Isocitrate lyase promotes Puccinia striiformis f. sp. tritici susceptibility in wheat (Triticum aestivum) by suppressing accumulation of glyoxylate cycle intermediates.

Plant fungal parasites manipulate host metabolism to support their own survival. Among the many central metabolic pathways altered during infection, the glyoxylate cycle is frequently upregulated in both fungi and their host plants. Here, we examined the response of the glyoxylate cycle in bread wheat (Triticum aestivum) to infection by the obligate biotrophic fungal pathogen Puccinia striiformis f. sp. tritici (Pst). Gene expression analysis revealed that wheat genes encoding the two unique enzymes of the glyoxylate cycle, isocitrate lyase (TaICL) and malate synthase, diverged in their expression between susceptible and resistant Pst interactions. Focusing on TaICL, we determined that the TaICL B homoeolog is specifically upregulated during early stages of a successful Pst infection. Furthermore, disruption of the B homoeolog alone was sufficient to significantly perturb Pst disease progression. Indeed, Pst infection of the TaICL-B disruption mutant (TaICL-BY400*) was inhibited early during initial penetration, with the TaICL-BY400* line also accumulating high levels of malic acid, citric acid, and aconitic acid. Exogenous application of malic acid or aconitic acid also suppressed Pst infection, with trans-aconitic acid treatment having the most pronounced effect by decreasing fungal biomass 15-fold. Thus, enhanced TaICL-B expression during Pst infection may lower accumulation of malic acid and aconitic acid to promote Pst proliferation. As exogenous application of aconitic acid and malic acid has previously been shown to inhibit other critical pests and pathogens, we propose TaICL as a potential target for disruption in resistance breeding that could have wide-reaching protective benefits for wheat and beyond.

A wheat kinase and immune receptor form host-specificity barriers against the blast fungus.

Since emerging in Brazil in 1985, wheat blast has spread throughout South America and recently appeared in Bangladesh and Zambia. Here we show that two wheat resistance genes, Rwt3 and Rwt4, acting as host-specificity barriers against non-Triticum blast pathotypes encode a nucleotide-binding leucine-rich repeat immune receptor and a tandem kinase, respectively. Molecular isolation of these genes will enable study of the molecular interaction between pathogen effector and host resistance genes.

Caveats of Using Bacterial Type Three Secretion Assays for Validating Fungal Avirulence Effectors in Wheat.

Functional characterization of effector proteins of fungal obligate biotrophic pathogens, especially confirmation of avirulence (Avr) properties, has been notoriously difficult, due to the experimental intractability of many of these organisms. Previous studies in wheat have shown promising data suggesting the type III secretion system (T3SS) of bacteria may be a suitable surrogate for delivery and detection of Avr properties of fungal effectors. However, these delivery systems were tested in the absence of confirmed Avr effectors. Here, we tested two previously described T3SS-mediated delivery systems for their suitability when delivering two confirmed Avr effectors from two fungal pathogens of wheat, Puccinia graminis f. sp. tritici and Magnaporthe oryzae pathotype tritici. We showed that both effectors (AvrSr50 and AvrRmg8) were unable to elicit a hypersensitive response on wheat seedlings with the corresponding resistance gene when expressed by the Pseudomonas fluorescens "Effector to Host Analyser" (EtHAn) system. Furthermore, we found the utility of Burkholderia glumae for screening Avr phenotypes is severely limited, as the wild-type strain elicits nonhost cell death in multiple wheat accessions. These results provide valuable insight into the suitability of these systems for screening fungal effectors for Avr properties that may help guide further development of surrogate bacterial delivery systems in wheat. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Next-generation phylogeography resolves post-glacial colonization patterns in a widespread carnivore, the red fox (Vulpes vulpes), in Europe.

