The application of CRISPR/Cas9 in hairy roots to explore the functions of AhNFR1 and AhNFR5 genes during peanut nodulation

Hongmei Shu,Jianping Wang,Ze Peng,Ziliang Luo

doi:10.1186/s12870-020-02614-x

Hongmei Shu, Jianping Wang + Show 2 more

Open Access

https://doi.org/10.1186/s12870-020-02614-x

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Abstract

BackgroundPeanut is an important legume crop growing worldwide. With the published allotetraploid genomes, further functional studies of the genes in peanut are very critical for crop improvement. CRISPR/Cas9 system is emerging as a robust tool for gene functional study and crop improvement, which haven’t been extensively utilized in peanut yet. Peanut plant forms root nodules to fix nitrogen through a symbiotic relationship with rhizobia. In model legumes, the response of plants to rhizobia is initiated by Nod factor receptors (NFRs). However, information about the function of NFRs in peanut is still limited. In this study, we applied the CRISPR/Cas9 tool in peanut hairy root transformation system to explore the function of NFR genes.ResultsWe firstly identified four AhNFR1 genes and two AhNFR5 genes in cultivated peanut (Tifrunner). The gene expression analysis showed that the two AhNFR1 and two AhNFR5 genes had high expression levels in nodulating (Nod+) line E5 compared with non-nodulating (Nod-) line E4 during the process of nodule formation, suggesting their roles in peanut nodulation. To further explore their functions in peanut nodulation, we applied CRISPR technology to create knock-out mutants of AhNFR1 and AhNFR5 genes using hairy root transformation system. The sequencing of these genes in transgenic hairy roots showed that the selected AhNFR1 and AhNFR5 genes were successfully edited by the CRISPR system, demonstrating its efficacy for targeted mutation in allotetraploid peanut. The mutants with editing in the two AhNFR5 genes showed Nod- phenotype, whereas mutants with editing in the two selected AhNFR1 genes could still form nodules after rhizobia inoculation.ConclusionsThis study showed that CRISPR-Cas9 could be used in peanut hairy root transformation system for peanut functional genomic studies, specifically on the gene function in roots. By using CRISPR-Cas9 targeting peanut AhNFR genes in hairy root transformation system, we validated the function of AhNFR5 genes in nodule formation in peanut.

Highlights

Peanut is an important legume crop growing worldwide
The two AhNFR5 genes had no intron while the four AhNFR1 genes had multiple introns (Table 1)
Combining the results of AhNFR5 transgenic hairy roots transformed with p201G/Cas9: NFR5AB1 + NFR5AB2 vector, these results showed that AhNFR5B genes’ sequence of all clones from one transgenic event were all edited, which could cause non-nodulation phenotype

Summary

Introduction

With the published allotetraploid genomes, further functional studies of the genes in peanut are very critical for crop improvement. CRISPR/Cas system is emerging as a robust tool for gene functional study and crop improvement, which haven’t been extensively utilized in peanut yet. We applied the CRISPR/Cas tool in peanut hairy root transformation system to explore the function of NFR genes. The genomes of the two ancestral species and two cultivated peanut cultivars (Tifrunner and Shitouqi) were fully sequenced and publicly available [1,2,3]. These reference genomes greatly facilitate peanut molecular and genetic studies. There is a need to update the arsenal of gene functional analysis for peanut genetic study

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