The phc Quorum-Sensing System in Ralstonia solanacearum Species Complex.

Ralstonia solanacearum species complex (RSSC) strains are devastating plant pathogens distributed worldwide. The primary cell density-dependent gene expression system in RSSC strains is phc quorum sensing (QS). It regulates the expression of about 30% of all genes, including those related to cellular activity, primary and secondary metabolism, pathogenicity, and more. The phc regulatory elements encoded by the phcBSRQ operon and phcA gene play vital roles. RSSC strains use methyl 3-hydroxymyristate (3-OH MAME) or methyl 3-hydroxypalmitate (3-OH PAME) as the QS signal. Each type of RSSC strain has specificity in generating and receiving its QS signal, but their signaling pathways might not differ significantly. In this review, I describe the genetic and biochemical factors involved in QS signal input and the regulatory network and summarize control of the phc QS system, new cell-cell communications, and QS-dependent interactions with soil fungi.

The Micacocidin Production-Related RSc1806 Deletion Alters the Quorum Sensing-Dependent Gene Regulation of Ralstonia pseudosolanacearum Strain OE1-1.

The soil-borne phytopathogenic gram-negative bacterium Ralstonia solanacearum species complex (RSSC) produces staphyloferrin B and micacocidin as siderophores that scavenge for trivalent iron (Fe3+) in the environment, depending on the intracellular divalent iron (Fe2+) concentration. The staphyloferrin B-deficient mutant reportedly retains its virulence, but the relationship between micacocidin and virulence remains unconfirmed. To elucidate the effect of micacocidin on RSSC virulence, we generated the micacocidin productivity-deficient mutant (ΔRSc1806) that lacks RSc1806, which encodes a putative polyketide synthase/non-ribosomal peptide synthetase, using the RSSC phylotype I Ralstonia pseudosolanacearum strain OE1-1. When incubated in the condition without Fe2+, ΔRSc1806 showed significantly lower Fe3+-scavenging activity, compared with OE1-1. Until 8 days after inoculation on tomato plants, ΔRSc1806 was not virulent, similar to the mutant (ΔphcA) missing phcA, which encodes the LysR-type transcriptional regulator PhcA that regulates the expression of the genes responsible for quorum sensing (QS)-dependent phenotypes including virulence. The transcriptome analysis revealed that RSc1806 deletion significantly altered the expression of more than 80% of the PhcA-regulated genes in the mutant grown in medium with or without Fe2+. Among the PhcA-regulated genes, the transcript levels of the genes whose expression was affected by the deletion of RSc1806 were strongly and positively correlated between the ΔRSc1806 and the phcA-deletion mutant. Furthermore, the deletion of RSc1806 significantly modified QS-dependent phenotypes, similar to the effects of the deletion of phcA. Collectively, our findings suggest that the deletion of micacocidin production-related RSc1806 alters the regulation of PhcA-regulated genes responsible for QS-dependent phenotypes including virulence as well as Fe3+-scavenging activity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Disproof of the Structures and Biosynthesis of Ergoynes, Gs-Polyyne-l-Ergothioneine Cycloadducts from Gynuella sunshinyii YC6258.

Some bacteria produce "bacterial polyynes" bearing a conjugated C≡C bond that starts with a terminal alkyne. Ergoynes A and B have been reported as sulfur-containing metabolites from Gynuella sunshinyii YC6258. These compounds were thought to be formed by cycloaddition between a bacterial polyyne (named Gs-polyyne) and l-ergothioneine. The biosynthetic gene clusters (BGCs), which may contribute to their synthesis, were present in the YC6258 genome. The biosynthetic origin of Gs-polyyne is interesting considering its rare 2-isopentyl fatty acyl skeleton. Here, the structures and biosynthesis of Gs-polyyne and ergoynes were verified by analytical, chemical, and genetic techniques. In the YC6258 extract, which was prepared considering their instability, Gs-polyyne was detected as a major LC peak, and ergoynes were not detected. The NMR data of the isolated Gs-polyyne contradicted the proposed structure and identified it as the previously reported protegenin A. The expression of Gs-polyyne BGC in Escherichia coli BL21(DE3) also yielded protegenin A. The cyclization between protegenin A and l-ergothioneine did not proceed during sample preparation; a base, such as potassium carbonate, was required. Overall, Gs-polyyne was identified as protegenin A, while ergoynes were determined to be artifacts. This cyclization may provide a derivatization to stabilize polyynes or create new chemical space.

