Bacterial blight of rice is an important disease in Asia and Africa. The pathogen,Xanthomonas oryzaepv.oryzae(Xoo), secretes one or more of six known transcription-activator-like effectors (TALes) that bind specific promoter sequences and induce, at minimum, one of the three host sucrose transporter genesSWEET11,SWEET13andSWEET14, the expression of which is required for disease susceptibility. We used CRISPR–Cas9-mediated genome editing to introduce mutations in all threeSWEETgene promoters. Editing was further informed by sequence analyses of TALe genes in 63Xoostrains, which revealed multiple TALe variants forSWEET13alleles. Mutations were also created inSWEET14, which is also targeted by two TALes from an AfricanXoolineage. A total of five promoter mutations were simultaneously introduced into the rice line Kitaake and the elite mega varieties IR64 and Ciherang-Sub1. Paddy trials showed that genome-editedSWEETpromoters endow rice lines with robust, broad-spectrum resistance.

Summary: Rice 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.

More than two billion people are micronutrient deficient. Polished grains of popular rice varieties have concentration of approximately 2 μg g⁻¹ iron (Fe) and 16 μg g⁻¹zinc (Zn). The HarvestPlus breeding programs for biofortified rice target 13 μg g⁻¹Fe and 28 μg g⁻¹Zn to reach approximately 30% of the estimated average requirement (EAR). Reports on engineering Fe content in rice have shown an increase up to 18 μg g⁻¹ in glasshouse settings; in contrast, under field conditions, 4 μg g⁻¹ was the highest reported concentration. Here, we report on selected transgenic events, field evaluated in two countries, showing 15 μg g⁻¹Fe and 45.7 μg g⁻¹Zn in polished grain. Rigorous selection was applied to 1,689 IR64 transgenic events for insert cleanliness and, trait and agronomic performances. Event NASFer-274 containing rice nicotianamine synthase (OsNAS2) and soybean ferritin (SferH-1) genes showed a single locus insertion without a yield penalty or altered grain quality. Endosperm Fe and Zn enrichment was visualized by X-ray fluorescence imaging. The Caco-2 cell assay indicated that Fe is bioavailable. No harmful heavy metals were detected in the grain. The trait remained stable in different genotype backgrounds.

Significance: This work reports discovery of a unique gene important for rice agriculture. A significant yield enhancement in rice modern cultivar was achieved by identification of a gene, SPIKELET NUMBER ( SPIKE ) in Indonesian rice landrace. The SPIKE increased grain yield of an indica cultivar IR64, which is widely grown in the tropics, over four seasons at the field level and improved plant architecture without changing grain quality or growth period, which are important for regional adaptability. These results indicate finding of SPIKE will be extremely valuable for contributing to increase grain production of indica rice cultivars.