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Endofungal Mycetohabitans (formerly Burkholderia ) spp. rely on a type III secretion system to deliver mostly unidentified effector proteins when colonizing their host fungus, Rhizopus microsporus . The one known secreted effector family from Mycetohabitans consists of homologues of transcription activator-like (TAL) effectors, which are used by plant pathogenic Xanthomonas and Ralstonia spp. to activate host genes that promote disease. These ' Burkholderia TAL-like (Btl)' proteins bind corresponding specific DNA sequences in a predictable manner, but their genomic target(s) and impact on transcription in the fungus are unknown. Recent phenotyping of Btl mutants of two Mycetohabitans strains revealed that the single Btl in one Mycetohabitans endofungorum strain enhances fungal membrane stress tolerance, while others in a Mycetohabitans rhizoxinica strain promote bacterial colonization of the fungus. The phenotypic diversity underscores the need to assess the sequence diversity and, given that sequence diversity translates to DNA targeting specificity, the functional diversity of Btl proteins. Using a dual approach to maximize capture of Btl protein sequences for our analysis, we sequenced and assembled nine Mycetohabitans spp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal-bacterial metagenomes. We show that btl genes are present across diverse Mycetohabitans strains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested that Mycetohabitans might contain more species than previously recognized. Within our data set, Btl proteins were more conserved across M. rhizoxinica strains than across M. endofungorum , but there was also evidence of greater overall strain diversity within the latter clade. Overall, the results suggest that Btl proteins contribute to bacterial-fungal symbioses in myriad ways.

Pathovars of Xanthomonas campestris cause distinct diseases on different brassicaceous hosts. The genomic relationships among pathovars as well as the genetic determinants of host range and tissue specificity remain poorly understood despite decades of research. Here, leveraging advances in multiplexed long-read technology, we fully sequenced the genomes of a collection of X. campestris strains isolated from cruciferous crops and weeds in New York and California as well as strains from global collections, to investigate pathovar relationships and candidate genes for host- and tissue-specificity. Pathogenicity assays and genomic comparisons across this collection and publicly available X. campestris genomes revealed a correlation between pathovar and genomic relatedness and provide support for X. campestris pv. barbareae , the validity of which had been questioned. Linking strain host range with type III effector repertoires identified AvrAC (also 'XopAC') as a candidate host-range determinant, preventing infection of Matthiola incana , and this was confirmed experimentally. Furthermore, the presence of a copy of the cellobiosidase gene cbsA with coding sequence for a signal peptide was found to correlate with the ability to infect vascular tissues, in agreement with a previous study of diverse Xanthomonas species; however, heterologous expression in strains lacking the gene gave mixed results, indicating that factors in addition to cbsA influence tissue specificity of X. campestris pathovars. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Xanthomonas campestris infections of nonnative, invasive garlic mustard populations have been recently reported in the eastern United States. Here, we report the genome sequence of the pathogenic X. campestris strain FDWSRU 18048. The genome is 4,978,509 bp and closely related to the genome of X. campestris pv. incanae strain CFBP2527.

Background: Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, a devastating disease of rice. Among the type-3 effectors secreted by Xoo to support pathogen virulence, the Transcription Activator-Like Effector (TALE) family plays a critical role. Some TALEs are major virulence factors that activate susceptibility (S) genes, overexpression of which contributes to disease development. Host incompatibility can result from TALE-induced expression of so-called executor (E) genes leading to a strong and rapid resistance response that blocks disease development. In that context, the TALE functions as an avirulence (Avr) factor. To date no such avirulence factors have been identified in African strains of Xoo., Results: With respect to the importance of TALEs in the Rice-Xoo pathosystem, we aimed at identifying those that may act as Avr factor within African Xoo. We screened 86 rice accessions, and identified 12 that were resistant to two African strains while being susceptible to a well-studied Asian strain. In a gain of function approach based on the introduction of each of the nine tal genes of the avirulent African strain MAI1 into the virulent Asian strain PXO99 A , four were found to trigger resistance on specific rice accessions. Loss-of-function mutational analysis further demonstrated the avr activity of two of them, talD and talI, on the rice varieties IR64 and CT13432 respectively. Further analysis of TalI demonstrated the requirement of its activation domain for triggering resistance in CT13432. Resistance in 9 of the 12 rice accessions that were resistant against African Xoo specifically, including CT13432, could be suppressed or largely suppressed by trans-expression of the truncTALE tal2h, similarly to resistance conferred by the Xa1 gene which recognizes TALEs generally independently of their activation domain., Conclusion: We identified and characterized TalD and TalI as two African Xoo TALEs with avirulence activity on IR64 and CT13432 respectively. Resistance of CT13432 against African Xoo results from the combination of two mechanisms, one relying on the TalI-mediated induction of an unknown executor gene and the other on an Xa1-like gene or allele., (© 2022. The Author(s).)

Prime editing is an adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase fusion and a guide RNA amended with template and primer binding site sequences to achieve RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of prime editing in plants, a variety of edits in rice and wheat were described, including insertions up to 15 bp. Several studies in rice quickly followed, but none reported a larger insertion. Here, we report easy-to-use vectors for prime editing in dicots as well as monocots, their validation in Nicotiana benthamiana , rice, and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Kaya, Zhang, Rai, Willmann, Carpenter, Read, Martin, Fei, Leach, Martin and Bogdanove.)

The giant sequoia ( Sequoiadendron giganteum ) of California are massive, long-lived trees that grow along the U.S. Sierra Nevada mountains. Genomic data are limited in giant sequoia and producing a reference genome sequence has been an important goal to allow marker development for restoration and management. Using deep-coverage Illumina and Oxford Nanopore sequencing, combined with Dovetail chromosome conformation capture libraries, the genome was assembled into eleven chromosome-scale scaffolds containing 8.125 Gbp of sequence. Iso-Seq transcripts, assembled from three distinct tissues, was used as evidence to annotate a total of 41,632 protein-coding genes. The genome was found to contain, distributed unevenly across all 11 chromosomes and in 63 orthogroups, over 900 complete or partial predicted NLR genes, of which 375 are supported by annotation derived from protein evidence and gene modeling. This giant sequoia reference genome sequence represents the first genome sequenced in the Cupressaceae family, and lays a foundation for using genomic tools to aid in giant sequoia conservation and management., (Copyright © 2020 Scott et al.)

