Publications

4794

ABSTRACT Insects of the suborder Auchenorrhyncha harbour multiple ancient endosymbionts that jointly produce essential nutrients lacking from the host's diet. Compared to cicadas, leafhoppers, and spittlebugs, our understanding of the multipartite symbioses among planthoppers, an extremely diverse insect group, is still very limited. Herein, we assembled the genomes of the primary endosymbionts of two planthopper species from the Cixiidae family, Cixius wagneri and Pentastiridius leporinus , both vectors of phytopathogenic Arsenophonus in Europe. Each species harboured a different tripartite endosymbiont consortium: while P. leporinus carried the well‐known combination ‘ Candidatus Karelsulcia muelleri’, ‘ Ca . Vidania fulgoroideae’, and ‘ Ca . Purcelliella pentastirinorum’, C. wagneri harboured a yet unknown Gammaproteobacterium in addition to Karelsulcia and Vidania . This new endosymbiont ‘ Ca . Mirabilia symbiotica’ is likely much older than Purcelliella , considering its extremely reduced genome. In both species, Karelsulcia and Vidania jointly produce the 10 essential amino acids, whereas Purcelliella and Mirabilia provide the non‐essential amino acid cysteine and slightly different gene sets encoding B vitamins. Our findings confirm the functional stability of multipartite planthopper endosymbiont consortia despite changing partners over evolutionary time. In addition, we describe a new Rickettsia strain from the Meloidae group colonising P. leporinus , highlighting the diversity of bacterial endosymbionts associated with planthoppers.

Abstract Background Candidatus Phytoplasma infection is among the most destructive plant diseases, characterized by phyllody, witches broom, virescence etc. Sesame (Sesamum indicum L.), one of the oldest cultivated oilseed crops, valued for its nutritional and medicinal properties, is highly susceptible to phytoplasma infection, causing substantial annual yield losses. The present study aimed to investigate the metabolomic and molecular alterations in sesame plants as a consequence of phytoplasma infection in two distinct tissue types, leaves and flowers, using a multi-omics approach. Results The study revealed that phytoplasma infection significantly alters the expression of floral homeobox and meristem identity genes in an antagonistic manner, resulting in the development of leaf-like traits in floral tissues. Integrated analyses of transcriptomic and metabolomic datasets showed that control leaves, control flowers, infected leaves, and infected flowers each displayed distinct metabolomic and transcriptomic profiles. Metabolomic profiling demonstrated major changes in pathways such as porphyrin metabolism, brassinosteroid metabolism, and phenylpropanoid biosynthesis. Complementary KEGG pathway enrichment analysis of transcriptome data confirmed strong enrichment of secondary metabolite biosynthesis in both tissue types upon infection. Tissue-specific responses were evident. Floral tissues accumulated green pigments due to increased porphyrin biosynthesis and reduced degradation, while leaves showed simultaneous upregulation of both biosynthesis and breakdown pathways of porphyrins. Floral tissues exhibited stronger stress-associated responses, including upregulation of genes related to stress enzymes, phenylpropanoids, and lignification-related metabolites. In contrast, certain compounds such as lignans were specifically accumulated in leaves upon infection. These observations were further supported by biochemical, histological, and qRT-PCR assays. Conclusion This study provides the first clear evidence of tissue-specific metabolic reprogramming in sesame under Ca. Phytoplasma infection through integrated transcriptomic and metabolomic analyses. These findings improve our understanding of host-pathogen interactions and offer a basis for strategies to reduce phytoplasma-induced yield losses.

ABSTRACTSesame phyllody, a destructive disease caused by phytoplasma infection, induces severe morphological abnormalities, including floral virescence, phyllody, witches' broom, leaf deformation, and stunted growth. This study aimed to characterize phytoplasma isolates from diverse regions of India, identifying them as Candidatus Phytoplasma asteris (16Sr‐I), Candidatus Phytoplasma citri (16Sr‐II), and Candidatus Phytoplasma australasia (16Sr‐II). Whole‐genome sequencing of Candidatus Phytoplasma asteris isolate SPGN revealed a genome size of 563,754 bp, encoding 542 proteins, including several genes associated with antibiotic resistance. Effector prediction analysis identified key virulence‐associated proteins, such as SAP50‐like, SAP34‐like, TENGU‐su inducer, and immunodominant membrane proteins, which manipulate host development and immune responses. Transcriptomic analysis of infected sesame plants revealed significant gene expression alterations, with upregulated genes linked to floral malformation, vascular tissue modifications, and stress responses, while the downregulated genes were associated with flavonoid metabolism and immune signaling. Phytoplasma infection disrupted hormonal pathways, leading to increased expression of auxin, cytokinin, and gibberellin‐related genes, suggesting hormonal dysregulation as a key factor in symptom development. Furthermore, immune suppression was evident through the downregulation of key defense‐related genes, including those involved in MAPK signaling and pathogenesis‐related protein families. These findings enhance our understanding of phytoplasma pathogenesis in sesame and provide potential targets for developing effective disease management strategies.

