In Vitro Uptake of140kDa 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*大众卫生报
State Key Laboratory of Agricultural Microbiology,College of Life Science and Technology,Huazhong Agricultural University,Wuhan,China
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 than28kDa.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 pro
teins(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 J2decreased with increasing of protein molecular mass,based on enzyme-linked immunosorbent assay analysis.Our discovery revealed140kDa nematicidal crystal proteins entered M.hapla J2via 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 of140kDa Bacillus thuringiensis Nematicidal Crystal Proteins by the Second Stage Juvenile of Meloidogyne hapla.PLoS ONE7(6):e38534.doi:10.1371/journal.pone.0038534
Editor:Carlos Eduardo Winter,Universidade de Sa˜o Paulo,Brazil
Received November18,2011;Accepted May7,2012;Published June,2012
Copyright:ß2012Zhang 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(2011AA10A203and2006AA02Z174), 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),China948Program 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.
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Competing Interests:The authors have declared that no competing interests exist.*E-mail:m98sun@mail.hzau.edu走近女局长
.These authors contributed equally to this work.
Introduction
Plant-parasitic nematodes(PPNs)are the primary pathogens of potato,sugar beet,soybean,tomato and other crops[1],and cause an estimated annual economic loss of$125billion worldwide[2]. This damage is mainly caused by cyst nematodes(Heterodera and Globodera spp)and root-knot nematod
es(Meloidogyne spp.).Both Meloidogyne hapla and Meloidogyne incognita are highly destructive root-knot nematode species,and their genomes have been sequenced[3].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[1].While chemical nematicides remain the most current means of controlling root-knot nematodes[4],their use is declining,because of their toxic effects towards humans and the environment[5].
Bacillus thuringiensis is a rod-shaped,Gram-positive,spore-forming bacterium that forms parasporal crystals during the stationary phase of growth[6].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[9]. Li ported that Cry6A expressed in transgenic roots significantly impaired the ability of M.incognita to reproduce[10]. In addition,a truncated79kDa Cry5B expressed in transgenic roots significantly reduced the number of M.incognita galls and reduced progeny levels by nearly3-fold[1].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[9](Table1).Bioassay results showed that these three crystal proteins were highly toxic to second-stage juveniles(J2)of M.hapla[9],and a combination of Cry6Aa and Cry55Aa showed significant synergistic toxicity against M.incognita[12].
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[13].In beet cyst nematode Heterodera schachtii this has been found to be28kDa and is referred to as the exclusion limit[14].However,to date,the exclusion limit of B.thuringiensis crystal proteins entering root-
21
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[14].In this study,we monitored the pathway of B.thuringiensis crystal proteins entering M.hapla J2by confocal laser scanning microscopy(CLSM).Then we detected the changes in the molecular mass of crystal proteins entered
M. hapla J2by Western blot.While,the uptake efficiency of the crystal proteins by the M.hapla J2was tested by enzyme-linked immunosorbent assay analysis(ELISA).
杠杆舞Results
Use of Resorcinol to Improve B.thuringiensis Crystal Protein Efficacy
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[17].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 be1m g/ml.
For Cry55Aa,the dose at which the intoxicated(%)is reduced to50%is10.0m g/ml in resorcinol,25.2m g/ml in TRE, 261.3m g/ml in ddH2O.For Cry6Aa,it is13.2m g/ml in resorcinol,32.6m g/ml in TRE,302.1m g/ml in ddH2O.For Cry5Ba,it is7.6m g/ml in resorcinol,16.1m g/ml in TRE, 156.3m g/ml in ddH2O(Figure1).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(45–140kDa)can Enter M.hapla J2via the Stylet
To confirm the entry pathway of nematicidal crystal proteins, M.hapla J2were incubated in rhodamine-labeled crystal proteins for different periods of time.To confirm whether the rhodamine labeled crystal proteins were active proteins,M.hapla J2were 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 J2compared with the non-labeled crystal proteins(Figure2).The rhodamine6G was used as a control and it showed no toxicity to M.hapla J2even at the concentration of 800nM(Figure2D).
The signals from rhodamine-labeled crystal proteins were then monitored by CLSM.M.hapla J2fed with rhodamine6G (400nM)alone were used as control.The results are shown in Figure3,Figure S1and 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 (45kDa)and Cry6Aa(54kDa)were initially selected to detect thei
r entry pathway.CLSM showed that Cry55Aa were initially detected in the stylet and esophageal lumen at12hours post ingested(hpi),and subsequently in the gut from36to72hpi 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.Rhodamine6G alone was detected in the stylet,esophageal lumen and gut of M. hapla J2at12hpi,and the fluorescence was more apparent in gut from36to72hpi(Figure S1D and Figure S2D).These observations demonstrated that the smaller molecular mass proteins Cry55Aa and Cry6Aa could enter M.hapla J2via the stylet.
