Ribosome-inactivating proteins are a class of toxins that target eukaryotic ribosomes, inhibit protein synthesis, and ultimately induce cell death. Several of these toxins pose significant clinical and public health threats. Among these, ricin, derived from the castor bean plant (Ricinus communis), is a highly potent biotoxin with recognized bioterrorism potential. Other ribosome-inactivating proteins, including Shiga toxin produced by pathogenic Shigella and Escherichia coli, as well as mucoricin from Mucorales fungi, contribute to disease severity and can lead to life-threatening complications. Despite these risks, no approved therapeutics are currently available. The development of effective inhibitors depends on robust and well-defined strategies to identify and validate small molecules that disrupt toxin–ribosome interactions. Efforts to target the catalytic active site have met with limited success, largely due to its broad, shallow, and highly polar architecture, which is not conducive to high-affinity binding by drug-like molecules. In contrast, the ribosome-binding interface represents a more tractable target, as it is essential for toxin recruitment and offers more structurally defined and druggable features. Inhibitors targeting this interface can also exert allosteric effects by disrupting long-range conformational coupling between the ribosome-binding region and the active site, thereby attenuating catalytic activity without directly engaging the catalytic pocket. In this review, we compile and evaluate biophysical and biochemical assays for the discovery and characterization of small-molecule inhibitors that target toxin–ribosome interactions. We examine in vitro binding approaches, including surface plasmon resonance-based fragment screening and fluorescence anisotropy assays for ranking inhibitory activity. We further review biochemical and molecular assays that assess ribosome protection from toxin-mediated depurination, along with complementary cell-based assays that evaluate functional rescue in cellular systems. Collectively, this review consolidates current screening methodologies and highlights opportunities to refine assay strategies, thereby supporting the advancement of targeted therapeutics.

Non-specific lipid transfer proteins (nsLTPs) are vital and versatile components of plant cellular systems. They are characterized by a conserved eight-cysteine motif and are increasingly recognized for their dual roles in direct defense and stress modulation. nsLTPs serve critical structural and signaling functions in plant immunity. In contrast, other lipid transfer proteins, which lack the conserved cysteine motif, are primarily localized at membrane contact sites, specialized inter-organelle junctions that act as central hubs for lipid trafficking and signaling. This review explores the diverse roles of nsLTPs from structural, functional, and evolutionary perspectives, and examines current classification methodologies for the plant nsLTP superfamily. Functionally, nsLTPs contribute to the formation of protective barriers by transporting cutin monomers and other lipids, while also possessing lipid-specific antimicrobial properties that disrupt pathogen membranes. They support redox balance by scavenging reactive oxygen species, thereby minimizing oxidative stress. Additionally, nsLTPs are involved in defense signaling by transporting lipid-derived molecules essential to systemic acquired resistance. Their structural adaptability enables binding to a wide range of lipid species, underpinning their involvement in cuticle integrity, immune responses, and abiotic stress tolerance. These attributes position nsLTPs as promising targets for engineering durable, broad-spectrum disease resistance in crops. However, significant knowledge gaps remain regarding their structure-function relationships, lipid transport mechanisms, and roles in defense signaling and pathogen resistance. Addressing these challenges through advanced molecular and genetic tools could unlock the potential of nsLTPs to enhance crop resilience and contribute significantly to global food security.

Ricin is a category B agent for bioterrorism, and Shiga toxins are the primary virulence factors of Shiga toxin (Stx) producing Escherichia coli. Ricin and Stxs bind the ribosomal P-stalk proteins to depurinate the sarcin/ricin loop on the eukaryotic ribosome and inhibit translation. Both toxins are prime targets for therapeutic intervention because no effective therapy exists for ricin intoxication or Shiga toxin producing Escherichia coli infection. Binding of ricin toxin A subunit (RTA) to the ribosomal P-stalk stimulates depurination of the sarcin/ricin loop by an unknown mechanism. We previously identified compounds that bind the P-stalk pocket of RTA and inhibit catalytic activity. Here we characterize a second-generation lead compound, which binds the P-stalk pocket of RTA with over 30-fold improved affinity relative to the original compound and inhibits the cytotoxicity of ricin holotoxin in Vero cells with no apparent cellular toxicity by itself. This compound also shows protection against Stx2A1. X-ray crystal structure of RTAinhibitor complexes suggests that the orientation of the carboxylic acid influences the inhibitor contacts at the P-stalk site of RTA and contributes to inhibitor potency. The structural changes triggered at the P-stalk site of RTA were validated by solution NMR-based chemical shift perturbation analysis. A key finding by NMR is that binding-induced conformational changes extend beyond the P-stalk site to residues in the active site cleft of RTA. Collectively, these results provide valuable new insight into the conformational flexibility in the C-terminal domain of RTA and its potential role in mediating the remarkable catalytic activity of ricin.

