Publications

4364

The lipopolysaccharides (LPS) of gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic ‘Candidatus Liberibacter’ spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on nonhost tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host orange (Citrus sinensis), confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione pool, callose deposition, and activation of the salicylic acid and azelaic acid (AzA) signaling networks. Transient expression of ‘Ca. L. asiaticus’ bacterioferritin comigratory protein (BCP) peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that ‘Ca. L. asiaticus’ BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. ‘Ca. L. asiaticus’ BCP peroxiredoxin (i) attenuates NO-mediated SAR signaling and (ii) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of ‘Ca. L. asiaticus’ acquisition and transmission by fecund psyllids throughout the limited flush period in citrus. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Phytoplasmas are insect-transmitted plant pathogens that cause substantial losses in agriculture. In addition to economic impact, phytoplasmas induce distinct disease symptoms in infected plants, thus attracting attention for research on molecular plant-microbe interactions and plant developmental processes. Due to the difficulty of establishing an axenic culture of these bacteria, culture-independent genome characterization is a crucial tool for phytoplasma research. However, phytoplasma genomes have strong nucleotide composition biases and are repetitive, which make it challenging to produce complete assemblies. In this study, we utilized Illumina and Oxford Nanopore sequencing technologies to obtain the complete genome sequence of ‘Candidatus Phytoplasma luffae’ strain NCHU2019 that is associated with witches’ broom disease of loofah (Luffa aegyptiaca) in Taiwan. The fully assembled circular chromosome is 769 kb in size and is the first representative genome sequence of group 16SrVIII phytoplasmas. Comparative analysis with other phytoplasmas revealed that NCHU2019 has a remarkably repetitive genome, possessing a pair of 75 kb repeats and at least 13 potential mobile units (PMUs) that account for ∼25% of its chromosome. This level of genome repetitiveness is exceptional for bacteria, particularly among obligate pathogens with reduced genomes. Our genus-level analysis of PMUs demonstrated that these phytoplasma-specific mobile genetic elements can be classified into three major types that differ in gene organization and phylogenetic distribution. Notably, PMU abundance explains nearly 80% of the variance in phytoplasma genome sizes, a finding that provides a quantitative estimate for the importance of PMUs in phytoplasma genome variability. Finally, our investigation found that in addition to horizontal gene transfer, PMUs also contribute to intra-genomic duplications of effector genes, which may provide redundancy for subfunctionalization or neofunctionalization. Taken together, this work improves the taxon sampling for phytoplasma genome research and provides novel information regarding the roles of mobile genetic elements in phytoplasma evolution.

Huanglongbing (HLB), caused by “Candidatus liberibacter asiaticus” (CaLas), is one of the most devastating diseases in citrus but its pathogenesis remains poorly understood. Here, we reported the role of the CaLasSDE115 (CLIBASIA_05115) effector, encoded by CaLas, during pathogen-host interactions. Bioinformatics analyses showed that CaLasSDE115 was 100% conserved in all reported CaLas strains but had sequence differences compared with orthologs from other “Candidatus liberibacter.” Prediction of protein structures suggested that the crystal structure of CaLasSDE115 was very close to that of the invasion-related protein B (IalB), a virulence factor from Bartonella henselae. Alkaline phosphatase (PhoA) assay in E. coli further confirmed that CaLasSDE115 was a Sec-dependent secretory protein while subcellular localization analyses in tobacco showed that the mature protein of SDE115 (mSDE115), without its putative Sec-dependent signal peptide, was distributed in the cytoplasm and the nucleus. Expression levels of CaLasSDE115 in CaLas-infected Asian citrus psyllid (ACP) were much higher (∼45-fold) than those in CaLas-infected Wanjincheng oranges, with the expression in symptomatic leaves being significantly higher than that in asymptomatic ones. Additionally, the overexpression of mSDE115 favored CaLas proliferation during the early stages (2 months) of infection while promoting the development of symptoms. Hormone content and gene expression analysis of transgenic plants also suggested that overexpressing mSDE115 modulated the transcriptional regulation of genes involved in systemic acquired resistance (SAR) response. Overall, our data indicated that CaLasSDE115 effector contributed to the early colonization of citrus by the pathogen and worsened the occurrence of Huanglongbing symptoms, thereby providing a theoretical basis for further exploring the pathogenic mechanisms of Huanglongbing disease in citrus.

A novel micro-organism designated AS10Twas isolated from dry salt collected from Aveiro saltern in the north of Portugal. Cells were Gram-stain-positive, non-motile, non-endospore-forming, rod-shaped and aerobic. Strain AS10Twas characterized by long filaments of rod-shaped cells, presenting also coccoid cellular forms at the end of the filaments, unveiling some pleomorphism. Rod-shaped cells varied from 0.3 to 0.6 µm wide and from 0.6 to 2 µm long. Growth of AS10Toccurred at 15–40 °C (optimum, 20–30 °C), 0–10% (w/v) NaCl (optimum, 2%) and pH 4.5–11.0 (optimum, pH 8.0–11.0). The peptidoglycan type was A1ϒ-type with 3-hydroxy-diaminopimelic acid. The major fatty acids were C16:0, iso-C14:0, C17:0and C14:0. The major respiratory quinone was MK-9(H4). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain AS10Twas similar to actinobacterial members of the classNitriliruptoria, withNitriliruptor alkaliphilusANL-iso2Tbeing the closest relative the species with a sequence pairwise similarity of 91.21%. Average nucleotide identity, average amino acid identity andin silicoDNA–DNA hybridization values between strain AS10TandN. alkaliphilusANL-iso2Twere 71.34, 53.57 and 18.90%, respectively. The genome DNA G+C content of AS10Twas 71.8 mol%. Based on genomic, phylogenetic, phenotypic and chemotaxonomic studies, we describe a new species of a novel genus represented by strain AS10T(=LMG 31937T=CECT 30148T) for which we propose the nameSalsipaludibacter albusgen. nov., sp. nov. We also propose that this organism represents a new family namedSalsipaludibacteraceaefam. nov. of a novel order namedSalsipaludibacteralesord. nov.

Abstract This study explored the microbial population dynamics of Accumulibacter (Acc) at low temperature and metabolic shift to limiting polyphosphate (Poly-P) in enhanced biological phosphorus removal (EBPR) system. The Accumulibacter-enriched EBPR systems, fed with acetate (HAc) and propionate (HPr) at 10 ± 1 °C respectively, were operated for 60 days in two identical SBR reactors (SBR-1 and SBR-2). The phosphorus removal performance in two systems was stable at 10 ± 1 °C, while the microbial community structure changed. Compared with the population structure in seed sludge, Accumulibacter clades reduced in the HAc system, while Acc I increased significantly in the HPr system. Low temperature was beneficial to the formation of granular sludge in the EBPR system, and the sludge granulation in the HAc system was more homogeneous than that in the HPr system. Accumulibacter in the HPr system can get ATP through glycogen accumulating metabolism (GAM) under limiting Poly-P condition at 10 ± 1 °C, while that in the HAc system cannot. This work suggests that poly-P levels can affect the metabolic pathway of Accumulibacter in EBPR systems under low temperature.