Novel alleles of rice eIF4G generated by CRISPR/Cas9-targeted mutagenesis confer resistance to Rice tungro spherical virus.

Anca Macovei,Daniel F Voytas,Tomáš Čermák,Inez Slamet‐Loedin,Gilda B Jonson,Christian Cantos,Il‐Ryong Choi,Prabhjit Chadha‐Mohanty,Neah R Sevilla

doi:10.1111/pbi.12927

Abstract

SummaryRice tungro disease (RTD) is a serious constraint in rice production across tropical Asia. RTD is caused by the interaction between Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus. RTSV resistance found in traditional cultivars has contributed to a reduction in the incidence of RTD in the field. Natural RTSV resistance is a recessive trait controlled by the translation initiation factor 4 gamma gene (eIF4G). The Y1059V1060V1061 residues of eIF4G are known to be associated with the reactions to RTSV. To develop new sources of resistance to RTD, mutations in eIF4G were generated using the CRISPR/Cas9 system in the RTSV‐susceptible variety IR64, widely grown across tropical Asia. The mutation rates ranged from 36.0% to 86.6%, depending on the target site, and the mutations were successfully transmitted to the next generations. Among various mutated eIF4G alleles examined, only those resulting in in‐frame mutations in SVLFPNLAGKS residues (mainly NL), adjacent to the YVV residues, conferred resistance. Furthermore, our data suggest that eIF4G is essential for normal development, as alleles resulting in truncated eIF4G could not be maintained in homozygous state. The final products with RTSV resistance and enhanced yield under glasshouse conditions were found to no longer contain the Cas9 sequence. Hence, the RTSV‐resistant plants with the novel eIF4G alleles represent a valuable material to develop more diverse RTSV‐resistant varieties.

Highlights

rice tungro disease (RTD) is caused by the interaction between Rice tungro spherical virus (RTSV), having a single-stranded RNA genome, and Rice tungro bacilliform virus (RTBV), having a double-stranded DNA genome (Hull, 1996)
Cultivars with resistance to RTSV can contribute to a reduction in RTD in large fields, as plants infected with RTBV alone do not serve as sources for secondary spread (Anjaneyulu et al, 1994)
Our results showed that targeted mutagenesis mediated by CRISPR/Cas9 was highly efficient and dependent on the gRNA sequence, and the targeted mutations were inherited in subsequent generations

Summary

Introduction

Achieving high crop yields with efficient and sustainable agricultural practices is essential to attain global food security. The escalation of biotic and abiotic stresses adds even more pressure to agricultural crop production. This especially applies in the case of rice as a species responsible for feeding billions of people, many of whom represent the low-income population from developing and underdeveloped countries in Asia and Africa (Seck et al, 2012). Rice tungro disease (RTD) causes severe disruption of rice production, affecting more than 350 000 ha throughout the main rice-producing Asian countries (Azzam and Chancellor, 2002; Chancellor et al, 2006; Muralidharan et al, 2003). RTBV is responsible for the development of the disease symptoms, while RTSV acts as a helper virus assisting transmission of RTBV by GLH and enhancing the symptoms (Hull, 1996). Cultivars with resistance to RTSV can contribute to a reduction in RTD in large fields, as plants infected with RTBV alone do not serve as sources for secondary spread (Anjaneyulu et al, 1994)

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