In vitro uptake of 140 kDa Bacillus thuringiensis nematicidal crystal proteins by the second stage juvenile of Meloidogyne hapla.
ABSTRACT Plant-parasitic nematodes (PPNs) are piercing/sucking pests, which cause severe damage to crops worldwide, and are difficult to control. The cyst and root-knot nematodes (RKN) are sedentary endoparasites that develop specialized multinucleate feeding structures from the plant cells called syncytia or giant cells respectively. Within these structures the nematodes produce feeding tubes, which act as molecular sieves with exclusion limits. For example, Heterodera schachtii is reportedly unable to ingest proteins larger than 28 kDa. However, it is unknown yet what is the molecular exclusion limit of the Meloidogyne hapla. Several types of Bacillus thuringiensis crystal proteins showed toxicity to M. hapla. To monitor the entry pathway of crystal proteins into M. hapla, second-stage juveniles (J2) were treated with NHS-rhodamine labeled nematicidal crystal proteins (Cry55Aa, Cry6Aa, and Cry5Ba). Confocal microscopic observation showed that these crystal proteins were initially detected in the stylet and esophageal lumen, and subsequently in the gut. Western blot analysis revealed that these crystal proteins were modified to different molecular sizes after being ingested. The uptake efficiency of the crystal proteins by the M. hapla J2 decreased with increasing of protein molecular mass, based on enzyme-linked immunosorbent assay analysis. Our discovery revealed 140 kDa nematicidal crystal proteins entered M. hapla J2 via the stylet, and it has important implications in designing a transgenic resistance approach to control RKN.
- SourceAvailable from: Raffi V Aroian[show abstract] [hide abstract]
ABSTRACT: Plant-parasitic nematodes are major pests of agricultural crops, and it is becoming increasing urgent to find new methods to control them. Bacillus thuringiensis (Bt) has been used for decades as a biological control agent against insect pests and occurs as a natural pathogen of nematodes, but its potential to control nematode pests has been largely ignored. To test whether Bt can be used for control against nematodes, we decided to express the active component of Bt, namely an ingestible crystal (Cry) protein, inside plant roots where the nematodes feed. Truncation studies here demonstrate that the first 2094 nucleotides of the nematicidal Cry protein, Cry5B, are sufficient for toxicity against a free-living nematode. Based on these results, a plant codon-modified cry5B gene was synthesized and transformed into tomato hairy roots. Cry5B expression in hairy roots was assessed at the mRNA level and quantified at the protein level. Characterization and quantification of root-knot nematode infections on control versus Cry5B-expressing roots indicate that Cry5B intoxicates the parasitic nematode as judged by inhibition of the nematode to establish or maintain root-knot sites and as judged by a nearly 3-fold reduction in progeny production. We demonstrate for the first time that a three-domain Cry protein can provide protection against a plant-endoparasitic nematode and that Cry proteins as a group have excellent potential as control agents of plant-parasitic nematodes.Biological Control. 01/2008;
- [show abstract] [hide abstract]
ABSTRACT: The recent de-registration of several chemical nematicides and the impending loss of methyl bromide from the pest-control market necessitate the development of new methods for controlling nematode-induced crop damage. One approach for developing novel target-specific controls is by exploiting fundamental differences between the biological processes of nematodes and their host plants. Researchers of the Agricultural Research Service (ARS) of the US Department of Agriculture are actively exploring these differences. Research accomplishments include the discovery of heat shock protein genes possibly involved in developmental arrest of the soybean cyst nematode, the identification of neuropeptides and female-specific proteins in the soybean cyst nematode, the disruption of nematode reproduction with inhibitors of nematode sterol metabolism, the development of novel morphological and molecular (heat shock protein genes and the D3 segment of large subunit ribosomal DNA) features useful for nematode identification and classification, and the elucidation of the population genetics of potato cyst nematode pathotypes. In addition, several ARS researchers are investigating biological determinants of nematode response to management strategies utilized in agricultural fields. These collective efforts should lead to new chemical and non-chemical alternatives to conventional nematode control strategies.Pest Management Science 01/2003; 59(6-7):748-53. · 2.59 Impact Factor
