Altered carbon status in Glycine max hairy roots induced by Agrobacterium rhizogenes.
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ABSTRACT:
SUBMITTER: Okamoto S
PROVIDER: S-EPMC9278451 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Aim:To develop a useful alternative approach to evaluate the gene function in hairy roots. Methods:Arabidopsis and tobacco (wild-type or mutant) were a host for Agrobacterium rhizogenes transformation. Results:The hairy roots formation efficiency ranged from 53 to 98% in tobacco and 53 to 66% in Arabidopsis. Hairy and intact roots showed similar gene expression pattern in response to salt and aluminum stress. Genomic polymerase chain reaction and fluorescent images showed high rate (>80%) of co-integration of T-DNAs and uniform cell transformation without use of any antibiotic selection. Whole processes of hairy roots were completed within 1 month after the infection of Agrobacterium. Conclusion:Aluminum-responsive orthologous gene function could be evaluated by NtSTOP1-KD and Atstop1 as a host for hairy roots transformation.
| S-EPMC6426172 | biostudies-literature
Project description:Agrobacterium rhizogenes is a pathogenic bacteria that causes hairy root disease by transferring bacterial DNA into the plant genome. It is an essential tool for industry and research due to its capacity to produce genetically modified roots and whole organisms. Here, we identified and characterized small RNAs generated from the transfer DNA (T-DNA) of A. rhizogenes in hairy roots of common bean (Phaseolus vulgaris). Distinct abundant A. rhizogenes T-DNA-derived small RNAs (ArT-sRNAs) belonging to several oncogenes were detected in hairy roots using high-throughput sequencing. The most abundant and diverse species of ArT-sRNAs were those of 21- and 22-nucleotides in length. Many T-DNA encoded genes constituted phasiRNA producing loci (PHAS loci). Interestingly, degradome analysis revealed that ArT-sRNAs potentially target genes of P. vulgaris. In addition, we detected low levels of ArT-sRNAs in the A. rhizogenes-induced calli generated at the wound site before hairy root emergence. These results suggest that RNA silencing targets several genes from T-DNA of A. rhizogenes in hairy roots of common bean. Therefore, the role of RNA silencing observed in this study has implications in our understanding and usage of this unique plant-bacteria interaction.
| S-EPMC5285386 | biostudies-literature
Project description:BackgroundSoybean mosaic virus (SMV), a Potyvirus, is the most prevalent viral pathogen of soybean that causes severe yield and seed quality reductions in world soybean production. So far, multiple resistance loci for different SMV strains have been fine-mapped while the candidate genes' functions need to be verified. However, identification of the resistance or susceptibility genes via stable genetic transformation is time-consuming and labor-intensive, which hinders further exploration of these genes' functions in soybean. Thus we tried to explore a rapid and efficient method for verification of SMV-related target gene function in soybean.ResultsAn Agrobacterium rhizogenes (A. rhizogenes) induced soybean hairy roots versus Soybean mosaic virus (ARISHR-SMV) pathosystem was established. The procedure is characterized with that (1) the soybean hairy roots that can be infected by SMV are induced by A. rhizogenes K599 using soybean cotyledons as explants, (2) the gene to be examined for its function, which may be the endogenous SMV-resistance or -susceptible gene or exogenous SMV-related gene, is transformed into soybean calluses mediated by A. rhizogenes, (3) the transformed calluses on explants further inoculated with the purified tester-SMV virions using pricking method under aseptic conditions, and (4) the measurement of the SMV content in positive hairy roots for evaluating the SMV-related target gene function. The procedure takes about 30 days for one cycle. Utilizing the established procedure, the soybean hairy roots induced by A. rhizogenes was efficiently infected by multiple different SMV strains, the SMV infectivity in soybean hairy roots can be retained at least twice successive transfer cultures and Tomato bushy stunt virus (TBSV) P19 promoting and SMV CP suppressing SMV infection in soybean hairy roots was demonstrated, respectively. This procedure can also be used for identification of resistance to SMV strains for soybean germplasms.ConclusionThe ARISHR-SMV is an efficient pathosystem that allows a quick and convenient identification of SMV-related target gene function in soybean.
| S-EPMC6534890 | biostudies-literature
Project description:Agrobacterium rhizogenes induce the production of the hairy root through the transformation of plant genomes. In this article, we executed the transcriptome of A. rhizogenes through RNA-sequencing. RNA-sequencing of A. rhizogenes generated a total of 2.6 Gb raw data with a 75 bp paired-end sequence. The raw data has been submitted to the SRA database of NCBI with accession number SRR5641651. Reads were generated 2946 unigenes and all unigenes were annotated in the database. The length of transcripts ranged from 90 to 6369 bp, with a median transcript length of 968. The transcripts were annotated through the number of databases to obtain information about SSRs, SNPs, Gene Ontology, Transcription factors, and pathways analysis .
