Publications

4498

Insect–microbe endosymbiotic associations are omnipresent in nature, wherein the symbiotic microbes often play pivotal biological roles for their host insects. In particular, insects utilizing nutritionally imbalanced food sources are dependent on specific microbial symbionts to compensate for the nutritional deficiency via provisioning of B vitamins in blood-feeding insects, such as tsetse flies, lice, and bedbugs. Bat flies of the family Nycteribiidae (Diptera) are blood-sucking ectoparasites of bats and shown to be associated with co-speciating bacterial endosymbiont “Candidatus Aschnera chinzeii,” although functional aspects of the microbial symbiosis have been totally unknown. In this study, we report the first complete genome sequence of Aschnera from the bristled bat fly Penicillidia jenynsii. The Aschnera genome consisted of a 748,020 bp circular chromosome and a 18,747 bp circular plasmid. The chromosome encoded 603 protein coding genes (including 3 pseudogenes), 33 transfer RNAs, and 1 copy of 16S/23S/5S ribosomal RNA operon. The plasmid contained 10 protein coding genes, whose biological function was elusive. The genome size, 0.77 Mbp, was drastically reduced in comparison with 4–6 Mbp genomes of free-living γ-proteobacteria. Accordingly, the Aschnera genome was devoid of many important functional genes, such as synthetic pathway genes for purines, pyrimidines, and essential amino acids. On the other hand, the Aschnera genome retained complete or near-complete synthetic pathway genes for biotin (vitamin B7), tetrahydrofolate (vitamin B9), riboflavin (vitamin B2), and pyridoxal 5'-phosphate (vitamin B6), suggesting that Aschnera provides these vitamins and cofactors that are deficient in the blood meal of the host bat fly. Similar retention patterns of the synthetic pathway genes for vitamins and cofactors were also observed in the endosymbiont genomes of other blood-sucking insects, such as Riesia of human lice, Arsenophonus of louse flies, and Wigglesworthia of tsetse flies, which may be either due to convergent evolution in the blood-sucking host insects or reflecting the genomic architecture of Arsenophonus-allied bacteria.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The phloem-restricted bacterium Candidatus Liberibacter asiaticus (CLas) is considered to be the main pathogen responsible for HLB. There is currently no effective practical strategy for the control of HLB. Our understanding of how pathogens cause HLB is limited because CLas has not been artificially cultured. In this study, 15 potential virulence factors were predicted from the proteome of CLas through DeepVF and PHI-base searches. One among them, FlgI, was found to inhibit yeast growth when expressed in Saccharomyces cerevisiae. The expression of the signal peptide of FlgI fused with PhoA in Escherichia coli resulted in the discovery that FlgI was a novel Sec-dependent secretory protein. We further found that the carboxyl-terminal HA-tagged FlgI was secreted via outer membrane vesicles in Sinorhizobium meliloti. Fluoresence localization of transient expression FlgI-GFP in Nicotiana benthamiana revealed that FlgI is mainly localized in the cytoplasm, cell periphery, and nuclear periphery of tobacco cells. In addition, our experimental results suggest that FlgI has a strong ability to induce callose deposition and cell necrosis in N. benthamiana. Finally, by screening a large library of compounds in a high-throughput format, we found that cyclosporin A restored the growth of FlgI-expressing yeast. These results confirm that FlgI is a novel Sec-dependent effector, enriching our understanding of CLas pathogenicity and helping to develop new and more effective strategies to manage HLB.

IntroductionThe potato psyllid Bactericera cockerelli is the insect vector of the fastidious bacterium ‘Candidatus Liberibacter solanacearum’. The bacterium infects both B. cockerelli and plant species, causing zebra chip (ZC) disease of potato and vein-greening disease of tomato. Temperatures are known to influence the initiation and progression of disease symptom in the host plant, and seasonal transitions from moderate to high temperatures trigger psyllid dispersal migration to facilitate survival.Methods‘Ca. L. solanacearum’ -infected and uninfected psyllids were reared at previously established ‘permissible’, optimal, and ‘non-permissible’ and temperatures of 18°C, 24°C, and 30°C, respectively. Gene expression profiles for ‘Ca. L. solanacearum’-infected and -uninfected adult psyllids reared at different temperatures were characterized by Illumina RNA-Seq analysis. Bacterial genome copy number was quantified by real-time quantitative-PCR (qPCR) amplification.ResultsRelative gene expression profiles varied in psyllids reared at the three experimental temperatures. Psyllids reared at 18°C and 30°C exhibited greater fold-change increased expression of stress- and ‘Ca. L. solanacearum’ invasion-related proteins. Quantification by qPCR of bacterial genome copy number revealed that ‘Ca. L. solanacearum’ accumulation was significantly lower in psyllids reared at 18°C and 30°C, compared to 24°C.DiscussionTemperature is a key factor in the life history of potato psyllid and multiplication/accumulation of ‘Ca. L. solanacearum’ in both the plant and psyllid host, influences the expression of genes associated with thermal stress tolerance, among others, and may have been instrumental in driving the co-evolution of the pathosystem.

