Genome editing in alfalfa (Medicago sativa) to hyper-accumulate phosphate

Abstract

Rock phosphate, the main source of phosphate (P) for crop fertilizers, is a finite resource that is predicted to be depleted in 50-100 years. P is a critical nutrient in agriculture and its application can dramatically improve plant productivity. However, many soils have excess amounts of P from application of animal manures and runoff of phosphate from agricultural lands is the major source of nonpoint water pollution in the Midwestern US. The goal of this project is to create mutations by gene editing in the ubiquitin E2 conjugating enzyme PHO2, involved in P signaling and P homeostasis in alfalfa so that plants hyper-accumulate phosphate. Such plants could be used to reduce soil P levels and reclaim P for use as a fertilizer. From a draft diploid Medicago sativa genome scaffold sequence and the alfalfa transcriptome database (AGED), three PHO2 genes were identified. The genes, two of which are >99% homologous (a/b), each have seven exons interspersed by six introns. The open reading frames are 912 amino acids except when an alternate splice site is used in a/b gene transcript resulting in a 902 amino acid sequence. Alfalfa plants grown under P limiting conditions expressed low levels of the a/b transcripts with higher levels seen for PHO2c, while application of higher P induced increased expression mainly of the a/b transcripts. Under high P conditions, roots and shoots accumulated 4.1x and 2.5x more P than in low P conditions, respectively. An initial CRISPR/Cas9/Cys4 reagent targeting all three genes was generated and used to transform alfalfa cv. RegenSY. A total of 67 verified transgenic plants were screened by acrylamide gel shift assays, cloning, and sequencing to identify plants with mutations. Mutations ranging from a 1 bp insertion to a 25 bp deletion were identified in a total of 10 plants and some plants had multiple targets hit. Recently, a second attempt at CRISPR/Cas9 mutation utilized a cassette vector system with either the tRNA or Cys4 splicing system and exonuclease components. Initial screening results indicate that the tRNA splicing system may have yielded greater numbers of mutations. TaqMan probes were designed to identify plants with changes in the target sites and were verified by restriction digestions, cloning, and sequencing. Data on inheritance of mutations and phosphate accumulation in edited plants will be presented. The results of these experiments demonstrate that editing of multiple targets can be accomplished in alfalfa, although the tetraploid inheritance of genes complicates analysis.