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1.
Syst Appl Microbiol ; 43(1): 126046, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31818496

RESUMO

The genus Agrobacterium was created a century ago by Conn who included it in the family Rhizobiaceae together with the genus Rhizobium. Initially, the genus Agrobacterium contained the non-pathogenic species Agrobacterium radiobacter and the plant pathogenic species Agrobacterium tumefaciens and Agrobacterium rhizogenes. At the end of the past century two new pathogenic species, Agrobacterium rubi and Agrobacterium vitis, were added to the genus. Already in the present century these species plus Agrobacterium larrymoorei were reclassified into genus Rhizobium. This reclassification was controversial and for a time both genus names were used when new species were described. Few years ago, after a taxonomic revision based on genomic data, the old species A. rhizogenes was maintained in the genus Rhizobium, the old species A. vitis was transferred to the genus Allorhizobium and several Rhizobium species were transferred to the genus Agrobacterium, which currently contains 14 species including the old species A. radiobacter, A. tumefaciens, A. rubi and A. larrymoorei. Most of these species are able to produce tumours in different plants, nevertheless the genus Agrobacterium also encompasses non-pathogenic species, one species able to nodulate legumes and one human pathogenic species. Taking into account that the species affiliations to five Agrobacterium genomospecies have not been determined yet, an increase in the number of species within this genus is expected in the near future.


Assuntos
Agrobacterium/classificação , Filogenia , Agrobacterium/genética , DNA Bacteriano/genética , Genes Bacterianos/genética , Genes Essenciais/genética , Genoma Bacteriano/genética , Humanos , Rhizobiaceae/classificação , Rhizobiaceae/genética , Rhizobium/classificação , Rhizobium/genética
2.
J Plant Physiol ; 243: 153055, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31639537

RESUMO

Food demands of increasing human population dictate intensification of livestock production, however, environmental stresses could jeopardize producers' efforts. Forage legumes suffer from yield losses and poor nutritional status due to salinity increase of agricultural soils. As tools aimed to reduce negative impacts of biotic or abiotic stresses, proteinase inhibitors (PIs) have been promoted for biotechnological improvements. In order to increase tolerance of Lotus corniculatus L. to salt stress, serine PI, BvSTI, was introduced into this legume using Agrobacterium rhizogenes, with final transformation efficiency of 4.57%. PCR, DNA gel-blot, RT-PCR and in-gel protein activity assays confirmed the presence and activity of BvSTI products in transformed lines. Plants from three selected transgenic lines (21, 73 and 109) showed significant alterations in overall phenotypic appearance, corresponding to differences in BvSTI accumulation. Lines 73 and 109 showed up to 7.3-fold higher number of tillers and massive, up to 5.8-fold heavier roots than in nontransformed controls (NTC). Line 21 was phenotypically similar to NTC, accumulated less BvSTI transcripts and did not exhibit an additional band of recombinant trypsin inhibitor as seen in lines 73 and 109. Exposure of the transgenic lines to NaCl revealed different levels of salt stress susceptibility. The NaCl sensitivity index, based on morphological appearance and chlorophyll concentrations showed that lines 73 and 109 were significantly less affected by salinity than NTC or line 21. High level of BvSTI altered morphology and delayed salt stress related senescence, implicating BvSTI gene as a promising tool for salinity tolerance improvement trials in L. corniculatus.


Assuntos
Beta vulgaris/fisiologia , Lotus/fisiologia , Proteínas de Plantas/genética , Inibidores de Serino Proteinase/genética , Agrobacterium/genética , Beta vulgaris/crescimento & desenvolvimento , Lotus/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/fisiologia , Tolerância ao Sal/genética , Inibidores de Serino Proteinase/metabolismo
3.
Plant Mol Biol ; 101(4-5): 415-437, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31542868

