-AIC3 in ExPASy PeptideMass for
confirming the already-known sequence and performing
coverage analysis.
In vitro inhibitory assays
Activity validation of transiently expressed α-AIC3
The inhibitory activity of N. benthamiana-expressed
α-AIC3 was first assessed and validated against cotton
boll weevil amylase (AGA) based on the comparative in-
hibitory activity of α-AIC3 previously expressed in A.
thaliana [26]. The colorimetric assay was performed by
measuring the AGA activity using the 3,5-dinitrosalicylic
acid (DNS) method adapted from Bernfeld [56] and
using 2% (m/v) starch as substrate. Gut extracts as
source of α-amylase were prepared by isolating gut from
adults of A. grandis using a steel blade and mixing with
AGA buffer (150 mM succinic acid, 20 mM CaCl
mM NaCl, and 1 mM PMSF, pH 5.0) to a concentration
of 0.5 g/mL. The assays were performed with a volume
of gut extract containing one unit of α-amylase, which
was defined as the amount of enzyme necessary to in-
crease the absorbance (OD550) within 20 min to an
amount between 0.11 and 0.15. Seed protein extracts
from transgenic and non-transgenic A. thaliana were
used as a control for α-AIC3 activity, whose transgenic
one expressed α-AIC3 at a level of around 0.2% TSP
[26]. These extracts were prepared by grinding seeds
using a mortar and pestle, mixing each mg of powder
with 7 μL of PBS-T buffer (10 mM sodium phosphate, 0.
15 M NaCl, 0.05% (v/v) Tween-20, pH 7.5). Crude seed
extracts were incubated on ice for 20 min, strongly
shaken for 20 min at 4 °C using a vortex and centrifuged
at 14000 gfor 30 min at 4 °C. TSP were recovered from
the supernatants and used for performing assays. Nega-
tive controls of digestion for all the samples were applied
by inactivating the enzyme at 95 °C for five minutes
before adding starch to the reaction system. Negative
controls were used to prove that the enzyme was
heat-inactivated and, thus, to give a background of in-
hibition to be used in calculations for inhibition level in
digestion systems without heat-inactivation. A. thaliana
seed extract controls were used to validate parameters of
the assay based on published data. This validation step
allows conclusions concerning N. benthamiana extracts,
such as the inhibition ability of AGA for the prepared
extracts, and the comparison of inhibition level for each
α-AIC3 against AGA. All of the reaction systems, i.e.,
digestions and negative controls, were performed in
three technical replicates. We used 100 μg of dried pro-
tein resuspended in 75 μL of AGA buffer containing 1
unit of AGA as a source of plant material for each reac-
tion. The absorbance values were recorded at 550 nm
using a SpectraMax 190 microplate reader (Molecular
Devices), and the samples were analysed using Excel
2007 software (Microsoft). Calculations were based on
discounting the absorbance values for respective
negative controls of digestion in each sample. Resulting
values were used as following: absorbance values for
samples containing α-AIC3 were discounted from the
values for samples without α-AIC3, and the mean of
triplicates indicated the level of activity remaining in
each system.
Biosafety analysis: Non-target species enzymes
Once the inhibitory activity of the N. benthamiana
extracts containing α-AIC3 was confirmed against AGA,
these samples were used for assaying the inhibitory
activity against enzymes of non-target species (Apis
mellifera amylase AMA and Spodoptera frugiperda
amylase SFA). Samples at concentrations of 0.5 g/mL
of ground whole insects were prepared using either
AMA buffer (150 mM succinic acid, 20 mM CaCl
mM NaCl, and 1 mM PMSF, pH 6.5) or SFA buffer (500
mM Tris-Cl, 20 mM CaCl
, 60 mM NaCl, and 1 mM
PMSF, pH 9.0) based on the recommended values of pH
for enzyme activity according to the literature [57,58].
The assays were performed following the same steps as
those of the AGA test, as well as 100 μg of protein from
the dialyzed N. benthamiana extracts was used. Since
there are no specific amylase inhibitors developed, set
and available for both insect species, comparison values
relative to the absence of activity for AMA and SFA
were exclusively derived from heat-inactivated enzyme
systems, similarly to the negative control for AGA. The
colour reactions were read at 550 nm, after which the
appropriate calculations were used to analyse samples
and inhibitory activities.