CRISPR-based technologies have revolutionized plant science by enabling precise modulation of gene function, including CRISPR activation (CRISPRa), a recently emerging strategy which shows particular promise for enhancing disease resistance through targeted gene upregulation. Unlike conventional CRISPR editing, which introduces double-stranded DNA breaks and permanent genomic changes, CRISPRa employs a deactivated Cas9 (dCas9) fused to transcriptional activators. This system allows quantitative and reversible gene activation without altering the DNA sequence, offering a gain-of-function (GOF) like enhanced blight resistance in staple crops. Despite its potential, the limited adoption of CRISPRa in plant biology to date underscores the need for future studies to fully harness its capabilities for crop improvement. This review addresses the groundbreaking and relatively underexplored potential of CRISPR activation (CRISPRa) systems for GOF studies in plant biology, and advocates for the adoption of CRISPRa to discover and harness genetic variation for enhancing disease resistance. We present recent advancements in CRISPRa technology, emphasizing its successful application in boosting plant immunity. Moreover, we discuss the synergistic potential of integrating CRISPRa with functional genomics tools such as genome-wide association studies (GWAS) and multi-omics approaches to identify and characterize key resistance genes. Additionally, we highlight ongoing progress in developing plant-specific programmable transcriptional activators (PTAs) to optimize CRISPRa efficiency. Challenges associated with achieving transgene-free overexpression and the deployment of alternative CRISPR systems are also explored. Together, these advances position CRISPRa as a transformative tool for future crop breeding strategies aimed at achieving durable, broad-spectrum disease resistance and sustainability in agriculture.

Ricin, a category-B agent for bioterrorism, and Shiga toxins (Stxs), which cause food poisoning bind to the ribosomal P-stalk to depurinate the sarcin/ricin loop. No effective therapy exists for ricin or Stx intoxication. Ribosome binding sites of the toxins have not been targeted by small molecules. We previously identified CC10501, which inhibits toxin activity by binding the P-stalk pocket of ricin toxin A subunit (RTA) remote from the catalytic site. Here, we developed a fluorescence polarization assay and identified a new class of compounds, which bind P-stalk pocket of RTA with higher affinity and inhibit catalytic activity with submicromolar potency. A lead compound, RU-NT-206, bound P-stalk pocket of RTA with similar affinity as a five-fold larger P-stalk peptide and protected cells against ricin and Stx2 holotoxins for the first time. These results validate the P-stalk binding site of RTA as a critical target for allosteric inhibition of the active site.

Ricin is one of the most toxic substances known and a type B biothreat agent. Shiga toxins (Stxs) produced by E. coli (STEC) and Shigella dysenteriae are foodborne pathogens. There is no effective therapy against ricin or STEC and there is an urgent need for inhibitors. Ricin toxin A subunit (RTA) and A1 subunit of Stx2a (Stx2A1) bind to the C-terminal domain (CTD) of the ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. Modulation of toxin-ribosome interactions has not been explored as a strategy for inhibition. Therefore, development of assays that detect inhibitors targeting toxin-ribosome interactions remains a critical need. Here we describe a fluorescence anisotropy (FA)-based competitive binding assay using a BODIPY-TMR labeled 11-mer peptide (P11) derived from the P-stalk CTD to measure the binding affinity of peptides ranging from 3 to 11 amino acids for the P-stalk pocket of RTA and Stx2A1. Comparison of the affinity with the surface plasmon resonance (SPR) assay indicated that although the rank order was the same by both methods, the FA assay could differentiate better between peptides that show nonspecific interactions by SPR. The FA assay detects only interactions that compete with the labeled P11 and can validate inhibitor specificity and mechanism of action.

