Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA.

DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A-to-G-editing TALEDs but not C-to-T-editing DdCBEs induce tens of thousands of transcriptome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants not only reduced RNA off-target edits by >99% but also minimized off-target mtDNA mutations and bystander edits at a target site. Unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.

DNA base editing in nuclear and organellar genomes.

Genome editing continues to revolutionize biological research. Due to its simplicity and flexibility, CRISPR/Cas-based editing has become the preferred technology in most systems. Cas nucleases tolerate fusion to large protein domains, thus allowing combination of their DNA recognition properties with new enzymatic activities. Fusion to nucleoside deaminase or reverse transcriptase domains has produced base editors and prime editors that, instead of generating double-strand breaks in the target sequence, induce site-specific alterations of single (or a few adjacent) nucleotides. The availability of protein-only genome editing reagents based on transcription activator-like effectors has enabled the extension of base editing to the genomes of chloroplasts and mitochondria. In this review, we summarize currently available base editing methods for nuclear and organellar genomes. We highlight recent advances with improving precision, specificity, and efficiency and discuss current limitations and future challenges. We also provide a brief overview of applications in agricultural biotechnology and gene therapy.

Targeted C•G-to-T•A base editing with TALE-cytosine deaminases in plants.

BACKGROUND: TALE-derived DddA-based cytosine base editors (TALE-DdCBEs) can perform efficient base editing of mitochondria and chloroplast genomes. They use transcription activator-like effector (TALE) arrays as programmable DNA-binding domains and a split version of the double-strand DNA cytidine deaminase (DddA) to catalyze C•G-to-T•A editing. This technology has not been optimized for use in plant cells. RESULTS: To systematically investigate TALE-DdCBE architectures and editing rules, we established a ß-glucuronidase reporter for transient assays in Nicotiana benthamiana. We show that TALE-DdCBEs function with distinct spacer lengths between the DNA-binding sites of their two TALE parts. Compared to canonical DddA, TALE-DdCBEs containing evolved DddA variants (DddA6 or DddA11) showed a significant improvement in editing efficiency in Nicotiana benthamiana and rice. Moreover, TALE-DdCBEs containing DddA11 have broader sequence compatibility for non-TC target editing. We have successfully regenerated rice with C•G-to-T•A conversions in their chloroplast genome, as well as N. benthamiana with C•G-to-T•A editing in the nuclear genome using TALE-DdCBE. We also found that the spontaneous assembly of split DddA halves can cause undesired editing by TALE-DdCBEs in plants. CONCLUSIONS: Altogether, our results refined the targeting scope of TALE-DdCBEs and successfully applied them to target the chloroplast and nuclear genomes. Our study expands the base editing toolbox in plants and further defines parameters to optimize TALE-DdCBEs for high-fidelity crop improvement.

TALE-based organellar genome editing and gene expression in plants.

KEY MESSAGE: TALE-based editors provide an alternative way to engineer the organellar genomes in plants. We update and discuss the most recent developments of TALE-based organellar genome editing in plants. Gene editing tools have been widely used to modify the nuclear genomes of plants for various basic research and biotechnological applications. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing platform is the most commonly used technique because of its ease of use, fast speed, and low cost; however, it encounters difficulty when being delivered to plant organelles for gene editing. In contrast, protein-based editing technologies, such as transcription activator-like effector (TALE)-based tools, could be easily delivered, expressed, and targeted to organelles in plants via Agrobacteria-mediated nuclear transformation. Therefore, TALE-based editors provide an alternative way to engineer the organellar genomes in plants since the conventional chloroplast transformation method encounters technical challenges and is limited to certain species, and the direct transformation of mitochondria in higher plants is not yet possible. In this review, we update and discuss the most recent developments of TALE-based organellar genome editing in plants.

Flexible TALEs for an expanded use in gene activation, virulence and scaffold engineering.

