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. Newly evolved Xanthomonas citri pv. malvacearum isolates triggers recent bacterial blight outbreaks in cotton. Here, the authors show that a recently evolved TALE, Tal7b, activates host susceptibility genes GhSWEET14a and GhSWEET14b rather than GhSWEET10 to confer pathogenicity in these new isolates.

To infect rice, Xanthomonas oryzae pv. oryzae (Xoo) deploys transcription activator-like effectors (TALEs) that specifically bind and upregulate host 'susceptibility' (S) genes. 34-amino acid (aa) repeats in TALEs interact one-to-one with DNA bases. Variation at positions 12 and 13 in each repeat, the repeat-variable diresidue (RVD), determine specificity. Some repeat variants shorter or longer than 34 aa can disengage to accommodate a single base deletion in the target sequence. OsSWEET11, 13, and 14 are key S genes, targeted by different TALEs from diverse Xoo strains. xa13 is a SWEET11 allele lacking the TALE binding site and thus conferring resistance. xa13 is overcome by TALEs that activate SWEET13 or SWEET14. We report here that an xa13-compatible Xoo strain, IX-221, from India, harbours an ortholog of the SWEET14-targeting TALE PthXo3 and two orthologs of the SWEET13-cognate PthXo2, each with one or two 36-aa repeats capable of disengaging. One of the PthXo2 orthologs, PthXo2BIX221, has a repeat region identical to the previously characterized PthXo2BPXO61, except for a two amino acid difference near the end of the 19th repeat. Like PthXo2BPXO61, PthXo2BIX221 upregulates SWEET13 in japonica rice and no SWEET in indica rice, but unlike PthXo2B PXO61 it nonetheless renders indica rice susceptible, pointing to an alternative S gene. Further, a designer TALE (dTALE) constructed using a standard, consensus sequence for each repeat and RVDs identical to those of PthXo2BIX221 failed to render indica rice susceptible. Alignment of the PthXo2BIX221 repeats shows a departure from the consensus in each of two repeats carrying the RVD 'NN': the sequence 'MAIAN' in place of 'VAIAS' beginning at position 7. Together, the PthXo2BIX221 results thus suggest that non-RVD sequence variation affects TALE targeting profiles. More broadly, the presence of the three aberrant repeat-harbouring TALEs in IX-221 suggests that widespread deployment of xa13 in India resulted in strains super-equipped to overcome it, capable of activating multiple SWEET genes and alleles as well as an apparent alternate S gene. ### Competing Interest Statement The authors have declared no competing interest.

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.

The type III secreted transcription activator-like effector Tal2g of the rice bacterial leaf streak (BLS) pathogen Xanthomonas oryzae pv. oryzicola promotes lesion development and bacterial exudation through stomata by binding to and upregulating a putative sulfate transporter gene in rice, OsSULTR3;6. To understand how OsSULTR3;6 contributes to disease development, we are characterizing its transport mechanics, subcellular localization, and potential substrates, and phenotyping OsSULTR3;6 knockout lines generated by genome editing. Following a brief introduction to the plant SULTR gene family, this chapter summarizes our findings so far and presents speculative functional models for the role of OsSULTR3;6 in BLS.

