Beauveria bassiana degenerates after repeated subcultures, demonstrating declined conidiation and insect virulence. The target of rapamycin (TOR) kinase conserved among eukaryotes is the master regulator of cellular physiology and is likely involved in culture degeneration. Indeed, the levels of TOR-associated proteins increase over successive subcultures. Here, CRISPR/Cas9 locus engineering introduced the inducible Tet-On promoter upstream of the TOR kinase 2 gene tor2 in B. bassiana. The mutant PTet-On tor2 ‘T41’ was verified for the Tet-On integration via PCR analyses and provided a model for evaluating the fungal phenotypes according to the tor2 expression levels, induced by doxycycline (Dox) concentrations. At 0 µg·mL−1 of Dox, T41 had 68% of the wild type’s (WT) tor2 expression level, hampered radial growth and relatively lower levels of oxidative stress tolerance, conidiation and virulence against Spodoptera exigua, compared to those under the presence of Dox. A low dose of Dox at 0.1–1 µg·mL−1 induced tor2 upregulation in T41 by up to 91% compared to 0 µg·mL−1 of Dox, resulting in significant increases in radial growth by 8–10% and conidiation by 8–27%. At 20 µg·mL−1 of Dox, which is 132% higher than T41’s tor2 expression level at 0 µg·mL−1 of Dox, T41 showed an increased oxidative stress tolerance and a decrease in growth inhibition under iron replete by 62%, but its conidiation significantly dropped by 47% compared to 0 µg·mL−1 of Dox. T41 at 20 µg·mL−1 of Dox had a strikingly increased virulence (1.2 day lower LT50) against S. exigua. The results reflect the crucial roles of TOR kinase in the vegetative growth, conidiation, pathogenicity and oxidative stress tolerance in B. bassiana. Since TOR upregulation is correlated with culture degeneration in multiple subcultures, our data suggest that TOR signaling at relatively low levels plays an important role in growth and development, but at moderate to high levels could contribute to some degenerated phenotypes, e.g., those found in successive subcultures. Beauveria bassiana has long been reported to be parallel to its widespread application in integrated pest management (IPM). Several fungi degenerate after successive subculturing in vitro, such as on potato dextrose agar (PDA) and Sabouraud dextrose agar (SDA). The degenerated cultures show a reduction or loss in virulence [The culture degeneration or attenuation of the entomopathogenic fungus irulence ,2, whichirulence ,4. Entomirulence .Beauveria bassiana cultures were recently unraveled via proteomics analysis. Late in vitro subcultures had increased levels of proteins, which were associated with oxidative stress response, autophagy, amino acid homeostasis and apoptosis, whilst decreased levels of proteins were associated with DNA repair, ribosome biogenesis, energy metabolism and virulence [B. bassiana degeneration.The physiological changes in degenerated irulence . The finCaenorhabditis elegans, Drosophila melanogaster and mice [TOR signaling is the master regulator of growth and is highly conserved in eukaryotes . It is mand mice ,11,12,13Beauveria bassiana BCC 2660 has a single copy of the TOR kinase gene, tor2, which serves as a common core component of TORC1 and TORC2, similarly to most eukaryotes. In this study, a B. bassiana PTet-On tor2 mutant was used to investigate exogenous tor2 expression induction [tor2 expression levels and the affected phenotypes as a result of culture degeneration. We aim to determine the contribution of TOR kinase in growth and development in the fungus, and to determine if the relevant contributions are dose dependent. We demonstrate the significance of tor2 in mycelial growth, conidiation, virulence and oxidative stress tolerance in this fungus. nduction , which hBeauveria bassiana BCC 2660 was acquired from BIOTEC Culture Collection (BCC), Thailand and was maintained on