Biology subjects

Kvitko, B. H.

Publications and source records attributed to Kvitko, B. H..

15 recordsLinked to original sources

bioRxiv2025

The two-component regulator CvsR has a small core regulon in planta and modulates Pseudomonas syringae global gene expression with some overlap to the pattern triggered immunity stimulon response.

Pattern-triggered immunity (PTI) provides broad-spectrum protection in plants by activating defense responses upon perception of conserved microbial signatures such as bacterial flagellin. In vitro transcriptome profiling revealed that the Pseudomonas syringae pv. tomato DC3000 two-component regulator CvsR mirrors some of the broader regulatory patterns observed under the exposure to PTI in planta. Our analyses indicated that during infection in planta, CvsR primarily governs a small core regulon centered on carbonic anhydrase and its associated transporter. Comparative RNA-seq analyses between the {Delta}cvsR and wild type strain further confirm this narrow regulatory scope. Moreover, the majority of bacterial transcriptional shifts appear to reflect indirect consequences of response to the host immune environment rather than direct CvsR-dependent regulation, including responses associated with sulfate starvation. Together, these findings suggest that PTI-driven bacterial transcriptional reprogramming is shaped predominantly by host immune status, with CvsR exerting modest, targeted control restricted to a limited set of genes.

microbiology↗

bioRxiv2025

The AltR transcription factor responds to plant thiosulfinates to regulate gene expression in a bacterial pathogen of onion.

Pantoea ananatis, the causative agent of onion center rot, encounters potent antimicrobial thiosulfinates, volatile organosulfur compounds released from damaged Allium tissues. The allicin tolerance (alt) gene cluster allows P. ananatis to overcome this chemical barrier. We demonstrate that AltR, a TetR-family transcriptional repressor, specifically regulates expression of the alt cluster and thus thiosulfinate tolerance in vitro and fitness in vivo. We identified a putative AltR binding box both in the altR promoter and elsewhere in the alt cluster, show that AltR-mediated repression is relieved in response to thiosulfinates. Using cysteine to serine substitutions, we demonstrate that AltR Cys100 is essential for thiosulfinate-responsive de-repression, while other AltR cysteine residues tune responsivity. Strains expressing AltR alleles with reduced thiosulfinate responsivity have reduced fitness in planta. Our findings uncover a regulatory mechanism by which a plant antimicrobial secondary metabolite acts as an environmental cue to modulate bacterial gene expression, enabling pathogen survival and virulence.

microbiology↗

bioRxiv2025

Ecologically Significant Genetic Loci of P. allii: Contributions to Pathogenesis and Competition

Pantoea allii, one of four Pantoea species known to cause onion center rot, is infrequently isolated from onion compared to its closely related onion-pathogenic sister taxa. To better understand the genomic diversity and genetic determinants of pathogenicity in this species, we analyzed a collection of 38 P. allii strains isolated from two primary ecological niches, plants and water, across three continents using comparative genomics and phylogenetic approaches. Core-genome phylogeny, average nucleotide identity (ANI), and gene presence-absence analyses revealed three genetically distinct lineages. All strains harbored conserved biosynthetic gene clusters (BGCs) for quorum sensing, carotenoid production, siderophores, and thiopeptides. In contrast, two phosphonate BGCs, key determinants of onion pathogenicity, exhibited lineage-specific distributions. Onion-associated strains from Lineages 1 and 2 carried the Halophos BGC associated with onion tissue necrosis, and onion isolates encoded the alt gene cluster conferring thiosulfinate tolerance. Lineage 3 strains, isolated from both onion and rainwater, either lacked a phosphonate BGC loci or carried the HiVir phosphonate BGC. In addition, Lineage 3 strains lacked the alt cluster altogether. The localization of these virulence genes in the genome varied, with Halophos integrated in the chromosome, HiVir encoded on the conserved Large Pantoea Plasmid, and alt located on a small, variable plasmids (plasmid B). The Type IV and Type VI secretion systems showed variable genomic architectures, with plasmid-borne T4SSs and two chromosomal T6SS loci differing in conservation and gene content. Additionally, conserved Pantailocin phage islands were detected in most genomes. Overall, this study reveals that while core metabolic and competitive traits are conserved across P. allii, virulence-associated loci display lineage-specific partitioning, reflecting ecological differentiation and evolutionary plasticity within the species. Impact StatementThis study presents a comprehensive comparative genomic analysis of available Pantoea allii genomes, a known onion pathogen. By analyzing 38 strains isolated from plant and water sources across three continents, we uncovered lineage-specific distributions of key virulence genes, alongside conserved genetic traits associated with competition and environmental resilience. These findings clarify the genetic basis of P. allii pathogenesis and highlight its potential as a biocontrol agent, offering broader insights into how ecologically significant loci contribute to the dual roles of plant-associated bacteria as both pathogens and beneficial microbes. Data SummaryPENDING

