Publications

4877

ABSTRACT Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Developing effective therapies remains a major challenge, as CLas cannot be cultured in vitro and colonizes the host phloem systemically. This study presents a rapid, reproducible, and cost-effective in vivo platform for screening bacteriostatic and bactericidal compounds using CLas-infected citron (Citrus medica (L.) Osbeck) stem cuttings. Among seven citrus genotypes tested, citron stem cuttings exhibited superior rooting performance and uniform vegetative growth. Four propagation protocols were developed and assessed based on the dynamics of rooting and shoot growth, CLas colonization, and the response to oxytetracycline (OTC) treatment. CLas+ stem cuttings were treated with OTC via drenching at different developmental root stages. The roots or new vegetative flushes were sampled for bacterial quantification by qPCR. In Protocol 2, in which treatments were applied and sampled 14 and 35 days after planting (DAP) respectively, OTC-treated roots achieved the highest suppression of CLas and the lower incidence of CLas+ rooted cuttings compared to non-treated roots. Time-course analysis showed that OTC delayed bacterial establishment in root tissues, with maximal suppression observed at 35 DAP. The proposed protocols simulate the natural progression of systemic infection in citrus plants, allow the assessment of phytotoxicity, and offer a scalable technology that does not underestimate the efficacy of future bactericidal candidates. This platform significantly reduces time and cost compared to traditional seedling or nursery tree experiments and enhances the early-phase screening of antimicrobial compounds. Altogether, this stem cutting-based approach represents a biologically relevant, scalable tool to accelerate therapeutic discovery and strengthen integrated HLB management strategies.

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.

AbstractBACKGROUNDAutophagy is a conserved mechanism by which eukaryotic organisms defend against pathogen infection. However, the molecular mechanisms underlying the role of autophagy in the interactions of insect vectors with the phloem‐limited bacterial pathogen remain unclear. The citrus Huanglongbing (HLB)‐associated pathogen ‘Candidatus Liberibacter asiaticus’ (CLas) seriously endangers development of the citrus industry. It spreads via Diaphorina citri in a persistent and propagative manner.RESULTSIn this study, a total of 30 autophagy‐related genes (ATG) were identified in the D. citri genome, among which multiple genes were significantly regulated after CLas infection. Concurrently, CLas infection also leads to an increased number of autophagosomes and enhanced accumulation of ATG8‐II. Ultrastructural observations revealed the presence of bacterial‐like structures within autophagosomes in the midgut of CLas‐infected D. citri. Furthermore, both activation and inhibition of autophagy significantly influenced CLas titers. However, autophagy cannot completely eliminate CLas in D. citri. We identified a CLas effector, SDE4040 (CLIBASIA_04040), that interacts with DcATG8 and co‐localizes on autophagosomes in D. citri. Co‐expression of SDE4040 and DcATG8 induces autophagy in Spodoptera frugiperda (Sf9) cells.CONCLUSIONTaken together, these results indicate that CLas infection activates the autophagy pathway in D. citri, contributing to a reduction in bacterial titer. Our data also revealed that CLas may trigger complex interactions with the insect. © 2025 Society of Chemical Industry.

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 Citrus Huanglongbing (HLB) is the most destructive disease in citriculture, mainly caused by Candidatus Liberibacter asiaticus (CLas). However, the immune response of citrus to CLas at the cellular level remains to be elucidated. In this study, the first single-cell atlas of rough lemon (Citrus jambhiri Lush.) root apexes were generated using single-nucleus RNA sequencing at 20 weeks post-inoculation with CLas. According to gene expression patterns, the single-cell atlas was partitioned into 20 transcriptionally distinct clusters, and five cell types were identified within these clusters. A significant number of defense-related genes were co-upregulated across the five cell types following CLas infection, whereas genes involved in signal transduction pathways, such as tubulin beta-6 chain (TUBB1) and the phospholipase D alpha 1 (PLD1), were concurrently downregulated. Based on pseudotime trajectory analysis, the key pathways and genes involved in the coordination of cell differentiation and resistance in citrus under CLas infection were characterized. Following CLas infection, the development of phloem cells was significantly delayed, and the differentiation of cambium cells into xylem cells was evident. The expression of genes associated with lignin synthesis was significantly upregulated in these cells. The reduction in phloem cell differentiation and the enhanced differentiation of cambium cells into defense-related xylem cells may represent the primary vascular immune mechanisms exhibited by citrus plants in response to CLas infection. Additionally, DNA-binding one zinc finger transcription factor DOF2.4 was found to potentially serve dual roles in regulating vascular cell development and inducing plant resistance against CLas. In conclusion, this study collectively provides insights into the cellular innate immunity responses of citrus to CLas infection. These findings hold significant implications for the sustainable development of citriculture amidst the ongoing global HLB epidemic, and offer novel insights into vascular immunity and plant defense responses.