Transcription Activator-like Effector Research Articles

Exploration of the Interactions Between Xanthomonas citri subsp. citri-Agrobacterium-Citrus to Improve Agrobacterium-Mediated Transient Expression in Plants.

Agrobacterium-mediated transient expression (AMTE) is an important tool in plant genetics study and biotechnology. AMTE remains problematic in citrus and many plant species. Previous study has shown that pretreatment of citrus leaves with Xanthomonas citri subsp. citri (Xcc), which causes citrus canker, significantly improves the AMTE efficacy. Here we have shown that Xcc promotes AMTE mainly through triggering cell division and upregulating plant cell wall degrading enzymes. We demonstrate that Xcc improves AMTE via PthA, a transcription activator-like effector (TALE) known to trigger cell division in citrus, and mutation of pthA4 abolished the promoting effect of Xcc. Mutation of the effector (PthA4) binding element (EBE) in the promoter region and coding region of CsLOB1, which is known to be directly activated by PthA4, significantly reduced Xcc promotion of AMTE. Mutation of PthA4 significantly reduced expression of cell division related genes (CDKA, CDKB1-2 and CDKB2-2) compared to wild type Xcc and the complemented strain. Cell division inhibitor mimosine but not colchicine also significantly decreased Xcc promoting of AMTE. In addition, PthA4 is known to upregulate plant growth hormones auxin (IAA), gibberellin and cytokinin, as well as cell wall degrading enzymes (e.g., cellulase). Exogenous application of IAA, cytokinin, and cellulase but not gibberellin significantly improved AMTE in leaves of sweet orange, pummelo, Meiwa kumquat, lucky bamboo and rose mallow. Our study provides a mechanistic understanding regarding how Xcc promotes AMTE and develops practical measures to improve AMTE via pretreatment with plant hormones (i.e., auxin and cytokinin) and cellulase.

Excision of HIV-1 Provirus in Human Primary Cells with Nanocapsuled TALEN Proteins.

Despite the tremendous success of combination antiretroviral therapy (ART) to treat human immunodeficiency virus (HIV) infection, the durability and persistence of latent reservoirs of HIV-infected cells in HIV-infected patients remain obstacles to achieving HIV cure. While technically challenging, the most direct means to eradicate latent reservoirs is to destroy the HIV provirus, thus ensuring that HIV virions are not produced while preserving resident cells. Transcription activator-like effector nucleases (TALEN)─a genome editing method with high DNA targeting efficiency─have been investigated as a potential gene therapy by disrupting the HIV-1 coreceptor CCR5 genes in HIV target cells or HIV proviral DNA in infected cells. However, the transduction and editing efficiencies are low in primary cells and vary by cell type. Using a nanotechnology platform, which we term nanocapsules, the TALEN protein can be effectively delivered into primary cells and escape from endosome/lysosome sequestration. We report that TALEN nanocapsules can effectively mutagenize the HIV-1 proviral DNA integrated into two primary HIV-1 reservoir cells─T cells and macrophages, such that replication and/or reactivation from latency is aborted. We envision that this study provides a useful platform to deliver a wide range of DNA-modifying enzymes for effective HIV therapy.

Precise modelling of mitochondrial diseases using optimized mitoBEs.

The development of animal models is crucial for studying and treating mitochondrial diseases. Here we optimized adenine and cytosine deaminases to reduce off-target effects on the transcriptome and the mitochondrial genome, improving the accuracy and efficiency of our newly developed mitochondrial base editors (mitoBEs)1. Using these upgraded mitoBEs (version 2 (v2)), we targeted 70 mouse mitochondrial DNA mutations analogous to human pathogenic variants2, establishing a foundation for mitochondrial disease mouse models. Circular RNA-encoded mitoBEs v2 achieved up to 82% editing efficiency in mice without detectable off-target effects in the nuclear genome. The edited mitochondrial DNA persisted across various tissues and was maternally inherited, resulting in F1 generation mice with mutation loads as high as 100% and some mice exhibiting editing only at the target site. By optimizing the transcription activator-like effector (TALE)binding site, we developed a single-base-editing mouse model for the mt-Nd5 A12784G mutation. Phenotypic evaluations led to the creation of mouse models for the mt-Atp6 T8591C and mt-Nd5 A12784G mutations, exhibiting phenotypes corresponding to the reduced heart rate seen in Leigh syndrome and the vision loss characteristic of Leber's hereditary optic neuropathy, respectively. Moreover, the mt-Atp6 T8591C mutation proved to be more deleterious than mt-Nd5 A12784G, affecting embryonic development and rapidly diminishing through successive generations. These upgraded mitoBEs offer a highly efficient and precise strategy for constructing mitochondrial disease models, laying a foundation for further research in this field.