Article: The genomes of root-knot nematodes.[show abstract] [hide abstract]
ABSTRACT: Plant-parasitic nematodes are the most destructive group of plant pathogens worldwide and are extremely challenging to control. The recent completion of two root-knot nematode genomes opens the way for a comparative genomics approach to elucidate the success of these parasites. Sequencing revealed that Meloidogyne hapla, a diploid that reproduces by facultative, meiotic parthenogenesis, encodes approximately 14,200 genes in a compact, 54 Mpb genome. Indeed, this is the smallest metazoan genome completed to date. By contrast, the 86 Mbp Meloidogyne incognita genome encodes approximately 19,200 genes. This species reproduces by obligate mitotic parthenogenesis and exhibits a complex pattern of aneuploidy. The genome includes triplicated regions and contains allelic pairs with exceptionally high degrees of sequence divergence, presumably reflecting adaptations to the strictly asexual reproductive mode. Both root-knot nematode genomes have compacted gene families compared with the free-living nematode Caenorhabditis elegans, and both encode large suites of enzymes that uniquely target the host plant. Acquisition of these genes, apparently via horizontal gene transfer, and their subsequent expansion and diversification point to the evolutionary history of these parasites. It also suggests new routes to their control.Annual Review of Phytopathology 02/2009; 47:333-51. · 10.23 Impact Factor
In Vitro Uptake of 140 kDa Bacillus thuringiensis
Nematicidal Crystal Proteins by the Second Stage
Juvenile of Meloidogyne hapla
Fengjuan Zhang., Donghai Peng., Xiaobo Ye, Ziquan Yu, Zhenfei Hu, Lifang Ruan, Ming Sun*
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
Plant-parasitic nematodes (PPNs) are piercing/sucking pests, which cause severe damage to crops worldwide, and are
difficult to control. The cyst and root-knot nematodes (RKN) are sedentary endoparasites that develop specialized
multinucleate feeding structures from the plant cells called syncytia or giant cells respectively. Within these structures the
nematodes produce feeding tubes, which act as molecular sieves with exclusion limits. For example, Heterodera schachtii is
reportedly unable to ingest proteins larger than 28 kDa. However, it is unknown yet what is the molecular exclusion limit of
the Meloidogyne hapla. Several types of Bacillus thuringiensis crystal proteins showed toxicity to M. hapla. To monitor the
entry pathway of crystal proteins into M. hapla, second-stage juveniles (J2) were treated with NHS-rhodamine labeled
nematicidal crystal proteins (Cry55Aa, Cry6Aa, and Cry5Ba). Confocal microscopic observation showed that these crystal
proteins were initially detected in the stylet and esophageal lumen, and subsequently in the gut. Western blot analysis
revealed that these crystal proteins were modified to different molecular sizes after being ingested. The uptake efficiency of
the crystal proteins by the M. hapla J2 decreased with increasing of protein molecular mass, based on enzyme-linked
immunosorbent assay analysis. Our discovery revealed 140 kDa nematicidal crystal proteins entered M. hapla J2 via the
stylet, and it has important implications in designing a transgenic resistance approach to control RKN.
Citation: Zhang F, Peng D, Ye X, Yu Z, Hu Z, et al. (2012) In Vitro Uptake of 140 kDa Bacillus thuringiensis Nematicidal Crystal Proteins by the Second Stage
Juvenile of Meloidogyne hapla. PLoS ONE 7(6): e38534. doi:10.1371/journal.pone.0038534
Editor: Carlos Eduardo Winter, Universidade de Sa ˜o Paulo, Brazil
Received November 18, 2011; Accepted May 7, 2012; Published June, 2012
Copyright: ? 2012 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the National High Technology Research and Development Program (863) of China (2011AA10A203 and 2006AA02Z174),
the National Basic Research Program (973) of China (2009CB118902), the National Natural Science Foundation of China (30870066), the Genetically Modified
Organisms Breeding Major Projects of China (2009ZX08009-032B), China 948 Program of Ministry of Agriculture (2011-G25) and Ministry of Forestry (2006-4-41)
and the Technology Program of Wuhan (200850731360). The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Plant-parasitic nematodes (PPNs) are the primary pathogens of
potato, sugar beet, soybean, tomato and other crops , and cause
an estimated annual economic loss of $125 billion worldwide .
