Genome editing of an African elite rice variety confers resistance against endemic and emerging Xanthomonas oryzae pv. oryzae strains
- Institute for Molecular Physiology, Heinrich Heine University Düsseldorf, Germany
- International Rice Research Institute, Philippines
- Plant Health Institute of Montpellier (PHIM), Université Montpellier, IRD, CIRAD, INRAE, Institut Agro, France
- Division of Plant Science and Technology, Bond Life Sciences Center, University of Missouri, United States
- Tanzania Agricultural Research Institute (TARI)-Uyole Centre, United Republic of Tanzania
- International Rice Research Institute, Eastern and Southern Africa Region, Kenya
- International Rice Research Institute (IRRI), Africa Regional Office, Kenya
- Donald Danforth Plant Science Center, United States
- Institute for Transformative Biomolecules, ITbM, Nagoya University, Japan
Figures
Figure 1 with 2 supplements
Detection of an outbreak and survey of BB in Tanzania.
(A) No reports of serious BB infections before 2019. (B) Detection in Dakawa; in 2019, (C) in Lukenge in 2021, (D–I) survey results from 2022 at different degrees of severity. Note that sampling and …
Figure 1—figure supplement 1
Photos of infected fields showing severe infection of the field in Dakawa, Morogoro, Tanzania, in 2019.
Figure 1—figure supplement 2
Photos of infected fields showing infection of the field in Lukenge, Morogoro, Tanzania, in 2021.
Figure 2 with 3 supplements
Resistance spectrum of wild-type Komboka rice against African Xoo strains.
Leaf-clip inoculation of wild-type Komboka rice plants with a panel of 21 Xoo strains originating from 8 African countries along with Asian reference strain PXO86 from the Philippines. Lesion length …
Figure 2—figure supplement 1
Comparison of the susceptibility of Komboka to Tanzanian strains iTzDak19-1and iTzLuk21-3.
Leaf-clip inoculation of wild-type Komboka rice plants with a panel of two Xoo strains from the Philippines (PXO99A and PXO61), one from Burkina Faso (BAI3) , one collected in 2019 in Dakawa, and …
Figure 2—figure supplement 2
Virulence of the African Xoo diversity panel on the susceptible rice line Azucena.
Leaf-clip inoculation of wildtype Azucena with a panel of 21 Xoo strains originating from 8 African countries along with the Asian reference strain PXO86. Lesion lengths were measured 14 days after …
Figure 2—figure supplement 3
Presence of bacterial blight R genes in select rice varieties.
(A) QTL profiling of Komboka and other selected varieties from IRRI QTL database (https://rbi.irri.org/resources-and-tools/qtl-profiles). +: presence, -: absence, n.d., not determined. (B-D). …
Figure 3 with 1 supplement
Analysis of the genomes of Tanzanian Xoo isolates.
(A) Core genome Xanthomonas oryzae phylogenetic tree. Only the names of Tanzanian isolates are indicated. Abbreviations: Xoo, X. oryzae pv. oryzae; Xoc, X. oryzae pv. oryzicola; Xol, X. oryzae pv. le…
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Figure 3—source data 1
Compressed archive with Fasta Sequence files of Xoo strains CIX4462, CIX4506 and CIX4462.
CIX4505, CIX4507, CIX4508, CIX4457, CIX4458, Xoo3-1.
- https://cdn.elifesciences.org/articles/84864/elife-84864-fig3-data1-v3.docx
Figure 3—figure supplement 1
Pairwise counts of SNPs in the core genome of the newly isolated X. oryzae pv. oryzae strains from Tanzania and the closest neighboring clade.
Counts were obtained by applying the dist.dna function from the ape package (with the value 'N' to the model parameter) to pairs of genomes in the multiple SNP alignment file generated by parsnp and …
Figure 4
Virulence of the new Tanzanian Xoo strains depends on the induction of SWEET11a.