Transcription activator-like effectors (TALEs) are bacterial proteins with a programmable DNA-binding domain, which turned them into exceptional tools for biotechnology. TALEs contain a central array of consecutive 34 amino acid long repeats to bind DNA in a simple one-repeat-to-one-nucleotide manner. However, a few naturally occurring aberrant repeat variants break this strict binding mechanism, allowing for the recognition of an additional sequence with a -1 nucleotide frameshift. The limits and implications of this extended TALE binding mode are largely unexplored. Here, we analyse the complete diversity of natural and artificially engineered aberrant repeats for their impact on the DNA binding of TALEs. Surprisingly, TALEs with several aberrant repeats can loop out multiple repeats simultaneously without losing DNA-binding capacity. We also characterized members of the only natural TALE class harbouring two aberrant repeats and confirmed that their target is the major virulence factor OsSWEET13 from rice. In an aberrant TALE repeat, the position and nature of the amino acid sequence strongly influence its function. We explored the tolerance of TALE repeats towards alterations further and demonstrate that inserts as large as GFP can be tolerated without disrupting DNA binding. This illustrates the extraordinary DNA-binding capacity of TALEs and opens new uses in biotechnology.

Binding of the transcription activator-like effector augments transcriptional regulation by another transcription factor.

DNA transcription is regulated by a range of diverse mechanisms and primarily by transcription factors that recruit the RNA polymerase complex to the promoter region on the DNA. Protein binding to DNA at nearby or distant sites can synergistically affect this process in a variety of ways, but mainly through direct interactions between DNA-binding proteins. Here we show that a Transcription Activator-Like Effector (TALE), which lacks an activation domain, can enhance transcription in mammalian cells when it binds in the vicinity of and without direct interaction with several different dimeric or monomeric transcription factors. This effect was observed for several TALEs regardless of the recognition sequences and their DNA-bound orientation. TALEs can exert an effect over the distance of tens of nucleotides and it also potentiated KRAB-mediated repression. The augmentation of transcriptional regulation of another transcription factor is characteristic of TALEs, as it was not observed for dCas9/gRNA, zinc finger, or Gal4 DNA-binding domains. We propose that this mechanism involves an allosteric effect exerted on DNA structure or dynamics. This mechanism could be used to modulate transcription but may also play a role in the natural context of TALEs.

Consequences of adaptation of TAL effectors on host susceptibility to Xanthomonas.

Transcription activator-like effectors (TALEs) are virulence factors of Xanthomonas that induce the expression of host susceptibility (S) genes by specifically binding to effector binding elements (EBEs) in their promoter regions. The DNA binding specificity of TALEs is dictated by their tandem repeat regions, which are highly variable between different TALEs. Mutation of the EBEs of S genes is being utilized as a key strategy to generate resistant crops against TALE-dependent pathogens. However, TALE adaptations through rearrangement of their repeat regions is a potential obstacle for successful implementation of this strategy. We investigated the consequences of TALE adaptations in the citrus pathogen Xanthomonas citri subsp. citri (Xcc), in which PthA4 is the TALE required for pathogenicity, whereas CsLOB1 is the corresponding susceptibility gene, on host resistance. Seven TALEs, containing two-to-nine mismatching-repeats to the EBEPthA4 that were unable to induce CsLOB1 expression, were introduced into Xcc pthA4:Tn5 and adaptation was simulated by repeated inoculations into and isolations from sweet orange for a duration of 30 cycles. While initially all strains failed to promote disease, symptoms started to appear between 9-28 passages in four TALEs, which originally harbored two-to-five mismatches. Sequence analysis of adapted TALEs identified deletions and mutations within the TALE repeat regions which enhanced putative affinity to the CsLOB1 promoter. Sequence analyses suggest that TALEs adaptations result from recombinations between repeats of the TALEs. Reintroduction of these adapted TALEs into Xcc pthA4:Tn5 restored the ability to induce the expression of CsLOB1, promote disease symptoms and colonize host plants. TALEs harboring seven-to-nine mismatches were unable to adapt to overcome the incompatible interaction. Our study experimentally documented TALE adaptations to incompatible EBE and provided strategic guidance for generation of disease resistant crops against TALE-dependent pathogens.

