Use of a Golden Gate Plasmid Set Enabling Scarless MoClo-Compatible Transcription Unit Assembly.

Golden Gate cloning has become a powerful and widely used DNA assembly method. Its modular nature and the reusability of standardized parts allow rapid construction of transcription units and multi-gene constructs. Importantly, its modular structure makes it compatible with laboratory automation, allowing for systematic and highly complex DNA assembly. Golden Gate cloning relies on type IIS enzymes that cleave an adjacent undefined sequence motif at a defined distance from the directed enzyme recognition motif. This feature has been used to define hierarchical Golden Gate assembly standards with defined overhangs ("fusion sites") for defined part libraries. The simplest Golden Gate standard would consist of three-part libraries, namely promoter, coding and terminator sequences, respectively. Each library would have defined fusion sites, allowing a hierarchical Golden Gate assembly to generate transcription units. Typically, type IIS enzymes are used, which generate four nucleotide overhangs. This results in small scar sequences in hierarchical DNA assemblies, which can affect the functionality of transcription units. However, there are enzymes that generate three nucleotide overhangs, such as SapI. Here we provide a step-by-step protocol on how to use SapI to assemble transcription units using the start and stop codon for scarless transcription unit assembly. The protocol also provides guidance on how to perform multi-gene Golden Gate assemblies with the resulting transcription units using the Modular Cloning standard. The transcription units expressing fluorophores are used as an example.

Modular Golden Gate Assembly of Linear DNA Templates for Cell-Free Prototyping.

Cell-free transcription and translation (TXTL) systems have emerged as a powerful tool for testing genetic regulatory elements and circuits. Cell-free prototyping can dramatically accelerate the design-build-test-learn cycle of new functions in synthetic biology, in particular when quick-to-assemble linear DNA templates are used. Here, we describe a Golden-Gate-assisted, cloning-free workflow to rapidly produce linear DNA templates for TXTL reactions by assembling transcription units from basic genetic parts of a modular cloning toolbox. Functional DNA templates composed of multiple parts such as promoter, ribosomal binding site (RBS), coding sequence, and terminator are produced in vitro in a one-pot Golden Gate assembly reaction followed by polymerase chain reaction (PCR) amplification. We demonstrate assembly, cell-free testing of promoter and RBS combinations, as well as characterization of a repressor-promoter pair. By eliminating time-consuming transformation and cloning steps in cells and by taking advantage of modular cloning toolboxes, our cell-free prototyping workflow can produce data for large numbers of new assembled constructs within a single day.

Golden Gate Assembly of Transcriptional Unit Libraries into a Rearrangeable Gene Cluster.

Both regulatory sequences and genome organization contribute to the production of diverse transcript isoforms, which can influence how genes, or sets of genes, are expressed. An efficient, modular approach is needed to generate the combinatorial complexity required to empirically test many combinations of different regulatory sequences and different gene orders. Golden Gate assembly provides such a tool for seamless one-pot cleavage and ligation, by using type IIS restriction enzymes, which cleave outside of their recognition site. In addition to reducing the number of steps, this one-pot reaction can improve correct assemblies by the continued cleavage of self-ligation products that retain the recognition site. Switching the specific restriction enzyme used between steps allows for modular assembly of several units. A protocol to perform modular assemblies with two type IIS restriction enzymes, namely BsaI-v2-HF and BsmBI-v2, is described here. This protocol includes a description for generating destination vectors that add loxPsym sites between transcriptional units, allowing for diversification of gene order, orientation, and spacing.

Activating transcription factor 4 plays a major role in shaping the transcriptional response to isoginkgetin in HCT116 cells.

Activating transcription factor 4 (ATF4) plays a central role in the integrated stress response (ISR) and one overlapping branch of the unfolded protein response (UPR). We recently reported that the splicing inhibitor isoginkgetin (IGG) induced ATF4 protein along with several known ATF4-regulated transcripts in a response that resembled the ISR and UPR. However, the contribution of ATF4-dependent and -independent transcriptional responses to IGG exposure was not known. Here we used RNA-sequencing in HCT116 colon cancer cells and an isogenic subline lacking ATF4 to investigate the contribution of ATF4 to IGG-induced changes in gene expression. Approximately 85% of the IGG-responsive DEGs in HCT116 cells were also differentially expressed in response to the ER stressor thapsigargin (Tg) and these were enriched for genes associated with the UPR and ISR. Most of these were positively regulated by IGG with impaired responses in the ATF4-deficient cells. Nonetheless, there were DEGs that responded similarly in both cell lines. The ATF4-independent IGG-induced DEGs included several metal responsive transcripts encoding metallothionines and a zinc transporter. Taken together, the predominant IGG response was ATF4-dependent in these cells and resembled the UPR and ISR while a second less prominent response involved the ATF4-independent regulation of metal responsive mRNAs.