To test whether larger molecular mass nematicidal crystal proteins could enter M.hapla,similarly experiments were performed by using Cry5Ba(140kDa).CLSM showed that Cry5Ba were initially detected in the stylet and esophageal lumen at22hpi,and subsequently in the gut from50to96hpi 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 J2via 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 J2were 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(Figure4B),approximately60-kDa at12hpi and 70-kDa at36hpi.Similarly,the molecular mass of Cry55Aa became larger as well(Figure4A),in addition to the main45-kD signal band,signal bands corresponding to approximately 90-kD and150-kD from12hpi till to72hpi were observed. The Cry5Ba was also modified after being ingested by M.hapla J2.An approximately60-kDa band was observed between 22hpi and50hpi.The main band subsequently increased to about90-kDa and250-kDa at96hpi(Figure4D).To determine whether the60-kDa Cry5Ba toxin formed before or after ingestion,total proteins were extracted from treated M. hapla at12hpi,an earlier time than the former22hpi.Western blot results indicated a140kDa band was present at this time (Figure4F),suggesting that a140kDa form of Cry5Ba entered M.hapla J2directly through the stylet.Based on the above information,we concluded that M.hapla J2can ingest140kDa proteins.
Uptake Efficiency of Crystal Proteins by the M.hapla J2Stylet 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 J2with 1500ng/ml purified Cry55Aa,Cry6Aa,and Cry5Ba for 96h.The protein concentra-tion after ingestion was then tested by ELISA.The calculated uptake efficiency by M.hapla J2of Cry55Aa (45kDa),Cry6Aa (54kDa),and Cry5Ba (140kDa)proteins were 78.3%,69.5%,and 17.2%,respectively (Figure 5).These data showed the uptake efficiency of crystal proteins by M.hapla J2stylet decreased with increasing protein molecular mass.
Discussion
The original bioassay for the detection of crystal proteins targeting M.hapla was conducted with the addition of TRE [9].TRE can attract nematodes to plant roots,induce stylet thrusting,release of secretions and increase in nematode mobility [16].In this study,we improved the B.thuringiensis
crystal proteins bioassay protocol for M.hapla J2by using resorcinol instead of TRE.Resorcinol was previously used to stimulate the uptake of dsRNA during in vitro RNAi of M.incognita J2[17].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 proteases during nematode infection [18].For example,Brevibacillus laterosporus secretes extracellular proteases that damage the nematode cuticle [18,19].Initially we conjectured that crystal proteins were able to enter M.hapla J2through nematode cuticle.However,in this in vitro study,our results confirmed that M.hapla J2could ingest a range of proteins sizes from 45kDa to 140kDa directly through the stylet (Figure 3and Figure 4).When the head of M.hapla J2was 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 S1C1and 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
阿贝原则
proteins
Figure 1.The dose response curves of Cry6Aa,Cry55Aa,and Cry5Ba against M.hapla J2in 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 J2were conducted in the presence of resorcinol (RES),or tomato root exudates (TRE),or ddH 2O (CK),respectively.A non-nematicidal crystal protein Cry1Ac (D)was treated as the same and used as control.The M.hapla J2were 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 60animals.The mortality was 3.3%in the absence of any toxins.doi:10.1371/journal.pone.0038534.g001
by M.hapla J2decreased 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 [13].About the uptake size of M.hapla J2,We found 140kDa Cry5B can enter M.hapla J2and the uptake efficiency was very low (17.2%).However,we did not assess proteins larger than 140kDa,maybe larger proteins could also enter M.hapla J2stylet.
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 [4].It is reported that the beet cyst nematode H.schachtii was unable to ingest proteins larger than 28kDa [14].Goverse ported that Globodera rostochiensis juveniles could ingest 32kDa proteins [22].While,Cry6A and a truncated 79kDa 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 79kDa in vivo .
In the experimental system described here,we demonstrated that different sized B.thuringiensis crystal proteins can enter M.hapla J2through the stylet.Although this in vitro experiment may not be applicable to feeding tubes produced in vivo ,it would suggest that 140kDa 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 J2within 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 e
xtracellular plant secretions.Our discovery has important implications in controlling M.hapla J2during the migratory phase of the second stage juvenile before the J2becomes 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 140kDa B.thuringiensis crystal proteins can enter M.hapla J2through the stylet.It has important implications for the design of any transgenic resistance approach against M.hapla .
Materials and Methods Ethics Statement
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
2012重庆
高考作文B.thuringiensis strains BMB0250,BMB0224,and BMB0215[9]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 J2were 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 J2were 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 60animals.Error bars represent the S.D.from the mean of averages over three independent experiments.doi:10.1371/journal.pone.0038534.g002
strains were maintained on Luria-Bertani (LB)agar plates and supplemented with appropriate antibiotics at 28u C [23].
M.hapla Rearing and Bioassay
The M.hapla bioassay procedure was undertaken according to the method described by Bischof et al [24].The toxicity of crystal proteins against M.hapla J2was tested by touching the worms
directly,typically 3times 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 [24].
Sixty M.hapla J2larvae 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 J2in the presence of resorcinol (Res)or tomato root exudates (TRE).M.hapla J2were 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 40.43m m.
doi:10.1371/journal.pone.0038534.g003
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