genomics↗

bioRxiv2025

The Proteomics Landscape of Pattern Triggered Immunity in the Arabidopsis Leaf Apoplast

The apoplast is a critical interface in plant-pathogen interactions particularly in the context of pattern-triggered immunity (PTI), which is initiated by recognition of microbe-associated molecular patterns (PAMPs). Our study characterizes the proteomic profile of the Arabidopsis apoplast during PTI induced by flg22, a 22 amino acid bacterial flagellin epitope, to elucidate the output of PTI. Apoplastic washing fluid (AWF) was extracted with minimal cytoplasmic contamination for LC-MS/MS analysis. We observed consistent identification of PTI enriched and depleted peptides across replicates with limited correlation between total protein abundance and transcript abundance. We observed topological bias in peptide recovery of receptor-like kinases with peptides predominantly recovered from their ectodomains. Notably, tetraspanin 8, an exosome marker, was enriched in PTI samples. We additionally confirmed increased concentrations of exosomes during PTI. This study enhances our understanding of the proteomic changes in the apoplast during plant immune responses and lays the groundwork for future investigations into the molecular mechanisms of plant defense under recognition of pathogen molecular patterns.

plant biology↗

bioRxiv2024

Distinct Virulence Mechanisms of Burkholderia gladioli in Onion Foliar and Bulb Scale Tissues

Slippery skin of onion caused by Burkholderia gladioli pv. alliicola (Bga) is a common bacterial disease reported from onion growing regions around the world. Despite the increasing attention in recent years, our understanding of the virulence mechanisms of this pathogen remains limited. In this study, we characterized the predicted genetic determinants of virulence in Bga strain 20GA0385 using reverse genetics approach. Using the closely related rice pathogen, B. glumae as a reference, comparative genomics analysis was performed to identify Bga candidate virulence factors and regulators. Marked and unmarked deletion mutants were generated using allelic exchange and the mutants were functionally validated using in vitro and in vivo assays. The role of mutants in pathogenic phenotypes was analyzed using onion foliar/seedling necrosis assays, the Red Scale Necrosis (RSN) assay and in planta bacterial population counts. The phytotoxin toxoflavin was a major contributor to foliar necrosis and bacterial populations whereas the type II and type III secretion system (T2SS/T3SS) were dispensable for foliar symptoms. In onion scale tissue, the T2SS single mutant gspC and its double and triple mutant derivatives all contributed to scale lesion area. Neither the lipocyclopeptide icosalide, toxoflavin, nor T3SS were required for scale symptoms. Our results suggest the quorum sensing tofIMR system in Bga regulates, toxoflavin, T2SS, and T3SS, contributing to onion symptom production. We show different virulence factors contribute to onion tissue-specific virulence patterns in Bga and that decreases in scale symptoms often do not result in decreased Bga populations in onion tissue.

microbiology↗

bioRxiv2024

Natural Language Processing-like Deep Learning Aided in Identification and Validation of Thiosulfinate Tolerance Clusters in Diverse Bacteria

