Multiome Perturb-seq unlocks scalable discovery of integrated perturbation effects on the transcriptome and epigenome.

Single-cell CRISPR screens link genetic perturbations to transcriptional states, but high-throughput methods connecting these induced changes to their regulatory foundations are limited. Here we introduce Multiome Perturb-seq, extending single-cell CRISPR screens to simultaneously measure perturbation-induced changes in gene expression and chromatin accessibility. We apply Multiome Perturb-seq in a CRISPRi screen of 13 chromatin remodelers in human RPE-1 cells, achieving efficient assignment of sgRNA identities to single nuclei via an improved method for capturing barcode transcripts from nuclear RNA. We organize expression and accessibility measurements into coherent programs describing the integrated effects of perturbations on cell state, finding that ARID1A and SUZ12 knockdowns induce programs enriched for developmental features. Pseudotime analysis of perturbations connects accessibility changes to changes in gene expression, highlighting the value of multimodal profiling. Overall, our method provides a scalable and simply implemented system to dissect the regulatory logic underpinning cell state.

Genome-wide CRISPR-Cas9 knockout screens identify DNMT1 as a druggable dependency in sonic hedgehog medulloblastoma.

Sonic hedgehog subgroup of medulloblastoma (SHH-MB) is characterized by aberrant activation of the SHH signaling pathway. An inhibition of the positive SHH regulator Smoothened (SMO) has demonstrated promising clinical efficacy. Yet, primary and acquired resistance to SMO inhibitors limit their efficacy. An understanding of underlying molecular mechanisms of resistance to therapy is warranted to bridge this unmet need. Here, we make use of genome-wide CRISPR-Cas9 knockout screens in murine SMB21 and human DAOY cells, in order to unravel genetic dependencies and drug-related genetic interactors that could serve as alternative therapeutic targets for SHH-MB. Our screens reinforce SMB21 cells as a faithful model system for SHH-MB, as opposed to DAOY cells, and identify members of the epigenetic machinery including DNA methyltransferase 1 (DNMT1) as druggable targets in SHH-dependent tumors. We show that Dnmt1 plays a crucial role in normal murine cerebellar development and is required for SHH-MB growth in vivo. Additionally, DNMT1 pharmacological inhibition alone and in combination with SMO inhibition effectively inhibits tumor growth in murine and human SHH-MB cell models and prolongs survival of SHH-MB mouse models by inhibiting SHH signaling output downstream of SMO. In conclusion, our data highlight the potential of inhibiting epigenetic regulators as a novel therapeutic avenue in SMO-inhibitor sensitive as well as resistant SHH-MBs.

DNA-binding factor footprints and enhancer RNAs identify functional non-coding genetic variants.

BACKGROUND: Genome-wide association studies (GWAS) have revealed a multitude of candidate genetic variants affecting the risk of developing complex traits and diseases. However, the highlighted regions are typically in the non-coding genome, and uncovering the functional causative single nucleotide variants (SNVs) is challenging. Prioritization of variants is commonly based on genomic annotation with markers of active regulatory elements, but current approaches still poorly predict functional variants. To address this, we systematically analyze six markers of active regulatory elements for their ability to identify functional variants. RESULTS: We benchmark against molecular quantitative trait loci (molQTL) from assays of regulatory element activity that identify allelic effects on DNA-binding factor occupancy, reporter assay expression, and chromatin accessibility. We identify the combination of DNase footprints and divergent enhancer RNA (eRNA) as markers for functional variants. This signature provides high precision, but with a trade-off of low recall, thus substantially reducing candidate variant sets to prioritize variants for functional validation. We present this as a framework called FINDER-Functional SNV IdeNtification using DNase footprints and eRNA. CONCLUSIONS: We demonstrate the utility to prioritize variants using leukocyte count trait and analyze variants in linkage disequilibrium with a lead variant to predict a functional variant in asthma. Our findings have implications for prioritizing variants from GWAS, in development of predictive scoring algorithms, and for functionally informed fine mapping approaches.

Functional genomic analysis of genes important for Candida albicans fitness in diverse environmental conditions.