Carnivores tend to exhibit a lack of (or less pronounced) genetic structure at continental scales in both a geographic and temporal sense and this can confound the identification of post-glacial colonization patterns in this group. In this study we used genome-wide data (using genotyping by sequencing [GBS]) to reconstruct the phylogeographic history of a widespread carnivore, the red fox (Vulpes vulpes), by investigating broad-scale patterns of genomic variation, differentiation and admixture amongst contemporary populations in Europe. Using 15,003 single nucleotide polymorphisms (SNPs) from 524 individuals allowed us to identify the importance of refugial regions for the red fox in terms of endemism (e.g., Iberia). In addition, we tested multiple post-glacial recolonization scenarios of previously glaciated regions during the Last Glacial Maximum using an Approximate Bayesian Computation (ABC) approach that were unresolved from previous studies. This allowed us to identify the role of admixture from multiple source population post-Younger Dryas in the case of Scandinavia and ancient land-bridges in the colonization of the British Isles. A natural colonization of Ireland was deemed more likely than an ancient human-mediated introduction as has previously been proposed and potentially points to a larger mammalian community on the island in the early post-glacial period. Using genome-wide data has allowed us to tease apart broad-scale patterns of structure and diversity in a widespread carnivore in Europe that was not evident from using more limited marker sets and provides a foundation for next-generation phylogeographic studies in other non-model species.

Occurrences of Threatened Species included in the Third Edition of the Red Data Book of the Komi Republic (Russia).

BACKGROUND: The purpose of the data paper was to introduce into scientific literature the results of scientific work carried out for the third edition of the 'Red Data Book of the Komi Republic'. The article reflects methodological approaches to the formation of a list of rare and in need of protection species and describes the corresponding datasets published in GBIF. NEW INFORMATION: Information about 7,187 occurrences of 438 rare species and infraspecies included in the third edition of the 'Red Data Book of the Komi Republic' have been published.

The branched-chain amino acid aminotransferase TaBCAT1 modulates amino acid metabolism and positively regulates wheat rust susceptibility.

Plant pathogens suppress defense responses to evade recognition and promote successful colonization. Although identifying the genes essential for pathogen ingress has traditionally relied on screening mutant populations, the post-genomic era provides an opportunity to develop novel approaches that accelerate identification. Here, RNA-seq analysis of 68 pathogen-infected bread wheat (Triticum aestivum) varieties, including three (Oakley, Solstice and Santiago) with variable levels of susceptibility, uncovered a branched-chain amino acid aminotransferase (termed TaBCAT1) as a positive regulator of wheat rust susceptibility. We show that TaBCAT1 is required for yellow and stem rust infection and likely functions in branched-chain amino acid (BCAA) metabolism, as TaBCAT1 disruption mutants had elevated BCAA levels. TaBCAT1 mutants also exhibited increased levels of salicylic acid (SA) and enhanced expression of associated defense genes, indicating that BCAA regulation, via TaBCAT1, has a key role in SA-dependent defense activation. We also identified an association between the levels of BCAAs and resistance to yellow rust infection in wheat. These findings provide insight into SA-mediated defense responses in wheat and highlight the role of BCAA metabolism in the defense response. Furthermore, TaBCAT1 could be manipulated to potentially provide resistance to two of the most economically damaging diseases of wheat worldwide.

A modular cloning toolkit for genome editing in plants.

BACKGROUND: CRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. Applying CRISPR/Cas often requires delivering multiple expression units into plant and hence there is a need for a quick and easy cloning procedure. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be easily interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs. RESULTS: Here we present an expanded cloning toolkit that contains 103 modules encoding a variety of CRISPR/Cas-based nucleases and their corresponding guide RNA backbones. Among other components, the toolkit includes a number of promoters that allow expression of CRISPR/Cas nucleases (or any other coding sequences) and their guide RNAs in monocots and dicots. As part of the toolkit, we present a set of modules that enable quick and facile assembly of tRNA-sgRNA polycistronic units without a PCR step involved. We also demonstrate that our tRNA-sgRNA system is functional in wheat protoplasts. CONCLUSIONS: We believe the presented CRISPR/Cas toolkit is a great resource that will contribute towards wider adoption of the CRISPR/Cas genome editing technology and modular cloning by researchers across the plant science community.

Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots.

Plant synthetic biology and cereal engineering depend on the controlled expression of transgenes of interest. Most engineering in plant species to date has relied heavily on the use of a few, well-established constitutive promoters to achieve high levels of expression; however, the levels of transgene expression can also be influenced by the use of codon optimization, intron-mediated enhancement and varying terminator sequences. Most of these alternative approaches for regulating transgene expression have only been tested in small-scale experiments, typically testing a single gene of interest. It is therefore difficult to interpret the relative importance of these approaches and to design engineering strategies that are likely to succeed in different plant species, particularly if engineering multigenic traits where the expression of each transgene needs to be precisely regulated. Here, we present data on the characterization of 46 promoters and 10 terminators in Medicago truncatula, Lotus japonicus, Nicotiana benthamiana and Hordeum vulgare, as well as the effects of codon optimization and intron-mediated enhancement on the expression of two transgenes in H. vulgare. We have identified a core set of promoters and terminators of relevance to researchers engineering novel traits in plant roots. In addition, we have shown that combining codon optimization and intron-mediated enhancement increases transgene expression and protein levels in barley. Based on our study, we recommend a core set of promoters and terminators for broad use and also propose a general set of principles and guidelines for those engineering cereal species.