potato dextrose agar  at 25–28 °C. To ensure the highest virulence, the fungus was first passaged through Spodoptera exigua following Jirakkakul et al.’s (2018) [7 conidia was spread on half-strength PDA and incubated for 7 days at 25–28 °C. Wild-type (WT) strain of B. bassiana BCC 2660 was subcultured from the 2nd to 20th subcultures for the examination of culture degeneration. For isolation of genomic DNA or total RNA, B. bassiana was shaken in potato dextrose broth (PDB) at 150 rpm at 25–28 °C for 4 days. The Tet-On inducer, doxycycline (Dox), at 0, 0.1, 0.5, 1 or 20 µg·mL−1 was also supplemented in culture medium for total RNA extraction. Escherichia coli DH5α was routinely used in molecular cloning. Culture media were supplemented with ampicillin for the recombinant DNA cloning of pCB1534  protocol and using the plasmids pFC332 and pFC334 [tor2-targeted sgRNA cassette. The first fragment was amplified with primers CSN389 (5′-GGGTTTAAUGCGTAAGCTCCCTAATTGGC-3′) and tor2sgRNA-r (5′-AGCTT ACUCGTTTCGTCCTCACGGACTCATCAGGTCACTCGGTGATGTCTGCTCAGCG-3′). The second fragment was amplified with primers tor2sgRNA-f (5′-AGTAAGCUCGTCGTCACTGAATTGGGAAACCGGTTTTAGAGCTAGAAATAGCAAGTTAAA-3′) and CSN390 (5′-GGTCTTAAUGAGCCAAGAGCGGATTCCTC-3′). The two fragments integrated the tor2-specific protospacer sequence  and homology-directed repair were adopted to introduce the Tet-On promoter just upstream of the d pFC334 . Two frator2 promoter sequences and the 5′ end of the tor2 coding sequence, respectively. For the construction of the donor DNA vector, the three DNA fragments were cloned into the vector pCB1534 via restriction cloning. The primers 5F-Bsi (5′-GATCGTACGCTGATGCTTCTCGAATCTGC-3′) and 5R-Nco (5′-GTACCATGG AAGCAAGCTTGCCCGCGCAA-3′) were used to amplify the 5′-HA from the genomic DNA of Beauveria bassiana BCC 2660. The primers TetF-Nco (5′-GAACCATGGAA GCTTCCCTCGGCTGGTCTGT-3′) and TetR-Age (5′-GACACCGGTGTTTAAAC GGTGATGTCTGCT-3′) were used to amplify the Tet-On sequence from pFW22.1, a plasmid vector that carries the Tet-On inducible gene expression system. Lastly, the primers 3F-Age (5′-GATACCGGTAACTGGCGAGGACAGTGTGCA-3′) and 3R-Avr (5′-GCGCCTAGGTTCTTGTTTCCAGGTGTAATGC-3′) were used to amplify 3′-HA from the B. bassiana genomic DNA. After PCR amplification, the 5′-HA fragment was introduced to pCB1534 via double digestion of both pCB1534 and 5′-HA by BsiWI and NcoI, followed by DNA ligation. This step generated the recombinant vector pCB-5HA. Next, the Tet-On sequence was introduced via double digestion of both pCB-5HA and the Tet-On fragment by NcoI and AgeI, followed by DNA ligation. The cloning generated pCB-5HA-Tet. Lastly, 3′-HA was introduced via double digestion of both pCB-5HA-Tet and 3′-HA by AgeI and AvrII and DNA ligation, hence generating pCB-5T3  and a 900 bp 3′-homology arm (3′-HA) that have sequences complementary to the  pCB-5T3 A. Both tBeauveria bassiana via PEG-mediated protoplast transformation with modifications from Srisuksam et al.’s (2015) protocol [8 conidia from 5-day culture was inoculated to 50 mL of PDB. The culture was shaken at 150 rpm at 25 °C for 16 h, and the young mycelia were harvested via centrifugation at 1500× g. For protoplast preparation, cell-well-degrading enzyme mix VinoTaste Pro  was added at a final concentration of 35 mg·mL−1 in 0.6 M MgSO4. The mix was incubated at 28 °C at 90 rpm until most of the mycelia were digested to protoplasts. Equal volumes of 0.6 M sorbitol and 100 mM Tris buffer (pH 7.0) were added, followed by centrifugation at 1500× g to collect the protoplast pellet. Protoplasts were then resuspended in 500 µL of 1 M sorbitol, 10 mM Tris buffer (pH 7.5) and 10 mM CaCl2. Then, the protoplasts were added to a transformation mixture comprising 50 µL of transformation solution , 0.6 M KCl , 50 mM Tris buffer (pH 8.0) , 50 mM CaCl2 , water ), CRISPR/Cas9 vector and donor vector (20 µg