Publications

4472

AbstractCandidatus Liberibacter asiaticus (Las) is an emergent bacterial pathogen that is associated with the devastating citrus Huanglongbing (HLB). Vectored by the Asian citrus psyllid, Las colonizes the phloem tissue of citrus, causing severe damage to infected trees. So far, cultivating pure Las culture in axenic media has not been successful and dual-transcriptome analyses aiming to profile gene expression in both Las and its host(s) have a low coverage of the Las genome due to the low abundance of bacterial RNA in total RNA extracts from infected tissues. Therefore, the lack of a Las transcriptome remains as a significant knowledge gap. Here, we used a bacterial cell enrichment procedure and confidently determined the expression profiles of approximately 84% of the Las genes. Genes that exhibited the highest expression levels in citrus include ion transporters, ferritin, outer membrane porins, and genes involved in phage-related functions, pilus formation, cell wall modification, and stress responses. One hundred and six genes were found to be differentially expressed in citrus vs psyllids. Genes related to transcription/translation and resilience to host defense response were upregulated in citrus; whereas genes involved in energy generation and the flagella system were expressed to higher levels in psyllids. We also determined the relative expression levels of potential Sec-dependent effectors, which are considered as key virulence factors of Las. This work advances our understanding of HLB biology and offers novel insight into the interactions of Las with its plant host and insect vector.

Citrus greening or Huanglongbing (HLB) is one of the main devastating citrus diseases worldwide. HLB causing infectious bacterium “Candidatus Liberibacter” is a phloem specific and fastidious agent, having three different species. A comparison of conventional PCR and real-time quantitative PCR (qPCR) was done by universal 16S rDNA marker. In the current study conventional PCR was used to early screening of Candidatus Liberibacter spp and real-time quantitative PCR used for the quantification of HLB causing agent in citrus trees. Total 35 citrus cultivars were selected from mandarins and sweet oranges groups from an orchard “Citrus Research Institute, Sargodha,” Pakistan. The DNA was extracted from HLB asymptomatic and symptomatic leaves. By conventional PCR, the HLB infectious bacterium has identified in 11 sweet oranges samples (31%) in which an amplicon of 1174 bp was generated. While qPCR subsequently reported to quantify the titer of Ca. L.asiaticus in host plant. In the present study qPCR results showed the pathogen could be detected with a Cq value getting in ranged of 26.58to 38.17. Samples with higher Cq ratio showed lower copy number of targeted gene. There was no amplification detected with healthy citrus samples and water control. A robust and sensitive qPCR assay can be used for the screening and quantify the Ca. L. asiaticus strain to confirm the HLB infection in Pakistan. This diagnostic test will be very helpful for implementing a wide range of diagnostics research programs aiming at ultimately developing strategies for the control of HLB in Pakistan. Keywords: Citrus greening, Conventional PCR, Candidatus Liberibacter asiaticus, Huanglongbing, cytochrome oxidase, real-time qPCR.

‘Candidatus Liberibacter asiaticus’ is known as the most pathogenic organism associated with citrus greening disease. Since its publicized emergence in Florida in 2005, ‘Ca. L. asiaticus’ remains unculturable. Currently, a limited number of potential disease effectors have been identified through in silico analysis. Therefore, these potential effectors remain poorly characterized and do not fully explain the complexity of symptoms observed in citrus trees infected with ‘Ca. L. asiaticus.’ LotP has been identified as a potential effector and have been partially characterized. This protein retains structural homology to the substrate binding domain of the Lon protease. LotP interacts with chaperones like GroEL, Hsp40, DnaJ, and ClpX and may exercise its biological role through interactions with different proteins involved in proteostasis networks. Here, we evaluate the interactome of LotP—revealing a new protein–protein interaction target (Lon-serine protease) and its effect on citrus plant tissue integrity. We found that via protein–protein interactions, LotP can enhance Lon protease activity, increasing the degradation rate of its specific targets. Infiltration of purified LotP strained citrus plant tissue causing photoinhibition and chlorosis after several days. Proteomics analysis of LotP tissues recovering after the infiltration revealed a large abundance of plant proteins associated with the stabilization and processing of mRNA transcripts, a subset of important transcription factors; and pathways associated with innate plant defense were highly expressed. Furthermore, interactions and substrate binding module of LotP suggest potential interactions with plant proteins, most likely proteases.

Summary Candidatus Dormibacterota is an uncultured bacterial phylum found predominantly in soil that is present in high abundances within cold desert soils. Here, we interrogate nine metagenome‐assembled genomes ( MAGs ), including six new MAGs derived from soil metagenomes obtained from two eastern Antarctic sites. Phylogenomic and taxonomic analyses revealed these MAGs represent four genera and five species, representing two order‐level clades within Ca . Dormibacterota. Metabolic reconstructions of these MAGs revealed the potential for aerobic metabolism, and versatile adaptations enabling persistence in the ‘extreme’ Antarctic environment. Primary amongst these adaptations were abilities to scavenge atmospheric H 2 and CO as energy sources, as well as using the energy derived from H 2 oxidation to fix atmospheric CO 2 via the Calvin–Bassham–Benson cycle, using a RuBisCO type IE . We propose that these allow Ca . Dormibacterota to persist using H 2 oxidation and grow using atmospheric chemosynthesis in terrestrial Antarctica. Fluorescence in situ hybridization revealed Ca . Dormibacterota to be coccoid cells, 0.3–1.4 μm in diameter, with some cells exhibiting the potential for a symbiotic or syntrophic lifestyle.

Specific scopeThis Standard describes a diagnostic protocol for ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’.1This Standard should be used in conjunction with PM 7/76 (5) Use of EPPO diagnostic protocols.Specific approval and amendmentFirst approved in 2014–09. Revised in 2021–04.

Summary We report on the genomic characterization of three novel classes in the phylum Desulfobacterota. One class (proposed name Candidatus ‘Anaeroferrophillalia’) was characterized by heterotrophic growth capacity, either fermentatively or utilizing polysulfide, tetrathionate or thiosulfate as electron acceptors. In the absence of organic carbon sources, autotrophic growth via the Wood–Ljungdahl (WL) pathway and using hydrogen or Fe(II) as an electron donor is also inferred for members of the ‘Anaeroferrophillalia’. The second class (proposed name Candidatus ‘Anaeropigmentia’) was characterized by its capacity for growth at low oxygen concentration, and the capacity to synthesize the methyl/alkyl carrier CoM, an ability that is prevalent in the archaeal but rare in the bacterial domain. Pigmentation is inferred from the capacity for carotenoid (lycopene) production. The third class (proposed name Candidatus ‘Zymogenia’) was characterized by fermentative heterotrophic growth capacity, broad substrate range and the adaptation of some of its members to hypersaline habitats. Analysis of the distribution pattern of all three classes showed their occurrence as rare community members in multiple habitats, with preferences for anaerobic terrestrial, freshwater and marine environments over oxygenated (e.g. pelagic ocean and agricultural land) settings. Special preference for some members of the class Candidatus ‘Zymogenia’ for hypersaline environments such as hypersaline microbial mats and lagoons was observed.