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

Abstract

Citrus 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 (EBE_PthA4 ), 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 EBE_PthA4 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 EBE_PthA4 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 EBE_PthA4 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.

Keywords: CsLOB1; CRISPR/Cas9; citrus canker; genome editing; resistance.

Figure 1

Genotype of the CsLOB1 promoter in Wanjincheng orange (Citrus sinensis Osbeck). (a) High‐resolution melting analysis of the CsLOB1 promoter in Wanjincheng orange (blue curve), using Chandler pummelo (C. grandis; red curve) and Satsuma mandarin (C. unshiu; grey curve) as controls. (b) Direct sequencing analysis of the CsLOB1 promoter in Wanjincheng orange. Chromatograms for CsLOB1 ^G /CsLOB1 ^G , CsLOB1 ^G /CsLOB1 ⁻ and CsLOB1 ^G /CsLOB1 ⁻ in Satsuma mandarin (SM), Wanjincheng orange (WJ) and Chandler pummelo (CP) are shown. The indel tested is indicated by a square and arrow.

Figure 2

CRISPR/Cas9‐mediated modification of the CsLOB1 promoter in Wanjincheng orange (Citrus sinensis Osbeck). (a) Schematic structure of CsLOB1. CsLOB1 contains two exons indicated by grey rectangles. The translation initiation codon (ATG) and termination codon (TGA) are shown. The putative TATA box is in bold. The PthA4 effector binding element (EBE) sequence is in italics. The indel distinguishing CsLOB1 ^G and CsLOB1 ⁻ is in blue. The directions of sgRNAs (S1, S2, S3, S4 and S5) are indicated by long thin arrows. The protospacer adjacent motif (PAM) sites are shown. (b) Schematic diagram of pCas9/CsLOB1sgRNA vectors. 35S, Cauliflower mosaic virus 35S promoter from tobacco; gus:npt‐II , fusion of β‐glucuronidase and neomycin phosphotransferase genes; nos, nos terminator; pcoCas9, plant codon‐optimized SpCas9 gene; AtU6, Arabidopsis U6‐1 polymerase III promoter; LB, left border; RB, right border. (c) Representative chromatograms of CsLOB1 promoter mutations. ‘⊿#bp’ indicates the number of deleted nucleotides; ‘*’ indicates an insertion; ‘CM’ indicates chimera mutations.

Figure 3

Expression characteristics of CsLOB1 in Wanjincheng orange (Citrus sinensis Osbeck) mutants. (a) Expression of CsLOB1 in mutant plants after Xanthomonas citri subsp. citri (Xcc) inoculation. At 1 day postinoculation (dpi), CsLOB1 transcripts in leaves were analysed by quantitative real‐time PCR (qPCR). (b) Time course of CsLOB1 expression in mutants after Xcc inoculation. Transcript levels of CsLOB1 in leaves were determined by qPCR at 1, 3, 5, 7 and 9 dpi. (c) Statistical analysis of transcripts of CsLOB1 ^G and CsLOB1 ⁻ in citrus mutants. At 5 dpi, CsLOB1 cDNA from infected leaves was amplified by PCR, cloned into the pGEM ^®‐T Easy vector and sequenced. Twenty clones per mutant line were sequenced. Frequency (%) indicates the percentage of each CsLOB1 mRNA out of the total mRNAs tested. Relative expression level of CsLOB1 was determined by comparing the CsLOB1 transcript levels after Xcc inoculation with that after water inoculation. Error bars (a and b) indicate standard deviation of three independent tests.

Figure 4

Identification of citrus canker resistance in Wanjincheng orange (Citrus sinensis Osbeck) mutants. (a) Representative sequences of CsLOB1 mutations induced by CRISPR/Cas9. The pthA4 effector binding element (EBE) is in bold italics. The sgRNAs are in upper‐case letters, and the protospacer adjacent motif (PAM) site is underlined. The indel distinguishing CsLOB1 ^G and CsLOB1 ⁻ is in blue. Red dashes indicate deleted nucleotides. Pink letters indicate inserted nucleotides. ‘G ()’and ‘– ()’indicate mutations of CsLOB1 ^G and CsLOB1 ⁻ , respectively. In parentheses, ‘d#’ and ‘i#’ indicate the number of nucleotides deleted and inserted, respectively. Frequency (%) was calculated based on the number of clones with the same mutation out of the total number of clones sequenced. More than forty clones per line were sequenced to investigate mutations. (b, c and d) Assay of resistance to Xanthomonas citri subsp. citri (Xcc) in mutant plants. Fully expanded leaves of mutant lines and the wild type were treated with 10⁵ CFU/mL Xcc. Citrus canker symptoms (b) were recorded by photographing 5 and 9 days postinoculation (dpi). Disease lesion area (c) and disease index (d) of leaves of each mutation line were investigated at 9 dpi. (e) Growth of Xcc in leaves of mutant plants. Values are expressed as means ± standard deviation of six independent experiments. Different letters above bars represent significant differences from the wild type based on Duncan's multiple range test (P < 0.05). WT, wild type. In (b), bars = 1 mm.

Figure 5

In vivo assay of citrus canker resistance in Wanjincheng orange (Citrus sinensis Osbeck) mutants. Leaves were infiltrated with Xanthomonas citri subsp. citri (Xcc) suspensions. At 6 days postinoculation (dpi), pustules were detected in wild type, but absent or significantly reduced in mutant plants. At 12 dpi, severe canker symptoms were detected in wild type, whereas markedly reduced symptoms were observed in S2‐5 and S2‐12. No canker symptoms were found in S2‐6 and S5‐13.