We set out to evaluate our hypothesis that editing known SWEET EBEs will endow rice with broad resistance to bacterial blight. The success of our approach depended on extant Xoo strains fitting the profile of the relatively few Xoo strains that have been genetically characterized. Indeed, analysis of a broad collection of 63 Xoo strains found that all strains targeted, or were predicted to target, SWEET11, SWEET13 and SWEET14, as previously reported8,10,27. Furthermore, most Xoo strains, if not all, target some combination of six short EBE sequences present in the promoters of the three SWEET genes. We applied multiplexed CRISPR–Cas9 genome editing to systematically interfere with SWEET gene induction at all known major TALe EBEs and engineer Kitaake rice resistant to all currently known strains of Xoo. Kitaake is an excellent cultivar for proof-of-concept owing to high regeneration and rapid flowering cycle. The lines generated here will serve as diagnostic tools for rapid evaluation of the virulence of novel Xoo isolates24. Although Kitaake can be used for breeding resistance genes into Japanese and Chinese varieties, the japonica cultivar is not optimal as breeding material in large parts of southeast Asia and Africa, which use mainly indica varieties. Therefore, we also applied the same editing approach in two mega varieties, IR6426 and Ciherang-Sub128. The edited mega variety lines grew normally, without yield suppression, and were resistant to three representative strains of bacterial blight carrying the known major TALes.

To devise an efficient genome editing strategy it was important to identify a panoply of TALes and cognate EBEs to guide engineering of broadly applicable bacterial blight resistance. Sequence analysis of the seven deviant Xoo strains from Asia and relatedness of TALes with close, but not identical, RVDs led us to propose that variants of PthXo2 target variant EBEs in SWEET13. These seven Asian strains retained virulence on the first-generation of genome-edited EBE lines in the Kitaake background, indicating that the variant strains either carry another major TALe targeting an undefined susceptibility gene or a variant major TALe targeting SWEET11, SWEET13 or SWEE14 at an alternate EBE. The two newly identified pthXo2 members (pthXo2B and pthXo2C) were predicted to target the SWEET13 promoters found in many japonica varieties, including Kitaake and Nipponbare. Indeed, PthXo2B conferred virulence to ME2, a strain lacking major TALe genes, on both Kitaake and Nipponbare, concomitantly with SWEET13 induction. Thus, the presence of PthXo2B or PthXo2C in the deviant strains is sufficient to explain their virulence on the TALEN-edited lines. Ultimately, several lines with CRISPR-mediated edits at the PthXo2B-cognate EBE of Kitaake were resistant to pthXo2B-containing strains, indicating that the Kitaake allele for SWEET13 was responsible for the susceptibility of line 52-1. Recent testing of all TALe genes of the strain PXO61 did not reveal any new major TALe other than PthXo3 and PthXo2B29. XooS strains carrying PthXo2B were obtained in the Philippines, where several bacterial blight outbreaks occurred. This sublineage is characterized as local races 1 and 9d, which have not been reported in other countries. At the same time, the Philippine archipelago may offer a safe geographic reservoir for less prevalent strains. Both strains from the Korean peninsula possess PthXo2C. The sampling of Korean strains was limited, and we are unable to state whether genes for PthXo2C are the rule or the exception. What is clear is that different variants of PthXo2 family are present in the global Xoo population, suggesting an ongoing adaptation to nucleotide variations in the SWEET13 promoter12,16,17.

African strains were shown to possess less TALe diversity and fall into two sublineages. All strains harbor the major virulence TALe, TalC, which targets SWEET14. Members of the second sublineage have TalC and TalF, the latter also targeting SWEET14. Another feature of the African strains is that loss of TalC function results in significantly reduced lesion length13. Xoo strains relying solely on TalC retained full virulence on Kitaake lines mutated in the TalC EBE within SWEET14 (ref. 19).

We show here that mutation of the TalC EBE in the promoter of SWEET14 in Kitaake led to only moderate resistance to the African strain AXO1947. By contrast, double knock out of SWEET13 and SWEET14 resulted in complete resistance24. Consistent with previous studies19,30, the dependence of African Xoo strains on SWEET gene expression to cause symptoms appears more complicated than anticipated. Alternatively, lesion length may not be strictly correlated with ecological fitness or disease severity, and variation in lesion length, particularly at lower value ranges, may be due to variant host defense responses to diverse pathogen lineages.

Broad resistance to bacterial blight at the SWEET promoters will not prevent adaptation of the pathogen, and the durability of this approach will depend on the ability of Xoo populations to adapt to recessive resistance alleles. As noted, TALe genes have limited diversity, and, when diversity is present, it comprises minor variations. We propose that creating polymorphisms that are larger than a single nucleotide change (ideally modifying the whole EBE) in the EBEs of SWEET genes is advisable. These changes will still capture minor TALe variants of the major, for example, for PthXo2 and AvrXa7/PthXo3. The promoter editing will also likely thwart TALe adaptation, assuming that ease of adaptation to new binding sites is inversely related to the number of novel nucleotides in the target sequence. EBEs must meet certain structural requirements for TALe binding within the available sequences of the susceptibility gene promoters. Another consideration is that breeders will likely combine these recessive resistance alleles with other locally effective resistance genes and these combinations will likely severely reduce disease pressure, further delaying strain adaptation.

A diagnostic kit that can both identify the promoter variants best suited to defeat the local pathogen population and monitor variant strain emergence is presented in an accompanying publication24. Breeders and pathologists can work together to provide selected lines to farmers, with an overall strategy of deploying multiple lines in a region to disfavor the evolution of novel strains.

Multiplex targeting using CRISPR–Cas9 has created the ability to simultaneously edit all EBEs present in any single rice line. However, this method is limited owing to the constraint of possible edits and problems with the delivery of editing complexes. For example, the most efficient genome editing is via DNA-mediated delivery, which confers regulatory concerns on edited plants. Genome editors such as CRISPR–Cpf1 (also called Cas12a, which allows for larger mutations), potent ribonucleoprotein (RNP) delivery and transgene-free genome editing may mitigate these limitations and improve the durability of disease resistance.

In conclusion, by using a combination of systematic analyses of diverse Xoo strains, an understanding of SWEET genes and genome editing, we were able to engineer broad-spectrum resistance in Kitaake and two mega varieties IR64 and Ciherang-Sub1. Genome editing can result in off-target effects and the transformation process often leads to mutations and somaclonal variation31,32, but on- and off-target analysis of rice genomes in NCBI with the Cas9 guide RNAs revealed that all the predicted off-target sites lack PAM sequences (an essential component for Cas9 recognition) and contain mismatches larger than 4 nucleotides to the guide sequences within the single guide RNA (sgRNA) genes (Supplementary Table 8). Furthermore, off-target mutations (if they are present), and other mutations derived from tissue culture and the regeneration process, will be eliminated in crosses during breeding. Full genome sequencing will be needed to identify possible off-target mutations and to ensure that the lines do not contain any T-DNA. Finally, our preliminary analysis of agronomic traits, while promising, is insufficient for direct deployment. Extensive field trials will be necessary to ensure performance of these edited resistant lines.