This damage is mainly caused by cyst nematodes (Heterodera and
Globodera spp) and root-knot nematodes (Meloidogyne spp.). Both
Meloidogyne hapla and Meloidogyne incognita are highly destructive
root-knot nematode species, and their genomes have been
sequenced . Both these groups of nematodes are sedentary
endoparasites and are difficult to control. They live underground
and spend most of their lives in the roots, which can offer
protection against chemical nematicides . While chemical
nematicides remain the most current means of controlling root-
knot nematodes , their use is declining, because of their toxic
effects towards humans and the environment .
Bacillus thuringiensis is a rod-shaped, Gram-positive, spore-
forming bacterium that forms parasporal crystals during the
stationary phase of growth . The crystal proteins produced by
some of B. thuringiensis are pore-forming toxins which are lethal
against insects and some nematodes [7,8]. Nematicidal activity has
been found in several families of B. thuringiensis crystal proteins,
such as Cry5, Cry6, Cry12, Cry13, Cry14, Cry21, and Cry55 .
Li et al. reported that Cry6A expressed in transgenic roots
significantly impaired the ability of M. incognita to reproduce .
In addition, a truncated 79 kDa Cry5B expressed in transgenic
roots significantly reduced the number of M. incognita galls and
reduced progeny levels by nearly 3-fold . Until now, our group
has isolated several specific B. thuringiensis strains which showed
high activity against plant-parasitic nematodes [9,11]. Subse-
quently, three nematicidal crystal protein encoding genes, cry6Aa2,
cry5Ba2, and cry55Aa1, were isolated from the highly nematicidal
B. thuringiensis strain YBT-1518  (Table 1). Bioassay results
showed that these three crystal proteins were highly toxic to
second-stage juveniles (J2) of M. hapla , and a combination of
Cry6Aa and Cry55Aa showed significant synergistic toxicity
against M. incognita .
Plant-parasitic nematodes (PPNs) feed using a specialized
stylet. During feeding a tube is produced that acts as a sieve
which can only permit the proteins of particular size and
dimension to enter the nematode . In beet cyst nematode
Heterodera schachtii this has been found to be 28 kDa and is
referred to as the exclusion limit . However, to date, the
exclusion limit of B. thuringiensis crystal proteins entering root-
PLoS ONE | www.plosone.org1 June 2012 | Volume 7 | Issue 6 | e38534
knot nematodes have not been reported. Investigating whether
or not crystal proteins can enter root-knot nematodes would
help to define the molecular exclusion limit and would facilitate
the design of a transgenic resistance approach to control root-
knot nematodes . In this study, we monitored the pathway
of B. thuringiensis crystal proteins entering M. hapla J2 by
confocal laser scanning microscopy (CLSM). Then we detected
the changes in the molecular mass of crystal proteins entered M.
hapla J2 by Western blot. While, the uptake efficiency of the
crystal proteins by the M. hapla J2 was tested by enzyme-linked
immunosorbent assay analysis (ELISA).
Use of Resorcinol to Improve B. thuringiensis Crystal
The previous bioassays used to assess crystal proteins targeting
M. hapla were conducted with the addition of tomato root exudates
(TRE), which potentially increases the frequency of stylet thrusting
[15,16]. In addition, resorcinol stimulates the uptake of double
stranded ribonucleic acid (dsRNA) during in vitro RNA interfer-
ence (RNAi) for M. incognita J2 . To monitor the role of
resorcinol during this bioassay, different concentrations of
resorcinol were evaluated to assess its toxicity against M. hapla
and its effects on stylet thrusting frequency stimulation (Data not
shown). The optimum final concentration of resorcinol was
determined to be 1 mg/ml.
