Biology subjects

Bogdanove, A. J.

Publications and source records attributed to Bogdanove, A. J..

15 recordsLinked to original sources

bioRxiv2026

Mobile genetic elements accompany the emergence of clonal crop lineages from weed-associated populations of Xanthomonas campestris

Xanthomonas campestris is a destructive pathogen of cruciferous vegetables and ornamentals and is frequently isolated from related weed species growing near agricultural land. However, the evolutionary relationships, genomic features, and biosecurity significance of weed-associated strains remain poorly understood. Here, we integrate phylogenomic and pangenomic analyses of X. campestris, including nine newly sequenced strains isolated from invasive garlic mustard (Alliaria petiolata), to compare strains from cultivated and non-cultivated hosts across multiple pathovars. Weed-isolated strains predominantly clustered within basal, genetically diverse lineages associated with broad host ranges, whereas crop-isolated strains were concentrated in host-associated clonal lineages. The emergence of specialized crop-associated lineages coincided with multiple inferred acquisitions of large mobile elements, including integrative conjugative and mobilizable elements and plasmids carrying genes previously associated with pathogenicity and host range. Although these elements were enriched in clonal crop-associated lineages, they also occurred in diverse weed-associated strains, indicating that genetic material associated with crop infection is distributed among divergent lineages and persists in wild and weedy systems. These findings identify invasive weeds as ancillary hosts that may harbor pathogen diversity and provide opportunities for genetic exchange, underscoring the importance of surveillance beyond agricultural fields to monitor pathogen diversification and emerging threats. Our pangenomic analysis also identified crop-associated pathogen factors and putative host resistance targets for future investigation, providing a foundation for developing critically needed black rot-resistant cultivars.

microbiology↗

bioRxiv2025

Lifestyle-associated variation in type IV secretion systems between phytopathogenic and environmental Ralstonia