each). After incubation on ice for 30 min, an additional 1 mL of transformation solution was added to the mix and it was left to incubate in RT for 45 min. Transformation mix was subsequently transferred to 15 mL of molten PDA supplemented with 0.8 M sucrose and 250 µg·mL−1 hygromycin B (hyg) before pouring into a Petri dish. The culture was incubated at 28 °C until emergence of fungal colonies. The CRISPR/Cas9 vector and the donor vector were co-introduced to protocol . Conidia−1 of hyg). Hyg selection was repeated for five generations. Afterwards, colonies were propagated on half PDA and then in PDB for genomic DNA isolation.Fungal colonies from transformation culture were isolated and transferred to new selection media (PDA with 250 µg mLTet primer pair HindIII_Tet_F (5′-AAGCTTCCCTCGGCTGGTCTGTCTTAC-3′) and Tet_plus_R (5′-ATCCAGATTGCACACTGTCCGTTTAAACGGTGATGTCTGCTC-3′). The second PCR verified Tet-On insertion downstream of tor2 promoter. The primer pair TorTet-seqF (5′-GCAATACCAGCCACACCTGCA-3′) and TorTet-seqR (5′-GCGAGTAACT AGCCAGGTCAG-3′) amplified partial Tet sequence and tor2 coding sequence. Transformants showing correct DNA band sizes in both PCR were subjected to Sanger DNA sequencing.Tet-On introduction in transformants was verified by two rounds of PCR. The first PCR verified the presence of Tet-On promoter in transformants, using PTet-On tor2 mutants induced with 0, 0.1, 0.5, 1 and 20 µg·mL−1 of Dox were determined for their tor2 expression levels using qPCR. Total RNA was extracted with Ambion TRIzol Reagent . Traces of DNA were removed using Ambion DNase I (Thermo Fisher Scientific). First-strand cDNA was then synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). All procedures were conducted according to the manufacturer’s protocol. First-strand cDNA from samples were verified via PCR using tor2-specific primer pair tor2-f (5′-GCAATGTCAGCGTCCAGAAT-3′) and tor2-r (5′-AAAAGTACCCGGAGCGT GTA-3′), as well as the housekeeping gene, M1PD (mannitol 1-phosphate dehydrogenase). The primer pairs amplified intron-inclusive coding sequence in genome so as to distinguish PCR product bands between tor2 cDNA and control (genomic DNA). qPCR was performed using SYBR Green Master Mix . Thermal cycle was as follows: 95 °C for 10 min; 39 cycles of 95 °C for 15 s; 55 °C for 30 s; and 72 °C for 30 s. M1PD was used as internal control. Expression fold change of PTet-On tor2 mutant treatments relative to WT was normalized and calculated using the 2–ΔΔCt method [t method . ExperimPTet-On tor2 mutant was conducted to determine optimal Dox concentration and to assess tor2-associated radial growth change. Conidial drop prepared as above was inoculated to PDA supplemented with 0, 0.1, 0.5, 1, 5, 50, 100, 200 and 500 µg·mL−1 of Dox. WT was used as comparative control. Colony diameter was measured daily. Each treatment consisted of 4 replicates, and the experiment was repeated twice.Radial growth assay for PTet-On tor2 mutant against oxidative stress under different tor2 inductions was assessed. Mutant was cultured with 0 or 20 µg·mL−1 of Dox and was compared to WT. Ferrous ion (Fe2+) was introduced as FeSO4 at 0 or 10 µM to induce oxidative stress. Ten-microliter drops of conidial suspension at 1 × 107 conidia·mL−1 were cultured on minimal media agar (0.02% (w/v) dextrose, 0.51% (w/v) (NH4)2SO4, 0.17% (w/v) yeast nitrogen base (YNB) without amino acids) and incubated at 25–28 °C for 12 days with daily examination and measurement of colony diameter. Each datum was averaged from two perpendicular measurements. Each treatment consisted of 3 replicate plates with 2 colonies per replicate, and the experiment was repeated twice.Tolerance of Beauveria bassiana WT or PTet-On tor2 mutant were cultured on PDA for 7 days. Mutant tor2 was induced by 0, 0.1, 0.5, 1 or 20 µg·mL−1 of Dox. For successive subcultures of WT, subcultures S2, S4, S8, S12, S16 