RESUMO

KEY MESSAGE: Naturally transgenic plant species occur on an unexpectedly large scale. Agrobacterium-mediated gene transfer leads to the formation of crown galls or hairy roots, due to expression of transferred T-DNA genes. Spontaneous regeneration of transformed cells can produce natural transformants carrying cellular T-DNA (cT-DNA) sequences of bacterial origin. This particular type of horizontal gene transfer (HGT) could play a role in plant evolution. However, the material available today is not enough for generalizations concerning the role of Agrobacterium in HGT from bacteria to plants. In this study, we searched for T-DNA-like genes in the sequenced genomes of dicots and monocots. We demonstrate the presence of cT-DNAs in 23 out of 275 dicot species, within genera Eutrema, Arachis, Nissolia, Quillaja, Euphorbia, Parasponia, Trema, Humulus, Psidium, Eugenia, Juglans, Azadirachta, Silene, Dianthus, Vaccinium, Camellia, and Cuscuta. Analysis of transcriptome data of 356 dicot species yielded 16 additional naturally transgenic species. Thus, HGT from Agrobacterium to dicots is remarkably widespread. Opine synthesis genes are most frequent, followed by plast genes. Species in the genera Parasponia, Trema, Camellia, Azadirachta, Quillaja, and Diospyros contain a combination of plast and opine genes. Some are intact and expressed, but the majority have internal stop codons. Among the sequenced monocot species, Dioscorea alata (greater yam) and Musa acuminata (banana) also contain T-DNA-like sequences. The identified examples are valuable material for future research on the role of Agrobacterium-derived genes in plant evolution, for investigations on Agrobacterium strain diversity, and for studies on the function and evolution of cT-DNA genes in natural transformants.


Assuntos
Agrobacterium/genética , Genoma de Planta , Transformação Genética , Evolução Biológica , DNA Bacteriano/química , DNA de Plantas/química , Transferência Genética Horizontal , Plantas Geneticamente Modificadas/genética , Análise de Sequência de DNA , Transcriptoma
4.
Biosens Bioelectron ; 142: 111504, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31401226

RESUMO

The wide cultivation of genetically modified (GM) crops has raised concerns on the risks to humans and the environment. 5-enolpyruvylshikimate-3-phosphate synthase isolated from Agrobacterium species strain CP4 (CP4-EPSPS) protein is most widely present in these crops. Therefore the measurement of CP4-EPSPS sensitively in a point-of-care testing (POCT) manner for the screening of transgenic plants is demanded. To date the development of quantitative POCT system has not yet been reported. In presented study, an electrochemical immunosensor towards CP4-EPSPS has been fabricated by integrating a portable bioanalytical device with a disposable screen-printed carbon electrode (SPCE) for POCT of GM crops. The dual-functionalized AuNPs were used as nanoprobes and prepared by simultaneously tagging horseradish peroxidase (HRP) and antibody on AuNPs with an exceptionally simple protocol. The sensitivity of the developed nanoprobe-based immunosensor was 62.5-fold higher than that using HRP-labeled antibody. As a result, the proposed immunosensor using SPCE could detect CP4-EPSPS down to 0.050 ng mL-1 with the linear range of 0.10-10 ng mL-1 within 65 min. In addition, the developed method has been validated with genuine GM crops and the results show a good correlation coefficient of 0.9909 compared with those of a commercial ELISA kit. Therefore, this portable electrochemical immunosensor is suitable for rapid and sensitive detection and provides a convenient and reliable platform for POCT assay.


Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/análise , Agrobacterium/enzimologia , Técnicas Biossensoriais/instrumentação , Produtos Agrícolas/genética , Plantas Geneticamente Modificadas/genética , 3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Agrobacterium/genética , Anticorpos Imobilizados/química , Técnicas Eletroquímicas/instrumentação , Desenho de Equipamento , Ouro/química , Imunoensaio/instrumentação , Nanopartículas Metálicas/química
5.
Int J Mol Sci ; 20(16)2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31443292

RESUMO

Agrobacterium-mediated transient expression and virus-induced gene silencing (VIGS) are very useful in functional genomics in plants. However, whether these methods are effective in cassava (Manihot esculenta), one of the most important tropical crops, remains elusive. In this study, we used green fluorescent protein (GFP) and ß-glucuronidase (GUS) as reporter genes in a transient expression assay. GFP or GUS could be detected in the infiltrated leaves at 2 days postinfiltration (dpi) and were evidenced by visual GFP and GUS assays, reverse-transcription PCR, and Western blot. In addition, phytoene desaturase (PDS) was used to show the silencing effect in a VIGS system. Both Agrobacterium GV3101 and AGL-1 with tobacco rattle virus (TRV)-MePDS-infiltrated distal leaves showed an albino phenotype at 20 dpi; in particular, the AGL-1-infiltrated plants showed an obvious albino area in the most distal leaves. Moreover, the silencing effect was validated by molecular identification. Notably, compared with the obvious cassava mosaic disease symptom infiltrated by African-cassava-mosaic-virus-based VIGS systems in previous studies, TRV-based VIGS-system-infiltrated cassava plants did not show obvious virus-induced disease symptoms, suggesting a significant advantage. Taken together, these methods could promote functional genomics in cassava.


