Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.
The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.
Plant genetic engineering is conventionally used for transferring beneficial traits to plants. In recent years, this technology has been applied to convert plants into bio-factories for the production of pharmaceutically important and commercially valuable proteins, many of which cannot be chemically synthesized and are very costly to produce using animal or microbial systems1. By the introduction of new genes, the plant’s own metabolism can be manipulated for the production of various biopharmaceuticals like antibodies, metabolic enzymes, hormones, antigens or vaccine2.
Established gene transfer technologies for plants are the Agrobacterium-mediated delivery3, bombardment with DNA-coated microprojectiles (biolistics)4, and electroporation5 or polyethylene glycol6 treatment of protoplasts. Techniques requiring protoplasts are generally avoided because they are time-consuming, cumbersome and yield inconsistent results7. As a result, virtually all plant modification work at present utilizes either Agrobacterium or microprojectiles for gene transfer. The Agrobacterium method is more extensively used but not applicable to many economically important plant species. Meanwhile, microprojectile bombardment is more versatile due to a broad range of susceptible plants but requires specialized equipment and often causes severe tissue damage. Furthermore, these methods involve either a random (biolistics) or complex (Agrobacterium) delivery mechanism and have variable transformation rates8. Hence, a novel plant transformation technology that is simple yet effective, and applicable to different plant types is required.
Currently, plants are subjected to genetic modification primarily by the delivery of exogenous DNA encoding a desired trait, rather than by direct delivery of a target protein. The higher stability of DNA over proteins is a prime advantage; nevertheless, potential problems associated with DNA delivery include the random insertion of exogenous DNA into the plant genome and unintended transmission of antibiotic resistance genes to pathogenic bacteria via horizontal gene transfer9. For genome-editing purposes, the ability to edit plant genomes without introducing foreign DNA into cells may circumvent regulatory concerns related to genetically modified plants. Thus, an alternative DNA-free strategy for the modification of plants by direct delivery of protein will be able to cater to these needs.
Here we introduce a peptide-based system, originally developed for human gene therapy10-14, for the targeted delivery of exogenous genes or proteins in intact plants. Peptides are able to protect DNA from nuclease degradation and can mediate gene transfer across cell as well as organellar membranes15-17. They also have diverse and tunable properties besides being non-cytotoxic18-20. More importantly, with the use of peptides, genes can be precisely targeted to intracellular organelles such as the mitochondria21 or plastids (chloroplasts)22 for expression-a task not achievable by biolistic or Agrobacterium-mediated transformation. Depending on the cargo type, this new plant modification technology can be exploited to deliver proteins23 and either express (plasmid21,24 or double-stranded DNA25) or down-regulate (double-stranded RNA26) specific genes within the plant, throughout its cytosolic space24-26 or within a specific organellar compartment21. The designed carrier peptides consist of a cationic domain in the form of either polylysine (K8) or polylysine-co-histidine (KH)9 for binding and/or condensation of negatively-charged cargoes, which is conjugated to cell penetrating (BP100 peptide) or mitochondria transit (Cytcox peptide) sequences.
Kritiske trin i den protokol, der er blevet identificeret empirisk diskuteres. Med sprøjte infiltration, er peptidbaserede formuleringer introduceres i luftrum inde i planten blad gennem spalteåbninger. For at sikre maksimal optagelse af løsningen, bør infiltration processen udføres når planterne er under forhold, der bidrager til stomatal åbning dvs., Forsynet med tilstrækkeligt vand og i lyset periode. Med hensyn til fremstillingen af transfektionskomplekser, for formuleringer, der involverer en kombination af to peptider (for mitokondrie-målrettet DNA levering), sekvensen for tilsætning af hver komponent er afgørende og bør ikke vendes.
Der er mange plausible ændringer af proceduren. En anden mulighed for infiltrationen af planteceller er anvendelsen af vakuum infiltration, som er i stand til at indføre den komplekse opløsning til hele planter og / eller partielle væv, herunderapikale dannelsesvæv. Til denne alternative procedure, er frøplanter nedsænket i transfektion opløsning, og baseret på det tryk, der genereres af vakuum, er peptid-cargo komplekser tvinges gennem stomata og i plantecellerne (Yoshizumi, T., upublicerede data).
