A new generation of Bt maize for control of fall armyworm (Spodoptera frugiperda).

BACKGROUND: The pyramided genetically modified maize (Zea mays [L.]) event MON 95379, expressing the Cry1B.868 and Cry1Da_7 proteins, was designed to protect against larval feeding damage by the fall armyworm, Spodoptera frugiperda (FAW). Here, we conducted laboratory, greenhouse, and field studies to assess the dose and field efficacy of MON 95379 against FAW and inform the development of insect resistance management plans. RESULTS: The Cry1B.868 and Cry1Da_7 proteins were active against susceptible FAW neonates in diet-incorporation bioassays: median lethal concentration [LC50 ] (95% CI) = 62.8 (42.6-87.6) µg/ml diet for Cry1B.868 and 9.4 (5.3-18.6) µg/ml diet for Cry1Da_7. In laboratory leaf disc bioassays, MON 95379 maize and experimental maize lines expressing the individual components were effective in controlling susceptible FAW. In whole-plant assays, MON 95379 controlled FAW resistant to the Cry1A.105 and Cry2Ab2 proteins. Likewise, under field conditions, MON 95379 maize expressing Cry1B.868 and Cry1Da_7 was highly effective at protecting plants against the larval feeding of FAW. CONCLUSIONS: The expression of Cry1B.868 and Cry1Da_7 in MON 95379 consistently protected maize plants against larval feeding by FAW and represents an alternative to manage trait resistance issues in South America. © 2021 Bayer Crop Science-US. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Cryptococcus neoformans UGT1 encodes a UDP-Galactose/UDP-GalNAc transporter.

Cryptococcus neoformans, an opportunistic fungal pathogen, produces a glycan capsule to evade the immune system during infection. This definitive virulence factor is composed mainly of complex polysaccharides, which are made in the secretory pathway by reactions that utilize activated nucleotide sugar precursors. Although the pathways that synthesize these precursors are known, the identity and the regulation of the nucleotide sugar transporters (NSTs) responsible for importing them into luminal organelles remain elusive. The UDP-galactose transporter, Ugt1, was initially identified by homology to known UGTs and glycan composition analysis of ugt1Δ mutants. However, sequence is an unreliable predictor of NST substrate specificity, cells may express multiple NSTs with overlapping specificities, and NSTs may transport multiple substrates. Determining NST activity thus requires biochemical demonstration of function. We showed that Ugt1 transports both UDP-galactose and UDP-N-acetylgalactosamine in vitro. Deletion of UGT1 resulted in growth and mating defects along with altered capsule and cellular morphology. The mutant was also phagocytosed more readily by macrophages than wild-type cells and cleared more quickly in vivo and in vitro, suggesting a mechanism for the lack of virulence observed in mouse models of infection.

Expression of a truncated ATHB17 protein in maize increases ear weight at silking.

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.

Cryptococcus neoformans dual GDP-mannose transporters and their role in biology and virulence.

Cryptococcus neoformans is an opportunistic yeast responsible for lethal meningoencephalitis in humans. This pathogen elaborates a polysaccharide capsule, which is its major virulence factor. Mannose constitutes over one-half of the capsule mass and is also extensively utilized in cell wall synthesis and in glycosylation of proteins and lipids. The activated mannose donor for most biosynthetic reactions, GDP-mannose, is made in the cytosol, although it is primarily consumed in secretory organelles. This compartmentalization necessitates specific transmembrane transporters to make the donor available for glycan synthesis. We previously identified two cryptococcal GDP-mannose transporters, Gmt1 and Gmt2. Biochemical studies of each protein expressed in Saccharomyces cerevisiae showed that both are functional, with similar kinetics and substrate specificities in vitro. We have now examined these proteins in vivo and demonstrate that cells lacking Gmt1 show significant phenotypic differences from those lacking Gmt2 in terms of growth, colony morphology, protein glycosylation, and capsule phenotypes. Some of these observations may be explained by differential expression of the two genes, but others suggest that the two proteins play overlapping but nonidentical roles in cryptococcal biology. Furthermore, gmt1 gmt2 double mutant cells, which are unexpectedly viable, exhibit severe defects in capsule synthesis and protein glycosylation and are avirulent in mouse models of cryptococcosis.

The pathogenic fungus Cryptococcus neoformans expresses two functional GDP-mannose transporters with distinct expression patterns and roles in capsule synthesis.