Transcriptional reprogramming in the entomopathogenic fungus Metarhizium brunneum and its aphid host Myzus persicae during the switch between saprophytic and parasitic lifestyles.

BACKGROUND: The fungus Metarhizium brunneum has evolved a remarkable ability to switch between different lifestyles. It develops as a saprophyte, an endophyte establishing mutualistic relationships with plants, or a parasite, enabling its use for the control of insect pests such as the aphid Myzus persicae. We tested our hypothesis that switches between lifestyles must be accompanied by fundamental transcriptional reprogramming, reflecting adaptations to different environmental settings. RESULTS: We combined high throughput RNA sequencing of M. brunneum in vitro and at different stages of pathogenesis to validate the modulation of genes in the fungus and its host during the course of infection. In agreement with our hypothesis, we observed transcriptional reprogramming in M. brunneum following conidial attachment, germination on the cuticle, and early-stage growth within the host. This involved the upregulation of genes encoding degrading enzymes and gene clusters involved in synthesis of secondary metabolites that act as virulence factors. The transcriptional response of the aphid host included the upregulation of genes potentially involved in antifungal activity, but antifungal peptides were not induced. We also observed the induction of a host flightin gene, which may be involved in wing formation and flight muscle development. CONCLUSIONS: The switch from saprophytic to parasitic development in M. brunneum is accompanied by fundamental transcriptional reprogramming during the course of the infection. The aphid host responds to fungal infection with its own transcriptional reprogramming, reflecting its inability to express antifungal peptides but featuring the induction of genes involved in winged morphs that may enable offspring to avoid the contaminated environment.

Heavy water inhibits DNA double-strand break repairs and disturbs cellular transcription, presumably via quantum-level mechanisms of kinetic isotope effects on hydrolytic enzyme reactions.

Heavy water, containing the heavy hydrogen isotope, is toxic to cells, although the underlying mechanism remains incompletely understood. In addition, certain enzymatic proton transfer reactions exhibit kinetic isotope effects attributed to hydrogen isotopes and their temperature dependencies, indicative of quantum tunneling phenomena. However, the correlation between the biological effects of heavy water and the kinetic isotope effects mediated by hydrogen isotopes remains elusive. In this study, we elucidated the kinetic isotope effects arising from hydrogen isotopes of water and their temperature dependencies in vitro, focusing on deacetylation, DNA cleavage, and protein cleavage, which are crucial enzymatic reactions mediated by hydrolysis. Intriguingly, the intracellular isotope effects of heavy water, related to the in vitro kinetic isotope effects, significantly impeded multiple DNA double-strand break repair mechanisms crucial for cell survival. Additionally, heavy water exposure enhanced histone acetylation and associated transcriptional activation in cells, consistent with the in vitro kinetic isotope effects observed in histone deacetylation reactions. Moreover, as observed for the in vitro kinetic isotope effects, the cytotoxic effect on cell proliferation induced by heavy water exhibited temperature-dependency. These findings reveal the substantial impact of heavy water-induced isotope effects on cellular functions governed by hydrolytic enzymatic reactions, potentially mediated by quantum-level mechanisms underlying kinetic isotope effects.

PRC2-RNA interactions: Viewpoint from YongWoo Lee and Jeannie T. Lee.

Here, we expound on the view that Xist RNA directly controls Polycomb repressive complex 2 (PRC2) recruitment, off-loading to chromatin, catalytic activity, and eviction from chromatin. RNA-PRC2 interactions also control RNA polymerase II transcription pausing. Dynamic RNA folding determines PRC2 activity. Disparate studies and interpretations abound but can be reconciled.

An RNA-centric view of transcription and genome organization.

