Biology subjects

Mueth, N. A.

Publications and source records attributed to Mueth, N. A..

5 recordsLinked to original sources

bioRxiv2024

Small DNA elements that act as both insulators and silencers in plants

Insulators are cis-regulatory elements that separate transcriptional units, whereas silencers are elements that repress transcription regardless of their position. In plants, these elements remain largely uncharacterized. Here, we use the massively parallel reporter assay Plant STARR-seq with short fragments of eight large insulators to identify more than 100 fragments that block enhancer activity. The short fragments can be combined to generate more powerful insulators that abolish the capacity of the strong viral 35S enhancer to activate the 35S minimal promoter. Unexpectedly, when tested upstream of weak enhancers, these fragments act as silencers and repress transcription. Thus, these elements are capable of both insulating or repressing transcription dependent upon regulatory context. We validate our findings in stable transgenic Arabidopsis, maize, and rice plants. The short elements identified here should be useful building blocks for plant biotechnology efforts.

plant biology↗

bioRxiv2024

The regulatory potential of transposable elements in maize

The genomes of flowering plants consist largely of transposable elements (TEs), some of which modulate gene regulation and function. However, the repetitive nature of TEs and difficulty of mapping individual TEs by short-read-sequencing have hindered our understanding of their regulatory potential. We demonstrate that long-read chromatin fiber sequencing (Fiber-seq) comprehensively identifies accessible chromatin regions (ACRs) and CpG methylation across the maize genome. We uncover stereotypical ACR patterns at young TEs that degenerate with evolutionary age, resulting in TE-enhancers preferentially marked by a novel plant-specific epigenetic feature: simultaneous hyper-CpG methylation and chromatin accessibility. We show that TE ACRs are co-opted as gene promoters and that ACR-containing TEs can facilitate gene amplification. Lastly, we uncover a pervasive epigenetic signature - hypo-5mCpG methylation and diffuse chromatin accessibility - directing TEs to specific loci, including the loci that sparked McClintocks discovery of TEs.

genomics↗

bioRxiv2023

Cooperativity and additivity in plant enhancers

Enhancers are cis-regulatory elements that shape gene expression in response to numerous developmental and environmental cues. In animals, several models have been proposed to explain how enhancers integrate the activity of multiple transcription factors. However, it remains largely unknown how plant enhancers integrate transcription factor activity. Here, we use Plant STARR-seq to characterize three light-responsive plant enhancers--AB80, Cab-1, and rbcS-E9--derived from genes active in photosynthesis. Saturation mutagenesis reveals mutations, many of which cluster in short regions, that strongly reduce enhancer activity in the light, in the dark or in both conditions. When tested in the light, these mutation-sensitive regions do not function on their own; rather, cooperative interactions with other such regions are required for full activity. Epistatic interactions occur between mutations in adjacent mutation-sensitive regions, and the spacing and order of mutation-sensitive regions in synthetic enhancers affects enhancer activity. In contrast, when tested in the dark, mutation-sensitive regions act independently and additively in conferring enhancer activity. Taken together, this work demonstrates that plant enhancers show evidence for both cooperative and additive interactions among their functional elements. This knowledge can be harnessed to design strong, condition-specific synthetic enhancers.

plant biology↗

bioRxiv2023

Features that Govern Terminator Strength in Plants

The 3 end of a gene, often called a terminator, modulates mRNA stability, localization, translation, and polyadenylation. Here, we adapted Plant STARR-seq, a massively parallel reporter assay, to measure the activity of over 50,000 terminators from the plants Arabidopsis thaliana and Zea mays. We characterize thousands of plant terminators, including many that outperform bacterial terminators commonly used in plants. Terminator activity is species-specific, differing in tobacco leaf and maize protoplast assays. While recapitulating known biology, our results reveal the relative contributions of polyadenylation motifs to terminator strength. We built a computational model to predict terminator strength and used it to conduct in silico evolution that generated optimized synthetic terminators. Additionally, we discover alternative polyadenylation sites across tens of thousands of terminators; however, the strongest terminators tend to have a dominant cleavage site. Our results establish features of plant terminator function and identify strong naturally occurring and synthetic terminators.

plant biology↗

bioRxiv2022

Small RNAs target native and cross-kingdom transcripts on both sides of the wheat stripe rust interaction

The wheat stripe rust fungus (Puccinia striiformis f.sp tritici) poses a significant challenge to global wheat production. Plant defense induction against pathogens is partly modulated by small RNA (sRNA) molecules that downregulate complementary protein-coding transcripts. Additionally, the two-way RNA exchange between host and microbe involves cross-kingdom gene silencing that impacts pathogen virulence, yet few examples are known among rust fungi. The purpose of this study was to discover small RNA-target pairs on each side of this interaction. We performed sRNA sequencing and parallel analysis of RNA ends (PARE) in infected and uninfected wheat seedlings and combined these data with gene expression information. Wheat 24 nt heterochromatic siRNA (hc-siRNA) sequences were suppressed upon infection, while specific 35 nt tRNA and rRNA fragments were strongly induced. Target transcripts were identified by the observation of high mRNA slicing frequency at the precise position of sRNA binding sites. Wheat small RNAs showed evidence of cleaving several fungal transcripts including a ribosomal protein-coding gene and a glycosyl hydrolase effector gene. In P. striiformis, we confirmed and expanded previous findings that sRNAs feature microRNA-like sequences and siRNAs originating from long inverted repeats near protein-coding genes. Long inverted repeat regions produced sets of phased sRNAs at 21 nt intervals. Fungal sRNAs were identified that target native transcripts coding for transposons and protein kinases. Cross-kingdom gene targets of pathogen sRNAs included wheat nucleotide-binding domain leucine-rich repeat receptors (NLRs) and multiple families of defense-related transcription factors. The identified target genes shed light on an intricately co-evolved interaction and provide useful prospects for the development of pathogen control biotechnology.

genomics↗