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae ( Xoo ) constrains production in major rice growing countries of Asia. Xoo injects transcription activator-like effectors (TALEs) that bind to and activate host "susceptibility" ( S ) genes that are important for disease. The bacterial blight resistance gene xa 5, which reduces TALE activity generally, has been widely deployed. However, strains defeating xa 5 have been reported in India and recently also in Thailand. We completely sequenced and compared the genomes of one such strain from each country and examined the encoded TALEs. The two genomes are nearly identical, including the TALE genes, and belong to a previously identified, highly clonal lineage. Each strain harbors a TALE known to activate the major S gene SWEET11 strongly enough to be effective even when diminished by xa 5. The findings suggest international migration of the xa5 -compatible pathotype and highlight the utility of whole genome sequencing and TALE analysis for understanding and responding to breakdown of resistance., (Copyright © 2020 Carpenter, Mishra, Ghoshal, Dash, Wang, Midha, Laha, Lore, Kositratana, Singh, Singh, Patil, Oliva, Patarapuwadol, Bogdanove and Rai.)

The Xo1 locus in the heirloom rice variety Carolina Gold Select confers resistance to bacterial leaf streak and bacterial blight, caused by Xanthomonas oryzae pv. oryzicola and X. oryzae pv. oryzae , respectively. Resistance is triggered by pathogen-delivered transcription activator-like effectors (TALEs) independent of their ability to activate transcription and is suppressed by truncated variants called truncTALEs, common among Asian strains. By transformation of the susceptible variety Nipponbare, we show that one of 14 nucleotide-binding, leucine-rich repeat (NLR) protein genes at the locus, with a zinc finger BED domain, is the Xo1 gene. Analyses of published transcriptomes revealed that the Xo1 -mediated response is more similar to those mediated by two other NLR resistance genes than it is to the response associated with TALE-specific transcriptional activation of the executor resistance gene Xa23 and that a truncTALE dampens or abolishes activation of defense-associated genes by Xo1 . In Nicotiana benthamiana leaves, fluorescently tagged Xo1 protein, like TALEs and truncTALEs, localized to the nucleus. And endogenous Xo1 specifically coimmunoprecipitated from rice leaves with a pathogen-delivered, epitope-tagged truncTALE. These observations suggest that suppression of Xo1-function by truncTALEs occurs through direct or indirect physical interaction. They further suggest that effector coimmunoprecipitation may be effective for identifying or characterizing other resistance genes.

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., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Crossovers (COs), that drive genetic exchange between homologous chromosomes, are strongly biased toward subtelomeric regions in plant species. Manipulating the rate and positions of COs to increase the genetic variation accessible to breeders is a longstanding goal. Use of genome editing reagents that induce double-stranded breaks (DSBs) or modify the epigenome at desired sites of recombination, and manipulation of CO factors, are increasingly applicable approaches for achieving this goal. These strategies for 'controlled recombination' have potential to reduce the time and expense associated with traditional breeding, reveal currently inaccessible genetic diversity, and increase control over the inheritance of preferred haplotypes. Considerable challenges to address include translating knowledge from models to crop species and determining the best stages of the breeding cycle at which to control recombination., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Long-read sequencing facilitates assembly of complex genomic regions. In plants, loci containing nucleotide-binding, leucine-rich repeat (NLR) disease resistance genes are an important example of such regions. NLR genes constitute one of the largest gene families in plants and are often clustered, evolving via duplication, contraction, and transposition. We recently mapped the Xo1 locus for resistance to bacterial blight and bacterial leaf streak, found in the American heirloom rice variety Carolina Gold Select, to a region that in the Nipponbare reference genome is NLR gene-rich. Here, toward identification of the Xo1 gene, we combined Nanopore and Illumina reads and generated a high-quality Carolina Gold Select genome assembly. We identified 529 complete or partial NLR genes and discovered, relative to Nipponbare, an expansion of NLR genes at the Xo1 locus. One of these has high sequence similarity to the cloned, functionally similar Xa1 gene. Both harbor an integrated zfBED domain, and the repeats within each protein are nearly perfect. Across diverse Oryzeae, we identified two sub-clades of NLR genes with these features, varying in the presence of the zfBED domain and the number of repeats. The Carolina Gold Select genome assembly also uncovered at the Xo1 locus a rice blast resistance gene and a gene encoding a polyphenol oxidase (PPO). PPO activity has been used as a marker for blast resistance at the locus in some varieties; however, the Carolina Gold Select sequence revealed a loss-of-function mutation in the PPO gene that breaks this association. Our results demonstrate that whole genome sequencing combining Nanopore and Illumina reads effectively resolves NLR gene loci. Our identification of an Xo1 candidate is an important step toward mechanistic characterization, including the role(s) of the zfBED domain. Finally, the Carolina Gold Select genome assembly will facilitate identification of other useful traits in this historically important variety., Competing Interests: The authors have declared that no competing interests exist.