Forbigående genekspression, baseret på undersøgelser under anvendelse af dyreceller, har vist sig at øges med højere DNA-koncentrationer 29-31. Det er vigtigt at bemærke, at forholdet mellem peptid og DNA påvirker de biofysiske egenskaber (størrelse, overfladeladning) af komplekser (figur 4), hvilket påvirker transfektionseffektiviteten; derfor behov for optimale forhold, der skal opretholdes, selv med øget DNA-koncentration.
En af de vigtigste fordele ved at anvende peptider som et gen / protein levering middel er, at dets sekvens er modtagelig for tuning at opfylde den ønskede funktion. For eksempel mitokondrie-targeting domæne af bæreren peptid beskrevet i denne study kan erstattes med chloroplast eller peroxisomale-targeting sekvenser til lokalisering til disse organeller. Mens chloroplast-transformation er muligt i flere anlæg ved hjælp biolistics 32-34, kunne hverken Agrobacterium eller biolistisk fremgangsmåde introducere gener i mitokondrierne eller andre organeller foruden kernen (tabel 2).
Der er ingen stive vært-range begrænsninger for peptidbaseret transfektion modsætning Agrobacterium -baseret fremgangsmåde (tabel 2). Hidtil har formuleringer til transfektion af A. thaliana, N. benthamiana og poppel blevet optimeret (tabel 1), men fremgangsmåden kan også anvendes til Nicotiana tabacum, tomat kultivar Micro-Tom, ris (baseret på indledende forsøg), og andre mono- og tokimbladede planter.
Endnu er der ingen kendt begrænsning i transgen størrelse, når peptider anvendes ens transfektion vektorer. I modsætning hertil er der fundet store DNA-molekyler til at reducere transformationseffektivitet på Agrobacterium-medieret metoder 35,36, og en øvre størrelsesgrænse for transgener på ca. 200 kb er blevet rapporteret 37-39. Brug af biolistics, på den anden side store DNA-fragmenter kan blive klippet under fremstillingen eller levering til planter 38. Selv om ingen øvre grænse er blevet bestemt for biolistisk transformation hidtil har fysisk begrænser vist sig at begrænse størrelsen af DNA, som kan overføres til meget mindre end 150 kb 40. De største fordele ved at anvende peptid-baseret system til genoverførsel i forhold til eksisterende fremgangsmåder, der anvender enten Agrobacterium eller mikroprojektilbombardement, diskuteret ovenfor, er opsummeret i tabel 2.
Et par begrænsninger findes for denne metode, som den er. For det første målrettet afgivelse af pDNA til specifikke organeller, såsom mitochondria, har vist sig muligt ved en simpel kombination af DNA-bindende, celle-gennemtrængende og organel-transit-sekvenser selv om effektiviteten var lav. Transgenekspression kunne påvises, ved konfokal mikroskopi, kun i en lille population af mitokondrier i cellerne. Derfor er det nødvendigt for yderligere ændringer: (i) øge translokation af flere komplekser over cellen / organellar membran, og (ii) at forbedre dissociation og overførsel af pDNA fra luftfartsselskabet peptid ind i målet organel for ekspression. For det andet ved anvendelse af dette DNA leveringssystem, transient ekspression af exogene reportergener blev succes opnået i den cytosoliske og mitokondriel rum i celler. Stabil inkorporering og ekspressionen af de indførte gener i atomkraftværket / organellar genom er endnu ikke fastlagt, men på grund af manglen på egnede selektionsstrategier.
Erkender, at der er områder til forbedringer eller videre ddvikling, peptid-afledte strategi beskrives her fortsat en enkel og alsidig teknik, der har banet vejen for levering af forskellige ladninger i forskellige anlægstyper.
The authors have nothing to disclose.
Forfatterne vil gerne anerkende finansiering fra Japan Science and Technology Agency sonderende forskning for Advanced Technology (JST-ERATO), den Nye Energi og Industrial Technology Development Organization (NEDO), og Cross-ministerielle Promotion Program Strategic Innovation (SIP), Japan .