Type IV secretion systems (T4SSs) are versatile machines with variable functions including DNA uptake and release, protein translocation, and DNA conjugation. However, the diversity, distribution, and functional roles of the T4SS in the Ralstonia genus remain poorly understood. The Ralstonia solanacearum species complex (RSSC) comprises three species of plant-pathogenic bacteria that cause bacterial wilt disease. The Ralstonia genus also includes non-RSSC species that are primarily environmental bacteria and rare opportunistic human pathogens. This study compared the diversity and phylogenetic distribution of T4SSs in the RSSC phytopathogens vs. non-RSSC environmentals. Phylogenetic analysis of VirB4 sequences and synteny analysis revealed 16 distinct T4SS clusters in Ralstonia, with ten clusters found in RSSC phytopathogen genomes, twelve in non-RSSC environmental genomes, and six clusters in both groups. Collectively, these gene clusters were more prevalent in non-RSSC environmental genomes. The presence of type IV coupling protein and relaxase genes suggests that at least 14 of these T4SS gene clusters could be putative DNA conjugation systems. The clusters were encoded on accessory plasmids of various sizes or as integrative and conjugative elements (ICEs) on the chromosome or megaplasmid. The putative regions of transfer for T4SS gene clusters in the RSSC phytopathogen genomes often contained type III effectors, type VI secretion toxin/antitoxin clusters, and hemagglutinin gene clusters. In contrast, the non-RSSC environmentals were enriched in heavy metal metabolism and resistance genes. One of the 16 T4SS clusters, cluster i, exhibited evidence of specialization for the RSSC phytopathogens. These findings shed light on the eco-evolutionary differences in the Ralstonia genus. Impact statementThe Ralstonia genus contains the Ralstonia solanacearum species complex (RSSC), a group of globally important plant pathogens that impact food security. These pathogens are known to have an expansive, open pangenome with high levels of gene flow. To shed light on the eco-evolutionary differences between RSSC phytopathogens and closely related environmental species, we explored the potential role of type IV secretion systems (T4SSs) in horizontal gene flow within the Ralstonia genus. Surprisingly, these mobile genetic elements were less common in the phytopathogens than the environmentals. Nevertheless, we identified a particular T4SS gene cluster encoded on an accessory plasmid that appears to be specialized to the pathogenic lifestyle of the RSSC. This specialized cluster harbors genes that drive RSSC host range (type III effectors) and others that likely allow these pathogens to antagonize the plant microbiota (type VI secretion toxins and hemagglutinin two partner secretion systems). Moreover, this study sheds light on the cryptic lifestyles of the poorly studied environmental Ralstonia species, revealing that their conjugative T4SSs frequently carry heavy metal metabolism and resistance genes. Overall, this study provides a foundation to investigate the functional roles of T4SS clusters and their cargo genes in the fitness of phytopathogenic and environmental Ralstonia. Data summarySupplemental Table S1 lists details of the RSSC phytopathogen, non-RSSC environmental, and Burkholderiaceae family genomes used in this study. Supplemental Table S2A is the final list of 501 VirB4 sequences collectively identified in 636 RSSC phytopathogen genomes and 143 non-RSSC environmental genomes. Supplemental Table S2B includes the annotations and NCBI accessions for every gene and protein sequence in reference clusters a-p. Supplemental Table S2C is the list of putative T4SS cargo genes identified in complete Ralstonia genomes. Supplemental Table S2D is the list of putative T4SS cargo genes identified in cluster i regions from draft and complete Ralstonia genomes. Supplemental File S1 is the multiple sequence alignment (MSA) input for HMMER, made from the 753 VirB4 protein sequences from the Burkholderiaceae family genomes. Supplemental Files S2 and S3 are PDF-format versions of the species trees displaying the presence of T4SS gene clusters in RSSC phytopathogens and non-RSSC environmentals, respectively. Supplemental File S4 is the species tree displaying the presence of RSp0179 and RSp1521 in the Ralstonia genus. Supplemental File S5 is the MSA input for FastTree 2, made from the VirB4 protein sequences from Ralstonia genomes. Supplemental Files S6-S9 are various formats of the VirB4 protein tree in Figure 1. Supplemental Files S10-S25 are the GBK files for reference clusters a-p. Supplemental File S26 is the clinker output comparing the reference clusters. Supplemental Files S27-S36 are the clinker outputs comparing the putative regions of transfer of T4SSs in complete Ralstonia genomes. Supplemental File S37 is an MSA analysis comparing RSp1521 to AcvB homologs. Supplemental File S38 is the clustered average nucleotide identity (ANI) matrix for the putative regions of transfer of cluster i T4SSs in draft and complete Ralstonia genomes. Supplemental File S39 is the clinker output comparing the cluster i regions in draft and complete Ralstonia genomes. All Supplemental Tables and Files are available through Zenodo (DOI: 10.5281/zenodo.14873518). O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/675681v2_fig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1640850org.highwire.dtl.DTLVardef@5de6f5org.highwire.dtl.DTLVardef@e605feorg.highwire.dtl.DTLVardef@1937bac_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Sequence diversity of the T4SS marker VirB4 in the Ralstonia genus. VirB4 protein sequences (n=496) were identified by a custom hidden Markov model (HMM) search, and an approximately-maximum-likelihood tree was built with FastTree 2 from a MUSCLE multiple sequence alignment (MSA) of the VirB4 sequences. Bootstrap values (0 to 1) from 1,000 resamples are shown for key nodes. The unrooted tree was visualized in iTOL and labeled using Affinity Designer. The 16 distinct T4SS gene clusters (a-p) are outlined and labeled. Circles denote clusters that are present in the RSSC phytopathogens and triangles denote clusters that are present in the non-RSSC environmentals. Colored clusters were found in RSSC phytopathogen genomes, and clusters with dashed outlines were not found in the RSSC phytopathogens. Supplemental files contain the virB4 gene list (Supplemental Table S2A), the VirB4 protein sequence MSA (Supplemental File S5), the Newick file for the tree (File S6), a PDF of the unrooted tree with branch labels (File S7), and PDFs of the rectangular tree with branch labels, without and with colors (Files S8 and S9). C_FIG This studys newly sequenced genomes, Gazipur 4 and Gazipur 5, are available on NCBI with the following accessions: GCF_049860735.1 and GCF_049860725.1, respectively. The two contigs containing the cluster j T4SS were submitted to NCBI through the Third Party Annotation (TPA) section of the DDBJ/ENA/GenBank databases with the following accessions: BK072068-BK072069. These contigs were assembled from SRA reads under the accession: SRR18649448.

microbiology↗

bioRxiv2025

A Xanthomonas effector protein contributes quantitatively to virulence by inducing at least two minor Susceptibility genes

