Publications

4938

Candidatus Liberibacter spp. can infect most citrus plants and rely entirely on phloem sieve tube cells of the host plant for survival. Candidatus Liberibacter primarily contains Ca. L. asiaticus (CLas), Ca. L. africanus (CLaf), and Ca. L. americanus (CLam). Among these, CLas is the most harmful and widely distributed and is the primary pathogen of the devastating citrus disease Huanglongbing (HLB). Effectors are among the core weapons secreted by pathogens into plant cells to attack the plant immune system. In this study, we focused on CLas-specific effectors and those that are highly expressed during the infection stage to identify essential virulence effectors. Using secretion signal peptide prediction analysis, 40 candidate effectors with potential secretory capabilities were identified. Transient expression of these candidate effectors in Nicotiana benthamiana revealed their impact on pattern-triggered immunity, including INF-induced cell death and microbial pattern-induced reactive oxygen species (ROS) bursts, and the resistance of N. benthamiana to the bacterial pathogen Pst DC3000. 10 candidate effectors capable of suppressing plant immunity were identified. The stable expression of these candidate effectors in Arabidopsis showed that several candidate effectors enhanced plant susceptibility to Pst DC3000 and inhibited flg22-induced ROS production and MAPK activation. Among the three candidate effectors that significantly suppressed ROS burst, one effector, E3 (CLIBASIA_03085), interacts with the plant NADPH oxidase RbohD, a key enzyme responsible for ROS production. This suggests that E3 likely inhibits ROS accumulation by directly targeting RbohD. Here, we identified multiple candidate effectors capable of suppressing microbial pattern-triggered immunity that may be essential virulence factors for CLas infection, enhancing our understanding of CLas pathogenesis.

Abstract Methanogens are strictly anaerobic archaea capable of energy conservation by methane production, yet their presence in oxic and arid environments challenges existing paradigms. In this study, we enriched and genomically characterized seven methanogenic cultures from desert biocrusts, affiliated with the genera Methanobacterium, Methanosarcina, and Methanocella. Six of these new enrichment cultures represent new species. Nonetheless, phylogenomic analyses revealed close genetic relationships with organisms from anoxic environments, indicating the absence of an evolutionary distinction. Comparative genomics exposed diverse though non-unique repertories of antioxidant (e.g. catalase, superoxide dismutase and desulfoferrodoxin), and desiccation-resistance genes (including genes for maintaining osmotic pressure and repair of cell wall and membrane), with Methanobacterium spp possessing the lowest gene abundance and diversity for oxygen and desiccation tolerance. Nevertheless, the occurrence of a Class I methanogen such as Methanobacterium in arid soils challenges the notion that members of this class are less oxygen tolerant than Class II. Pangenome analysis further uncovered unique genes enriched in membrane-associated functions and potentially non-functional stress-related genes. Via a global metagenomic survey we find that methanogens are underdetected in dryland soils, likely due to sequencing depth limitations. Our findings highlight previously overlooked methanogen diversity and ecological plasticity in oxic and desiccated habitats, and emphasize the need for further studies to elucidate their survival strategies.

‘Candidatus Phytoplasma solani’ of the 16SrXII group is an emerging vector-borne pathogen in European crop production. The cixiid planthopper Hyalesthes obsoletus transmits 16SrXII-A stolbur phytoplasmas that are associated with diseases in grapevine, potato, and various weeds. While 16SrXII-P genomes transmitted by Pentastiridius leporinus are available, no genome of an H. obsoletus-transmissible 16SrXII-A phytoplasma has been reported from Germany. Here, we present insights into the phylogenetic position and pathogen–host interactions through the functional reconstruction of the complete 832,614 bp genome of the H. obsoletus transmissible ‘Ca. P. solani’ 16SrXII-A strain POT from a potato field. Phylogenetic analyses highlight the heterogeneity within the stolbur group using whole-genome alignment and a BUSCO-based core gene analysis approach. The POT chromosome shares highest average nucleotide identity with Italian bindweed-associated genomes and displays strong synteny with the c5 strain. Consistent with the typical phytoplasma architecture, the POT genome combines mobile-element-driven instability with a conserved core metabolism. Virulence factors include transposon-linked effectors but lack pathogenicity island organisation. POT further differs from other 16SrXII-group phytoplasmas through unique collagen-like proteins that could contribute to virulence. These findings provide a robust genomic framework that improves diagnostics, enables strain-level resolution and supports the assessment of breeding materials under stolbur phytoplasma pressure, thereby refining our understanding of stolbur phytoplasma diversity and highlighting the evolutionary divergence within the 16SrXII subgroup.

Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is the most destructive citrus disease worldwide, severely reducing yield and fruit quality. Although no naturally resistant cultivars are available, citrus plants overexpressing Arabidopsis NPR1 ( At NPR1) display enhanced tolerance, yet the molecular mechanisms underlying this phenotype remain insufficiently understood. To uncover early transcriptional events associated with tolerance, we performed comparative RNA-seq and phytohormone profiling of susceptible wild-type (WT) and tolerant At NPR1-overexpressing ( At NPR1-OE) Duncan grapefruit at 0, 6, and 24 hours post infection (hpi). Before infection, At NPR1 plants downregulated genes involved in cytoskeleton organization, cell wall biogenesis, and receptor signaling, suggesting a primed basal defense state. After CLas exposure, At NPR1 plants exhibited stronger and earlier transcriptional reprogramming, with substantially more differentially expressed genes at 6 hpi than WT. At 24 hpi, At NPR1 plants showed suppression of callose synthase genes and selective induction of β-1,3-glucanases, indicating more controlled phloem callose regulation. Concurrently, attenuated expression of respiratory burst oxidase homologs and ROS-associated genes suggested a moderated and less damaging oxidative burst. At NPR1 plants maintained stable levels of salicylic acid, and gibberellins while preventing the CLas-induced induction of abscisic acid observed in WT. Collectively, these findings reveal that At NPR1 overexpression enhances HLB tolerance by integrating early transcriptional reprogramming with balanced structural, oxidative, and hormonal responses. This study provides a mechanistic framework for understanding NPR1-mediated tolerance to CLas during the initial stages of infection.

Abstract Ferdinand Cohn observed abundant filamentous bacteria in drinking water wells in 1870 that he named Crenothrix polyspora. Subsequent research has revealed the methanotrophic metabolism of Crenothrix bacteria and their disproportionately high activity in stratified lakes compared to unicellular methanotrophs, yet laboratory cultivation has proven elusive, leaving the ecophysiology of Crenothrix bacteria largely unknown. Here we report the isolation of two methanotrophic strains of the “lacustrine Crenothrix” clade from an iron-rich wetland and reveal their unique cell biology and ecology. We demonstrate that the strains are microaerobic and grow as filaments of cells, which are connected by unidirectionally oriented structures. The strains have broad genomic repertoires for addressing O2 limitation that are uniquely associated with lacustrine Crenothrix compared to related clades based on genome data. Aligning with laboratory observations, we identify lacustrine Crenothrix bacteria along potential redox gradients in the wetland at iron-rich snow sites, and we also detect such bacteria in diverse global ecosystems based on public metagenome searches. Together, our data strongly point to an ecophysiology of lacustrine Crenothrix bacteria that is tightly linked to O2 limitation, and we propose that the strains uniquely store or share metabolic intermediates between cells in filaments to thrive under such conditions. Our cultivation-based findings for these strains, which we name Allocrenothrix methanica, provide new insights into the diversity, evolution, and ecology of filamentous methanotrophs, connecting over 150 years of microbiology research and opening vast new opportunities to investigate bacteria contributing to the global methane cycle under O2 limitation.

