Genetics

AbstractIncreasing incidences of phytoplasma infestations in Almond trees warrants the better management approach to prevent yield losses. Disease management rely on identification of the pathogen based on molecular profiling. The present study aimed, to identify the phytoplasma agent in almond trees and to measure the biochemical responses it causes in the host. Direct and Nested PCRs performed using phytoplasma specific primer pairs 16S rRNA, detected the presence of phytoplasma agent in symptomatic trees but not in symptomless trees. Phylogeny based on the sequence analysis revealed that the infecting agent was closely related to ‘Candidatus Phytoplasma phoenicium’ (16SrIX‐B subgroup). Then the study was carried out to determine the responses of physiological and biochemical mechanisms in almond trees infected with phytoplasma. Total chlorophyll and protein contents of infected almond trees were lower compared to healthy control, on the other hand, the levels of catalase and peroxidase activity increased in infected trees. Higher levels of stress‐related metabolites such as proline (20.53–39.23 μmol/g), phenol (3.00–4.44 mg GAE/g), salicylic (94.96–138.22 ng SA/mg protein) and jasmonic acid (965.86–1465.10 ng JA/mg protein) were observed in infected trees compared to asymptomatic trees, respectively in healthy and infected trees. The results showed that the Ca. P. phoenicium, which was detected for the first time in Türkiye, was able to change the physiological and biochemical mechanisms. The pathogenic agent could possess a potential danger in almond production areas. It is crucially important that this agent should be considered in certification programs.

ABSTRACT Filamentous Chloroflexota are abundant in activated sludge wastewater treatment plants (WWTPs) worldwide and are occasionally associated with poor solid-liquid separation or foaming, but most of the abundant lineages remain undescribed. Here, we present a comprehensive overview of Chloroflexota abundant in WWTPs worldwide, using high-quality metagenome-assembled genomes (MAGs) and 16S rRNA amplicon data from 740 Danish and global WWTPs. Many novel taxa were described, encompassing 4 families, 13 genera, and 29 novel species. These were widely distributed across most continents, influenced by factors such as climate zone and WWTP process design. Visualization by fluorescence in situ hybridization (FISH) confirmed their high abundances in many WWTPs based on the amplicon data and showed a filamentous morphology for nearly all species. Most formed thin and short trichomes integrated into the floc structure, unlikely to form the typical inter-floc bridging that hinders activated sludge floc settling. Metabolic reconstruction of 53 high-quality MAGs, representing most of the novel genera, offered further insights into their versatile metabolisms and suggested a primary role in carbon removal and involvement in nitrogen cycling. The presence of glycogen reserves, detected by FISH-Raman microspectroscopy, seemed widespread across the phylum, demonstrating that these bacteria likely utilize glycogen as energy storage to survive periods with limited resources. This study gives a broad overview of the Chloroflexota community in global activated sludge WWTPs and improves our understanding of their roles in these engineered ecosystems. IMPORTANCE Chloroflexota are often abundant members of the biomass in wastewater treatment plants (WWTPs) worldwide, typically with a filamentous morphology, forming the backbones of the activated sludge floc. However, their overgrowth can often cause operational issues connected to poor settling or foaming, impairing effluent quality and increasing operational costs. Despite their importance, few Chloroflexota genera have been characterized so far. Here, we present a comprehensive overview of Chloroflexota abundant in WWTPs worldwide and an in-depth characterization of their morphology, phylogeny, and ecophysiology, obtaining a broad understanding of their ecological role in activated sludge.

Abstract Objectives “Candidatus Liberibacter asiaticus” (CLas) is an un-culturable α-proteobacterium that caused citrus Huanglongbing (HLB), a destructive disease threatening citrus production worldwide. In China, the presence of HLB was first reported in Chaoshan region of Guangdong province, China around a century ago. Thus, whole genome information of CLas strains from Chaoshan area become the most important resource to understand the population diversity and evaluation of CLas in China. Data description CLas strain GDCZ was originally from Chaozhou city (Chaoshan area) and sequenced using PacBio Sequel long-read sequencing and Illumina short-read sequencing. The genome of strain GDCZ comprised of 1,230,507 bp with an average G + C content of 36.4%, along with a circular CLasMV1 phage: CLasMV1_GDCZ (8,869 bp). The CLas strain GDCZ contained a Type 2 prophage (37,452 bp) and encoded a total of 1,057 open reading frames and 53 RNA genes. The whole genome sequence of CLas strain GDCZ from the historical HLB endemic region in China will serve as a useful resource for further analyses of CLas evolution and HLB epidemiology in China and world.

