Development of blast resistant rice plants using CRISPR / Cas9 system for genome editing

Abstract

Rice (Oryza sativa L.) is the main food crop for more than half of the world population but unfortunately, it is severely affected by blast, one of the most widespread and devastating plant diseases, caused by the fungus Magnaporthe oryzae. Hence, the development of rice cultivars with greater resistance to blast is one of the main focuses of breeding programs. However, due to the complex biology of the pathogen, rice cultivars genetically resistant to the fungus become susceptible in a short period of time. In this context, the knockout of rice susceptibility genes represents a flourishing approach to obtain rice cultivars with a broader and longer-lasting resistance to M. oryzae. The present study aimed to use the genomic editing technology - CRISPR/Cas9 system, for knocking-out genes engaged with rice susceptibility to fungal infection. From previous transcriptomics results of two semi-isogenic rice lines - NILs infected by M. oryzae, potential rice-blast susceptibility genes were selected. The prospection of candidate genes for gene editing was complemented by a comparative shotgun proteomic analysis of the protein profile of the interaction between IRBLi-F5 (susceptible) and IRBL5-M (resistant) NILs in early stages of M. oryzae infection, that revealed a specific set of proteins potentially associated with susceptibility. After the characterization and validation of gene expression by RT-qPCR of the most prominent candidates, the target genes OsDjA2, OsERF104 and OsPyl5 were selected and submitted to a functional validation via gene silencing in planta, using antisense oligonucleotides (ASO), in which a clear reduction of leaf symptoms was observed in the compatible identification. Subsequently, the model japonica rice variety Nipponbare was transformed with simplex CRISPR/Cas9 vectors aiming to the independent knockout of each target gene. The T1 progeny of rice-edited plants, homozygous for the null (loss of function)-mutation were tested for blast resistance. As expected, mutant plants showed a decrease of disease symptoms in comparison with control lines (transformant non-edited plants). The results obtained in this study can contribute for the development of rice cultivars resistant to blast disease, besides shedding light on new potential rice-blast susceptibility genes.