The transcription activator-like effector Tal12a is widely conserved among Xanthomonas campestris pv. campestris strains that cause black rot in Brassica crops. However, its role in disease remains unclear. To investigate how Tal12a contributes to pathogenesis, we combined transcriptomic profiling of cauliflower leaves infected with Tal12a-expressing strains, prediction of TALE-binding elements, and heterologous expression assays in Nicotiana benthamiana. The aim of this approach was to identify candidate susceptibility genes. Artificial TALEs were further employed to validate the contribution of these candidate targets to disease development. Tal12a enhanced both virulence and bacterial growth in cauliflower. Transcriptome analysis revealed 380 genes that were induced upon infection, nine of which were prioritised as candidates. Seven of these were confirmed as direct Tal12a targets, while the induction of the sugar transporter genes BoSWEET13 and BoSWEET14c likely occurred indirectly. Functional assays demonstrated that these two SWEET genes and the BoIAA7c gene, which encodes auxin-dependent transcriptional regulator, contribute to disease development. These findings identify the first susceptibility genes in cauliflower and reveal that Tal12a promotes disease through a complex transcriptional reprogramming, involving direct and indirect target induction, with several genes functioning as minor susceptibility factors.

plant biology↗

bioRxiv2024

A PthXo2B ortholog in Xanthomonas oryzae pv oryzae strain IX-221 acts as a major virulence factor on indica rice without activating a Clade III SWEET gene.

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, PthXo2BIX-221, 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, PthXo2BIX-221 upregulates SWEET13 in japonica rice and no SWEET in indica rice, but unlike PthXo2BPXO61 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 PthXo2BIX-221 failed to render indica rice susceptible. Alignment of the PthXo2BIX-221 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 PthXo2BIX-221 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.

plant biology↗

bioRxiv2023

Prevalence and diversity of TAL effector-like proteins in fungal endosymbiotic Mycetohabitans spp.

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 homologs 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 M. endofungorum strain enhances fungal membrane stress tolerance, while others in a M. 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 Mycetohabitans rhizoxinica strains than across Mycetohabitans 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. Impact StatementMany Mucoromycota fungi harbor endosymbiotic bacteria, including Rhizopus spp. that are food fermenters and pathogens of plants and immunocompromised people. Rhizopus microsporus has endofungal Mycetohabitans (formerly Burkholderia) spp. that deploy proteins related to DNA-binding transcription activator-like effectors of plant pathogens, which enter plant nuclei and activate disease susceptibility genes. By sequencing isolated bacteria and mining fungal holobiont sequences, we found Btl proteins in diverse Mycetohabitans strains, varying in predicted DNA binding specificity, thus in potential host targets. Btl proteins were more conserved within M. rhizoxinica, suggesting distinctions among the two named species. The results in the context of phenotypic differences observed in other studies suggest that Btl proteins contribute to symbiosis in diverse ways, providing insight into effector evolution and arguing for functional characterization of additional Btl proteins to understand establishment and maintenance of these important fungal-bacterial interactions.

microbiology↗

bioRxiv2021

TAL effectors with avirulence activity in African strains of Xanthomonas oryzae pv. oryzae

BackgroundXanthomonas oryzae pv. oryzae causes bacterial leaf blight, a devastating disease of rice. Among the type-3 effectors secreted by Xanthomonas oryzae pv. oryzae 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 Xanthomonas oryzae pv. oryzae. ResultsWith 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 PXO99A, 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. ConclusionWe 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.

plant biology↗

bioRxiv2021

An atypical class of non-coding small RNAs produced in rice leaves upon bacterial infection

Non-coding small RNAs (sRNA) act as mediators of gene silencing and regulate plant growth, development and stress responses. Early insights into plant sRNAs established a role in antiviral defense and they are now extensively studied across plant-microbe interactions. Here, sRNA sequencing discovered a class of sRNA in rice (Oryza sativa) specifically associated with foliar diseases caused by Xanthomonas oryzae bacteria. Xanthomonas-induced small RNAs (xisRNAs) loci were distinctively upregulated in response to diverse virulent strains at an early stage of infection producing a single duplex of 20-22nt sRNAs. xisRNAs production was dependent on the Type III secretion system, a major bacterial virulence factor for host colonization. xisRNA loci overlap with annotated transcripts sequences often encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and some xisRNA loci coincide with the coding sequence of a conserved kinase motif. xisRNAs exhibit features of small interfering RNAs and their biosynthesis depend on canonical components OsDCL1 and OsHEN1. xisRNA induction possibly mediates post-transcriptional gene silencing but they do not broadly suppress cis-genes expression on the basis of mRNA-seq data. Overall, our results identify a group of unusual sRNAs with a potential role in plant-microbe interactions.

molecular biology↗

bioRxiv2020

Spelling changes and fluorescent tagging with prime editing vectors for plants

Prime editing (PE) is a recent adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase (RT) fusion and a guide RNA (pegRNA) amended with template and primer binding site (PBS) sequences to achieve RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of PE 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 PE in dicots and monocots, their validation in Nicotiana benthamiana, rice and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging.

