Anthocyanin, a novel and user-friendly reporter for convenient, non-destructive, low cost, directly visual selection of transgenic hairy roots in the study of rhizobia-legume symbiosis.
ABSTRACT: Background:Agrobacterium rhizogenes-mediated hairy root transformation provides a powerful tool for investigating the functions of plant genes involved in rhizobia-legume symbiosis. However, in the traditional identification methods of transgenic hairy roots based on reporter genes, an expensive chemical substrate or equipment is required. Results:Here, we report a novel, low cost, and robust reporter for convenient, non-destructive, and directly visual selection of transgenic hairy roots by naked eye, which can be used in the study of rhizobia-legume symbiosis. The reporter gene AtMyb75 in Arabidopsis, encoding an R2R3 type MYB transcription factor, was ectopically expressed in hairy roots-mediated by A. rhizogenes, which induced purple/red colored anthocyanin accumulation in crop species like soybean (Glycine max (L.) Merr.) and two model legume species, Lotus japonicas and Medicago truncatula. Transgenic hairy roots of legumes containing anthocyanin can establish effective symbiosis with rhizobia. We also demonstrated the reliability of AtMyb75 as a reporter gene by CRISPR/Cas9-targeted mutagenesis of the soybean resistance to nodulation Rfg1 gene in the soybean PI377578 (Nod-) inoculated with Sinorhizobium fredii USDA193. Without exception, mature nitrogen-fixation nodules, were formed on purple transgenic hairy roots containing anthocyanin. Conclusions:Anthocyanin is a reliable, user-friendly, convenient, non-destructive, low cost, directly visual reporter for studying symbiotic nitrogen-fixing nodule development and could be widely applied in broad leguminous plants.
Project description:The development and functioning of the nitrogen fixing symbiosis between legume plants and soil bacteria collectively called rhizobia requires continuous chemical dialogue between the partners using different molecules such as flavonoids, lipo-chitooligosaccharides, polysaccharides and peptides. Agrobacterium rhizogenes mediated hairy root transformation of legumes is widely used to study the function of plant genes involved in the process. The identification of transgenic plant tissues is based on antibiotics/herbicide selection and/or the detection of different reporter genes that usually require special equipment such as fluorescent microscopes or destructive techniques and chemicals to visualize enzymatic activity. Here, we developed and efficiently used in hairy root experiments binary vectors containing the MtLAP1 gene driven by constitutive and tissue-specific promoters that facilitate the production of purple colored anthocyanins in transgenic tissues and thus allowing the identification of transformed roots by naked eye. Anthocyanin producing roots were able to establish effective symbiosis with rhizobia. Moreover, it was shown that species-specific allelic variations and a mutation preventing posttranslational acetyl modification of an essential nodule-specific cysteine-rich peptide, NCR169, do not affect the symbiotic interaction of Medicago truncatula cv. Jemalong with Sinorhizobium medicae strain WSM419. Based on the experiments, it could be concluded that it is preferable to use the vectors with tissue-specific promoters that restrict anthocyanin production to the root vasculature for studying biotic interactions of the roots such as symbiotic nitrogen fixation or mycorrhizal symbiosis.
Project description:<h4>Background</h4> Strigolactones (SLs) play a key role in modulating plant root growth, shoot branching, and plant-symbiont interaction. However, despite their significance, the components of SL biosynthesis and signaling in soybean and their role in soybean-rhizobia interaction is unknown. <h4>Methods</h4> In this study, we identified and functionally characterized the GmD53a from soybean. The GmD53a ORFs were amplified from root cDNA using primers for GmD53a RNA interference. To induce transgenic hairy roots of soybean, electric shock was used to transform pB7WG1WG2 vectors containing GmD53a knockdown and GUS into K599 strains of Agrobacterium rhizogenes. The hairy roots and nodules were collected and examined for root nodules ratio and RNA was extracted after 4 weeks of rhizobia inoculation. <h4>Results</h4> A tissue-specific expression assay showed that GmD53a was differentially expressed in plant parts, predominantly in the stem and nodule. Furthermore, its expression was significantly up-regulated during rhizobia infection and varied with nodule formation. The GmD53a-knockdown chimerical plants were produced to further check its role in soybean nodulation in comparison with control GUS. In knockdown lines, the GmD53a (suppressor of strigolactone MAX2) has a higher number of nodules compared to control lines. Furthermore, the expression levels of several nodulation genes essential for initiation and formation of nodules were altered in GmD53a-knockdown lines. <h4>Conclusion</h4> The results revealed that SL biosynthesis and signaling are not conserved but also have close interaction between SL and legume rhizobia.
Project description:<h4>Background</h4>Agrobacterium 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.<h4>Results</h4>Here, 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.<h4>Conclusions</h4>We 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:<h4>Background</h4>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.<h4>Results</h4>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.<h4>Conclusion</h4>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.