HomeMolecular Plant-Microbe Interactions®Vol. 35, No. 4Complete Genome Resource of Xanthomonas oryzae pv. oryzicola GX01 Isolated in South China PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseComplete Genome Resource of Xanthomonas oryzae pv. oryzicola GX01 Isolated in South ChinaXiang-Na Niu, Yiming Li, Sara C. D. Carpenter, Xue Dan, Tianjiao Li, Qiaozhi Wu, Li Wang, Wei Jiang, Sheng Huang, Ji-Liang Tang, Adam J. Bogdanove, and Yong-Qiang HeXiang-Na NiuState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources and College of Life Science and Technology, 100 Daxue Road, Nanning, Guangxi 530004, China, Yiming LiState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources and College of Life Science and Technology, 100 Daxue Road, Nanning, Guangxi 530004, ChinaNational Demonstration Center for Experimental Plant Science Education, College of Agriculture, Guangxi University, Nanning 530004, Guangxi, China, Sara C. D. CarpenterPlant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, U.S.A., Xue DanNational Demonstration Center for Experimental Plant Science Education, College of Agriculture, Guangxi University, Nanning 530004, Guangxi, China, Tianjiao LiState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources and College of Life Science and Technology, 100 Daxue Road, Nanning, Guangxi 530004, ChinaNational Demonstration Center for Experimental Plant Science Education, College of Agriculture, Guangxi University, Nanning 530004, Guangxi, China, Qiaozhi WuState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources and College of Life Science and Technology, 100 Daxue Road, Nanning, Guangxi 530004, China, Li WangPlant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, U.S.A., Wei JiangState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources and College of Life Science and Technology, 100 Daxue Road, Nanning, Guangxi 530004, China, Sheng HuangState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources and College of Life Science and Technology, 100 Daxue Road, Nanning, Guangxi 530004, China, Ji-Liang TangState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources and College of Life Science and Technology, 100 Daxue Road, Nanning, Guangxi 530004, China, Adam J. Bogdanovehttps://orcid.org/0000-0003-1683-4117Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, U.S.A., and Yong-Qiang He†Corresponding author: Y.-Q. He; E-mail Address: yqhe@gxu.edu.cnhttps://orcid.org/0000-0001-6803-0431State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources and College of Life Science and Technology, 100 Daxue Road, Nanning, Guangxi 530004, ChinaNational Demonstration Center for Experimental Plant Science Education, College of Agriculture, Guangxi University, Nanning 530004, Guangxi, ChinaPlant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, U.S.A. AffiliationsAuthors and Affiliations Xiang-Na Niu1 Yiming Li1 2 Sara C. D. Carpenter3 Xue Dan2 Tianjiao Li1 2 Qiaozhi Wu1 Li Wang3 Wei Jiang1 Sheng Huang1 Ji-Liang Tang1 Adam J. Bogdanove3 Yong-Qiang He1 2 3 † 1State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources and College of Life Science and Technology, 100 Daxue Road, Nanning, Guangxi 530004, China 2National Demonstration Center for Experimental Plant Science Education, College of Agriculture, Guangxi University, Nanning 530004, Guangxi, China 3Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, U.S.A. Published Online:14 Mar 2022https://doi.org/10.1094/MPMI-10-21-0259-AAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Genome AnnouncementThe phytopathogenic bacterium Xanthomonas oryzae pv. oryzicola causes rice bacterial leaf streak (BLS), an emerging and destructive disease in rice growing regions of Asia and Africa. BLS was first reported in the Philippines in 1918. The earliest documented occurrence of BLS in China was in 1953 in the Guangdong and Guangxi provinces of South China, the region where double-cropping of rice is most prevalent. Although complete genome sequences of some X. oryzae pv. oryzicola isolates are publicly available, none are from strains isolated in South China. We previously isolated X. oryzae pv. oryzicola GX01, which carries a plasmid, from South China and here report the complete genome sequence of GX01, generated using PacBio long-read technology.X. oryzae pv. oryzicola is a plant-pathogenic bacterium that causes bacterial leaf streak (BLS) in rice and some Oryzeae weeds (Fang et al. 1957; Niño-Liu et al. 2006). X. oryzae pv. oryzicola infects host leaves, mainly through stomata or wounds, and colonizes the apoplast of mesophyll cells, resulting in interveinal necrotic lesions (Fang et al. 1957). BLS (then named bacterial stripe) was first discovered in the Philippines in 1918 (Ou 1972), but its causal agent was not elucidated until 1957 (Fang et al. 1957). In China, the first emergence of BLS was reported in 1953 in Guangdong and Guangxi provinces in South China, one of the longest-season rice-growing areas in China, having two cropping times annually (Zhang et al. 2014). Since it was first reported, BLS has expanded northward (Zhang et al. 2014). The disease has become a major constraint for rice production in the vast rice-growing areas south of the Yellow River, the northernmost frontier of BLS spread in China (Zhang et al. 2014).In 2005, we isolated an X. oryzae pv. oryzicola strain, which we designated as GX01, from a BLS-infected rice field in Hezhou, at the junction of Guangdong and Guangxi provinces (Xiao et al. 2011). Unlike previously characterized X. oryzae pv. oryzicola strains, GX01 carries a plasmid, pXOCgx01 (Niu et al. 2015). Since GX01 is amenable to genetic manipulation, it has been used in research to probe molecular mechanisms of BLS and as a representative strain in rice breeding for resistance to BLS. We sequenced the complete genome of GX01 to provide better insight into the pathogenicity, adaptation, and evolution of this phytopathogenic bacterium.Sequencing the genome of an X. oryzae pv. oryzicola strain can be challenging; X. oryzae pv. oryzicola is armed with large clusters of tal genes—nearly identical, tandem repeat–filled sequences coding for transcription activator-like effector proteins, which play important roles in disease by directly activating corresponding host genes—and these sequences are impossible to resolve using short-read sequencing. Therefore, to sequence the whole genome of GX01, total genomic DNA was isolated from two isolates of GX01 and was submitted for sequencing on two and three cells of a PacBio RS II instrument, as previously described (Booher et al. 2015) but using