Biology subjects

Lunde, C.

Publications and source records attributed to Lunde, C..

6 recordsLinked to original sources

bioRxiv2026

Tracking Transgenes with Color: RUBY as a Visual Marker in CRISPR-Edited Mutant Plants in Two Triticum Species

CRISPR-Cas9 is a powerful tool for precise genome editing in plants, but the presence of foreign DNA, such as T-DNA, raises regulatory concerns and complicates mutant screening and field studies of edited material. Detecting plants with good transgene expression and later removing the T-DNA from edited plants is both time-consuming and costly. To address this, we developed a system that uses the non-destructive RUBY reporter, linked to the CRISPR-Cas9 cassette, and expressed under the ZmUbi1 promoter. To assess the applicability of the system, it was tested on two Triticum species, targeting three genes in either tetraploid or hexaploid wheat. Strong correlations were observed in both T0 and T1 plants between betalain content and Cas9 expression, allowing for the quick identification of plants likely to be edited. Furthermore, the RUBY reporter could be used to select against the transgenic CRISPR-Cas9 cassette in subsequent generations at both the seed and seedling stages, thereby reducing the number of plants that need to be screened to identify edited lines without a T-DNA. This approach, using a nondestructive reporter, enabled rapid distinction between transgene expression in primary transgenics and served as a negative selection in the T1 generation, streamlining selection towards edited and T-DNA-free progeny.

plant biology↗

bioRxiv2025

Natural variation in regulatory code revealed through Bayesian analysis of plant pan-genomes and pan-transcriptomes

Understanding the genetic code of cis-regulatory elements (CREs) is essential for engineering gene expression and modulating agronomic traits in crops. In plants, CREs underlying rapid evolution of gene expression often overlap with structural variation in promoters, making them undetectable using single-reference genomes. Here, we develop K-PROB (K-mer-based in silico PROmoter Bashing), a computational tool that learns from intraspecies promoter sequence and gene expression variation in pan-genomes and pan-transcriptomes to identify CREs controlling gene expression. K-PROB deploys a k-mer-based Bayesian variable selection framework to prioritize causal variable identification. We demonstrate the effectiveness of our approach in maize and soybean, two staple crops species. Applying K-PROB to genes with the most highly variable promoter sequences and the most diverse patterns of expression, such as nucleotide-binding leucine-rich repeat receptors, we identified k-mers enriched for bona fide transcription factor binding sequences, and overlapping with open chromatin regions and DAP-seq binding sites. Notably, multiple significant k-mers are located within presence/absence structural variants, highlighting structural variation in promoters as key drivers of transcriptional diversity of highly variable genes. We further validated the regulatory effects of identified k-mers on gene expression using luciferase reporter assays. Our results showcase a high-throughput and pangenomic approach for probing natural intraspecies cis-regulatory diversity, discovering new causative cis-elements, and facilitating future expression engineering across plant species. Significance StatementUnderstanding which DNA sequences control gene expression is essential for crop improvement. Current methods for identifying regulatory elements rely on expensive, specialized biochemical datasets typically limited to a single genotype. We developed a computational tool that links natural sequence variation and gene expression variation to identify functional regulatory sequences. Our tool employs a statistical framework that prioritizes causality over correlation, in contrast to most genome-wide association studies. Applying it to maize and soybean, two staple crops, we uncovered known and novel regulatory elements and validated them with molecular assays. Our approach is scalable, cost-effective, and efficiently utilizes natural variation from existing pangenomic datasets, opening new avenues for future crop engineering and studying gene regulation in diverse plant species.

bioinformatics↗

bioRxiv2024

Wheat Enhanced Disease Resistance EMS-Mutants Include Lesion-mimics With Adult Plant Resistance to Stripe Rust

Tetraploid durum wheat Triticum turgidum subsp. durum cv Kronos has extensive genetic variation resources, including a sequenced and cataloged ethyl methanesulfonate (EMS) mutagenized population. To utilize this allelic diversity, we screened over 2,000 mutant lines and identified over 30 enhanced disease resistance (EDR) mutants in a forward genetic field screen against stripe rust. Sixteen of the EDR lines have persistent resistance to stripe rust after four years, and several mutants showed differential disease responses against other fungal pathogens, indicating that the lines possess diverse alleles that affect multiple routes of pathogen suppression. Five of these 16 lines showed spontaneous lesion formation in the absence of pathogens. Only one showed a reduction in kernel weight under pathogen pressure, a testimony to the high mutational density that wheat can tolerate. Phenotypic selection for resistance at the adult stage identified useful EMS alleles for stripe rust resistance. The mutations in the 16 EDR lines were newly mapped to a recently released long-read Kronos genome to enhance their utility in molecular breeding for fungal resistance and for fundamental studies of plant-pathogen interactions.