and S20 were analyzed. Two hundred microliters of conidial suspension at 5 × 107 conidia·mL−1 was spread on PDA plate and incubated at 28 °C for 7 days. Conidia were then harvested by rinsing with 10 mL of 0.1% Tween 20 and counted. Each treatment consisted of 3 replicates, and the experiment was repeated twice.Subcultures of tor2 induction were prepared similarly as above. For B. bassiana WT, S2, S8, S12 and S20 were analyzed. Conidial suspension was prepared with saline (0.85% NaCl) and was adjusted to 1 × 105 conidia·mL−1. Conidia were introduced to the larvae of Spodoptera exigua via intrahemocoelic injection, adapted from Toopaang et al.’s (2017) protocol [S. exigua) larvae were obtained from the Viral Technology for Biocontrol Research Team . Three microliters of the conidial suspension was injected to the insect larvae with a specialized 33-gauge microliter syringe set . Injected larvae were then transferred to individual wells of a 24-well plate coated with mung-bean-based semi-synthetic diet (in 1 L: (w/v) 13% mung bean, 1% brewer’s yeast, 0.3% ascorbic acid, 0.125% sorbic acid, 0.25% methyl paraben, 0.3% casein, 0.15% vitamin E, 0.05% choline chloride and 1.2% agar; (v/v) 0.3% vitamin stock and 0.08% formalin). Control larvae were injected with saline. Each treatment consisted of ten larvae. Cumulative insect mortality was recorded for 7 days on a daily basis. The experiment was repeated twice. Median lethal time (LT50) was determined using SPSS’s probit analysis.Fungal culture and protocol . Fourth-50 in insect bioassay was calculated using Probit regression analysis. LT50 was determined by recording the days required for killing half of the beet armyworm population. All significance tests were completed using SPSS package version 23 .Experiments in this study were conducted either two or three times with three or four replicates for each treatment. Statistical significance was determined using one-way ANOVA. Univariate linear model was used, and Duncan’s test was used as the post hoc test. The significance letters represent the different homogenous subsets in Duncan’s post hoc test. LTPTet-On tor2 model for the following experiments. The DNA sequencing confirmed the correct integration of the Tet-On cassette in the transformant.Eighty transformants were initially recovered under hygromycin selection after PEG-mediated protoplast transformation. Four additional rounds of repeated selections using hygromycin screened out 71 colonies of ectopic integration, whereas the remaining 9 colonies were subject to PCR. In the first PCR, the transformant T41 displayed the correct Tet-On PCR product size of 2581 bp, and the transformant T54 also showed the expected product with less intensity B,C. In tBeauveria bassiana WT and the mutant PTet-On tor2 in response to the Dox concentrations. Under the culture without Dox, the mutant had a markedly decreased growth compared to the WT  of inhibition at the same incubation time  subculture at 2.8 × 109 conidia per Petri dish, suggesting a notable decrease of 51% . Nonetheless, an even higher concentration of Dox at 50 µg·mL−1 led to lower mortalities in T41 than at 20 µg·mL−1, suggesting that an optimal tor2 expression level, induced by a moderate concentration of Dox, supports the high insect mortality in Beauveria bassiana. A too-high level of tor2 expression resulted in a lower insect mortality, as we also observed in the late subcultures.The virulence of the f the WT A. The LTectively . In cont−1 of Dox had the lowest LT50 at 3.08 days, which was 1.2 days (28.8 h) shorter than T41 in the absence of Dox . The results overall reflect the contribution of tor2 in B. bassiana’s virulence against insects.For the culture degeneration in the  culture B, consiscultures . CompareFusarium fujikuroi, Aspergillus fumigatus, Trichoderma atroviridae, Verticilium dahliae [Beauveria bassiana