Allicin tolerance (alt) clusters in phytopathogenic bacteria, which provide resistance to thiosulfinates like allicin, are challenging to find using conventional approaches due to their varied architecture and the paradox of being vertically maintained within genera despite likely being horizontally transferred. This results in significant sequential diversity that further complicates their identification. Natural language processing (NLP) - like techniques, such as those used in DeepBGC, offers a promising solution by treating gene clusters like a language, allowing for identifying and collecting gene clusters based on patterns and relationships within the sequences. We curated and validated alt-like clusters in Pantoea ananatis 97-1R (PA), Burkholderia gladioli pv. gladioli FDAARGOS 389 (BG), and Pseudomonas syringae pv. tomato DC3000 -(PTO). Leveraging sequences from the RefSeq bacterial database, we conducted comparative analyses of gene synteny, gene/protein sequences, protein structures, and predicted protein interactions. This approach enabled the discovery of several novel alt-like clusters previously undetectable by other methods, which were further validated experimentally. Our work highlights the effectiveness of NLP-like techniques for identifying underrepresented gene clusters and expands our understanding of the diversity and utility of alt-like clusters in diverse bacterial genera. This work demonstrates the potential of these techniques to simplify the identification process and enhance the applicability of biological data in real-world scenarios. Significance StatementThiosulfinates, like allicin, are potent antifeedants and antimicrobials produced by Allium species and pose a challenge for phytopathogenic bacteria. Phytopathogenic bacteria have been shown to utilize an allicin tolerance (alt) gene cluster to circumvent this host response, leading to economically significant yield losses. Due to the complexity of mining these clusters, we applied techniques akin to natural language processing to analyze Pfam domains and gene proximity. This approach led to the identification of novel alt-like gene clusters, showcasing the potential of artificial intelligence to reveal elusive and underrepresented genetic clusters and enhance our understanding of their diversity and role across various bacterial genera.

pathology↗

bioRxiv2024

Plasmids encode and can mobilize onion pathogenicity in Pantoea agglomerans

Pantoea agglomerans is one of four Pantoea species for which strains have been reported in the United States to cause bacterial rot of onion bulbs. However, not all P. agglomerans strains are pathogenic to onion. We characterized onion-associated strains of P. agglomerans to elucidate the genetic and genomic signatures of onion-pathogenic P. agglomerans. We collected >300 P. agglomerans strains associated with symptomatic onion plants and bulbs from public culture collections, research laboratories, and a multi-year survey in 11 states in the USA. Genome assemblies were generated for 87 P. agglomerans strains that showed a range in onion virulence phenotypes. Combining the 87 genome assemblies with 100 high-quality, public P. agglomerans genome assemblies identified two well-represented and well-supported P. agglomerans phylogroups. Strains causing severe symptoms on onion leaves and bulbs were only identified in Phylogroup II and encoded the HiVir biosynthetic cluster for the phytotoxin pantaphos, supporting the role of HiVir as a crucial pathogenicity factor. Using a MASH-based plasmid classification system, the P. agglomerans HiVir cluster was determined to be encoded in two distinct plasmid contexts: 1) as an accessory gene cluster on a conserved P. agglomerans plasmid (pAggl), or 2) on a mosaic cluster of plasmids common among onion strains (pOnion). Analysis of closed genomes of P. agglomerans revealed that the pOnion plasmids harbored alt genes responsible for encoding tolerance to the thiosulfinate defensive chemistry in Allium spp. Additionally, many of these pOnion plasmids harbored cop gene clusters, which confer resistance to copper. However, the pOnion plasmids encoded the HiVir cluster less frequently. We demonstrated that the pOnion plasmid pCB1C, encoding HiVir and alt clusters as well as an intact conjugative type IV secretion system (T4SS), can act as a natively mobilizable pathogenicity plasmid that transforms P. agglomerans Phylogroup I strains, including environmental strains, into virulent pathogens of onion. This work indicates a central role for plasmids and plasmid ecology in mediating P. agglomerans interactions with onion plants, with potential implications for onion bacterial disease management.

microbiology↗

bioRxiv2024

Thiosulfinate tolerance gene clusters are common features of Burkholderia onion pathogens.