size-selected 10-kb SMRTbell libraries. Reads from the single-molecule real-time (SMRT) cells were assembled de novo separately and in combination, using HGAP 2.0 or HGAP 3.0 (Chin et al. 2013), and the resulting assemblies were compared. Most assemblies consisted of single contigs, but some had two contigs corresponding to the chromosome and the plasmid that we had isolated and Sanger-sequenced previously (Niu et al. 2015). A two-contig HGAP 2.0 assembly of the genome of one isolate sequenced with three SMRT cells was selected for finishing; the assembly was trimmed, rotated, and polished once with Quiver, using the resequencing pipeline in SMRTAnalysis 2.3. The assembly was polished once more using Arrow, and methylation and motifs were analyzed using SMRTAnalysis 7.0. The tal gene sequences were separately assembled using PBX for in silico assembly verification (Booher et al. 2015).The final assembly of GX01 consists of a 4,811,977-bp chromosome and a 53,205-bp plasmid with GC contents of 64.02 and 61.25%, respectively, and was sequenced to a mean coverage depth of 339×. The genome of GX01 (chromosome and plasmid) was annotated by the National Center for Biotechnology Information prokaryotic genome annotation pipeline (Tatusova et al. 2016). The pipeline detected 4,432 protein coding genes, 499 pseudogenes, two each of 5S, 16S, and 23S rRNA genes, 55 transfer RNA genes, and 163 small RNA genes. The PacBio-sequenced plasmid was nearly identical to the Sanger-sequenced reference (GenBank accession KR071788.1), differing by only 2 bp. Analysis by PBHoney (English et al. 2014) determined three clusters of long reads that support three different breakpoints for plasmid integration into the chromosome within an approximately 18-kb region shared between the chromosome and plasmid. Long reads spanning this region also supported the separate, two-contig assembly. The evidence for chromosomal integration (and excision) and mobilization machinery on the plasmid suggests that it may be an integrative conjugative element capable of transferring to other bacteria and of integrating into the chromosome as well as replicating independently.The annotated GX01 complete genome assembly will serve as a resource for further insight into the genetic diversity, pathogenicity, and virulence evolution of Xanthomonas spp., facilitating discovery of novel chemical targets and other measures for disease control.Data AvailabilityThe complete genome sequence of X. oryzae pv. oryzicola GX01 has been deposited in GenBank under the accession numbers CP080589.1 and CP080588.1 for the chromosome and plasmid, respectively. Raw reads and methylation data have been deposited in the Sequence Read Archive under SRR16155239/SRR16155240/SRR16155241.AcknowledgmentsThe authors thank R. P. Sebra from Icahn Institute for Genomics and Multiscale Biology and Department of Genetics & Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York for his kind help in genome sequencing.The author(s) declare no conflict of interest.Literature CitedBooher, N. J., Carpenter, S. C. D., Sebra, R. P., Wang, L., Salzberg, S. L., Leach, J. E., and Bogdanove, A. J. 2015. Single molecule real-time sequencing of Xanthomonas oryzae genomes reveals a dynamic structure and complex TAL (transcription activator-like) effector gene relationships. Microb. Genom. 1:e000032. https://doi.org/10.1099/mgen.0.000032 Crossref, ISI, Google ScholarChin, C. S., Alexander, D. H., Marks, P., Klammer, A. A., Drake, J., Heiner, C., Clum, A., Copeland, A., Huddleston, J., Eichler, E. E., Turner, S. W., and Korlach, J. 2013. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat. Methods 10:563-569. https://doi.org/10.1038/nmeth.2474 Crossref, Medline, ISI, Google ScholarEnglish, A. C., Salerno, W. J., and Reid, J. G. 2014. PBHoney: Identifying genomic variants via long-read discordance and interrupted mapping. BMC Bioinformatics 15:180. https://doi.org/10.1186/1471-2105-15-180 Crossref, Medline, ISI, Google ScholarFang, C. T., Ren, H. C., Chen, T. K., Chu, Y. K., Faan, H. C., and Wu, S. C. 1957. A comparison of rice bacterial leaf blight organism with the bacterial leaf streak organism of rice and Leersia hexandra Swartz. Acta Phytopathol. Sin. 3:99-124. Google ScholarNiño-Liu, D. O., Ronald, P. C., and Bogdanove, A. J. 2006. Xanthomonas oryzae pathovars: Model pathogens of a model crop. Mol. Plant Pathol. 7:303-324. https://doi.org/10.1111/j.1364-3703.2006.00344.x Crossref, Medline, ISI, Google ScholarNiu, X. N., Wei, Z. Q., Zou, H. F., Xie, G. G., Wu, F., Li, K. J., Jiang, W., Tang, J. L., and He, Y. Q. 2015. Complete sequence and detailed analysis of the first indigenous plasmid from Xanthomonas oryzae pv. oryzicola. BMC Microbiol. 15:233. https://doi.org/10.1186/s12866-015-0562-x Crossref, Medline, ISI, Google ScholarOu, S. H. 1972. Rice Diseases. Commonwealth Mycological Institute. Kew, Surrey, U.K. Google ScholarTatusova, T., DiCuccio, M., Badretdin, A., Chetvernin, V., Nawrocki, E. P., Zaslavsky, L., Lomsadze, A., Pruitt, K. D., Borodovsky, M., and Ostell, J. 2016. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 44:6614-6624. https://doi.org/10.1093/nar/gkw569 Crossref, Medline, ISI, Google ScholarXiao, Y. S., Wei, X. X., Gao, H. P., Niu, X. N., Cen, Z. L., Huang, P., Wu, Y., and He, Y. Q. 2011. Characterization of a Xanthomonas oryzae pv. oryzicola strain and the establishment of its genetic manipulation system. (In Chinese) Genomics Appl. Biol. 30:1211-1217. Google ScholarZhang, R., Chen, Z., and Liu, Y. 2014. Advances in rice bacterial leaf streak research. (In Chinese) Jiangsu J. Agric. Sci. 30:901-908. Google ScholarXiang-Na Niu and Yiming Li contributed equally.Funding: The work was supported by the National Key R&D Program of China (2018YFD0200302) and the Natural Science Foundation of Guangxi (2019GXNSFAA245052).The author(s) declare no conflict of interest. Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.DetailsFiguresLiterature CitedRelated Vol. 35, No. 4 April 2022ISSN:0894-0282e-ISSN:1943-7706 Download Metrics Downloaded 740 times Article History Issue Date: 12 Apr 2022Published: 14 Mar 2022Accepted: 12 Jan 2022 Pages: 357-359 InformationCopyright © 2022 The Author(s).This is an open access article distributed under the CC BY-NC-ND 4.0 International license.FundingNational Key R&D Program of ChinaGrant/Award Number: 2018YFD0200302Natural Science Foundation of GuangxiGrant/Award Number: 2019GXNSFAA245052Keywordsgenometranscription activator-like effector (TALE)PacBio sequencingXanthomonas oryzae pv. oryzicolaThe author(s) declare no conflict of interest.PDF download