Trichothecene mycotoxins such as deoxynivalenol (DON) are virulence factors of Fusarium graminearum, which causes Fusarium head blight, one of the most important diseases of small grain cereals. We previously identified a nonspecific lipid transfer protein (nsLTP) gene, AtLTP4.4, which was overexpressed in an activation-tagged Arabidopsis line resistant to trichothecin, a type B trichothecene in the same class as DON. Here we show that overexpression of AtLTP4.4 in transgenic wheat significantly reduced F. graminearum growth in 'Bobwhite' and 'RB07' lines in the greenhouse and reduced fungal lesion size in detached leaf assays. Hydrogen peroxide accumulation was attenuated on exposure of transgenic wheat plants to DON, indicating that AtLTP4.4 may confer resistance by inhibiting oxidative stress. Field testing indicated that disease severity was significantly reduced in two transgenic 'Bobwhite' lines expressing AtLTP4.4. DON accumulation was significantly reduced in four different transgenic 'Bobwhite' lines expressing AtLTP4.4 or a wheat nsLTP, TaLTP3, which was previously shown to have antioxidant activity. Recombinant AtLTP4.4 purified from Pichia pastoris exhibited potent antifungal activity against F. graminearum. These results demonstrate that overexpression of AtLTP4.4 in transgenic wheat suppresses DON accumulation in the field. Suppression of DON-induced reactive oxygen species by AtLTP4.4 might be the mechanism by which fungal spread and mycotoxin accumulation are inhibited in transgenic wheat plants.

Ricin interacts with the ribosomal P stalk to cleave a conserved adenine from the α-sarcin/ricin loop (SRL) of the rRNA. Ricin toxin A chain (RTA) uses Arg235 as the most critical arginine for binding to the P stalk through electrostatic interactions to facilitate depurination. Structural analysis showed that a P2 peptide binds to a hydrophobic pocket on RTA and the last two residues form hydrogen bonds with Arg235. The importance of hydrophobic residues relative to Arg235 in the interaction with the P stalk in vivo and on the toxicity of RTA is not known. Here, we mutated residues in the hydrophobic pocket to analyze their contribution to toxicity and depurination activity in yeast and in mammalian cells. We found that Leu232, Tyr183 and Phe240 contribute cumulatively to toxicity, with Leu232 being the most significant. A quadruple mutant, Y183A/L232A/R235A/F240A, which combined mutations in critical hydrophobic residues with R235A completely abolished the activity of RTA, indicating that Arg235 and hydrophobic residues are required for full biological activity. Y183A and F240A mutants had reduced activity on RNA, but higher activity on ribosomes compared with R235A in vitro, suggesting that they could partially regain activity upon interaction with ribosomes. These results expand the region of interaction between RTA and the P stalk critical for cellular activity to include the hydrophobic pocket and provide the first evidence that interaction of P stalk with the hydrophobic pocket promotes a conformational rearrangement of RTA to correctly position the active site residues for catalytic attack on the SRL.

Ricin undergoes retrograde transport to the endoplasmic reticulum (ER), and ricin toxin A chain (RTA) enters the cytosol from the ER. Previous reports indicated that RTA inhibits activation of the unfolded protein response (UPR) in yeast and in mammalian cells. Both precursor (preRTA) and mature form of RTA (mRTA) inhibited splicing of HAC1(u) (u for uninduced) mRNA, suggesting that UPR inhibition occurred on the cytosolic face of the ER. Here, we examined the role of ribosome binding and depurination activity on inhibition of the UPR using mRTA mutants. An active-site mutant with very low depurination activity, which bound ribosomes as WT RTA, did not inhibit HAC1(u) mRNA splicing. A ribosome-binding mutant, which showed reduced binding to ribosomes but retained depurination activity, inhibited HAC1(u) mRNA splicing. This mutant allowed separation of the UPR inhibition by RTA from cytotoxicity because it reduced the rate of depurination. The ribosome-binding mutant inhibited the UPR without affecting IRE1 oligomerization or cleavage of HAC1(u) mRNA at the splice site junctions. Inhibition of the UPR correlated with the depurination level, suggesting that ribosomes play a role in splicing of HAC1(u) mRNA. We show that HAC1(u) mRNA is associated with ribosomes and does not get processed on depurinated ribosomes, thereby inhibiting the UPR. These results demonstrate that RTA inhibits HAC1(u) mRNA splicing through its depurination activity on the ribosome without directly affecting IRE1 oligomerization or the splicing reaction and provide evidence that IRE1 recognizes HAC1(u) mRNA that is associated with ribosomes.

