Enhanced Rice Blast Resistance by CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor Gene OsERF922.

Richard A Wilson,Chunlian Wang,Wei Hao,Piqing Liu,Ying Gao,Kaijun Zhao,Cailin Lei,Yao-Guang Liu,Fujun Wang

doi:10.1371/journal.pone.0154027

Richard A Wilson, Chunlian Wang + Show 7 more

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https://doi.org/10.1371/journal.pone.0154027

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Abstract

Rice blast is one of the most destructive diseases affecting rice worldwide. The adoption of host resistance has proven to be the most economical and effective approach to control rice blast. In recent years, sequence-specific nucleases (SSNs) have been demonstrated to be powerful tools for the improvement of crops via gene-specific genome editing, and CRISPR/Cas9 is thought to be the most effective SSN. Here, we report the improvement of rice blast resistance by engineering a CRISPR/Cas9 SSN (C-ERF922) targeting the OsERF922 gene in rice. Twenty-one C-ERF922-induced mutant plants (42.0%) were identified from 50 T0 transgenic plants. Sanger sequencing revealed that these plants harbored various insertion or deletion (InDel) mutations at the target site. We showed that all of the C-ERF922-induced allele mutations were transmitted to subsequent generations. Mutant plants harboring the desired gene modification but not containing the transferred DNA were obtained by segregation in the T1 and T2 generations. Six T2 homozygous mutant lines were further examined for a blast resistance phenotype and agronomic traits, such as plant height, flag leaf length and width, number of productive panicles, panicle length, number of grains per panicle, seed setting percentage and thousand seed weight. The results revealed that the number of blast lesions formed following pathogen infection was significantly decreased in all 6 mutant lines compared with wild-type plants at both the seedling and tillering stages. Furthermore, there were no significant differences between any of the 6 T2 mutant lines and the wild-type plants with regard to the agronomic traits tested. We also simultaneously targeted multiple sites within OsERF922 by using Cas9/Multi-target-sgRNAs (C-ERF922S1S2 and C-ERF922S1S2S3) to obtain plants harboring mutations at two or three sites. Our results indicate that gene modification via CRISPR/Cas9 is a useful approach for enhancing blast resistance in rice.

Highlights

Rice (Oryza sativa L.) is one of the most important food crops in the world, feeding nearly 50% of the world’s population
The disease-susceptibility gene and the sucrose efflux transporter gene OsSWEET14, which aids in pathogen survival and virulence, was mutated by transcription activator-like effector nucleases (TALENs) to produce disease-resistant rice with normal phenotypes [56]; using TALENs to target the OsBADH2 gene produced a generation of fragrant rice that contain 2-acetyl-1-pyrroline (2AP), a major fragrance compound [60]
Knocking out all three MILDEW-RESISTANCE LOCUS (MLO) alleles in bread wheat using one pair of TALENs resulted in the creation of stable mutant lines exhibiting broad-spectrum resistance to powdery mildew [47]

Summary

Introduction

Rice (Oryza sativa L.) is one of the most important food crops in the world, feeding nearly 50% of the world’s population. Enhancing the resistance of rice to M. oryzae has been shown to be the most economical and effective approach for controlling rice blast [3, 4]. Over the course of evolution, plants have evolved sophisticated mechanisms to resist pathogen infection. Surface-localized pattern recognition receptors (PRRs) rapidly perceive pathogen-associated molecular patterns (PAMPs) [5] and activate a battery of defense mechanisms [6]. PAMP-triggered immunity (PTI) is considered a conserved and ancient form of plant immunity that acts as the first line of inducible defense to various pathogens [6, 7]. The plant hormones abscisic acid, salicylic acid, jasmonic acid and ethylene play important roles in this defense response [8,9,10]

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