Insight into resistance to 'Candidatus Liberibacter asiaticus,' associated with Huanglongbing, in Oceanian citrus genotypes.

Huanglongbing (HLB), the most destructive citrus disease, is associated with unculturable, phloem-limited Candidatus Liberibacter species, mainly Ca. L. asiaticus (Las). Las is transmitted naturally by the insect Diaphorina citri. In a previous study, we determined that the Oceanian citrus relatives Eremocitrus glauca, Microcitrus warburgiana, Microcitrus papuana, and Microcitrus australis and three hybrids among them and Citrus were full-resistant to Las. After 2 years of evaluations, leaves of those seven genotypes remained Las-free even with their susceptible rootstock being infected. However, Las was detected in their stem bark above the scion-rootstock graft union. Aiming to gain an understanding of the full-resistance phenotype, new experiments were carried out with the challenge-inoculated Oceanian citrus genotypes through which we evaluated: (1) Las acquisition by D. citri fed onto them; (2) Las infection in sweet orange plants grafted with bark or budwood from them; (3) Las infection in sweet orange plants top-grafted onto them; (4) Las infection in new shoots from rooted plants of them; and (5) Las infection in new shoots of them after drastic back-pruning. Overall, results showed that insects that fed on plants from the Oceanian citrus genotypes, their canopies, new flushes, and leaves from rooted cuttings evaluated remained quantitative real-time polymerase chain reaction (qPCR)-negative. Moreover, their budwood pieces were unable to infect sweet orange through grafting. Furthermore, sweet orange control leaves resulted infected when insects fed onto them and graft-receptor susceptible plants. Genomic and morphological analysis of the Oceanian genotypes corroborated that E. glauca and M. warburgiana are pure species while our M. australis accession is an M. australis × M. inodora hybrid and M. papuana is probably a M. papuana × M. warburgiana hybrid. E. glauca × C. sinensis hybrid was found coming from a cross between E. glauca and mandarin or tangor. Eremocitrus × Microcitrus hybrid is a complex admixture of M. australasica, M. australis, and E. glauca while the last hybrid is an M. australasica × M. australis admixture. Confirmation of consistent full resistance in these genotypes with proper validation of their genomic parentages is essential to map properly genomic regions for breeding programs aimed to generate new Citrus-like cultivars yielding immunity to HLB.

Isotachophoresis for rapid transformation of Escherichia coli.

A frequent limitation of electroporation (EP) and chemical transformation (CT) are the need of tedious and time-consuming procedures for inducing transformation competence, the substantial number of cells required, and the low transformation yields typically achieved. Here, we show a new and rapid electrokinetic method for transformation of small number of noncompetent Escherichia coli TOP10 cells (2-3 × 105 ) at room temperature. Escherichia coli TOP10 cells and plasmid DNA are sequentially injected into a 50 µm ID capillary and focused into 11.5 nL by isotachophoresis (ITP) induced by application of high DC voltage (-16 kV). Through ITP, a large excess of plasmid DNA is brought in contact with the cell surface, with the contact time adjusted by application of a counter-pressure (1.3 psi) opposing the ITP movement. The transformation rate was more than 1000-fold higher compared to EP and CT at survival rates greater than 60%.

Resistance to 'Candidatus Liberibacter asiaticus,' the Huanglongbing Associated Bacterium, in Sexually and/or Graft-Compatible Citrus Relatives.

Huanglongbing (HLB) is the most destructive, yet incurable disease of citrus. Finding sources of genetic resistance to HLB-associated 'Candidatus Liberibacter asiaticus' (Las) becomes strategic to warrant crop sustainability, but no resistant Citrus genotypes exist. Some Citrus relatives of the family Rutaceae, subfamily Aurantioideae, were described as full-resistant to Las, but they are phylogenetically far, thus incompatible with Citrus. Partial resistance was indicated for certain cross-compatible types. Moreover, other genotypes from subtribe Citrinae, sexually incompatible but graft-compatible with Citrus, may provide new rootstocks able to restrict bacterial titer in the canopy. Use of seedlings from monoembryonic species and inconsistencies in previous reports likely due to Las recalcitrance encouraged us to evaluate more accurately these Citrus relatives. We tested for Las resistance a diverse collection of graft-compatible Citrinae species using an aggressive and consistent challenge-inoculation and evaluation procedure. Most Citrinae species examined were either susceptible or partially resistant to Las. However, Eremocitrus glauca and Papua/New Guinea Microcitrus species as well as their hybrids and those with Citrus arose here for the first time as full-resistant, opening the way for using these underutilized genotypes as Las resistance sources in breeding programs or attempting using them directly as possible new Las-resistant Citrus rootstocks or interstocks.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2016-2018).

