an-efficient-reproducible-method-for-producing-composite-plants

An Efficient and Reproducible Method for Producing Composite Plants by <em>Agrobacterium rhizogenes</em>-Based Hairy Root Transformation

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a ...

Liaocheng University

 is a fast-growing rhizobial species that can establish a nitrogen-fixing symbiosis with a wide range of legume species including soybeans (). In soybeans, this interaction shows a high level of specificity such that particular  strains nodulate only a limited set of plant genotypes. Here we report the identification of a dominant gene in soybeans that restricts nodulation with  USDA193. Genetic mapping in an F2 population revealed co-segregation of the underlying locus with the previously cloned  gene. The  allele encodes a member of the Toll-interleukin receptor/nucleotide-binding site/leucine-rich repeat class of plant resistance proteins that restricts nodulation by  strains USDA257 and USDA205, and an allelic variant of this gene also restricts nodulation by  USDA122. By means of complementation tests and CRISPR/Cas9-mediated gene knockouts, we demonstrate that the  allele also is responsible for resistance to nodulation by  USDA193. Therefore, the  allele likely provides broad-spectrum resistance to nodulation by many  and  strains in soybeans.

The Soybean  Gene Restricts Nodulation by  USDA193.

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.

One-step generation of composite soybean plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation.

-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.

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.

-mediated (ARM) transformation is an efficient and powerful tool to generate transgenic roots to study root-related biology. For loss-of-function studies, transgenic-root-induced indel mutations by CRISPR/Cas9 only with homozygous/biallelic mutagenesis can exhibit mutant phenotype(s) (excluding recessive traits). However, a low frequency of homozygous mutants was produced by a constitutive promoter to drive  expression. Here, we identified a highly efficient  gamma- promoter, termed , with strong activity in the region where the root meristem will initiate and in the whole roots in broad eudicots species.  achieved higher homozygous/biallelic mutation efficiency than the most widely used  promoter in driving  expression in soybean, , and tomato roots. Using the p-Cas9 system, the average homozygous/biallelic mutation frequency is 1.7-fold and 8.3-fold higher than the p-Cas9 system for single and two target site(s) in the genome, respectively. Our results demonstrate the advantage of the p-Cas9 system used in ARM transformation, especially its great potential in diploids with multiple-copy genes targeted mutations and polyploid plants with multiplex genome editing.  is conservatively active in various eudicots species, suggesting that  might be applied in a wide range of dicots species.

 promoter-driven clustered regularly interspaced short palindromic repeats/Cas9 highly efficiently generates homozygous/biallelic mutations in the transformed roots by -mediated transformation.

With the advent of multiple omics and Genome-Wide Association Studies (GWAS) technology, genome-scale functional analysis of candidate genes is to be conducted in diverse plant species. Construction of plant binary expression vectors is the prerequisite for gene function analysis. Therefore, it is of significance to develop a set of plant binary expression vectors with highly efficient, inexpensive, and convenient cloning method, and easy-to-use in screening of positive recombinant in . In this study, we developed a set of plant binary expression vectors, termed pBTR vectors, based on Golden Gate cloning using I restriction site. Foreign DNA fragment of interest (FDI) can be cloned into the destination pBTR by one-step digestion-ligation reaction in a single tube, and even the FDI contains internal I site(s). Markedly, in one digestion-ligation reaction, multiple FDIs (exemplified by cloning four soybean , , , and  genes) can be cloned into the pBTR vector to generate multiple corresponding expression constructs (each expression vector carrying an FDI). In addition, the pBTR vectors carry the visual marker, a brightness monomeric red fluorescent protein mScarlet-I, that can be observed with the unaided eye in screening of positive recombinants without the use of additional reagents/equipment. The reliability of the pBTR vectors was validated in plants by overexpression of / in tomato and  in soybean roots -mediated transformation, promoter activity analysis of  in -mediated transformation, and protein subcellular localization of the  VvCEB1 in tobacco, respectively. These results demonstrated that the pBTR vectors can be used in analysis of gene (over)expression, promoter activity, and protein subcellular localization. These vectors will contribute to speeding up gene function analysis and the process of plant molecular breeding.

Development of a set of novel binary expression vectors for plant gene function analysis and genetic transformation.

Composite plants containing transgenic hairy roots produced with Agrobacterium rhizogenes-mediated transformation have become an important method to study the interaction between plants and arbuscular mycorrhizal fungi (AMF). Not all hairy roots induced by A. rhizogenes are transgenic, however, which leads to requirement of a binary vector to carry a reporter gene to distinguish transgenic roots from non-transformed hairy roots. The beta-glucuronidase gene (GUS) and fluorescent protein gene often are used as reporter markers in the process of hairy root transformation, but they require expensive chemical reagents or imaging equipment. Alternatively, AtMYB75, an R2R3 MYB transcription factor from Arabidopsis thaliana, recently has been used as a reporter gene in hairy root transformation in some leguminous plants and can cause anthocyanin accumulation in transgenic hairy roots. Whether AtMYB75 can be used as a reporter gene in the hairy roots of tomato and if the anthocyanins accumulating in the roots will affect AMF colonization, however, are still unknown. In this study, the one-step cutting method was used for tomato hairy root transformation by A.rhizogenes. It is faster and has a higher transformation efficiency than the conventional method. AtMYB75 was used as a reporter gene in tomato hairy root transformation. The results showed that the overexpression of AtMYB75 caused anthocyanin accumulation in the transformed hairy roots. Anthocyanin accumulation in the transgenic hairy roots did not affect their colonization by the arbuscular mycorrhizal fungus, Funneliformis mosseae strain BGC NM04A, and there was no difference in the expression of the AMF colonization marker gene SlPT4 in AtMYB75 transgenic roots and wild-type roots. Hence, AtMYB75 can be used as a reporter gene in tomato hairy root transformation and in the study of symbiosis between tomato and AMF.

Shanhua Lyu

College of Agriculture

Liaocheng University

An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

Shanhua Lyu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}College of Agriculture

Liaocheng University

An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

College of Agriculture