ACN: Acetonitrile; AGA: Anthonomus grandis α-amylase; AMA: Apis millefera
amylase; BSA: Bovine serum albumin; Bt:Bacillus thuringiensis; Cry: Crystal
toxin; DNS: 3,5-dinitrosalicylic acid; FW: Fresh weight; IR: Insect resistance;
MWCO: Molecular weight cut-off; PopMV: Poplar mosaic virus; PVX: Potato
virus; SFA: Spodoptera frugiperda amylase; TSP: Total soluble proteins; X
SEC: Size exclusion chromatography; α-AI: α-amylase inhibitor
Authors thank Dr. Leonardo Lima Pepino de Macedo (Embrapa Genetic
Resources and Biotechnology) for the experimental support and the kind
Prado et al. BMC Biotechnology (2019) 19:15 Page 11 of 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
provision of the bacterially derived β-subunit of the α-AIC3 for use as a
standard molecule in the ELISAs. Authors thanks Pr Jacques Simporé for
financial support.
GS was supported by the Coordination for the Improvement of Higher
Education Personnel (CAPES, CfP AFCAPES 201203). The research was co-
financed by the Agropolis Fondation under the reference ID 1203005 through
the Investissements davenirprogramme (Labex Agro: ANR-10-LABX-0001-01)
and by the Embrapa Genetic Resources and Biotechnology lab.
Availability of data and materials
All the data and material presented in the article are available from the
corresponding author upon reasonable request.
GS, JPB, MP and SL designed the experiments. GS, PK, VO, JA, JPB, MB and SL
performed the experiments and collected the data. FXG, MC, MP, CB, DS, SL
and MF supervised the experiments. GS, PK and SL interpreted the data and
wrote the article. MC, MP and MF supervised and complemented the
writing. All authors have read and approved the manuscript.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
Embrapa Genetic Resources and Biotechnology, Brasília, DF, Brazil.
University of Brasília, Brasília, DF, Brazil.
IRD, CIRAD, Université Montpellier,
Interactions Plantes Microorganismes et Environnement (IPME), Montpellier,
INERA/LMI Patho-Bios, Institut de LEnvironnement et de Recherches
Agricoles (INERA), Laboratoire de Virologie et de Biotechnologies Végétales,
Ouagadougou, Burkina Faso.
Received: 16 August 2018 Accepted: 4 March 2019
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... However, as compared to stable transgenic production of recombinant proteins in plants, a transient production system offers a faster and higher level of production (Yamamoto et al. 2018). Various industrially important proteins have already been successfully produced via transient expression in plants such as α-amylase (Prado et al. 2019), Interleukin-6 (Nausch et al. 2012), antibodies (Vézina et al. 2009), HIV-1 Nef (Lombardi et al. 2009), eVLPs (Sainsbury and Lomonossoff 2008), and even COVID-19 proteins (Lindsay et al. 2020). Despite its benefits, the plant expression platform has yet to overcome its major challenges in terms of mass production and profitability (Habibi et al. 2017). ...
Full-text available
The human basic fibroblast growth factor (bFGF) is a protein that plays a pivotal role in cellular processes like cell proliferation and development. As a result, it has become an important component in cell culture systems, with applications in biomedical engineering, cosmetics, and research. Alternative production techniques, such as transient production in plants, are becoming a feasible option as the demand continues to grow. High-level bFGF production was achieved in this study employing an optimized Agrobacterium-mediated transient expression system, which yielded about a 3-fold increase in production over a conventional system. This yield was further doubled at about 185 µg g⁻¹ FW using a mutant protease-resistant version that degraded/aggregated at a three-fold slower rate in leaf crude extracts. To achieve a pure product, a two-step purification technique was applied. The capacity of the pure protease-resistant bFGF (PRbFGF) to stimulate cell proliferation was tested and was found to be comparable to that of E. coli-produced bFGF in HepG2 and CHO-K1 cells. Overall, this study demonstrates a high-level transient production system of functional PRbFGF in N. benthamiana leaves as well as an efficient tag-less purification technique of leaf crude extracts. Fullsize Image
... AvrRpt2-HA and AvrRpt2 C122A -HA, expressed under the control of a 35S promoter, were a gift from Dr. Kee Hoon Sohn and have been described previously (Prokchorchik et al. 2020). p19 (silencing suppressor) expressed under the control of a 35S promoter has been described previously (Hamilton et al. 2002;Prado et al. 2019). RFP-OsRac1 expressed under the control of 35S promoter (pGDR vector backbone) was a gift from Dr. Guo-Liang Wang. ...