BCC2660 (unpublished data). In this study, we selected the TOR kinase 2 gene ‘tor2’ to target both the TOR complexes that generally exist in most organisms, TORC1 and TORC2. The Tet-On introduction was precisely placed just upstream of the tor2 coding sequence. The native tor2 promoter remained at the locus since it is likely shared with an adjacent gene. The CRISPR/Cas9-mediated Tet-On promoter introduction enabled exogenous tor2 expression induction via Dox. Here, our data from various characterizations of the PTet-On tor2 mutant demonstrate the significance of the TOR kinase in B. bassiana’s mycelial growth, conidiation, virulence and oxidative stress tolerance. It is also interesting how this essential developmental program may contribute to the culture degeneration of B. bassiana.The morphological changes in degenerated fungal cultures include the attenuation of sporulation and impairment in sexuality, fruiting body formation and secondary metabolism . It is i dahliae ,24,25,26Beauveria bassiana conidia attach to the host cuticle. Secondly, these cells germinate, grow ‘vegetatively’ and form the cuticle-penetrating appressorium for hemolymph access. The fungus then gradually infects and colonizes the host by exploiting the host’s nutrients for proliferation, releasing insecticidal compounds whilst evading the host’s immune response. The fungus later emerges from the host cadaver and releases conidia, where the infection cycle repeats [Fungal conidiation, vegetative growth and oxidative stress tolerance predominately determine virulence against insect hosts. In nature, firstly, the fungal dispersal is principally carried out by means of ‘conidiation’ and subsequent release into the surrounding area. The dispersed  repeats . ConidiaBeauveria bassiana through an intricate regulation network, as supported by knockdown studies. TORC1 inhibition via rapamycin often leads to a serious or complete loss of fungal sporulation. The antisense RNA knockdown of the TOR kinase gene in B. bassiana increased the fungus’s sensitivity to osmotic stress, heat stress, cell-wall-perturbing agents and oxidative stress [B. bassiana [V. dahliae, TORC1 inhibition via rapamycin significantly inhibits fungal mycelial growth and pathogenicity [Fusarium graminearum, the TOR downstream component Tap42 interacts with different types of 2A phosphatases Pp2A, Sit4 and Ppg1, which regulate the cell wall integrity, mycelial growth, mycotoxin production and virulence [T. atroviridae [Thirdly, ‘oxidative stress tolerance’ plays an important role as the stress response when exposed to the host’s ‘oxidative burst’ upon microbial detection  or to ade stress . The TORbassiana , which abassiana ,32, whilgenicity . Similarirulence . TOR alsoviridae , which iPTet-On tor2 ‘T41’ serves as a tool for evaluating the fungal phenotypes according to the TOR expression levels. Our results from these phenotypic characterizations support TOR’s significance in the growth, survival and pathogenesis of the fungus. tor2’s downregulation from the absence of Dox resulted in the hampering of PTet-On tor2’s radial growth, a lower oxidative stress tolerance and a decrease in conidiation and virulence against S. exigua compared to those under the presence of Dox. The upregulation of tor2 induced by Dox resulted in different extents of improvement in these phenotypes depending on the Dox concentration. A low range of concentrations of Dox at 0.1–1 µg·mL−1 induced tor2 upregulation by up to 30%, resulting in an increasing gradient of conidiation and radial growth. However, at a relatively higher Dox concentration at 20 µg·mL−1, the oxidative stress tolerance increased, but the conidiation significantly dropped. The conidiation decrease in T41 is akin to the deteriorating conidiation in the Beauveria bassiana WT after successive subcultures in vitro [S. exigua of T41 with 20 µg·mL−1 