Cryptococcus neoformans is a fungal pathogen that is responsible for life-threatening disease, particularly in the context of compromised immunity. This organism makes extensive use of mannose in constructing its cell wall, glycoproteins, and glycolipids. Mannose also comprises up to two-thirds of the main cryptococcal virulence factor, a polysaccharide capsule that surrounds the cell. The glycosyltransfer reactions that generate cellular carbohydrate structures usually require activated donors such as nucleotide sugars. GDP-mannose, the mannose donor, is produced in the cytosol by the sequential actions of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase. However, most mannose-containing glycoconjugates are synthesized within intracellular organelles. This topological separation necessitates a specific transport mechanism to move this key precursor across biological membranes to the appropriate site for biosynthetic reactions. We have discovered two GDP-mannose transporters in C. neoformans, in contrast to the single such protein reported previously for other fungi. Biochemical studies of each protein expressed in Saccharomyces cerevisiae show that both are functional, with similar kinetics and substrate specificities. Microarray experiments indicate that the two proteins Gmt1 and Gmt2 are transcribed with distinct patterns of expression in response to variations in growth conditions. Additionally, deletion of the GMT1 gene yields cells with small capsules and a defect in capsule induction, while deletion of GMT2 does not alter the capsule. We suggest that C. neoformans produces two GDP-mannose transporters to satisfy its enormous need for mannose utilization in glycan synthesis. Furthermore, we propose that the two proteins have distinct biological roles. This is supported by the different expression patterns of GMT1 and GMT2 in response to environmental stimuli and the dissimilar phenotypes that result when each gene is deleted.

Loss of cell wall alpha(1-3) glucan affects Cryptococcus neoformans from ultrastructure to virulence.

Yeast cell walls are critical for maintaining cell integrity, particularly in the face of challenges such as growth in mammalian hosts. The pathogenic fungus Cryptococcus neoformans additionally anchors its polysaccharide capsule to the cell surface via alpha(1-3) glucan in the wall. Cryptococcal cells disrupted in their alpha glucan synthase gene were sensitive to stresses, including temperature, and showed difficulty dividing. These cells lacked surface capsule, although they continued to shed capsule material into the environment. Electron microscopy showed that the alpha glucan that is usually localized to the outer portion of the cell wall was absent, the outer region of the wall was highly disorganized, and the inner region was hypertrophic. Analysis of cell wall composition demonstrated complete loss of alpha glucan accompanied by a compensatory increase in chitin/chitosan and a redistribution of beta glucan between cell wall fractions. The mutants were unable to grow ina mouse model of infection, but caused death in nematodes. These studies integrate morphological and biochemical investigations of the role of alpha glucan in the cryptococcal cell wall.

Eukaryotic UDP-galactopyranose mutase (GLF gene) in microbial and metazoal pathogens.

Galactofuranose (Gal(f)) is a novel sugar absent in mammals but present in a variety of pathogenic microbes, often within glycoconjugates that play critical roles in cell surface formation and the infectious cycle. In prokaryotes, Gal(f) is synthesized as the nucleotide sugar UDP-Gal(f) by UDP-galactopyranose mutase (UGM) (gene GLF). Here we used a combinatorial bioinformatics screen to identify a family of candidate eukaryotic GLFs that had previously escaped detection. GLFs from three pathogens, two protozoa (Leishmania major and Trypanosoma cruzi) and one fungus (Cryptococcus neoformans), had UGM activity when expressed in Escherichia coli and assayed in vivo and/or in vitro. Eukaryotic GLFs are closely related to each other but distantly related to prokaryotic GLFs, showing limited conservation of core residues around the substrate-binding site and flavin adenine dinucleotide binding domain. Several eukaryotes not previously investigated for Gal(f) synthesis also showed strong GLF homologs with conservation of key residues. These included other fungi, the alga Chlamydomonas and the algal phleovirus Feldmannia irregularis, parasitic nematodes (Brugia, Onchocerca, and Strongyloides) and Caenorhabditis elegans, and the urochordates Halocynthia and Cionia. The C. elegans open reading frame was shown to encode UGM activity. The GLF phylogenetic distribution suggests that Gal(f) synthesis may occur more broadly in eukaryotes than previously supposed. Overall, GLF/Gal(f) synthesis in eukaryotes appears to occur with a disjunct distribution and often in pathogenic species, similar to what is seen in prokaryotes. Thus, UGM inhibition may provide an attractive drug target in those eukaryotes where Gal(f) plays critical roles in cellular viability and virulence.

UDP-glucose dehydrogenase plays multiple roles in the biology of the pathogenic fungus Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic fungus surrounded by an elaborate polysaccharide capsule that is strictly required for its virulence in humans and other mammals. Nearly half of the sugar residues in the capsule are derived from UDP-glucuronic acid or its metabolites. To examine the role of these nucleotide sugars in C. neoformans, the gene encoding UDP-glucose dehydrogenase was disrupted. Mass spectrometry analysis of nucleotide sugar pools showed that the resulting mutant lacked both UDP-glucuronic acid and its downstream product, UDP-xylose, thus confirming the effect of the knockout and indicating that an alternate pathway for UDP-glucuronic acid production was not used. The mutant was dramatically affected by the lack of specific sugar donors, demonstrating altered cell integrity, temperature sensitivity, lack of growth in an animal model of cryptococcosis, and morphological defects. Additionally, the polysaccharide capsule could not be detected on the mutant cells, although the possibility remains that abbreviated forms of capsule components are made, possibly without proper surface display. The capsule defect is largely independent of the other observed changes, as cells that are acapsular because of mutations in other genes show lack of virulence but do not exhibit alterations in cell integrity, temperature sensitivity, or cellular morphology. All of the observed alterations were reversed by correction of the gene disruption.

