Biology subjects

Kvitko, B.

Publications and source records attributed to Kvitko, B..

9 recordsLinked to original sources

bioRxiv2026

Evidence for Burkholderia gladioli pv. alliicola Extracellular Detoxification of Thiosulfinates

Onion tissues produce antimicrobial thiosulfinates after tissue damage and cellular decompartmentalization. Burkholderia gladioli pv. alliicola (Bga), a common onion pathogen, encodes a thiosulfinate tolerance gene (TTG) cluster that protects the bacterium during thiosulfinate exposure. Previous work showed that the TTG cluster contributes to foliar infection but has little effect on infection of onion bulb tissue. To further examine Bga-thiosulfinate interactions in foliar and bulb tissues, we used a thiosulfinate-responsive PaltR-Lux reporter strain to determine when and where Bga encounters thiosulfinates. In leaves, Bga-induced necrosis was associated with de-repression of the PaltR-Lux reporter and coincided with a contribution of the TTG cluster to bacterial population size, indicating thiosulfinate exposure during foliar infection. In contrast, TTG mutants and wild-type (WT) strains showed similar growth in scales, and PaltR-Lux signal declined as scale necrosis progressed, suggesting limited thiosulfinate exposure during bulb colonization. However, when necrosis was induced by the non-native toxin pantaphos, PaltR-Lux was de-repressed and recovery of the TTG mutant was reduced. These results indicate that Bga encounters thiosulfinates during foliar infection but largely avoids exposure during bulb infection. Preconditioning the TTG mutant in onion scale tissue did not alter its thiosulfinate sensitivity in vitro, arguing against an infection-associated thiosulfinate exclusion mechanism. In contrast, partial rescue of the TTG mutant by the WT strain in zone-of-inhibition co-plating assays suggests extracellular thiosulfinate detoxification. Together, these findings indicate that Bga detoxifies thiosulfinates released during bulb necrosis, limiting thiosulfinate exposure during onion bulb infection. The molecular basis for detoxification and tissue specificity remain unresolved.

microbiology↗

bioRxiv2023

Pantailocins: Phage-derived Bacteriocins from Pantoea ananatis and Pantoea stewartii subsp. indologenes

Phage-derived bacteriocins are highly specific and effective antimicrobial molecules which have successfully been used as prophylactic treatments to prevent phytopathogen infections. Given the specificity of tailocins, a necessary step for broadening the tailocin catalog and for extending applicability across systems and diseases is the screening of new clades of phytopathogens for production of molecules with tailocin-like killing activity. Here, we describe production by and sensitivity of strains to tailocins produced by Pantoea ananatis and Pantoea stewartii subsp. indologenes. Phylogenetic evidence suggests that these tailocins are derived from Myoviridae family phage like many previously described R type syringacins and R type pyocins, but also suggests that cooption from phage occurred independently of previously described tailocins. Since these tailocin encoding loci are present in the same genomic locations across multiple strains of both species and display a level of divergence that is consistent with other shared regions between the genomes and with vertical inheritance of the locus, we refer to them broadly as pantailocins.

microbiology↗

bioRxiv2023

A critical role of a eubiotic microbiota in gating proper immunocompetence in Arabidopsis

Although many studies have shown that microbes can ectopically stimulate or suppress plant immune responses, the fundamental question of whether the entire preexisting microbiota is indeed required for proper development of plant immune response remains unanswered. Using a recently developed peat-based gnotobiotic plant growth system we found that Arabidopsis grown in the absence of a natural microbiota lacked age-dependent maturation of plant immune response and were defective in several aspects of pattern-triggered immunity. Axenic plants exhibited hypersusceptibility to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and the fungal pathogen Botrytis cinerea. Microbiota-mediated immunocompetence was suppressed by rich nutrient conditions, indicating a tripartite interaction between the host, microbiota, and abiotic environment. A synthetic microbiota composed of 48 culturable bacterial strains from the leaf endosphere of healthy Arabidopsis plants was able to substantially restore immunocompetence similar to plants inoculated with a soil-derived community. In contrast, a 52-member dysbiotic synthetic leaf microbiota overstimulated the immune transcriptome. Together, these results provide evidence for a causal role of a eubiotic microbiota in gating proper immunocompetence and age-dependent immunity in plants.

plant biology↗

bioRxiv2023

Shoot maturation strengthens FLS2-mediated resistance to Pseudomonas syringae

A temporal-spatial regulation of immunity components is essential for properly activating plant defense response. Flagellin-sensing 2 (FLS2) is a surface-localized receptor that recognizes bacterial flagellin. The immune function of FLS2 is compromised in early stages of shoot development. However, the underlying mechanism for the age-dependent FLS2 signaling is not clear. Here, we show that the reduced basal immunity of juvenile leaves against Pseudomonas syringae pv. tomato DC3000 is independent of FLS2. The flg22-induced marker gene expression and ROS activation were comparable in juvenile and adult stage, but callose deposition was more evident in the adult stage than that of juvenile stage. We further demonstrated that microRNA156, a master regulator of plant aging, suppressed callose deposition in juvenile leaves in response to flg22 but not the expression of FLS2 and FRK1 (Flg22-induced receptor-like kinase 1). Altogether, we revealed an intrinsic mechanism that regulates the amplitude of FLS2-mediated resistance during aging.