Burkholderia gladioli pv. alliicola, B.cepacia, and B. orbicola are common bacterial pathogens of onion. Onions produce organosulfur thiosulfinate defensive compounds after cellular decompartmentalization. Using whole genome sequencing and in silico analysis, we identified putative thiosulfinate tolerance gene (TTG) clusters in multiple onion-associated Burkholderia species similar to those characterized in other Allium-associated bacterial endophytes and pathogens. Sequence analysis revealed the presence of three Burkholderia TTG cluster types with both Type A and Type B being broadly distributed in B. gladioli, B. cepacia, and B. orbicola in both the chromosome and plasmids. Based on isolate natural variation and generation of isogenic strains, we determined the in vitro and in vivo contribution of TTG clusters in B. gladioli, B. cepacia, and B. orbicola. The Burkholderia TTG clusters contributed to enhanced allicin tolerance and improved growth in filtered onion extract by all three species. TTG clusters also made clear contributions to B gladioli foliar necrosis symptoms and bacterial populations. Surprisingly, the TTG cluster did not contribute to bacterial populations in onion bulb scales by these three species. Based on our findings, we hypothesize onion-associated Burkholderia may evade or inhibit the production of thiosulfinates in onion bulb tissues.

microbiology↗

bioRxiv2022

The genetic requirements for HiVir-mediated onion necrosis by Pantoea ananatis, a necrotrophic plant pathogen

Pantoea ananatis is an unusual bacterial pathogen that lacks typical virulence determinants yet causes extensive necrosis in onion foliage and bulb tissues. The onion necrosis phenotype is dependent on the expression of a phosphonate toxin, pantaphos that is catalyzed by putative enzymes encoded by the HiVir gene cluster. The genetic contributions of individual hvr genes in HiVir-mediated onion necrosis remain largely unknown except for the first gene hvrA (phosphoenolpyruvate mutase, pepM) whose deletion resulted in the loss of onion pathogenicity. In this study, using gene deletion mutation and complementation, we report that of the ten remaining genes, hvrB-hvrF are also strictly required for the HiVir-mediated onion necrosis and in planta bacterial growth whereas hvrG-hvrJ partially contributed to these phenotypes. As the HiVir gene cluster is a common genetic feature shared among the onion-pathogenic P. ananatis strains, and as it could serve as a useful diagnostic marker of onion pathogenicity, we sought to understand the genetic basis of HiVir positive yet phenotypically deviant (non-pathogenic) strains. We identified and genetically characterized inactivating single nucleotide polymorphisms (SNPs) in essential hvr genes of six phenotypically deviant P. ananatis strains. Finally, inoculation of the cell-free spent medium of Ptac-driven HiVir strain caused P. ananatis-characteristic red onion scale necrosis (RSN) as well as cell death symptoms in tobacco. The co-inoculation of the spent medium with essential hvr mutant strains restored strains in planta populations to the wild-type level, suggesting that necrosis is important for proliferation of P. ananatis in onion tissue.

microbiology↗

bioRxiv2022

In planta transcriptomics reveals conflicts between pattern-triggered immunity and the AlgU sigma factor regulon

In previous work, we determined the transcriptomic impacts of flg22 pre-induced Pattern Triggered Immunity (PTI) in Arabidopsis thaliana on the pathogen Pseudomonas syringae pv. tomato DC3000 (Pto). During PTI exposure we observed expression patterns in Pto reminiscent of those previously observed in a Pto algU mutant. AlgU is a conserved extracytoplasmic function sigma factor which has been observed to regulate over 950 genes in Pto in vitro. We sought to identify the AlgU regulon in planta.and which PTI-regulated genes overlapped with AlgU-regulated genes. In this study, we analyzed transcriptomic data from RNA-sequencing to identify the AlgU in planta regulon and its relationship with PTI. Our results showed that approximately 224 genes are induced by AlgU, while another 154 genes are downregulated by AlgU in Arabidopsis during early infection. Both stress response and virulence-associated genes were induced by AlgU, while the flagellar motility genes are downregulated by AlgU. Under the pre-induced PTI condition, more than half of these AlgU-regulated genes have lost induction/suppression in contrast to naive plants, and almost all function groups regulated by AlgU were affected by PTI.

microbiology↗

bioRxiv2021

Genome context influences evolutionary flexibility of nearly identical type III effectors in two phytopathogenic Pseudomonads

