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

In eudicotyledonous model plants, the B-function genes encode a pair of partner MADS-domain proteins, APETALA3 (AP3) and PISTILLATA (PI) in Arabidopsis and DEFICIENS (DEF) and GLOBOSA (GLO) in Antirrhinum. These proteins, which must form heterodimers to function, are required to specify petal and stamen identity during flower development. Here, we report cloning and characterization of TrPI (Taihangia rupestris PISTILLATA), a PI/GLO-like gene from the core eudicot species Taihangia rupestris (Rosaceae). DNA gel blot analysis showed that TrPI is a single copy gene in the T. rupestris genome. Quantitative RT-PCR and in situ hybridization analyses revealed that TrPI is transcribed in both the vegetative and reproductive organs at different levels. Ectopic expression of TrPI in Arabidopsis caused severe modifications in vegetative plant architecture, including rosette leaves and cauline leaves arranged in a non-spiral phyllotaxy, and a flattened primary inflorescence stem that produced two or three offshoots at the base, middle or top. Moreover, we show that the TrPI gene is capable of rescuing pi-1 mutant phenotypes. Yeast two-hybrid assays showed that TrPI forms homodimers. Taken together, these results show that TrPI might function in regulating plant architecture in addition to its function as a floral organ identity gene in T. rupestris, suggesting that the TrPI protein has biochemical features that distinguish it from the well-studied orthologs, PI and GLO.

Ectopic expression of TrPI, a Taihangia rupestris (Rosaceae) PI ortholog, causes modifications of vegetative architecture in Arabidopsis.

In this study, intraspecific responses of 12 winter wheat cultivars to different doses of ultraviolet-B radiation (UV-B) were analyzed and compared. The results showed that the low UV-B dose of 3.24kJm(-2)d(-1) generally inhibited the plant height, but promoted the dry weight and photochemical reflectance index (PRI). The high UV-B dose of 5.40kJm(-2)d(-1) inhibited most of the indexes, especially plant height and fresh weight. Under the treatments of two UV-B doses, the response indexes (RIs) of plant height, dry weight, fresh weight, carotenoid, and anthocyanin were all significantly correlated with the cumulative stress response index (CSRI). The RIs of carotenoid and anthocyanin exhibited higher correlations with dry weight and fresh weight, indicating that these indexes were vital to UV-B tolerance. By comparing the correlations of the seven indexes between two doses of UV-B radiation, the responses of 12 cultivars' plant height and dry weight to different doses of UV-B were very significant (P<0.01). Thus, when comparing the UV-B tolerance of different winter wheat seedlings, no matter using high dose or low dose UV-B, the index of plant height should be concerned first and dry weight could be used secondarily. Among 12 winter wheat cultivars, Nongda 6081 exhibited significant resistance to two doses of UV-B radiation while others were variable. Differences in the accumulation of UV-absorbing compounds induced by UV-B in leaves may be the main and direct reason for the intraspecific differences between resistant and sensitive cultivars.

Comparing intraspecific responses of 12 winter wheat cultivars to different doses of ultraviolet-B radiation.

The majority of plant disease resistance (R) genes encode proteins that share common structural features. However, the transcription activator-like effector (TALE) associated executor type R genes show no considerable sequence homology to any known R genes. We adopted a map-based cloning approach and TALE-based technology to isolate and characterize Xa23, a new executor R gene derived from the wild rice (Oryza rufipogon) that confers an extremely broad spectrum of resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Xa23 encodes a 113-amino acid protein that shares 50% identity to the known executor R protein XA10. The predicted transmembrane helices in XA23 also overlap with those of XA10. Unlike Xa10, however, Xa23 transcription is specifically activated by AvrXa23, a TALE present in all examined Xoo field isolates. Moreover, the susceptible xa23 allele has an identical open reading frame of Xa23, but differs in promoter region by lacking the TALE binding-element (EBE) for AvrXa23. XA23 can trigger strong hypersensitive response in rice, tobacco and tomato. Our results provide the first evidence that plant genomes have an executor R gene family in which members execute their function and spectrum of disease resistance by recognizing the cognate TALEs in pathogen.

XA23 is an executor R protein and confers broad-spectrum disease resistance in rice.

 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.

Isoflavones are not involved in rhizobial signaling in red clover, but likely play a role in defense in the rhizosphere. Red clover (Trifolium pratense) is a high-quality forage legume, well suited for grazing and hay production in the temperate regions of the world. Like many legumes, red clover produces a number of phenylpropanoid compounds including anthocyanidins, flavan-3-ols, flavanols, flavanones, flavones, and isoflavones. The study of isoflavone biosynthesis and accumulation in legumes has come into the forefront of biomedical and agricultural research due to potential for medicinal, antimicrobial, and environmental implications. CRISPR/Cas9 was used to knock out the function of a key enzyme in the biosynthesis of isoflavones, isoflavone synthase (IFS1). A hemizygous plant carrying a 9-bp deletion in the IFS1 gene was recovered and was intercrossed to obtain homozygous mutant plants. Levels of the isoflavones formononetin, biochanin A and genistein were significantly reduced in the mutant plants. Wild-type and mutant plants were inoculated with rhizobia to test the effect of the mutation on nodulation, but no significant differences were observed, suggesting that these isoflavones do not play important roles in nodulation. Gene expression profiling revealed an increase in expression of the upstream genes producing the precursors for IFS1, namely, phenylalanine ammonium lyase and chalcone synthase, but there were no significant differences in IFS1 gene expression or in the downstream genes in the production of specific isoflavones. Higher expression in genes involved in ethylene response was observed in the mutant plants. This response is normally associated with biotic stress, suggesting that the plants may have been responding to cues in the surrounding rhizosphere due to lower levels of isoflavones.

Isoflavone levels, nodulation and gene expression profiles of a CRISPR/Cas9 deletion mutant in the isoflavone synthase gene of red clover.

-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.

Low temperature injury in spring has seriously destabilized the production and grain quality of common wheat. However, the molecular mechanisms underlying spring frost tolerance remain elusive. In this study, we investigated the response of a frost-tolerant wheat variety Zhongmai8444 to freezing stress at the meiotic stage. Transcriptome profiles over a time course were subsequently generated by high-throughput sequencing. Our results revealed that the prolonged freezing temperature led to the significant reductions in plant height and seed setting rate. Cell wall thickening in the vascular tissue was also observed in the stems. RNA-seq analyses demonstrated the identification of 1010 up-regulated and 230 down-regulated genes shared by all time points of freezing treatment. Enrichment analysis revealed that gene activity related to hormone signal transduction and cell wall biosynthesis was significantly modulated under freezing. In addition, among the identified differentially expressed genes, 111 transcription factors belonging to multiple gene families exhibited dynamic expression pattern. This study provided valuable gene resources beneficial for the breeding of wheat varieties with improved spring frost tolerance.

Transcriptomic profiling of wheat stem during meiosis in response to freezing stress.

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.

Yinglun Fan

College of Agriculture

Liaocheng University

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

Yinglun Fan

.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