Assuntos
Agrobacterium/genética , Inativação Gênica/fisiologia , Manihot/genética , Manihot/virologia , Vírus de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Oxirredutases/genética
6.
Appl Microbiol Biotechnol ; 103(16): 6657-6672, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31273398

RESUMO

Rhizogenic agrobacteria induce extensive root proliferation, in several economically valuable, dicotyledonous plant species, a phenomenon referred to as "hairy roots." Besides their pathogenic nature, agrobacteria have proven to be a valuable asset in biotechnology and molecular plant breeding. To assess the potential of frequently used rhizogenic strains, growth in yeast extract glucose broth and antibiotic resistance was analyzed. Growth curves were established for Arqua1, NCPPB2659, LMG150, LMG152, and ATCC15834; and regression analysis of the exponential growth phase resulted in a reliable and standardized method for preparation of a bacterial suspension for inoculation. Cell density did not correlate with the timing of hairy root emergence. The highest number of hairy roots was obtained with an inoculum of 1 × 108 CFU ml-1 for Arqua1, NCPPB2659, and LMG152. Cell density of ATCC15834 did not affect the number of hairy roots formed. The identity of the rhizogenic strains for plant transformation was verified in phylogenetic analysis using average nucleotide identity (ANI), which also provided insight in their genetic diversity within the Rhizobium taxon.


Assuntos
Agrobacterium/genética , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Transformação Genética , Agrobacterium/crescimento & desenvolvimento , Daucus carota/genética , Daucus carota/microbiologia , Genes Bacterianos , Loci Gênicos
7.
J Plant Res ; 132(5): 695-703, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31325057

RESUMO

The development and functioning of the nitrogen fixing symbiosis between legume plants and soil bacteria collectively called rhizobia requires continuous chemical dialogue between the partners using different molecules such as flavonoids, lipo-chitooligosaccharides, polysaccharides and peptides. Agrobacterium rhizogenes mediated hairy root transformation of legumes is widely used to study the function of plant genes involved in the process. The identification of transgenic plant tissues is based on antibiotics/herbicide selection and/or the detection of different reporter genes that usually require special equipment such as fluorescent microscopes or destructive techniques and chemicals to visualize enzymatic activity. Here, we developed and efficiently used in hairy root experiments binary vectors containing the MtLAP1 gene driven by constitutive and tissue-specific promoters that facilitate the production of purple colored anthocyanins in transgenic tissues and thus allowing the identification of transformed roots by naked eye. Anthocyanin producing roots were able to establish effective symbiosis with rhizobia. Moreover, it was shown that species-specific allelic variations and a mutation preventing posttranslational acetyl modification of an essential nodule-specific cysteine-rich peptide, NCR169, do not affect the symbiotic interaction of Medicago truncatula cv. Jemalong with Sinorhizobium medicae strain WSM419. Based on the experiments, it could be concluded that it is preferable to use the vectors with tissue-specific promoters that restrict anthocyanin production to the root vasculature for studying biotic interactions of the roots such as symbiotic nitrogen fixation or mycorrhizal symbiosis.


Assuntos
Antocianinas/fisiologia , Medicago truncatula/fisiologia , Fixação de Nitrogênio , Raízes de Plantas/fisiologia , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Simbiose , Agrobacterium/genética , Biomarcadores/análise , Medicago truncatula/genética , Medicago truncatula/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/fisiologia , Nódulos Radiculares de Plantas/microbiologia , Transformação Genética
8.
Transgenic Res ; 28(Suppl 2): 61-64, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31321685

RESUMO

Processes of traditional trait development in plants depend on genetic variations derived from spontaneous mutation or artificial random mutagenesis. Limited availability of desired traits in crossable relatives or failure to generate the wanted phenotypes by random mutagenesis led to develop innovative breeding methods that are truly cross-species and precise. To this end, we devised novel methods of precise genome engineering that are characterized to use pre-assembled CRISPR/Cas9 ribonucleoprotein (RNP) complex instead of using nucleic ands or Agrobacterium. We found that our methods successfully engineered plant genomes without leaving any foreign DNA footprint in the genomes. To facilitate introduction of RNP into plant nucleus, we first obtained protoplasts after removing the transfection barrier, cell wall. Whole plants were regenerated from the single cell of protoplasts that has been engineered with the RNP. Pending the improved way of protoplast regeneration technology especially in crop plants, our methods should help develop novel traits in crop plants in relatively short time with safe and precise way.


