Publications

4372

Tripartite symbioses between anaerobic ciliated protists and their intracellular archaeal and bacterial symbionts are not uncommon, but most reports have been based mainly on microscopic observations. Deeper insights into the function, ecology, and evolution of these fascinating symbioses involving partners from all three domains of life have been hampered by the difficulties of culturing anaerobic ciliates in the laboratory and the frequent loss of their prokaryotic partners during long-term cultivation. In the present study, we report the isolation of an anaerobic scuticociliate, strain GW7, which has been stably maintained in our laboratory for more than 3 years without losing either of its endosymbionts. Unexpectedly, molecular characterization of the endosymbionts revealed that the bacterial partner of GW7 is phylogenetically related to intranuclear endosymbionts of aerobic ciliates. This strain will enable future genomic, transcriptomic, and proteomic analyses of the interactions in this tripartite symbiosis and a comparison with endosymbioses in aerobic ciliates.

Huanglongbing (HLB) or greening currently is the most devastating citrus disease worldwide. The fastidious phloem-colonizing bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) is the causal agent of citrus HLB in Florida. Bactericides containing the active ingredient oxytetracycline (OTC) have been used in foliar spray to control citrus HLB in Florida since 2016. However, the minimum concentration of OTC required to suppress CLas in planta remains unknown. We developed a new method for evaluating the effects of OTC treatment on CLas titers in infected plants and determined the relationship between OTC residue levels and control levels achieved for CLas using mathematical modeling in greenhouse and field experiments. In both greenhouse and field, OTC spray did not reduce the titers of CLas, and it produced undetectable or mild levels of OTC residue in leaves within 7 days post-application (DPA). In greenhouse, OTC injection at 0.05 g per tree decreased CLas titers to an undetectable level (cycle threshold value ≥ 36.0) from 7 to 30 DPA and produced a residue level of OTC at 0.68 to 0.73 µg/g of fresh tissue over this period. In the field, OTC injection at 0.50 g per tree resulted in the decline of CLas titers by 1.52 log reduction from 14 to 60 DPA, with residue levels of OTC at 0.27 to 0.33 µg/g of fresh tissue. In both trials, a first-order compart model of OTC residue dynamics in leaves of trunk-injected trees was specified for estimating the retention of effective concentrations. Furthermore, nonlinear modeling revealed significant positive correlations between OTC residue levels in leaves and the control levels for CLas achieved. The results suggested that the minimum concentrations of OTC required to suppress CLas populations in planta to below the detection limit are 0.68 and 0.86 µg/g and that the minimum concentrations of OTC required for initial inhibition of CLas growth in planta are ∼0.17 and ∼0.215 µg/g in leaf tissues under greenhouse and field conditions, respectively. This finding highlights that a minimum concentration of OTC should be guaranteed to be delivered to target CLas in infected plants for effective control of citrus HLB.

AbstractBackgroundThe symbiosis between the olive fruit fly,Bactrocera oleae, andCandidatusErwinia dacicola has been demonstrated as essential for the fly’s larval development and adult physiology. The mass rearing of the olive fruit fly has been hindered by several issues, including problems which could be related to the lack of the symbiont, presumably due to preservatives and antibiotics currently used during rearing under laboratory conditions. To better understand the mechanisms underlying symbiont removal or loss during the rearing of lab colonies of the olive fruit fly, we performed experiments that focused on bacterial transfer from wild female flies to their eggs. In this research, eggs laid by wild females were treated with propionic acid solution, which is often used as an antifungal agent, a mixture of sodium hypochlorite and Triton X, or water (as a control). The presence of the bacterial symbiont on eggs was evaluated by real-time PCR and scanning electron microscopy.ResultsDGGE analysis showed a clear band with the same migration behavior present in all DGGE profiles but with a decreasing intensity. Molecular analyses performed by real-time PCR showed a significant reduction inCa. E. dacicola abundance in eggs treated with propionic acid solution or a mixture of sodium hypochlorite and Triton X compared to those treated with water. In addition, the removal of bacteria from the surfaces of treated eggs was highlighted by scanning electron microscopy.ConclusionsThe results clearly indicate how the first phases of the colony-establishment process are important in maintaining the symbiont load in laboratory populations and suggest that the use of products with antimicrobial activity should be avoided. The results also suggest that alternative rearing procedures for the olive fruit fly should be investigated.