To compare overall genome structure and transcription activator-like effector content, we completely sequenced Xanthomonas axonopodis pv. glycines strain 12-2, isolated in 1992 in Thailand, and strain EB08, isolated in 2008 in the United States (Iowa) using PacBio technology. We reassembled the genome sequence for a second US strain, 8ra, derived from a 1980 Iowa isolate, from existing PacBio reads. Despite geographic and temporal separation, the three genomes are highly syntenous, and their transcription activator-like effector repertoires are highly conserved., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The Pseudomonas syringae cysteine protease AvrPphB activates the Arabidopsis resistance protein RPS5 by cleaving a second host protein, PBS1. AvrPphB induces defense responses in other plant species, but the genes and mechanisms mediating AvrPphB recognition in those species have not been defined. Here, we show that AvrPphB induces defense responses in diverse barley cultivars. We also show that barley contains two PBS1 orthologs, that their products are cleaved by AvrPphB, and that the barley AvrPphB response maps to a single locus containing a nucleotide-binding leucine-rich repeat (NLR) gene, which we termed AvrPphB Response 1 ( Pbr1 ). Transient coexpression of PBR1 with wild-type AvrPphB but not with a protease inactive mutant triggered defense responses, indicating that PBR1 detects AvrPphB protease activity. Additionally, PBR1 coimmunoprecipitated with barley and Nicotiana benthamiana PBS1 proteins, suggesting mechanistic similarity to detection by RPS5. Lastly, we determined that wheat cultivars also recognize AvrPphB protease activity and contain two putative Pbr1 orthologs. Phylogenetic analyses showed, however, that Pbr1 is not orthologous to RPS5 . Our results indicate that the ability to recognize AvrPphB evolved convergently and imply that selection to guard PBS1-like proteins occurs across species. Also, these results suggest that PBS1-based decoys may be used to engineer protease effector recognition-based resistance in barley and wheat.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae ( Xoo ) injects transcription activator-like effectors (TALEs) that bind and activate host "susceptibility" ( S ) genes important for disease. Clade III SWEET genes are major S genes for bacterial blight. The resistance genes xa5 , which reduces TALE activity generally, and xa13 , a SWEET11 allele not recognized by the cognate TALE, have been effectively deployed. However, strains that defeat both resistance genes individually were recently reported in India and Thailand. To gain insight into the mechanism(s), we completely sequenced the genome of one such strain from each country and examined the encoded TALEs. Strikingly, the two strains are clones, sharing nearly identical TALE repertoires, including a TALE known to activate SWEET11 strongly enough to be effective even when diminished by xa5 . We next investigated SWEET gene induction by the Indian strain. The Indian strain induced no clade III SWEET in plants harboring xa13 , indicating a pathogen adaptation that relieves dependence on these genes for susceptibility. The findings open a door to mechanistic understanding of the role SWEET genes play in susceptibility and illustrate the importance of complete genome sequence-based monitoring of Xoo populations in developing varieties with effective disease resistance.

Xanthomonas transcription activator-like effectors (TALEs) are injected inside plant cells to promote host susceptibility by enhancing transcription of host susceptibility genes. TALE-encoding (tal) genes were thought to be absent from Brassicaceae-infecting Xanthomonas campestris (Xc) genomes based on four reference genomic sequences. We discovered tal genes in 26 of 49 Xc strains isolated worldwide and used a combination of single molecule real time (SMRT) and tal amplicon sequencing to yield a near-complete description of the TALEs found in Xc (Xc TALome). The 53 sequenced tal genes encode 21 distinct DNA binding domains that sort into seven major DNA binding specificities. In silico analysis of the Brassica rapa promoterome identified a repertoire of predicted TALE targets, five of which were experimentally validated using quantitative reverse transcription polymerase chain reaction. The Xc TALome shows multiple signs of DNA rearrangements that probably drove its evolution from two ancestral tal genes. We discovered that Tal12a and Tal15a of Xcc strain Xca5 contribute together in the development of disease symptoms on susceptible B. oleracea var. botrytis cv Clovis. This large and polymorphic repertoire of TALEs opens novel perspectives for elucidating TALE-mediated susceptibility of Brassicaceae to black rot disease and for understanding the molecular processes underlying TALE evolution., (© 2018 The Authors New Phytologist © 2018 New Phytologist Trust.)

Most Xanthomonas species translocate Transcription Activator-Like (TAL) effectors into plant cells where they function like plant transcription factors via a programmable DNA-binding domain. Characterized strains of rice pathogenic X. oryzae pv. oryzae harbor 9-16 different tal effector genes, but the function of only a few of them has been decoded. Using sequencing of entire genomes, we first performed comparative analyses of the complete repertoires of TAL effectors, herein referred to as TALomes, in three Xoo strains forming an African genetic lineage different from Asian Xoo. A phylogenetic analysis of the three TALomes combined with in silico predictions of TAL effector targets showed that African Xoo TALomes are highly conserved, genetically distant from Asian ones, and closely related to TAL effectors from the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Nine clusters of TAL effectors could be identified among the three TALomes, including three showing higher levels of variation in their repeat variable diresidues (RVDs). Detailed analyses of these groups revealed recombination events as a possible source of variation among TAL effector genes. Next, to address contribution to virulence, nine TAL effector genes from the Malian Xoo strain MAI1 and four allelic variants from the Burkinabe Xoo strain BAI3, thus representing most of the TAL effector diversity in African Xoo strains, were expressed in the TAL effector-deficient X. oryzae strain X11-5A for gain-of-function assays. Inoculation of the susceptible rice variety Azucena lead to the discovery of three TAL effectors promoting virulence, including two TAL effectors previously reported to target the susceptibility (S) gene OsSWEET14 and a novel major virulence contributor, TalB. RNA profiling experiments in rice and in silico prediction of EBEs were carried out to identify candidate targets of TalB, revealing OsTFX1, a bZIP transcription factor previously identified as a bacterial blight S gene, and OsERF#123, which encodes a subgroup IXc AP2/ERF transcription factor. Use of designer TAL effectors demonstrated that induction of either gene resulted in greater susceptibility to strain X11-5A. The induction of OsERF#123 by BAI3Δ1, a talB knockout derivative of BAI3, carrying these designer TAL effectors increased virulence of BAI3Δ1, validating OsERF#123 as a new, bacterial blight S gene., Competing Interests: The authors have declared that no competing interests exist.

Protein engineering is used to generate novel protein folds and assemblages, to impart new properties and functions onto existing proteins, and to enhance our understanding of principles that govern protein structure. While such approaches can be employed to reprogram protein-protein interactions, modifying protein-DNA interactions is more difficult. This may be related to the structural features of protein-DNA interfaces, which display more charged groups, directional hydrogen bonds, ordered solvent molecules and counterions than comparable protein interfaces. Nevertheless, progress has been made in the redesign of protein-DNA specificity, much of it driven by the development of engineered enzymes for genome modification. Here, we summarize the creation of novel DNA specificities for zinc finger proteins, meganucleases, TAL effectors, recombinases and restriction endonucleases. The ease of re-engineering each system is related both to the modularity of the protein and the extent to which the proteins have evolved to be capable of readily modifying their recognition specificities in response to natural selection. The development of engineered DNA binding proteins that display an ideal combination of activity, specificity, deliverability, and outcomes is not a fully solved problem, however each of the current platforms offers unique advantages, offset by behaviors and properties requiring further study and development.

