Application of CRISPR/CAS9-mediated genome editing for studying soybean resistance to soybean cyst nematode

Abstract

Soybean is one of the world's most important crops, providing billions of people with food, energy resources, and industrial materials. The value of soybean has increased to 41 billion dollars in the United States. Soybean cyst nematode (Heterodera glycines; SCN) has become one of the most economically important pathogens of soybean in the world. SCN causes more than 1.2 billion dollars in yield losses annually to soybean farmers in the US alone. The main strategy for management of SCN is planting resistant cultivars. Most of the resistant cultivars on the market are derived from plant introductions (PI) 88788, 548402 (Peking) and 437654. Currently, the molecular basis of soybean resistance to SCN is not fully elucidated. Since the discovery of the first quantitative trait loci (QTL) for resistance to Heterodera glycines (Rhg) in the early 1960s, researchers have been working to understand the molecular basis of SCN resistance in soybean. Rhg1 and Rhg4 are two major QTL conferring resistance to SCN. The PI 88788 source of resistance requires Rhg1, whereas Peking and PI 437654 resistance is bigenic requiring both Rhg1 and Rhg4. At the Rhg4 locus, a gene encoding a serine hydroxymethyltransferase (SHMT) has been confirmed to play a role in resistance to SCN. SHMT is ubiquitous in nature. This enzyme functions in the simultaneous interconversion of serine to glycine and tetrahydrofolate to 5, 10-methylenetetrahydrofolate. However, the pathways and mechanisms leading to SCN resistance remain to be elucidated. Although a combination of reverse genetic methods including RNAi (RNA interference), TILLING (Targeting Induced Local Lesions in Genomes), and VIGS (Virus-Induced Gene Silencing) have been used effectively to study SCN resistance genes in soybean, limitations such as off-target effects, incomplete silencing, and background mutations can complicate analysis. More recent genome editing technologies have become appealing for studies of gene function in soybean. A new, simpler genome editing method called bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats /Cas9 (CRISPR-associated) immune system has recently emerged. The CRISPR/Cas9 system only requires a Cas9 nuclease and a single guide RNA (sgRNA) to perform the genome editing. This project was designed to apply CRISPR/Cas9 methodology to test soybean genes for a role in SCN resistance, and to further characterize the function of Rhg4 (SHMT) in SCN resistance.