ABSTRACT Autophagy plays an important role against pathogen infection in many organisms; however, little has been done with regard to vector-borne plant and animal pathogens, that sometimes replicate and cause deleterious effects in their vectors. Candidatus Liberibacter solanacearum (CLso) is a fastidious gram-negative phloem-restricted plant pathogen and vectored by the carrot psyllid, Bactericera trigonica . The plant disease caused by this bacterium is called carrot yellows and has recently gained much importance due to worldwide excessive economical losses. Here, we demonstrate that calcium ATPase, cytosolic calcium, and most importantly Beclin-1 have a role in regulating autophagy and its association with Liberibacter inside the psyllid. The presence of CLso generates reactive oxygen species and induces the expression of detoxification enzymes in the psyllid midguts, a main site for bacteria transmission. CLso also induces the expression of both sarco/endoplasmic reticulum Ca2+pump (SERCA) and 1,4,5-trisphosphate receptors (ITPR) in midguts, resulting in high levels of calcium in the cellular cytosol. Silencing these genes individually disrupted the calcium levels in the cytosol and resulted in direct effects on autophagy and subsequently on Liberibacter persistence and transmission. Inhibiting Beclin1-phosphorylation through different calcium-induced kinases altered the expression of autophagy and CLso titers and persistence. Based on our results obtained from the midgut, we suggest the existence of a direct correlation between cytosolic calcium levels, autophagy, and CLso persistence and transmission by the carrot psyllid. IMPORTANCE Plant diseases caused by vector-borne Liberibacter species are responsible for the most important economic losses in many agricultural sectors. Preventing these diseases relies mostly on chemical sprays against the insect vectors. Knowledge-based interference with the bacteria-vector interaction remains a promising approach as a sustainable solution. For unravelling how Liberibacter exploits molecular pathways in its insect vector for transmission, here, we show that the bacterium manipulates calcium levels on both sides of the endoplasmic reticulum membrane, resulting in manipulating autophagy. Silencing genes associated with these pathways disrupted the calcium levels in the cytosol and resulted in direct effects on autophagy and Liberibacter transmission. These results demonstrate major pathways that could be exploited for manipulating and controlling the disease transmission.

ABSTRACT Bacterial cells can vary greatly in size, from a few hundred nanometers to hundreds of micrometers in diameter. Filamentous cable bacteria also display substantial size differences, with filament diameters ranging from 0.4 to 8 µm. We analyzed the genomes of cable bacterium filaments from 11 coastal environments of which the resulting 23 new genomes represent 10 novel species-level clades of Candidatus Electrothrix and two clades that putatively represent novel genus-level diversity. Fluorescence in situ hybridization with a species-level probe showed that large-sized cable bacteria belong to a novel species with the proposed name Ca . Electrothrix gigas. Comparative genome analysis suggests genes that play a role in the construction or functioning of large cable bacteria cells: the genomes of Ca . Electrothrix gigas encode a novel actin-like protein as well as a species-specific gene cluster encoding four putative pilin proteins and a putative type II secretion platform protein, which are not present in other cable bacteria. The novel actin-like protein was also found in a number of other giant bacteria, suggesting there could be a genetic basis for large cell size. This actin-like protein (denoted big bacteria protein, Bbp) may have a function analogous to other actin proteins in cell structure or intracellular transport. We contend that Bbp may help overcome the challenges of diffusion limitation and/or morphological complexity presented by the large cells of Ca . Electrothrix gigas and other giant bacteria. IMPORTANCE In this study, we substantially expand the known diversity of marine cable bacteria and describe cable bacteria with a large diameter as a novel species with the proposed name Candidatus Electrothrix gigas. In the genomes of this species, we identified a gene that encodes a novel actin-like protein [denoted big bacteria protein (Bbp)]. The bbp gene was also found in a number of other giant bacteria, predominantly affiliated to Desulfobacterota and Gammaproteobacteria, indicating that there may be a genetic basis for large cell size. Thus far, mostly structural adaptations of giant bacteria, vacuoles, and other inclusions or organelles have been observed, which are employed to overcome nutrient diffusion limitation in their environment. In analogy to other actin proteins, Bbp could fulfill a structural role in the cell or potentially facilitate intracellular transport.

Abstract The most damaging citrus diseases are Huanglongbing (HLB) and citrus canker, which are caused by Candidatus Liberibacter asiaticus (CaLas) and Xanthomonas citri pv. citri (Xcc), respectively. Endolysins from bacteriophages are a possible option for disease resistance in plant breeding. Here, we report improvement of citrus resistance to HLB and citrus canker using the LasLYS1 and LasLYS2 endolysins from CaLas. LasLYS2 demonstrated bactericidal efficacy against several Rhizobiaceae bacteria and Xcc, according to inhibition zone analyses. The two genes, driven by a strong promoter from Cauliflower mosaic virus, 35S, were integrated into Carrizo citrange via Agrobacterium-mediated transformation. More than 2 years of greenhouse testing indicated that LasLYS2 provided substantial and long-lasting resistance to HLB, allowing transgenic plants to retain low CaLas titers and no obvious symptoms while also clearing CaLas from infected plants in the long term. LasLYS2 transgenic plants with improved HLB resistance also showed resistance to Xcc, indicating that LasLYS2 had dual resistance to HLB and citrus canker. A microbiome study of transgenic plants revealed that the endolysins repressed Xanthomonadaceae and Rhizobiaceae populations in roots while increasing Burkholderiaceae and Rhodanobacteraceae populations, which might boost the citrus defense response, according to transcriptome analysis. We also found that Lyz domain 2 is the key bactericidal motif of LasLYS1 and LasLYS2. Four endolysins with potential resistance to HLB and citrus canker were found based on the structures of LasLYS1 and LasLYS2. Overall, the work shed light on the mechanisms of resistance of CaLas-derived endolysins, providing insights for designing endolysins to develop broad-spectrum disease resistance in citrus.