Key Message: A major gene conferring resistance to bacterial leaf streak was mapped to chromosome 5R in triticale. Bacterial leaf streak (BLS), caused by Xanthomonas translucens pv. undulosa (Xtu), is an important disease of wheat and triticale around the world. Although resistance to BLS is limited in wheat, several triticale accessions have high levels of resistance. To characterize the genetic basis of this resistance, we developed triticale mapping populations using a resistant accession (Siskiyou) and two susceptible accessions (UC38 and Villax St. Jose). Bulked segregant analysis in an F 2 population derived from the cross of Siskiyou × UC38 led to the identification of a simple sequence repeat (SSR) marker (XSCM138) on chromosome 5R that co-segregated with the resistance gene. The cross of Siskiyou × Villax St. Jose was advanced into an F 2:5 recombinant inbred line population and evaluated for BLS reaction. Genetic linkage maps on this population were assembled with markers generated using genotyping-by-sequencing as well as several SSR markers previously identified on 5R. Quantitative trait locus (QTL) mapping revealed a single major QTL on chromosome 5R, underlined by the same SSR marker as in the Siskiyou × UC38 population. The F 1 hybrids of the two crosses were highly resistant to BLS, indicating that resistance is largely dominant. This work will facilitate introgression of this rye-derived BLS resistance gene into the wheat genome by molecular marker-mediated chromosome engineering.

Bacterial leaf streak caused by Xanthomonas translucens pv. undulosa (Xtu) is an important disease of wheat (Triticum aestivum) and barley (Hordeum vulgare) worldwide. Transcription activator-like effectors (TALEs) play determinative roles in many of the plant diseases caused by the different species and pathovars of Xanthomonas , but their role in this disease has not been characterized. ICMP11055 is a highly virulent Xtu strain from Iran. The aim of this study was to better understand genetic diversity of Xtu and to assess the role of TALEs in bacterial leaf streak of wheat by comparing the genome of this strain to the recently completely sequenced genome of a U.S. Xtu strain, and to several other draft X. translucens genomes, and by carrying out mutational analyses of the TALE (tal) genes the Iranian strain might harbor. The ICMP11055 genome, including its repeat-rich tal genes, was completely sequenced using single molecule, real-time technology (Pacific Biosciences). It consists of a single circular chromosome of 4,561,583 bp, containing 3,953 genes. Whole genome alignment with the genome of the United States Xtu strain XT4699 showed two major re-arrangements, nine genomic regions unique to ICMP11055, and one region unique to XT4699. ICMP110055 harbors 26 non-TALE type III effector genes and seven tal genes, compared to 25 and eight for XT4699. The tal genes occur singly or in pairs across five scattered loci. Four are identical to tal genes in XT4699. In addition to common repeat-variable diresidues (RVDs), the tal genes of ICMP11055, like those of XT4699, encode several RVDs rarely observed in Xanthomonas , including KG, NF, Y ∗ , YD, and YK. Insertion and deletion mutagenesis of ICMP11055 tal genes followed by genetic complementation analysis in wheat cv. Chinese Spring revealed that Tal2 and Tal4b of ICMP11055 each contribute individually to the extent of disease caused by this strain. A largely conserved ortholog of tal2 is present in XT4699, but for tal4b , only a gene with partial, fragmented RVD sequence similarity can be found. Our results lay the foundation for identification of important host genes activated by Xtu TALEs as targets for the development of disease resistant varieties.

Transcription activator-like effectors (TALEs) recognize their DNA targets via tandem repeats, each specifying a single nucleotide base in a one-to-one sequential arrangement. Due to this modularity and their ability to bind long DNA sequences with high specificity, TALEs have been used in many applications. Contributions of individual repeat-nucleotide associations to affinity and specificity have been characterized. Here, using in vitro binding assays, we examined the relationship between the number of repeats in a TALE and its affinity, for both target and non-target DNA. Each additional repeat provides extra binding energy for the target DNA, with the gain decaying exponentially such that binding energy saturates. Affinity for non-target DNA also increases non-linearly with the number of repeats, but with a slower decay of gain. The difference between the effect of length on affinity for target versus non-target DNA manifests in specificity increasing then diminishing with increasing TALE length, peaking between 15 and 19 repeats. Modeling across different hypothetical saturation levels and rates of gain decay, reflecting different repeat compositions, yielded a similar range of specificity optima. This range encompasses the mean and median length of native TALEs, suggesting that these proteins as a group have evolved for maximum specificity., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Transcription activator-like (TAL) effectors from Xanthomonas citri subsp. malvacearum (Xcm) are essential for bacterial blight of cotton (BBC). Here, by combining transcriptome profiling with TAL effector-binding element (EBE) prediction, we show that GhSWEET10, encoding a functional sucrose transporter, is induced by Avrb6, a TAL effector determining Xcm pathogenicity. Activation of GhSWEET10 by designer TAL effectors (dTALEs) restores virulence of Xcm avrb6 deletion strains, whereas silencing of GhSWEET10 compromises cotton susceptibility to infections. A BBC-resistant line carrying an unknown recessive b6 gene bears the same EBE as the susceptible line, but Avrb6-mediated induction of GhSWEET10 is reduced, suggesting a unique mechanism underlying b6-mediated resistance. We show via an extensive survey of GhSWEET transcriptional responsiveness to different Xcm field isolates that additional GhSWEETs may also be involved in BBC. These findings advance our understanding of the disease and resistance in cotton and may facilitate the development cotton with improved resistance to BBC.

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.)