Assuntos
Sistemas CRISPR-Cas/genética , DNA/genética , Edição de Genes/tendências , Ribonucleoproteínas/genética , Agrobacterium/genética , Genoma de Planta/genética , Mutação , Protoplastos/metabolismo
9.
BMC Plant Biol ; 19(1): 311, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307375

RESUMO

BACKGROUND: CRISPR/Cas9 gene editing is now revolutionizing the ability to effectively modify plant genomes in the absence of efficient homologous recombination mechanisms that exist in other organisms. However, soybean is allotetraploid and is commonly viewed as difficult and inefficient to transform. In this study, we demonstrate the utility of CRISPR/Cas9 gene editing in soybean at relatively high efficiency. This was shown by specifically targeting the Fatty Acid Desaturase 2 (GmFAD2) that converts the monounsaturated oleic acid (C18:1) to the polyunsaturated linoleic acid (C18:2), therefore, regulating the content of monounsaturated fats in soybean seeds. RESULTS: We designed two gRNAs to guide Cas9 to simultaneously cleave two sites, spaced 1Kb apart, within the second exons of GmFAD2-1A and GmFAD2-1B. In order to test whether the Cas9 and gRNAs would perform properly in transgenic soybean plants, we first tested the CRISPR construct we developed by transient hairy root transformation using Agrobacterium rhizogenesis strain K599. Once confirmed, we performed stable soybean transformation and characterized ten, randomly selected T0 events. Genotyping of CRISPR/Cas9 T0 transgenic lines detected a variety of mutations including large and small DNA deletions, insertions and inversions in the GmFAD2 genes. We detected CRISPR- edited DNA in all the tested T0 plants and 77.8% of the events transmitted the GmFAD2 mutant alleles to T1 progenies. More importantly, null mutants for both GmFAD2 genes were obtained in 40% of the T0 plants we genotyped. The fatty acid profile analysis of T1 seeds derived from CRISPR-edited plants homozygous for both GmFAD2 genes showed dramatic increases in oleic acid content to over 80%, whereas linoleic acid decreased to 1.3-1.7%. In addition, transgene-free high oleic soybean homozygous genotypes were created as early as the T1 generation. CONCLUSIONS: Overall, our data showed that dual gRNA CRISPR/Cas9 system offers a rapid and highly efficient method to simultaneously edit homeologous soybean genes, which can greatly facilitate breeding and gene discovery in this important crop plant.


Assuntos
Ácidos Graxos Dessaturases/genética , Edição de Genes/métodos , Genes de Plantas , RNA Guia , Soja/genética , Ácido alfa-Linoleico/genética , Agrobacterium/genética , Sistemas CRISPR-Cas , Marcadores Genéticos , Vetores Genéticos , Técnicas de Genotipagem , Padrões de Herança , Plantas Geneticamente Modificadas
10.
BMC Res Notes ; 12(1): 457, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31345264

RESUMO

OBJECTIVE: GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA). We examined the ability of the GAANTRY Agrobacterium rhizogenes ArPORT1 '10-stack' strain to generate transgenic potato plants. RESULTS: The 28.5 kilobase 10-stack T-DNA, was introduced into Lenape potato plants with a 32% transformation efficiency. Molecular and phenotypic characterization confirmed that six of the seven tested independent transgenic lines carried the entire desired construct, demonstrating that the GAANTRY 10-stack strain can be used can be used in a tissue culture-based callus transformation method to efficiently generate transgenic potato plants. Analysis using droplet digital PCR showed that most of the characterized events carry one or two copies of the 10-stack transgenes and that 'backbone' DNA from outside of the T-DNA was absent in the transgenic plants. These results demonstrate that the GAANTRY system efficiently generates high quality transgenic potato plants with a large construct of stacked transgenes.