Foundational models of transcriptional regulation involve the assembly of protein complexes at DNA elements associated with specific genes. These assemblies, which can include transcription factors, cofactors, RNA polymerase, and various chromatin regulators, form dynamic spatial compartments that contribute to both gene regulation and local genome architecture. This DNA-protein-centric view has been modified with recent evidence that RNA molecules have important roles to play in gene regulation and genome structure. Here, we discuss evidence that gene regulation by RNA occurs at multiple levels that include assembly of transcriptional complexes and genome compartments, feedback regulation of active genes, silencing of genes, and control of protein kinases. We thus provide an RNA-centric view of transcriptional regulation that must reside alongside the more traditional DNA-protein-centric perspectives on gene regulation and genome architecture.

Nuclear sorting of short RNA polymerase II transcripts.

Mammalian genomes produce an abundance of short RNA. This is, to a large extent, due to the genome-wide and spurious activity of RNA polymerase II (RNAPII). However, it is also because the vast majority of initiating RNAPII, regardless of the transcribed DNA unit, terminates within a ∼3-kb early "pausing zone." Given that the resultant RNAs constitute both functional and non-functional species, their proper sorting is critical. One way to think about such quality control (QC) is that transcripts, from their first emergence, are relentlessly targeted by decay factors, which may only be avoided by engaging protective processing pathways. In a molecular materialization of this concept, recent progress has found that both "destructive" and "productive" RNA effectors assemble at the 5' end of capped RNA, orchestrated by the essential arsenite resistance protein 2 (ARS2) protein. Based on this principle, we here discuss early QC mechanisms and how these might sort short RNAs to their final fates.

The zinc-finger transcription factor Sfp1 imprints specific classes of mRNAs and links their synthesis to cytoplasmic decay.

To function effectively as an integrated system, the transcriptional and post-transcriptional machineries must communicate through mechanisms that are still poorly understood. Here, we focus on the zinc-finger Sfp1, known to regulate transcription of proliferation-related genes. We show that Sfp1 can regulate transcription either by binding to promoters, like most known transcription activators, or by binding to the transcribed regions (gene bodies), probably via RNA polymerase II (Pol II). We further studied the first mode of Sfp1 activity and found that, following promoter binding, Sfp1 binds to gene bodies and affects Pol II configuration, manifested by dissociation or conformational change of its Rpb4 subunit and increased backtracking. Surprisingly, Sfp1 binds to a subset of mRNAs co-transcriptionally and stabilizes them. The interaction between Sfp1 and its client mRNAs is controlled by their respective promoters and coincides with Sfp1's dissociation from chromatin. Intriguingly, Sfp1 dissociation from the chromatin correlates with the extent of the backtracked Pol II. We propose that, following promoter recruitment, Sfp1 accompanies Pol II and regulates backtracking. The backtracked Pol II is more compatible with Sfp1's relocation to the nascent transcripts, whereupon Sfp1 accompanies these mRNAs to the cytoplasm and regulates their stability. Thus, Sfp1's co-transcriptional binding imprints the mRNA fate, serving as a paradigm for the cross-talk between the synthesis and decay of specific mRNAs, and a paradigm for the dual-role of some zinc-finger proteins. The interplay between Sfp1's two modes of transcription regulation remains to be examined.

The ability to fine-tune the production of proteins in a cell is essential for organisms to exist. An imbalance in protein levels can be the cause of various diseases. Messenger RNA molecules (mRNA) link the genetic information encoded in DNA and the produced proteins. Exactly how much protein is made mostly depends on the amount of mRNA in the cell's cytoplasm. This is controlled by two processes: the synthesis of mRNA (also known as transcription) and mRNA being actively degraded. Although much is known about mechanisms regulating transcription and degradation, how cells detect if they need to degrade mRNA based on the levels of its synthesis and vice versa is poorly understood. In 2013, researchers found that proteins known as 'RNA decay factors' responsible for mRNA degradation are actively moved from the cell's cytoplasm into its nucleus to instruct the transcription machinery to produce more mRNA. Kelbert, Jordán-Pla, de-Miguel-Jiménez et al. ­ including some of the researchers involved in the 2013 work ­ investigated how mRNA synthesis and degradation are coordinated to ensure a proper mRNA level. The researchers used advanced genome engineering methods to carefully manipulate and measure mRNA production and degradation in yeast cells. The experiments revealed that the protein Sfp1 ­ a well-characterized transcription factor for stimulating the synthesis of a specific class of mRNAs inside the nucleus ­ can also prevent the degradation of these mRNAs outside the nucleus. During transcription, Sfp1 bound directly to mRNA. The investigators could manipulate the co-transcriptional binding of Sfp1 to a certain mRNA, thereby changing the mRNA stability in the cytoplasm. This suggests that the ability of Sfp1 to regulate both the production and decay of mRNA is dependent on one another and that transcription can influence the fate of its transcripts. This combined activity can rapidly change mRNA levels in response to changes in the cell's environment. RNA plays a key role in ensuring correct levels of proteins. It can also function as an RNA molecule, independently of its coding capacity. Many cancers and developmental disorders are known to be caused by faulty interactions between transcription factors and nucleic acids. The finding that some transcription factors can directly regulate both mRNA synthesis and its destruction introduces new angles for studying and understanding these diseases.

