A Variant of LbCas12a and Elevated Incubation Temperatures Enhance the Rate of Gene Editing in the Oomycete Phytophthora infestans.

CRISPR-Cas editing systems have proved to be powerful tools for functional genomics research, but their effectiveness in many non-model species remains limited. In the potato and tomato pathogen Phytophthora infestans, an editing system was previously developed that expresses the Lachnospiracae bacterium Cas12a endonuclease (LbCas12a) and guide RNA from a DNA vector. However, the method works at low efficiency. Based on a hypothesis that editing is constrained by a mismatch between the optimal temperatures for P. infestans growth and endonuclease catalysis, we tested two strategies that increased the frequency of editing of two target genes by about 10-fold. First, we found that editing was boosted by a mutation in LbCas12a (D156R) that had been reported to expand its catalytic activity over a broader temperature range. Second, we observed that editing was enhanced by transiently incubating transformed tissue at a higher temperature. These modifications should make CRISPR-Cas12a more useful for interrogating gene and protein function in P. infestans and its relatives, especially species that grow optimally at lower temperatures. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Emerging forest-peatland bistability and resilience of European peatland carbon stores.

Northern peatlands store large amounts of carbon. Observations indicate that forests and peatlands in northern biomes can be alternative stable states for a range of landscape settings. Climatic and hydrological changes may reduce the resilience of peatlands and forests, induce persistent shifts between these states, and release the carbon stored in peatlands. Here, we present a dynamic simulation model constrained and validated by a wide set of observations to quantify how feedbacks in water and carbon cycling control resilience of both peatlands and forests in northern landscapes. Our results show that 34% of Europe (area) has a climate that can currently sustain existing rainwater-fed peatlands (raised bogs). However, raised bog initiation and restoration by water conservation measures after the original peat soil has disappeared is only possible in 10% of Europe where the climate allows raised bogs to initiate and outcompete forests. Moreover, in another 10% of Europe, existing raised bogs (concerning ∼20% of the European raised bogs) are already affected by ongoing climate change. Here, forests may overgrow peatlands, which could potentially release in the order of 4% (∼24 Pg carbon) of the European soil organic carbon pool. Our study demonstrates quantitatively that preserving and restoring peatlands requires looking beyond peatland-specific processes and taking into account wider landscape-scale feedbacks with forest ecosystems.

Genomic and physiological analyses reveal that extremely thermophilic Caldicellulosiruptor changbaiensis deploys uncommon cellulose attachment mechanisms.

The genus Caldicellulosiruptor is comprised of extremely thermophilic, heterotrophic anaerobes that degrade plant biomass using modular, multifunctional enzymes. Prior pangenome analyses determined that this genus is genetically diverse, with the current pangenome remaining open, meaning that new genes are expected with each additional genome sequence added. Given the high biodiversity observed among the genus Caldicellulosiruptor, we have sequenced and added a 14th species, Caldicellulosiruptor changbaiensis, to the pangenome. The pangenome now includes 3791 ortholog clusters, 120 of which are unique to C. changbaiensis and may be involved in plant biomass degradation. Comparisons between C. changbaiensis and Caldicellulosiruptor bescii on the basis of growth kinetics, cellulose solubilization and cell attachment to polysaccharides highlighted physiological differences between the two species which are supported by their respective gene inventories. Most significantly, these comparisons indicated that C. changbaiensis possesses uncommon cellulose attachment mechanisms not observed among the other strongly cellulolytic members of the genus Caldicellulosiruptor.

Complete Genome Sequence of Caldicellulosiruptor changbaiensis CBS-Z, an Extremely Thermophilic, Cellulolytic Bacterium Isolated from a Hot Spring in China.

Here, we describe the complete genome sequence of Caldicellulosiruptor changbaiensis, isolated from a hot spring in the Changbai Mountain Range of China. Currently, only one other genome sequence representing a Caldicellulosiruptor species from China is available. Assembly of a continuous single contig used both Oxford Nanopore and Illumina sequencing data.

A unified nomenclature for quantification and description of water conducting properties of sapwood xylem based on Darcy's law.

The literature dealing with the water conducting properties of sapwood xylem in trees is inconsistent in terminology, symbols and units. This has resulted from confusion in the use of either an analogy to Ohm's law or Darcy's law as the basis for nomenclature. Ohm's law describes movement of electricity through a conductor, whereas Darcy's law describes movement of a fluid (liquid or gas) through a porous medium. However, it is generally not realized that, in their full notation, these laws are mathematically equivalent. Despite this, plant physiologists have failed to agree on a convention for nomenclature. As a result, the study of water movement through sapwood xylem is confusing, especially for scientists entering the field. To improve clarity, we suggest the adoption of a single nomenclature that can be used by all plant physiologists when describing water movement in xylem. Darcy's law is an explicit hydraulic relationship and the basis for established theories that describe three-dimensional saturated and unsaturated flow in porous media. We suggest, therefore, that Darcy's law is the more appropriate theoretical framework on which to base nomenclature describing sapwood hydraulics. Our proposed nomenclature is summarized in a table that describes conventional terms, with their formulae, dimensions, units and symbols; the table also lists the many synonyms found in recent literature that describe the same concepts. Adoption of this proposal will require some changes in the use of terminology, but a common rigorous nomenclature is needed for efficient and clear communication among scientists.

Characterizing geochemical reactions in unsaturated mine waste-rock piles using gaseous O2, CO2, 12CO2, and 13CO2.

In situ determinations of geochemical reaction rates in mine waste-rock piles remain a challenge. Depth-profiles of field O2 and CO2 pore-gas concentrations, delta13C(CO2) values, and moisture contents were used to characterize and quantify geochemical reaction rates in two waste-rock piles at the Key Lake Uranium Mine in northern Saskatchewan, Canada. Traditionally, the presence of O2 concentrations less than atmospheric in waste-rock piles has been attributed to mineral oxidation. This study showed that the interpretation of O2 and CO2 concentration profiles alone could not be used to identify the depths of dominant geochemical reactions in the piles and could lead to erroneous estimates of reaction rates. Modeling of the delta13C(CO2) depth profiles clearly showed that the gas concentration profiles present in the piles were the result of the oxidation of organic matter present below the piles, a mechanism not previously reported in the literature. Based on these findings, the rates of reactions in the organic zone were determined. The oxidation of organic matter at the base of waste-rock piles should be considered in future mine-waste pore-gas studies, in addition to sulfide oxidation and carbonate buffering.