Abstract Citrus greening disease, caused by ‘Candidatus Liberibacter asiaticus’, severely impacts citrus production worldwide. The development of sustainable control strategies for this disease is restricted by the unavailability of the bacterium in pure culture. Herein, the metabolic profile of the waxworm larvae, Galleria mellonella, was compared to that of Diaphorina citri, the vector of ‘Ca. L. asiaticus’. Our findings showed that G. mellonella larvae possess the nutritional needs to host ‘Ca. L. asiaticus’, supporting its short-term persistence, and responds to infection with a visible immune reaction by producing melanin upon bacterial invasion. The inoculated larvae exhibit detectable bacterial titers for up to four days when inoculated with infected citrus phloem sap or D. citri haemolymph, after which bacterial titers decline, and infected larvae show reduced survival compared to mock-treated and ‘Ca. L. asiaticus’-free controls. Metabolic profiling of G. mellonella, D. citri, and honeybees (Apis mellifera) reveals distinct chemical compositions in their haemolymph. G. mellonella contains higher levels of amino acids, organic acids, nucleotides, and sugar-nucleotides, providing essential nutrients for ‘Ca. L. asiaticus’, while D. citri is enriched in monosaccharides and sugar-alcohols. Citric acid was detected exclusively in the haemolymph of G. mellonella. These findings suggest G. mellonella as a convenient model that can transiently host ‘Ca. L. asiaticus’ for short-term use, which would facilitate high-throughput screening of antimicrobial compounds against ‘Ca. L. asiaticus’, as well as exploring host-pathogen interactions. This model could accelerate the development of effective treatments against citrus greening and inform broader strategies for managing vector-borne plant diseases.

Abstract Autophagy, a cellular process involved in the degradation and recycling of cellular components, has emerged as a pivotal mechanism for maintaining cellular homeostasis and combating pathogen invasion. Here, we provide evidence that the overexpression of CsATG8c inhibits CLas proliferation in citrus. CsATG8c directly interacts with the CLas effector protein SDE4040, leading to its degradation via the autophagic pathway. The SDE4040 protein acts as a virulence effector, and transgenic citrus plants expressing SDE4040 promote CLas proliferation by suppressing the hypersensitive response (HR) of the host and reducing the levels of jasmonic acid (JA). Collectively, these results demonstrate that ATG8-mediated selective autophagy limits CLas infection by degrading a virulence effector protein, which provides the first evidence for the role of autophagy in combating CLas infection.

ABSTRACT Polyphosphate-accumulating organisms (PAOs) are the main bacteria responsible for phosphorus removal and recovery in full-scale wastewater treatment plants (WWTPs). They encompass members of the genera Candidatus Accumulibacter, Azonexus (formerly Dechloromonas ), and Candidatus Phosphoribacter (formerly Tetrasphaera ), with most studies focusing on Ca . Accumulibacter, primarily using lab-scale enrichment cultures. Although members from the three genera often co-exist in full-scale WWTPs, the metabolic capabilities and traits that determine the niche differentiation of the specific species are still unknown. We retrieved 214 high-quality metagenome-assembled genomes from a full-scale plant with phosphorus removal and examined the polyphosphate-related metabolic pathways using genome-resolved metatranscriptomics in the different process tanks in situ and by using short-term incubations ex situ . We observed the co-existence of nine uncultured PAO species from the three genera with clear niche differentiation in the utilization of different carbon sources and involvement in the denitrification process. Additionally, we observed several physiological differences among species of the same genus, indicating variations in niche specialization. This suggests that biological P removal and other processes in full-scale WWTPs are carried out by a complex and diverse PAO community that together ensures stable plant performance. IMPORTANCE The current understanding of the ecology and physiology of polyphosphate-accumulating organisms (PAOs) is mostly based on Candidatus Accumulibacter, primarily studied in enriched lab-scale studies. Recent taxonomic reclassification revealed that the most studied Ca . Accumulibacter species are either not present or present in low abundance in full-scale wastewater treatment plants (WWTPs). This raises concerns that knowledge from lab-scale studies may not apply to species in full-scale plants. Additionally, the indication of a distinct PAO physiology in Candidatus Phosphoribacter compared to Ca . Accumulibacter and the other abundant PAO Ca . Azonexus poses further questions about the accuracy of the current PAO model. Here, we show that in full-scale plant species from Ca . Accumulibacter, Ca . Azonexus, and Ca . Phosphoribacter always co-exist, and they have distinct niche separations in terms of carbon source utilization and the use of electron acceptors. This co-existence and metabolic diversity indicate that a complex microbial community is crucial for efficient phosphorus removal in full-scale WWTPs.

Phytoplasmas (‘Candidatus Phytoplasma’) are intracellular pleomorphic plant pathogens belonging to the class Mollicutes. They colonize both plant hosts and insect vectors in their life cycle. Apple proliferation (AP) is one of the most important phytoplasmoses present in Europe, causing significant economic losses in apple production. The causal agent, ‘Ca. P. mali’, was identified in apple and Cacopsylla picta samples using both real-time PCR and nested PCR based on the amplification of 16S rDNA. The objective of this study was to gain deeper insights into the epidemiology of apple proliferation in Croatia. Variability of genetic markers other than 16S rRNA was used for characterization of strains. Four molecular markers differing in level of conservation, aceF, pnp, imp, and secY, were selected in line with previously typed fruit tree phytoplasmas. New genotypes were discerned for each genetic marker, and 20 different sequence types were revealed in the Croatian strains of ‘Ca. P. mali’. On the basis of this comprehensive analysis, the founder sequence type ST1 (A13–P10–S12–I21) can be proposed. This is the first extensive research and multigene typing performed on Croatian ‘Ca. P. mali’ strains. Obtained results reveal considerable genetic diversity of epidemiological relevance limited to only two locations in north-western Croatia. Additionally, novel primers were constructed to amplify fragments larger than the entire coding region for all four genes in order to further expand the phytoplasma multi-locus sequence typing scheme.