‘Candidatus Phytoplasma meliae’ is a pathogen associated with chinaberry yellowing disease, which has become a major phytosanitary problem for chinaberry forestry production in Argentina. Despite its economic impact, no genome information of this phytoplasma has been published, which has hindered its characterization at the genomic level. In this study, we used a metagenomics approach to analyze the draft genome of the ‘Ca. P. meliae’ strain ChTYXIII. The draft assembly consisted of twenty-one contigs with a total length of 751.949 bp, and annotation revealed 669 CDSs, 34 tRNAs, and 1 set of rRNA operons. The metabolic pathways analysis showed that ChTYXIII contains the complete core genes for glycolysis and a functional Sec system for protein translocation. Our phylogenomic analysis based on 133 single-copy genes and genome-to-genome metrics supports the classification as unique ‘Ca. P. species’ within the MPV clade. We also identified 31 putative effectors, including a homolog to SAP11 and others that have only been described in this pathogen. Our ortholog analysis revealed 37 PMU core genes in the genome of ‘Ca. P. meliae’ ChTYXIII, leading to the identification of 2 intact PMUs. Our work provides important genomic information for ‘Ca. P. meliae’ and others phytoplasmas for the 16SrXIII (MPV) group.

Abstract Historically, Xanthomonas species are primarily known for their pathogenicity against plants, but recently, there have been more findings of non-pathogenic xanthomonads. In the present study, we report isolates from healthy rice seeds that belong to a new species, Xanthomonas protegens, a protector of the rice plants against a serious pathogenic counterpart, i.e. X. oryzae pv. oryzae upon leaf clip co-inoculation. The new member species is non-pathogenic to rice and lacks a type III secretion system. The pangenome investigation revealed a large number of unique genes, including a novel lipopolysaccharide biosynthetic gene cluster, that might be important in its adaptation. The phylo-taxonogenomic analysis revealed that X. protegens is a taxonomic outlier species of X. sontii, a core, vertically transmitted rice seed endophyte with numerous probiotic properties. Interestingly, X. sontii is also reported as a keystone species of healthy rice seed microbiome. The findings and resources will help in the development of unique gene markers and evolutionary studies of X. sontii as a successful symbiont and X. oryzae as a serious pathogen. Here, we propose X. protegens sp. nov. as a novel species of the genus Xanthomonas with PPL118 = MTCC 13396 = CFBP 9164 = ICMP 25181 as the type strain. PPL117, PPL124, PPL125, and PPL126 are other strains of the species.

Abstract Five halophilic archaeal strains, XH8T, CK5-1T, GDY1T, HW8-1T, and XH21T, were isolated from commercial coarse salt produced in different regions of China. Their 16S rRNA and rpoB′ gene sequences indicated that four of the strains (CK5-1T, GDY1T, HW8-1T, and XH21T) represent distinct species within the genus Haloplanus (family Haloferacaceae), while strain XH8T represents a novel genus within the same family. These assignments were supported by phylogenetic and phylogenomic analyses, which showed that strains CK5-1T, GDY1T, HW8-1T, and XH21T cluster with the current species of the genus Haloplanus, while strain XH8T forms a separate branch from the genus Haloplanus. The digital DNA–DNA hybridization and average amino acid identity (AAI) values among these four strains and the current members of the genus Haloplanus were 23.1%–35.2% and 75.9%–83.8%, respectively; and those values between strain XH8T and other genera in the family Haloferacaceae were 18.8%–33.6% and 59.8%–66.6%, respectively, much lower than the threshold values for species demarcation. Strain XH8T may represent a novel genus of the family Haloferacaceae according to the cut-off value of AAI (≤72.1%) proposed to differentiate genera within the family Haloferacaceae. These five strains could be distinguished from the related species according to differential phenotypic characteristics. Based on these results, it is proposed that strain XH8T represents a novel genus within the family Haloferacaceae, and strains CK5-1T, GDY1T, HW8-1T, and XH21T represent four novel species of the genus Haloplanus, respectively. Additionally, these five strains possess genes encoding enzymes critical for the fermentation process in salt-fermented foods, indicating their potential as starter cultures for these applications.

