Biology subjects

Helm, M.

Publications and source records attributed to Helm, M..

34 records · Page 2Linked to original sources

bioRxiv2023

Uncovering the Infection Strategy of Phyllachora maydis during Maize Colonization: A Comprehensive Analysis

Tar spot, a disease caused by the ascomycete fungal pathogen Phyllachora maydis, is considered one of the most significant yield-limiting diseases of maize (Zea mays L.) within the United States. P. maydis may also be found in association with other fungi, forming a disease complex with characteristic fish eye lesions. Understanding how P. maydis colonizes maize leaf cells is essential for developing effective disease control strategies. Here, we used histological approaches to elucidate how P. maydis infects and multiplies within susceptible maize leaves. We collected tar spot-infected maize leaf samples from four different fields in northern Indiana at three different time points during the growing season. Samples were chemically fixed and paraffin-embedded for high-resolution light and scanning electron microscopy. We observed a consistent pattern of disease progression in independent leaf samples collected across different geographical regions. Each stromata contained a central pycnidium that produced asexual spores. Perithecia with sexual spores developed in the stomatal chambers adjacent to the pycnidia, and a cap of spores formed over the stromata. P. maydis reproductive structures formed around but not within the vasculature. In our samples containing fish eye lesions, P. maydis is associated with two additional fungi, one of which is likely a member of the Paraphaeospheria genus; the other is an unknown fungi. Our data provide fundamental insights into how this pathogen colonizes and spreads within maize leaves. This knowledge can inform new approaches to managing tar spot, which could help mitigate the significant economic losses caused by this disease.

plant biology↗

bioRxiv2023

Analysis of cell death induction by the barley NLR immune receptor PBR1

The barley (Hordeum vulgare subsp. vulgare) disease resistance protein AvrPphB Response 1 (PBR1) mediates recognition of the Pseudomonas syringae effector, AvrPphB. PBR1 belongs to the coiled-coil nucleotide-binding leucine-rich repeat (CNL) family. However, little is known about the molecular mechanisms that lead to PBR1-dependent cell death (hypersensitive reaction; HR) in response to AvrPphB. Here, we investigated PBR1 immune signaling after Agrobacterium-mediated transient expression in Nicotiana benthamiana. We found that co-expression of PBR1 with AvrPphB resulted in robust cell death, confirming previous observations that PBR1 is indeed the cognate NLR that recognizes AvrPphB. The N-terminal tagging of PBR1 with super Yellow Fluorescent Protein (sYFP) abolished PBR1-mediated cell death, demonstrating that an N-terminal epitope tag disrupts PBR1-mediated immune signaling. Furthermore, none of the individual protein domains or truncations of PBR1 induced a HR-like cell death response as strong as full-length PBR1 when co-expressed with AvrPphB, indicating that the individual domains and fragments of PBR1 are insufficient to trigger HR. Intriguingly, introducing the typically auto-activating D496V mutation within NB-ARC-containing fragments of PBR1 does not activate immune signaling revealing PBR1-mediated immune signaling requires cooperation of all domains in cis. Using co-immunoprecipitation and split-luciferase assays, we also show full-length PBR1 self-associates in the absence of AvrPphB and such self-association is not dependent on a functional P-loop/Walker A motif. Collectively, these findings provide valuable insights into PBR1-mediated disease resistance and extends upon our understanding of NLR-mediated immune signaling.

plant biology↗

bioRxiv2022

NanopoReaTA: a user-friendly tool for nanopore-seq real-time transcriptional analysis

SummaryOxford Nanopore Technologies (ONT) sequencing platform offers an excellent opportunity to perform real-time analysis during sequencing. This feature allows for early insights into experimental data and accelerates a potential decision-making process for further analysis, which can be particularly relevant in the clinical context. Although some tools for the real-time analysis of DNA-sequencing data already exist, there is currently no application available for differential transcriptome data analysis designed for scientists or physicians with limited bioinformatics knowledge. Here we introduce NanopoReaTA, a user-friendly real-time analysis toolbox for RNA sequencing data from ONT. Sequencing results from a running or finished experiment are processed through an R Shiny-based graphical user interface (GUI) with an integrated Nextflow pipeline for whole transcriptome or gene-specific analyses. NanopoReaTA provides visual snapshots of a sequencing run in progress, thus enabling interactive sequencing and rapid decision-making that could also be applied to clinical cases. Availabilityhttps://github.com/AnWiercze/NanopoReaTA Contactbuttamer@uni-mainz.de and sugerber@uni-mainz.de

bioinformatics↗

bioRxiv2022

Peroxisomal β-oxidation enzyme, DECR2, regulates lipid metabolism and promotes treatment resistance in advanced prostate cancer