New, Complete Circularized Genomes of Xanthomonas citri pv. mangiferaeindicae Produced from Short- and Long-Read Co-Assembly Shed Light on Strains that Emerged a Decade Ago on Mango and Cashew in Burkina Faso.

We report high-quality genomes of three strains of Xanthomonas citri pv. mangiferaeindicae, the causal agent of mango bacterial canker disease, including the pathotype strain of this pathovar and two strains from Burkina Faso that emerged a decade ago. These strains hosted two to three plasmids of sizes ranging from 19 to 86 kb. Genome mining revealed the presence of several secretion systems and effectors involved in the virulence of xanthomonads with (i) a type I secretion system of the hlyDB group; (ii) xps and xcs type II secretion systems; (iii) a type III secretion system with several type III effectors, including transcription activator-like effectors; (iv) several type IV secretion systems associated with plasmid or integrative conjugative elements mobility; (v) three type V secretion system subclasses (Va, Vb, and Vc); and (vi) a single i3* type VI secretion system. The two strains isolated in Burkina Faso from mango (Mangifera indica) and cashew (Anacardium occidentale) differed by only 14 single-nucleotide polymorphisms and shared identical secretion systems and type III effector repertoires. Several transcription activator-like effectors were identified in each strain, some of which may target plant genes previously found implicated in disease development in other xanthomonad-associated pathosystems. These results support the emergence in Burkina Faso a decade ago of very closely related strains that became epidemic on mango and cashew (i.e., two distinct host genera of a same plant family). These new genomic resources will contribute to better understanding the biology and evolution of this agriculturally major crop pathogen.

TALome and phenotypic analysis of Pakistani Xanthomonas oryzae pv. oryzae population revealed novel virulent TALEs contributing to bacterial blight of rice

Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo), is an important disease in rice-growing countries, including Pakistan, where it was first reported in the mid-1970s. Transcription activator-like effectors (TALEs) play vital roles in many plant diseases caused by Xanthomonas spp.; however, Pakistani Xoo TALome diversity and their contribution to pathogenicity is largely unknown. In this study, 101 Xoo strains were screened using specific PCR primers. The genomic DNA from these strains underwent BamHI digestion and hybridized with the internal SphI fragment of PthXo1. Southern blot analysis revealed 16 to 20 putative tale fragments among the tested strains. These strains were further classified into 11 genotypes based on the number and size of the hybridizing bands. Genotypes 1, 2, 3, and 4 represented 24, 2, 51, and 17 strains, respectively. Pathogenicity assays on near-isogenic lines (NILs) containing different resistance (R) genes exhibited that CBB23 was incompatible with all tested Pakistani-Xoo genotypes, whereas IRBB5 and IRBB4 showed resistance against specific genotypes. In contrast, paddy trails on NILs containing single, double, and triple mutants of OsSWEET11a, OsSWEET13, and OsSWEET14 in the effector binding elements (EBEs) of cv. Kitaake revealed that KP-22 and LD-5 harbor novel virulent TAL effector/s. Interestingly, the expression analysis of six clade-III OsSWEET genes suggests that novel TALE/s targeting unidentified susceptibility gene/s. Altogether, this study highlights gene-for-gene relationships between tested rice lines and Pakistani-Xoo strains. This is the first report providing the diversity of TALEs and their relationship to R and S (susceptibility) genes. Further identification of novel virulent TALE/s and their cognate target/s is warranted to precisely elucidate their role in BB.

Activation of three targets by a TAL effector confers susceptibility to bacterial blight of cotton

Bacterial transcription activator-like effectors (TALEs) promote pathogenicity by activating host susceptibility (S) genes. To understand the pathogenicity and host adaptation of Xanthomonas citri pv. malvacearum (Xcm), we assemble the genome and the TALE repertoire of three recent Xcm Texas isolates. A newly evolved TALE, Tal7b, activates GhSWEET14a and GhSWEET14b, different from GhSWEET10 targeted by a TALE in an early Xcm isolate. Activation of GhSWEET14a and GhSWEET14b results in water-soaked lesions. Transcriptome profiling coupled with TALE-binding element prediction identify a pectin lyase gene as an additional Tal7b target, quantitatively contributing to Xcm virulence alongside GhSWEET14a/b. CRISPR-Cas9 gene editing supports the function of GhSWEETs in cotton bacterial blight and the promise of disrupting the TALE-binding site in S genes for disease management. Collectively, our findings elucidate the rapid evolution of TALEs in Xanthomonas field isolates and highlight the virulence mechanism wherein TALEs induce multiple S genes to promote pathogenicity.