For Cry55Aa, the dose at which the intoxicated (%) is reduced
to 50% is 10.0 mg/ml in resorcinol, 25.2 mg/ml in TRE,
261.3 mg/ml in ddH2O. For Cry6Aa, it is 13.2 mg/ml in
resorcinol, 32.6 mg/ml in TRE, 302.1 mg/ml in ddH2O. For
Cry5Ba, it is 7.6 mg/ml in resorcinol, 16.1 mg/ml in TRE,
156.3 mg/ml in ddH2O (Figure 1). These data indicate that,
compared with TRE, resorcinol improved the nematicidal activity
of crystal proteins in our M. hapla bioassay.
NHS-rhodamine Labeled B. thuringiensis Crystal Proteins
with Different Molecular Mass (45–140 kDa) can Enter
M. hapla J2 via the Stylet
To confirm the entry pathway of nematicidal crystal proteins,
M. hapla J2 were incubated in rhodamine-labeled crystal proteins
for different periods of time. To confirm whether the rhodamine
labeled crystal proteins were active proteins, M. hapla J2 were
exposed to crystal protein and rhodamine labeled crystal protein
respectively in the presence of resorcinol. We found that
rhodamine labeled Cry55Aa, Cry6Aa, and Cry5Ba has reduced
toxicity to M. hapla J2 compared with the non-labeled crystal
proteins (Figure 2). The rhodamine 6G was used as a control and
it showed no toxicity to M. hapla J2 even at the concentration of
800 nM (Figure 2D).
The signals from rhodamine-labeled crystal proteins were then
monitored by CLSM. M. hapla J2 fed with rhodamine 6G
(400 nM) alone were used as control. The results are shown in
Figure 3, Figure S1 and Figure S2. The photographs were
captured under fluorescence illumination (left), bright-field (mid-
dle), and merge (right). Due to the molecular exclusion limits of the
nematode, two smaller nematicidal crystal proteins Cry55Aa
(45 kDa) and Cry6Aa (54 kDa) were initially selected to detect
their entry pathway. CLSM showed that Cry55Aa were initially
detected in the stylet and esophageal lumen at 12 hours post
ingested (hpi), and subsequently in the gut from 36 to 72 hpi in the
presence of resorcinol (Figure S1A) or TRE (Figure S2A). The
movement of the Cry6Aa toxin through M. hapla (Figure S1B and
Figure S2B) was identical to that for Cry55Aa. Rhodamine 6G
alone was detected in the stylet, esophageal lumen and gut of M.
hapla J2 at 12 hpi, and the fluorescence was more apparent in gut
from 36 to 72 hpi (Figure S1D and Figure S2D). These
observations demonstrated that the smaller molecular mass
proteins Cry55Aa and Cry6Aa could enter M. hapla J2 via the
To test whether larger molecular mass nematicidal crystal
proteins could enter M. hapla, similarly experiments were
performed by using Cry5Ba (140 kDa). CLSM showed that
Cry5Ba were initially detected in the stylet and esophageal lumen
at 22 hpi, and subsequently in the gut from 50 to 96 hpi in the
presence of resorcinol (Figure S1C) or TRE (Figure S2C). These
results demonstrated that the larger molecular mass proteins
Cry5Ba could also enter M. hapla J2 via the stylet.
The Molecular Mass of Nematicidal Crystal Proteins
become Larger After Ingested by M. hapla J2
To monitor the changes in the nematicidal crystal proteins after
ingestion, M. hapla J2 were fed purified Cry6Aa, Cry55Aa, and
Cry5Ba proteins in the presence of resorcinol at different times.
Total proteins were then extracted from crystal protein treated
nematodes, separated by SDS-PAGE, and subjected to Western
blot analysis using an anti-crystal proteins antibody.
Western blot revealed that the molecular mass of Cry6Aa
became larger (Figure 4B), approximately 60-kDa at 12 hpi and
70-kDa at 36 hpi. Similarly, the molecular mass of Cry55Aa
became larger as well (Figure 4A), in addition to the main 45-
kD signal band, signal bands corresponding to approximately
90-kD and 150-kD from 12 hpi till to 72 hpi were observed.
