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 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.

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 Gene Silencing by Expression of Hairpin RNA in Lotus japonicus Roots and Root Nodules

2003 ◽  
Vol 16 (8) ◽  
pp. 663-668 ◽  
Author(s):  
Hirotaka Kumagai ◽  
Hiroshi Kouchi
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Rna Silencing ◽  
Root Nodules ◽  
Lotus Japonicus ◽  
Loss Of Function ◽  
Coding Region ◽  
Hairpin Rna ◽  
Model Legume ◽  
Root Transformation

We investigated the efficacy of self-complementary hairpin RNA (hpRNA) expression to induce RNA silencing in the roots and nodules of model legume Lotus japonicus, using hairy root transformation mediated by Agrobacterium rhizogenes. Transgenic lines that express β-glucuronidase (GUS) by constitutive or nodule-specific promoters were supertransformed by infection of A. rhizogenes harboring constructs for the expression of hpRNAs with sequences complementary to the GUS coding region. GUS activity in more than 60% of the hairy roots was decreased or silenced almost completely. Silencing of the GUS gene was also observed in symbiotic nodules formed on hairy roots in both early and late stages of nodule organogenesis. These results indicate that transient RNA silencing by hairy root transformation provides a powerful tool for loss-of-function analyses of genes that function in roots and root nodules.

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.

Hairy Root Transformation in Lotus japonicus

BIO-PROTOCOL ◽  
2013 ◽  
Vol 3 (12) ◽  
Author(s):  
Satoru Okamoto ◽  
Emiko Yoro ◽  
Takuya Suzaki ◽  
Masayoshi Kawaguchi
Keyword(s):  
Hairy Root ◽  
Lotus Japonicus ◽  
Root Transformation

Detection of Target Sites Edited in Upland Cotton By The CRISPR/Cas9 System Mediated By Agrobacterium Rhizogenes

2021 ◽  
Author(s):  
Lili Zhou ◽  
Yali Wang ◽  
Peilin Wang ◽  
Jiamin Wang ◽  
Hongmei Cheng
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Upland Cotton ◽  
Gene Editing ◽  
Target Site ◽  
Shoot Meristem ◽  
Multiple Target ◽  
Root Transformation ◽  
Target Sites

Abstract Background CRIPSR/Cas9 gene editing has the ability to effectively modify plant genomes. Multiple target sites usually were designed and the effective target sites were selected for editing. However, upland cotton is allotetraploid and is commonly considered as difficult and inefficient to transform. Therefore, it’s important to quickly identify feasibility of the target site. In this study, we use Agrobacterium rhizogenes K599 strain to infect cotton shoot meristem and induce them to grow hairy roots to detect the feasibility of a selected target designed in GhMYB25-like gene. Results We designed a sgRNA within the second exons of GhMYB25-likeA and GhMYB25-likeD and constructed the CRISPR vector. Transient hairy root transformation using A. rhizogenes K599 with four OD600s (0.4, 0.6,0.8, 1.0) was performed in Coker 312 (R15). The results show that A. rhizogenes at OD600 = 0.6–0.8 is the best concentration range for inducing cotton hairy roots. The other three cultivars (TM-1, Lumian 21, Zhongmian 49) were injected using A. rhizogenes K599 with OD600 = 0.6-0.8 and all produced hairy roots. We characterized ten R15 plants with hairy roots and detected different degrees of base deletions and insert at the target site in five R15 plants. Conclusion Overall, our data show A. rhizogenes-mediated transient hairy root transformation offers a rapid and efficient method to detect sgRNA feasibility in cotton.

Evaluation of influence of different strains of Agrobacterium rhizogenes on efficiency of hairy root induction in Rauwolfia serpentina

2020 ◽  
Vol 79 (04) ◽  
Author(s):  
Pratima Bhagat ◽  
Sachin Kumar Verma ◽  
Amit Kumar Singh ◽  
Gajendra Kumar Aseri ◽  
Neeraj Khare
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Transformation Efficiency ◽  
Effective Means ◽  
Root Induction ◽  
Infection Time ◽  
Rauwolfia Serpentina ◽  
Cultivation Period ◽  
Significant Difference ◽  
Hairy Root Induction

An improved protocol was established for hairy root induction in Rauwolfia serpentina using two strains of Agrobacterium rhizogenes MTCC 532 and 2364. Hairy root induction efficiency was standardized based on infection time, co-cultivation period, OD600 and acetosyringone concentration. Higher transformation efficiency was established using MTCC 532 (31%) and MTCC 2364 (24%) with 30 min infection time, 72 h co-cultivation period and 0.6 OD600. Transformation efficiency was further enhanced to 55 % with 125 μM acetosyringone. MTCC 532 was proven a better strain over the MTCC 2364 in all the tested factors. Putative transgenic hairy roots were confirmed by polymerase chain reaction using rolA specific primers. Biomass of randomly selected 5 hairy root lines was also significantly enhanced. No significant difference of growth was recorded among the lines except line no 4. In the present study, an enhanced system for Agrobacterium rhizogenesmediated hairy root culture was established which offer an effective means to attain improved transformation efficiency and ultimately beneficial for the industrial scale in vitro production of the secondary metabolite.

scholarly journals Induction of transgenic hairy roots in soybean genotypes by Agrobacterium rhizogenes‑mediated transformation

2011 ◽  
Vol 46 (9) ◽  
pp. 1070-1075 ◽  
Author(s):  
Ricardo Luís Mayer Weber ◽  
Maria Helena Bodanese‑Zanettini
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Fluorescent Protein ◽  
Root Culture ◽  
Transformation Frequency ◽  
Plant Production ◽  
Uniform Size ◽  
Soybean Genotypes

The objective of this work was to perform the screening of soybean genotypes as to their ability to respond to the induction of hairy roots by Agrobacterium rhizogenes‑mediated transformation. Four Brazilian soybean cultivars (BRSMG 68 Vencedora, BRS 137, Embrapa 48, and MG/BR 46 Conquista) and two North American ones adapted to Brazilian cropping conditions (Bragg and IAS‑5) were screened for their capacity to respond to A. rhizogenes in protocols for in vitro hairy root culture and ex vitro composite plant production. Four‑day‑old seedlings with uniform size were injected with A. rhizogenes harboring the plasmid p35S‑GFP. Seedlings expressing green fluorescent protein (GFP) in at least one hairy root were used to determine the transformation frequency. Using an axenic in vitro protocol, excised cotyledons from four‑day‑old seedlings were infected with A. rhizogenes harboring the pCAMBIA1301 plasmid, containing the gusA reporter gene. The transformation frequency and the number of days for hairy root emergence after bacterial infection (DAI) were evaluated. The transformation frequency and DAI varied according to the genotype. Cultivars MG/BR 46 Conquista and BRSMG 68 Vencedora are more susceptible to A. rhizogenes and can be recommended for transformation experiments.

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