Determinants of Subtype-Selectivity of the Anthelmintic Paraherquamide A on Caenorhabditis elegans Nicotinic Acetylcholine Receptors.

The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. SIGNIFICANCE STATEMENT: Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.

Structures and Biosynthesis of Caryoynencins, Unstable Bacterial Polyynes from Pseudomonas protegens Recombinant Expressing the cayG Gene.

Bacteria in certain genera can produce "bacterial polyynes" that contain a conjugated C≡C bond starting from a terminal alkyne. Protegenin A is a derivative of octadecanoic acid that contains an ene-tetrayne moiety. It was discovered in Pseudomonas protegens Cab57 and exhibits strong antioomycete and moderate antifungal activity. By introducing cayG, a cytochrome P450 gene from Burkholderia caryophylli, into P. protegens Cab57, protegenin A was converted into more complex polyynes, caryoynencins A-E. A purification method that minimized the degradation and isomerization of caryoynencins was established. For the first time, as far as we know, the 1H and 13C{1H} NMR signals of caryoynencins were completely assigned by analyzing the NMR data of the isolated compounds and protegenin A enriched with [1-13C]- or [2-13C]-acetate. Through the structural analysis of caryoynencins D/E and bioconversion experiments, we observed that CayG constructs the allyl alcohol moiety of caryoynencins A-C through sequential hydroxylation, dehydration, and hydroxylation. The recombinant strain exhibited a stronger antioomycete activity compared to the wild-type strain. This paper proposes a stable purification and structural determination method for various bacterial polyynes, and P. protegens Cab57 holds promise as an engineering host for the production of biologically active polyynes.

The behavior of Ralstonia pseudosolanacearum strain OE1-1 and morphological changes of cells in tomato roots.

The soil-borne Gram-negative ß-proteobacterium Ralstonia solanacearum species complex (RSSC) infects tomato roots through the wounds where secondary roots emerge, infecting xylem vessels. Because it is difficult to observe the behavior of RSSC by a fluorescence-based microscopic approach at high magnification, we have little information on its behavior at the root apexes in tomato roots. To analyze the infection route of a strain of phylotype I of RSSC, R. pseudosolanacearum strain OE1-1, which invades tomato roots through the root apexes, we first developed an in vitro pathosystem using 4 day-old-tomato seedlings without secondary roots co-incubated with the strain OE1-1. The microscopic observation of toluidine blue-stained longitudinal semi-thin resin sections of tomato roots allowed to detect attachment of the strain OE1-1 to surfaces of the meristematic and elongation zones in tomato roots. We then observed colonization of OE1-1 in intercellular spaces between epidermis and cortex in the elongation zone, and a detached epidermis in the elongation zone. Furthermore, we observed cortical and endodermal cells without a nucleus and with the cell membrane pulling away from the cell wall. The strain OE1-1 next invaded cell wall-degenerated cortical cells and formed mushroom-shaped biofilms to progress through intercellular spaces of the cortex and endodermis, infecting pericycle cells and xylem vessels. The deletion of egl encoding ß-1,4-endoglucanase, which is one of quorum sensing (QS)-inducible plant cell wall-degrading enzymes (PCDWEs) secreted via the type II secretion system (T2SS) led to a reduced infectivity in cortical cells. Furthermore, the QS-deficient and T2SS-deficient mutants lost their infectivity in cortical cells and the following infection in xylem vessels. Taking together, infection of OE1-1, which attaches to surfaces of the meristematic and elongation zones, in cortical cells of the elongation zone in tomato roots, dependently on QS-inducible PCDWEs secreted via the T2SS, leads to its subsequent infection in xylem vessels.