AbstractBackgroundAlthough archaea are widespread in terrestrial environments, little is known about the selection forces that shape their composition, functions, survival, and proliferation strategies in the rhizosphere. The ammonia-oxidizing archaea (AOA), which are abundant in soil environments, catalyze the first step of nitrification and have the potential to influence plant growth and development significantly.ResultsBased on archaeal 16S rRNA andamoAgene (encoding the ammonia monooxygenase subunit A) amplicon sequencing analysis, distinct archaeal communities dominated by AOA were found to be associated with the root systems of pepper (Capsicum annuumL.) and ginseng (Panax ginsengC.A. Mey.) plants compared to bulk soil not penetrated by roots. AOA related to “CandidatusNitrosocosmicus”, which, unlike most other AOA, harbor genes encoding manganese catalase (MnKat), dominated rhizosphere soils, and thus contributed to the development of distinct archaeal communities in rhizospheres. Accordingly, for both plant species, the copy number ratios of AOA MnKat genes toamoAgenes were significantly higher in rhizosphere soils than in bulk soils. In contrast to MnKat-negative strains from other AOA clades, the catalase activity of a representative isolate of “Ca.Nitrosocosmicus” was demonstrated. Members of this clade were enriched in H2O2-amended bulk soils, and constitutive expression of their MnKat gene was observed in both bulk and rhizosphere soils.ConclusionsDue to their abundance, “Ca.Nitrosocosmicus” members can be considered key players mediating the nitrification process in rhizospheres. The selection of this MnKat-containing AOA in rhizospheres of several agriculturally important plants hints at a previously overlooked AOA-plant interaction. For additional mechanistic analyses of the interaction, this key clade of AOA with cultured representatives can be employed.

Abstract The evolutionary trajectory of Methylophilaceae includes habitat transitions from freshwater sediments to freshwater and marine pelagial that resulted in genome reduction (genome-streamlining) of the pelagic taxa. However, the extent of genetic similarities in the genomic structure and microdiversity of the two genome-streamlined pelagic lineages (freshwater “Ca. Methylopumilus” and the marine OM43 lineage) has so far never been compared. Here, we analyzed complete genomes of 91 “Ca. Methylopumilus” strains isolated from 14 lakes in Central Europe and 12 coastal marine OM43 strains. The two lineages showed a remarkable niche differentiation with clear species-specific differences in habitat preference and seasonal distribution. On the other hand, we observed a synteny preservation in their genomes by having similar locations and types of flexible genomic islands (fGIs). Three main fGIs were identified: a replacement fGI acting as phage defense, an additive fGI harboring metabolic and resistance-related functions, and a tycheposon containing nitrogen-, thiamine-, and heme-related functions. The fGIs differed in relative abundances in metagenomic datasets suggesting different levels of variability ranging from strain-specific to population-level adaptations. Moreover, variations in one gene seemed to be responsible for different growth at low substrate concentrations and a potential biogeographic separation within one species. Our study provides a first insight into genomic microdiversity of closely related taxa within the family Methylophilaceae and revealed remarkably similar dynamics involving mobile genetic elements and recombination between freshwater and marine family members.

Abstract Insects engage in manifold interactions with bacteria that can shift along the parasitism–mutualism continuum. However, only a small number of bacterial taxa managed to successfully colonize a wide diversity of insects, by evolving mechanisms for host-cell entry, immune evasion, germline tropism, reproductive manipulation, and/or by providing benefits to the host that stabilize the symbiotic association. Here, we report on the discovery of an Enterobacterales endosymbiont (Symbiodolus, type species Symbiodolus clandestinus) that is widespread across at least six insect orders and occurs at high prevalence within host populations. Fluorescence in situ hybridization in several Coleopteran and one Dipteran species revealed Symbiodolus’ intracellular presence in all host life stages and across tissues, with a high abundance in female ovaries, indicating transovarial vertical transmission. Symbiont genome sequencing across 16 host taxa revealed a high degree of functional conservation in the eroding and transposon-rich genomes. All sequenced Symbiodolus genomes encode for multiple secretion systems, alongside effectors and toxin-antitoxin systems, which likely facilitate host-cell entry and interactions with the host. However, Symbiodolus-infected insects show no obvious signs of disease, and biosynthetic pathways for several amino acids and cofactors encoded by the bacterial genomes suggest that the symbionts may also be able to provide benefits to the hosts. A lack of host-symbiont cospeciation provides evidence for occasional horizontal transmission, so Symbiodolus’ success is likely based on a mixed transmission mode. Our findings uncover a hitherto undescribed and widespread insect endosymbiont that may present valuable opportunities to unravel the molecular underpinnings of symbiosis establishment and maintenance.