Location: Plant Science Research
2023 Annual Report
Objectives
The overall goal of this project is to reduce nutrient inputs, particularly nitrogen (N) and phosphorus (P), in legume crops through the identification of germplasm having root architectural diversity and the discovery of genes that may contribute to that diversity. Desired outcomes from the research proposed herein include identification of unique germplasm with altered root morphology that may reduce costly fertilizer inputs, novel genes that regulate root development and function, and fundamental insight into the biochemical processes that affect nutrient acquisition. To achieve these goals and outcomes, three integrated objectives will be pursued.
Objective 1: Phenotype and evaluate root architecture changes in soybean, common bean and Medicago mutants, determine relationships between root architecture and improved nutrient acquisition, and define genome lesions.
Objective 2: Evaluate whole genome transcript analysis of common bean and alfalfa through RNA-seq analysis of roots, root nodules, leaves and seeds to compare wild-type and mutants.
Objective 3: Identify genes contributing to root architecture and nutrient acquisition in legumes and determine their function.
Approach
Identify mutant plants derived from fast neutron and Tnt1 mutagenized populations which affect root architecture and development, and define genetic lesions through next generation sequencing. Conduct RNA-seq transcript expression studies for the organs of wild type and mutant legume species such as alfalfa, common bean, and soybean to identify genes involved in unique adaptations displayed by these species. Utilize RNAi, zinc finger nuclease modification and/or antisense constructs to silence expression of selected root-specific/enhanced genes affecting root architecture and/or nutrient acquisition.
Progress Report
Three objectives were pursued. For Objective 1, targeted mutagenesis of six Medicago truncatula candidate genes associated with root and nodule traits was achieved using CRISPR/Cas9. Successful gene knock-out, heritable transmission of target mutations, and removal of the transgene were demonstrated. A manuscript reporting these results is in preparation. Additionally, mutant plants related to root meristem activity and root nodule symbiosis were generated for collaborators. Genotype analysis confirmed successful gene knock-out and heritable transmission of mutations in these candidate genes. Objective 2 involved genome assembly and annotation of the M. truncatula accession HM078, which is resistant to the Phoma medicaginis fungal pathogen. RNA-seq analysis of mock and inoculated tissues identified candidate genes associated with pathogen defense and root architecture. Reagents were generated, function-tested in hairy-root assays, and are being used for whole plant transformation assays. A manuscript describing this work is being prepared. For Objective 3, phenotyping of 600 root samples from 260 M. truncatula accessions was performed using laser ablation tomography (LAT) technology. This led to the identification of six promising candidate genes associated with various root architecture traits. Genome-wide association study (GWAS), gene expression, and ortholog analysis were used to refine gene candidates. Reagents for whole plant transformation have been constructed.
In preparation for the new project plan starting in March 2023, several tasks have been accomplished: 1) construction and delivery of phloem mobile gRNA reagents to M. truncatula and soybean scions expressing Cas9, with targeted mutagenesis detected in scion plant leaves; 2) optimization of alfalfa gene editing reagent efficiency through testing of five Cas9 enzymes expressed by a legume-specific promoter. This will be followed by the selection of the most efficient one for whole plant transformation; 3) improvement of alfalfa transformation using the RegenSY27x genotype and an Agrobacterium strain with a helper ternary vector; and 4) successful transformation of soybean using a novel embryonic axis protocol, though the efficiency of the selected base editor was found to be low. Efforts are underway to optimize legume base editing by testing two new adenine base editors with higher efficiencies in hairy-root assays before evaluating their function and efficiency in whole plant transformation. These incremental improvements and hold promise for enhancing transformation of elite alfalfa genotypes and improving the efficiency of base editing in soybean.
Accomplishments
1. Generation of mutant plants associated with metal ion uptake and accumulation. Heavy metal contamination of soil and its entry into the food chain via plant uptake is an agricultural problem that needs to be addressed. Understanding the genetic foundations of metal accumulation and tolerance in plants is crucial and will assist efforts to use plants for removal of heavy metals from soil. Several promising candidate genes involved in metal uptake were recently identified by genome wide association studies. To validate these genes, ARS scientists in St. Paul, Minnesota, generated five mutated Medicago truncatula plants by CRISPR/Cas9 gene editing. The mutants were screened for targeted mutations and the heritable transmission of the mutations was demonstrated in the next generation along with removal of the foreign genes by genetic segregation. The plants were analyzed under different concentrations of metal ions and their accumulation was quantified by x-ray fluorescence microscopy. This work identified mutants capable of accumulating toxic metal ions and are promising for use in removal of toxic metals from contaminated farmland. Farmers and environmental managers can utilize these plants or other species with these mutations to reduce the negative impact these contaminants have on the environment and consumer.
2. Characterization of a Medicago truncatula accession resistant to spring black stem and leaf spot. Ascochyta medicaginicola is a fungal plant pathogen that infects alfalfa and M. truncatula and is the causative agent of spring black stem and leaf spot. The disease is a major cause of yield loss, stand decline, and forage quality in alfalfa. Identifying genetic resistance will be key to controlling the destruction by this pathogen. To understand the genetic mechanisms that leads to disease resistance, ARS scientists in St. Paul, Minnesota, identified an M. truncatula accession (Hm078) with robust resistance to the pathogen and sequenced its genome using long-read DNA sequencing technologies. In addition, analysis of gene transcripts of pathogen-infected and non-infected tissue in both the resistant and a susceptible accession was carried out to assist in identifying genes involved in disease resistance and several candidate genes were identified. Plant breeders can introduce these genes into susceptible alfalfa or M. truncatula accessions to create new disease resistant plants as a means of reducing damage from the disease.
Review Publications
Huertas, R., Torres-Jerez, I., Curtin, S.J., Scheible, W., Udvardi, M. 2023. Medicago truncatula PHO2 genes have distinct roles in phosphorus homeostasis and symbiotic nitrogen fixation. Frontiers in Plant Science. 14. https://doi.org/10.3389/fpls.2023.1211107.
Ahn, E.J., Fall, C., Botkin, J., Curtin, S.J., Prom, L.K., Magill, C.W. 2023. Inoculation and screening methods for major sorghum diseases caused by fungal pathogens: Claviceps africana, Colletotrichum sublineola, Sporisorium reilianum, Peronosclerospora sorghi and Macrophomina phaseolina. Plants. 12(9). Article 1906. https://doi.org/10.3390/plants12091906.
Ahn, E.J., Botkin, J., Curtin, S.J., Zsogon, A. 2023. Ideotype breeding and genome engineering for legume crop improvement. Current Opinion in Biotechnology. 82(8). Article 102961.. https://doi.org/10.1016/j.copbio.2023.102961.