Advances in modern osteotomies around the knee : Report on the Association of Sports Traumatology, Arthroscopy, Orthopaedic surgery, Rehabilitation (ASTAOR) Moscow International Osteotomy Congress 2017.

Corrective lower limb osteotomies are innovative and efficient therapeutic procedures for restoring axial alignment and managing unicompartmental knee osteoarthritis. This review presents critical insights into the up-dated clinical knowledge on osteotomies for complex posttraumatic or congenital lower limb deformities with a focus on high tibial osteotomies, including a comprehensive overview of basic principles of osteotomy planning, biomechanical considerations of different implants for osteotomies and insights in specific bone deformity correction techniques. Emphasis is placed on complex cases of lower limb osteotomies associated with ligament and multiaxial instability including pediatric cases, computer-assisted navigation, external fixation for long bone deformity correction and return to sport after such osteotomies. Altogether, these advances in the experimental and clinical knowledge of complex lower limb osteotomies allow generating improved, adapted therapeutic regimens to treat congenital and acquired lower limb deformities.

Speed breeding is a powerful tool to accelerate crop research and breeding.

The growing human population and a changing environment have raised significant concern for global food security, with the current improvement rate of several important crops inadequate to meet future demand 1 . This slow improvement rate is attributed partly to the long generation times of crop plants. Here, we present a method called 'speed breeding', which greatly shortens generation time and accelerates breeding and research programmes. Speed breeding can be used to achieve up to 6 generations per year for spring wheat (Triticum aestivum), durum wheat (T. durum), barley (Hordeum vulgare), chickpea (Cicer arietinum) and pea (Pisum sativum), and 4 generations for canola (Brassica napus), instead of 2-3 under normal glasshouse conditions. We demonstrate that speed breeding in fully enclosed, controlled-environment growth chambers can accelerate plant development for research purposes, including phenotyping of adult plant traits, mutant studies and transformation. The use of supplemental lighting in a glasshouse environment allows rapid generation cycling through single seed descent (SSD) and potential for adaptation to larger-scale crop improvement programs. Cost saving through light-emitting diode (LED) supplemental lighting is also outlined. We envisage great potential for integrating speed breeding with other modern crop breeding technologies, including high-throughput genotyping, genome editing and genomic selection, accelerating the rate of crop improvement.

Haplotype dictionary for the Rht-1 loci in wheat.

The introduction of Reduced height (Rht)-B1b and Rht-D1b into bread wheat (Triticum aestivum) varieties was a key component of the 'green revolution' and today these alleles are the primary sources of semi-dwarfism in wheat. The Rht-1 loci encode DELLA proteins, which are transcription factors that affect plant growth and stress tolerance. In bread wheat, Rht-D1b and Rht-B1b influence resistance to the disease Fusarium Head Blight. To identify Rht-1 variants, locus specific primers were developed and used to sequence the entire open reading frame (ORF) and 1.7 kb of the 5' and 0.5 kb of the 3' flanking regions of Rht-A1 (Rht-A1+f), Rht-B1 (Rht-B1+f), and Rht-D1 (Rht-D1+f) in bread wheat (36 sequences from each genome) and tetraploid and diploid wheat (TDW) (one to three sequences from each genome). Among the bread wheat accessions, the Rht-A1+f and Rht-D1+f sequences contained relatively low genetic diversity and few haplotypes relative to the Rht-B1+f sequences. The TDW accessions were relatively rich in genetic diversity and contained the majority of the polymorphic sites. Novel polymorphisms, relative to 'Chinese Spring', discovered among the accessions include 160 and 197 bp insertions 5' of Rht-B1 and a frameshift in the Rht-B1 ORF. Quantitative real-time PCR using shoot and leaf tissue from 5-day-old seedlings of genotypes lacking or containing the 5' insertions revealed no major effect on Rht-B1 transcript accumulation. This research provides insights into the genetic diversity present at the Rht-1 loci in modern bread wheat and in relation to ancestral wheat accessions.

