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Agrobacterium rhizogenes-mediated transformation of Pisum sativum L. roots as a tool for studying the mycorrhizal and root nodule symbioses.

ABSTRACT: In this study, we demonstrated the successful transformation of two pea (Pisum sativum L.) cultivars using Agrobacterium rhizogenes, whereby transgenic roots in the resulting composite plants showed expression of the gene encoding the green fluorescent protein. Subsequent to infection with A. rhizogenes, approximately 70%-80% of pea seedlings developed transgenic hairy roots. We found out that the transgenic roots can be efficiently nodulated by Rhizobium leguminosarum bv. viciae and infected by the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis. The morphology of nodules in the transgenic roots was found to be identical to that of nodules observed in wild-type roots, and we also observed the effective induction of markers typical of the symbiotic association with AM fungi. The convenient protocol for highly efficient A. rhizogenes-mediated transformation developed in this study would be a rapid and effective tool for investigating those genes involved in the development of the two types of symbioses found in pea plants.

SUBMITTER: Leppyanen IV

PROVIDER: S-EPMC6408910 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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.

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Agrobacterium rhizogenes-mediated transformation of Pisum sativum L. roots as a tool for studying the mycorrhizal and root nodule symbioses.

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