Developmental and housekeeping transcriptional programs display distinct modes of enhancer-enhancer cooperativity in Drosophila.

Genomic enhancers are key transcriptional regulators which, upon the binding of sequence-specific transcription factors, activate their cognate target promoters. Although enhancers have been extensively studied in isolation, a substantial number of genes have more than one simultaneously active enhancer, and it remains unclear how these cooperate to regulate transcription. Using Drosophila melanogaster S2 cells as a model, we assay the activities of more than a thousand individual enhancers and about a million enhancer pairs toward housekeeping and developmental core promoters with STARR-seq. We report that housekeeping and developmental enhancers show distinct modes of enhancer-enhancer cooperativity: while housekeeping enhancers are additive such that their combined activity mirrors the sum of their individual activities, developmental enhancers are super-additive and combine multiplicatively. Super-additivity between developmental enhancers is promiscuous and neither depends on the enhancers' endogenous genomic contexts nor on specific transcription factor motif signatures. However, it can be further boosted by Twist and Trl motifs and saturates for the highest levels of enhancer activity. These results have important implications for our understanding of gene regulation in complex multi-enhancer developmental loci and genomically clustered housekeeping genes, providing a rationale to interpret the transcriptional impact of non-coding mutations at different loci.

[Regulation of Transcription by RNA Polymerase III Promotors in the Norm and Pathology].

RNA polymerase III synthesizes a wide range of noncoding RNAs shorter than 400 nucleotides in length. These RNAs are involved in protein synthesis (tRNA, 5S rRNA, and 7SL RNA), maturation, and splicing of different types of RNA (RPR, MRP RNA, and U6 snRNA), regulation of transcription (7SK RNA), replication (Y RNA), and intracellular transport (vault RNA). BC200 and BC1 RNA genes are transcribed by RNA polymerase III in neurons only where these RNAs regulate protein synthesis. Mutations in the regulatory elements of the genes transcribed by RNA polymerase III as well as in transcription factors of this RNA polymerase are associated with the development of a number of diseases, primarily oncological and neurological. In this regard, the mechanisms of regulation of the expression of the genes containing various RNA polymerase III promoters were actively studied. This review describes the structural and functional classification of polymerase III promoters, as well as the factors involved in the regulation of promoters of different types. A number of examples demonstrate the role of the described factors in the pathogenesis of human diseases.

ZIC2 and ZIC3 promote SWI/SNF recruitment to safeguard progression towards human primed pluripotency.

The primed epiblast acts as a transitional stage between the relatively homogeneous naïve epiblast and the gastrulating embryo. Its formation entails coordinated changes in regulatory circuits driven by transcription factors and epigenetic modifications. Using a multi-omic approach in human embryonic stem cell models across the spectrum of peri-implantation development, we demonstrate that the transcription factors ZIC2 and ZIC3 have overlapping but essential roles in opening primed-specific enhancers. Together, they are essential to facilitate progression to and maintain primed pluripotency. ZIC2/3 accomplish this by recruiting SWI/SNF to chromatin and loss of ZIC2/3 or degradation of SWI/SNF both prevent enhancer activation. Loss of ZIC2/3 also results in transcriptome changes consistent with perturbed Polycomb activity and a shift towards the expression of genes linked to differentiation towards the mesendoderm. Additionally, we find an intriguing dependency on the transcriptional machinery for sustained recruitment of ZIC2/3 over a subset of primed-hESC specific enhancers. Taken together, ZIC2 and ZIC3 regulate highly dynamic lineage-specific enhancers and collectively act as key regulators of human primed pluripotency.