of Dox did not exhibit a similar drop to that found in conidiation suggests that the regulatory spectrum by the TOR signaling pathway may be different depending on the phenotypes. Alternatively, the TOR pathway may not be the sole factor for the declined insect virulence; instead, this may be caused by multiple factors that were not determined in our PTet-On tor2 model.In this study, the mutant in vitro , supportBeauveria bassiana’s culture degeneration [C. elegans, TOR inhibition during development is growth inhibitory; yet, inhibition starting from the first day of adulthood lengthens its lifespan [B. bassiana could attest to TOR’s potential role in culture degeneration at ‘adulthood’, which we speculate to be a representation of the restriction of B. bassiana to a saprophytic mode of nutrition during cultivation on axenic media.Apart from its role in virulence, the TOR signaling pathway was reported to be associated with neration . TOR negneration . Temporalifespan . Our expM. anisopliae, the development of a sterile sector in A. nidulans as well as the altered energy metabolism in Beauveria bassiana [TOR’s contribution to culture degeneration could stem from the failure to select longevity-promoting traits in natural selection . Under ibassiana ,20,37. NThe culture degeneration of entomopathogenic fungi probably results from a combination of inter-connected factors , where s


StPIP1_1, encodes a protein of 76 amino acids with sequence homology to PAMP-induced secreted peptides (PIPs) from Arabidopsis thaliana. Expression of StPIP1_1 is also induced in response to infection with Phytophthora infestans, the causal agent of late blight disease. Apoplastic localization of StPIP1_1-mCherry fusion proteins is dependent on the presence of the predicted signal peptide. A synthetic peptide corresponding to the last 13 amino acids of StPIP1_1 elicits the expression of the StPIP1_1 gene itself, as well as that of pathogenesis related genes. The oxidative burst induced by exogenously applied StPIP1_1 peptide in potato leaf disks is dependent on functional StSERK3A/B, suggesting that StPIP1_1 perception occurs via a receptor complex involving the co-receptor StSERK3A/B. Moreover, StPIP1_1 induces expression of FRK1 in Arabidopsis in an RLK7-dependent manner. Expression of an RLK from potato with high sequence homology to AtRLK7 is induced by StPIP1_1, by Pep-13 and in response to infection with P. infestans. These observations are consistent with the hypothesis that, upon secretion, StPIP1_1 acts as an endogenous peptide required for amplification of the defense response.Treatment of potato plants with the pathogen-associated molecular pattern Pep-13 leads to the activation of more than 1200 genes. One of these,  Similar to PAMPs, these damage-associated molecular patterns (DAMPs) bind to plasma membrane-localized receptor kinases, leading to the activation of immune responses. DAMPs can be released passively upon cell disruption, as is the case for cell wall fragments and extracellular plant-derived compounds such as ATP4, rapid alkalinization factor RALF5, plant elicitor peptides (PEPs6), as well as the serine rich endogenous peptides (SCOOP7) and PAMP-induced peptides, PIPs8. These peptides, termed phytocytokines11), are perceived by plasma membrane receptors14, thus activating immune responses such as cytosolic Ca2+ elevation, reactive oxygen species (ROS) burst, MAP kinase activation and transcriptional reprogramming16.In contrast, there are endogenous danger signals that are actively processed and released into the apoplastic space. These include peptides that are produced from preproproteins, such as systemin17, thus emphasizing the dual role of phytocytokines in immunity and development. Apoplastic localization has been shown for Arabidopsis AtPIP18, SCOOP1018 and IDA-like7 (IDL719), where they are postulated to bind to