Publications

3681

Phytoplasmas are linked to diseases in hundreds of economically important crops, including carrots. In carrots, phytoplasmosis is associated with leaf chlorosis and necrosis, coupled with inhibited root system development, ultimately leading to significant economic losses. During a field study conducted in Baden-Württemberg (Germany), two strains of the provisional taxon ‘Candidatus Phytoplasma asteris’ were identified within a carrot plot. For further analysis, strains M8 and M33 underwent shotgun sequencing, utilising single-molecule-real-time (SMRT) long-read sequencing and sequencing-by-synthesis (SBS) paired-end short-read sequencing techniques. Hybrid assemblies resulted in complete de novo assemblies of two genomes harboring circular chromosomes and two plasmids. Analyses, including average nucleotide identity and sequence comparisons of established marker genes, confirmed the phylogenetic divergence of ‘Ca. P. asteris’ and a different assignment of strains to the 16S rRNA subgroup I-A for M33 and I-B for M8. These groups exhibited unique features, encompassing virulence factors and genes, associated with the mobilome. In contrast, pan-genome analysis revealed a highly conserved gene set related to metabolism across these strains. This analysis of the Aster Yellows (AY) group reaffirms the perception of phytoplasmas as bacteria that have undergone extensive genome reduction during their co-evolution with the host and an increase of genome size by mobilome.

ABSTRACT Bacterial endosymbionts of eukaryotic hosts typically experience massive genome reduction, but the underlying evolutionary processes are often obscured by the lack of free-living relatives. Endomicrobia, a family-level lineage of host-associated bacteria in the phylum Elusimicrobiota that comprises both free-living representatives and endosymbionts of termite gut flagellates, are an excellent model to study evolution of intracellular symbionts. We reconstructed 67 metagenome-assembled genomes (MAGs) of Endomicrobiaceae among more than 1,700 MAGs from the gut microbiota of a wide range of termites. Phylogenomic analysis confirmed a sister position of representatives from termites and ruminants, and allowed to propose eight new genera in the radiation of Endomicrobiaceae . Comparative genome analysis documented progressive genome erosion in the new genus Endomicrobiellum , which comprises all flagellate endosymbionts characterized to date. Massive gene losses were accompanied by the acquisition of new functions by horizontal gene transfer, which led to a shift from a glucose-based energy metabolism to one based on sugar phosphates. The breakdown of glycolysis and many anabolic pathways for amino acids and cofactors in several subgroups was compensated by the independent acquisition of new uptake systems, including an ATP/ADP antiporter, from other gut microbiota. The putative donors are mostly flagellate endosymbionts from other bacterial phyla, including several, hitherto unknown lineages of uncultured Alphaproteobacteria , documenting the importance of horizontal gene transfer in the convergent evolution of these intracellular symbioses. The loss of almost all biosynthetic capacities in some lineages of Endomicrobiellum suggests that their originally mutualistic relationship with flagellates is on its decline. IMPORTANCE Unicellular eukaryotes are frequently colonized by bacterial and archaeal symbionts. A prominent example are the cellulolytic gut flagellates of termites, which harbor diverse but host-specific bacterial symbionts that occur exclusively in termite guts. One of these lineages, the so-called Endomicrobia, comprises both free-living and endosymbiotic representatives, which offers the unique opportunity to study the evolutionary processes underpinning the transition from a free-living to an intracellular lifestyle. Our results revealed a progressive gene loss in energy metabolism and biosynthetic pathways, compensated by the acquisition of new functions via horizontal gene transfer from other gut bacteria, and suggest the eventual breakdown of an initially mutualistic symbiosis. Evidence for convergent evolution of unrelated endosymbionts reflects adaptations to the intracellular environment of termite gut flagellates.

Abstract Background ‘Candidatus Phytoplasma mali’, the causal agent of apple proliferation disease, exerts influence on its host plant through various effector proteins, including SAP11CaPm which interacts with different TEOSINTE BRANCHED1/ CYCLOIDEA/ PROLIFERATING CELL FACTOR 1 and 2 (TCP) transcription factors. This study examines the transcriptional response of the plant upon early expression of SAP11CaPm. For that purpose, leaves of Nicotiana occidentalis H.-M. Wheeler were Agrobacterium-infiltrated to induce transient expression of SAP11CaPm and changes in the transcriptome were recorded until 5 days post infiltration. Results The RNA-seq analysis revealed that presence of SAP11CaPm in leaves leads to downregulation of genes involved in defense response and related to photosynthetic processes, while expression of genes involved in energy production was enhanced. Conclusions The results indicate that early SAP11CaPm expression might be important for the colonization of the host plant since phytoplasmas lack many metabolic genes and are thus dependent on metabolites from their host plant.

Cottony ash psyllid (CAP, Phyllopsis discrepans) is an important, invasive insect pest of ash trees in North America where it has established populations and is the host of a newly identified strain of 'Candidatus Liberibacter solanacearum'. However, not much is known about the diversity of its introduced population. In this study, a CAP mitochondrial genome (mitogenome) sequence was obtained from a collection in Saskatoon, Saskatchewan, Canada. The CAP mitogenome is a circular DNA of 18,824 bp, encoding 13 protein-coding genes, 21 transfer RNA genes, and 2 ribosomal RNA genes. BLAST search using the CAP mitogenome as query against GenBank sequence database showed the mitogenome of Euphyllura phillyreae (15,202 bp) was the most similar (Query Coverage = 77%; Percentage Identity = 78.90%). The CAP mitogenome is significantly different from other known psyllid mitogenomes with the presence of a 4,357 bp control region (CR). The mitogenome sequence will further genomic understand of CAP.

Huanglongbing (HLB), which is associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas), is a devastating disease that affects citrus trees worldwide. Because of the pervasiveness of the bacteria and psyllid vector, the disease is considered endemic in Florida. Although the effects of CLas on tree growth and physiology have been investigated for decades, most studies compared infected and noninfected trees under greenhouse conditions. This study used newly planted field-grown ‘Valencia’ sweet orange (Citrus sinensis) trees on two different rootstocks to monitor the distribution and accumulation of CLas in aboveground and belowground tissues following natural psyllid colonization and assess tree physiological responses and biomass reductions under HLB-endemic conditions. Trees were transplanted into the field with individual protective covers (IPCs), which are used to exclude psyllids and prevent infection. Openings were cut in the IPCs of half of the trees; to promote infection, these IPCs were temporarily removed during the main vegetative flushing period when psyllid populations were high. All trees that were exposed to psyllids became infected and displayed the symptoms typically associated with HLB. Throughout the study, higher levels of CLas were detected in the leaves compared with those in the fibrous roots. Trees that were not exposed to psyllids remained noninfected and healthy. After 18 months, a subset of trees was excavated to assess biomass differences between infected and noninfected trees. Infected trees had root system reductions of 37% and shoot system reductions of 20%, thereby significantly reducing the belowground-to-aboveground biomass ratio. Fibrous root loss was 49% and more severe than the loss of the rest of the root tissue. This study is the first to demonstrate the full extent of damage caused by CLas infection under natural HLB-endemic conditions. The results confirm previous observations that suggested fibrous root loss as one of the major consequences of infection and colonization with CLas. They also reinforce the benefits of using IPCs to prevent infection of young citrus trees during the first years of growth in the field.