Publications

4539

Abstract ‘Candidatus Liberibacter asiaticus’ (CLas), the causal agent of citrus huanglongbing, is transmitted by the Asian citrus psyllid Diaphorina citri. While CLas-positive (CLas+) females exhibit increased fecundity and metabolic demands, their neuroendocrine regulation remains unclear. We propose CLas manipulates dopamine (DA) signaling to enhance psyllid fecundity and CLas proliferation. Metabolomics revealed elevated DA in CLas+ females. Silencing DA synthesis genes and receptor DcDop2 via RNAi reduced lipid reserves, fecundity, and ovarian CLas titers. Through combined in vivo and in vitro experiments, we demonstrated that the microRNA miR-31a suppresses DcDop2 expression by binding to its 3’ untranslated region. Overexpression of miR-31a resulted in decreased DcDop2 expression and CLas titers in the ovaries, eliciting phenotypic defects akin to DcDop2 knockdown. Furthermore, DcDop2 knockdown and miR-31a overexpression reduced juvenile hormone (JH) levels and adipokinetic hormone (AKH) signaling in fat bodies and ovaries. Consequently, CLas hijacks the DA/DcDop2-miR-31a-AKH-JH signaling cascade to improve D. citri lipid metabolism and fecundity, while simultaneously promoting its replication. These findings suggest a mutualistic interaction between CLas proliferation and ovarian development within the insect host that enrich our understanding of the molecular interplay between plant pathogens and vector insects and offer novel targets and strategies for the field management of HLB.

ABSTRACT “ Candidatus Liberibacter solanacearum” (Lso) is a highly destructive plant pathogen within the alpha-proteobacteria group. Multiple Lso haplotypes occur worldwide, each uniquely associated with a specific psyllid vector. Haplotypes A and B, found in the United States, cause serious damage to solanaceous crops and are transmitted by Bactericera cockerelli , known as the potato psyllid in a circulative and persistent manner. The psyllid gut is the first organ encountered by Lso and may act as a barrier to its transmission; however, the immune response of the gut to Lso infection remains largely uncharacterized. In this study, we examined autophagic responses in the gut of potato psyllids at early, mid, and late infection stages. Based on gene expression analyses, microscopic observations, and Western blotting, we found no clear evidence of autophagy induction despite Lso infection. Nevertheless, during mid and late phases of LsoB infection, we observed downregulation of the mechanistic target of rapamycin and decreased accumulation of ATG8-II. Notably, inducing autophagy with rapamycin significantly reduced LsoA titers in the psyllid guts after a 5-day acquisition access period and lowered transmission efficiency, while LsoB remained unaffected. These findings suggest that modulating autophagy in the gut of potato psyllids could be a promising strategy to limit LsoA acquisition and transmission, while highlighting LsoA and LsoB possess distinct molecular regulatory mechanisms in the psyllid gut. IMPORTANCE Liberibacters are devastating plant pathogens transmitted by psyllids. Because these bacteria are fastidious, the study of the molecular mechanisms involved in plant infection and transmission is difficult. Here, we determined that inducing autophagy in the potato psyllid can affect the acquisition and transmission of Lso haplotype A but not haplotype B. Comparing the host and vector responses to different liberibacters can help identify their transmission and infection mechanisms and find targets to disrupt these processes.

Abstract‘Candidatus Phytoplasma (Ca. P.) solani’ is associated with Bois noir (BN) of grapevine and stolbur of solanaceous plants and is primarily transmitted by Hyalesthes obsoletus Signoret. Four tuf‐a and five tuf‐b1 ‘Ca. P. solani’ strains were transmitted to tomato plants (cv. Micro‐Tom) to set the basis for studying molecular interactions between different strains of the pathogen and host plants. The strains were acquired by using bait‐plants and by capturing H. obsoletus adults on bindweed and stinging nettle in vineyards of Friuli Venezia Giulia (northeastern Italy) with a high prevalence of BN. Captured insects were forced to feed on healthy tomato plants to induce infection. All strains obtained from symptomatic plants and confirmed by real‐time PCR were maintained on tomato through grafting. Successively, the strains were characterised by macroscopic and microscopic symptoms induced in the host, Multi‐Locus Sequence Typing (MLST) based on tuf, secY, stamp, and vmp1 genes, in‐planta spread and multiplication patterns. Molecular typing distinguished the strains into five lineages comprised in three clusters: one including strains of tuf‐a genotype and two including strains of tuf‐b1 genotype. Quite different symptoms were induced on tomatoes by strains belonging to the two tuf genotypes; infection by tuf‐a strains resulted in plant decline around 95–100 days after grafting and absence of cauliflower‐like inflorescence with symptoms of phyllody and virescence, which were usually associated with tuf‐b1 strains. The different symptoms, the outcome of disease, and the ultrastructural observation performed on sieve elements suggested a higher virulence of tuf‐a strains in tomato. Overall, our results propose that genomic variability of ‘Ca. P. solani’ strains should be extensively explored to determine possible associations with type of symptoms and strain virulence.

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.

‘Candidatus Liberibacter asiaticus’ (CLas), the agent associated with the Huanglongbing citrus disease, is commonly detected using quantitative PCR (qPCR) with hydrolysis probes. Internal standards are typically included in the qPCR assays to reduce the risk of false negatives caused by inhibitors. When the internal standard is detected but CLas is not, it is generally assumed that the pathogen is absent from the tested sample. However, our study shows that trace amounts of CLas may go undetected if the internal standard is either overly abundant or too dissimilar to CLas. To overcome these limitations, we developed a synthetic internal standard (IS) that uses the same primer as the CLas target sequence, along with three to four downstream nucleotides, but with a unique internal sequence derived from smooth hammerhead shark (Sphyrna zygaena; IS-SHK). This sequence matches the guanine-cytosine content and melting temperature of the CLas target and was specifically selected to minimize potential interference from other nucleic acid materials in citrus samples. To minimize competition between the IS-SHK standard and the CLas target, an average of only 21 molecules of IS-SHK standard is added to each qPCR reaction. Therefore, when IS-SHK standard is detected at expected levels and CLas is not, the absence of CLas is confirmed rather than inhibition of the detection. Conversely, the absence of both IS-SHK standard and CLas suggests the presence of a qPCR inhibitor, warranting retesting of the sample.

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.