| S-EPMC7334392 | biostudies-literature
Project description:CRIPSR/Cas9 gene editing system is an effective tool for genome modification in plants. Multiple target sites are usually designed and the effective target sites are selected for editing. Upland cotton (Gossypium hirsutum L., hereafter cotton) is allotetraploid and is commonly considered as difficult and inefficient to transform, it is important to select the effective target sites that could result in the ideal transgenic plants with the CRISPR-induced mutations. In this study, Agrobacterium rhizogenes-mediated hairy root method was optimized to detect the feasibility of the target sites designed in cotton phytoene desaturase (GhPDS) gene. A. rhizogenes showed the highest hairy root induction (30%) when the bacteria were cultured until OD600 reached to 0.8. This procedure was successfully applied to induce hairy roots in the other three cultivars (TM-1, Lumian-21, Zhongmian-49) and the mutations were detected in GhPDS induced by CRISPR/Cas9 system. Different degrees of base deletions at two sgRNAs (sgRNA5 and sgRNA10) designed in GhPDS were detected in R15 hairy roots. Furthermore, we obtained an albino transgenic cotton seeding containing CRISPR/Cas9-induced gene editing mutations in sgRNA10. The hairy root transformation system established in this study is sufficient for selecting sgRNAs in cotton, providing a technical basis for functional genomics research of cotton.
| S-EPMC9832336 | biostudies-literature
Project description:BackgroundAgrobacterium-mediated genetic transformation is a widely used and efficient technique for gene functional research in crop breeding and plant biology. While in some plant species, including soybean, genetic transformation is still recalcitrant and time-consuming, hampering the high-throughput functional analysis of soybean genes. Thus we pursue to develop a rapid, simple, and highly efficient hairy root system induced by Agrobacterium rhizogenes (A. rhizogenes) to analyze soybean gene function.ResultsIn this report, a rapid, simple, and highly efficient hairy root transformation system for soybean was described. Only sixteen days were required for the whole workflow and the system was suitable for various soybean genotypes, with an average transformation frequency of 58-64%. Higher transformation frequency was observed when wounded cotyledons from 1-day-germination seeds were inoculated and co-cultivated with A. rhizogenes in 1/2 B5 (Gamborg' B-5) medium. The addition of herbicide selection to root production medium increased the transformation frequency to 69%. To test the applicability of the hairy root system for gene functional analysis, we evaluated the protein expression and subcellular localization in transformed hairy roots. Transgenic hairy roots exhibited significantly increased GFP fluorescence and appropriate protein subcellular localization. Protein-protein interactions by BiFC (Bimolecular Fluorescent Complimentary) were also explored using the hairy root system. Fluorescence observations showed that protein interactions could be observed in the root cells. Additionally, hairy root transformation allowed soybean target sgRNA screening for CRISPR/Cas9 gene editing. Therefore, the protocol here enables high-throughput functional characterization of candidate genes in soybean.ConclusionA rapid, simple, and highly efficient A. rhizogenes-mediated hairy root transformation system was established for soybean gene functional analysis, including protein expression, subcellular localization, protein-protein interactions and gene editing system evaluation.
| S-EPMC8272327 | biostudies-literature
Project description:BackgroundLitchi chinensis Sonn. is an economically important fruit tree in tropical and subtropical regions. However, litchi functional genomics is severely hindered due to its recalcitrance to regeneration and stable transformation. Agrobacterium rhizogenes-mediated hairy root transgenic system provide an alternative to study functional genomics in woody plants. However, the hairy root transgenic system has not been established in litchi.ResultsIn this study, we report a rapid and highly efficient A. rhizogenes-mediated co-transformation system in L. chinensis using Green Fluorescent Protein (GFP) gene as a marker. Both leaf discs and stem segments of L. chinensis cv. 'Fenhongguiwei' seedlings were able to induce transgenic hairy roots. The optimal procedure involved the use of stem segments as explants, infection by A. rhizogenes strain MSU440 at optical density (OD600) of 0.7 for 10 min and co-cultivation for 3 days, with a co-transformation efficiency of 9.33%. Furthermore, the hairy root transgenic system was successfully used to validate the function of the key anthocyanin regulatory gene LcMYB1 in litchi. Over-expression of LcMYB1 produced red hairy roots, which accumulated higher contents of anthocyanins, proanthocyanins, and flavonols. Additionally, the genes involving in the flavonoid pathway were strongly activated in the red hairy roots.ConclusionWe first established a rapid and efficient transformation system for the study of gene function in hairy roots of litchi using A. rhizogenes strain MSU440 by optimizing parameters. This hairy root transgenic system was effective for gene function analysis in litchi using the key anthocyanin regulator gene LcMYB1 as an example.