Molecular characterization of Rht-1 dwarfing genes in hexaploid wheat.

The introduction of the Reduced height (Rht)-B1b and Rht-D1b semidwarfing genes led to impressive increases in wheat (Triticum aestivum) yields during the Green Revolution. The reduction in stem elongation in varieties containing these alleles is caused by a limited response to the phytohormone gibberellin (GA), resulting in improved resistance to stem lodging and yield benefits through an increase in grain number. Rht-B1 and Rht-D1 encode DELLA proteins, which act to repress GA-responsive growth, and their mutant alleles Rht-B1b and Rht-D1b are thought to confer dwarfism by producing more active forms of these growth repressors. While no semidwarfing alleles of Rht-A1 have been identified, we show that this gene is expressed at comparable levels to the other homeologs and represents a potential target for producing novel dwarfing alleles. In this study, we have characterized additional dwarfing mutations in Rht-B1 and Rht-D1. We show that the severe dwarfism conferred by Rht-B1c is caused by an intragenic insertion, which results in an in-frame 90-bp insertion in the transcript and a predicted 30-amino acid insertion within the highly conserved amino-terminal DELLA domain. In contrast, the extreme dwarfism of Rht-D1c is due to overexpression of the semidwarfing Rht-D1b allele, caused by an increase in gene copy number. We show also that the semidwarfing alleles Rht-B1d and Rht-B1e introduce premature stop codons within the amino-terminal coding region. Yeast two-hybrid assays indicate that these newly characterized mutations in Rht-B1 and Rht-D1 confer "GA-insensitive" dwarfism by producing DELLA proteins that do not bind the GA receptor GA INSENSITIVE DWARF1, potentially compromising their targeted degradation.

Mechanisms for shaping, orienting, positioning and patterning plant secondary cell walls.

Xylem vessels are cells that develop a specifically ornamented secondary cell wall to ensure their vascular function, conferring both structural strength and impermeability. Further plasticity is given to these vascular cells by a range of different patterns described by their secondary cell walls that-as for the growth of all plant organs-are developmentally regulated. Microtubules and their associated proteins, named MAPs, are essential to define the shape, the orientation, the position and the overall pattern of these secondary cell walls. Key actors in this process are the land-plant specific MAP70 proteins which not only allow the secondary cell wall to be positioned at the cell cortex but also determine the overall pattern described by xylem vessel secondary cell walls. 

Transmission of 112 Gb/s PM-QPSK signals over up to 635 km of multimode optical fiber.

We investigate transmission of 112 Gb/s PM-QPSK signals over 50 µm core diameter OM3 multimode fiber using the center launch approach. We demonstrate successful transmission of 16 DWDM channels over a distance of 635 km for a capacity-distance product of 1016 Tb/s-km. The limiting impairment appears due to mode coupling and multipath interference effects.

The microtubule-associated protein AtMAP70-5 regulates secondary wall patterning in Arabidopsis wood cells.

Xylem tracheary elements (TEs) form hollow, sap-conducting tubes kept open by thickened ribs of secondary cell wall that provide the major structural element in wood. These ribs are enriched with cellulose and lignin, molecules that utilize more atmospheric CO(2) than any other biopolymer on Earth. The thickenings form characteristic patterns (e.g., spiral and pitted) that depend upon the bundling of underlying microtubules [1, 2]. To identify microtubule-associated proteins (MAPs) involved in patterning microtubules, we optimized an in vitro system for triggering single Arabidopsis cells to differentiate synchronously into TEs. From more than 200 microtubule-implicated proteins, AtMAP70-5 was the only MAP upregulated upon, and specific to, TE differentiation. It lines the borders of each microtubule bundle and forms C-shaped "spacers" between adjacent bundles. Manipulating levels of AtMAP70-5 and its binding partner AtMAP70-1 by overexpression or RNA interference (RNAi) silencing shifted the balance between the characteristic patterns. RNAi silencing produced stunted plants with disorganized vascular bundles. In culture, RNAi knockdown caused ribs of secondary cell wall, surrounded by microtubules, to invaginate and fall into the cytoplasm. These results suggest that AtMAP70-5 and AtMAP70-1 are essential for defining where secondary cell wall polymers are applied at the cell cortex in wood-forming cells.

AtMAP70-5, a divergent member of the MAP70 family of microtubule-associated proteins, is required for anisotropic cell growth in Arabidopsis.