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.

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.

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 xa5, which reduces TALE activity generally, has been widely deployed. However, strains defeating xa5 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 xa5. 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.

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 wildtype 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.

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 micro-sporus, 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 se-quenced 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 differen-tially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a com-plemented 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 sup-port 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.

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.

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. Author Summary The ability of most Xanthomonas plant pathogenic bacteria to infect their hosts relies on the action of a specific family of proteins called TAL effectors, which are transcriptional activators injected into the plant by the bacteria. TAL effectors enter the plant cell nucleus and bind to the promoters of specific plant genes. Genes that when induced can benefit pathogen multiplication or disease development are called susceptibility ( S ) genes. Here, we perform a comparative analysis of the TAL effector repertoires of three strains of X. oryzae pv. oryzae , which causes bacterial leaf blight of rice, a major yield constraint in this staple crop. Using sequencing of entire genomes, we compared the large repertoires of TAL effectors in three African Xoo strains which form a genetic lineage distinct from Asian strains. We assessed the individual contribution to pathogen virulence of 13 TAL effector variants represented in the three strains, and identified one that makes a major contribution. By combining host transcriptome profiling and TAL effector binding sites prediction, we identified two targets of this TAL effector that function as S genes, one previously identified, and one, new S gene. We validated the new S gene by functional characterization using designer TAL effectors. Both S genes encode transcription factors and can therefore be considered as susceptibility hubs for pathogen manipulation of the host transcriptome. Our results provide new insights into the diversified strategies underlying the roles of TAL effectors in promoting plant disease.