Xanthomonas spp. reduce crop yields and quality worldwide. During infection of their plant hosts, many strains secrete transcription activator-like (TAL) effectors, which enter the host cell nucleus and activate specific corresponding host genes at effector binding elements (EBEs) in the promoter. TAL effectors may contribute to disease by activating the expression of susceptibility genes or trigger resistance associated with the hypersensitive reaction (HR) by activating an executor resistance (R) gene. The rice bacterial leaf streak pathogen X. oryzae pv. oryzicola (Xoc) is known to suppress host resistance, and no host R gene has been identified against it, despite considerable effort. To further investigate Xoc suppression of host resistance, we conducted a screen of effectors from BLS256 and identified Tal2a as an HR elicitor in rice when delivered heterologously by a strain of the closely related rice bacterial blight pathogen X. oryzae pv. oryzae (Xoo) or by the soybean pathogen X. axonopodis pv. glycines. The HR required the Tal2a activation domain, suggesting an executor R gene. Tal2a activity was differentially distributed among geographically diverse Xoc isolates, being largely conserved among Asian isolates. We identified four genes induced by Tal2a in next-generation RNA sequencing experiments and confirmed them using quantitative real-time reverse transcription-polymerase chain reaction (qPCR). However, neither individual nor collective activation of these genes by designer TAL effectors resulted in HR. A tal2a knockout mutant of BLS256 showed virulence comparable with the wild-type, but plasmid-based overexpression of tal2a at different levels in the wild-type reduced virulence in a directly corresponding way. Overall, the results reveal that host resistance suppression by Xoc plays a critical role in pathogenesis. Further, the dose-dependent avirulence activity of Tal2a and the apparent lack of a single canonical target that accounts for HR point to a novel, activation domain-dependent mode of action, which might involve, for example, a non-coding gene or a specific pattern of activation across multiple targets., (© 2016 BSPP and John Wiley & Sons Ltd.)

Delivered into plant cells by type III secretion from pathogenic Xanthomonas species, TAL (transcription activator-like) effectors are nuclear-localized, DNA-binding proteins that directly activate specific host genes. Targets include genes important for disease, genes that confer resistance, and genes inconsequential to the host-pathogen interaction. TAL effector specificity is encoded by polymorphic repeats of 33-35 amino acids that interact one-to-one with nucleotides in the recognition site. Activity depends also on N-terminal sequences important for DNA binding and C-terminal nuclear localization signals (NLS) and an acidic activation domain (AD). Coding sequences missing much of the N- and C-terminal regions due to conserved, in-frame deletions are present and annotated as pseudogenes in sequenced strains of Xanthomonas oryzae pv. oryzicola (Xoc) and pv. oryzae (Xoo), which cause bacterial leaf streak and bacterial blight of rice, respectively. Here we provide evidence that these sequences encode proteins we call "truncTALEs," for "truncated TAL effectors." We show that truncTALE Tal2h of Xoc strain BLS256, and by correlation truncTALEs in other strains, specifically suppress resistance mediated by the Xo1 locus recently described in the heirloom rice variety Carolina Gold. Xo1 -mediated resistance is triggered by different TAL effectors from diverse X. oryzae strains, irrespective of their DNA binding specificity, and does not require the AD. This implies a direct protein-protein rather than protein-DNA interaction. Similarly, truncTALEs exhibit diverse predicted DNA recognition specificities. And, in vitro , Tal2h did not bind any of several potential recognition sites. Further, a single candidate NLS sequence in Tal2h was dispensable for resistance suppression. Many truncTALEs have one 28 aa repeat, a length not observed previously. Tested in an engineered TAL effector, this repeat required a single base pair deletion in the DNA, suggesting that it or a neighbor disengages. The presence of the 28 aa repeat, however, was not required for resistance suppression. TruncTALEs expand the paradigm for TAL effector-mediated effects on plants. We propose that Tal2h and other truncTALEs act as dominant negative ligands for an immune receptor encoded by the Xo1 locus, likely a nucleotide binding, leucine-rich repeat protein. Understanding truncTALE function and distribution will inform strategies for disease control.

Understanding the processes that shaped contemporary pathogen populations in agricultural landscapes is quite important to define appropriate management strategies and to support crop improvement efforts. Here, we took advantage of an historical record to examine the adaptation pathway of the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo) in a semi-isolated environment represented in the Philippine archipelago. By comparing genomes of key Xoo groups we showed that modern populations derived from three Asian lineages. We also showed that diversification of virulence factors occurred within each lineage, most likely driven by host adaptation, and it was essential to shape contemporary pathogen races. This finding is particularly important because it expands our understanding of pathogen adaptation to modern agriculture.

The rice pathogens Xanthomonas oryzae pathovar (pv.) oryzae and pv. oryzicola produce numerous transcription activator-like (TAL) effectors that increase bacterial virulence by activating expression of host susceptibility genes. Rice resistance mechanisms against TAL effectors include polymorphisms that prevent effector binding to susceptibility gene promoters, or that allow effector activation of resistance genes. This study identifies, in the heirloom variety Carolina Gold Select, a third mechanism of rice resistance involving TAL effectors. This resistance manifests through strong suppression of disease development in response to diverse TAL effectors from both X. oryzae pathovars. The resistance can be triggered by an effector with only 3.5 central repeats, is independent of the composition of the repeat variable di-residues that determine TAL effector binding specificity, and is independent of the transcriptional activation domain. We determined that the resistance is conferred by a single dominant locus, designated Xo1, that maps to a 1.09 Mbp fragment on chromosome 4. The Xo1 interval also confers complete resistance to the strains in the African clade of X. oryzae pv. oryzicola, representing the first dominant resistance locus against bacterial leaf streak in rice. The strong phenotypic similarity between the TAL effector-triggered resistance conferred by Xo1 and that conferred by the tomato resistance gene Bs4 suggests that monocots and dicots share an ancient or convergently evolved mechanism to recognize analogous TAL effector epitopes., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

Transcription activator-like effector nucleases (TALENs) can be exquisitely specific and highly effective genome editing reagents. Specificity and efficacy depend however on good design for minimal off-targeting and strong binding. Several online tools are accessible to aid in this process. Here, we tabulate those tools, noting their functions and key features.

Pathogen-injected, direct transcriptional activators of host genes, TAL (transcription activator-like) effectors play determinative roles in plant diseases caused by Xanthomonas spp. A large domain of nearly identical, 33-35 aa repeats in each protein mediates DNA recognition. This modularity makes TAL effectors customizable and thus important also in biotechnology. However, the repeats render TAL effector ( tal ) genes nearly impossible to assemble using next-generation, short reads. Here, we demonstrate that long-read, single molecule real-time (SMRT) sequencing solves this problem. Taking an ensemble approach to first generate local, tal gene contigs, we correctly assembled de novo the genomes of two strains of the rice pathogen X. oryzae completed previously using the Sanger method and even identified errors in those references. Sequencing two more strains revealed a dynamic genome structure and a striking plasticity in tal gene content. Our results pave the way for population-level studies to inform resistance breeding, improve biotechnology and probe TAL effector evolution.