Project description:Background:Soybean 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. Results:An 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. Conclusion:The ARISHR-SMV is an efficient pathosystem that allows a quick and convenient identification of SMV-related target gene function in soybean.
Project description:The <i>Agrobacterium rhizogenes</i> hairy root transformation system is widely used in symbiotic studies of model legumes. It typically relies on fluorescent reporters, such as DsRed, for identification of transgenic roots. The MtLAP1 transcription factor has been utilized as a reporter system in <i>Medicago truncatula</i> based on production of anthocyanin pigment. Here, we describe a version of this reporter driven by a root-cap specific promoter for direct observation of anthocyanin accumulation in root tips, which allows the identification of transgenic hairy roots by the naked eye. Results from our analysis suggest that the reporter had no significant effects on nodulation of <i>M. truncatula</i>. This approach, by virtue of its strong and specific expression in root cap cells, greatly reduces false positives and false negatives, and its use of an easily scored visible pigment should allow greater versatility and efficiency in root biology studies.
Project description:Plants fix CO<sub>2</sub> into carbohydrates through photosynthesis, and various organisms interact with plants to obtain carbohydrates. <i>Agrobacterium rhizogenes</i> is a soil bacterium known as a plant pathogen that induces hairy root disease. Through <i>A. rhizogenes</i>-plant interactions, transfer-DNA (T-DNA) of the Ri plasmid is inserted into the host plant genome, leading to excessive formation of hairy roots and the synthesis of opines that are carbon and nitrogen sources for <i>A. rhizogenes</i>. In this study, we analyzed the carbohydrate contents in soybean (<i>Glycine max</i>) hairy roots. We found that the starch content was strongly increased in hairy roots, whereas the glucose was significantly decreased. On the other hand, no significant differences were observed in sucrose levels between the main roots and hairy roots of <i>A. rhizogenes</i>-inoculated plants. This result suggests that <i>A. rhizogenes</i> infection caused a change in primary carbon metabolism in the host plant cells.
Project description:As an essential nutrient element, phosphorus (P) plays an important role in plant growth and development. Low P availability is a limiting factor for crop production, especially for legume crops (e.g. soybean), which require additional P to sustain nitrogen fixation through symbiotic associations with rhizobia. Although PHOSPHATE STARVATION RESPONSE 1 (PHR1) or PHR1-like is considered as a central regulator of phosphate (Pi) homeostasis in several plant species, it remains undefined in soybean. In this study, 35 GmPHR members were cloned from the soybean genome and expression patterns in soybean were assayed under nitrogen (N) and P deficiency conditions. GmPHR25, which is up-regulated in response to Pi starvation, was then overexpressed in soybean hairy roots in vitro and in vivo to investigate its functions. The results showed that overexpressing GmPHR25 increased Pi concentration in transgenic soybean hairy roots under normal conditions, accompanied with a significant decrease in hairy root growth. Furthermore, transcripts of 11 out of 14 high-affinity Pi transporter (GmPT) members as well as five other Pi starvation-responsive genes were significantly increased in soybean hairy roots with GmPHR25 overexpression. Taken together, this study suggests that GmPHR25 is a vital regulator in the P signaling network, and controls Pi homeostasis in soybean.
Project description:<h4>Background</h4>Litchi chinensis Sonn. is an economically important fruit tree in tropical and subtropical regions. However, litchi functional genomics is severely hindered due to its recalcitrance to regeneration and stable transformation. Agrobacterium rhizogenes-mediated hairy root transgenic system provide an alternative to study functional genomics in woody plants. However, the hairy root transgenic system has not been established in litchi.<h4>Results</h4>In this study, we report a rapid and highly efficient A. rhizogenes-mediated co-transformation system in L. chinensis using Green Fluorescent Protein (GFP) gene as a marker. Both leaf discs and stem segments of L. chinensis cv. 'Fenhongguiwei' seedlings were able to induce transgenic hairy roots. The optimal procedure involved the use of stem segments as explants, infection by A. rhizogenes strain MSU440 at optical density (OD<sub>600</sub>) of 0.7 for 10 min and co-cultivation for 3 days, with a co-transformation efficiency of 9.33%. Furthermore, the hairy root transgenic system was successfully used to validate the function of the key anthocyanin regulatory gene LcMYB1 in litchi. Over-expression of LcMYB1 produced red hairy roots, which accumulated higher contents of anthocyanins, proanthocyanins, and flavonols. Additionally, the genes involving in the flavonoid pathway were strongly activated in the red hairy roots.<h4>Conclusion</h4>We first established a rapid and efficient transformation system for the study of gene function in hairy roots of litchi using A. rhizogenes strain MSU440 by optimizing parameters. This hairy root transgenic system was effective for gene function analysis in litchi using the key anthocyanin regulator gene LcMYB1 as an example.
Project description:<h4>Background</h4>Agrobacterium-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.<h4>Results</h4>In 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.<h4>Conclusion</h4>A 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.