Full-text available
Key message RIN4 homologs from important crop species differ in their ability to prevent ectopic activity of the nucleotide binding-leucine rich repeat resistance protein, RPS2. Abstract Pathogens deploy virulence effectors to perturb host processes. Plants utilize intracellular resistance (R) proteins to recognize pathogen effectors either by direct interaction or indirectly via effector-mediated perturbations of host components. RPM1-INTERACTING PROTEIN4 (RIN4) is a plant immune regulator that mediates the indirect activation of multiple, independently evolved R-proteins by multiple, unrelated effector proteins. One of these, RPS2 (RESISTANT TO P. SYRINGAE2), is activated upon cleavage of Arabidopsis (At)RIN4 by the Pseudomonas syringae effector AvrRpt2. To gain insight into the AvrRpt2-RIN4-RPS2 defense-activation module, we compared the function of AtRIN4 with RIN4 homologs present in a diverse range of plant species. We selected seven homologs containing conserved features of AtRIN4, including two NOI (Nitrate induced) domains, each containing a predicted cleavage site for AvrRpt2, and a C-terminal palmitoylation site predicted to mediate membrane tethering of the proteins. Palmitoylation-mediated tethering of AtRIN4 to the plasma membrane and cleavage by AvrRpt2 are required for suppression and activation of RPS2, respectively. While all seven homologs are localized at the plasma membrane, only four suppress RPS2 when transiently expressed in Nicotiana benthamiana. All seven homologs are cleaved by AvrRpt2 and, for those homologs that are able to suppress RPS2, cleavage relieves suppression of RPS2. Further, we demonstrate that the membrane-tethered, C-terminal AvrRpt2-generated cleavage fragment is sufficient for the suppression of RPS2. Lastly, we show that the membrane localization of RPS2 is unaffected by its suppression or activation status.
... The efficiency of transient expression is largely affected by both A. tumefaciens strains and host tobacco species (Nausch et al. 2012;Prado et al. 2019;Shamloul et al. 2014). rGA733-2 protein was expressed at similar levels in N. tabacum plants, regardless of the A. tumefaciens strains used for transient expressions. ...
The colorectal carcinoma-associated protein GA733-2 is one of the representative candidate protein for the development of plant-derived colorectal cancer vaccine. Despite of its significant importance for colorectal vaccine development, low efficiency of GA733-2 production limits its wide applications. To improve productivity of GA733-2 in plants, we here tested multiple factors that affect expression of recombinant GA733-2 (rGA733-2) and rGA733 fused to fragment crystallizable (Fc) domain (rGA733-Fc) protein. The rGA733-2 and rGA733-Fc proteins were highly expressed when the pBINPLUS vector system was used for transient expression in tobacco plants. In addition, the length of interval between rGA733-2 and left border of T-DNA affected the expression of rGA733 protein. Transient expression analysis using various combinations of Agrobacterium tumefaciens strains (C58C1, LBA4404, and GV3101) and tobacco species (Nicotiana tabacum cv. Xanthi nc and Nicotiana benthamiana) revealed that higher accumulation of rGA733-2 and rGA733-Fc proteins were obtained by combination of A. tumefaciens LBA4404 and Nicotiana benthamiana. Transgenic plants generated by introduction of the rGA733-2 and rGA733-Fc expression cassettes also significantly accumulated corresponding recombinant proteins. Bioactivity and stability of the plant-derived rGA733 and rGA733-Fc were evaluated by further in vitro assay, western blot and N-glycosylation analysis. Collectively, we here suggest the optimal condition for efficient production of functional rGA733-2 protein in tobacco system.
... [61] Jongedijk et al. elucidated the biosynthesis pathway of MePA and reconstituted it in Nicotiana benthamiana (a more suitable expression system), including the expression of SdPAOMT. [61,62] Chemical oxidation of MePA was readily carried out in two steps to give TA. [60] Future expression of the pathway in E. coli or another suitable host would allow large scale production of Scheme 4. LAMT catalyses carboxyl group methylation to give loganin which is a biosynthetic precursor to both anticancer compounds vinblastine and vincristine. ...