The interaction of barley, Hordeum vulgare L., with the powdery mildew fungus Blumeria graminis f. sp. hordei is a well-developed model to investigate resistance and susceptibility to obligate biotrophic pathogens. The 130-Mb Blumeria genome encodes approximately 540 predicted effectors that are hypothesized to suppress or induce host processes to promote colonization. Blumeria effector candidate (BEC)1019, a single-copy gene encoding a putative, secreted metalloprotease, is expressed in haustorial feeding structures, and host-induced gene silencing of BEC1019 restricts haustorial development in compatible interactions. Here, we show that Barley stripe mosaic virus-induced gene silencing of BEC1019 significantly reduces fungal colonization of barley epidermal cells, demonstrating that BEC1019 plays a central role in virulence. In addition, delivery of BEC1019 to the host cytoplasm via Xanthomonas type III secretion suppresses cultivar nonspecific hypersensitive reaction (HR) induced by Xanthomonas oryzae pv. oryzicola, as well as cultivar-specific HR induced by AvrPphB from Pseudomonas syringae pv. phaseolicola. BEC1019 homologs are present in 96 of 241 sequenced fungal genomes, including plant pathogens, human pathogens, and free-living nonpathogens. Comparative analysis revealed variation at several amino acid positions that correlate with fungal lifestyle and several highly conserved, noncorrelated motifs. Site-directed mutagenesis of one of these, ETVIC, compromises the HR-suppressing activity of BEC1019. We postulate that BEC1019 represents an ancient, broadly important fungal protein family, members of which have evolved to function as effectors in plant and animal hosts.

Xanthomonas oryzae pv. oryzicola (Xoc) causes the increasingly important disease bacterial leaf streak of rice (BLS) in part by type III delivery of repeat-rich transcription activator-like (TAL) effectors to upregulate host susceptibility genes. By pathogen whole genome, single molecule, real-time sequencing and host RNA sequencing, we compared TAL effector content and rice transcriptional responses across 10 geographically diverse Xoc strains. TAL effector content is surprisingly conserved overall, yet distinguishes Asian from African isolates. Five TAL effectors are conserved across all strains. In a prior laboratory assay in rice cv. Nipponbare, only two contributed to virulence in strain BLS256 but the strict conservation indicates all five may be important, in different rice genotypes or in the field. Concatenated and aligned, TAL effector content across strains largely reflects relationships based on housekeeping genes, suggesting predominantly vertical transmission. Rice transcriptional responses did not reflect these relationships, and on average, only 28% of genes upregulated and 22% of genes downregulated by a strain are up- and down- regulated (respectively) by all strains. However, when only known TAL effector targets were considered, the relationships resembled those of the TAL effectors. Toward identifying new targets, we used the TAL effector-DNA recognition code to predict effector binding elements in promoters of genes upregulated by each strain, but found that for every strain, all upregulated genes had at least one. Filtering with a classifier we developed previously decreases the number of predicted binding elements across the genome, suggesting that it may reduce false positives among upregulated genes. Applying this filter and eliminating genes for which upregulation did not strictly correlate with presence of the corresponding TAL effector, we generated testable numbers of candidate targets for four of the five strictly conserved TAL effectors.

Both plants and fungi produce ent-kaurene as a precursor to the gibberellin plant hormones. A number of rhizobia contain functionally conserved, sequentially acting ent-copalyl diphosphate and ent-kaurene synthases (CPS and KS, respectively), which are found within a well-conserved operon that may lead to the production of gibberellins. Intriguingly, the rice bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc) contains a homologous operon. Here, we report biochemical characterization of the encoded CPS and KS, and the impact of insertional mutagenesis on virulence and the plant defense response for these genes, as well as that for one of the cytochromes P450 (CYP112) found in the operon. Activity of the CPS and KS found in this phytopathogen was verified - that is, Xoc is capable of producing ent-kaurene. Moreover, knocking out CPS, KS or CYP112 led to mutant Xoc that exhibited reduced virulence. Investigation of the effect on marker gene transcript levels suggests that the Xoc diterpenoid affects the plant defense response, most directly that mediated by jasmonic acid (JA). Xoc produces an ent-kaurene-derived diterpenoid as a virulence factor, potentially a gibberellin phytohormone, which is antagonistic to JA, consistent with the recent recognition of opposing effects for these phytohormones on the microbial defense response., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

TAL effectors are transcription factors injected into plant cells by pathogenic bacteria during infection. They find their specific DNA targets via a string of contiguous, structural repeats that individually recognize single nucleotides (with some degeneracy) by virtue of polymorphisms at residue 13. The number of repeats and sequence of the amino acids at position 13 determine the nucleotide sequence of the DNA target. Due to this modularity, TAL effectors are readily engineered and have been used alone or as molecular fusions for targeted gene activation, gene repression, chromatin modification, chromatin tagging, and most broadly, for genome editing as TAL effector nucleases (TALENs). Several moderate and high-throughput cloning methods are in place for assembling TAL effector-based genetic constructs. Targeting is complicated to an extent by a general requirement for thymine to precede the DNA target, a requirement of TALENs to bind paired opposing sites separated by a defined range of distances, differential contributions of different repeat types to overall affinity, and a polarity to mismatch tolerance. Several computational tools are available online to aid in design and the identification of candidate off-target binding sites, as well as assembly and implementation. These tools vary in their approaches, capabilities, and relative utility for different types of TAL effector applications. Accuracy of off-target prediction is not well characterized yet for any of the tools and will require a better understanding of the qualitative and quantitative variation in the nucleotide preferences of individual repeats., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Recent advances in DNA targeting allow unprecedented control over gene function and expression. Targeting based on TAL effectors is arguably the most promising for systems biology and metabolic engineering. Multiple, orthogonal TAL-effector reagents of different types can be used in the same cell. Furthermore, variation in base preferences of the individual structural repeats that make up the TAL effector DNA recognition domain makes targeting stringency tunable. Realized applications range from genome editing to epigenome modification to targeted gene regulation to chromatin labeling and capture. The principles that govern TAL effector DNA recognition make TAL effectors well suited for applications relevant to plant physiology and metabolism. TAL effector targeting has merits that are distinct from those of the RNA-based DNA targeting CRISPR/Cas9 system., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Precise genome editing in complex genomes is enabled by engineered nucleases that can be programmed to cleave in a site-specific manner. Here, we fused the small, sequence-tolerant monomeric nuclease domain from the homing endonuclease I-TevI to transcription-like activator effectors (TALEs) to create monomeric Tev-TALE nucleases (Tev-mTALENs). Using the PthXo1 TALE scaffold to optimize the Tev-mTALEN architecture, we found that choice of the N-terminal fusion point on the TALE greatly influenced activity in yeast-based assays, and that the length of the linker used affected the optimal spacing of the TALE binding site from the I-TevI cleavage site, specified by the motif 5'-CNNNG-3'. By assaying activity on all 64 possible sequence variants of this motif, we discovered that in the Tev-mTALEN context, I-TevI prefers A/T-rich triplets over G/C-rich ones at the cleavage site. Profiling of nucleotide requirements in the DNA spacer that separates the CNNNG motif from the TALE binding site revealed substantial, but not complete, tolerance to sequence variation. Tev-mTALENs showed robust mutagenic activity on an episomal target in HEK 293T cells consistent with specific cleavage followed by nonhomologous end-joining repair. Our data substantiate the applicability of Tev-mTALENs as genome-editing tools but highlight DNA spacer and cleavage site nucleotide preferences that, while enhancing specificity, do confer moderate targeting constraints., (Copyright © 2014 B. P. Kleinstiver et al.)

