Publications

4861

A novel hyperthermophilic, heterotrophic and anaerobic archaeon, designated strain SLHALR2 T , was isolated from the ‘Cooking Pots’ hot springs at Hell’s Gate, Tikitere, New Zealand. During the exponential growth phase, cells of strain SLHALR2 T appear as long, thin rods to filaments, measuring 0.25–0.4 µm in width and 3.0–7.0 µm in length. Strain SLHALR2 T grows at temperatures ranging from 60 to 97 °C (optimum 80–85 °C), at pH values between 5.0 and 7.3 (optimum pH 6.0) and with 0–8 g l −1 sodium chloride concentration (optimum 0–1.0 g l −1 ). Elemental sulphur is reduced to hydrogen sulphide. Strain SLHALR2 T is an obligate organotroph capable of utilizing a variety of proteinaceous substrates and a limited range of sugars. Archaeal cell extracts improve growth but are not mandatory for growth. Phylogenetic analysis based on 16S rRNA gene sequence comparisons indicates that strain SLHALR2 T belongs to the genus Infirmifilum within the family Thermofilaceae , order Thermofilales (phylum Thermoproteota ). The strain shares 98.4–99% 16S rRNA gene sequence similarity with described Infirmifilum species. Despite this high level of 16S rRNA gene similarity with recently described Infirmifilum species, both average nucleotide identity (71–74%), average amino acid identity (67–75.5%) values and digital DNA–DNA hybridization values support the designation of strain SLHALR2 T as a distinct species within Infirmifilum . Based on phylogenomic analyses and the physiological characteristics of the novel isolate, strain SLHALR2 T is proposed as the type strain of a new species, Infirmifilum tikiterense sp. nov. (=OCM2026=UBOCC M-3571), and an emended description of the genus Infirmifilum is proposed.

Abstract Syndrome Basses Richesses (SBR) is a phloem-limited disease of sugar beet caused by ‘ Candidatus Arsenophonus phytopathogenicus’ (ARSEPH), a bacterium currently considered a facultative endosymbiont of its insect vector, Pentastiridius leporinus . Due to increasing disease incidence and the need for reliable vector-free resistance screening methods, this study evaluated alternative ARSEPH transmission routes using chip grafting and the parasitic plant dodder ( Cuscuta campestris ). Given that Arsenophonus species are unculturable and their insect vectors have complex life cycles so laboratory propagation is labor intensive, the development of vector-independent systems is desirable for controlled pathogen transmission and host–pathogen interaction studies. Chip grafting enabled successful ARSEPH transmission to sugar beet, though with low efficiency (~ 9.6%) and delayed pathogen detection. While C. campestris acquired and maintained high ARSEPH titers, it failed to transmit the bacterium to healthy plants, indicating limited systemic movement. In contrast, vector-mediated transmission via P. leporinus achieved 100% efficiency within 25 days, confirming a strong biological compatibility. These findings underscore the limitations of non-vector-based approaches for phloem-restricted pathogens and highlight the need for methodological optimization. The study provides key insights into ARSEPH’s complex transmission biology and supports a functionally specialized association with its insect vector.

ABSTRACT Citrus greening disease, associated with the bacterium Candidatus Liberibacter asiaticus ( C Las), has severely impacted citrus yields on a global scale. C Las is vectored by the Asian citrus psyllid, Diaphorina citri Kuwayama, in a circulative and propagative manner. Extensive application of chemical insecticides for suppression of psyllid populations has resulted in widespread resistance. Despite considerable effort and expenditure, citrus greening disease continues to impair citrus production. For transmission to occur, C Las must bind and infect the psyllid vector via the gut epithelium, but little is known of the specific molecular interactions involved. By screening a phage display library, we selected ten psyllid gut binding peptides, a subset of which have sequence similarity to C Las surface proteins. Peptide binding to the surface of the psyllid gut epithelium was confirmed by identification of gut proteins bound by specific peptides. We evaluated whether these peptides could interfere with the interaction between C Las and the gut epithelium of D. citri , thereby impeding pathogen transmission. Peptide DcNy2 significantly reduced C Las acquisition and inoculation by D. citri compared to control treatments. Nymphs fed specific peptides exhibited reduced C Las acquisition and infection of both the alimentary canal and salivary glands without negatively impacting psyllid fecundity, development, or survival. These results support peptide-mediated disruption of C Las transmission by D. citri as a potential component of integrated strategies to manage citrus greening disease. IMPORTANCE Citrus greening disease, or huanglongbing, causes billions of dollars in losses and represents one of the greatest challenges facing global citrus production. Despite significant investment on multiple fronts, effective disease management strategies are lacking. This study shows for the first time that short peptides that bind to the surface of the gut of the Asian citrus psyllid vector interfere with C Las association with the gut. This critical first gut binding step is required for C Las to infect and propagate within the psyllid vector. The downstream consequence of this peptide-mediated interference is reduced acquisition and transmission of the citrus pathogen. This work provides the foundation for new transgenic or paratransgenic tools for use in the management of citrus greening disease.