Assuntos
Agrobacterium/genética , DNA Bacteriano/genética , Técnicas de Transferência de Genes , Plasmídeos/metabolismo , Solanum tuberosum/genética , Transgenes , Agrobacterium/metabolismo , DNA Bacteriano/metabolismo , Dosagem de Genes , Expressão Gênica , Genes Reporter , Glucuronidase/genética , Glucuronidase/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fenótipo , Folhas de Planta/genética , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Plasmídeos/química , Reação em Cadeia da Polimerase/métodos , Solanum tuberosum/microbiologia
11.
World J Microbiol Biotechnol ; 35(7): 109, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31280382

RESUMO

Echinocandin B (ECB) is an important lipohexapeptide used for chemical manufacture of the antifungal agent anidulafungin. Sterigmatocystin (ST) is a polyketide mycotoxin produced by certain species of Aspergillus such as Aspergillus delacroxii SIPIW15, which could produce both ECB and ST. However, the presence of the potent carcinogen ST will greatly affect the quality and safety of ECB production. Therefore, it is essential to eliminate the ST biosynthesis and increase ECB titers in Asp. delacroxii SIPIW15. In this study, the polyketide synthase gene (stcA) required for biosynthesis of ST and its flanking region in Asp. delacroxii SIPIW15 were cloned, sequenced and analyzed firstly. Based on Agrobacterium-mediated transformation, the ΔstcA mutant AMT-1 was obtained and its yield of ECB was increased by 40% without ST detected at the same time as compared to the original strain. The results of the fed-batch experiments showed that the ECB yield of the ΔstcA strain AMT-1 was increased to 2163 ± 31 mg/l and no ST was detected in the 50 l bioreactor. This work suggested that the ΔstcA strain AMT-1 has the potential for application in ECB production improvement, and more importantly, to eliminate ST-related environmental pollution in ECB fermentation industry.


Assuntos
Aspergillus/genética , Aspergillus/metabolismo , Equinocandinas/biossíntese , Equinocandinas/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Policetídeo Sintases/genética , Esterigmatocistina/biossíntese , Agrobacterium/genética , Anidulafungina , Antifúngicos , Sequência de Bases , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , DNA Fúngico/isolamento & purificação , Fermentação , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Metabolismo Secundário/genética , Transformação Genética
12.
J Agric Food Chem ; 67(31): 8493-8499, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31310523

RESUMO

The ginsenosides Rh2 and Rg3 induce tumor cell apoptosis, inhibit tumor cell proliferation, and restrain tumor invasion and metastasis. Despite Rh2 and Rg3 having versatile pharmacological activities, contents of them in natural ginseng are extremely low. To produce ginsenosides Rh2 and Rg3, the saponin-producing capacity of endophytic bacteria isolated from Panax ginseng was investigated. In this work, 81 endophytic bacteria isolates were taken from ginseng roots by tissue separation methods. Among them, strain PDA-2 showed the highest capacity to produce the rare ginsenosides; the concentrations of rare ginsenosides Rg3 and Rh2 reached 62.20 and 18.60 mg/L, respectively. On the basis of phylogenetic analysis, it was found that strain PDA-2 belongs to the genus Agrobacterium and was very close to Agrobacterium rhizogenes.


Assuntos
Bactérias/metabolismo , Endófitos/metabolismo , Ginsenosídeos/biossíntese , Panax/microbiologia , Agrobacterium/classificação , Agrobacterium/genética , Agrobacterium/isolamento & purificação , Agrobacterium/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Endófitos/classificação , Endófitos/genética , Endófitos/isolamento & purificação , Filogenia , Raízes de Plantas/microbiologia
13.
Mol Biotechnol ; 61(9): 663-673, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31228008

RESUMO

The prevalence of insect resistance against Bt toxins has led to the idea of enhancing demethylation from cell wall pectin by pectin methylesterase enzyme for overproduction of methanol which is toxic to insects pests. The AtPME and AnPME fragments ligated into pCAMBIA1301 vector were confirmed through restriction digestion with EcoR1 and BamH1. Excision of 3363 bp fragment from 11,850 bp vector confirmed the ligation of both fragments into pCAMBIA1301 vector. Transformation of pectin methylesterase-producing genes, i.e., AtPME and AnPME from Arabidopsis thaliana and Aspergillus niger cloned in plant expression vector pCAMBIA1301 under 35S promoter into cotton variety CEMB-33 harboring two Bt genes Cry1Ac and Cry2A, respectively, was done by using shoot apex-cut Agrobacterium-mediated transformation method. The plantlets were screened on MS medium supplemented with hygromycin on initial basis. Amplification of 412 and 543 bp, respectively, through gene-specific primer has been obtained which confirmed the successful introduction of pCAMBIA AtPME and AnPME genes into cotton variety CEMB 33. Relative expression of AtPME and AnPME genes through real-time PCR determined the expression level of both gene ranges between 3- and 3.5-fold in different transgenic cotton lines along with quantity of methanol ranging from 0.8 to 0.9% of maximum while 0.5% to 0.6% of minimum but no expression was obtained in negative non-transgenic control cotton plant with least quantity of methanol, i.e., 0.1%. Almost 100% mortality was observed in insect bioassay for Helicoverpa armigera on detached leaves bioassay and 63% for Pink Bollworm (Pectinophora gossypiella) on growing transgenic cotton bolls as compared to positive control transgenic cotton with double Bt genes where mortality was found to be 82% for H. armigera and 50% for P. gossypiella while 0% in negative control non-transgenic plants.