Abstract Background ‘Candidatus Phytoplasma solani’ is endemic in Europe and infects a wide range of weeds and cultivated plants. Phytoplasmas are prokaryotic plant pathogens that colonize the sieve elements of their host plant, causing severe alterations in phloem function and impairment of assimilate translocation. Typical symptoms of infected plants include yellowing of leaves or shoots, leaf curling, and general stunting, but the molecular mechanisms underlying most of the reported changes remain largely enigmatic. To infer a possible involvement of Fe in the host-phytoplasma interaction, we investigated the effects of ‘Candidatus Phytoplasma solani’ infection on tomato plants (Solanum lycopersicum cv. Micro-Tom) grown under different Fe regimes. Results Both phytoplasma infection and Fe starvation led to the development of chlorotic leaves and altered thylakoid organization. In infected plants, Fe accumulated in phloem tissue, altering the local distribution of Fe. In infected plants, Fe starvation had additive effects on chlorophyll content and leaf chlorosis, suggesting that the two conditions affected the phenotypic readout via separate routes. To gain insights into the transcriptional response to phytoplasma infection, or Fe deficiency, transcriptome profiling was performed on midrib-enriched leaves. RNA-seq analysis revealed that both stress conditions altered the expression of a large (> 800) subset of common genes involved in photosynthetic light reactions, porphyrin / chlorophyll metabolism, and in flowering control. In Fe-deficient plants, phytoplasma infection perturbed the Fe deficiency response in roots, possibly by interference with the synthesis or transport of a promotive signal transmitted from the leaves to the roots. Conclusions ‘Candidatus Phytoplasma solani’ infection changes the Fe distribution in tomato leaves, affects the photosynthetic machinery and perturbs the orchestration of root-mediated transport processes by compromising shoot-to-root communication.

‘Candidatus Liberibacter asiaticus’ (CLas) is one of the causal agents of citrus Huanglongbing (HLB), a bacterial disease of citrus trees that greatly reduces fruit yield and quality. CLas strains produce an array of currently uncharacterized Sec-dependent secretory proteins. In this study, the conserved chromosomally encoded protein CLIBASIA_03875 was identified as a novel Sec-dependent secreted protein. We show that CLIBASIA_03875 contains a putative Sec- secretion signal peptide (SP), a 29 amino acid residue located at the N-terminus, with a mature protein (m3875) of 22 amino acids found to localize in multiple subcellular components of the leaf epidermal cells of Nicotiana benthamiana. When overexpressed via a Potato virus X (PVX)-based expression vector in N. benthamiana, m3875 suppressed programmed cell death (PCD) and the H2O2 accumulation triggered by the pro-apoptotic mouse protein BAX and the Phytophthora infestans elicitin INF1. Overexpression also resulted in a phenotype of dwarfing, leaf deformation and mosaics, suggesting that m3875 has roles in plant immune response, growth, and development. Substitution mutagenesis of the charged amino acid (D7, R9, R11, and K22) with alanine within m3875 did not recover the phenotypes for PCD and normal growth. In addition, the transiently overexpressed m3875 regulated the transcriptional levels of N. benthamiana orthologs of CNGCs (cyclic nucleotide-gated channels), BI-1 (Bax-inhibitor 1), and WRKY33 that are involved in plant defense mechanisms. To our knowledge, m3875 is the first PCD suppressor identified from CLas. Studying the function of this protein provides insight as to how CLas attenuates the host immune responses to proliferate and cause Huanglongbing disease in citrus plants.

Disease caused by the bacterial pathogen “Candidatus Liberibacter solanacearum” (Lso) represents a serious threat to solanaceous crop production. Insecticide applications to control the psyllid vector, Bactericera cockerelli Šulc (Hemiptera: Triozidae) has led to the emergence of resistance in psyllids populations. Efforts to select natural resistant cultivars have been marginally successful and have been complicated by the presence of distinct Lso haplotypes (LsoA, LsoB) differing in symptoms severity on potato and tomato. A potentially promising management tool is to boost host resistance to the pathogen and/or the insect vector by promoting mycorrhization. Here we tested the hypothesis that mycorrhizal fungi can mitigate the effect of Lso infection on tomato plants. The presence of mycorrhizal fungi substantially delayed and reduced the incidence of Lso-induced symptoms on tomato as compared to non-mycorrhized plants. However, PCR with specific Lso primers revealed that mycorrhization did not prevent Lso transmission or translocation to newly formed leaves. Mycorrhization significantly reduced oviposition by psyllids harboring LsoA and survival of nymphs from these eggs. However, mycorrhization had no effect on oviposition by psyllids harboring LsoB or the survival of nymphs from parents harboring LsoB. These findings indicate the use of mycorrhizal fungi is a promising strategy for the mitigation of disease caused by both LsoA and LsoB and warrants additional field testing.