Fungal pathogens such as Candida albicans pose a significant threat to human health with limited treatment options available. One strategy to expand the therapeutic target space is to identify genes important for pathogen growth in host-relevant environments. Here, we leverage a pooled functional genomic screening strategy to identify genes important for fitness of C. albicans in diverse conditions. We identify an essential gene with no known Saccharomyces cerevisiae homolog, C1_09670C, and demonstrate that it encodes subunit 3 of replication factor A (Rfa3). Furthermore, we apply computational analyses to identify functionally coherent gene clusters and predict gene function. Through this approach, we predict the cell-cycle-associated function of C3_06880W, a previously uncharacterized gene required for fitness specifically at elevated temperatures, and follow-up assays confirm that C3_06880W encodes Iml3, a component of the C. albicans kinetochore with roles in virulence in vivo. Overall, this work reveals insights into the vulnerabilities of C. albicans.

Comprehensive network modeling approaches unravel dynamic enhancer-promoter interactions across neural differentiation.

BACKGROUND: Increasing evidence suggests that a substantial proportion of disease-associated mutations occur in enhancers, regions of non-coding DNA essential to gene regulation. Understanding the structures and mechanisms of the regulatory programs this variation affects can shed light on the apparatuses of human diseases. RESULTS: We collect epigenetic and gene expression datasets from seven early time points during neural differentiation. Focusing on this model system, we construct networks of enhancer-promoter interactions, each at an individual stage of neural induction. These networks serve as the base for a rich series of analyses, through which we demonstrate their temporal dynamics and enrichment for various disease-associated variants. We apply the Girvan-Newman clustering algorithm to these networks to reveal biologically relevant substructures of regulation. Additionally, we demonstrate methods to validate predicted enhancer-promoter interactions using transcription factor overexpression and massively parallel reporter assays. CONCLUSIONS: Our findings suggest a generalizable framework for exploring gene regulatory programs and their dynamics across developmental processes; this includes a comprehensive approach to studying the effects of disease-associated variation on transcriptional networks. The techniques applied to our networks have been published alongside our findings as a computational tool, E-P-INAnalyzer. Our procedure can be utilized across different cellular contexts and disorders.

Nutritional Genomics and Cancer Prevention.

The scientific innovations have emphasized the importance of diet for one's health and wellbeing. The genetic revolution has enhanced our understanding about the effect of nutrients on genomic and transcriptomic profiles and gene-nutrition interactions (nutritional genomics). Furthermore, the contribution of micronutrient insufficiencies and macronutrient excess is evident in the development and progression of many diseases, especially cancer. It is speculated that nutrients have capacity to implicitly affect the physiological and pathophysiological processes via gene expression various regulatory processes. Moreover, the nutrients are known to affect the cellular networks involved in cancer progression and cancer inhibitory mechanisms targeting apoptosis or impaired angiogenesis. The interplay of regulatory processes in physiological systems and nutrients provides basis for the nutrigenomics. The functional genomics data further argue that cellular and molecular processes involved in the cancer progression are possibly programed genes during early development which may persist into adulthood and become detrimental. The incorporation of the functional interactions between nutrients and the genome has revolutionized the field of personalized medicine and provided the foundation for targeted cancer therapy through nutrients. There is growing evidence on the beneficial impacts of eating habits on lowering the risk of cancer, even if it can be difficult to pinpoint the precise role of nutrients. The nutrigenomic information may provide bases to develop disease prevention and treatment via nutrition, at the molecular level.

The Metabolism of Leuconostoc Genus Decoded by Comparative Genomics.

Leuconostoc encompasses a number of species that frequently appear in foods where they play different roles, ranging from ripening to spoiling. The number of available Leuconostoc genomes has recently increased and enabled the precise taxonomic and phylogenetic delineation of species. Nonetheless, a thorough investigation of the functions and the metabolic potential of Leuconostoc species has never been accomplished. In this study, all the currently available 553 Leuconostoc genomes were downloaded from NCBI GenBank and annotated utilizing specific tools in order to reconstruct the metabolic potential of the genus in terms of carbohydrate hydrolysis and fermentative pathways, transporters, and anabolic potential. The analysis revealed that species cluster based on their metabolic potential, showing unique adaptation and ecological roles. Pentose phosphate and phosphoketolase pathways were highlighted as the main ones of central metabolism. The various identified PTS and ABC transporters showed adaptability to different sugars. The metabolic diversity described in this study not only supports the role of Leuconostoc spp. in natural ecosystems but also highlights their potential in industrial applications, particularly in the fermentation industry where their ability to metabolize a wide range of substrates can be harnessed for the production of various fermented foods and bioproducts.

Functional Genomics in Psoriasis.