Peroxisomes are central metabolic organelles that have key roles in fatty acid homeostasis, including {beta}-oxidation, and emerging evidence has linked aberrant peroxisome metabolism to cancer development and progression. While targeting mitochondrial {beta}-oxidation in prostate cancer (PCa) has gained significant attention in recent years, the contribution of peroxisomal {beta}-oxidation (perFAO) to PCa tumorigenesis is comparatively unexplored. Herein, we explored the therapeutic efficacy of targeting perFAO in PCa cells and clinical prostate tumours, and subsequently identified peroxisomal 2,4-dienoyl CoA reductase 2 (DECR2), as a key therapeutic target. DECR2 is markedly upregulated in clinical PCa, most notably in metastatic castrate-resistant PCa. Depletion of DECR2 significantly suppressed proliferation, migration, and 3D growth of a range of CRPC and enzalutamide-resistant PCa cell lines, and inhibited LNCaP tumour growth and proliferation in vivo. Using transcriptomic and lipidomic analyses, we determined that DECR2 influences cell cycle progression and lipid metabolism to enable tumour cell proliferation. We further demonstrated a novel role for perFAO in driving resistance to standard-of-care androgen receptor pathway inhibition, using genetic and pharmacological approaches to alter DECR2/perFAO in treatment-resistant PCa cells. Our findings highlight a need to focus on peroxisomes to suppress tumour cell proliferation and reveal new therapeutic targets for advanced, treatment-resistant PCa.

cancer biology↗

bioRxiv2022

ACSM1 and ACSM3 regulate prostate cancer fatty acid metabolism to promote tumour growth and constrain ferroptosis

Prostate tumours are highly reliant on lipids for energy, growth and survival. Activity of the androgen receptor (AR) is associated with reprogramming of lipid metabolic processes in prostate cancer, although the molecular underpinnings of this relationship remain to be fully elucidated. Here, we identified Acyl-CoA Synthetase Medium Chain Family Members 1 and 3 (ACSM1 and ACSM3) as AR-regulated mediators of prostate cancer metabolism and growth. ACSM1 and ACSM3 are upregulated in prostate tumours compared to non-malignant tissues and other cancer types. Both enzymes enhanced proliferation and protected PCa cells from death in vitro, while silencing ACSM3 led to reduced tumour growth in an orthotopic xenograft model. We show that ACSM1 and ACSM3 are major regulators of the PCa lipidome and enhance energy production via fatty acid oxidation. Metabolic dysregulation caused by loss of ACSM1/3 led to mitochondrial oxidative stress, lipid peroxidation and cell death by ferroptosis. Conversely, over-expression of ACSM1/3 enabled PCa cells to survive toxic doses of medium chain fatty acids and promoted resistance to ferroptosis-inducing drugs and AR antagonists. Collectively, these studies uncover a new link between AR and lipid metabolism and position ACSM1 and ACSM3 as key players in prostate cancer progression and therapy resistance.

cancer biology↗

bioRxiv2022

ANTHRACNOSE RESISTANCE GENE2 confers fungal resistance in sorghum

Sorghum is an important food and feed crop globally; its production is hampered by anthracnose disease caused by the fungal pathogen Colletotrichum sublineola (Cs). Here, we report identification and characterization of ANTHRACNOSE RESISTANCE GENE 2 (ARG2) encoding a nucleotide-binding leucine-rich repeat (NLR) protein that confers race-specific resistance to Cs strains. ARG2 is one of a cluster of several NLR genes in the sorghum differential line SC328C that is resistant to some Cs strains. This cluster shows structural and copy number variations in different sorghum genotypes. Different sorghum variants carrying independent ARG2 alleles provided the genetic validation for the identity of the ARG2 gene. ARG2 expression is induced by Cs, and chitin induce ARG2 expression in resistant but not in susceptible lines. ARG2-mediated resistance is accompanied by higher expression of defense and secondary metabolite genes at early stages of infection, and anthocyanin and zeatin metabolisms are upregulated in resistant plants. Interestingly, ARG2 localizes to the plasma membrane when transiently expressed in Nicotiana benthamiana. Importantly, ARG2 plants produced higher shoot dry matter than near-isogenic lines carrying the susceptible allele suggesting absence of an ARG2 associated growth trade-off. Further, ARG2-mediated resistance is stable at a wide range of temperatures. Our observations open avenues for resistance breeding and for dissecting mechanisms of resistance.