(A) Lesion lengths were measured 14 days after leaf-clipping inoculation of Kitaake individual sweet knock out lines (ossweet11a, ossweet13 and ossweet14) in the cultivar Kitaake (Eom et al., 2019) …
Figure 5 with 3 supplements
Resistance of EBE-edited Komboka lines against six representative Xoo strains.
(A) Reactions of WT and six edited Komboka lines to infection by Xoo strains (PXO99A, PXO61, PXO86, BAI3, and MAI1) harboring PthXo1, PthXo2 PthXo3, AvrXa7, TalC and/or TalF. ME2 is a PXO99A mutant …
Figure 5—figure supplement 1
Comparison of EBE sequences for the SWEET11a, 13 and 14 promoters between Komboka and Kitaake.
Kitaake and Komboka have the same EBE sequences for PthXo1, TalC, TalF, AvrXa7, PthXo3. The promoter sequence of SWEET13 in wild type Komboka corresponds to the EBE for PthXo2A, while for Kitaake …
Figure 5—figure supplement 2
Hybrid CRISPR-Cas9/Cpf1 system.
(A) Guide RNA design. Four guide RNAs were designed to target six known EBEs within OsSWEET11a, 13 and 14 promoters. EBE sequences are bolded, and the respective TALEs are indicated. Arrowheads …
Figure 5—figure supplement 3
Genotypes of T0 lines with biallelic mutations at six targeted EBEs.
TATAA box is labeled in red. LbCpf1 and SpCas9 PAM sequences are labeled in blue and purple, respectively. EBE sequences are indicated (e.g. PthXo1).
Figure 6
Genotypes and phenotypes of two EBE-edited lines generated in a second transformation experiment.
(A) Mutations at the EBEs for PthXo1, PthXo2, TalC, TalF, PthXo3, and AvrXa7 in two Komboka edited lines, 14_19 and 14_65. (B). Reactions of wild-type Komboka and the two edited lines to the …
Figure 7 with 1 supplement
Komboka SWEET promoter edited lines are fully resistant against Tanzanian strains of Xoo.
Lesion lengths measured 14 days after leaf-clip inoculation of wild-type Komboka and the two multi-edited lines 1.2_40 and 1.5_19 with Xoo strains PXO99A (PthXo1), BAI3 (TalC), PXO61 (PthXo2B, …
Figure 7—figure supplement 1
qRT-PCR analysis of SWEET11a mRNA accumulation of edited Komboka lines after infection with Tanzanian Xoo strains.
Relative mRNA levels (2-ΔCt) of SWEET11a in Komboka wild type and the multi-EBE-edited lines 1.2_40 and 1.5_19 upon infection by PXO99A (PthXo1), BAI3 (TalC) and Tanzanian PthXo1B-dependent Xoo …
Tables
Table 1
Efficiency of resistance genes in IRBB rice lines towards a diversity panel of 26 African Xoo strains.