Psoriasis is an autoimmune cutaneous condition that significantly impacts quality of life and represents a burden on society due to its prevalence. Genome-wide association studies (GWASs) have pinpointed several psoriasis-related risk loci, underlining the disease's complexity. Functional genomics is paramount to unveiling the role of such loci in psoriasis and disentangling its complex nature. In this review, we aim to elucidate the main findings in this field and integrate our discussion with gold-standard techniques in molecular biology-i.e., Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-and high-throughput technologies. These tools are vital to understanding how disease risk loci affect gene expression in psoriasis, which is crucial in identifying new targets for personalized treatments in advanced precision medicine.

A wound-induced differentiation trajectory for neurons.

Animals capable of whole-body regeneration can replace any missing cell type and regenerate fully functional new organs, including new brains, de novo. The regeneration of a new brain requires the formation of diverse neural cell types and their assembly into an organized structure with correctly wired circuits. Recent work in various regenerative animals has revealed transcriptional programs required for the differentiation of distinct neural subpopulations, however, how these transcriptional programs are initiated in response to injury remains unknown. Here, we focused on the highly regenerative acoel worm, Hofstenia miamia, to study wound-induced transcriptional regulatory events that lead to the production of neurons and subsequently a functional brain. Footprinting analysis using chromatin accessibility data on a chromosome-scale genome assembly revealed that binding sites for the Nuclear Factor Y (NFY) transcription factor complex were significantly bound during regeneration, showing a dynamic increase in binding within one hour upon amputation specifically in tail fragments, which will regenerate a new brain. Strikingly, NFY targets were highly enriched for genes with neuronal function. Single-cell transcriptome analysis combined with functional studies identified soxC+ stem cells as a putative progenitor population for multiple neural subtypes. Further, we found that wound-induced soxC expression is likely under direct transcriptional control by NFY, uncovering a mechanism for the initiation of a neural differentiation pathway by early wound-induced binding of a transcriptional regulator.

Insights from different reproductive gene knockdowns via RNA interference in the lady beetle Eriopis connexa: Establishing a new model for molecular studies on natural enemies.

Insect pest control can be achieved by the application of RNA interference (RNAi), a key molecular tool in functional genomics. Whereas most RNAi research has focused on insect pests, few studies have been performed on natural enemies. Validating the efficacy of RNAi in natural enemies is crucial for assessing its safety and enabling molecular research on these organisms. Here, we assessed the efficacy of RNAi in the ladybird beetle Eriopis connexa Germar (Coleoptera: Coccinellidae), focusing on genes related to reproduction, such as vitellogenin (Vg) and its receptor (VgR). In the transcriptome of E. connexa, we found one VgR (EcVgR) and two Vg genes (EcVg1 and EcVg2). These genes have been validated by in silico analyses of functional domains and evolutionary relationships. Five-day-old females were injected with 500 ng/µL of a specific double-stranded RNA (dsRNA) (dsEcVg1, dsEcVg2, or dsEcVgR) for RNAi tests, while nonspecific dsRNA (dsGFP or dsAgCE8.1) was used as a control. Interestingly, dsEcVg2 was able to knockdown both Vg genes, while dsEcVg1 could silence only EcVg1. Additionally, the viability of the eggs was significantly reduced when both Vg genes were knocked down at the same time (after treatment with dsEcVg2 or "dsEcVg1+dsEcVg2"). Ultimately, malformed, nonviable eggs were produced when EcVgR was silenced. Interestingly, no dsRNA treatment had an impact on the quantity of eggs laid. Therefore, the feasibility of RNAi in E. connexa has been confirmed, suggesting that this coccinellid is an excellent Neotropical model for molecular research on natural enemies and for studying RNAi nontarget effects.

Unlocking biological mechanisms with integrative functional genomics approaches.

Reverse genetics offers precise functional insights into genes through the targeted manipulation of gene expression followed by phenotypic assessment. While these approaches have proven effective in model organisms such as Saccharomyces cerevisiae, large-scale genetic manipulations in human cells were historically unfeasible due to methodological limitations. However, recent advancements in functional genomics, particularly clustered regularly interspaced short palindromic repeats (CRISPR)-based screening technologies and next-generation sequencing platforms, have enabled pooled screening technologies that allow massively parallel, unbiased assessments of biological phenomena in human cells. This review provides a comprehensive overview of cutting-edge functional genomic screening technologies applicable to human cells, ranging from short hairpin RNA screens to modern CRISPR screens. Additionally, we explore the integration of CRISPR platforms with single-cell approaches to monitor gene expression, chromatin accessibility, epigenetic regulation, and chromatin architecture following genetic perturbations at the omics level. By offering an in-depth understanding of these genomic screening methods, this review aims to provide insights into more targeted and effective strategies for genomic research and personalized medicine.