Integrative Conjugative Elements (ICEs) are replicons that can insert and excise from chromosomal locations in a site specific manner, can conjugate across strains, and which often carry a variety of genes useful for bacterial growth and survival under specific conditions. Although ICEs have been identified and vetted within certain clades of the agricultural pathogen Pseudomonas syringae, the impact of ICE carriage and transfer across the entire P. syringae species complex remains underexplored. Here we identify and vet an ICE (PmaICE-DQ) from P. syringae pv. maculicola ES4326, a strain commonly used for laboratory virulence experiments, demonstrate that this element can excise and conjugate across strains, and contains loci encoding multiple type III effector proteins. Moreover, genome context suggests that another ICE (PmaICE-AOAB) is highly similar in comparison with and found immediately adjacent to PmaICE-DQ within the chromosome of strain ES4326, and also contains multiple type III effectors. Lastly, we present passage data from in planta experiments that suggests that genomic plasticity associated with ICEs may enable strains to more rapidly lose type III effectors that trigger R-gene mediated resistance in comparison to strains where nearly isogenic effectors are not present in ICEs. Taken together, our study sheds light on a set of ICE elements from P. syringae pv. maculicola ES4326 and highlights how genomic context may lead to different evolutionary dynamics for shared virulence genes between strains.

microbiology↗

bioRxiv2021

Elucidating the patterns of seed-to-seedling transmission of Xanthomonas citri pv. malvacearum, the causal agent of cotton bacterial blight

Cotton bacterial blight (CBB) was a major disease of cotton in the United States in the early part of the 20th century. The recent reemergence of CBB, caused by Xanthomonas citri pv. malvacearum (Xcm) revealed many gaps in our understanding of this important disease. In this study, we employed a field isolate of Xcm from Georgia USA (WT) to generate a non- pathogenic, hrcV mutant lacking a functional Type III Secretion System (T3SS-). We tagged the WT and T3SS- strains with an auto-bioluminescent Tn7 reporter and compared colonization patterns of susceptible and resistant cotton seedlings using macroscopic image analysis and bacterial load enumeration. Wildtype and T3SS- Xcm strains colonized cotton cotyledons of resistant and susceptible cotton cultivars. However, Xcm populations were significantly higher in susceptible seedlings inoculated with the WT strain. Additionally, WT and T3SS- Xcm strains systemically colonized true leaves, although at different rates. Finally, we observed that seed-to-seedling transmission of Xcm may involve systemic spread through the vascular tissue of cotton plants. These findings yield novel insights into potential Xcm reservoirs for CBB outbreaks.

plant biology↗

bioRxiv2020

The distribution of onion virulence gene clusters among Pantoea spp.

Pantoea ananatis is a gram-negative bacterium and the primary causal agent of center rot of onions in Georgia. Previous genomic studies identified two virulence gene clusters, HiVir and alt, associated with center rot. The HiVir gene cluster is required to induce necrosis on onion tissues via synthesis of a predicted small molecule toxin. The alt gene cluster aids in tolerance to thiosulfinates generated during onion tissue damage. Whole genome sequencing of other Pantoea species suggest that these gene clusters are present outside of P. ananatis. To assess the distribution of these gene clusters, two PCR primer sets were designed to detect the presence of HiVir and alt. Two hundred fifty-two strains of Pantoea spp. were phenotyped using the red onion scale necrosis (RSN) assay and were assayed using PCR for the presence of these virulence genes. A diverse panel of strains from three distinct culture collections comprised of 24 Pantoea species, 41 isolation sources, and 23 countries, collected from 1946-2019, were tested. There is a significant association between the alt PCR assay and Pantoea strains recovered from symptomatic onion (P<0.001). There is also a significant association of a positive HiVir PCR and RSN assay among P. ananatis strains but not among Pantoea spp., congeners. This may indicate a divergent HiVir cluster or different pathogenicity and virulence mechanisms. Last, we describe natural alt positive [RSN+/HiVir+/alt+] P. ananatis strains, which cause extensive bulb necrosis in a neck-to-bulb infection assay compared to alt negative [RSN+/HiVir+/alt-] P. ananatis strains. A combination of assays that include PCR of virulence genes [HiVir and alt] and an RSN assay can potentially aid in identification of onion-bulb-rotting pathogenic P. ananatis strains.