plant biology↗

bioRxiv2022

Protein-based Virtual Screening Tools applied for RNA-Ligand Docking identify new Binders of the preQ1-Riboswitch

Targeting RNA with small molecules is an emerging field. While several ligands for different RNA targets are reported, structure-based virtual screenings against RNAs are still rare. Here, we elucidated the general capabilities of protein-based docking programmes to reproduce native binding modes of small molecule RNA ligands and to discriminate known binders from decoys by the scoring function. The programmes were found to perform similar compared to the RNA-based docking tool rDOCK and the faced challenges during docking, namely protomer and tautomer selection, target dynamics and explicit solvent, do not largely differ from challenges in conventional protein-ligand docking. A prospective virtual screening with the Bacillus subtilis preQ1-riboswitch aptamer domain performed with FRED, HYBRID and FlexX, followed by microscale thermophoresis assays identified 6 active compounds out of 23 tested virtual screening hits with potencies between 29.5 nM and 11.0 M. The hits were selected not solely based on their docking score, but for resembling key interactions of the native ligand. Therefore, this study demonstrates the general feasibility to perform structure-based virtual screenings against RNA targets, while at the same time it highlights pitfalls and their potential solutions when executing RNA-ligand docking.

bioinformatics↗

bioRxiv2022

Candidate effector proteins from the maize tar spot pathogen Phyllachora maydis localize to diverse plant cell compartments

Most fungal pathogens secrete effector proteins into host cells to modulate their immune responses, thereby promoting pathogenesis and fungal growth. One such fungal pathogen is the ascomycete Phyllachora maydis, which causes tar spot disease on leaves of maize (Zea mays). Sequencing of the P. maydis genome revealed 462 putatively secreted proteins of which 40 contain expected effector-like sequence characteristics. However, the subcellular compartments targeted by P. maydis effector candidate (PmECs) proteins remain unknown and it will be important to prioritize them for further functional characterization. To test the hypothesis that PmECs target diverse subcellular compartments, cellular locations of super Yellow Fluorescent Protein (sYFP)-tagged P. maydis effector candidate proteins were identified using a Nicotiana benthamiana-based heterologous expression system. Immunoblot analyses showed that most of the PmEC-fluorescent protein fusions accumulated protein in N. benthamiana, indicating the candidate effectors could be expressed in dicot leaf cells. Laser-scanning confocal microscopy of N. benthamiana epidermal cells revealed most of the P. maydis putative effectors localized to the nucleus and cytosol. One candidate effector, PmEC01597, localized to multiple subcellular compartments including the nucleus, nucleolus, and plasma membrane while an additional putative effector, PmEC03792, preferentially labelled both the nucleus and nucleolus. Intriguingly, one candidate effector, PmEC04573, consistently localized to the stroma of chloroplasts as well as stroma-containing tubules (stromules). Collectively, these data suggest effector candidate proteins from P. maydis target diverse cellular organelles and may thus provide valuable insights into their putative functions as well as host processes potentially manipulated by this fungal pathogen.

plant biology↗

bioRxiv2022

Human tRNA methyltransferase ALKBH8 targets wider repertoire of tRNAs and non-coding RNAs

Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U containing tRNAs obtain 5-methoxycarbonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by Alkylation Repair Homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP-seq and RIP-seq analyses, we uncover ALKBH8-bound RNAs. It targets an additional wobble U-containing tRNA tRNALys(UUU). More interestingly, the spectrum of bound RNAs includes other types of non-coding RNAs, such as C/D box snoRNAs, 7SK RNA or some miRNAs, respectively.

