Publications

4372

Abstract Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits Candidatus Liberibacter asiaticus (Las), the bacterial pathogen putatively responsible for citrus huanglongbing. Multiple studies have shown psyllids acquire Las more frequently, and are more likely to inoculate susceptible plants, when they acquire Las as nymphs. Understanding the transmission of Las to nymphs is critical to the Las lifecycle. The objective of this study was to determine the transmission Las by female D. citri to their offspring. Two transmission pathways were quantified: horizontal transmission (acquisition of Las via feeding at the oviposition site) and vertical transmission (transovarial). Eggs of individual, infected females were transferred to an uninfected seedling to assess vertical transmission. In a second experiment, horizontal transmission was evaluated by replacing eggs laid by infected females with uninfected nymphs. Nymphs exposed to Las via horizontal transmission of the oviposition site were more likely to acquire Las than from vertical transmission. Las deposited in flush by an infected adult female feeding during oviposition was sufficient for infecting nymphs. Combined results of both experiments suggest that vertical transmission allows Las to spread in low amounts even when infected plant hosts are not available and that inoculation of the oviposition site provides a source of Las to developing nymphs via the plant phloem. These data support the hypothesis that transmission through infected plant material via maternal inoculation is a primary pathway of Las transmission between vector and host.

Summary A hallmark of the SUP05 clade of marine Gammaproteobacteria is the ability to use energy obtained from reduced inorganic sulfur to fuel autotrophic fixation of carbon using RuBisCo. However, some SUP05 also have the genetic potential for heterotrophic growth, raising questions about the roles of SUP05 in the marine carbon cycle. We used genomic reconstructions, physiological growth experiments and proteomics to characterize central carbon and energy metabolism in Candidatus Thioglobus singularis strain PS1, a representative from the SUP05 clade that has the genetic potential for autotrophy and heterotrophy. Here, we show that the addition of individual organic compounds and 0.2 μm filtered diatom lysate significantly enhanced the growth of this bacterium. This positive growth response to organic substrates, combined with expression of a complete TCA cycle, heterotrophic pathways for carbon assimilation, and methylotrophic pathways for energy conversion demonstrate strain PS1's capacity for heterotrophic growth. Further, our inability to verify the expression of RuBisCO suggests that carbon fixation was not critical for growth. These results highlight the metabolic diversity of the SUP05 clade that harbours both primary producers and consumers of organic carbon in the oceans and expand our understanding of specific pathways of organic matter oxidation by the heterotrophic SUP05.

In recent decades, ‘Candidatus Liberibacter spp.’ have emerged as a versatile group of psyllid-vectored plant pathogens and endophytes capable of infecting a wide range of economically important plant hosts. The most notable example is ‘Candidatus Liberibacter asiaticus’ (CLas) associated with Huanglongbing (HLB) in several major citrus-producing areas of the world. CLas is a phloem-limited α-proteobacterium that is primarily vectored and transmitted among citrus species by the Asian citrus psyllid (ACP) Diaphorina citri. HLB was first detected in North America in Florida (USA) in 2005, following introduction of the ACP to the State in 1998. HLB rapidly spread to all citrus growing regions of Florida within three years, with severe economic consequences to growers and considerable expense to taxpayers of the state and nation. Inability to establish CLas in culture (except transiently) remains a significant scientific challenge toward effective HLB management. Lack of axenic cultures has restricted functional genomic analyses, transfer of CLas to either insect or plant hosts for fulfillment of Koch’s postulates, characterization of host-pathogen interactions and effective screening of antibacterial compounds. In the last decade, substantial progress has been made toward CLas culturing: (i) three reports of transient CLas cultures were published, (ii) a new species of Liberibacter was identified and axenically cultured from diseased mountain papaya (Liberibacter crescens strain BT-1), (iii) psyllid hemolymph and citrus phloem sap were biochemically characterized, (iv) CLas phages were identified and lytic genes possibly affecting CLas growth were described, and (v) genomic sequences of 15 CLas strains were made available. In addition, development of L. crescens as a surrogate host for functional analyses of CLas genes, has provided valuable insights into CLas pathogenesis and its physiological dependence on the host cell. In this review we summarize the conclusions from these important studies.

‘Candidatus Liberibacter solanacearum’ is an unculturable α-proteobacterium that is the causal agent of zebra chip disease of potato—a major problem in potato-growing areas, because it affects growth and yield. Developing effective treatments for ‘Ca. L. solanacearum’ has been hampered by the difficulty in functionally characterizing the proteins of this organism, largely because they are not easily expressed and purified in standard expression systems. ‘Ca. L. solanacearum’ has a reduced genome and its proteins are predicted to be prone to instability and aggregation. Among intracellular-dwelling bacteria, chaperone proteins are conserved and overexpressed to buffer against problems in protein folding. We mimicked this approach for expressing and purifying ‘Ca. L. solanacearum’ proteins in Escherichia coli by coexpressing them with chaperones. Neither of the representative ‘Ca. L. solanacearum’ enzymes, dihydrodipicolinate synthase (key in lysine biosynthesis) and pyruvate kinase (involved in glycolysis), were overexpressed in standard E. coli expression plasmids or strains. However, soluble dihydrodipicolinate synthase was successfully coexpressed with GroEL/GroES, while soluble pyruvate kinase was successfully coexpressed with either GroEL/GroES, dnaK/dnaJ/grpE, or a trigger factor. Both enzymes, believed to be key proteins for the organism, were purified by a combination of affinity chromatography and size-exclusion chromatography. Additionally, both ‘Ca. L. solanacearum’ enzymes are active and have the canonical tetrameric oligomeric structure in solution, consistent with other bacterial orthologs. This is the first study to successfully isolate and functionally characterize proteins from ‘Ca. L. solanacearum’. Thus, we provide a general strategy for characterizing its proteins, enabling new research and drug discovery programs to study and manage the pathogenicity of the organism.