The Cry5Ba was also modified after being ingested by M. hapla
J2. An approximately 60-kDa band was observed between
22 hpi and 50 hpi. The main band subsequently increased to
about90-kDa and 250-kDa at 96 hpi (Figure
determine whether the 60-kDa Cry5Ba toxin formed before
or after ingestion, total proteins were extracted from treated M.
hapla at 12 hpi, an earlier time than the former 22 hpi. Western
blot results indicated a 140 kDa band was present at this time
(Figure 4F), suggesting that a 140 kDa form of Cry5Ba entered
M. hapla J2 directly through the stylet. Based on the above
information, we concluded that M. hapla J2 can ingest 140 kDa
Table 1. The information of Cry55Aa, Cry6Aa, and Cry5Ba used in this study.
StrainCrystal proteinsMolecular mass Susceptible hostSource
BMB0250Cry55Aa45 kDaM. hapla, M. incognita, and C. elegans [9,12]
BMB0215Cry6Aa 54 kDaM. hapla, M. incognita, and C. elegans [9,11,12]
BMB0224 Cry5Ba140 kDaM. hapla, M. incognita, and C. elegans[9,12]
Meloidogyne Can Ingest 140 kDa Proteins
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Uptake Efficiency of Crystal Proteins by the M. hapla J2
Stylet Decreases with Increasing Protein Molecular Mass
The time, taken for B. thuringiensis crystal proteins to enter the
stylet and esophageal lumen or move into the nematode gut, was
different between small and large molecular mass crystal proteins
(Figure S1, Figure S2, and Figure 3). We further confirmed the
relationship between uptake efficiency and molecular mass of
crystal proteins by feeding M. hapla J2 with 1500 ng/ml purified
Cry55Aa, Cry6Aa, and Cry5Ba for 96 h. The protein concentra-
tion after ingestion was then tested by ELISA. The calculated
uptake efficiency by M. hapla J2 of Cry55Aa (45 kDa), Cry6Aa
(54 kDa), and Cry5Ba (140 kDa) proteins were 78.3%, 69.5%,
and 17.2%, respectively (Figure 5). These data showed the uptake
efficiency of crystal proteins by M. hapla J2 stylet decreased with
increasing protein molecular mass.
The original bioassay for the detection of crystal proteins
targeting M. hapla was conducted with the addition of TRE .
TRE can attract nematodes to plant roots, induce stylet
thrusting, release of secretions and increase in nematode
mobility . In this study, we improved the B. thuringiensis
crystal proteins bioassay protocol for M. hapla J2 by using
resorcinol instead of TRE. Resorcinol was previously used to
stimulate the uptake of dsRNA during in vitro RNAi of M.
incognita J2 . Compared with TRE, resorcinol is simple and
more stable, and may improve the crystal nematicidal activity
during the M. hapla bioassay (Figure 1).
The pathway of B. thuringiensis crystal proteins entering M.
hapla is still not clear. Cuticle penetration is predominately
believed to be the primary action mode of the extracellular
damage the nematode cuticle [18,19]. Initially we conjectured
that crystal proteins were able to enter M. hapla J2 through
nematode cuticle. However, in this in vitro study, our results
confirmed that M. hapla J2 could ingest a range of proteins sizes
from 45 kDa to 140 kDa directly through the stylet (Figure 3
and Figure 4). When the head of M. hapla J2 was magnified, the
signal of Cry5Ba around stylet and esophageal was also
detected, but it was weaker in comparison with that in stylet
and esophageal lumen (Figure S1C1 and Figure S1C2). So Cry
protein could enter mainly through the stylet, it can also enter
through the mouth and flowing around the stylet but still
entering. Also, we found the uptake efficiency of crystal proteins
Figure 1. The dose response curves of Cry6Aa, Cry55Aa, and Cry5Ba against M. hapla J2 in the presence of resorcinol (RES) or
tomato root exudates (TRE). The bioassay of three nematicidal crystal protein Cry6Aa (A), Cry55Aa (B), Cry5Ba (C) against M. hapla J2 were
conducted in the presence of resorcinol (RES), or tomato root exudates (TRE), or ddH2O (CK), respectively. A non-nematicidal crystal protein Cry1Ac
(D) was treated as the same and used as control. The M. hapla J2 were exposed to five doses of each crystal proteins. Data shown represent the
percentage of animals that were intoxicated when fed crystal proteins. Error bars represent the S.D. from the mean of averages over three
independent experiments. Each data point represents the average size of 60 animals. The mortality was 3.3% in the absence of any toxins.