The Complete Genome Resource of Xanthomonas oryzae pv. oryzae CIX2779 Includes the First Sequence of a Plasmid for an African Representative of This Rice Pathogen.

The bacterial plant pathogen Xanthomonas oryzae pv. oryzae is responsible for the foliar rice bacterial blight disease. Genetically contrasted, continent-specific, sublineages of this species can cause important damages to rice production both in Asia and Africa. We report on the genome of the CIX2779 strain of this pathogen, previously named NAI1 and originating from Niger. Oxford Nanopore long reads assembly and Illumina short reads polishing produced a genome sequence composed of a 4,725,792-bp circular chromosome and a 39,798-bp-long circular plasmid designated pCIX2779_1. The chromosome structure and base-level sequence are highly related to reference strains of African X. oryzae pv. oryzae and encode identical transcription activator-like effectors for virulence. Importantly, our in silico analysis strongly indicates that pCIX2779_1 is a genuine conjugative plasmid, the first indigenous one sequenced from an African strain of the X. oryzae species. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Assembling highly repetitive Xanthomonas TALomes using Oxford Nanopore sequencing.

BACKGROUND: Most plant-pathogenic Xanthomonas bacteria harbor transcription activator-like effector (TALE) genes, which function as transcriptional activators of host plant genes and support infection. The entire repertoire of up to 29 TALE genes of a Xanthomonas strain is also referred to as TALome. The DNA-binding domain of TALEs is comprised of highly conserved repeats and TALE genes often occur in gene clusters, which precludes the assembly of TALE-carrying Xanthomonas genomes based on standard sequencing approaches. RESULTS: Here, we report the successful assembly of the 5 Mbp genomes of five Xanthomonas strains from Oxford Nanopore Technologies (ONT) sequencing data. For one of these strains, Xanthomonas oryzae pv. oryzae (Xoo) PXO35, we illustrate why Illumina short reads and longer PacBio reads are insufficient to fully resolve the genome. While ONT reads are perfectly suited to yield highly contiguous genomes, they suffer from a specific error profile within homopolymers. To still yield complete and correct TALomes from ONT assemblies, we present a computational correction pipeline specifically tailored to TALE genes, which yields at least comparable accuracy as Illumina-based polishing. We further systematically assess the ONT-based pipeline for its multiplexing capacity and find that, combined with computational correction, the complete TALome of Xoo PXO35 could have been reconstructed from less than 20,000 ONT reads. CONCLUSIONS: Our results indicate that multiplexed ONT sequencing combined with a computational correction of TALE genes constitutes a highly capable tool for characterizing the TALomes of huge collections of Xanthomonas strains in the future.

Development of a Transcriptional Activator-Like Effector Protein to Accurately Discriminate Single Nucleotide Difference.

Transcriptional activator-like effector (TALE), a DNA-binding protein, is widely used in genome editing. However, the recognition of the target sequence by the TALE is adversely affected by the number of mismatches. Therefore, the association constant of DNA-TALE complex formation can be controlled by appropriately introducing a mismatch into the TALE recognition sequence. This study aimed to construct a TALE that can distinguish a single nucleotide difference. Our results show that a single mismatch present in repeats 2 or 3 of TALE did not interfere with the complex formation with DNA, whereas continuous mismatches present in repeats 2 and 3 significantly reduced association with the target DNA. Based on these findings, we constructed a detection system of the one nucleotide difference in gene with high accuracy and constructed a TALE-nuclease (TALEN) that selectively cleaves DNA with a single mismatch.

High-efficiency prime editing enables new strategies for broad-spectrum resistance to bacterial blight of rice.