plant biology↗

bioRxiv2022

Convergent Gene Expression Patterns During Compatible Interactions Between Two Pseudomonas syringae Pathovars and a Common Host (Nicotiana benthamiana)

Summary/Abstract Pseudomonas syringae is a diverse phytopathogenic species complex, and includes strains that can cause disease across a wide variety of plant species. Much previous research into the molecular basis of immunity and infection has focused on pathogen and plant responses in a handful of model strains and hosts, and with a tacit assumption that early steps in infection and host resistance are generalizable to the species complex and across plant hosts as a whole. Here, we provide a test of this assumption by measuring the dual pathogen and host transcriptomes of two distinct pathogenic lineages of P. syringae during compatible infection of a shared model host (Nicotiana benthamiana). Our results demonstrate that, with a handful of exceptions, host plants largely respond in a similar way to both pathogenic lineages and both bacterial pathogens possess highly similar transcriptional responses at 5 hours post inoculation. However, we also highlight that subsets of genes with differential expression patterns in both bacteria and host which likely represent strain-specific responses.

plant biology↗

bioRxiv2022

A novel biosynthetic gene cluster across the Pantoea species complex is important for pathogenicity in onion

Onion center rot is caused by at least four species of Pantoea (P. ananatis, P. agglomerans, P. allii, and P. stewartii subsp. indologenes). Critical onion pathogenicity determinants for P. ananatis were recently described but whether those determinants are common among other onion-pathogenic Pantoea species remains unknown. In this work, we report onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii. We identified two distinct secondary metabolite biosynthetic gene clusters in different strains of onion pathogenic P. stewartii subsp. indologenes. One cluster is similar to the previously described HiVir phosphonate biosynthetic cluster of P. ananatis and another is a novel putative phosphonate biosynthetic gene cluster, which we name "Halophos". The Halophos gene cluster was also identified in P. allii strains. Both clusters are predicted to be phosphonate biosynthetic clusters based on the presence of a characteristic phosphenolpyruvate phosphomutase (pepM) gene. The deletion of pepM gene from either the P. stewartii subsp. indologenes HiVir or Halophos clusters caused loss of necrosis on onion leaves and red onion scales, and resulted in significantly lower bacterial populations compared to the corresponding wildtype and complemented strains. Seven (halB-halH) out of eleven genes (halA-halK) in the Halophos gene cluster are required for onion necrosis phenotypes. The onion non-pathogenic strain PNA15-2 gained the capacity to cause foliar necrosis on onion via exogenous expression of a minimal seven gene Halophos cluster (halB -halH). Furthermore, cell-free culture filtrates of PNA14-12 expressing the intact Halophos-gene cluster caused necrosis on onion leaves consistent with the presence of a secreted toxin. Together, these observations indicated that pepM genes in both phosphonate biosynthetic gene clusters (HiVir and Halophos) are important for Pantoea spp. onion pathogenicity and the biosynthetic product of the Halophos cluster causes necrosis on onion leaf tissue. Overall, this is the first report of onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii. Author summaryOnion center rot is caused by multiple Pantoea species including P. stewartii subsp. indologenes and P. allii. We identified two distinct secondary metabolite biosynthetic clusters associated with onion pathogenic strains, the validated HiVir phosphonate cluster and a putative phosphonate biosynthetic cluster that we named as Halophos based on the associated "halo" phenotype on the red onion scales. We found that pepM genes from each cluster (HiVir and Halophos) are required for onion infection by P. stewartii subsp. indologenes and P. allii but not for millet infection by P. stewartii subsp. indologenes. Conversely, the T3SS was important for millet infection by P. stewartii subsp. indologenes but not onion infection. Induction of the intact Halophos cluster was associated with the accumulation of a necrosis-inducing factor in culture, which suggests it might be a secreted phytotoxin. Seven of the eleven Halophos cluster genes are required for onion necrosis phenotypes and expression of this minimal cluster conferred a limited onion necrosis phenotype to an onion-non-pathogenic Pantoea strain. We provide evidence of a Halophos biosynthetic gene cluster to be associated with onion pathogenicity in strains of P. stewartii subsp. indologenes and P. allii.

microbiology↗

bioRxiv2021

Pseudomonas syringae effector AvrE associates with plant membrane nanodomains and binds phosphatidylinositides in vitro

Bacterial phytopathogens deliver effector proteins into host cells as key virulence weapons to cause disease. Extensive studies revealed diverse functions and biochemical properties of different effector proteins from pathogens. In this study, we show that the Pseudomonas syringae effector AvrE, the founding member of a broadly conserved and pathologically important bacterial effector family, binds to phosphatidylinositides (PIPs) in vitro and shares some properties with eukaryotic PROPPINs ({beta}-propellers that bind polyphosphoinositides). In planta pull down experiments with transgenic Arabidopsis plants expressing AvrE revealed that AvrE is associated with several plant proteins including plasma membrane lipid-raft proteins. These results shed new light on the properties of a bacterial effector that is crucial for bacterial virulence in plants.