Fusarium head blight (FHB) or scab is one of the most important plant diseases worldwide, affecting wheat, barley and other small grains. Trichothecene mycotoxins such as deoxynivalenol (DON) accumulate in the grain, presenting a food safety risk and health hazard to humans and animals. Despite considerable breeding efforts, highly resistant wheat or barley cultivars are not available. We screened an activation tagged Arabidopsis thaliana population for resistance to trichothecin (Tcin), a type B trichothecene in the same class as DON. Here we show that one of the resistant lines identified, trichothecene resistant 1 (trr1) contains a T-DNA insertion upstream of two nonspecific lipid transfer protein (nsLTP) genes, AtLTP4.4 and AtLTP4.5. Expression of both nsLTP genes was induced in trr1 over 10-fold relative to wild type. Overexpression of AtLTP4.4 provided greater resistance to Tcin than AtLTP4.5 in Arabidopsis thaliana and in Saccharomyces cerevisiae relative to wild type or vector transformed lines, suggesting a conserved protection mechanism. Tcin treatment increased reactive oxygen species (ROS) production in Arabidopsis and ROS stain was associated with the chloroplast, the cell wall and the apoplast. ROS levels were attenuated in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls. Exogenous addition of glutathione and other antioxidants enhanced resistance of Arabidopsis to Tcin while the addition of buthionine sulfoximine, an inhibitor of glutathione synthesis, increased sensitivity, suggesting that resistance was mediated by glutathione. Total glutathione content was significantly higher in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls, highlighting the importance of AtLTP4.4 in maintaining the redox state. These results demonstrate that trichothecenes cause ROS accumulation and overexpression of AtLTP4.4 protects against trichothecene-induced oxidative stress by increasing the glutathione-based antioxidant defense.

Trichothecene mycotoxins are natural contaminants of small grain cereals and are encountered in the environment, posing a worldwide threat to human and animal health. Their mechanism of toxicity is poorly understood, and little is known about cellular protection mechanisms against trichothecenes. We previously identified inhibition of mitochondrial protein synthesis as a novel mechanism for trichothecene-induced cell death. To identify cellular functions involved in trichothecene resistance, we screened the Saccharomyces cerevisiae deletion library for increased sensitivity to nonlethal concentrations of trichothecin (Tcin) and identified 121 strains exhibiting higher sensitivity than the parental strain. The largest group of sensitive strains had significantly higher reactive oxygen species (ROS) levels relative to the parental strain. A dose-dependent increase in ROS levels was observed in the parental strain treated with different trichothecenes, but not in a petite version of the parental strain or in the presence of a mitochondrial membrane uncoupler, indicating that mitochondria are the main site of ROS production due to toxin exposure. Cytotoxicity of trichothecenes was alleviated after treatment of the parental strain and highly sensitive mutants with antioxidants, suggesting that oxidative stress contributes to trichothecene sensitivity. Cotreatment with rapamycin and trichothecenes reduced ROS levels and cytotoxicity in the parental strain relative to the trichothecene treatment alone, but not in mitophagy deficient mutants, suggesting that elimination of trichothecene-damaged mitochondria by mitophagy improves cell survival. These results reveal that increased mitophagy is a cellular protection mechanism against trichothecene-induced mitochondrial oxidative stress and a potential target for trichothecene resistance.

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: This project involves the synthesis and characterization of novel ionic liquid (IL) electrolytes for high-performance dye sensitized solar cells (DSSCs). Since most commonly used organic liquid electrolytes of DSSCs are volatile and flammable, they limit the long-term stability and high temperature operations of these cells. In our present work, we have addressed these issues through the development of non-volatile, non-flammable and polymerizable IL electrolytes. Our IL characterization efforts have focused on thermo-physical and electrochemical properties that are relevant for DSSC-specific applications. Computer simulations have also been performed to understand the structural properties of the new electrolytes/nano-composite systems.