Using genetic resistance against bacterial blight (BB) caused by Xanthomonas oryzae pathovar oryzae (Xoo) is a major objective in rice breeding programmes. Prime editing (PE) has the potential to create novel germplasm against Xoo. Here, we use an improved prime-editing system to implement two new strategies for BB resistance. Knock-in of TAL effector binding elements (EBE) derived from the BB susceptible gene SWEET14 into the promoter of a dysfunctional executor R gene xa23 reaches 47.2% with desired edits including biallelic editing at 18% in T0 generation that enables an inducible TALE-dependent BB resistance. Editing the transcription factor TFIIA gene TFIIAγ5 required for TAL effector-dependent BB susceptibility recapitulates the resistance of xa5 at an editing efficiency of 88.5% with biallelic editing rate of 30% in T0 generation. The engineered loci provided resistance against multiple Xoo strains in T1 generation. Whole-genome sequencing detected no OsMLH1dn-associated random mutations and no off-target editing demonstrating high specificity of this PE system. This is the first-ever report to use PE system to engineer resistance against biotic stress and to demonstrate knock-in of 30-nucleotides cis-regulatory element at high efficiency. The new strategies hold promises to fend rice off the evolving Xoo strains and protect it from epidemics.

A promoter trap in transgenic citrus mediates recognition of a broad spectrum of Xanthomonas citri pv. citri TALEs, including in planta-evolved derivatives.

Citrus bacterial canker (CBC), caused by Xanthomonas citri subsp. citri (Xcc), causes dramatic losses to the citrus industry worldwide. Transcription activator-like effectors (TALEs), which bind to effector binding elements (EBEs) in host promoters and activate transcription of downstream host genes, contribute significantly to Xcc virulence. The discovery of the biochemical context for the binding of TALEs to matching EBE motifs, an interaction commonly referred to as the TALE code, enabled the in silico prediction of EBEs for each TALE protein. Using the TALE code, we engineered a synthetic resistance (R) gene, called the Xcc-TALE-trap, in which 14 tandemly arranged EBEs, each capable of autonomously recognizing a particular Xcc TALE, drive the expression of Xanthomonas avrGf2, which encodes a bacterial effector that induces plant cell death. Analysis of a corresponding transgenic Duncan grapefruit showed that transcription of the cell death-inducing executor gene, avrGf2, was strictly TALE-dependent and could be activated by several different Xcc TALE proteins. Evaluation of Xcc strains from different continents showed that the Xcc-TALE-trap mediates resistance to this global panel of Xcc isolates. We also studied in planta-evolved TALEs (eTALEs) with novel DNA-binding domains and found that these eTALEs also activate the Xcc-TALE-trap, suggesting that the Xcc-TALE-trap is likely to confer durable resistance to Xcc. Finally, we show that the Xcc-TALE-trap confers resistance not only in laboratory infection assays but also in more agriculturally relevant field studies. In conclusion, transgenic plants containing the Xcc-TALE-trap offer a promising sustainable approach to control CBC.

CRISPRi in Xanthomonas demonstrates functional convergence of transcription activator-like effectors in two divergent pathogens.

Functional analysis of large gene families in plant pathogens can be cumbersome using classical insertional mutagenesis. Additionally, Cas9 toxicity has limited the application of CRISPR-Cas9 for directed mutagenesis in bacteria. Here, we successfully applied a CRISPR interference strategy to investigate the cryptic role of the transcription activator-like effector (tale) multigene family in several plant-pathogenic Xanthomonas bacterial species, owing to their contribution to pathogen virulence. Single guide RNAs (sgRNAs) designed against Xanthomonas phaseoli pv manihotis tale conserved gene sequences efficiently silenced expression of all tales, with concomitant decrease in virulence and TALE-induced host gene expression. The system is readily translatable to other Xanthomonas species infecting rice, citrus, Brassica, and cassava, silencing up to 16 tales in a given strain using a single sgRNA. Complementation with plasmid-borne designer tales lacking the sgRNA-targeted sequence restored molecular and virulence phenotypes in all pathosystems. Our results evidenced that X. campestris pv campestris CN08 tales are relevant for symptom development in cauliflower. They also show that the MeSWEET10a sugar transporter is surprisingly targeted by the nonvascular cassava pathogen X. cassavae, highlighting a new example of TALE functional convergence between phylogenetically distant Xanthomonas. Overall, this novel technology provides a platform for discovery and rapid functional understanding of highly conserved gene families.

