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Agrobacterium rhizogenes-mediated hairy roots transformation as a tool for exploring aluminum-responsive genes function.

ABSTRACT: 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.

SUBMITTER: Daspute AA

PROVIDER: S-EPMC6426172 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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<i>Agrobacterium rhizogenes</i>-mediated hairy roots transformation as a tool for exploring aluminum-responsive genes function.

Daspute Abhijit A AA Yunxuan Xian X Gu Minghua M Kobayashi Yuriko Y Wagh Sopan S Panche Archana A Koyama Hiroyuki H

Future science OA 20190208 3

<h4>Aim</h4>To develop a useful alternative approach to evaluate the gene function in hairy roots.<h4>Methods</h4><i>Arabidopsis</i> and tobacco (wild-type or mutant) were a host for <i>Agrobacterium rhizogenes</i> transformation.<h4>Results</h4>The hairy roots formation efficiency ranged from 53 to 98% in tobacco and 53 to 66% in <i>Arabidopsis</i>. Hairy and intact roots showed similar gene expression pattern in response to salt and aluminum stress. Genomic polymerase chain reaction and fluore ...[more]

PMID: 30906565

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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.

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.

Dataset Information

Agrobacterium rhizogenes-mediated hairy roots transformation as a tool for exploring aluminum-responsive genes function.

Publications

<i>Agrobacterium rhizogenes</i>-mediated hairy roots transformation as a tool for exploring aluminum-responsive genes function.

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