scholarly journals Agrobacterium rhizogenes Transformation of the Phaseolus spp.: A Tool for Functional Genomics

2006 ◽  
Vol 19 (12) ◽  
pp. 1385-1393 ◽  
Author(s):  
Georgina Estrada-Navarrete ◽  
Xochitl Alvarado-Affantranger ◽  
Juan-Elías Olivares ◽  
Claudia Díaz-Camino ◽  
Olivia Santana ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Agrobacterium Rhizogenes ◽  
Fluorescent Protein ◽  
Phaseolus Vulgaris L ◽  
Rhizobium Tropici ◽  
Transgenic Roots ◽  
Root Transformation ◽  
Microbe Interactions ◽  
Wild Accessions ◽  
Green Fluorescent

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: P. coccineus, P. lunatus, and P. acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and β-glu-curonidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root–microbe interactions.

scholarly journals Use of the rhizobial type III effector gene nopP to improve Agrobacterium rhizogenes-mediated transformation of Lotus japonicus

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yan Wang ◽  
Feng Yang ◽  
Peng-Fei Zhu ◽  
Asaf Khan ◽  
Zhi-Ping Xie ◽  
...  
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Fluorescent Protein ◽  
Transformation Efficiency ◽  
Lotus Japonicus ◽  
Nodule Bacterium ◽  
Transgenic Roots ◽  
Model Legume ◽  
Effector Gene ◽  
Root Transformation

Abstract Background Protocols for Agrobacterium rhizogenes-mediated hairy root transformation of the model legume Lotus japonicus have been established previously. However, little efforts were made in the past to quantify and improve the transformation efficiency. Here, we asked whether effectors (nodulation outer proteins) of the nodule bacterium Sinorhizobium sp. NGR234 can promote hairy root transformation of L. japonicus. The co-expressed red fluorescent protein DsRed1 was used for visualization of transformed roots and for estimation of the transformation efficiency. Results Strong induction of hairy root formation was observed when A. rhizogenes strain LBA9402 was used for L. japonicus transformation. Expression of the effector gene nopP in L. japonicus roots resulted in a significantly increased transformation efficiency while nopL, nopM, and nopT did not show such an effect. In nopP expressing plants, more than 65% of the formed hairy roots were transgenic as analyzed by red fluorescence emitted by co-transformed DsRed1. A nodulation experiment indicated that nopP expression did not obviously affect the symbiosis between L. japonicus and Mesorhizobium loti. Conclusion We have established a novel protocol for hairy root transformation of L. japonicus. The use of A. rhizogenes LBA9402 carrying a binary vector containing DsRed1 and nopP allowed efficient formation and identification of transgenic roots.

scholarly journals Agrobacterium rhizogenes—mediated transformation ofPisum sativumL. roots as a tool for studying the mycorrhizal and root nodule symbioses

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6552 ◽  
Author(s):  
Irina V. Leppyanen ◽  
Anna N. Kirienko ◽  
Elena A. Dolgikh
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Rhizobium Leguminosarum ◽  
Fluorescent Protein ◽  
Arbuscular Mycorrhizal ◽  
Am Fungi ◽  
Symbiotic Association ◽  
Gene Encoding ◽  
Transgenic Roots ◽  
Pea Seedlings ◽  
Green Fluorescent

In this study, we demonstrated the successful transformation of two pea (Pisum sativumL.) cultivars usingAgrobacterium rhizogenes, whereby transgenic roots in the resulting composite plants showed expression of the gene encoding the green fluorescent protein. Subsequent to infection withA. rhizogenes, approximately 70%–80% of pea seedlings developed transgenic hairy roots. We found out that the transgenic roots can be efficiently nodulated byRhizobium leguminosarumbv.viciaeand infected by the arbuscular mycorrhizal (AM) fungusRhizophagus 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 efficientA. 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.

scholarly journals The Nod Factor–Independent Symbiotic Signaling Pathway: Development of Agrobacterium rhizogenes–Mediated Transformation for the Legume Aeschynomene indica