plant biology↗

bioRxiv2020

Cloning of the rice Xo1 resistance gene and interaction of the Xo1 protein with the defense-suppressing Xanthomonas effector Tal2h

The Xo1 locus in the heirloom rice variety Carolina Gold Select confers resistance to bacterial leaf streak and bacterial blight, caused by Xanthomonas oryzae pvs. oryzicola and oryzae, respectively. Resistance is triggered by pathogen-delivered transcription activator-like effectors (TALEs) independent of their ability to activate transcription, and is suppressed by 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 zfBED domain, is the Xo1 gene. Analyses of published transcriptomes revealed that the Xo1-mediated response is similar to those of NLR resistance genes Pia and Rxo1 and distinct from that associated with induction 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 co-immunoprecipitated 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 co-immunoprecipitation may be effective for identifying or characterizing other resistance genes.

plant biology↗

bioRxiv2020

The giant sequoia genome and proliferation of disease resistance genes

The giant sequoia (Sequoiadendron giganteum) of California are massive, long-lived trees that grow along the U.S. Sierra Nevada mountains. As they grow primarily in isolated groves within a narrow range, conservation of existing trees has been a national goal for over 150 years. Genomic data are limited in giant sequoia, and the assembly and annotation of the first giant sequoia genome has been an important goal to allow marker development for restoration and management. Using Illumina and Oxford Nanopore sequencing combined with Dovetail chromosome conformation capture libraries, 8.125 Gbp of sequence was assembled into eleven chromosome-scale scaffolds. This giant sequoia assembly 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. Beyond conservation and management applications, the giant sequoia assembly is a resource for answering questions about the life history of this enigmatic and robust species. Here we provide an example by taking an inventory of the large and complex family of NLR type disease resistance genes.

genomics↗

bioRxiv2020

A TAL effector-like protein of symbiotic Mycetohabitans increases stress tolerance and alters the transcriptome of the fungal host Rhizopus microsporus

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.

microbiology↗

bioRxiv2019

Genome assembly and characterization of a complex zfBED-NLR gene-containing disease resistance locus in Carolina Gold Select rice with Nanopore sequencing

BackgroundLong-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 make up 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 rich in NLR genes.\n\nResultsToward identification of the Xo1 gene, we combined Nanopore and Illumina reads to generate a high-quality genome assembly for Carolina Gold Select. We identified 529 full or partial NLR genes and discovered, relative to the reference, an expansion of NLR genes at the Xo1 locus. One NLR gene at Xo1 has high sequence similarity to the cloned, functionally similar Xa1 gene. Both harbor an integrated zfBED domain and near-identical, tandem, C-terminal repeats. Across diverse Oryzeae, we identified two sub-clades of such NLR genes, varying in the presence of the zfBED domain and the number of repeats.\n\nConclusionsWhole genome sequencing combining Nanopore and Illumina reads effectively resolves NLR gene loci, providing context as well as content. Our identification of an Xo1 candidate is an important step toward mechanistic characterization, including the role(s) of the zfBED domain. Further, the Carolina Gold Select genome assembly will facilitate identification and exploitation of other useful traits in this historically important rice variety.

plant biology↗

bioRxiv2018

A strain of an emerging Indian pathotype of Xanthomonas oryzae pv. oryzae defeats the rice bacterial blight resistance gene xa13 without inducing a clade III SWEET gene and is nearly identical to a recent Thai isolate

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 harbouring 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.

pathology↗

bioRxiv2018

Convergent evolution of effector protease recognition by Arabidopsis and barley

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 show also 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 Resistance 1 (Pbr1). Transient co-expression of PBR1 with wild-type AvrPphB, but not a protease inactive mutant, triggered defense responses, indicating that PBR1 detects AvrPphB protease activity. Additionally, PBR1 co-immunoprecipitated with barley and N. benthamiana PBS1 proteins, suggesting mechanistic similarity to detection by RPS5. Lastly, we determined that wheat cultivars also recognize AvrPphB protease activity and contain a Pbr1 ortholog. 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 is ancient. Also, the results suggest that PBS1-based decoys may be used to engineer protease effector recognition-based resistance in barley and wheat.

plant biology↗

bioRxiv2018

Functional analysis of African Xanthomonas oryzae pv. oryzae TALomes reveals a new susceptibility gene in bacterial leaf light of rice

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 BAI3{Delta}1, a talB knockout derivative of BAI3, carrying these designer TAL effectors increased virulence of BAI3{Delta}1 validating OsERF#123 as a new, bacterial blight S gene.\n\nAuthor SummaryThe 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.

plant biology↗