Competitive chrodrimanin B interactions with rat brain GABAA receptors revealed by radioligand binding assays.

Meroterpenoid compounds chrodrimanins produced by Talaromyces sp. YO-2 have been shown to act as competitive antagonists of silkworm larval GABAA receptors using electrophysiology, yet no further evidence has been provided to support such an action. We have investigated the actions of chrodrimanin B on rat brain GABAA receptors by binding assays with non-competitive ligand of GABAA receptors [3H]EBOB and competitive ligands [3H]gabazine and [3H]muscimol. Chrodrimanin B did not significantly affect the binding of [3H]EBOB while reducing the binding of [3H]gabazine and [3H]muscimol to the rat membrane preparations. Chrodrimanin B increased the dissociation constant Kd of [3H]gabazine and [3H]muscimol without significantly affecting the maximum binding, pointing to competitive interactions of chrodrimanin B with rat GABAA receptors in support of our previous observation that the compound acts as a competitive antagonist on the silkworm larval GABA receptor.

Oleamide rescues tibialis anterior muscle atrophy of mice housed in small cages.

Skeletal muscle atrophy causes decreased physical activity and increased risk of metabolic diseases. We investigated the effects of oleamide (cis-9,10-octadecanamide) treatment on skeletal muscle health. The plasma concentration of endogenous oleamide was approximately 30 nm in male ddY mice under normal physiological conditions. When the stable isotope-labelled oleamide was orally administered to male ddY mice (50 mg/kg), the plasma concentration of exogenous oleamide reached approximately 170 nm after 1 h. Male ddY mice were housed in small cages (one-sixth of normal size) to enforce sedentary behaviour and orally administered oleamide (50 mg/kg per d) for 4 weeks. Housing in small cages decreased tibialis anterior (TA) muscle mass and the cross-sectional area of the myofibres in TA muscle. Dietary oleamide alleviated the decreases in TA muscle and resulted in plasma oleamide concentration of approximately 120 nm in mice housed in small cages. Housing in small cages had no influence on the phosphorylation levels of Akt serine/threonine kinase (Akt), mechanistic target of rapamycin (mTOR) and ribosomal protein S6 kinase (p70S6K) in TA muscle; nevertheless, oleamide increased the phosphorylation levels of the proteins. Housing in small cages increased the expression of microtubule-associated protein 1 light chain 3 (LC3)-II and sequestosome 1 (p62), but not LC3-I, in TA muscle, and oleamide reduced LC3-I, LC3-II and p62 expression levels. In C2C12 myotubes, oleamide increased myotube diameter at ≥100 nm. Furthermore, the mTOR inhibitor, Torin 1, suppressed oleamide-induced increases in myotube diameter and protein synthesis. These results indicate that dietary oleamide rescued TA muscle atrophy in mice housed in small cages, possibly by activating the phosphoinositide 3-kinase/Akt/mTOR signalling pathway and restoring autophagy flux.

Disproof of the Proposed Structures of Bradyoxetin, a Putative Bradyrhizobium japonicum Signaling Molecule, and HMCP, a Putative Ralstonia solanacearum Quorum-Sensing Molecule.

First, we revisited the reported NMR data of bradyoxetin, a putative cell density factor of Bradyrhizobium japonicum, and found some inconsistencies in the proposed structure. To elucidate the correct structure, we synthesized model oxetane compounds and confirmed that the NMR data of the synthetic compounds did not match those of the reported bradyoxetin. After reinterpreting the reported NMR data, we concluded that bradyoxetin must be chloramphenicol. Next, some derivatives of 2-hydroxy-4-((methylamino)(phenyl)methyl)cyclopentanone (HMCP), which is a putative quorum-sensing molecule of Ralstonia solanacearum, were synthesized. The NMR spectra of the synthesized compounds were completely different from those of the reported natural products. Based on theoretical studies, including the estimation of 1H and 13C NMR chemical shifts using density functional theory calculations, we confirmed the correctness of the structure of the synthesized compound. These results strongly suggest that the proposed structure of HMCP could be incorrect.

2-Oxo-histidine-containing dipeptides are functional oxidation products.