Abstract Extreme acidic environments represent natural laboratories for investigating the mechanisms of microbial community assembly, yet the ecological processes structuring these communities remain incompletely understood. Here, we investigate how spatial partitioning, hydrodynamics, and colonization history shape microbial succession in a unique sulfur-rich, acidic river of volcanic origin in northern Patagonia. We combined 16S rRNA gene profiling and shotgun metagenomics with a multi-scale experimental framework encompassing water column fractionation and colonization assays under native and controlled conditions. Microbial diversity was strongly influenced by spatial fractionation, with free-living communities exhibiting higher richness and temporal variability than particle-associated assemblages. Water flow modulated community structure, increasing evenness in free-living fractions under high-flow conditions, but had limited impact on particle-attached communities. Colonization of sulfur-beads followed a structured successional trajectory, with autotrophic sulfur oxidizers dominating early stages and heterotrophs adapted to biofilm lifestyles increasing over time. Ex situ recolonization assays revealed strong priority effects, with initial colonizers determining successional trajectories. Turnover analyses revealed that the balance among stochastic and deterministic assembly processes shifted across communities with pronounced stochasticity in the water column and flow-dependent effects in free-living communities, while biofilm associated communities on sulfur-beads exhibited stronger contribution of deterministic selection. These ecological patterns were mirrored by functional differentiation, with gene enrichment analyses revealing adaptive signatures of substrate attachment and resource acquisition. By integrating fine-scale environmental variation with colonization dynamics, this study reveals how microscale habitat structure and temporal fluxes jointly modulate microbial community assembly rules, offering a nuanced framework to dissect ecological processes in extreme systems.

Abstract Members of the phylum Deferribacterota inhabit diverse environments, but their symbiosis with protists has never been reported. We discovered an ectosymbiotic clade of Deferribacterota specifically associated with spirotrichonymphid protists in the guts of the termites Reticulitermes speratus and Hodotermopsis sjostedti and trichonymphid protists in the gut of the wood-feeding cockroach Cryptocercus punctulatus. The ectosymbiotic Deferribacterota were spiral shaped and attached to 16%–91% of the host protist cells. These formed a monophyletic cluster within an uncultured insect gut-associated family-level clade, which is sister to the vertebrate gut-associated family Mucispirillaceae. The complete genome of an ectosymbiotic Deferribacterota was obtained from a Trichonympha acuta cell in a C. punctulatus gut and analyzed together with a single-cell amplified genome of another ectosymbiotic Deferribacterota associated with Holomastigotes sp. in the gut of R. speratus. Genome analyses suggest that these Deferribacterota ferment monosaccharides and conduct fumarate and oxidative respiration with H2 as an electron donor. They thus possibly contribute to the removal of hydrogen and oxygen to protect the fermentative activity of the protist hosts. The ectosymbionts possess reduced signal transduction gene repertoires, implying that the association has provided a relatively stable environment for these bacteria. The ectosymbionts likely possess flagella with an unusually expanded number of flagellin variants up to 40, which may reflect an adaptation to their ectosymbiotic lifestyle. We propose a novel genus, Termitispirillum, for these ectosymbionts and a novel family, Termitispirillaceae, for the insect-gut clade, under SeqCode. Our findings provide new insights into the ecology and evolution of Deferribacterota.

Abstract Nitrobacter strain NHB1 is a nitrite-oxidizing bacterium previously demonstrated to form a consortium capable of nitrification under acidic conditions when cocultivated with a neutrophilic ammonia-oxidizing bacterium. Here, we characterize the growth of isolated NHB1 under different pH and nitrite (NO2−) concentrations, as well as its influence on the activity of obligately acidophilic soil ammonia-oxidizing archaea (AOA) isolated from acidic soils when grown in coculture. NHB1 is acidotolerant with optimal growth at pH 6.0 (range: 5.0–7.5) at an initial NO2− concentration of 500 μM. However, at lower NO2− concentrations, closer to those found in soil, its pH optimum decreases to 5.0 and with detectable growth extended to pH 3.5. In coculture, NHB1 enhances the growth of the acidophilic AOA Nitrosotalea devaniterrae Nd1 and Nitrosotalea sinensis Nd2, which are highly sensitive to NO2-derived compounds and typically oxidize only ~200 to 300 μM ammonia (NH3) when grown in batch cultures as isolates. However, in coculture with NHB1, both strains oxidized up to ~3 mM NH3, limited only by the buffering capacity of the medium, and their pH range was also extended downward by ~0.5 units. NHB1 also possesses a cyanase, enabling reciprocal cross-feeding through cyanate-derived NH3 production while utilizing AOA-derived NO2−. These findings suggest that NO2− removal is essential for ammonia oxidizer growth in acidic soils and emphasize the importance of considering substrate and metabolic product concentrations when characterizing ecophysiology. Genome analysis reveals that NHB1 is distinct from validated species, and we propose the name “Nitrobacter laanbroekii.”