Quantitative scores of lesion length produced upon leaf-clip inoculation of a diversity panel consisting of 26 Xanthomonas oryzae pv. oryzae strains from West and East Africa on IRBB …
| Strain name | CIX code | Country | Azucena | IR24 | IRBB1 | IRBB3 | IRBB4 | IRBB5 | IRBB7 | IRBB23 | Komboka |
|---|---|---|---|---|---|---|---|---|---|---|---|
| NatiPark | 607 | Benin | S | R | R | R | R | R | R | R | R |
| Karfiguela13 | 705 | Burkina Faso | S | R | R | R | R | R | R | R | R |
| N2-4 | 4482 | Niger | S | R | R | R | R | R | R | R | R |
| Tanguieta3 | 609 | Benin | S | MR | R | MR | R | R | MR | R | R |
| Toula20 | 629 | Niger | S | MR | R | MR | R | R | MR | MR | R |
| BAI250 | 4127 | Burkina Faso | S | MR | R | MR | MR | R | R | MR | R |
| S62-2-22 | 2374 | Senegal | S | MR | R | MS | R | R | MR | R | R |
| CII-1 | 4083 | Ivory Coast | S | MS | MR | MS | R | R | MR | MR | R |
| CII-2 | 1042 | Ivory Coast | S | MS | MR | MS | MR | R | MR | MR | R |
| NAI9 | 2787 | Niger | S | MS | MR | MS | MR | MR | MS | S | R |
| AXO1947 | 1917 | Cameroon | S | S | R | S | R | R | MS | MS | R |
| MAI145 | 894 | Mali | S | S | R | S | MR | MS | S | MR | R |
| BAI3 | 4092 | Burkina Faso | S | MS | MR | S | MR | MR | MS | S | R |
| CFBP1948 | 2801 | Cameroon | S | MS | MR | S | MR | MR | S | S | R |
| NAI5 | 4099 | Niger | S | S | MR | S | MR | MR | S | S | R |
| MAI73 | 4079 | Mali | S | S | R | S | MS | MR | S | MR | R |
| S82-4-3 | 2976 | Senegal | S | MS | MR | S | MS | MR | MS | S | R |
| MAI132 | 4517 | Mali | S | S | R | S | S | MS | S | MR | R |
| iTzDak19-1 | 4457 | Tanzania | S | S | S | S | S | S | S | R | S |
| iTzDak19-2 | 4458 | Tanzania | S | S | S | S | S | S | S | R | S |
| iTzDak19-3 | 4462 | Tanzania | S | S | S | S | S | S | S | R | S |
| iTzLuk21-1 | 4506 | Tanzania | nd | nd | nd | nd | nd | nd | nd | nd | nd |
| iTzLuk21-2 | 4509 | Tanzania | nd | nd | nd | nd | nd | nd | nd | nd | nd |
| iTzLuk21-3 | 4505 | Tanzania | nd | nd | nd | nd | nd | nd | nd | nd | S |
| iTzLuk21-4 | 4507 | Tanzania | nd | nd | nd | nd | nd | nd | nd | nd | S |
| iTzLuk21-5 | 4508 | Tanzania | nd | nd | nd | nd | nd | nd | nd | nd | nd |
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n.d. not determined.
Additional files
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Supplementary file 1
Supplementary file with additional data.
(a) List of Xoo strains used in this study (b) Disease survey in multiple rice growing areas in Tanzania in 2022. (c) Sequences of Xa1 and Xa4 genes from Oryza sativa cv. Komboka. (d) Characteristics of the TALome of Xanthomonas oryzae pv. oryzae Tanzanian strains. (e) OsSWEET promoter sequences for the rice varieties Komboka and Kitaake. (f) Guide RNA sequences. (g) EBE sequences in select OsSWEET promoters of Komboka wild-type and CRISPR-edited lines. (h) List of SNPs and Indels in OsSWEET promoters after editing of the rice varieties of Komboka and Kitaake (i) List of T0 lines with biallelic mutations at all targeted EBEs. (j) Screening of the T1 generation for homozygous mutations in all six SWEET EBEs. (k) List of T1 lines with homozygous mutations of all targeted EBEs. n.d. not determined. (l) List of primers used in this study (m) Alignment of OsSWEET11a promoters from the rice cv. Komboka and Kitaake.
- https://cdn.elifesciences.org/articles/84864/elife-84864-supp1-v3.docx
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Supplementary file 2
Accession numbers and main features of genome assemblies used for strain phylogeny inference.
- https://cdn.elifesciences.org/articles/84864/elife-84864-supp2-v3.xlsx
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Supplementary file 3
SWEET11a-targeting RVD sequences of the iTz strains.
- https://cdn.elifesciences.org/articles/84864/elife-84864-supp3-v3.xlsx
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Source data 1
Summary of raw data files available at dryad https://doi.org/10.5061/dryad.xpnvx0kk3.
- https://cdn.elifesciences.org/articles/84864/elife-84864-data1-v3.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/84864/elife-84864-mdarchecklist1-v3.pdf
