Engineering canker‐resistant plants through CRISPR/Cas9‐targeted editing of the susceptibility gene CsLOB1 promoter in citrus

Abstract

SummaryCitrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is severely damaging to the global citrus industry. Targeted editing of host disease‐susceptibility genes represents an interesting and potentially durable alternative in plant breeding for resistance. Here, we report improvement of citrus canker resistance through CRISPR/Cas9‐targeted modification of the susceptibility gene CsLOB1 promoter in citrus. Wanjincheng orange (Citrus sinensis Osbeck) harbours at least three copies of the CsLOB1 G allele and one copy of the CsLOB1 − allele. The promoter of both alleles contains the effector binding element (EBEP thA4), which is recognized by the main effector PthA4 of Xcc to activate CsLOB1 expression to promote citrus canker development. Five pCas9/CsLOB1sgRNA constructs were designed to modify the EBEP thA4 of the CsLOB1 promoter in Wanjincheng orange. Among these constructs, mutation rates were 11.5%–64.7%. Homozygous mutants were generated directly from citrus explants. Sixteen lines that harboured EBEP thA4 modifications were identified from 38 mutant plants. Four mutation lines (S2‐5, S2‐6, S2‐12 and S5‐13), in which promoter editing disrupted CsLOB1 induction in response to Xcc infection, showed enhanced resistance to citrus canker compared with the wild type. No canker symptoms were observed in the S2‐6 and S5‐13 lines. Promoter editing of CsLOB1 G alone was sufficient to enhance citrus canker resistance in Wanjincheng orange. Deletion of the entire EBEP thA4 sequence from both CsLOB1 alleles conferred a high degree of resistance to citrus canker. The results demonstrate that CRISPR/Cas9‐mediated promoter editing of CsLOB1 is an efficient strategy for generation of canker‐resistant citrus cultivars.

CRISPR‐LbCas12a‐mediated modification of citrus

“…Here, we employed the CaMV 35S promoter and the Yao promoter to drive LbCas12a expression (Figure b and c). However, in transgenic citrus, both CaMV 35S and AtU6‐1 were successfully used to drive sgRNAs for CRISPR‐SpCas9 (Jia et al ., ; Peng et al ., ) and for CRISPR‐SaCas9 (Jia et al ., ). To guarantee that the crRNA could be efficiently expressed in LbCas12a‐crRNA‐lobp‐transformed citrus, both CaMV 35S and AtU6‐1 were employed to drive crRNA (Figure b and c).…”

“…The longer deletions could be attributable to 5′ overhangs resulting from the stagger cutting of Cas12a at sites distal to the PAM (Tang et al ., ; Zetsche et al ., ). Interestingly, all of the Type II CsLOBP mutations generated by LbCas12a were 1 bp deletions among the colonies that were sequenced (Figure b and c, Figure S2A), and they are similar to the short indels (1–2 bp) induced by SpCas9 in citrus (Jia et al ., , ; Peng et al ., ; Zhang et al ., ).…”

Bacterial Citrus Diseases: Major Threats and Recent Progress

“…The number and type of LOB1 alleles differ among citrus genomes. For instance, Duncan grapefruit has two allele types in a ratio 1:1 [87] and Wanjincheng orange has two in a ratio 3:1 [88], whereas Satsuma mandarin and Chandler pummelo harbor only one [89,90].…”

“…Since grapefruit resulted from hybridization between sweet orange and pummelo (C. maxima) [139], it contains CsLOB1 alleles from both sweet orange (type I) and pummelo (type II). Later, Peng et al [88] showed that Cas9/ sgRNA-mediated editing of the EBEs of both CsLOB1 alleles in Wanjincheng orange (C. sinensis) conferred high levels of resistance against CCK. Reduced susceptibility was also obtained when the coding sequence of CsLOB1 in Duncan grapefruit was edited using Cas9/sgRNA technology [140].…”

Genetic Structure of Diploid Gametes for the Production of Triploid Citrus Hybrids

“…Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9/single guide RNA (sgRNA) have been already successfully used to obtain genetically modified rice, wheat, Arabidopsis, tobacco and sorghum (Nekrasov et al, 2013;Shan et al, 2013). In citrus, genetic modification has been used either for general objectives like gene function detection (Jia and Wang, 2014) or for specific objectives as citrus canker resistance (Peng et al, 2017).…”