pathology↗

bioRxiv2020

Pan-genome-wide analysis of Pantoea ananatis identified genes linked to pathogenicity in onion

Pantoea ananatis is a member of a Pantoea spp. complex that causes center rot of onion, which significantly affects onion yield and quality. This pathogen does not have typical virulence factors like type II or type III secretion systems but appears to require a biosynthetic gene-cluster, HiVir/PASVIL (located chromosomally), for a phosphonate secondary metabolite, and the onion-virulence regions, OVR (localized on a megaplasmid), for onion pathogenicity and virulence, respectively. We conducted a deep pan-genome-wide association study (pan-GWAS) to predict additional genes associated with pathogenicity in P. ananatis using a panel of diverse strains (n = 81). We utilized a red-onion scale necrosis assay as an indicator of pathogenicity. Based on this assay, we differentiated pathogenic (n = 51)- vs. non-pathogenic (n = 30)-strains phenotypically. Pan-GWAS revealed a large core genome of 3,153 genes and a flexible accessory genome of [≤]5,065 genes. Phylogenomic analysis using pan-GWAS and presence and absence variants (PAVs) distinguished red-scale necrosis inducing (pathogenic) strains from non-scale necrosis inducing (non-pathogenic) strains of P. ananatis. The pan-GWAS also predicted 42 genes, including 14 from the previously identified HiVir/PASVIL cluster associated with pathogenicity, and 28 novel genes that were not previously associated with pathogenicity in onion. Of the 28 novel genes identified, eight have annotated functions of site-specific tyrosine kinase, N-acetylmuramoyl-L-alanine amidase, TraR/DksA family transcriptional regulator, and HTH-type transcriptional regulator. The remaining 20 genes are currently hypothetical. This is the first report of using pan-GWAS on P. ananatis for the prediction of novel genes contributing to pathogenicity in onion, which will be utilized for further functional analyses. Pan-genomic differences (using PAVs) differentiated onion pathogenic from non-pathogenic strains of P. ananatis, which has been difficult to achieve using single or multiple gene-based phylogenetic analyses. The pan-genome analysis also allowed us to evaluate the presence and absence of HiVir/PASVIL genes and 11 megaplasmid-borne OVR-A genes regarded as the alt cluster that aid in P. ananatis colonization in onion bulbs. We concluded that HiVir/PASVIL genes are associated with pathogenic P. ananatis strains and the alt gene cluster alone is not responsible for pathogenicity on onion. The pan-genome also provides clear evidence of constantly evolving accessory genes in P. ananatis that may contribute to host-range expansion and niche-adaptation. Author summaryPantoea ananatis is a major bacterial pathogen that causes center rot of onion and diseases of a number of other plant species. In order to understand the genome architecture and identify genes responsible for pathogenicity in onion, a pan-genome analysis was performed. We used 81 strains of P. ananatis collected over 20 years from different regions of the state of Georgia, USA. The pan-genome study identified a core genome with a conserved set of genes and an accessory genome that displayed variation among strains. We conducted pan-GWAS (pan-genome-wide association study) using presence and absence variants (PAVs) in the genomes and associated onion-pathogenic phenotypes based on a red-onion scale necrosis assay. The study resulted in identification of genes, including a cluster of chromosomal HiVir/PASVIL genes, that are significantly associated with the onion pathogenic phenotype. In addition, we identified 28 novel genes, a majority of which (n = 20) have hypothetical functions. We concluded and further substantiated earlier findings that a cluster of genes is responsible for pathogenicity on onion. The pan-genome analysis also allowed us to evaluate the presence and absence of HiVir/PASVIL genes and 11 megaplasmid-borne OVR-A genes regarded as the alt cluster that aid in bacterial colonization of onion bulbs by P. ananatis strains. We concluded that HiVir/PASVIL genes are associated with onion-pathogenic strains, and the alt gene cluster alone is not responsible for pathogenicity on onion. This study also provides potential evidence of constantly evolving accessory genes in P. ananatis which may help in host range expansion and adaptation to diverse niches.

genomics↗

bioRxiv2017

Validation of RT-qPCR approaches to monitor Pseudomonas syringae gene expression during infection and exposure to pattern-triggered immunity

Pseudomonas syringae pv. tomato DC3000 (DC3000) is an important model plant pathogen, with a fully annotated genome and multiple compatible plant hosts. Very few studies have examined the regulation of DC3000 gene expression in vivo. We developed a RT-qPCR assay to monitor transcriptional changes in DC3000 inoculated into Arabidopsis thaliana leaves during disease and exposure to pattern-triggered immunity (PTI). In our approach, bacterial RNA concentrations in total tissue RNA are standardized using P.syringae-specific16S ribosomal RNA primers. We validated multiple stable reference genes for normalization in calculating the relative expression of genes of interest. We used empirically derived rates of amplification efficiency to calculate relative expression of key marker genes for virulence-associated regulation. We demonstrated that exposure to PTI alters DC3000 expression of Type III secretion system, coronatine synthesis genes and flagellar marker genes.

microbiology↗