Imidazole-containing dipeptides (IDPs), such as carnosine and anserine, are found exclusively in various animal tissues, especially in the skeletal muscles and nerves. IDPs have antioxidant activity because of their metal-chelating and free radical-scavenging properties. However, the underlying mechanisms that would fully explain IDP antioxidant effects remain obscure. Here, using HPLC-electrospray ionization-tandem MS analyses, we comprehensively investigated carnosine and its related small peptides in the soluble fractions of mouse tissue homogenates and ubiquitously detected 2-oxo-histidine-containing dipeptides (2-oxo-IDPs) in all examined tissues. We noted enhanced production of the 2-oxo-IDPs in the brain of a mouse model of sepsis-associated encephalopathy. Moreover, in SH-SY5Y human neuroblastoma cells stably expressing carnosine synthase, H2O2 exposure resulted in the intracellular production of 2-oxo-carnosine, which was associated with significant inhibition of the H2O2 cytotoxicity. Notably, 2-oxo-carnosine showed a better antioxidant activity than endogenous antioxidants such as GSH and ascorbate. Mechanistic studies indicated that carnosine monooxygenation is mediated through the formation of a histidyl-imidazole radical, followed by the addition of molecular oxygen. Our findings reveal that 2-oxo-IDPs are metal-catalyzed oxidation products present in vivo and provide a revised paradigm for understanding the antioxidant effects of the IDPs.

A Unique Combination of Two Different Quorum Sensing Systems in the ß-Rhizobium Cupriavidus taiwanensis.

Cupriavidus taiwanensis LMG19424, a ß-rhizobial symbiont of Mimosa pudica, harbors phc and tqs quorum sensing (QS), which are the homologous cell-cell communication systems previously identified from the plant pathogen Ralstonia solanacearum and the human pathogen Vibrio cholerae, respectively. However, there has been no experimental evidence reported that these QS systems function in C. taiwanensis LMG19424. We identified (R)-methyl 3-hydroxymyristate (3-OH MAME) and (S)-3-hydroxypentadecan-4-one (C15-AHK) as phc and tqs QS signals, respectively, and characterized these QS systems. The expression of the signal synthase gene phcB and tqsA in E. coli BL21(DE3) resulted in the high production of 3-OH MAME and C15-AHK, respectively. Their structures were elucidated by comparison of EI-MS data and GC/chiral LC retention times with synthetic standards. The deletion of phcB reduced cell motility and increased biofilm formation, and the double deletion of phcB/tqsA caused the accumulation of the metal chelator coproporphyrin III in its mutant culture. Although the deletion of phcB and tqsA slightly reduced its ability to nodulate on aseptically grown seedlings of M. pudica, there was no significant difference in nodule formation between LMG19424 and its QS mutants when commercial soils were used. Taken together, this is the first example of the simultaneous production of 3-OH MAME/C15-AHK as QS signals in a bacterial species, and the importance of the phc/tqs QS systems in the saprophytic stage of C. taiwanensis LMG19424 is suggested.

Actinomycins inhibit the production of the siderophore pyoverdines in the plant pathogen Pseudomonas cichorii SPC9018.

Pyoverdines, a group of peptide siderophores produced by Pseudomonas species, function not only in iron acquisition, but also in their virulence in hosts. Thus, chemical inhibition of pyoverdine production may be an effective strategy to control Pseudomonas virulence. In the plant pathogen Pseudomonas cichorii SPC9018 (SPC9018), pyoverdine production is required for virulence on eggplant. We screened microbial culture extracts in a pyoverdine-production inhibition assay of SPC9018 and found Streptomyces sp. RM-32 as a candidate-producer. We isolated two active compounds from RM-32 cultures, and elucidated their structures to be actinomycins X2 and D. Actinomycins X2 and D inhibited pyoverdine production by SPC9018 with IC50 values of 17.6 and 29.6 µM, respectively. Furthermore, pyoverdine production in other Pseudomonas bacteria, such as the mushroom pathogen P. tolaasii, was inhibited by the actinomycins. Therefore, these actinomycins may be useful as chemical tools to examine pyoverdine functions and as seed compounds for anti-Pseudomonas virulence agents.