Full-text available
The use of methyltransferases (MTs) in industrial biotechnology to replace toxic alkylating agents is of increasing interest. Carboxyl MTs (CMTs) are a subgroup of MTs that methylate the hydroxyl oxygen of carboxylic acids. Research initially focussed on their natural functions in protein regulation and production of volatile methyl esters in plants. In this review we highlight this potentially valuable group of enzymes that show promise for formation of a wide range of structurally diverse methyl esters from the parent acids under aqueous conditions. CMTs have been used to generate intermediates for biofuels, bioplastics and pharmaceuticals. These biocatalysts could also be integrated into cascades with other enzymes such as acyltransferases that function under aqueous conditions. Recent approaches for regenerating the required cofactor S‐adenosylmethionine (SAM) are discussed including in vitro recycling, improvement in in vivo production and the use of more stable analogues. Advances in these areas will further improve the potential to use carboxyl MTs in industrial biotechnology. Carboxyl methyltransferases (CMTs) methylate the hydroxyl oxygen of carboxylic acids. They are potentially useful biocatalysts in industrial biotechnology, facilitating the formation of methyl esters under aqueous conditions and could be integrated into multistep enzyme cascades, such as coupling with acyltransferases. CMTs have shown potential for the production of bioplastics, biodiesel and pharmaceuticals.
Plants subjected to phytopathogens can synthesize peptides with antimicrobial properties. In the last few decades, there has been an impressive increase in the prospection of antimicrobial peptides (AMPs) due to continuous demand for the development and manufacture of new antibiotics. In this setting, plants have attracted scientific and pharmaceutical interest and are considered promising AMPs biofactories. However, it is a great challenge to explore the diversity of actions needed to obtain a pharmaceutical product with AMPs derived from plants on a large scale. This review presents the last 5 years main findings on plants used as AMPs biofactories. Published works in this period were reviewed, and perspectives are presented on recombinant AMPs for drug production that appear in a plant‐based system, as well as products available on the market.
Full-text available
In recent years, plants have been shown to be an efficient alternative expression system for high-value pharmaceuticals such as vaccines. However, constitutive expression of recombinant protein remains uncertain on their level of production and biological activity. To overcome these problems, transitory expression systems have been developed. Here, a series of experiments were performed to determine the most effective conditions to enhance vaccine antigen transient accumulation in Nicotiana benthamiana leaves using the promastigote surface antigen (PSA) from the parasitic protozoan Leishmania infantum. This protein has been previously identified as the major antigen of a licensed canine anti-leishmaniasis vaccine. The classical prokaryote Escherichia coli biosystem failed in accumulating PSA. Consequently, the standard plant system based on N.benthamiana has been optimized for the production of putatively active PSA. First, the RNA silencing defense mechanism set up by the plant against PSA ectopic expression was abolished by using three viral suppressors acting at different steps of the RNA silencing pathway. Then, we demonstrated that the signal peptide at the N-terminal side of the PSA is required for its accumulation. The PSA ER signaling and retention with the PSA signal peptide and the KDEL motif, respectively were optimized to significantly increase its accumulation. Finally, we demonstrate that the production of recombinant PSA in N.benthamiana leaves allows the conservation of its immunogenic property. These approaches demonstrate that based on these optimizations, plant based systems can be used to effectively produce the biological active PSA protein.
Full-text available
Background Development of chimeric Cry toxins by protein engineering of known and validated proteins is imperative for enhancing the efficacy and broadening the insecticidal spectrum of these genes. Expression of novel Cry proteins in food crops has however created apprehensions with respect to the safety aspects. To clarify this, premarket evaluation consisting of an array of analyses to evaluate the unintended effects is a prerequisite to provide safety assurance to the consumers. Additionally, series of bioinformatic tools as in silico aids are being used to evaluate the likely allergenic reaction of the proteins based on sequence and epitope similarity with known allergens. Results In the present study, chimeric Cry toxins developed through protein engineering were evaluated for allergenic potential using various in silico algorithms. Major emphasis was on the validation of allergenic potential on three aspects of paramount significance viz., sequence-based homology between allergenic proteins, validation of conformational epitopes towards identification of food allergens and physico-chemical properties of amino acids. Additionally, in vitro analysis pertaining to heat stability of two of the eight chimeric proteins and pepsin digestibility further demonstrated the non-allergenic potential of these chimeric toxins. Conclusions The study revealed for the first time an all-encompassing evaluation that the recombinant Cry proteins did not show any potential similarity with any known allergens with respect to the parameters generally considered for a protein to be designated as an allergen. These novel chimeric proteins hence can be considered safe to be introgressed into plants. Electronic supplementary material The online version of this article (doi:10.1186/s12896-017-0384-z) contains supplementary material, which is available to authorized users.
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Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologs. To efficiently achieve site/gene-specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has exerted broad site-specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2)and an endogenous gene GhCLA1 were chosen as targets. The DsRed2 edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7% to 100% .The mutation genotype were checked for both genes with Sanger sequencing. Barcode-based high-throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1 edited T0 plants and it matched well with Sanger sequencing results. No off-target editing was detected at the potential off-target sites. These results proves that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing. This article is protected by copyright. All rights reserved.