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.

TAL effectors are re-targetable transcription factors used for tailored gene regulation and, as TAL effector-nuclease fusions (TALENs), for genome engineering. Their hallmark feature is a customizable central string of polymorphic amino acid repeats that interact one-to-one with individual DNA bases to specify the target. Sequences targeted by TAL effector repeats in nature are nearly all directly preceded by a thymine (T) that is required for maximal activity, and target sites for custom TAL effector constructs have typically been selected with this constraint. Multiple crystal structures suggest that this requirement for T at base 0 is encoded by a tryptophan residue (W232) in a cryptic repeat N-terminal to the central repeats that exhibits energetically favorable van der Waals contacts with the T. We generated variants based on TAL effector PthXo1 with all single amino acid substitutions for W232. In a transcriptional activation assay, many substitutions altered or relaxed the specificity for T and a few were as active as wild type. Some showed higher activity. However, when replicated in a different TAL effector, the effects of the substitutions differed. Further, the effects differed when tested in the context of a TALEN in a DNA cleavage assay, and in a TAL effector-DNA binding assay. Substitution of the N-terminal region of the PthXo1 construct with that of one of the TAL effector-like proteins of Ralstonia solanacearum, which have arginine in place of the tryptophan, resulted in specificity for guanine as the 5' base but low activity, and several substitutions for the arginine, including tryptophan, destroyed activity altogether. Thus, the effects on specificity and activity generated by substitutions at the W232 (or equivalent) position are complex and context dependent. Generating TAL effector scaffolds with high activity that robustly accommodate sites without a T at position 0 may require larger scale re-engineering.

Transcription activator-like (TAL) effectors are transcription factors injected into plant cells by pathogenic bacteria of the genus Xanthomonas. They function as virulence factors by activating host genes important for disease, or as avirulence factors by turning on genes that provide resistance. DNA-binding specificity is encoded by polymorphic repeats in each protein that correspond one-to-one with different nucleotides. This code has facilitated target identification and opened new avenues for engineering disease resistance. It has also enabled TAL effector customization for targeted gene control, genome editing, and other applications. This article reviews the structural basis for TAL effector-DNA specificity, the impact of the TAL effector-DNA code on plant pathology and engineered resistance, and recent accomplishments and future challenges in TAL effector-based DNA targeting., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The functional study of Y chromosome genes has been hindered by a lack of mouse models with specific Y chromosome mutations. We used transcription activator-like effector nuclease (TALEN)-mediated gene editing in mouse embryonic stem cells (mESCs) to produce mice with targeted gene disruptions and insertions in two Y-linked genes--Sry and Uty. TALEN-mediated gene editing is a useful tool for dissecting the biology of the Y chromosome.

Obligate biotrophic pathogens of plants must circumvent or counteract defenses to guarantee accommodation inside the host. To do so, they secrete a variety of effectors that regulate host immunity and facilitate the establishment of pathogen feeding structures called haustoria. The barley powdery mildew fungus Blumeria graminis f. sp. hordei produces a large number of proteins predicted to be secreted from haustoria. Fifty of these Blumeria effector candidates (BEC) were screened by host-induced gene silencing (HIGS), and eight were identified that contribute to infection. One shows similarity to β-1,3 glucosyltransferases, one to metallo-proteases, and two to microbial secreted ribonucleases; the remainder have no similarity to proteins of known function. Transcript abundance of all eight BEC increases dramatically in the early stages of infection and establishment of haustoria, consistent with a role in that process. Complementation analysis using silencing-insensitive synthetic cDNAs demonstrated that the ribonuclease-like BEC 1011 and 1054 are bona fide effectors that function within the plant cell. BEC1011 specifically interferes with pathogen-induced host cell death. Both are part of a gene superfamily unique to the powdery mildew fungi. Structural modeling was consistent, with BEC1054 adopting a ribonuclease-like fold, a scaffold not previously associated with effector function.

The plant cell wall has many significant structural and physiological roles, but the contributions of the various components to these roles remain unclear. Modification of cell wall properties can affect key agronomic traits such as disease resistance and plant growth. The plant cell wall is composed of diverse polysaccharides often decorated with methyl, acetyl, and feruloyl groups linked to the sugar subunits. In this study, we examined the effect of perturbing cell wall acetylation by making transgenic Arabidopsis (Arabidopsis thaliana) and Brachypodium (Brachypodium distachyon) plants expressing hemicellulose- and pectin-specific fungal acetylesterases. All transgenic plants carried highly expressed active Aspergillus nidulans acetylesterases localized to the apoplast and had significant reduction of cell wall acetylation compared with wild-type plants. Partial deacetylation of polysaccharides caused compensatory up-regulation of three known acetyltransferases and increased polysaccharide accessibility to glycosyl hydrolases. Transgenic plants showed increased resistance to the fungal pathogens Botrytis cinerea and Bipolaris sorokiniana but not to the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. These results demonstrate a role, in both monocot and dicot plants, of hemicellulose and pectin acetylation in plant defense against fungal pathogens.