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.

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.

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.

ABSTRACT Onion ( Allium cepa L.) lines resistant to Botrytis leaf blight (BLB) were produced by backcrossing the Bs1 BLB resistance of Allium roylei Stearn into cultivated onion. This study evaluated important issues for the commercial use of BLB‐resistant onions: the relative degree of BLB control possible when Bs1 is homozygous rather than heterozygous, the control achieved against multiple pathogen isolates, and the potential for negative traits associated with transfer of Bs1 . Homozygous BLB‐resistant onions tested in inoculated growth chamber screens with five distinct Botrytis squamosa Walker isolates and in naturally infested regional field trials showed significantly lower BLB symptoms than susceptible commercial hybrid controls. F 1 hybrids heterozygous for the Bs1 gene showed levels of BLB symptoms between those of the homozygous resistant and susceptible controls, confirming previous reports of partial dominance. The difference in BLB control in plants heterozygous for Bs1 compared with homozygous plants was more noticeable under heavier disease pressure. Hybrids heterozygous for Bs1 had similar level of BLB control in most of the field trials as their parental homozygous BLB‐resistant lines, indicating that either heterozygous or homozygous BLB resistant hybrids could be used commercially for BLB control. There were no differences in the degree of pathogenicity across the five B. squamosa isolates, which were genetically different based on polymorphism for DNA markers, or among the naturally occurring pathogens present at the six different field locations used. No obvious association was noted between presence of the Bs1 resistance gene with unfavorable characteristics such as reduced bulb size or yield.

Nematode effector proteins originating from esophageal gland cells play central roles in suppressing plant defenses and in formation of the plant feeding cells that are required for growth and development of cyst nematodes. A gene (GrUBCEP12) encoding a unique ubiquitin carboxyl extension protein (UBCEP) that consists of a signal peptide for secretion, a mono-ubiquitin domain, and a 12 amino acid carboxyl extension protein (CEP12) domain was cloned from the potato cyst nematode Globodera rostochiensis. This GrUBCEP12 gene was expressed exclusively within the nematode's dorsal esophageal gland cell, and was up-regulated in the parasitic second-stage juvenile, correlating with the time when feeding cell formation is initiated. We showed that specific GrUBCEP12 knockdown via RNA interference reduced nematode parasitic success, and that over-expression of the secreted Gr(Δ) (SP) UBCEP12 protein in potato resulted in increased nematode susceptibility, providing direct evidence that this secreted effector is involved in plant parasitism. Using transient expression assays in Nicotiana benthamiana, we found that Gr(Δ) (SP) UBCEP12 is processed into free ubiquitin and a CEP12 peptide (GrCEP12) in planta, and that GrCEP12 suppresses resistance gene-mediated cell death. A target search showed that expression of RPN2a, a gene encoding a subunit of the 26S proteasome, was dramatically suppressed in Gr(Δ) (SP) UBCEP12 but not GrCEP12 over-expression plants when compared with control plants. Together, these results suggest that, when delivered into host plant cells, Gr(Δ) (SP) UBCEP12 becomes two functional units, one acting to suppress plant immunity and the other potentially affecting the host 26S proteasome, to promote feeding cell formation.

DspA/E is a type III effector of Erwinia amylovora, the bacterial pathogen that causes fire blight disease in roseaceous plants. This effector is indispensable for disease development, and it is translocated into plant cells. A DspA/E-specific chaperone, DspB/F, is necessary for DspA/E secretion and possibly for its translocation. In this work, DspB/F-binding sites and secretion and translocation signals in the DspA/E protein were determined. Based on yeast two-hybrid assays, DspB/F was found to bind DspA/E within the first 210 amino acids of the protein. Surprisingly, both DspB/F and OrfA, the putative chaperone of Eop1, also interacted with the C-terminal 1059 amino acids of DspA/E; this suggests another chaperone-binding site. Secretion and translocation assays using serial N-terminal lengths of DspA/E fused with the active form of AvrRpt2 revealed that at least the first 109 amino acids, including the first N-terminal chaperone-binding motif and DspB/F, were required for efficient translocation of DspA/E, although the first 35 amino acids were sufficient for its secretion and the presence of DspB/F was not required. These results indicate that secretion and translocation signals are present in the N terminus of DspA/E, and that at least one DspB/F-binding motif is required for efficient translocation into plant cells.