2010 ◽  
Vol 23 (12) ◽  
pp. 1537-1544 ◽  
Author(s):  
Katia Bonaldi ◽  
Hassen Gherbi ◽  
Claudine Franche ◽  
Géraldine Bastien ◽  
Joël Fardoux ◽  
...  
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Molecular Basis ◽  
Central Zone ◽  
Cauliflower Mosaic Virus ◽  
Promoter Activation ◽  
Transgenic Roots ◽  
Root Transformation ◽  
Invaluable Tool ◽  
Cell Layers ◽  
Spatio Temporal

The nitrogen-fixing symbiosis between Aeschynomene indica and photosynthetic bradyrhizobia is the only legume-rhizobium association described to date that does not require lipochito-oligosaccharide Nod factors (NF). To assist in deciphering the molecular basis of this NF-independent interaction, we have developed a protocol for Agrobacterium rhizogenes-mediated transformation of A. indica. The cotransformation frequency (79%), the nodulation efficiency of transgenic roots (90%), and the expression pattern of the 35S Cauliflower mosaic virus promoter in transgenic nodules were all comparable to those obtained for model legumes. We have made use of this tool to monitor the heterologous spatio-temporal expression of the pMtENOD11-β-glucuronidase fusion, a widely used molecular reporter for rhizobial infection and nodulation in both legumes and actinorhizal plants. While MtENOD11 promoter activation was not observed in A. indica roots prior to nodulation, strong reporter-gene expression was observed in the invaded cells of young nodules and in the cell layers bordering the central zone of older nodules. We conclude that pMtENOD11 expression can be used as an infection-related marker in A. indica and that Agrobacterium rhizogenes–mediated root transformation of Aeschynomene spp. will be an invaluable tool for determining the molecular basis of the NF-independent symbiosis.

scholarly journals Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation

Plants ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 51
Author(s):  
Alexey S. Kiryushkin ◽  
Elena L. Ilina ◽  
Elizaveta D. Guseva ◽  
Katharina Pawlowski ◽  
Kirill N. Demchenko
Keyword(s):  
Genome Editing ◽  
Hairy Root ◽  
Fluorescent Protein ◽  
Effective Means ◽  
Gene Encoding ◽  
Transgenic Roots ◽  
Translational Enhancer ◽  
Current State ◽  
Root Transformation ◽  
Agrobacterium Rhizogenes Strains

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.

A Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins

2019 ◽  
Author(s):  
Chi-Yun Lin ◽  
Matthew Romei ◽  
Luke Oltrogge ◽  
Irimpan Mathews ◽  
Steven Boxer
Keyword(s):  
Driving Force ◽  
Fluorescent Protein ◽  
Charge Transfer Band ◽  
Photophysical Properties ◽  
Polymethine Dyes ◽  
Emission Rates ◽  
Unified Framework ◽  
Underlying Factor ◽  
Green Fluorescent ◽  
Protein Environment

Green fluorescent protein (GFPs) have become indispensable imaging and optogenetic tools. Their absorption and emission properties can be optimized for specific applications. Currently, no unified framework exists to comprehensively describe these photophysical properties, namely the absorption maxima, emission maxima, Stokes shifts, vibronic progressions, extinction coefficients, Stark tuning rates, and spontaneous emission rates, especially one that includes the effects of the protein environment. In this work, we study the correlations among these properties from systematically tuned GFP environmental mutants and chromophore variants. Correlation plots reveal monotonic trends, suggesting all these properties are governed by one underlying factor dependent on the chromophore's environment. By treating the anionic GFP chromophore as a mixed-valence compound existing as a superposition of two resonance forms, we argue that this underlying factor is defined as the difference in energy between the two forms, or the driving force, which is tuned by the environment. We then introduce a Marcus-Hush model with the bond length alternation vibrational mode, treating the GFP absorption band as an intervalence charge transfer band. This model explains all the observed strong correlations among photophysical properties; related subtopics are extensively discussed in Supporting Information. Finally, we demonstrate the model's predictive power by utilizing the additivity of the driving force. The model described here elucidates the role of the protein environment in modulating photophysical properties of the chromophore, providing insights and limitations for designing new GFPs with desired phenotypes. We argue this model should also be generally applicable to both biological and non-biological polymethine dyes.<br>

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