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Genome editing has emerged as a technology with a potential to revolutionize plant breeding. In this study, we report on generating, in less than ten months, Tomelo, a non- transgenic tomato variety resistant to the powdery mildew fungal pathogen using the CRISPR/ Cas9 technology. We used wholegenome sequencing to show that Tomelo does not carry any foreign DNA sequences but only carries a deletion that is indistinguishable from naturally occurring mutations. We also present evidence for CRISPR/ Cas9 being a highly precise tool, as we did not detect off- target mutations in Tomelo. Using our pipeline, mutations can be readily introduced into elite or locally adapted tomato varieties in less than a year with relatively minimal effort and investment.
Full-text available
The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system was first identified in bacteria and archaea and can degrade exogenous substrates. It was developed as a gene editing technology in 2013. Over the subsequent years, it has received extensive attention owing to its easy manipulation, high efficiency, and wide application in gene mutation and transcriptional regulation in mammals and plants. The process of CRISPR/Cas is optimized constantly and its application has also expanded dramatically. Therefore, CRISPR/Cas is considered a revolutionary technology in plant biology. Here, we introduce the mechanism of the type II CRISPR/Cas called CRISPR/Cas9, update its recent advances in various applications in plants, and discuss its future prospects to provide an argument for its use in the study of medicinal plants.
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Abstract The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genome engineering that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing method, summarize the recent advances in CRISPR/Cas9 technology, and discuss their implications for plant research. To date, targeted gene knockout using the Cas9/gRNA system has been established in many plant species, and the targeting efficiency and capacity of Cas9 has been improved by optimizing its expression and that of its gRNA. The CRISPR/Cas9 system can also be used for sequence-specific mutagenesis/integration and transcriptional control of target genes. We also discuss off-target effects and the constraint that the protospacer-adjacent motif (PAM) puts on CRISPR/Cas9 genome engineering. To address these problems, a number of bioinformatic tools are available to help design specific gRNAs, and new Cas9 variants and orthologs with high fidelity and alternative PAM specificities have been engineered. Owing to these recent efforts, the CRISPR/Cas9 system is becoming a revolutionary and flexible tool for genome engineering. Adoption of the CRISPR/Cas9 technology in plant research would enable the investigation of plant biology at an unprecedented depth and create innovative applications in precise crop breeding.
To increase the tolerance of Chinese cabbage (Brassica campestris L. ssp. pekinensis) to Pieris rapae, we introduced a modified cowpea trypsin inhibitor (CpTI) gene, SCK, into various cultivars. SCK was derived from CpTI, an insect-resistance gene. The protein accumulating capacity of CpTI can be improved by adding a signal peptide sequence at the 5' end and an endoplasmic reticulum-detained signal sequence at the 3' end. Using an optimized Agrobacterium tumefaciens-mediated transformation system in Chinese cabbage, we obtained a maximum transformation efficiency of ~6.83%. Insect resistance tests and CpTI enzymatic assays showed that most of the transgenic plants had significant resistance to cabbage worm (Pieris rapae) larvae and that the plants with the highest levels of insect resistance had the greatest CpTI-related capacity, indicating a high correlation between SCK expression and insect resistance. An evaluation of segregation patterns in the independent transgenic line with the highest insect resistance, ‘ZB-08-04’, showed that kanamycin resistant versus sensitive plants segregated in a 3:1 Mendelian fashion. This study provides a potential germplasm resource for Chinese cabbage breeding in the future.
Genome editing in plants has been boosted tremendously by the development of the CRISPR/Cas9 technology. This powerful tool allows substantial improvement of plant traits in addition to those provided by classical breeding. Here we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) by utilizing Cas9/sgRNA technology to disrupt the recessive eIF4E gene function. Cas9/sgRNA constructs were targeted to the N' and C' terminus of the eIF4E gene. Small deletions and SNPs were observed in the eIF4E gene targeted sites of T1 generation transformed cucumber plants, but not in putative off-target sites. Non-transgenic heterozygous eIF4E mutant plants were selected for production of non-transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eIF4E sites exhibited immunity to Cucumber vein yellowing virus (ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W. In contrast, heterozygous-mutant and non-mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in the cucumber crop, non-transgenically, not visibly affecting plant development, and without long-term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants. This article is protected by copyright. All rights reserved.