Androgen signaling expands ß-cell mass in male rats and ß-cell androgen receptor is degraded under high-glucose conditions.

A deficient pancreatic ß-cell mass increases the risk of type 2 diabetes mellitus. Here, we investigated the effects of testosterone on the development of pancreatic ß-cell mass in male rats. The ß-cell mass of male rats castrated at 6 wk of age was reduced to ~30% of that of control rats at 16 wk of age, and castration caused glucose intolerance. Loss of ß-cell mass occurred because of decreases in islet density per pancreas and ß-cell cluster size. Castration was negatively associated with the number of Ki-67-positive ß-cells and positively associated with the number of TUNEL-positive ß-cells. These ß-cell changes could be prevented by testosterone treatment. In contrast, castration did not affect ß-cell mass in male mice. Androgen receptor (AR) localized differently in mouse and rat ß-cells. Testosterone enhanced the viability of INS-1 and INS-1 #6, which expresses high levels of AR, in rat ß-cell lines. siRNA-mediated AR knockdown or AR antagonism with hydroxyflutamide attenuated this enhancement. Moreover, testosterone did not stimulate INS-1 ß-cell viability under high d-glucose conditions. In INS-1 ß-cells, d-glucose dose dependently (5.5-22.2 mM) downregulated AR protein levels both in the presence and absence of testosterone. The intracellular calcium chelator (BAPTA-AM) could prevent this decrease in AR expression. AR levels were also reduced by a calcium ionophore (A23187), but not by insulin, in the absence of the proteasome inhibitor MG132. Our results indicate that testosterone regulates ß-cell mass, at least in part, by AR activation in the ß-cells of male rats and that the ß-cell AR is degraded under hyperglycemic conditions.

Bacterial quorum sensing in symbiotic and pathogenic relationships with hosts.

Gram-negative bacteria communicate with each other by producing and sensing diffusible signaling molecules. This mechanism is called quorum sensing (QS) and regulates many bacterial activities from gene expression to symbiotic/pathogenic interactions with hosts. Therefore, the elucidation and control of bacterial QS systems have been attracted increasing attention over the past two decades. The most common QS signals in Gram-negative bacteria are N-acyl homoserine lactones (AHLs). There are also bacteria that employ different QS systems, for example, the plant pathogen Ralstonia solanacearum utilizes 3-hydroxy fatty acid methyl esters as its QS signals. The QS system found in the endosymbiotic bacterium associated with the fungus Mortierella alpina, the development of an affinity pull-down method for AHL synthases, and the elucidation of a unique QS circuit in R. solanacearum are discussed herein.

An L319F mutation in transmembrane region 3 (TM3) selectively reduces sensitivity to okaramine B of the Bombyx mori l-glutamate-gated chloride channel.

Okaramines produced by Penicillium simplicissimum AK-40 activate l-glutamate-gated chloride channels (GluCls) and thus paralyze insects. However, the okaramine binding site on insect GluCls is poorly understood. Sequence alignment shows that the equivalent of residue Leucine319 of the okaramine B sensitive Bombyx mori (B. mori) GluCl is a phenylalanine in the okaramine B insensitive B. mori γ-aminobutyric acid-gated chloride channel of the same species. This residue is located in the third transmembrane (TM3) region, a location which in a nematode GluCl is close to the ivermectin binding site. The B. mori GluCl containing the L319F mutation retained its sensitivity to l-glutamate, but responses to ivermectin were reduced and those to okaramine B were completely blocked.

Studies on the biosynthesis of ralfuranones in Ralstonia solanacearum.

Ralfuranones, aryl-furanone secondary metabolites, are involved in the virulence of Ralstonia solanacearum in solanaceous plants. Ralfuranone I (6) has been suggested as a biosynthetic precursor for other ralfuranones; however, this conversion has not been confirmed. We herein investigate the biosynthesis of ralfuranones using feeding experiments with ralfuranone I (6) and its putative metabolite, ralfuranone B (2). The results obtained demonstrated that the biosynthesis of ralfuranones proceeded in enzymatic and non-enzymatic manners.