Multi-omic analysis reveals genes and proteins integral to bioactivity of Echinochrome A isolated from the waste stream of the sea urchin industry in Aotearoa New Zealand.

Evechinus chloroticus (commonly known as kina) is a sea urchin species endemic to New Zealand. Its roe is a culinary delicacy to the indigenous Maori and a globally exported food product. Echinochrome A (Ech A) is a bioactive compound isolated from the waste product of kina shells and spines; however, the molecular mechanisms of Ech A bioactivity are not well understood, partly due to Ech A never being studied using unbiased genome-wide analysis. To explore the high-value pharmaceutical potential of kina food waste, we obtained unbiased functional genomic and proteomic profiles of yeast cells treated with Echinochrome A. Abundance was measured for 4100 proteins every 30 min for four hours using fluorescent microscopy, resulting in the identification of 92 proteins with significant alterations in protein abundance caused by Ech A treatment that were over-represented with specific changes in DNA replication, repair and RNA binding after 30 min, followed by specific changes in the metabolism of metal ions (specifically iron and copper) from 60-240 min. Further analysis indicated that Ech A chelated iron, and that iron supplementation negated the growth inhibition caused by Ech A. Via a growth-based genome-wide analysis of 4800 gene deletion strains, 20 gene deletion strains were sensitive to Ech A in an iron-dependent manner. These genes were over-represented in the cellular response to oxidative stress, suggesting that Ech A suppressed growth inhibition caused by oxidative stress. Unexpectedly, genes integral to cardiolipin and inositol phosphate biosynthesis were required for Ech A bioactivity. Overall, these results identify genes, proteins, and cellular processes mediating the bioactivity of Ech A. Moreover, we demonstrate unbiased genomic and proteomic methodology that will be useful for characterizing bioactive compounds in food and food waste.

Functional genomics reveals an off-target dependency of drug synergy in gastric cancer therapy.

BACKGROUND: Integrating molecular-targeted agents into combination chemotherapy is transformative for enhancing treatment outcomes in cancer. However, realizing the full potential of this approach requires a clear comprehension of the genetic dependencies underlying drug synergy. While the interactions between conventional chemotherapeutics are well-explored, the interplay of molecular-targeted agents with conventional chemotherapeutics remains a frontier in cancer treatment. Hence, we leveraged a powerful functional genomics approach to decode genomic dependencies that drive synergy in molecular-targeted agent/chemotherapeutic combinations in gastric adenocarcinoma, addressing a critical need in gastric cancer therapy. METHODS: We screened pharmacological interactions between fifteen molecular-targeted agent/conventional chemotherapeutic pairs in gastric adenocarcinoma cells, and examined the genome-scale genetic dependencies of synergy integrating genome-wide CRISPR screening with the shRNA-based signature assay. We validated the synergy in cell death using fluorescence-based and lysis-dependent inference of cell death kinetics assay, and validated the genetic dependencies by single-gene knockout experiments. RESULTS: Our combination screen identified SN-38/erlotinib as the drug pair with the strongest synergism. Functional genomics assays unveiled a genetic dependency signature of SN-38/erlotinib identical to SN-38. Remarkably, the enhanced cell death with improved kinetics induced by SN-38/erlotinib was attributed to erlotinib's off-target effect, inhibiting ABCG2, rather than its on-target effect on EGFR. CONCLUSION: In the era of precision medicine, where emphasis on primary drug targets prevails, our research challenges this paradigm by showcasing a robust synergy underpinned by an off-target dependency. Further dissection of the intricate genetic dependencies that underlie synergy can pave the way to developing more effective combination strategies in gastric cancer therapy.

PARP4 interacts with hnRNPM to regulate splicing during lung cancer progression.