Super-resolved analysis of colocalization between replication and transcription along the cell cycle in a model of oncogene activation.

To understand how oncogenes affect genome organization, it is essential to visualize fundamental processes such as DNA replication and transcription at high resolution in intact cells. At the same time, it is important to determine the progression of the cell along the cell cycle, as cell cycle regulation is crucial for the control of cell proliferation and oncogenesis. Here, we present a super-resolution imaging-based method to analyze single cell nuclei sorted according to specific phases of the cell cycle. The sorting is based on the evaluation of the number and the intensity of pixels in the replication foci image and the colocalization analysis is based on image cross-correlation spectroscopy (ICCS). We evaluate the colocalization between replication and transcription, at different cell cycle phases, in a model of PML-RARα oncogene activation. We find that colocalization between replication and transcription is higher in cells in early S phase compared to cells in middle and late S phase. When we turn on the PML-RARα oncogene, this colocalization pattern is preserved but we detect an increase of colocalization between replication and transcription in the early S phase which points to an effect of the PML-RARα oncogene on the coordination between replication and transcription.

Transcriptional repression by HDAC3 mediates T cell exclusion from Kras mutant lung tumors.

Histone Deacetylase 3 (HDAC3) function in vivo is nuanced and directed in a tissue-specific fashion. The importance of HDAC3 in Kras mutant lung tumors has recently been identified, but HDAC3 function in this context remains to be fully elucidated. Here, we identified HDAC3 as a lung tumor cell-intrinsic transcriptional regulator of the tumor immune microenvironment. In Kras mutant lung cancer cells, we found that HDAC3 is a direct transcriptional repressor of a cassette of secreted chemokines, including Cxcl10. Genetic and pharmacological inhibition of HDAC3 robustly up-regulated this gene set in human and mouse Kras, LKB1 (KL) and Kras, p53 (KP) mutant lung cancer cells through an NF-κB/p65-dependent mechanism. Using genetically engineered mouse models, we found that HDAC3 inactivation in vivo induced expression of this gene set selectively in lung tumors and resulted in enhanced T cell recruitment at least in part via Cxcl10. Furthermore, we found that inhibition of HDAC3 in the presence of Kras pathway inhibitors dissociated Cxcl10 expression from that of immunosuppressive chemokines and that combination treatment of entinostat with trametinib enhanced T cell recruitment into lung tumors in vivo. Finally, we showed that T cells contribute to in vivo tumor growth control in the presence of entinostat and trametinib combination treatment. Together, our findings reveal that HDAC3 is a druggable endogenous repressor of T cell recruitment into Kras mutant lung tumors.

Simultaneous probing of transcription, G-quadruplex, and R-loop.

DNA and RNA can form various non-canonical secondary structures, including G-quadruplex (G4) and R-loops. These structures are considered transcriptional regulatory elements due to their enrichment at regulatory regions. During transcription, G-rich sequences in the non-template strand promote R-loop formation in the DNA template strand. These R-loops induce G4 structures in the non-template DNA strand, further stabilizing them. Additionally, the high rG: dC base-pairing within the R-loop contributes to the stability of DNA/RNA hybridization. Our previous study investigated the interplay between G4s and R-loops and its impact on transcription. We employed two techniques to demonstrate transcription-mediated G4 and R-loop formation. The single-molecule method allows us to detect intricate details of transcription initiation, elongation, and co-transcriptional R-loop and G4 formation. It provides a high-resolution view of the dynamic processes involved in transcriptional regulation. As an orthogonal approach, a gel-based assay enables the detection of the transcription-mediated R-loops and the RNA product. We can measure the progressive formation of R-loop and total RNA produced from transcription by analyzing gel electrophoresis patterns. In summary, these techniques provide valuable insights into the non-canonical nucleic acid structures and their impact on gene expression.

Studying the intersection of nucleoside modifications and SARS-CoV-2 RNA-dependent RNA transcription using an in vitro reconstituted system.