Meloidogyne Can Ingest 140 kDa Proteins
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by M. hapla J2 decreased with increasing protein molecular
mass. This situation was similar to the previous reports that
fluorescent molecule diffusion speed in the syncytium was
dependent upon its size . About the uptake size of M. hapla
J2, We found 140 kDa Cry5B can enter M. hapla J2 and the
uptake efficiency was very low (17.2%). However, we did not
assess proteins larger than 140 kDa, maybe larger proteins
could also enter M. hapla J2 stylet.
It’s known that the secretions of cyst, root-knot and a few other
sedentary endoparasitic nematodes produced a feeding tube at the
interface between the syncytial cytoplasm and the nematode’s
stylet [20,21]. Differences in molecular exclusion limits of the cyst
nematode H. schachtii and the root-knot nematodes may be due to
the variation in the ultra-structure and size of their feeding tubes
. It is reported that the beet cyst nematode H. schachtii was
unable to ingest proteins larger than 28 kDa . Goverse et al.
reported that Globodera rostochiensis juveniles could ingest 32 kDa
proteins . While, Cry6A and a truncated 79 kDa Cry5B
expressed in transgenic roots significantly impaired the ability of
M. incognita to reproduce [1,10], indicating the feeding tube of M.
incognita can uptake a protein of 79 kDa in vivo.
In the experimental system described here, we demonstrated
that different sized B. thuringiensis crystal proteins can enter M. hapla
J2 through the stylet. Although this in vitro experiment may not be
applicable to feeding tubes produced in vivo, it would suggest that
140 kDa cry proteins if produced as extracellular secreted protein
by a transgenic plant could be taken up by the nematode. Stylet
thrusting is a natural phennomenon induced by TRE. One could
imagine that during the migrating phase of the J2 within the root
system it is exposed to extracellular root secretions most if not all of
which are also present in TRE. Therefore, it can be envisaged that
cry proteins can be expressed as extracellular plant secretions. Our
discovery has important implications in controlling M. hapla J2
during the migratory phase of the second stage juvenile before the
J2 becomes sedentary and sets up its feed site and the subsequent
formation of its feeding tube.
In summary, we demonstrated in an in vitro system that 140 kDa
B. thuringiensis crystal proteins can enter M. hapla J2 through the
stylet. It has important implications for the design of any
transgenic resistance approach against M. hapla.
Materials and Methods
All the procedures related to animal housing, handling, care and
treatment in this study were approved by the Laboratory Animal
Monitoring Committee of Hubei province of China and
performed accordingly, the approval ID: SYXK 2005–0029.
Bacterial Strains and Media
B. thuringiensis strains BMB0250, BMB0224, and BMB0215 
were used for the preparation of nematicidal crystal proteins
Cry55Aa, Cry5Ba, and Cry6Aa, respectively. All B. thuringiensis
Figure 2. Activities of rhodamine labeled Cry6Aa, Cry55Aa, and Cry5Ba against M. hapla J2. M. hapla J2 were exposed to three doses of
non-labeled crystal protein or rhodamine labeled Cry6Aa (A), Cry55Aa (B), Cry5Ba (C), and rhodamine-6G (D) in the presence of resorcinol. M. hapla J2
were exposed to three doses of crystal protein. Data shown represent the percentage of animals that were intoxicated when fed crystal proteins or
rhodamine labeled crystal protein. Each data point represents the average size of 60 animals. Error bars represent the S.D. from the mean of averages
over three independent experiments.
