Phloem transport limitation in Huanglongbing affected sweet orange is dependent on phloem-limited bacteria and callose

Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is a phloem-limited disease which disrupts citrus production in affected areas. In HLB-affected plants, phloem sieve plate pores accumulate callose, and leaf carbohydrate export is reduced. However, whether HLB causes a reduction in carbohydrate phloem translocation speed, and the quantitative relationships among callose, CLas population, and phloem translocation are still unknown. In this work, a procedure was developed to concurrently measure sugar transport, callose deposition, and relative pathogen population at different locations throughout the stem. Increasing quantities of CLas genetic material were positively correlated with quantity and density of callose deposits, and negatively correlated with phloem translocation speed. Callose deposit quantity was site- and rootstock dependent, and were negatively correlated with phloem translocation speed, suggesting a localized relationship. Remarkably, callose accumulation and phloem translocation disruption in the scion was dependent on rootstock genotype. Regression results suggested that the interaction of Ct values and number of phloem callose depositions, but not their size or density, explained the effects on translocation speed. Sucrose, starch, and sink 14C label allocation data support the interpretation of a transport pathway limitation by CLas infection. This work shows that the interaction of local accumulation of callose and CLas affect phloem transport. Further, the extent of this accumulation is attenuated by the rootstock and provides important information about the disease mechanism of phloem-inhabiting bacteria. Together, these results constitute the first example of a demonstrated transport limitation of phloem function by a microbial infection.

DNAzyme Sensor for the Detection of Ca2+ Using Resistive Pulse Sensing

DNAzymes are DNA oligonucleotides that can undergo a specific chemical reaction in the presence of a cofactor. Ribonucleases are a specific form of DNAzymes where a tertiary structure undergoes cleavage at a single ribonuclease site. The cleavage is highly specificity to co-factors, which makes them excellent sensor recognition elements. Monitoring the change in structure upon cleavage has given rise to many sensing strategies; here we present a simple and rapid method of following the reaction using resistive pulse sensors, RPS. To demonstrate this methodology, we present a sensor for Ca2+ ions in solution. A nanoparticle was functionalised with a Ca2+ DNAzyme, and it was possible to follow the cleavage and rearrangement of the DNA as the particles translocate the RPS. The binding of Ca2+ caused a conformation change in the DNAzyme, which was monitored as a change in translocation speed. A 30 min assay produced a linear response for Ca2+ between 1–9 μm, and extending the incubation time to 60 min allowed for a concentration as low as 0.3 μm. We demonstrate that the signal is specific to Ca2+ in the presence of other metal ions, and we can quantify Ca2+ in tap and pond water samples.

Potassium application to alleviate drought stress in cassava production: A growth chamber based carbon-13 pulse labelling experiment

<p>It is predicted that climate change will cause an increase in frequency and duration of dry spells in Central Africa. This will lower yields of cassava (<em>Manihot esculenta</em> Crantz), a starchy root crop consumed daily by almost 800 million people in the tropics. Potassium has been considered as an important plant nutrient in mitigating the impact of drought stress because of its critical role in stomatal regulation, as an osmolyte, as well as in starch synthesis and assimilate translocation. This study aims to quantify the effects of potassium fertilizer on water use efficiency and translocation speed of new assimilates in water-stressed cassava plants at early bulking stage.</p><p>Cassava cuttings (Bailo variety), originating from the Eastern Democratic Republic of Congo, were grown in pots filled with 5 kg of calcium carbonate free sand substrate and fertilized with a complete nutrient solution either high (+K; 1.437 mM K<sup>+</sup>) or low (-K; 0.359 mM K<sup>+</sup>) in potassium. All pots were weighed every other to each day to monitor water use and were watered to field capacity. A drought treatment was imposed on half of the plants two months after planting by reducing irrigation amounts by half. Plants were put in an airtight walk-in growth chamber enriched with <sup>13</sup>C-CO<sub>2</sub> (for 8 h) to trace the translocation of new assimilates. One, nine and twenty-four days after labelling, plants were harvested and δ<sup>13</sup>C values for different plant organs were analysed.</p><p>Plant water use was higher in plants under low potassium nutrition (-K) in the period prior to imposition of drought. Data on biomass production and δ<sup>13</sup>C and δ<sup>18</sup>O values of these plants will further help understand whether the observed difference in water use also leads to a difference in water use efficiency. Further, a <sup>13</sup>C mass balance will be composed. These data, to be presented at EGU 2020, will provide information on the translocation speed of new assimilates from shoot to root and confirm whether potassium positively affects this process under dry conditions.</p>

