Effects of Abolishing Whi2 on the Proteome and Nitrogen Catabolite Repression-Sensitive Protein Production

In yeast physiology, a commonly used reference condition for many experiments, including those involving Nitrogen Catabolite Repression (NCR), is growth in Synthetic Complete (SC) medium. Four SC formulations, SCCSH, 1990, SCCSH, 1994, SCCSH, 2005 and SCME, have been used interchangeably as the nitrogen-rich medium of choice (Cold Spring Harbor Yeast Course Manuals, SCCSH, and a formulation in The Methods in Enzymology SCME). It has been tacitly presumed that all of these formulations support equivalent responses. However, Chen et al. (2018) concluded that: (i) TorC1 activity is down regulated by the lower concentration of primarily leucine in SCME relative to SCCSH. (ii) The Whi2-Psr1/2 complex is responsible for this down regulation. TorC1 is a primary nitrogen-responsive regulator in yeast. Among its downstream targets is control of NCR-sensitive transcription activators Gln3 and Gat1. They in turn control production of catabolic transporters and enzymes needed to scavenge poor nitrogen sources (e.g., Proline) and activate autophagy (ATG14). One of the reporters used in Chen et al. was an NCR-sensitive DAL80-GFP promoter fusion. This intrigued us because we expected minimal if any DAL80 expression in SC medium. Therefore, we investigated the source of the Dal80-GFP production and the proteomes of wild type and whi2Δ cells cultured in SCCSH and SCME. We found a massive and equivalent reorientation of amino acid biosynthetic proteins in both wild type and whi2Δ cells even though both media contained high overall concentrations of amino acids. Gcn2 appears to play a significant regulatory role in this reorientation. NCR-sensitive DAL80 expression and overall NCR-sensitive protein production were only marginally affected by the whi2Δ. In contrast, the levels of 58 proteins changed by an absolute value of log2 between 3 and 8) when Whi2 was abolished relative to wild type. Surprisingly, with only two exceptions could those proteins be related in GO analyses, i.e., GO terms associated with carbohydrate metabolism and oxidative stress after shifting a whi2Δ from SCCSH to SCME for 6 hours. What was conspicuously missing were proteins related by TorC1- and NCR-associated GO terms.

A giant NLR gene confers broad-spectrum resistance to Phytophthora sojae in soybean

Phytophthora root and stem rot caused by P. sojae is a destructive soybean soil-borne disease found worldwide. Discovery of genes conferring broad-spectrum resistance to the pathogen is a need to prevent the outbreak of the disease. Here, we show that soybean Rps11 is a 27.7-kb nucleotide-binding site-leucine-rich repeat (NBS-LRR or NLR) gene conferring broad-spectrum resistance to the pathogen. Rps11 is located in a genomic region harboring a cluster of large NLR genes of a single origin in soybean, and is derived from rounds of unequal recombination. Such events result in promoter fusion and LRR expansion that may contribute to the broad resistance spectrum. The NLR gene cluster exhibits drastic structural diversification among phylogenetically representative varieties, including gene copy number variation ranging from five to 23 copies, and absence of allelic copies of Rps11 in any of the non-Rps11-donor varieties examined, exemplifying innovative evolution of NLR genes and NLR gene clusters.

A giant chimeric NLR gene confers extreme broad-spectrum resistance to a plant pathogen

Phytophthora root and stem rot (PRSR) caused by Phytophthora sojae is the most destructive soybean soil-borne disease worldwide. Discovery of genes conferring broad-spectrum resistance to the pathogen is an urgent need to prevent the outbreak of the disease. Here we show that soybean Rps11 is a 27.7-kb nucleotide-binding site-leucine-rich repeat (NBS-LRR or NLR) gene conferring extreme broad-spectrum resistance to the pathogen. Rps11 is located in a genomic region harboring a cluster of unusually large NLR genes belonging to a single evolutionary lineage that is distinct from all other lineages in the soybean genome, and was derived from rounds of intergenic and intragenic unequal recombination. Such recombination events have resulted in promoter fusion and expansion of the LRR domain that presumably explains such broadness of the resistance spectrum. The NLR gene cluster exhibits drastic structural diversification among phylogenetically representative varieties, including gene copy number variation ranging from five to 23 copies, and absence of allelic copies of Rps11 (i.e., rps11) in any of the non-Rps11-donor varieties examined. Our study thus exemplifies innovative evolution of NLR genes and NLR gene clusters and will accelerate the deployment of Rps11 for soybean protection.