There is growing recognition that viral RNA genomes possess enzymatically incorporated modified nucleosides. These small chemical changes are analogous to epigenomic modifications in DNA and have the potential to be similarly important modulators of viral transcription and evolution. However, the molecular level consequences of individual sites of modification remain to be broadly explored. Here we describe an in vitro assay to examine the impact of nucleoside modifications on the rate and fidelity of SARS-CoV-2 RNA transcription. Establishing the role of modified nucleotides in SARS-CoV-2 is of interest both for advancing fundamental knowledge of RNA modifications in viruses, and because modulating the modification-landscape of SARS-CoV-2 may represent a therapeutic strategy to interfere with viral RNA replication. Our approach can be used to assess the influence both of modifications present in a template RNA, as well nucleotide analog inhibitors. These methods provide a reproducible guide for generating active SARS-CoV-2 replication/transcription complexes capable of establishing how RNA modifications influence the pre-steady state rate constants of nucleotide addition by RNA-dependent RNA polymerases.

Diversity and transcription of genes involved in respiratory As(V) reduction and As(III) methylation in Japanese paddy soils.

BACKGROUND: Arsenic (As) metabolism by soil microorganisms has an impact on As geochemical cycling in paddy soils, which in turn affects As uptake in rice. However, little is known about the key microorganisms involved in this process in Japanese paddy soil. RESULTS: Total RNA was extracted from Japanese paddy soils with different levels of dissolved As under flooded conditions, and the transcription of As metabolic genes (arrA, ttrA and arsM) was analyzed via a metatranscriptomic approach. The results showed that ttrA was the predominant respiratory arsenate reductase gene transcribed in these soils rather than arrA, suggesting that ttrA contributes to the reductive dissolution of As. The predominant taxa expressing ttrA differed among soils but were mostly associated with genera known for their iron- and/or sulfate-reduction activity. In addition, a wide variety of microorganisms expressed and upregulated arsM approximately 5.0- to 13.2-fold at 9 d compared with 3 d of incubation under flooded conditions in flasks. CONCLUSIONS: Our results support the involvement of microbial activity in the geochemical cycling of As in Japanese paddy soils and suggest that ttrA may be one of the key genes involved in the formation of arsenite, an inorganic species taken up by rice.

Free-swimming bacteria transcriptionally respond to shear flow.

Surface-attached cells can sense and respond to shear flow, but planktonic (free-swimming) cells are typically assumed to be oblivious to any flow that carries them. Here, we find that planktonic bacteria can transcriptionally respond to flow, inducing expression changes that are beneficial in flow. Specifically, we use microfluidic experiments and quantitative modeling to show that in the presence of flow, planktonic Pseudomonas aeruginosa induce shear rate-dependent genes that promote growth in low-oxygen environments. Untangling this mechanism revealed that in flow, motile P. aeruginosa spatially redistribute, leading to cell density changes that activate quorum sensing, which in turn enhances the oxygen uptake rate. In diffusion-limited environments, including those commonly encountered by bacteria, flow-induced cell density gradients also independently generate oxygen gradients that alter gene expression. Mutants deficient in this flow-responsive mechanism exhibit decreased fitness in flow, suggesting that this dynamic coupling of biological and mechanical processes can be physiologically significant.

Low molecular weight acids differentially impact Fusarium verticillioides transcription.

Fusarium verticillioides is both an endophyte and pathogen of maize. During growth on maize, the fungus often synthesizes the mycotoxins fumonisins, which have been linked to a variety of diseases, including cancer in some animals. How F. verticillioides responds to other fungi, such as Fusarium proliferatum, Aspergillus flavus, Aspergillus niger, and Penicillium oxalicum, that coinfect maize, has potential to impact mycotoxin synthesis and disease. We hypothesize that low molecular weight acids produced by these fungi play a role in communication between the fungi in planta/nature. To address this hypothesis, we exposed 48-h maize kernel cultures of F. verticillioides to oxalic acid, citric acid, fusaric acid, or kojic acid and then compared transcriptomes after 30 min and 6 h. Transcription of some genes were affected by multiple chemicals and others were affected by only one chemical. The most significant positive response was observed after exposure to fusaric acid which resulted in >2-fold upregulation of 225 genes, including genes involved in fusaric acid synthesis. Exposure of cultures to the other three chemicals increased expression of only 3-15 genes. The predicted function and frequent co-localization of three sets of genes support a role in protecting the fungus from the chemical or a role in catabolism. These unique transcriptional responses support our hypothesis that these chemicals can act as signaling molecules. Studies with gene deletion mutants will further indicate if the initial transcriptional response to the chemicals benefit F. verticillioides.