(KH)9-BP100 peptide | Custom-synthesized by RIKEN Brain Science Institute | N/A | Sequence: KHKHKHKHKHKHKHKH- KHKKLFKKILKYL |
(BP100)2-K8 peptide | Custom-synthesized by RIKEN Brain Science Institute | N/A | Sequence: KKLFKKILKYLKKLFKKIL- KYLKKKKKKKK |
BP100 peptide | Custom-synthesized by RIKEN Brain Science Institute | N/A | Sequence: KKLFKKILKYL |
Cytcox-(KH)9 peptide | Custom-synthesized by RIKEN Brain Science Institute | N/A | Sequence: MLSLRQSIRFFKKHKHKH- KHKHKHKHKHKH |
P35S-GFP(S65T)-TNOS and P35S-RLuc-TNOS | N/A | N/A | Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of CaMV 35S constitutive promoter (Ref: Lakshmanan et al. Biomacromolecules 2013, 14, 10) |
pDONR-cox2:gfp and pDONR-cox2:rluc | N/A | N/A | Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of cox2 mitochondrial-specific promoter (Ref: Chuah et al. Sci Rep 2015, 5, 7751) |
dsDNA (PCR-amplified from pBI221-P35S-Rluc-TNOS) | N/A | N/A | Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of CaMV 35S constitutive promoter (Ref: Lakshmanan et al. Plant Biotechnol 2015, 32, 39) |
GFP5 siRNA | N/A | N/A | For RNA interference-mediated silencing of green fluorescent protein (Ref: Numata et al. Plant Biotechnol J 2014, 12, 1027) |
CHS siRNA | N/A | N/A | For RNA interference-mediated silencing of chalcone synthase (Ref: Numata et al. Plant Biotechnol J 2014, 12, 1027) |
1 mL and 10 mL Plastic Syringes | TERUMO Corporation | SS-01T, SS-10ESZ | |
1.5 mL Microcentrifuge Tube | AS ONE Corporation | 151212 | |
96-Well Flat-Bottom Plate | Asahi Glass Co., Ltd. | 3860-096 | |
Adhesive Tape | Sekisui Chemical Co., Ltd. | No. 835 | |
Alcohol Dehydrogenase | Sigma-Aldrich Co., LLC. | A-7011 | |
Atomic Force Microscope | Seiko Instruments Inc. | SPI3800, SPA 300HV | |
Atomic Force Microscope | Hitachi High-Tech Science Corporation | AFM5300E | |
BCA Protein Assay Kit | Thermo Fisher Scientific Inc. | 23227 | |
Cantilever | Hitachi High-Tech Science Corporation | K-A102001593 | |
Confocal Laser Scanning Microscope | Carl Zeiss | LSM700 | |
Cork Borer | Sigma-Aldrich Co., LLC. | Z165220 | For excision of leaves into 1-cm diameter disks |
Coverslip | Matsunami Glass Ind., Ltd. | C02261 | |
Cuvette | Sarstedt | 759116 | |
Folded Capillary Cell | Malvern Instruments, Ltd. | DTS1070 | |
Forceps | Shimizu Akira Inc. | Stainless pincet 150 | |
Homogenization Pestle | Ieda Trading Corp. | 9993 | |
Mica | Nisshin EM Co., Ltd. | LC23Z | |
Microcentrifuge | Beckman Coulter | BKA46472 | |
Microplate Reader | Molecular Devices Corporation | Spectra MAX M3 | |
Microscope Slide | Matsunami Glass Ind., Ltd. | S011120 | |
Pipette | Eppendorf Research® plus | 3120000909 | |
Pipette Tips | AS ONE Corporation | 2-5138-01, 2-5138-02, 2-5138-03 | |
Plastic Petri Dish | AS ONE Corporation | 1-7484-01 | |
Renilla Luciferase Assay Kit | Promega corporation | E2810 | |
Rhodamine B Isothiocyanate | Sigma-Aldrich Co., LLC. | 283924 | |
RNase-Free Water | Qiagen | 129112 | |
Sodium Carbonate | Wako Pure Chemical Industries, Ltd. | 199-01585 | |
Surgical Blade and Handle | FEATHER Safety Razor Co., Ltd. | Stainless steel No. 14 (blade), No. 3L (handle) | |
Syringe Tip Cap | Musashi Engineering Inc. | NC-3E | |
Weighing Balance | Sartorius | CPA225D | |
Zeta Potentiometer | Malvern Instruments, Ltd. | Zetasizer Nano-ZS |