Adam Joseph Bogdanove

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, 9 of which were prioritized 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 2 SWEET genes and the BoIAA7c gene, which encodes an 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.

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 10 clusters found in RSSC phytopathogen genomes, 12 in non-RSSC environmental genomes, and 6 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 are putative DNA-conjugation systems. The clusters were encoded on accessory plasmids of various sizes or as integrative and conjugative elements 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 haemagglutinin 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 within the genus 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 statement: The 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 summary: Supplemental 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). This study’s 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.

Plant susceptibility (S) genes exploited by pathogenic bacteria play critical roles in disease development, collectively contributing to symptoms, pathogen proliferation, and spread. S genes may support pathogen establishment within the host, suppress host immunity, regulate host physiology or development, or function in other ways. S genes can be passive, e.g., involved in pathogen attraction or required for pathogen effector localization or activity, or active, contributing directly to symptoms or pathogen proliferation. Knowledge of S genes is important for understanding disease and other aspects of plant biology. It is also useful for disease management, as nonfunctional alleles can slow or prevent disease and, because they are often quantitative, can exert less selection on pathogens than dominant resistance genes, allowing greater durability. In this review, we discuss bacterial exploitation of S genes, S-gene functional diversity, approaches for identifying S genes, translation of S-gene knowledge for disease control, and future perspectives on this exciting area of plant pathology.

Summary: Transcription Activator-Like Effector (TALE) Tal12a is widespread in strains of Xanthomonas campestris pv. campestris ( Xcc ) causing black rot on Brassica crops. We sought to determine whether and how Tal12a contributes to disease. Transcriptomic analysis of cauliflower leaves infected with Xcc strains expressing Tal12a was combined with TALE-binding element prediction and heterologous expression assays in Nicotiana benthamiana in order to identify candidate Susceptibility ( S ) genes. Artificial TALEs (arTALEs) were used to validate the contribution of candidate target genes to susceptibility. Tal12a enhances bacterial virulence and growth in cauliflower. Transcriptomic analysis revealed 380 upregulated cauliflower genes, from which nine were selected as candidate targets. Expression of sugar transporter genes BoSWEET13 and BoSWEET14c was induced though likely indirectly. Five genes were confirmed as direct targets of Tal12a. Functional assays showed that BoIAA7c auxin-dependent transcriptional regulators and BoSWEET14c each independently contribute to disease, but not to bacterial proliferation. This work identifies the first susceptibility genes in cauliflower. Tal12a enhances susceptibility in cauliflower thanks to a complex transcriptional reprogramming comprising both direct and indirect tal12a targets, some of which act as minor S genes.

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.

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

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.

Xanthomonas spp. employ transcription activator-like effectors (TALEs) to promote pathogenicity by activating host susceptibility ( S ) genes. Cotton GhSWEET10 is an S gene targeted by a TALE in an early isolate of Xanthomonas citri pv. malvacearum ( Xcm ), but not by recent field Xcm isolates. To understand the pathogenicity shift in Xcm and its adaptation to cotton, we assembled the whole genome and the TALE repertoire of three recent Xcm Texas field isolates. A newly evolved TALE, Tal7b, activated different GhSWEET genes, GhSWEET14a and GhSWEET14b . Simultaneous activation of GhSWEET14a and GhSWEET14b resulted in pronounced water-soaked lesions. Transcriptome profiling coupled with TALE-binding element prediction identified a pectin lyase as an additional Tal7b target, quantitatively contributing to Xcm virulence alongside GhSWEET14a/b . CRISPR-Cas9-based gene editing supported the function of GhSWEETs as S genes in cotton bacterial blight and the promise of disrupting the TALE-binding site in these genes to control the disease. Collectively, our findings elucidate the rapid evolution of TALEs in Xanthomonas field isolates and highlight the virulence mechanism wherein TALEs induce multiple S genes simultaneously to promote pathogenicity.