BACKGROUND: The identification of cancer driver genes from sequencing data has been crucial in deepening our understanding of tumor biology and expanding targeted therapy options. However, apart from the most commonly altered genes, the mechanisms underlying the contribution of other mutations to cancer acquisition remain understudied. Leveraging on our whole-exome sequencing of the largest Asian lung adenocarcinoma (LUAD) cohort (n = 302), we now functionally assess the mechanistic role of a novel driver, PARP4. METHODS: In vitro and in vivo tumorigenicity assays were used to study the functional effects of PARP4 loss and mutation in multiple lung cancer cell lines. Interactomics analysis by quantitative mass spectrometry was conducted to identify PARP4's interaction partners. Transcriptomic data from cell lines and patient tumors were used to investigate splicing alterations. RESULTS: PARP4 depletion or mutation (I1039T) promotes the tumorigenicity of KRAS- or EGFR-driven lung cancer cells. Disruption of the vault complex, with which PARP4 is commonly associated, did not alter tumorigenicity, indicating that PARP4's tumor suppressive activity is mediated independently. The splicing regulator hnRNPM is a potentially novel PARP4 interaction partner, the loss of which likewise promotes tumor formation. hnRNPM loss results in splicing perturbations, with a propensity for dysregulated intronic splicing that was similarly observed in PARP4 knockdown cells and in LUAD cohort patients with PARP4 copy number loss. CONCLUSIONS: PARP4 is a novel modulator of lung adenocarcinoma, where its tumor suppressive activity is mediated not through the vault complex-unlike conventionally thought, but in association with its novel interaction partner hnRNPM, thus suggesting a role for splicing dysregulation in LUAD tumorigenesis.

Knock down to level up: Reframing RNAi for invertebrate ecophysiology.

Comparative ecophysiologists strive to understand physiological problems in non-model organisms, but molecular tools such as RNA interference (RNAi) are under-used in our field. Here, we provide a framework for invertebrate ecophysiologists to use RNAi to answer questions focused on physiological processes, rather than as a tool to investigate gene function. We specifically focus on non-model invertebrates, in which the use of other genetic tools (e.g., genetic knockout lines) is less likely. We argue that because RNAi elicits a temporary manipulation of gene expression, and resources to carry out RNAi are technically and financially accessible, it is an effective tool for invertebrate ecophysiologists. We cover the terminology and basic mechanisms of RNA interference as an accessible introduction for "non-molecular" physiologists, include a suggested workflow for identifying RNAi gene targets and validating biologically relevant gene knockdowns, and present a hypothesis-testing framework for using RNAi to answer common questions in the realm of invertebrate ecophysiology. This review encourages invertebrate ecophysiologists to use these tools and workflows to explore physiological processes and bridge genotypes to phenotypes in their animal(s) of interest.

Live Cell Protein Imaging of Tandem Complemented-GFP11-Tagged Coiled-Coil Domain-Containing Protein-124 Identifies this Factor in G3BP1-Induced Stress-Granules.

Coiled-coil domain-containing 124 protein is a multifunctional RNA-binding factor, and it was previously reported to interact with various biomolecular complexes localized at diverse subcellular locations, such as the ribosome, centrosome, midbody, and nucleoli. We aimed to better characterize the subcellular CCDC124 translocation by labelling this protein with a fluorescent tag, followed by laser scanning confocal microscopy methods. As traditional GFP-tagging of small proteins such as CCDC124 often faces limitations like potential structural perturbations of labeled proteins, and interference of the fluorescent-tag with their endogenous cellular functions, we aimed to label CCDC124 with the smallest possible split-GFP associated protein-tagging system (GFP11/GFP1-10) for better characterization of its subcellular localizations and its translocation dynamics. By recombinant DNA techniques we generated CCDC124-constructs labelled with either single of four tandem copies of GFP11 (GFP11 × 1::CCDC124, GFP11 × 4::CCDC124, or CCDC124::GFP11 × 4). We then cotransfected U2OS cells with these split-GFP constructs (GFP11 × 1(or X4)::CCDC124/GFP1-10) and analyzed subcellular localization of CCDC124 protein by laser scanning confocal microscopy. Tagging CCDC124 with four tandem copies of a 16-amino acid short GFP-derived peptide-tag (GFP11 × 4::CCDC124) allowed better characterization of the subcellular localization of CCDC124 protein in our model human bone osteosarcoma (U2OS) cells. Thus, by this novel methodology we successfully identified GFP11 × 4::CCDC124 molecules in G3BP1-overexpression induced stress-granules by live cell protein imaging for the first time. Our findings propose CCDC124 as a novel component of the stress granule which is a membraneless organelle involved in translational shut-down in response to cellular stress.

Systematic identification of transcriptional activation domains from non-transcription factor proteins in plants and yeast.