Assuntos
Hidrolases de Éster Carboxílico/genética , Proteínas Fúngicas/genética , Gossypium/genética , Larva/efeitos dos fármacos , Metanol/toxicidade , Mariposas/efeitos dos fármacos , Proteínas de Plantas/genética , Agrobacterium/genética , Agrobacterium/metabolismo , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Aspergillus niger/genética , Aspergillus niger/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Parede Celular/parasitologia , Clonagem Molecular , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Gossypium/parasitologia , Herbivoria/efeitos dos fármacos , Herbivoria/fisiologia , Inseticidas/química , Inseticidas/toxicidade , Larva/patogenicidade , Metanol/metabolismo , Mariposas/patogenicidade , Células Vegetais/metabolismo , Células Vegetais/parasitologia , Folhas de Planta/genética , Folhas de Planta/parasitologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transgenes
14.
Genetics ; 212(4): 1163-1179, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31243056

RESUMO

Malassezia encompasses a monophyletic group of basidiomycetous yeasts naturally found on the skin of humans and other animals. Malassezia species have lost genes for lipid biosynthesis, and are therefore lipid-dependent and difficult to manipulate under laboratory conditions. In this study, we applied a recently-developed Agrobacterium tumefaciens-mediated transformation protocol to perform transfer (T)-DNA random insertional mutagenesis in Malassezia furfur A total of 767 transformants were screened for sensitivity to 10 different stresses, and 19 mutants that exhibited a phenotype different from the wild type were further characterized. The majority of these strains had single T-DNA insertions, which were identified within open reading frames of genes, untranslated regions, and intergenic regions. Some T-DNA insertions generated chromosomal rearrangements while others could not be characterized. To validate the findings of our forward genetic screen, a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was developed to generate targeted deletion mutants for two genes identified in the screen: CDC55 and PDR10 This system is based on cotransformation of M. furfur mediated by A. tumefaciens, to deliver both a CAS9-gRNA construct that induces double-strand DNA breaks and a gene replacement allele that serves as a homology-directed repair template. Targeted deletion mutants for both CDC55 and PDR10 were readily generated with this method. This study demonstrates the feasibility and reliability of A. tumefaciens-mediated transformation to aid in the identification of gene functions in M. furfur, through both insertional mutagenesis and CRISPR/Cas9-mediated targeted gene deletion.


Assuntos
Sistemas CRISPR-Cas , Malassezia/genética , Mutagênese , Genética Reversa/métodos , Agrobacterium/genética , Farmacorresistência Fúngica/genética , Deleção de Genes , Mutagênese Insercional , Transformação Genética
15.
Genes (Basel) ; 10(6)2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31208028

RESUMO

Hairpin-structured (hp) RNA has been widely used to induce RNA interference (RNAi) in plants and animals, and an in vivo expression system for hpRNA is important for large-scale RNAi applications. Bacterial expression systems have so far been developed for in vivo expression of hpRNA or double-stranded (ds) RNA, but the structure of the resulting RNAi molecules has remained unclear. Here we report that long hpRNAs expressed in the bacteria Escherichia coli and Sinorhizobium meliloti were largely processed into shorter dsRNA fragments with no or few full-length molecules being present. A loss-of-function mutation in the dsRNA-processing enzyme RNase III, in the widely used E. coli HT115 strain, did not prevent the processing of hpRNA. Consistent with previous observations in plants, the loop sequence of long hpRNA expressed in Agrobacterium-infiltrated Nicotiana benthamiana leaves was excised, leaving no detectable levels of full-length hpRNA molecule. In contrast to bacteria and plants, long hpRNAs expressed in the budding yeast Saccharomyces cerevisiae accumulated as intact, full-length molecules. RNA extracted from hpRNA-expressing yeast cells was shown to be capable of inducing RNAi against a ß-glucuronidase (GUS) reporter gene in tobacco leaves when applied topically on leaf surfaces. Our results indicate that yeast can potentially be used to express full-length hpRNA molecules for RNAi and perhaps other structured RNAs that are important in biological applications.