Eliminating predictable DNA off-target effects of cytosine base editor by using dual guiders including sgRNA and TALE.

Predictable DNA off-target effect is one of the major safety concerns for the application of cytosine base editors (CBEs). To eliminate Cas9-dependent DNA off-target effects, we designed a novel effective CBE system with dual guiders by combining CRISPR with transcription activator-like effector (TALE). In this system, Cas9 nickase (nCas9) and cytosine deaminase are guided to the same target site to conduct base editing by single-guide RNA (sgRNA) and TALE, respectively. However, if nCas9 is guided to a wrong site by sgRNA, it will not generate base editing due to the absence of deaminase. Similarly, when deaminase is guided to a wrong site by TALE, base editing will not occur due to the absence of single-stranded DNA. In this way, Cas9- and TALE-dependent DNA off-target effects could be completely eliminated. Furthermore, by fusing TALE with YE1, a cytidine deaminase with minimal Cas9-independent off-target effect, we established a novel CBE that could induce efficient C-to-T conversion without detectable Cas9- or TALE-dependent DNA off-target mutations.

Insights into the Diversity of Transcription Activator-Like Effectors in Indian Pathotype Strains of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is a major rice pathogen, and its genome harbors extensive inter-strain and inter-lineage variations. The emergence of highly virulent pathotypes of Xoo that can overcome major resistance (R) genes deployed in rice breeding programs is a grave threat to rice cultivation. The present study reports on a long-read Oxford nanopore-based complete genomic investigation of Xoo isolates from 11 pathotypes that are reported based on their reaction toward 10 R genes. The investigation revealed remarkable variation in the genome structure in the strains belonging to different pathotypes. Furthermore, transcription activator-like effector (TALE) proteins secreted by the type III secretion system display marked variation in content, genomic location, classes, and DNA-binding domain. We also found the association of tal genes in the vicinity of regions with genome structural variations. Furthermore, in silico analysis of the genome-wide rice targets of TALEs allowed us to understand the emergence of pathotypes compatible with major R genes. Long-read, cost-effective sequencing technologies such as nanopore can be a game changer in the surveillance of major and emerging pathotypes. The resource and findings will be invaluable in the management of Xoo and in appropriate deployment of R genes in rice breeding programs.

The Dynamic Transcription Activator-Like Effector Family of Xanthomonas.

Transcription activator-like effectors (TALEs) are bacterial proteins that are injected into the eukaryotic nucleus to act as transcriptional factors and function as key virulence factors of the phytopathogen Xanthomonas. TALEs are translocated into plant host cells via the type III secretion system and induce the expression of host susceptibility (S) genes to facilitate disease. The unique modular DNA binding domains of TALEs comprise an array of nearly identical direct repeats that enable binding to DNA targets based on the recognition of a single nucleotide target per repeat. The very nature of TALE structure and function permits the proliferation of TALE genes and evolutionary adaptations in the host to counter TALE function, making the TALE-host interaction the most dynamic story in effector biology. The TALE genes appear to be a relatively young effector gene family, with a presence in all virulent members of some species and absent in others. Genome sequencing has revealed many TALE genes throughout the xanthomonads, and relatively few have been associated with a cognate S gene. Several species, including Xanthomonas oryzae pv. oryzae and X. citri pv. citri, have near absolute requirement for TALE gene function, while the genes appear to be just now entering the disease interactions with new fitness contributions to the pathogens of tomato and pepper among others. Deciphering the simple and effective DNA binding mechanism also has led to the development of DNA manipulation tools in fields of gene editing and transgenic research. In the three decades since their discovery, TALE research remains at the forefront of the study of bacterial evolution, plant-pathogen interactions, and synthetic biology. We also discuss critical questions that remain to be addressed regarding TALEs.