Methyl 3-Hydroxymyristate, a Diffusible Signal Mediating phc Quorum Sensing in Ralstonia solanacearum.

Ralstonia solanacearum, a plant pathogenic bacterium causing "bacterial wilt" on crops, uses a quorum sensing (QS) system consisting of phc regulatory elements to control its virulence. Methyl 3-hydroxypalmitate (3-OH PAME) was previously identified as the QS signal in strain AW1. However, 3-OH PAME has not been reportedly detected from any other strains, and this suggests that they produce another unknown QS signal. Here we identify (R)-methyl 3-hydroxymyristate [(R)-3-OH MAME] as a new QS signal that regulates the production of virulence factors and secondary metabolites. (R)-3-OH MAME was synthesized by the methyltransferase PhcB and sensed by the histidine kinase PhcS. The phylogenetic trees of these proteins from R. solanacearum strains were divided into two groups, according to their QS signal types--(R)-3-OH MAME or (R)-3-OH PAME. These results demonstrate that (R)-3-OH MAME is another crucial QS signal and highlight the unique evolution of QS systems in R. solanacearum.

Involvement of ralfuranone production in the virulence of Ralstonia solanacearum OE1-1.

Ralstonia solanacearum causes a destructive disease called "bacterial wilt" in numerous plant species. Its virulence is controlled by the transcriptional regulator PhcA, the activity of which is, in turn, regulated in a cell-density dependent manner, termed quorum sensing. We herein described the identification and characterization of ralfuranones J-L, new PhcA-regulated secondary metabolites, and the known derivatives, ralfuranones A and B, from R. solanacearum strain OE1-1. Their structures were determined by spectroscopic and chemical methods. These ralfuranones were also detected in vascular exudates from host plants infected with OE1-1. Deletion of ralA, which encodes an enzyme for ralfuranone biosynthesis, reduced the virulence of OE1-1 in tomato plants. Virulence was restored by complementation of the ralA gene. The results suggest that ralfuranones play important roles in the virulence of OE1-1.

Total synthesis and biological evaluation of PF1171A, C, F, and G, cyclic hexapeptides with insecticidal activity.

The total synthesis of the cyclic hexapeptides PF1171A, C, F, and G has been achieved by solid-phase synthesis of a linear precursor and solution-phase macrolactamization. The synthesis includes a solid-phase peptide coupling with the weakly nucleophilic amino group of an anthranilic acid residue. This was efficiently achieved by in situ generation of an Fmoc-amino acid chloride using triphosgene. The natural products exhibit potent paralytic activities against silkworm larvae, whereas epi-PF1171A and epi-PF1171C, bearing l-Ala instead of d-Ala, were relatively inactive. X-ray crystallographic analysis indicates that intramolecular hydrogen bonds in PF1171 peptides are critical for maintaining their active conformations.

Cycloheximide in the nanomolar range inhibits seed germination of Orobanche minor.

From the 992 samples of culture extracts of microorganisms isolated from soil in Japan, we found that the extract of Streptomyces sp. no. 226 inhibited Orobanche minor seed germination without significantly affecting the seed germination of Trifolium pratense and the growth of Aspergillus oryzae and Escherichia coli. Using ESI-MS, 1H-NMR, and 13C-NMR, we identified the active compound as cycloheximide. Cycloheximide had half-maximum inhibitory concentrations of 2.6 ng/mL for the inhibition of seed germination of O. minor and 2.5 µg/mL for that of the conidial germination of A. oryzae. Since cycloheximide is known to inhibit translation by interacting with ribosomal protein L28 (RPL28) in yeast, we investigated whether RPL protein of O. minor plays a critical role in the inhibition of O. minor seed germination. Our data suggested that O. minor RPL27A was not sensitive to cycloheximide by comparing it to the strain expressing S. cerevisiae RPL28. These findings suggest the presence of an unidentified mechanism by which cycloheximide hinders O. minor seed germination.