Transcription factors can promote gene expression through activation domains. Whole-genome screens have systematically mapped activation domains in transcription factors but not in non-transcription factor proteins (e.g., chromatin regulators and coactivators). To fill this knowledge gap, we employed the activation domain predictor PADDLE to analyze the proteomes of Arabidopsis thaliana and Saccharomyces cerevisiae. We screened 18,000 predicted activation domains from >800 non-transcription factor genes in both species, confirming that 89% of candidate proteins contain active fragments. Our work enables the annotation of hundreds of nuclear proteins as putative coactivators, many of which have never been ascribed any function in plants. Analysis of peptide sequence compositions reveals how the distribution of key amino acids dictates activity. Finally, we validated short, "universal" activation domains with comparable performance to state-of-the-art activation domains used for genome engineering. Our approach enables the genome-wide discovery and annotation of activation domains that can function across diverse eukaryotes.

Accelerated drug-resistant variant discovery with an enhanced, scalable mutagenic base editor platform.

Personalized cancer therapeutics bring directed treatment options to patients based on their tumor's genetic signature. Unfortunately, tumor genomes are remarkably adaptable, and acquired resistance through gene mutation frequently occurs. Identifying mutations that promote resistance within drug-treated patient populations can be cost, resource, and time intensive. Accordingly, base editing, enabled by Cas9-deaminase domain fusions, has emerged as a promising approach for rapid, large-scale gene variant screening in situ. Here, we adapt and optimize a conditional activation-induced cytidine deaminase (AID)-dead Cas9 (dCas9) system, which demonstrates greater heterogeneity of edits with an expanded footprint compared to the most commonly utilized cytosine base editor, BE4. In combination with a custom single guide RNA (sgRNA) library, we identify individual and compound variants in epidermal growth factor receptor (EGFR) and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) that confer resistance to established EGFR inhibitors. This system and analytical pipeline provide a simple, highly scalable platform for cis or trans drug-modifying variant discovery and for uncovering valuable insights into protein structure-function relationships.

Identification of pathways required for Salmonella to colonize alfalfa using TraDIS-Xpress.

Enteropathogenic bacteria, such as Salmonella, have been linked to numerous fresh produce outbreaks, posing a significant public health threat. The ability of Salmonella to persist on fresh produce for extended periods is partly attributed to its capacity to form biofilms, which pose a challenge to food decontamination and can increase pathogenic bacterial load in the food chain. Preventing Salmonella colonization of food products and food processing environments is crucial for reducing the incidence of foodborne outbreaks. Understanding the mechanisms of establishment on fresh produce will inform the development of decontamination approaches. We used Transposon-Directed Insertion site Sequencing (TraDIS-Xpress) to investigate the mechanisms used by Salmonella enterica serovar Typhimurium to colonize and establish on fresh produce over time. We established an alfalfa colonization model and compared the findings to those obtained from glass surfaces. Our research identified distinct mechanisms required for Salmonella establishment on alfalfa compared with glass surfaces over time. These include the type III secretion system (sirC), Fe-S cluster assembly (iscA), curcumin degradation (curA), and copper tolerance (cueR). Shared pathways across surfaces included NADH hydrogenase synthesis (nuoA and nuoB), fimbrial regulation (fimA and fimZ), stress response (rpoS), LPS O-antigen synthesis (rfbJ), iron acquisition (ybaN), and ethanolamine utilization (eutT and eutQ). Notably, flagellum biosynthesis differentially impacted the colonization of biotic and abiotic environments over time. Understanding the genetic underpinnings of Salmonella establishment on both biotic and abiotic surfaces over time offers valuable insights that can inform the development of targeted antibacterial therapeutics, ultimately enhancing food safety throughout the food processing chain. IMPORTANCE: Salmonella is the second most costly foodborne illness in the United Kingdom, accounting for £0.2 billion annually, with numerous outbreaks linked to fresh produce, such as leafy greens, cucumbers, tomatoes, and alfalfa sprouts. The ability of Salmonella to colonize and establish itself in fresh produce poses a significant challenge, hindering decontamination efforts and increasing the risk of illness. Understanding the key mechanisms of Salmonella to colonize plants over time is key to finding new ways to prevent and control contamination of fresh produce. This study identified genes and pathways important for Salmonella colonization of alfalfa and compared those with colonization of glass using a genome-wide screen. Genes with roles in flagellum biosynthesis, lipopolysaccharide production, and stringent response regulation varied in their significance between plants and glass. This work deepens our understanding of the requirements for plant colonization by Salmonella, revealing how gene essentiality changes over time and in different environments. This knowledge is key to developing effective strategies to reduce the risk of foodborne disease.