Ribosome-inactivating proteins (RIPs) are toxic because they bind to 28S rRNA and depurinate a specific adenine residue from the α-sarcin/ricin loop (SRL), thereby inhibiting protein synthesis. Shiga-like toxins (Stx1 and Stx2), produced by Escherichia coli, are RIPs that cause outbreaks of foodborne diseases with significant morbidity and mortality. Ricin, produced by the castor bean plant, is another RIP lethal to mammals. Currently, no US Food and Drug Administration-approved vaccines nor therapeutics exist to protect against ricin, Shiga-like toxins, or other RIPs. Development of effective small-molecule RIP inhibitors as therapeutics is challenging because strong electrostatic interactions at the RIP•SRL interface make drug-like molecules ineffective in competing with the rRNA for binding to RIPs. Herein, we report small molecules that show up to 20% cell protection against ricin or Stx2 at a drug concentration of 300 nM. These molecules were discovered using the doorstop approach, a new approach to protein•polynucleotide inhibitors that identifies small molecules as doorstops to prevent an active-site residue of an RIP (e.g., Tyr80 of ricin or Tyr77 of Stx2) from adopting an active conformation thereby blocking the function of the protein rather than contenders in the competition for binding to the RIP. This work offers promising leads for developing RIP therapeutics. The results suggest that the doorstop approach might also be applicable in the development of other protein•polynucleotide inhibitors as antiviral agents such as inhibitors of the Z-DNA binding proteins in poxviruses. This work also calls for careful chemical and biological characterization of drug leads obtained from chemical screens to avoid the identification of irrelevant chemical structures and to avoid the interference caused by direct interactions between the chemicals being screened and the luciferase reporter used in screening assays.

Fusarium head blight (FHB) reduces crop yield and results in contamination of grains with trichothecene mycotoxins. We previously showed that mitochondria play a critical role in the toxicity of a type B trichothecene. Here, we investigated the direct effects of type A and type B trichothecenes on mitochondrial translation and membrane integrity in Saccharomyces cerevisiae. Sensitivity to trichothecenes increased when functional mitochondria were required for growth, and trichothecenes inhibited mitochondrial translation at concentrations, which did not inhibit total translation. In organello translation in isolated mitochondria was inhibited by type A and B trichothecenes, demonstrating that these toxins have a direct effect on mitochondrial translation. In intact yeast cells trichothecenes showed dose-dependent inhibition of mitochondrial membrane potential and reactive oxygen species, but only at doses higher than those affecting mitochondrial translation. These results demonstrate that inhibition of mitochondrial translation is a primary target of trichothecenes and is not secondary to the disruption of mitochondrial membranes.

Trichothecene mycotoxins synthesized by Fusarium species are potent inhibitors of eukaryotic translation. They are encountered in both the environment and in food, posing a threat to human and animal health. They have diverse roles in the cell that are not limited to the inhibition of protein synthesis. To understand the trichothecene mechanism of action, we screened the yeast knockout library to identify genes whose deletion confers resistance to trichothecin (Tcin). The largest group of resistant strains affected mitochondrial function, suggesting a role for fully active mitochondria in trichothecene toxicity. Tcin inhibited mitochondrial translation in the wild-type strain to a greater extent than in the most resistant strains, implicating mitochondrial translation as a previously unrecognized site of action. The Tcin-resistant strains were cross-resistant to anisomycin and chloramphenicol, suggesting that Tcin targets the peptidyltransferase center of mitochondrial ribosomes. Tcin-induced cell death was partially rescued by mutants that regulate mitochondrial fusion and maintenance of the tubular morphology of mitochondria. Treatment of yeast cells with Tcin led to the fragmentation of the tubular mitochondrial network, supporting a role for Tcin in disruption of mitochondrial membrane morphology. These results provide genome-wide insight into the mode of action of trichothecene mycotoxins and uncover a critical role for mitochondrial translation and membrane maintenance in their toxicity.