A PthXo2B ortholog in Xanthomonas oryzae pv oryzae strain IX-221 acts as a major virulence factor on indica rice without activating a Clade III SWEET gene

To infect rice, Xanthomonas oryzae pv. oryzae ( Xoo ) deploys transcription a ctivator-like e ffectors (TALEs) that specifically bind and upregulate host “susceptibility” (S) genes. 34-amino acid (aa) repeats in TALEs interact one-to-one with DNA bases. Variation at positions 12 and 13 in each repeat, the repeat-variable diresidue (RVD), determine specificity. Some repeat variants shorter or longer than 34 aa can disengage to accommodate a single base deletion in the target sequence. OsSWEET11 , 13 , and 14 are key S genes, targeted by different TALEs from diverse Xoo strains. xa13 is a SWEET11 allele lacking the TALE binding site and thus conferring resistance. xa13 is overcome by TALEs that activate SWEET13 or SWEET14 . We report here that an xa13 -compatible Xoo strain, IX-221, from India, harbours an ortholog of the SWEET14 -targeting TALE PthXo3 and two orthologs of the SWEET13 -cognate PthXo2, each with one or two 36-aa repeats capable of disengaging. One of the PthXo2 orthologs, PthXo2B _IX-221 , has a repeat region identical to the previously characterized PthXo2B _PXO61 , except for a two amino acid difference near the end of the 19 ^th repeat. Like PthXo2B _PXO61 , PthXo2B _IX-221 upregulates SWEET13 in japonica rice and no SWEET in indica rice, but unlike PthXo2B _PXO61 it nonetheless renders indica rice susceptible, pointing to an alternative S gene. Further, a designer TALE (dTALE) constructed using a standard, consensus sequence for each repeat and RVDs identical to those of PthXo2B _IX-221 failed to render indica rice susceptible. Alignment of the PthXo2B _IX-221 repeats shows a departure from the consensus in each of two repeats carrying the RVD ‘NN’: the sequence ‘MAIAN’ in place of ‘VAIAS’ beginning at position 7. Together, the PthXo2B _IX-221 results thus suggest that non-RVD sequence variation affects TALE targeting profiles. More broadly, the presence of the three aberrant repeat-harbouring TALEs in IX-221 suggests that widespread deployment of xa13 in India resulted in strains super-equipped to overcome it, capable of activating multiple SWEET genes and alleles as well as an apparent alternate S gene.