One of the most cited limitations of capillary and microchip electrophoresis is the poor sensitivity. This review continues to update this series of biannual reviews, first published in Electrophoresis in 2007, on developments in the field of online/in-line concentration methods in capillaries and microchips, covering the period July 2016-June 2018. It includes developments in the field of stacking, covering all methods from field-amplified sample stacking and large-volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to online or in-line extraction methods that have been used for electrophoresis.

Separation of superparamagnetic magnetite nanoparticles by capillary zone electrophoresis using non-complexing and complexing electrolyte anions and tetramethylammonium as dispersing additive.

Separations of bare superparamagnetic magnetite nanoparticles (BSPMNPs, approx. 11 nm diameter) was performed using non-complexing (nitrate) and complexing (chloride, citrate and phosphate) electrolyte ions with additions of tetramethylammonium hydroxide (TMAOH), which is commonly applied to control the synthesis of stable iron oxides. The use of TMAOH as a background electrolyte (BGE) additive for capillary electrophoresis (CE) separations provided for the first time electropherograms of BSPMNPs exhibiting symmetrical and highly reproducible peaks, free of spurious spikes characteristic of nanoparticle clusters. Consequently, accurate determination of the electrophoretic effective mobility of BSPMNPs was possible, yielding a value of -3.345E-08 m2 V-1 s-1 (relative standard deviation (RSD) of 0.500%). The obtained mobilities of BSPMNPs in the presence of various electrolyte ions show that the degree of complexation with the surface of BSPMNPs follows the order chloride < citrate < phosphate, correlating with the stabilities of Fe(III) complexes with the respective anions. Finally, bare and carboxylated iron oxide nanoparticles were successfully separated in only 10 min using 10 mM Tris-nitrate containing 20 mM of TMAOH as electrolyte. Our findings show that simple and rapid CE experiments are an excellent tool to characterise and monitor properties and interactions of iron oxide nanoparticles with other molecules for surface modification purposes.

Unraveling the electron transfer processes of a nanowire protein from Geobacter sulfurreducens.

The extracellular electron transfer metabolism of Geobacter sulfurreducens is sustained by several multiheme c-type cytochromes. One of these is the dodecaheme cytochrome GSU1996 that belongs to a new sub-class of c-type cytochromes. GSU1996 is composed by four similar triheme domains (A­D). The C-terminal half of the molecule encompasses the domains C and D, which are connected by a small linker and the N-terminal half of the protein contains two domains (A and B) that form one structural unit. It was proposed that this protein works as an electrically conductive device in G. sulfurreducens, transferring electrons within the periplasm or to outer-membrane cytochromes. In this work, a novel strategy was applied to characterize in detail the thermodynamic and kinetic properties of the hexaheme fragment CD of GSU1996. This characterization revealed the electron transfer process of GSU1996 for the first time, showing that a heme at the edge of the C-terminal of the protein is thermodynamic and kinetically competent to receive electrons from physiological redox partners. This information contributes towards understanding how this new sub-class of cytochromes functions as nanowires, and also increases the current knowledge of the extracellular electron transfer mechanisms in G. sulfurreducens.

Characterization of the periplasmic redox network that sustains the versatile anaerobic metabolism of Shewanella oneidensis MR-1.

The versatile anaerobic metabolism of the Gram-negative bacterium Shewanella oneidensis MR-1 (SOMR-1) relies on a multitude of redox proteins found in its periplasm. Most are multiheme cytochromes that carry electrons to terminal reductases of insoluble electron acceptors located at the cell surface, or bona fide terminal reductases of soluble electron acceptors. In this study, the interaction network of several multiheme cytochromes was explored by a combination of NMR spectroscopy, activity assays followed by UV-visible spectroscopy and comparison of surface electrostatic potentials. From these data the small tetraheme cytochrome (STC) emerges as the main periplasmic redox shuttle in SOMR-1. It accepts electrons from CymA and distributes them to a number of terminal oxidoreductases involved in the respiration of various compounds. STC is also involved in the electron transfer pathway to reduce nitrite by interaction with the octaheme tetrathionate reductase (OTR), but not with cytochrome c nitrite reductase (ccNiR). In the main pathway leading the metal respiration STC pairs with flavocytochrome c (FccA), the other major periplasmic cytochrome, which provides redundancy in this important pathway. The data reveals that the two proteins compete for the binding site at the surface of MtrA, the decaheme cytochrome inserted on the periplasmic side of the MtrCAB-OmcA outer-membrane complex. However, this is not observed for the MtrA homologues. Indeed, neither STC nor FccA interact with MtrD, the best replacement for MtrA, and only STC is able to interact with the decaheme cytochrome DmsE of the outer-membrane complex DmsEFABGH. Overall, these results shown that STC plays a central role in the anaerobic respiratory metabolism of SOMR-1. Nonetheless, the trans-periplasmic electron transfer chain is functionally resilient as a consequence of redundancies that arise from the presence of alternative pathways that bypass/compete with STC.