Two TAL Effectors of Xanthomonas citri pv. malvacearum Induce Water Soaking by Activating GhSWEET14 Genes in Cotton.

Bacterial blight of cotton (BBC) caused by Xanthomonas citri pv. malvacearum (Xcm) is an important and destructive disease affecting cotton plants. Transcription activator-like effectors (TALEs) released by the pathogen regulate cotton resistance to the susceptibility. In this study, we sequenced the whole genome of Xcm Xss-V2-18 and identified eight tal genes: seven on the plasmids and one on the chromosome. Deletion and complementation experiments of Xss-V2-18 tal genes demonstrated that Tal1b is required for full virulence on cotton. Transcriptome profiling coupled with TALE-binding element prediction revealed that Tal1b targets GhSWEET14A04/D04 and GhSWEET14D02 simultaneously. Expression analysis confirmed the independent inducibility of GhSWEET14A04/D04 and GhSWEET14D02 by Tal1b, whereas GhSWEET14A04/D04 is additionally targeted by Tal1. Moreover, β-glucuronidase and Xa10-mediated hypersensitive response assays indicated that the effector-binding element (EBEs) are required for the direct and specific activation of the candidate targets by Tal1 and Ta1b. These insights enhance our understanding of the underlying mechanisms of bacterial blight in cotton and might lead to improved resistance through EBEs disruption or a TALE-trap strategy.

Breast Cancer Subtypes and Current Promising Genetic Engineering Tools for Breast Cancer Treatment - An Overview

Abstract: Breast cancer poses a significant global health challenge, and if current trends persist, the burden of breast cancer is projected to escalate, yielding over 3 million new cases and 1 million fatalities annually by the year 2040. Breast cancer is a highly heterogeneous disease, presenting a spectrum of subtypes, each characterized by unique clinical behaviors and responses to treatments. Understanding these breast cancer subtypes is of paramount importance in the fields of oncology and personalized medicine. In addition to conventional breast cancer treatments, such as surgery, chemotherapy, radiotherapy, hormonal therapy, and immunotherapy, recent scientific advancements have introduced a range of genetic engineering tools with noteworthy potential. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR), and small interfering RNA (siRNA) have emerged as promising components of breast cancer treatment. These tools offer encouraging applications due to their precision in targeting and manipulating genes. This review presents a comprehensive exploration of the various subtypes of breast cancer, along with an examination of the current promising genetic engineering tools in treating breast cancer. It sheds light on their roles in the evolving landscape of breast cancer treatment.

Exploring Ethical Frontiers: A Content Analysis of Perceptions Surrounding Genetic Editing in Mental Health Treatments Over Time

Genetic editing has become a pivotal biotechnology tool in healthcare, enabling precise manipulation of DNA through insertions, deletions, modifications, or replacements. Its applications have expanded from treating monogenic diseases to addressing polygenic conditions, including potential mental health therapies. This study examines public sentiment and ethical considerations surrounding genetic editing systems like CRISPR- Cas9, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). Analyzing a decade of medical literature, the research highlights a growing public acceptance of these technologies despite ethical concerns, such as off-target effects and unintended consequences. Positive sentiment trends indicate that social acceptance may support the integration of genetic editing in mental health treatments, driving advancements in healthcare. Addressing risks, including long-term impacts and ethical dilemmas, remains critical for responsible implementation. As public attitudes continue to improve, genetic editing may become a widely accepted therapeutic approach, fostering innovation in healthcare. Future research should focus on mitigating risks and enhancing the safety and efficacy of genetic editing to ensure its sustainable development and adoption.

Sequence-Specific Minor Groove Binders in Labeling and Single-Molecule Analysis of DNA.