DNAzyme Sensor for the Detection of Metal Ions Using Resistive Pulse Sensing

<b>DNAzymes are DNA based catalysts that can undergo cleavage upon binding of the target analyte. The cleavage reaction is highly specific, and DNAzymes exists for a wide range of metal ions. The change of structure upon binding of a specific metal ion has given rise to many sensing strategies, but few exist with nanopore sensors. Resistive Pulse Sensing, RPS, is a platform that has emerged in recent years capable of identifying changes in DNA structure and sequence. Here we develop the use of DNAzymes with RPS technologies for the detection of Ca2+ ions in solution. Ca2+ plays an important role in biological processes, critical for cell signally, protein folding and catalysis. Extreme concentrations of Ca2+ within drinking water have also been linked to problems with corrosion, scaling and the taste of water. Using DNAzyme functionalised nanocarriers and RPS, it was possible follow the Ca2+ ions binding to the DNAzyme. The binding of Ca2+ caused a conformation change in the DNAzyme which was monitored as a change in translocation speed. By following the changes to the translocation speed, it is hypothesised that RPS can verify the changes in structure. In addition, the assay allowed the quantification of Ca2+ between 1 – 9 μM, and due its catalytic nature, increasing incubation time from 30 to 90 minutes allowed lower detection limits, down to 0.3 μM. We demonstrate that the speed changes are specific to Ca2+ in the presence of other metal ions, and we can quantify Ca2+ in tap and pond water samples.</b><br>

DNAzyme Sensor for the Detection of Metal Ions Using Resistive Pulse Sensing

<b>DNAzymes are DNA based catalysts that can undergo cleavage upon binding of the target analyte. The cleavage reaction is highly specific, and DNAzymes exists for a wide range of metal ions. The change of structure upon binding of a specific metal ion has given rise to many sensing strategies, but few exist with nanopore sensors. Resistive Pulse Sensing, RPS, is a platform that has emerged in recent years capable of identifying changes in DNA structure and sequence. Here we develop the use of DNAzymes with RPS technologies for the detection of Ca2+ ions in solution. Ca2+ plays an important role in biological processes, critical for cell signally, protein folding and catalysis. Extreme concentrations of Ca2+ within drinking water have also been linked to problems with corrosion, scaling and the taste of water. Using DNAzyme functionalised nanocarriers and RPS, it was possible follow the Ca2+ ions binding to the DNAzyme. The binding of Ca2+ caused a conformation change in the DNAzyme which was monitored as a change in translocation speed. By following the changes to the translocation speed, it is hypothesised that RPS can verify the changes in structure. In addition, the assay allowed the quantification of Ca2+ between 1 – 9 μM, and due its catalytic nature, increasing incubation time from 30 to 90 minutes allowed lower detection limits, down to 0.3 μM. We demonstrate that the speed changes are specific to Ca2+ in the presence of other metal ions, and we can quantify Ca2+ in tap and pond water samples.</b><br>

Osmotically Driven and Detected DNA Translocations

A salinity gradient propels a DNA molecule through a solid-state nanopore and generates an ionic current whose change allows for the detection of the translocation. Measurements and theoretical analyses reveal the role of diffusio-osmosis in driving these phenomena: After accounting for known salinity-dependent electrode effects, the measured current change caused by the presence of a DNA molecule inside the nanopore and the DNA translocation speed through it both increase with the magnitude of the applied salinity gradients. The effects are consistent with the theory of diffuisio-osmosis and strong enough to enable DNA translocations to overcome an applied retarding potential of tens of millivolts. This work illustrates how salinity gradients can be used to power and operate a nanopore sensor.

Silicon nitride nanopore created by dielectric breakdown with a divalent cation: deceleration of translocation speed and identification of single nucleotides

Controlled dielectric breakdown with a divalent metal cation provides a silicon nitride nanopore with the ability to decelerate single-stranded DNA speed.