molecular biology↗

bioRxiv2021

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic cores of mammalian TET enzymes favor CpGs embedded within bHLH and bZIP transcription factor binding sites, with 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intra-substrate interactions and CpG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germline. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and OCT4, respectively, illuminating TET function in transcriptional responses and pluripotency support. One-Sentence SummaryThe catalytic domains of the enzymes that facilitate passive and drive active DNA demethylation have intrinsic sequence preferences that target DNA demethylation to bHLH and bZIP transcription factor binding sites.

molecular biology↗

bioRxiv2020

Translational adaptation to heat stress is mediated by 5-methylcytosine RNA modification in Caenorhabditis elegans

Methylation of carbon-5 of cytosines (m5C) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m5C-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, using Caenorhabditis elegans, we generated the first organism devoid of m5C in RNA, demonstrating that this modification is non-essential. We determined the localisation and enzymatic specificity of m5C sites in RNA in vivo and showed that animals devoid of m5C are sensitive to temperature stress. At the molecular level, we showed that loss of m5C specifically impacts decoding of leucine and proline thus reducing the translation efficiency of transcripts enriched in these amino acids. Finally, we found translation of leucine UUG codons to be the most strongly affected upon heat shock, suggesting a role of m5C tRNA wobble methylation in the adaptation to heat stress.

genetics↗

bioRxiv2020

Optimizing the PBS1 Decoy System to Confer Resistance to Potyvirus Infection in Arabidopsis and Soybean

The Arabidopsis resistance protein RPS5 is activated by proteolytic cleavage of the protein kinase PBS1 by the Pseudomonas syringae effector protease AvrPphB. We have previously shown that replacing seven amino acids at the cleavage site of PBS1 with a motif cleaved by the NIa protease of turnip mosaic virus (TuMV) enables RPS5 activation upon TuMV infection. However, this engineered resistance conferred a trailing necrosis phenotype indicative of a cell death response too slow to contain the virus. We theorized this could result from a positional mismatch within the cell between PBS1TuMV, RPS5 and the NIa protease. To test this, we re-localized PBS1TuMV and RPS5 to cellular sites of NIa accumulation. These experiments revealed that relocation of RPS5 away from the plasma membrane compromised RPS5-dependent cell death in N. benthamiana, even though PBS1 was efficiently cleaved. As an alternative approach, we tested whether overexpression of plasma membrane-localized PBS1TuMV would enhance RPS5 activation by TuMV. Significantly, over-expressing the PBS1TuMV decoy protein conferred complete resistance to TuMV when delivered by either Agrobacterium or by aphid transmission, showing that RPS5-mediated defense responses are effective against bacterial and viral pathogens. Lastly, we have now extended this PBS1 decoy approach to soybean by modifying a soybean PBS1 ortholog to be cleaved by the NIa protease of soybean mosaic virus (SMV). Transgenic overexpression of this soybean PBS1 decoy conferred immunity to SMV, demonstrating that we can use endogenous PBS1 proteins in crop plants to engineer economically relevant disease resistant traits.

plant biology↗

bioRxiv2018

Engineering a decoy substrate in soybean to enable recognition of the Soybean Mosaic Virus NIa protease

In Arabidopsis, recognition of the AvrPphB effector protease from Pseudomonas syringae is mediated by the disease resistance (R) protein RPS5, which is activated by AvrPphB-induced cleavage of the Arabidopsis protein kinase PBS1. The recognition specificity of RPS5 can be altered by substituting the AvrPphB cleavage site within PBS1 with cleavage sequences for other proteases, including proteases from viruses. AvrPphB also activates defense responses in soybean (Glycine max), suggesting that soybean may contain an R protein analogous to RPS5. It was unknown, however, whether this response is mediated by cleavage of a soybean PBS1-like protein. Here we show that soybean contains three PBS1 orthologs and that their products are cleaved by AvrPphB. Further, transient expression of soybean PBS1 derivatives containing a five-alanine insertion at their AvrPphB cleavage sites activated cell death in soybean protoplasts, demonstrating that soybean likely contains an AvrPphB-specific resistance protein that is activated by a conformational change in soybean PBS1 proteins. Significantly, we show that a soybean PBS1 decoy protein modified to contain a cleavage site for the Soybean mosaic virus (SMV) NIa protease triggers cell death in soybean protoplasts when cleaved by this protease, indicating that the PBS1 decoy approach will work in soybean using endogenous PBS1 genes. Lastly, we show that activation of the AvrPphB-dependent cell death response effectively inhibits systemic spread of SMV in soybean. These data also indicate that decoy engineering may be feasible in other crop plant species that recognize AvrPphB protease activity.