The ability to address specific sequences within DNA is of tremendous interest in biotechnology and biomedicine. Various technologies have been established over the past few decades, such as nicking enzymes and methyltransferase-directed sequence-specific labeling, transcription activator-like effector nucleases (TALENs), the CRISPR-Cas9 system, and polyamides of heterocycles as sequence-specific DNA minor groove binders. Pyrrole-imidazole polyamides have been reported to recognize predetermined DNA sequences, and some successful attempts have demonstrated their potential in regulating gene expression. However, few studies on single-molecule labeling and analysis of DNA have been explored, particularly at single-targeting sites. In this study, we rationally designed and synthesized a set of functional minor groove binders, varying in structures, sequence information addressed, and methods of dye introduction. Their potential for sequence-specific labeling and single-molecule DNA analysis was evaluated through chromatographic and on-surface optical assays. First results indicated that, while they yielded excellent imaging output, the labeling specificity of the hairpin polyamides for single-molecule use was hindered by single-mismatch sites. To address this issue, and in an unprecedented approach, we devised a competitive binding strategy that utilizes ethidium bromide as a nonspecific binder to competitively block the mismatches, significantly enhancing the labeling specificity. These findings provide valuable insights into the use of hairpin polyamides as sequence-programmable and enzyme-free DNA labelers in the field of sequence-specific DNA recognition and modification.

Evolutionary and Epidemiological Insights from Historical and Modern Genomes of Xanthomonas oryzae pv. oryzicola, the Causal Agent of Bacterial Leaf Streak of Rice.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) of rice. This disease represents a major constraint for rice production, which is a crop feeding more than half of the world's population. Xoc was first described in 1918 in the Philippines and is prevalent in southeast Asia. Today, BLS is also omnipresent in both East- and West-Africa, where the disease was first reported in the early 1980s. The appearance of Xoc in Africa decades after its first report in Asia suggests that the disease could have been introduced from Asia to Africa. Strict conservation of five transcription activator-like (TAL) effectors in whole-genome sequences of 10 strains of Xoc including three from West-Africa and seven from Asia also support this hypothesis. East-Africa, especially Madagascar, where the disease was first described in 1985 is located at the interface between Asia and Africa, hence representing an interesting region to explore the link between strains from Asia and West-Africa. In this study, we did the following: (i) reconstructed the genome of a historical Xoc strain from a herbarium specimen of rice showing symptoms of BLS that was sampled in Madagascar in 1931, 50 years before the first description of the disease, and (ii) sequenced nine new modern strains, including five from Madagascar and East-Africa. The analysis of those new genomes along with previously published ones shed light within the evolutionary and epidemiological history of Xoc. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Complete genome of Xanthomonas citri pv. anacardii strain CCRMTAQ13 (causal agent of angular leaf spot in cashew) from Brazil using long-read Nanopore technology.

Here, we present a single contiguous genome sequence of Xanthomonas citri pv. anacardii (Xca; strain CCRMTAQ13), the causal agent of angular leaf spot in cashew, an important commodity crop in Brazil. Oxford Nanopore Sequencing Technology was used to assemble the genome of the Xca in a single contig of 5,086,757 bp with a 64.53% GC content. This genome sequence will provide a useful resource for studies on virulence mechanisms, including the identification of a single transcription activator-like effector gene of 3,359 bp.

Engineering cold resilience: implementing gene editing tools for plant cold stress tolerance.

This paper highlights the need for innovative approaches to enhance cold tolerance. It underscores how genome-editing tools can deepen our understanding of genes involved in cold stress. Cold stress is a significant abiotic factor in high-altitude regions, adversely affecting plant growth and limiting crop productivity. Plants have evolved various mechanisms in response to low temperatures that enable resistance at both physiological and molecular levels during chilling and freezing stress. Several cold-inducible genes have been isolated and characterized, with most playing key roles in providing tolerance against low-temperature stress. However, many plants fail to survive at low temperatures due to the absence of cold acclimatization mechanisms. Conventional breeding techniques, such as inter-specific or inter-genic hybridization, have had limited effectiveness in enhancing the cold resistance of essential crops. Thus, it is crucial to develop crops with improved adaptability, high yields and resistance to cold stress using advanced genomic approaches. The current availability of gene editing tools offers the opportunity to introduce targeted modifications in plant genomes efficiently, thereby developing cold-tolerant varieties. This review discusses advancements in gene editing tools, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)/Cas12a(Cpf1), prime editing (PE) and retron library recombineering (RLR). We focus specifically on the CRISPR/Cas system, which has garnered significant attention in recent years as a groundbreaking tool for genome editing across various species. These techniques will enhance our understanding of molecular interactions under low-temperature stress response and highlight the progress of genome editing in designing future climate-resilient crops.