AtMAP70-5 is the most divergent of a recently described multigene family of plant-specific microtubule-associated proteins (MAPs). It is significantly smaller than other members and has several isoform-specific sequence features. To confirm that this protein still functions as a MAP we show that it directly binds microtubules in vitro and decorates microtubules in vivo. When added to tubulin polymerization assays, AtMAP70-5 increases the length distribution profile of microtubules indicating that it stabilizes microtubule dynamics. The overexpressed fusion protein perturbs cell polarity in cell suspensions by inducing extra poles for growth. Similarly, in Arabidopsis plants the overexpression of AtMAP70-5 causes epidermal cells to swell; it also stunts growth and induces right-handed organ twisting. RNAi-mediated downregulation of AtMAP70-5 results in reduced inflorescence stem length and diameter and individual cells are inhibited in their capacity for expansion. These observations suggest that the control over AtMAP70-5 expression levels is important in order to maintain axial polarity and to ensure regular extension of plant organs.

Identification of a novel family of 70 kDa microtubule-associated proteins in Arabidopsis cells.

Most plant microtubule-associated proteins (MAPs) have homologues across the phylogenetic spectrum. To find potential plant-specific MAPs that will have evaded bioinformatic searches we devised a low stringency method for isolating proteins from an Arabidopsis cell suspension on endogenous taxol-microtubules. By tryptic peptide mass fingerprinting we identified 55 proteins that were enriched on taxol-microtubules. Amongst a range of known MAPs, such as kinesins, MAP65 isoforms and MOR1, we detected 'unknown' 70 kDa proteins that belong to a family of five closely related Arabidopsis proteins having no known homologues amongst non-plant organisms. To verify that AtMAP70-1 associates with microtubules in vivo, it was expressed as a GFP fusion. This confirmed that the protein decorates all four microtubule arrays in both transiently infected Arabidopsis and stably transformed tobacco BY-2 suspension cells. Microtubule-directed drugs perturbed the localization of AtMAP70-1 but cytochalasin D did not. AtMAP70-1 contains four predicted coiled-coil domains and truncation studies identified a central domain that targets the fusion protein to microtubules in vivo. This study therefore introduces a novel family of plant-specific proteins that interact with microtubules.

Experimental observation of orthogonally polarized time-delayed optical soliton trapping in birefringent fibers.

Propagation of two orthogonally polarized time-delayed optical solitons in low-birefringence optical fiber is studied experimentally. We demonstrate soliton trapping and collisions and also the ability to control the separation and shape of soliton pulses by varying the power at the input of the fiber.

Electronic Structure of Luminescent d(0) Niobium and Tantalum Imido Compounds cis,mer-M(NR)Cl(3)L(2).

Electronic absorption and emission spectra are reported for luminescent d(0) monoimido group 5 compounds M(NR)Cl(3)L(2) (M = Nb, Ta; R = alkyl, aryl; L = dme, Cl(-), py). These compounds display weak (epsilon < 200 M(-)(1) cm(-)(1)), well-resolved lowest-energy transitions in the high-energy visible and near-UV regions (20 000 < E(abs) < 29 000 cm(-)(1)). The energy of this absorption band depends strongly on the nature of the imido substituent, with a significant decrease observed when aryl groups are present. Excitation into this transition results in long-lived luminescent excited states. Long emission lifetimes (50 ns to 17 &mgr;s) and high quantum yields (0.001-0.24) are observed, decreasing primarily as a function of the alkyl substituent, being lowest in the aryl imidos. Good overlap is observed with absorption, excitation, and emission mirror spectra, indicating absorption into and emission from the same excited state. The data are consistent with absorption into and emission from a (3)(nb, pi) state, or d(xy)() <-- Ta-N pi. Semiempirical molecular orbital calculations are presented which suggest that the imido compounds may be considered as having highly mixed but localized Ta=N pi-bonding. A significant difference is noted in [Ta(NPh)Cl(5)](2)(-), in which there is appreciable aryl character in Ta=N pi-type orbitals. This accounts for the difference in electronic properties of the aryl imidos compared to the alkyl imidos. An analysis of radiative and nonradiative excited-state deactivation pathways is presented. Significantly, an energy gap law correlation is observed for nonradiative decay in the imido compounds as a group, but a corresponding correlation of radiative rates with emission energy is not observed when aryl and alkyl imidos are compared, evidence of electronic perturbation by the aryl substituent.