Altering transcription factor binding reveals comprehensive transcriptional kinetics of a basic gene.

Transcription is a vital process activated by transcription factor (TF) binding. The active gene releases a burst of transcripts before turning inactive again. While the basic course of transcription is well understood, it is unclear how binding of a TF affects the frequency, duration and size of a transcriptional burst. We systematically varied the residence time and concentration of a synthetic TF and characterized the transcription of a synthetic reporter gene by combining single molecule imaging, single molecule RNA-FISH, live transcript visualisation and analysis with a novel algorithm, Burst Inference from mRNA Distributions (BIRD). For this well-defined system, we found that TF binding solely affected burst frequency and variations in TF residence time had a stronger influence than variations in concentration. This enabled us to device a model of gene transcription, in which TF binding triggers multiple successive steps before the gene transits to the active state and actual mRNA synthesis is decoupled from TF presence. We quantified all transition times of the TF and the gene, including the TF search time and the delay between TF binding and the onset of transcription. Our quantitative measurements and analysis revealed detailed kinetic insight, which may serve as basis for a bottom-up understanding of gene regulation.

A TAL effector-like protein of an endofungal bacterium increases the stress tolerance and alters the transcriptome of the host.

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. A btl19-13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in the M. rhizoxinica-R. microsporus symbiosis.

TAL effectors and the predicted host targets of pomegranate bacterial blight pathogen Xanthomonas citri pv. punicae.

The molecular mechanism of pomegranate susceptibility to bacterial blight, a serious threat to pomegranate production in India, is largely unknown. In the current study, we have used PacBio and Illumina sequencing of Xanthomonas citri pv. punicae (Xcp) strain 119 genome to identify tal genes and RNA-Seq analysis to identify putative host targets in the susceptible pomegranate variety Bhagwa challenged with Xcp119. Xcp119 genome encodes seven transcription activator-like effectors (TALEs), three of which are harbored by a plasmid. RVD-based phylogenetic analysis of TALEs of Xanthomonas citri pathovars indicate the TALEs of Xcp as evolutionarily and functionally close to Xanthomonas citri pv. malvacearum and Xanthomonas citri pv. glycines. Comparative RNA-Seq of Xcp and mock-inoculated leaf tissues revealed Xcp-induced pomegranate transcription modulation. The prediction of TALE binding elements (EBEs) in the promoters of up-regulated genes identified a set of TALE-targeted candidate genes in pomegranate-Xcp interaction. The predicted candidate susceptibility genes include two oxoglutarate-dependent dioxygenase gene, ethylene-responsive transcription factor and flavanone 3-hydroxylase-like gene, and the further characterization of these would enable blight resistance engineering in pomegranate.

Tal2b targets and activates the expression of OsF3H03g to hijack OsUGT74H4 and synergistically interfere with rice immunity.

Transcription activator-like (TAL) effectors are major virulence factors secreted by the type III secretion systems of Xanthomonas oryzae pv. oryzicola (Xoc) and X. oryzae pv. oryzae (Xoo), causing bacterial leaf streak and bacterial blight, respectively, in rice. However, the knowledge of Xoc TAL effector function in promoting bacterial virulence remains limited. Here, we isolated the highly virulent Xoc strain HGA4 from the outbreak region of Huanggang (Hubei, China), which contains four TAL effectors not found in the Chinese model strain RS105. Among these, Tal2b was selected for introduction into RS105, which resulted in a longer lesion length than that in the control. Tal2b directly binds to the promoter region of the gene and activates the expression of OsF3H03g , which encodes 2-oxoglutarate-dependent dioxygenase in rice. OsF3H03g negatively regulates salicylic acid (SA)-related defense by directly reducing SA, and it plays a positive role in susceptibility to both Xoc and Xoo in rice. OsF3H03g interacts with a uridine diphosphate-glycosyltransferase protein (OsUGT74H4), which positively regulates bacterial leaf streak susceptibility and may inactivate SA via glycosylation modification.