plant biology↗

bioRxiv2018

The RNA-binding protein AATF coordinates rRNA maturation

AATF is a central regulator of the cellular outcome upon p53 activation, a finding that has primarily been attributed to its function as a transcription factor. Recent data showed that AATF is essential for ribosome biogenesis and plays a role in rRNA maturation. AATF has been implicated to fulfil this role through direct interaction with rRNA and was identified in several RNA-interactome capture experiments. Here, we provide a first comprehensive analysis of the RNA bound by AATF using CLIP-sequencing. Interestingly, this approach shows predominant binding of the 45S pre-ribosomal RNA precursor molecules. Furthermore, AATF binds to mRNAs encoding for ribosome biogenesis factors as well as snoRNAs. These findings are complemented by an in-depth analysis of the protein interactome of AATF containing a large set of proteins known to play a role in rRNA maturation with an emphasis on the protein-RNA-complexes known to be required for the generation of the small ribosomal subunit (SSU). In line with this finding, the binding sites of AATF within the 45S rRNA precursor localize in close proximity to the SSU cleavage sites. Consequently, our multilayer analysis of the protein-RNA interactome of AATF reveals this protein to be an important hub for protein and RNA interactions involved in ribosome biogenesis.

molecular biology↗

bioRxiv2018

Convergent evolution of effector protease recognition by Arabidopsis and barley

The Pseudomonas syringae cysteine protease AvrPphB activates the Arabidopsis resistance protein RPS5 by cleaving a second host protein, PBS1. AvrPphB induces defense responses in other plant species, but the genes and mechanisms mediating AvrPphB recognition in those species have not been defined. Here, we show that AvrPphB induces defense responses in diverse barley cultivars. We show also that barley contains two PBS1 orthologs, that their products are cleaved by AvrPphB, and that the barley AvrPphB response maps to a single locus containing a nucleotide-binding leucine-rich repeat (NLR) gene, which we termed AvrPphB Resistance 1 (Pbr1). Transient co-expression of PBR1 with wild-type AvrPphB, but not a protease inactive mutant, triggered defense responses, indicating that PBR1 detects AvrPphB protease activity. Additionally, PBR1 co-immunoprecipitated with barley and N. benthamiana PBS1 proteins, suggesting mechanistic similarity to detection by RPS5. Lastly, we determined that wheat cultivars also recognize AvrPphB protease activity and contain a Pbr1 ortholog. Phylogenetic analyses showed however that Pbr1 is not orthologous to RPS5. Our results indicate that the ability to recognize AvrPphB evolved convergently, and imply that selection to guard PBS1-like proteins is ancient. Also, the results suggest that PBS1-based decoys may be used to engineer protease effector recognition-based resistance in barley and wheat.

plant biology↗

bioRxiv2017

Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs

Cytosine-5 RNA methylation plays an important role in several biologically and pathologically relevant processes. However, owing to methodological limitations, the transcriptome-wide distribution of this mark has remained largely unknown. We previously established RNA bisulfite sequencing as a method for the analysis of RNA cytosine-5 methylation patterns at single-base resolution. More recently, next-generation sequencing has provided opportunities to establish transcriptome-wide maps of this modification. Here we present a computational approach that integrates tailored filtering and data-driven statistical modeling to eliminate many of the artifacts that are known to be associated with bisulfite sequencing. Using RNAs from mouse embryonic stem cells we performed a comprehensive methylation analysis of mouse tRNAs, rRNAs and mRNAs. Our approach identified all known methylation marks in tRNA and two previously unknown but evolutionary conserved marks in 28S rRNA. In addition, mRNAs were found to be very sparsely methylated or not methylated at all. Finally, the tRNA-specific activity of the DNMT2 methyltransferase could be resolved at single-base resolution, which provided important further validation. Our approach can be used to profile cytosine-5 RNA methylation patterns in many experimental contexts and will be important for understanding the function of cytosine-5 RNA methylation in RNA biology and in human disease.

bioinformatics↗