plant biology↗

bioRxiv2020

RpoS Contributes to Successful Opportunistic Colonization by Human Enteric Pathogens during Plant Disease

With an increase in foodborne illnesses associated with the consumption of fresh produce, it is important to understand the interactions between human bacterial enteric pathogens and plants. It was previously established that diseased plants can create a permissive environment for opportunistic endophytic colonization of enteric pathogens. However, the factors that contribute to the colonization of enteric pathogens during plant disease are largely unknown. Here, we show that both strain and plant host factors contribute to significantly increased populations of enteric pathogens when co-inoculated with the plant pathogen, P. syringae pv. tomato. The two Salmonella enterica strains DM10000 and 14028S, differ in their ability to metabolize host-derived apoplastic carbohydrates dependent on the sigma factor RpoS. The rpoS gene is an important strain factor for endophytic colonization by S. enterica during plant disease. Our results suggest that rpoS plays a crucial role during in planta colonization, balancing nutrient metabolism and stress responses. ImportanceFoodborne illnesses caused by the bacterial human enteric pathogens, E. coli O157:H7 and S. enterica, often results in vomiting and diarrhea. If left untreated, this illness can cause dehydration and sometimes death of a patient. Both E. coli O157:H7 and S. enterica have caused repeated fresh produce-associated epidemics. Crop disease could promote the ability of plants to act as reservoirs for produce-borne outbreaks. Plant pathogens dampen plant immunity, which allows for a more permissive environment for human enteric pathogens to grow. These internalized enteric pathogen populations are especially dangerous since they cannot be removed by washing alone. Therefore, the need to understand the factors that contribute to the opportunistic colonization of human enteric pathogens during plant disease is apparent. Our research has identified host and strain factors that contribute to opportunistic colonization of diseased plants, which will inform the development of future management strategies to mitigate future outbreaks.

pathology↗

bioRxiv2020

Pantoea ananatis defeats Allium chemical defenses with a plasmid-borne virulence gene cluster

Onion (Allium. cepa L), garlic (A. sativum L.), and other members of the Allium genus produce volatile antimicrobial thiosulfinates upon cellular damage. Allicin has been known since the 1950s as the primary antimicrobial thiosulfinate compound and odorant produced by garlic. However, the roles of endogenous thiosulfinate production in host-bacterial pathogen interactions have not been described. The bacterial onion pathogen Pantoea ananatis, which lacks both the virulence Type III and Type II Secretion Systems, induces necrotic symptoms and extensive cell death in onion tissues dependent on a proposed secondary metabolite synthesis chromosomal gene cluster. We found strong correlation between the genetic requirements for P. ananatis to colonize necrotized onion tissue and its capacity for tolerance to the thiosulfinate allicin based on the presence of an eleven gene, plasmid-borne, virulence cluster of sulfur/redox genes. We have designated them alt genes for allicin tolerance. We show that allicin and onion thiosulfinates restrict bacterial growth with similar kinetics. The alt gene cluster is sufficient to confer allicin tolerance and protects the glutathione pool during allicin treatment. Independent alt genes make partial phenotypic contributions indicating that they function as a collective cohort to manage thiol stress. Our work implicates endogenous onion thiosulfinates produced during cellular damage as mediators of interactions with bacteria. The P. ananatis-onion pathosystem can be modeled as a chemical arms race of pathogen attack, host chemical counter-attack, and pathogen resistance. Significance StatementAlliums (e.g. onion and garlic), after sustaining cellular damage, produce potent antimicrobial thiosulfinates that react with cellular thiols. The bacterial onion pathogen Pantoea ananatis, which lacks the virulence Type III and Type II Secretion Systems, induces cell death and necrotic symptoms on onions. We have identified a plasmid-borne cluster of sulfur/redox virulence genes that 1) are required for P. ananatis to colonize necrotized onion tissue, 2) are sufficient for tolerance to the thiosulfinates, and, 3) protect the glutathione pool during thiosulfinate treatment. We propose that the thiosulfinate production potential of Allium spp. governs Allium-bacterial interaction outcomes and that the P. ananatis-onion pathosystem can be modeled as a chemical arms race of attack and counterattack between the pathogen and host.

plant biology↗