plant biology↗

bioRxiv2022

Liguleless narrow and narrow odd dwarf act in overlapping pathways to regulate maize development & physiology

narrow odd dwarf (nod) and Liguleless narrow (Lgn) are pleiotropic maize mutants that both encode plasma membrane proteins, cause similar developmental patterning defects, and constitutively induce stress signaling pathways. To investigate how these mutants coordinate maize development and physiology, we screened for protein interactors of NOD by affinity purification. LGN was identified by this screen as a strong candidate interactor, and we confirmed the NOD-LGN molecular interaction through orthogonal experiments. We further demonstrated that LGN, a receptor-like kinase, can phosphorylate NOD in vitro, hinting that they could act in intersecting signal transduction pathways. To test this hypothesis, we generated Lgn-R;nod mutants in two backgrounds (B73 and A619) and found that these mutations enhance each other, causing more severe developmental defects than either single mutation on its own, with phenotypes including very narrow leaves, increased tillering, and failure of the main shoot. Transcriptomic and metabolomic analyses of the single and double mutants in the two genetic backgrounds revealed widespread induction of pathogen defense genes and a shift in resource allocation away from primary metabolism in favor of specialized metabolism. These effects were similar in each single mutant and heightened in the double mutant, leading us to conclude that NOD and LGN act cumulatively in overlapping signaling pathways to coordinate growth-defense tradeoffs in maize.

plant biology↗

bioRxiv2022

A draft genome assembly for the heterozygous wild tomato Solanum habrochaites highlights haplotypic structural variations of intracellular immune receptors

Solanum habrochaites LA1353 is a self-incompatible, highly heterozygous wild tomato that is a useful germplasm resource for the study of metabolism, reproduction and disease resistance. We generated a draft genome assembly with PacBio HiFi reads and genome annotations, which underscored the expansion of gene families associated with metabolite-production, self-incompatibility, DNA regulation and immunity. After manually curating intracellular nucleotide-binding leucine-rich repeat immune receptors (NLRs), we found that S. habrochaites LA1353 has a larger NLR inventory than other wild tomato species. A great number of heterozygous local copy number variations (CNVs) driven by haplotypic structural variations further expands the inventory, both enhancing NLR diversity and providing more opportunities for sequence evolution. The NLRs associated with local CNVs predominantly appear in the helper NLR (NRC)-related phylogenetic clades and are concentrated in a few physical NLR gene clusters. Synteny analysis points out that these genomic regions correspond to the known NLR clusters from which experimentally validated, functional NLRs, such as Hero, Mi-1.2 and Rpi-amr1, have been identified. Producing and incorporating Resistance Gene Enrichment Sequencing (RenSeq) data across wild tomato species, we reveal that the regions with local CNVs might have been shaped nearly equally by recent NLR gains and losses, along with enhanced sequence diversification that diminishes one-to-one orthology between heterozygous alleles. Our analysis suggests that these genomic regions may have accelerated evolutionary dynamics for NLR diversity generation in S. habrochaites LA1353.

plant biology↗

bioRxiv2020

Degradation of mixed-linkage (1,3;1,4)-β-D-glucan in maize is mediated by the CAL1 licheninase

The presence of mixed-linkage (1,3;1,4)-{beta}-D-glucan (MLG) in plant cell walls is a key feature of grass species such as cereals - the main source of calorie intake for humans and cattle. Accumulation of this polysaccharide involves the coordinated regulation of biosynthetic and metabolic machineries. While several components of the MLG biosynthesis machinery have been identified in diverse plant species, degradation of MLG is poorly understood. A large-scale forward genetic maize screen for mutants with altered cell wall polysaccharide structural properties resulted in the identification of candy-leaf1 (cal1). Cell walls of CAL1-deficient plants contain higher amounts of MLG in several tissues, including adult leaves and senesced organs, where only trace amounts of MLG are usually detected. In addition, cal1 plants exhibit increased saccharification yields upon enzymatic digestion. Stacking cal1 with lignin-deficient mutations results in synergistic saccharification increases. Identification of the causative mutation revealed that CAL1 encodes a GH17 licheninase. Maize plants overexpressing CAL1 exhibit a 90% reduction in MLG content, indicating that CAL1 is not only required, but its expression sufficient to degrade MLG. CAL1 specifically hydrolyzes (1,3;1,4)-{beta}-D-Glucans in vitro, and the single CAL1E262K amino acid substitution is able to block all detectable activity. Time profiling experiments indicate that wall MLG content is modulated during day/night cycles inversely correlating with CAL1 transcript accumulation. This cycling is absent in the cal1 mutant, suggesting that the mechanism involved requires MLG degradation that may in turn regulate CAL1 gene expression. One sentence summary Mixed-linkage glucan is degraded by the CAL1 licheninase in maize

plant biology↗