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TAL effectors delivered by phytopathogenic Xanthomonas species are DNA-sequence-specific transcriptional activators of host susceptibility genes and sometimes resistance genes. The modularity of DNA recognition by TAL effectors makes them important also as tools for gene targeting and genome editing. Effector binding elements (EBEs) recognized by native TAL effectors in plants have been identified only on the forward strand of target promoters. Here, we demonstrate that TAL effectors can drive plant transcription from EBEs on either strand and in both directions. Furthermore, we show that a native TAL effector from Xanthomonas oryzae pv. oryzicola drives expression of a target with an EBE on each strand of its promoter. By inserting that promoter and derivatives between two reporter genes oriented head to head, we show that the TAL effector drives expression from either EBE in the respective orientations, and that activity at the reverse-strand EBE also potentiates forward transcription driven by activity at the forward-strand EBE. Our results reveal new modes of action for TAL effectors, suggesting the possibility of yet unrecognized targets important in plant disease, expanding the search space for off-targets of custom TAL effectors, and highlighting the potential of TAL effectors for probing fundamental aspects of plant transcription., (Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.)

Bacterial leaf streak of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an increasingly important yield constraint in this staple crop. A mesophyll colonizer, Xoc differs from X. oryzae pv. oryzae (Xoo), which invades xylem to cause bacterial blight of rice. Both produce multiple distinct TAL effectors, type III-delivered proteins that transactivate effector-specific host genes. A TAL effector finds its target(s) via a partially degenerate code whereby the modular effector amino acid sequence identifies nucleotide sequences to which the protein binds. Virulence contributions of some Xoo TAL effectors have been shown, and their relevant targets, susceptibility (S) genes, identified, but the role of TAL effectors in leaf streak is uncharacterized. We used host transcript profiling to compare leaf streak to blight and to probe functions of Xoc TAL effectors. We found that Xoc and Xoo induce almost completely different host transcriptional changes. Roughly one in three genes upregulated by the pathogens is preceded by a candidate TAL effector binding element. Experimental analysis of the 44 such genes predicted to be Xoc TAL effector targets verified nearly half, and identified most others as false predictions. None of the Xoc targets is a known bacterial blight S gene. Mutational analysis revealed that Tal2g, which activates two genes, contributes to lesion expansion and bacterial exudation. Use of designer TAL effectors discriminated a sulfate transporter gene as the S gene. Across all targets, basal expression tended to be higher than genome-average, and induction moderate. Finally, machine learning applied to real vs. falsely predicted targets yielded a classifier that recalled 92% of the real targets with 88% precision, providing a tool for better target prediction in the future. Our study expands the number of known TAL effector targets, identifies a new class of S gene, and improves our ability to predict functional targeting.

Xanthomonas transcription activator-like (TAL) effectors promote disease in plants by binding to and activating host susceptibility genes. Plants counter with TAL effector-activated executor resistance genes, which cause host cell death and block disease progression. We asked whether the functional specificity of an executor gene could be broadened by adding different TAL effector binding elements (EBEs) to it. We added six EBEs to the rice Xa27 gene, which confers resistance to strains of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) that deliver the TAL effector AvrXa27. The EBEs correspond to three other effectors from Xoo strain PXO99(A) and three from strain BLS256 of the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Stable integration into rice produced healthy lines exhibiting gene activation by each TAL effector, and resistance to PXO99(A) , a PXO99(A) derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains virulent toward wildtype Xa27 plants. Transcripts initiated primarily at a common site. Sequences in the EBEs were found to occur nonrandomly in rice promoters, suggesting an overlap with endogenous regulatory sequences. Thus, executor gene specificity can be broadened by adding EBEs, but caution is warranted because of the possible coincident introduction of endogenous regulatory elements., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)