| S-EPMC8502350 | biostudies-literature
Project description:Composite plants consisting of a wild-type shoot and a transgenic root are frequently used for functional genomics in legume research. Although transformation of roots using Agrobacterium rhizogenes leads to morphologically normal roots, the question arises as to whether such roots interact with arbuscular mycorrhizal (AM) fungi in the same way as wild-type roots. To address this question, roots transformed with a vector containing the fluorescence marker DsRed were used to analyse AM in terms of mycorrhization rate, morphology of fungal and plant subcellular structures, as well as transcript and secondary metabolite accumulations. Mycorrhization rate, appearance, and developmental stages of arbuscules were identical in both types of roots. Using Mt16kOLI1Plus microarrays, transcript profiling of mycorrhizal roots showed that 222 and 73 genes exhibited at least a 2-fold induction and less than half of the expression, respectively, most of them described as AM regulated in the same direction in wild-type roots. To verify this, typical AM marker genes were analysed by quantitative reverse transcription-PCR and revealed equal transcript accumulation in transgenic and wild-type roots. Regarding secondary metabolites, several isoflavonoids and apocarotenoids, all known to accumulate in mycorrhizal wild-type roots, have been found to be up-regulated in mycorrhizal in comparison with non-mycorrhizal transgenic roots. This set of data revealed a substantial similarity in mycorrhization of transgenic and wild-type roots of Medicago truncatula, validating the use of composite plants for studying AM-related effects.
| S-EPMC2736893 | biostudies-literature
Project description:BackgroundAgrobacterium rhizogenes-mediated (ARM) transformation is a highly efficient technique for generating composite plants composed of transgenic roots and wild-type shoot, providing a powerful tool for studying root biology. The ARM transformation has been established in many plant species, including soybean. However, traditional transformation of soybean, transformation efficiency is low. Additionally, the hairy roots were induced in a medium, and then the generated composite plants were transplanted into another medium for growth. This two-step operation is not only time-consuming, but aggravates contamination risk in the study of plant-microbe interactions.ResultsHere, we report a one-step ARM transformation method with higher transformation efficiency for generating composite soybean plants. Both the induction of hairy roots and continuous growth of the composite plants were conducted in a single growth medium. The primary root of a 7-day-old seedling was decapitated with a slanted cut, the residual hypocotyl (maintained 0.7-1 cm apical portion) was inoculated with A. rhizogenes harboring the gene construct of interest. Subsequently, the infected seedling was planted into a pot with wet sterile vermiculite. Almost 100% of the infected seedlings could produce transgenic positive roots 16 days post-inoculation in 7 tested genotypes. Importantly, the transgenic hairy roots in each composite plant are about three times more than those of the traditional ARM transformation, indicating that the one-step method is simpler in operation and higher efficiency in transformation. The reliability of the one-step method was verified by CRISPR/Cas9 system to knockout the soybean Rfg1, which restricts nodulation in Williams 82 (Nod-) by Sinorhizobium fredii USDA193. Furthermore, we applied this method to analyze the function of Arabidopsis YAO promoter in soybean. The activity of YAO promoter was detected in whole roots and stronger in the root tips. We also extended the protocol to tomato.ConclusionsWe established a one-step ARM transformation method, which is more convenient in operation and higher efficiency (almost 100%) in transformation for generating composite soybean plants. This method has been validated in promoter functional analysis and rhizobia-legume interactions. We anticipate a broad application of this method to analyze root-related events in tomato and other plant species besides soybean.
| S-EPMC7333419 | biostudies-literature
Project description:Hairy roots are made after the integration of a small set of genes from Agrobacterium rhizogenes in the plant genome. Little is known about how this small set is linked to their hormone profile, which determines development, morphology, and levels of secondary metabolite production. We used C. asiatica hairy root line cultures to determine the putative links between the rol and aux gene expressions with morphological traits, a hormone profile, and centelloside production. The results obtained after 14 and 28 days of culture were processed via multivariate analysis and machine-learning processes such as random forest, supported vector machines, linear discriminant analysis, and neural networks. This allowed us to obtain models capable of discriminating highly productive root lines from their levels of genetic expression (rol and aux genes) or from their hormone profile. In total, 12 hormones were evaluated, resulting in 10 being satisfactorily detected. Within this set of hormones, abscisic acid (ABA) and cytokinin isopentenyl adenosine (IPA) were found to be critical in defining the morphological traits and centelloside content. The results showed that IPA brings more benefits to the biotechnological platform. Additionally, we determined the degree of influence of each of the evaluated genes on the individual hormone profile, finding that aux1 has a significant influence on the IPA profile, while the rol genes are closely linked to the ABA profile. Finally, we effectively verified the gene influence on these two specific hormones through feeding experiments that aimed to reverse the effect on root morphology and centelloside content.
| S-EPMC9479193 | biostudies-literature