Editing SWEET Genes for Understanding Their Roles in Citrus Susceptibility to Huanglongbing Disease

Citrus greening, also known as Huanglongbing (HLB), is a devastating bacterial disease threatening citrus industries worldwide. There is an urgent need to develop a long-term, effective genetic solution to this threat. Various gene expression and gene editing approaches are being explored to achieve HLB resistance in citrus. Several disease susceptibility genes have been identified in model and crop plants. Among them are the sugars will eventually be exported transporters (SWEETs) genes. In rice, SWEET11, SWEET13 and SWEET14 genes are involved in rice susceptibility to Xanthomonas oryzae oryzae (Xoo). Knocking-out of these genes increased rice resistance to Xoo. Different gene expression studies in citrus suggest that SWEET genes may be involved in citrus susceptibility to HLB. To understand the roles of citrus SWEET genes in HLB susceptibility, we targeted SWEET1 and SWEET10 genes in two citrus genotypes (‘Duncan’ grapefruit and ‘Carrizo’ citrange) and edited them using the CRISPR/Cas9 system. Six different types of insertion and deletion of nucleotides were observed in different transgenic lines, ranging from addition of a single nucleotide to the deletion of up to 23 nucleotides. Most of the mutations were either addition or deletion of a single nucleotide at the targeted sites. Deep sequencing of the targeted region revealed that the mutation ranged from 2% to 98% in SWEET1-edited lines and up to 100% in SWEET10 edited lines. These gene-edited plants will be evaluated for their susceptibility/resistance to HLB and other bacterial diseases.