Assuntos
Conformação de Ácido Nucleico , RNA de Cadeia Dupla/química , Saccharomyces cerevisiae/química , Agrobacterium/química , Agrobacterium/genética , Escherichia coli/química , Escherichia coli/genética , Mutação com Perda de Função , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , Ribonuclease III/química , Ribonuclease III/genética , Saccharomyces cerevisiae/genética , Tabaco/química , Tabaco/genética
16.
BMC Plant Biol ; 19(1): 237, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170915

RESUMO

BACKGROUND: The Agrobacterium-mediated transient transformation is a versatile and indispensable way of rapid analyzing gene function in plants. Despite this transient expression system has been successfully applied in a number of plant species, it is poorly developed in Caragana intermedia. RESULTS: In this study, we established an Agrobacterium-mediated transient expression system in C. intermedia leaves and optimized the effect of different Agrobacterial strains, several surfactants and the concentration of Silwet L-77, which would affect transient expression efficiency. Among the 5 Agrobacterial strains examined, GV3101 produced the highest GUS expression level. Besides, higher level of transient expression was observed in plants infiltrated with Silwet L-77 than with Triton X-100 or Tween-20. Silwet L-77 at a concentration of 0.001% greatly improved the level of GUS transient expression. Real-time PCR showed that expression of CiDREB1C was highly up-regulated in transiently expressed plants and reached the highest level at the 2nd day after infiltration. Based on this optimized transient transformation method, we characterized CiDREB1C function in response to drought, salt and ABA treatment. The results showed that transiently expressed CiDREB1C in C. intermedia leaves could enhance the survival rate and chlorophyll content, and reduce the lodging rate compared with the control seedlings under drought, salt and ABA treatments. Furthermore, the rate of leaf shedding of CiDREB1C transient expression seedlings was lower than that of the control under ABA treatment. CONCLUSIONS: The optimized transient expression condition in C. intermedia leaves were infiltrated with Agrobacterial strains GV3101 plus Silwet L-77 at a concentration of 0.001% added into the infiltration medium. Transiently expressed CiDREB1C enhanced drought, salt and ABA stress tolerance, indicated that it was a suitable and effective tool to determine gene function involved in abiotic stress response in C. intermedia.


Assuntos
Agrobacterium/genética , Caragana/fisiologia , Regulação da Expressão Gênica de Plantas , Engenharia Genética/métodos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/fisiologia , Fatores de Transcrição/genética , Proteínas de Arabidopsis , Caragana/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Fatores de Transcrição/metabolismo
17.
Prep Biochem Biotechnol ; 49(8): 775-782, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31124740

RESUMO

Glucosinolates (GSLs) are sulfur- and nitrogen-containing secondary metabolites that function in plant defense and provide benefits to human health. In this study, using Agrobacterium rhizogenes R1000, green and red kale hairy roots were established. The expression levels of GSLs biosynthesis genes and their accumulation in both kale hairy roots were analyzed by quantitative real-time PCR and HPLC. The results showed that the expression of most indolic GSLs biosynthesis genes was higher in the hairy roots of green kale than in that of red kale. In contrast, the expression of BoCYP83A1 and BoSUR1 encoding key enzymes aromatic GSL biosynthesis was significantly higher in red kale hairy root. The HPLC analysis identified six GSLs. The levels of 4-methoxyglucobrassicin, glucobrassicin, and 4-hydroxyglucobrassicin were 6.21, 5.98, and 2 times higher, respectively, in green kale than in red kale, whereas the levels of neoglucobrassicin and gluconasturtiin were 16.2 and 3.48 times higher, respectively, in red kale than in green kale. Our study provides insights into the underlying mechanisms of GSLs biosynthesis in kale hairy roots and can be potentially used as "biological factories" for producing bioactive substances such as GSLs.


