Dataset Information

Small RNAs Derived from the T-DNA of Agrobacterium rhizogenes in Hairy Roots of Phaseolus vulgaris.

ABSTRACT: 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.

SUBMITTER: Pelaez P

PROVIDER: S-EPMC5285386 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Small RNAs Derived from the T-DNA of Agrobacterium rhizogenes in Hairy Roots of Phaseolus vulgaris.

Peláez Pablo P Hernández-López Alejandrina A Estrada-Navarrete Georgina G Sanchez Federico F

Frontiers in plant science 20170201

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 ...[more]

PMID: 28203245

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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.

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.

Dataset Information

Small RNAs Derived from the T-DNA of Agrobacterium rhizogenes in Hairy Roots of Phaseolus vulgaris.

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Small RNAs Derived from the T-DNA of <i>Agrobacterium rhizogenes</i> in Hairy Roots of <i>Phaseolus vulgaris</i>.

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