Publications

4862

Abstract Phytoplasmas are wall-less plant pathogens characterized by highly reduced genomes and limited metabolic capabilities, leading to an obligate host dependency. Since phytoplasma axenic cultivation has not yet been achieved, genomic approaches are essential to unravelling their biological and pathogenic traits. However, such insights are currently hindered by the scarcity of complete genomes and the lack of data for several clades, as is the case for the quarantine-relevant ‘ Candidatus Phytoplasma phoenicium’ (group 16SrIX-B), the causal agent of the severe stone-fruit disease almond witches’ broom (AlmWB). In this study, we present the complete genome sequence of the Lebanese strain F1A, which consists of a 552,248 bp chromosome with a 24.29% GC content and encodes 450 protein-coding genes. Comparative analyses with 16SrIX group draft genomes revealed high conservation among Lebanese AlmWB strains; however, comparisons with related 16SrIX-C phytoplasmas were limited by their low assembly completeness. Beyond providing a complete reference genome for this phytoplasma group, the F1A chromosome revealed distinct metabolic features. Strain F1A lacks the upper glycolytic pathway, indicating a metabolism that relies on glycerol-3-phosphate uptake and carboxylic acid fermentation. Notably, in addition to the conserved malate-to-acetate pathway, F1A encodes a complete citrate lyase complex, suggesting the potential for citrate utilization. Phylogenetic analysis of the associated 2-hydrocarboxylate symporter revealed the widespread occurrence of two phylogenetically distinct variants in phytoplasmas. Apart from the deduced metabolic capacities, the predicted effector repertoire shows similarities with those of other phytoplasmas, including proteins associated with branch proliferation and witches’ broom symptoms. Overall, these findings link genome reduction to host-dependent carboxylic acid metabolism in a quarantine-relevant phytoplasma, establishing a framework for comparative and functional studies of 16SrIX phytoplasmas and providing a basis for future investigations of AlmWB ecology and pathogenicity.

The Asian citrus psyllid, Diaphorina citri, transmits the putative causal agent of citrus greening disease, ‘ Candidatus Liberibacter asiaticus’. The transmission occurs in a propagative, circulative manner. Specific protein–protein interactions are required for the accomplishment of the transmission process. We employed the far-Western technique, also known as protein overlay assay, and mass spectrometry to identify D. citri proteins that specifically recognize membrane proteins of ‘ Ca. L. asiaticus’. Whole-body protein extracts of D. citri were separated by electrophoresis and blotted onto polyvinylidene difluoride membranes. The membranes were overlayed with sonicated leaf midrib extracts from ‘ Ca. L. asiaticus’-infected citrus plants. The protein complexes were detected using different antibodies against ‘ Ca. L. asiaticus’ membrane proteins and peptidoglycans associated with lipoproteins. By comparison, spots were extracted from a polyacrylamide gel for liquid chromatography-tandem mass spectrometry analysis. Identified proteins included ATP synthase alpha and beta subunits, actin, alpha- and beta-tubulins, transitional endoplasmic reticulum ATPase TER94, 78 kDa glucose-regulated protein, and arginine kinase. These findings emphasize the involvement of energy machinery and muscle proteins in the ‘ Ca. L. asiaticus’– D. citri interactions. Understanding the pathogen–vector interactions will lead to better-designed control strategies based on the interference with specific interactions and/or blocking transmission.

ABSTRACT The selective autophagy receptor NEIGHBOUR OF BRCA1 (NBR1) is a key regulator of plant immunity, but its role against bacterial pathogens remains poorly understood. Here, we demonstrated that overexpression of CsNBR1 confers citrus resistance to huanglongbing (HLB) caused by ‘ Candidatus Liberibacter asiaticus’ ( C Las). CsNBR1 can interact with multiple virulence effector proteins of C Las, including SDE5115, SDE1, SDE19 and SDE5, leading to their degradation via the autophagic pathway. In addition, expression of CsNBR1 in Nicotiana benthamiana mitigated the leaf curling and dwarfing phenotypes triggered by SDE1 by reducing the protein content of SDE1. These findings reveal that CsNBR1 positively regulates citrus defence against C Las by degrading its effector proteins via selective autophagy, providing both mechanistic insights and a potential target for resistance breeding.

A phytoplasma associated with poinsettia has previously been reported in Korea, but the report primarily focused on stem flattening symptoms and was based solely on partial 16S ribosomal RNA (rRNA) gene sequence analysis. In this study, phytoplasma-infected poinsettias collected in Jinan and Jeonju, Korea were examined for diverse symptom expression and additional molecular characterization of the associated phytoplasma. All seven plants showed a free-branching phenotype. Symptom severity ranged from low freebranching, with larger leaves and long internodes, to severe free-branching, with shortened internodes and small, irregularly twisted leaves. Based on 16S rRNA gene sequence and iPhyClassifier analyses, the phytoplasma detected in this study was assigned to subgroup 16SrIII-A of the X-disease phytoplasma group (16SrIII). Although this assignment differs from the previous Korean report, which classified poinsettia-associated phytoplasmas subgroup 16SrIII-H, the present study interpreted the classification using an updated 16SrIII subgroup framework. Sequence analyses of more variable regions, including the 16S-23S intergenic spacer region, ribosomal protein (rp) operon, and secA gene, showed 99.46-99.55% nucleotide identity with ‘Candidatus Phytoplasma pruni’-related reference strains. Phylogenetic analyses showed that the poinsettia-associated phytoplasmas in Korea were closely related to the subgroup 16SrIII-A. These results expand the known symptom spectrum of phytoplasma-infected poinsettias and provide multilocus molecular characterization supporting the phylogenetic placement of poinsettia-associated phytoplasmas in Korea.

Periploca aphylla witches’ broom (PapWB) is a phytoplasma-associated disease first reported in south-western Iran in 2010. Recent observations showed that the disease is spreading to new areas. PapWB phytoplasmas, detected in symptomatic P. aphylla plants, exhibited identical nucleotide sequences in the 1.8 kb PCR amplicon encompassing the 16S rRNA gene, the 16S-23S spacer region and the 5′-end of the 23S rRNA gene. When compared with the reference strains of previously described ‘ Candidatus Phytoplasma’ species, the selected representative strain PapWB-S exhibited the highest 16S rRNA gene sequence identity (98.15%) with salt cedar witches’ broom (SCWB1) phytoplasma, the reference strain of ‘ Ca . P. tamaricis’. Moreover, the sequence identity values for the tufB, rplV-rpsC and secY genes of PapWB-S strain, when compared with those of reference strains of previously described ‘ Ca . Phytoplasma’ species, were below the thresholds adopted for describing new species. Phylogenetic analyses confirmed the close relationship of the strain PapWB-S with SCWB1 followed by other ‘ Ca . Phytoplasma’ species belonging to the ribosomal groups 16SrX, 16SrXX and 16SrXXXIII. Following the revised guidelines for describing ‘ Ca . Phytoplasma’ species, ‘ Ca . P. periplocae’ sp. nov., associated with PapWB disease, is proposed, and the 16S rRNA gene sequence (accessible at GenBank/NCBI accession number OQ819043) is its nomenclatural type.