Meloidogyne Can Ingest 140 kDa Proteins
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strains were maintained on Luria-Bertani (LB) agar plates and
supplemented with appropriate antibiotics at 28uC .
M. hapla Rearing and Bioassay
The M. hapla bioassay procedure was undertaken according to
the method described by Bischof et al . The toxicity of crystal
proteins against M. hapla J2 was tested by touching the worms
directly, typically 3 times or so, and then looking for motility. A
visibly moving nematode was marked as alive. Nematodes that
were not moving were gently touched with a platinum pick and
watched for movement. Nematodes that failed to respond after
several touches were marked as dead or intoxicated .
Sixty M. hapla J2 larvae were individually placed into each
well of 96-well microtiter plates (Corning, 3513). Resorcinol
Figure 3. The pathway of nematicidal crystal proteins entering M. hapla J2. The confocal laser scanning microscope image showed the
ingestion manner and process of Cry55Aa (A), Cry6Aa (B), or Cry5Ba (C) by M. hapla J2 in the presence of resorcinol (Res) or tomato root exudates
(TRE). M. hapla J2 were incubated in rhodamine-labeled crystal toxins for three different times then imaged using a merged image. The rhodamine 6G
(D) was treated as the same and used as a control. Toxin was detected inside the treated M. hapla J2, but not in the control (CK). The anterior of M.
hapla is positioned within the upper region. Abbreviation: s=stylet; el=esophageal lumen; h=head of M. hapla J2. The scale bar of all the images is
Meloidogyne Can Ingest 140 kDa Proteins
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(1 mg/ml)  or TRE [15,16] was used to induce an increase
in stylet pulsing frequency. Cry1Ac was used as the control.
The dose at which the intoxicated (%) is reduced to 50% of the
crystal toxin against M. hapla J2 was evaluated using SAS 8.0
Crystal Protein Purification and Labeling
Cry55Aa, Cry5Ba, and Cry6Aa proteins were purified accord-
ing to the method described by Guo et al. . All purified protein
samples were then solubilized in 20 mM HEPES (Calbiochem
BB0364) (pH 8.0), quantified , and stored at 280uC. Purified
proteins were labeled with N-hydroxysuccinimide–rhodamine
(Pierce 46102) according to the method described by Griffitts
et al. .
Confocal Laser Scanning Microscope (CLSM) Detection
M. hapla were incubated in M9 medium  containing 2 mg/
ml proteins for each time period and washed five times in M9
medium before confocal laser scanning microscopy (CLSM; Zeiss
LSM 510) imaging . Resorcinol (1 mg/ml) or TRE was added
to induce stylet pulsing frequency. Images were captured using a
406objective. Fluorescence was monitored at an excitation wave-
Figure 4. Detection of size changes of Cry55Aa, Cry6Aa, and Cry5Ba in M. hapla J2 by Western blot analysis. M. hapla J2 were incubated
with Cry55Aa protein (Panel A) and Cry6Aa protein (Panel B), and then detected by Western blot at 0, 12, 36, and 72 hpi, using an anti-crystal
antibody; M. hapla J2 were incubated with Cry5Ba protein (Panel D, F) and then detected by Western blot at 0, 12, 22, 50, and 96 hpi, using Cry5Ba
protein antibody. Line CK: Controls of crystal protein without being incubated by M. hapla J2. Panel C: SDS-PAGE showed the molecular mass of
purified Cry5Ba. Panel E: the Cry5Ba was incubated by M. hapla J2 for 12 h, centrifuged at 12000 rpm for 10 min to remove M. hapla J2, and then
detected by Western blot using Cry5Ba antibody.
Figure 5. Enzyme-linked immunosorbent assay analysis of the
uptake efficiency of nematicidal crystal proteins by M. hapla J2.
The uptake efficiency was determined by subtracting the percentage
crystal proteins uptake in the absence of resorcinol from that in the
presence of resorcinol. Each bar value represents the mean SD of
Meloidogyne Can Ingest 140 kDa Proteins
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length of 543 nm and a high pass filter (LP 560). Images were
merged and data were stored.
