Use of CRISPR/Cas9 for Symbiotic Nitrogen Fixation Research in Legumes

Nitrogen-fixing rhizobia have established a symbiotic relationship with the legume family through more than 60 million years of evolution. Hundreds of legume host genes are involved in the SNF (symbiotic nitrogen fixation) process, such as recognition of the bacterial partners, nodulation signaling and nodule development, maintenance of highly efficient nitrogen fixation within nodules, regulation of nodule numbers, and nodule senescence. However, investigations of SNF-related gene functions and dissecting molecular mechanisms of the complicated signaling crosstalk on a genomic scale were significantly restricted by insufficient mutant resources of several representative model legumes. Targeted genome-editing technologies, including ZFNs, TALENs, and CRISPR-Cas systems, have been developed in recent years and rapidly revolutionized biological research in many fields. These technologies were also applied to legume plants, and significant progress has been made in the last several years. Here, we summarize the applications of these genome-editing technologies, especially CRISPR-Cas9, toward the study of SNF in legumes, which should greatly advance our understanding of the basic mechanisms underpinning the legume-rhizobia interactions and guide the engineering of the SNF pathway into nonlegume crops to reduce the dependence on the use of nitrogen fertilizers for sustainable development of modern agriculture.