Xanthomonas oryzae pv. oryzicola causes bacterial leaf streak of rice. A mutant disrupted in wxocB, predicted to encode an enzyme for lipopolysaccharide (LPS) synthesis, was previously shown to suffer reduced virulence. Here, we confirm a role for wxocB in virulence and demonstrate its requirement for LPS O-chain assembly. Structure analysis indicated that wild-type LPS contains a polyrhamnose O chain with irregular, variant residues and a core oligosaccharide identical to that of other Xanthomonas spp. and that the wxocB mutant lacks the O chain. The mutant also showed moderate impairment in exopolysaccharide (EPS) production, but comparison with an EPS-deficient mutant demonstrated that this impairment could not account entirely for the reduced virulence. The wxocB mutant was not detectably different from the wild type in its induction of pathogenesis-related rice genes, type II secretion competence, flagellar motility, or resistance to two phytoalexins or resveratrol, and it was more, not less, resistant to oxidative stress and a third phytoalexin, indicating that none of these properties is involved. The mutant was more sensitive to SDS and to novobiocin, so increased sensitivity to some host-derived antimicrobials cannot be ruled out. However, the mutant showed a marked decrease in type III secretion into plant cells. This was not associated with any change in expression of genes for type III secretion or the ability to attach to plant cells in suspension. Thus, virulence of the wxocB mutant is likely reduced due primarily to a direct, possibly structural, effect of the loss of the O chain on type III delivery of effector proteins.

TAL effectors are proteins secreted by bacterial pathogens into plant cells, where they enter the nucleus and activate expression of individual genes. TAL effectors display a modular architecture that includes a central DNA-binding region comprising a tandem array of nearly identical repeats that are almost all 34 residues long. Residue number 13 in each TAL repeat (one of two consecutive polymorphic amino acids that are termed 'repeat variable diresidues', or 'RVDs') specifies the identity of a single base; collectively the sequential repeats and their RVDs dictate the recognition of sequential bases along one of the two DNA strands. The modular architecture of TAL effectors has facilitated their extremely rapid development and application as artificial gene targeting reagents, particularly in the form of site-specific nucleases. Recent crystallographic and biochemical analyses of TAL effectors have established the structural basis of their DNA recognition properties and provide clear directions for future research., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The ability to precisely engineer plant genomes offers much potential for advancing basic and applied plant biology. Here, we describe methods for the targeted modification of plant genomes using transcription activator-like effector nucleases (TALENs). Methods were optimized using tobacco (Nicotiana tabacum) protoplasts and TALENs targeting the acetolactate synthase (ALS) gene. Optimal TALEN scaffolds were identified using a protoplast-based single-strand annealing assay in which TALEN cleavage creates a functional yellow fluorescent protein gene, enabling quantification of TALEN activity by flow cytometry. Single-strand annealing activity data for TALENs with different scaffolds correlated highly with their activity at endogenous targets, as measured by high-throughput DNA sequencing of polymerase chain reaction products encompassing the TALEN recognition sites. TALENs introduced targeted mutations in ALS in 30% of transformed cells, and the frequencies of targeted gene insertion approximated 14%. These efficiencies made it possible to recover genome modifications without selection or enrichment regimes: 32% of tobacco calli generated from protoplasts transformed with TALEN-encoding constructs had TALEN-induced mutations in ALS, and of 16 calli characterized in detail, all had mutations in one allele each of the duplicate ALS genes (SurA and SurB). In calli derived from cells treated with a TALEN and a 322-bp donor molecule differing by 6 bp from the ALS coding sequence, 4% showed evidence of targeted gene replacement. The optimized reagents implemented in plant protoplasts should be useful for targeted modification of cells from diverse plant species and using a variety of means for reagent delivery.

Genomes of the rice (Oryza sativa) xylem and mesophyll pathogens Xanthomonas oryzae pv. oryzae (Xoo) and pv. oryzicola (Xoc) encode numerous secreted transcription factors called transcription activator-like (TAL) effectors. In a few studied rice varieties, some of these contribute to virulence by activating corresponding host susceptibility genes. Some activate disease resistance genes. The roles of X. oryzae TAL effectors in diverse rice backgrounds, however, are poorly understood. Xoo TAL effectors that promote infection by activating SWEET sucrose transporter genes were expressed in TAL effector-deficient X. oryzae strain X11-5A, and assessed in 21 rice varieties. Some were also tested in Xoc on variety Nipponbare. Several Xoc TAL effectors were tested in X11-5A on four rice varieties. Xoo TAL effectors enhanced X11-5A virulence on most varieties, but to varying extents depending on the effector and variety. SWEET genes were activated in all tested varieties, but increased virulence did not correlate with activation level. SWEET activators also enhanced Xoc virulence on Nipponbare. Xoc TAL effectors did not alter X11-5A virulence. SWEET-targeting TAL effectors contribute broadly and non-tissue-specifically to virulence in rice, and their function is affected by host differences besides target sequences. Further, the utility of X11-5A for characterizing individual TAL effectors in rice was established., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

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.)

Transcription activator-like (TAL) effectors are repeat-containing proteins used by plant pathogenic bacteria to manipulate host gene expression. Repeats are polymorphic and individually specify single nucleotides in the DNA target, with some degeneracy. A TAL effector-nucleotide binding code that links repeat type to specified nucleotide enables prediction of genomic binding sites for TAL effectors and customization of TAL effectors for use in DNA targeting, in particular as custom transcription factors for engineered gene regulation and as site-specific nucleases for genome editing. We have developed a suite of web-based tools called TAL Effector-Nucleotide Targeter 2.0 (TALE-NT 2.0; https://boglab.plp.iastate.edu/) that enables design of custom TAL effector repeat arrays for desired targets and prediction of TAL effector binding sites, ranked by likelihood, in a genome, promoterome or other sequence of interest. Search parameters can be set by the user to work with any TAL effector or TAL effector nuclease architecture. Applications range from designing highly specific DNA targeting tools and identifying potential off-target sites to predicting effector targets important in plant disease.