Lipid nanoparticle delivery of TALEN mRNA targeting LPA causes gene disruption and plasma lipoprotein(a) reduction in transgenic mice

Lipoprotein(a), or Lp(a), is encoded by the LPA gene and is a causal genetic risk factor for cardiovascular disease. Individuals with high Lp(a) are at risk for cardiovascular morbidity and are refractory to standard lipid-lowering agents. Lp(a)-lowering therapies currently in clinical development require repetitive dosing, while a gene editing approach presents an opportunity for a single-dose treatment. In this study, mRNAs encoding Transcription Activator-Like Effector Nucleases (TALENs) were designed to target human LPA for gene disruption and permanent Lp(a) reduction. TALEN mRNAs were screened in vitro and found to cause on-target gene editing and target protein reduction with minimal off-target editing. TALEN mRNAs were then encapsulated with LUNAR®, a proprietary lipid nanoparticle (LNP), and administered to transgenic mice that expressed a human LPA transgene. A single dose of TALEN mRNA-LNPs reduced plasma Lp(a) levels in mice by over 80%, which was sustained for at least 5 weeks. Moreover, both standard and long-read next generation sequencing confirmed the presence of gene-inactivating deletions at LPA transgene loci. Overall, this study serves as a proof-of-concept for using TALEN-mediated gene editing to disrupt LPA in vivo, paving the way for the development of a feasible gene editing therapy for patients with high Lp(a).

A strategy for improving the mechanical properties of silk fibers through the combination of genetic manipulation and zinc ion crosslinking

Silk fiber is generally considered an excellent biological material due to its good biocompatibility, morphological plasticity and biodegradability. Previously, the construction of silkworm silk gland bioreactors based on the piggyBac transposon has been optimized. However, the inserted exogenous genes have problems such as position uncertainty, and expression is not strictly controlled. Here, we applied transcription activator-like effector nuclease (TALEN)-mediated homology-directed repair (HDR) to precisely insert histidine-rich cuticular protein (CP) into silkworm Sericin1 (Ser1) gene. The Ser1-CP fusion protein was successfully secreted into cocoon shell. Subsequently, based on the metal coordination ability of the histidine imidazole group, we crosslinked cocoon with metal ions in vitro. In this strategy, the mechanical properties of the fused silk fibers with crosslinked Zn2+ improved, and the maximum breaking stress of the crosslinked Zn2+-fused silk fibers was 23.5 % greater than that of the wild-type fibers. Analysis of the secondary structure of the silk protein showed that the fused silk fibers crosslinked with Zn2+ had more β-sheet structures. This study pioneered a method of improving the mechanical properties of silk fibers by crosslinking metal ions with fused exogenous proteins and expanded the application value of silk gland bioreactors in the development of novel biomaterials.

The Arabidopsis amino acid transporter UmamiT20 confers Botrytis cinerea susceptibility.

Pathogens infect plants to gain access to their nutrient resources, enabling the pathogens to cause disease and reproduce efficiently. Here we find that an amino acid transporter constitutes a susceptibility factor for the fungal pathogen B. cinerea .

Unconstrained Precision Mitochondrial Genome Editing with αDdCBEs.

DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C•G-to-T•A conversions in mitochondrial DNA (mtDNA). DdCBEs work in pairs, with each arm composed of a transcription activator-like effector (TALE), a split double-stranded DNA deaminase half, and a uracil glycosylase inhibitor. This pioneering technology has helped improve our understanding of cellular processes involving mtDNA and has paved the way for the development of models and therapies for genetic disorders caused by pathogenic mtDNA variants. Nonetheless, given the intrinsic properties of TALE proteins, several target sites in human mtDNA are predicted to remain out of reach to DdCBEs and other TALE-based technologies. Specifically, due to the conventional requirement for a thymine immediately upstream of the TALE target sequences (i.e., the 5'-T constraint), over 150 loci in the human mitochondrial genome are presumed to be inaccessible to DdCBEs. Previous attempts at circumventing this requirement, either by developing monomeric DdCBEs or utilizing DNA-binding domains alternative to TALEs, have resulted in suboptimal specificity profiles with reduced therapeutic potential. Here, aiming to challenge and elucidate the relevance of the 5'-T constraint in the context of DdCBE-mediated mtDNA editing, and to expand the range of motifs that are editable by this technology, we generated DdCBEs containing TALE proteins engineered to recognize all 5' bases. These modified DdCBEs are herein referred to as αDdCBEs. Notably, 5'-T-noncompliant canonical DdCBEs efficiently edited mtDNA at diverse loci. However, they were frequently outperformed by αDdCBEs, which exhibited significant improvements in activity and specificity, regardless of the most 5' bases of their TALE binding sites. Furthermore, we showed that αDdCBEs are compatible with the enhanced DddAtox variants DddA6 and DddA11, and we validated TALE shifting with αDdCBEs as an effective approach to optimize base editing outcomes. Overall, αDdCBEs enable efficient, specific, and unconstrained mitochondrial base editing.