An efficiently regulated promoter system for Cryptococcus neoformans utilizing the CTR4 promoter.

Cryptococcus neoformans is an opportunistic fungal pathogen responsible for serious meningitis. Although many useful molecular tools have been developed for its study, there are currently few inducible promoters available for general use. To address this need, we have constructed expression plasmids incorporating upstream elements of the C. neoformans copper transporter gene CTR4, and tested them in C. neoformans serotypes A and D. In response to copper deprivation, these plasmids mediate high-level expression of a reporter protein. This expression can be completely repressed using physiologically low concentrations of copper. Notably, this new family of copper-sensing promoters demonstrates excellent expression in serotype A, contrasting with other available promoters. These plasmids therefore offer efficient and regulated expression for both serotypes A and D, and should be valuable tools for the C. neoformans research community.

Biosynthesis of UDP-GlcA, a key metabolite for capsular polysaccharide synthesis in the pathogenic fungus Cryptococcus neoformans.

UDP-glucose dehydrogenase catalyses the conversion of UDP-glucose into UDP-GlcA, a critical precursor for glycan synthesis across evolution. We have cloned the gene encoding this important enzyme from the opportunistic pathogen Cryptococcus neoformans. In this fungus, UDP-GlcA is required for the synthesis of capsule polysaccharides, which in turn are essential for virulence. The gene was expressed in Escherichia coli and the 51.3-kDa recombinant protein from wild-type and five mutants was purified for analysis. The cryptococcal enzyme is strongly inhibited by UDP-xylose and NADH, has highest activity at pH 7.5 and demonstrates Km (app) values of 0.1 and 1.5 mM for NAD+ and UDP-glucose respectively. Its activity was significantly decreased by mutations in the putative sites of NAD+ and UDP-glucose binding. Unlike previously reported eukaryotic UDP-glucose dehydrogenases, which are hexamers, the cryptococcal enzyme is a dimer.

Evidence that protein disulfide isomerase (PDI) is involved in DNA-nuclear matrix anchoring.

DNA-nuclear matrix (NM) anchoring plays a critical role in the organization of DNA within the nucleus and in functional access to DNA for transcription, replication, and DNA repair. The cellular response to oxidative stress involves both gene expression and DNA repair. We, therefore, determined if changes in the oxidative-reductive environment can affect DNA-NM anchoring. The present study used two approaches to study the effect of the reducing agent DTT on DNA-NM anchoring. First, the relative stringency of the DNA-NM attachment was determined by measuring the ability of NM attached DNA loops to undergo supercoiling changes. Second, the effects of DTT on the association of nuclear proteins with DNA were determined by cisplatin crosslinking. When nucleoids (nuclear matrices with attached DNA loops) were prepared from HeLa cells with 1 mM dithiothreitol (DTT), supercoiled DNA loops unwound more efficiently compared with control in the presence of increasing propidium iodide (PI) concentrations. In addition, the rewinding of DNA supercoils in nucleoids treated with DTT was inhibited. Both effects on DNA supercoiling ability were reversed by diamide suggesting that they are dependent on the oxidation state of the protein thiols. When DTT treated nucleoids were isolated from gamma-irradiated cells, the inhibition of DNA supercoil rewinding was equal to the sum of the inhibition due to DTT and gamma-rays alone. Nucleoids isolated from heat-shocked cells with DTT, showed no inhibition of DNA rewinding, except a small inhibition at high PI concentrations. Nuclear DNA in DTT-treated nuclei was digested faster by DNase I than in untreated nuclei. These results suggest that DTT is altering DNA-NM anchoring by affecting the protein component(s) of the anchoring complex. Extracting NM with increasing concentrations of DTT did not solubilize any protein to a significant extent until measurable NM disintegration occurred. Therefore, we determined if 1 mM DTT affected the ability of 1 mM cisplatin to crosslink proteins to DNA. Isolated nuclei were treated with 1 mM DTT for 30 min or left untreated prior to crosslinking with 1 mM cisplatin for 2 h at 4 degrees C. The ability of capsulation to crosslink DNA to proteins per se, did not appear to be affected by 1 mM DTT because relative amounts of at least four proteins, 69, 60, 40, and 35 kDa, were crosslinked to DNA to the same extent in DTT-treated and untreated nuclei. However, protein disulfide isomerase (PDI) crosslinked to DNA in untreated nuclei, but did not crosslink DNA in nuclei that were treated with 1 mM DTT; 1 mM DTT did not affect the intranuclear localization of PDI. Thus, DTT appears to alter the conformation of PDI, as suggested by the DTT-induced change in DNA association, but not its NM association. These results also imply that DNA-NM anchoring involves the redox state of protein sulfhydryl groups.