The E. coli NudL enzyme is a Nudix hydrolase that cleaves CoA and its derivatives

The process of bacterial coenzyme A (CoA) degradation has remained unknown despite the otherwise detailed characterization of the CoA synthesis pathway over 30 years ago. Numerous enzymes capable of CoA degradation have been identified in other domains of life that belong to the Nudix superfamily of hydrolases, but the molecule responsible for this process in the model bacterial system of E. coli remains a mystery. We report here that E. coli contains two such Nudix enzymes capable of CoA degradation into 4’-phosphopantetheine and 3’,5’-adenosine monophosphate. The E. coli enzymes NudC and NudL were cloned in various promoter-fusion constructs in order to purify them as soluble active enzymes and characterize their ability to catalyze the phosphohydrolysis of CoA. NudC, an enzyme known to hydrolyze NADH as its principal substrate, demonstrated the ability to hydrolyze CoA, among other coenzymes, at comparable rates to eukaryotic Nudix hydrolases. NudL, a previously uncharacterized enzyme, demonstrated the ability to cleave only CoA and CoA-related molecules at a rate orders of magnitude slower than its eukaryotic orthologs. NudC and NudL therefore represent a previously uncharacterized pathway of CoA degradation in the highly studied E. coli system. While the two enzymes display some substrate overlap, their respective activities imply that NudC may play a role as a general coenzyme hydrolase, while NudL specifically targets CoA. These data further suggest a role for these enzymes in the regulation of bacterial CoA-RNA.

Codon-optimized TDP-43-mediated neurodegeneration in a Drosophila model for ALS/FTLD

ABSTRACTTransactive response DNA binding protein-43 (TDP-43) is known to mediate neurodegeneration associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The exact mechanism by which TDP-43 exerts toxicity in the brains of affected patients remains unclear. In a novel Drosophila melanogaster model, we report gain-of-function phenotypes due to misexpression of insect codon-optimized version of human wild-type TDP-43 (CO-TDP-43) using both the binary GAL4/UAS system and direct promoter fusion constructs. The CO-TDP-43 model showed robust tissue specific phenotypes in the adult eye, wing, and bristles in the notum. Compared to non-codon optimized transgenic flies, the CO-TDP-43 flies produced increased amount of high molecular weight protein, exhibited pathogenic phenotypes, and showed cytoplasmic aggregation with both nuclear and cytoplasmic expression of TDP-43. Further characterization of the adult retina showed a disruption in the morphology and function of the photoreceptor neurons with the presence of acidic vacuoles that are characteristic of autophagy. Based on our observations, we propose that TDP-43 has the propensity to form toxic protein aggregates via a gain-of-function mechanism, and such toxic overload leads to activation of protein degradation pathways such as autophagy. The novel codon optimized TDP-43 model is an excellent resource that could be used in genetic screens to identify and better understand the exact disease mechanism of TDP-43 proteinopathies and find potential therapeutic targets.

GhHB12, a HD-ZIP I Transcription Factor, Negatively Regulates the Cotton Resistance to Verticillium dahliae

The homeodomain-leucine zipper (HD-ZIP) is a plant-specific transcription factor family that plays important roles in plant developmental processes in response to multiple stressors. We previously isolated a cotton HD-ZIP class I transcription factor gene, GhHB12, which is regulated by the circadian clock and photoperiodism. Furthermore, it regulates cotton architecture, phase transition, and photoperiod sensitivity. Here we report that GhHB12 was induced by methyl jasmonate (MeJA) and Verticillium dahliae infection. Additionally, stress-responsive elements were found in the GhHB12 promoter. Promoter fusion analysis showed that GhHB12 was predominantly expressed in primary roots and that it was induced by mechanical damage. Overexpression of GhHB12 increased susceptibility of the cotton plant to the fungal pathogens Botrytis cinerea and V. dahliae, which was coupled with suppression of the jasmonic acid (JA)-response genes GhJAZ2 and GhPR3. Our results suggest that GhHB12, a cotton stress-responsive HD-ZIP I transcription factor, negatively regulates cotton resistance to V. dahliae by suppressing JA-response genes.

Global transcriptomic responses of Escherichia coli K-12 to volatile organic compounds

Volatile organic compounds (VOCs) are commonly used as solvents in various industrial settings. Many of them present a challenge to receiving environments, due to their toxicity and low bioavailability for degradation. Microorganisms are capable of sensing and responding to their surroundings and this makes them ideal detectors for toxic compounds. This study investigates the global transcriptomic responses of Escherichia coli K-12 to selected VOCs at sub-toxic levels. Cells grown in the presence of VOCs were harvested during exponential growth, followed by whole transcriptome shotgun sequencing (RNAseq). The analysis of the data revealed both shared and unique genetic responses compared to cells without exposure to VOCs. Results suggest that various functional gene categories, for example, those relating to Fe/S cluster biogenesis, oxidative stress responses and transport proteins, are responsive to selected VOCs in E. coli. The differential expression (DE) of genes was validated using GFP-promoter fusion assays. A variety of genes were differentially expressed even at non-inhibitory concentrations and when the cells are at their balanced-growth. Some of these genes belong to generic stress response and others could be specific to VOCs. Such candidate genes and their regulatory elements could be used as the basis for designing biosensors for selected VOCs.