Precision Genome Editing Techniques in Gene Therapy: Current State and Future Prospects.

Precision genome editing is a rapidly evolving field in gene therapy, allowing for the precise modification of genetic material. The CRISPR and Cas systems, particularly the CRISPRCas9 system, have revolutionized genetic research and therapeutic development by enabling precise changes like single-nucleotide substitutions, insertions, and deletions. This technology has the potential to correct disease-causing mutations at their source, allowing for the treatment of various genetic diseases. Programmable nucleases like CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs) can be used to restore normal gene function, paving the way for novel therapeutic interventions. However, challenges, such as off-target effects, unintended modifications, and ethical concerns surrounding germline editing, require careful consideration and mitigation strategies. Researchers are exploring innovative solutions, such as enhanced nucleases, refined delivery methods, and improved bioinformatics tools for predicting and minimizing off-target effects. The prospects of precision genome editing in gene therapy are promising, with continued research and innovation expected to refine existing techniques and uncover new therapeutic applications.

Development and Validation of an SNP Marker for Identifying Xanthomonas oryzae pv. oryzae Thai Isolates That Break xa5-Mediated Bacterial Blight Resistance in Rice.

Xanthomonas oryzae pv. oryzae (Xoo) is a pathogenic bacterium responsible for bacterial blight (BB) disease in rice, primarily mediated by the interaction between the plant and pathogen. The virulence mechanism involves the activation of the Sugars Will Eventually be Exported Transporter (SWEET) gene family in rice by transcription activator-like effectors derived from Xoo. The BB resistance gene xa5 has been identified as one of the most effective genes against Thai Xoo isolates, but xa5-mediated resistance-breaking Xoo strains have emerged. This study aimed to develop a single nucleotide polymorphism (SNP) marker for precise identification of xa5-mediated resistance-breaking Xoo. Comparative genomics of Thai Xoo isolates Xoo16PK001 and Xoo16PK002, which were incompatible and compatible with rice variety IRBB5 carrying xa5, respectively, identified eight SNP positions for the development of an SNP marker. The SNP marker XooE6 yields a specific 1,143 bp PCR product unique to Xoo16PK002. Screening 61 Thai isolates using XooE6 identified two positives: Xoo20PL010 and Xoo20UT002. Inoculation tests on rice varieties IRBB5 and IRBB13 demonstrated compatibility with IRBB5 and incompatibility with IRBB13, which bears Xa5 and xa13. Xoo16PK001 (XooE6-negative) showed different virulence. Inoculation on IRBB21 harboring Xa5, Xa13, and Xa21 resulted in partial resistance to both XooE6-positive and -negative strains. XooE6-positive strains up-regulated SWEET11 and suppressed SWEET14 in IRBB5, while Xoo16PK001 slightly induced SWEET11 but activated SWEET14 in IRBB13. This highlights the potential of XooE6 to identify xa5-mediated resistance-breaking Xoo strains and elucidate their pathogenic mechanisms through the upregulation of SWEET11.

Editing of the MeSWEET10a promoter yields bacterial blight resistance in cassava cultivar SC8.

Cassava starch is a widely used raw material for industrial production and food source for people. However, cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) results in severe yield losses and is the most destructive bacterial disease in all worldwide cassava-growing regions. Xam11 is a highly pathogenic subspecies from China that infects the Chinese local cassava South China No. 8 (SC8) cultivar with marked symptoms. This study showed that the transcription activator-like effector TALE20Xam11 of Xam11 strain regulates the expression of disease-susceptibility gene MeSWEET10a by binding to the EBETALE20 region of the MeSWEET10a promoter in cassava cultivar SC8. CRISPR/Cas9-generated mutations of the EBETALE20 region resulted in a significant reduction in MeSWEET10a expression after infection by Xam11, correlating with reduced disease symptoms, smaller lesion sizes and decreased bacterial proliferation compared with the wild type. Importantly, the edited plants maintained normal growth, development and yield characteristics under greenhouse conditions. The results lay a research foundation for breeding resistant cassava cultivar SC8 to bacterial blight.