Assuntos
Vias Biossintéticas , Brassica/metabolismo , Glucosinolatos/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Agrobacterium/genética , Brassica/genética , Regulação da Expressão Gênica de Plantas , Engenharia Genética , Glucosinolatos/análise , Glucosinolatos/genética , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/genética
18.
Mol Genet Genomics ; 294(5): 1251-1261, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31129735

RESUMO

Conventional methods for gene function study in Brassica campestris have lots of drawbacks, which greatly hinder the identification of important genes' functions and molecular breeding. The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is a versatile tool for genome editing that has been widely utilized in many plant species and has many advantages over conventional methods for gene function study. However, the application of CRISPR/Cas9 system in B. campestris remains unreported. The pectin-methylesterase genes Bra003491, Bra007665, and Bra014410 were selected as the targets of the CRISPR/Cas9 system. A single-targeting vector and a multitargeting vector were constructed. Different types of mutations were detected in T0 generation through Agrobacterium transformation. The mutation rate of the three designed sgRNA seeds varied from 20 to 56%. Although the majority of T0 mutants were chimeric, four homozygous mutants were identified. Transformation with the multitargeting vector generated one line with a large fragment deletion and one line with mutations in two target genes. Mutations in Bra003491 were stable and inherited by T1 and T2 generations. Nine mutants which did not contain T-DNA insertions were also obtained. No mutations were detected in predicted potential off-target sites. Our work demonstrated that CRISPR/Cas9 system is efficient on single and multiplex genome editing without off-targeting in B. campestris and that the mutations are stable and inheritable. Our results may greatly facilitate gene functional studies and the molecular breeding of B. campestris and other plants.


Assuntos
Brassica/genética , Sistemas CRISPR-Cas/genética , Genoma de Planta/genética , Agrobacterium/genética , Cruzamento/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Genes de Plantas/genética , Homozigoto , Mutação/genética , Taxa de Mutação , Fenótipo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética
19.
Science ; 364(6437): 292-295, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30819931

RESUMO

Cytosine and adenine base editors (CBEs and ABEs) are promising new tools for achieving the precise genetic changes required for disease treatment and trait improvement. However, genome-wide and unbiased analyses of their off-target effects in vivo are still lacking. Our whole-genome sequencing analysis of rice plants treated with the third-generation base editor (BE3), high-fidelity BE3 (HF1-BE3), or ABE revealed that BE3 and HF1-BE3, but not ABE, induce substantial genome-wide off-target mutations, which are mostly the C→T type of single-nucleotide variants (SNVs) and appear to be enriched in genic regions. Notably, treatment of rice with BE3 or HF1-BE3 in the absence of single-guide RNA also results in the rise of genome-wide SNVs. Thus, the base-editing unit of BE3 or HF1-BE3 needs to be optimized in order to attain high fidelity.


Assuntos
Citosina , Análise Mutacional de DNA/métodos , Edição de Genes/métodos , Oryza/genética , Sequenciamento Completo do Genoma/métodos , Adenina , Agrobacterium/genética , Mutação , Oryza/microbiologia , Polimorfismo de Nucleotídeo Único
20.
Viruses ; 11(2)2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30791535

RESUMO

Higher plants exploit posttranscriptional gene silencing as a defense mechanism against virus infection by the RNA degradation system. Plant RNA viruses suppress posttranscriptional gene silencing using their encoded proteins. Three important motifs (F-box-like motif, G139/W140/G141-like motif, and C-terminal conserved region) in P0 of Potato leafroll virus (PLRV) were reported to be essential for suppression of RNA silencing activity. In this study, Agrobacterium-mediated transient experiments were carried out to screen the available amino acid substitutions in the F-box-like motif and G139/W140/G141-like motif that abolished the RNA silencing suppression activity of P0, without disturbing the P1 amino acid sequence. Subsequently, four P0 defective mutants derived from a full-length cDNA clone of PLRV (L76F and W87R substitutions in the F-box-like motif, G139RRR substitution in the G139/W140/G141-like motif, and F220R substitution in the C-terminal conserved region) were successfully generated by reverse PCR and used to investigate the impact of these substitutions on PLRV infectivity. The RT-PCR and western blot analysis revealed that these defective mutants affected virus accumulation in inoculated leaves and systemic movement in Nicotiana benthamiana as well as in its natural hosts, potato and black nightshade. These results further demonstrate that the RNA silencing suppressor of PLRV is required for PLRV accumulation and systemic infection.


Assuntos
Inativação Gênica , Luteoviridae/genética , Mutação , Tabaco/virologia , Proteínas Virais/genética , Agrobacterium/genética , Substituição de Aminoácidos , Motivos F-Box/genética , Doenças das Plantas/virologia , Vírus de Plantas/genética , Solanum tuberosum/virologia
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