Western Blot Analysis
M. hapla treated with purified crystal proteins for different times
were harvested and subsequently washed five times in M9 medium
. Treated M. hapla samples were grinded with liquid nitrogen,
and then subjected to sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and Western blot analysis .
Antisera of Cry55Aa, Cry5Ba, and Cry6Aa were prepared
according to the following procedure: purified crystal proteins
were separated by SDS-PAGE and the gels were stained and
destained. Purified crystal proteins bands were then excised from
the gel, washed three times with water for 5 min each time, and
used to immunize rabbits for antibody development according to
standard protocols . The protocol used to immunize the
rabbits was described in the supporting method (see Protocol S1).
Enzyme-linked immunosorbent assay (ELISA) was conducted
according to the protocol described by Huang et al.  with
some modifications. ELISA plates were incubated at 4uC for 12 h
with different concentrations of crystal proteins in 20 mM HEPES
(pH 8.0) and washed five times with PBST (135 mM NaCl,
2 mM KCl, 10 mM Na2HPO4, 1.7 mM KH2PO4, pH 7.5, 0.1%
Tween-20). Plates were then blocked in 200 ml PBST plus 2%
BSA for 2 h at room temperature (RT), and washed five times
with PBST. ELISA plates were incubated with anti-crystal protein
antibody (1:1000) for 2 h at RT, followed by a secondary goat-
anti-rabbit-horseradish peroxidase (HRP) antibody for 2 h at RT.
The HRP enzymatic activity was determined using a freshly
prepared substrate (100 ml TMB) at RT for 40 min. Then the
enzymatic reaction was stopped with 100 ml 2 M H2SO4, and the
absorbance was read at 450 nm.
Detection of Crystal Proteins Uptake Efficiency
M. hapla J2 were fed on 1500 ng/ml purified crystal proteins in
the presence or absence of resorcinol for 96 h. Crystal protein
concentration after ingestion was determined from standard curves
by ELISA. Uptake efficiency was determined by subtracting
crystal proteins uptake percentage in the absence of resorcinol
from that in the presence of resorcinol.
entering M. hapla J2 in the presence of resorcinol.
Confocal laser scanning microscope image showing ingestion of
Cry55Aa (A), Cry6Aa (B), Cry5Ba (C), or rhodamine 6G (D) in
treated M. hapla J2 in the presence of resorcinol. M. hapla J2 were
incubated in rhodamine-labeled crystal toxins for three different
times, then imaged using the bright-field to visualize the M. hapla
(Middle), the rhodamine channel to visualize toxin (Left) and
merged image (Right). Toxin was detected inside the treated M.
hapla, but not in the control (CK). The anterior of M. hapla is
positioned within the upper region. s=stylet; el=esophageal
lumen; h=head of M. hapla J2; C1 and C2: the magnification of
head of M. hapla J2. The scale bar of C1 and C2 is 5.93 mm. The
scale bar of other images is 40.43 mm.
The pathway of nematicidal crystal proteins
entering M. hapla J2 in the presence of tomato root
exudates. Confocal laser scanning microscope image showing
ingestion of Cry55Aa (A), Cry6Aa (B), Cry5Ba (C), or rhodamine
6G (D) in treated M. hapla J2 in the presence of tomato root
exudates. M. hapla J2 were incubated in rhodamine-labeled crystal
toxins for different times, then imaged using the bright-field to
visualize the M. hapla (Middle), the rhodamine channel to visualize
toxin (Left), and merged image (Right). Toxin was detected inside
the treated M. hapla, but not in the control (CK). The anterior of
M. hapla was positioned within the upper region. The scale bar of
all the images is 40.43 mm.
The pathway of nematicidal crystal proteins
Supporting Method. Preparation of antiserum.
Conceived and designed the experiments: FZ MS. Performed the
experiments: FZ XY ZY ZH. Analyzed the data: FZ DP MS. Contributed
reagents/materials/analysis tools: MS. Wrote the paper: FZ DP LR MS.
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