Abstract Huanglongbing (HLB), primarily induced by the phloem-restricted bacterium Candidatus Liberibacter asiaticus (CLas), poses a significant threaten to citrus production worldwide. CLas deploys Sec-dependent effectors (SDEs) to manipulate host cellular processes, yet the underlying virulence mechanisms remain incompletely understood. Here, we identify SDE4580, a conserved CLas effector localized to the plant plasma membrane, as a key virulence factor that promotes pathogen proliferation and suppresses host immunity across citrus and Arabidopsis. Transcriptomic analysis reveals that SDE4580 reprograms membrane-associated biological processes and defense responses. Through yeast two-hybrid screening and biochemical validation, we demonstrate that SDE4580 physically interacts with the citrus vesicle trafficking regulator CsPRA1.E, a Prenylated Rab Acceptor 1 family protein. Functional assays establish CsPRA1.E as a positive regulator of immunity that promotes apoplastic secretion of defense-related proteins and enhances resistance to Xanthomonas citri subsp. citri. Crucially, SDE4580 antagonizes CsPRA1.E-mediated resistance by suppressing defense protein secretion without inducing target degradation. These findings uncover a novel pathogenic strategy whereby CLas subverts host endomembrane trafficking to disarm plant immunity. Targeting the SDE4580-CsPRA1.E interface may offer a promising avenue for developing durable HLB control strategies.

Peptidyl-tRNA hydrolase (Pth2) hydrolyzes peptidyl-tRNA, an immature product of aborted translation, into peptide and tRNA, thereby maintaining cellular protein synthesis through peptide release and tRNA recycling. Here, we present the crystal structure of Pth2 from Candidatus Lokiarchaeum sp. GC14_75 ( Loki Pth2) at 2.12 Å resolution. This is the first structure of Pth2 from a lineage within Promethearchaeati, a kingdom of archaea closely related to eukaryotes. The structure reveals that Loki Pth2 forms a homodimer and closely resembles Pth2 structures from other species. However, Loki Pth2 exhibits two prominent structural differences: a short helix around the catalytic center, which is absent in other Pth2s, and a distinct orientation of the C-terminal helix. Detailed comparative structural analysis suggests that these regions may regulate enzymatic activity and substrate binding, respectively. Furthermore, the corresponding regions in other Pth2s also exhibit high flexibility, suggesting that similar mechanisms may be conserved among Pth2s. To gain insights into the growth environment of Candidatus Lokiarchaeum sp. GC14_75, we assess the optimal temperature for the catalytic reaction of Loki Pth2, which suggests that Candidatus Lokiarchaeum sp. GC14_75 inhabits moderately thermophilic environments.

‘Candidatus Liberibacter solanacearum’ (Lso) haplotype D, transmitted by the carrot psyllid Bactericera trigonica, is a major threat to carrot production, particularly in the Mediterranean Basin and the Middle East. We investigated the impact of plant age at the time of inoculation on symptom development and yield under greenhouse and outdoor conditions. In greenhouse trials, 1- and 2-month-old plants were inoculated with psyllids harboring Lso. Both age groups showed reduced taproot weight compared with controls, but younger plants were more severely affected, exhibiting 90% yield reduction, while 2-month-old plants showed a 30% reduction. Witches’ broom symptoms appeared earlier in plants inoculated at 1 month (5 weeks postinoculation); however, within 2 weeks, plants inoculated at 2 months reached comparable levels of symptom incidence and severity. Additional greenhouse experiments using Lso-free psyllids confirmed that yield losses and disease symptoms were specifically due to Lso infection and not psyllid feeding. In outdoor trials, we tested the effects of inoculation at 7, 11, 15, and 19 weeks postsowing (wps) on disease incidence and yield. Significant yield losses occurred only in plants inoculated at 7 and 11 wps, with up to 45% reduction by harvest time. Symptoms began to appear at 20 wps in plants inoculated at 7 and 11 wps. At 23 wps, symptoms also appeared in plants inoculated at 15 wps, with a similar incidence to that observed in the earlier inoculation groups. Molecular detection of Lso at harvest (23 wps) confirmed that all plants inoculated at 19 wps or earlier were infected. These results demonstrate that carrots are highly susceptible to Lso-induced yield losses at the earlier stages of growth and highlight the importance of managing early season vector pressure to minimize economic damage.