their respective receptors RLK78, MIK220 and HAESA (HAE)/HAESA-LIKE2 (HSL217).The family of SGP-rich secreted endogenous peptides comprises not only the immunity-related PIPs and SCOOPs, but also Inflorescence Deficient in Abscission (IDA) peptides, which are involved in floral abscission21, AtPIP1 induces stomatal closure22, suggesting a role in stomatal immunity. Recently, the PAMP-induced peptides StPIP1 to StPIP4 were described in potato (Solanum tuberosum23). Although exogenous application of chemically synthesized StPIP1 to leaves does not induce increases in the cytosolic Ca2+ concentrations nor ROS production, transgenic potato plants overexpressing StPIP1 show enhanced defense gene expression upon virus infection, suggesting a role of StPIP in eliciting defense responses23.In Arabidopsis, PIPs are encoded by 11 genes. Similar to PEP1Solanum tuberosum) is late blight, caused by the hemibiotrophic oomycete Phytophthora infestans. Treatment of susceptible plants with the PAMP Pep-13, a 13 amino acid oligopeptide from an extracellular transglutaminase from Phytophthora species, results in the activation of salicylic acid and jasmonic acid-dependent immune responses, such as an oxidative burst, defense gene activation and hypersensitive cell death26). Perception of Pep-13 requires the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1), encoded by two genes in potato, the SOMATIC EMBRYOGENESIS RECEPTOR KINASEs StSERK3A and StSERK3B27. Transgenic potato plants with down-regulated expression of StSERK3A/B are unable to mount an oxidative burst or to activate MAP kinases in response to Pep-13 treatment27.One of the most devastating diseases of potato  and PGSC0003DMG400006400 (StPIP1_2), predicted to encode 76 and 75 amino acid proteins, respectively, was enhanced in potato leaves after Pep-13 infiltration, compared to infiltration of the nearly inactive analogue W2A  and StTHT (TYRAMINE HYDROXYCINNAMOYL-COA TRANSFERASE31) was enhanced in response to StPIP1_1 . The coding region of StPIP1_1 was cloned in front of an intron-containing mCherry gene to ensure that mCherry signals are derived from plant-expressed transcripts. To address the importance of the signal peptide, we included a variant lacking the first 87 nucleotides, encoding the predicted signal peptide of 29 amino acids (ΔSP_StPIP1_1-mCh). As plasma membrane marker, Arabidopsis sulfate transporter SULTR1;2-GFP was co-expressed33. Confocal microscopy imaging revealed localization of StPIP1_1-mCherry fluorescence in the apoplastic space that lies between GFP-SULTR1;2-GFP labeled plasma membranes . Consistent with published data, transcripts of FRK1 accumulated in response to AtPIP18. Interestingly, application of StPIP1_1 induced FRK1 expression to a similar level  did not accumulate in response to Pep-13 treatment in infiltrated potato leaves, while RLK7-like 2  transcript levels were elevated  7 and to activate defense responsesvel Fig. A. In theted Fig. . To veriPhytophthora species. As StPIP1_1, the amidated peptide was converted to the acidic form within 30 min  to analyze immune responses. This peptide has highest sequence homology to the first motif in AtPIP2 , and less to the C-terminal motifs in AtPIP2 (9 out of 23) or AtPIP1 (7 out of 23). Hou et al.8 used a peptide comprising the last 13 amino acids of AtPIP1. StPIP1_1 and StPIP1_2 have 11 and 10 identical amino acids, respectively, out of these 13 amino acids comprising the SGPS motif of AtPIP1. Moreover, AtPIP1, AtPIP2 and the two PIPs identified in our study represent the C-terminal amino acids, whereas StPIP1-4 have additional amino acids downstream of the GPS motif. Thus, we conclude that StPIP1_1 identified in this study is the potato PIP family member with the highest amino acid sequence homology to AtPIP1.The potato genome contains genes encoding small secreted proteins which are induced by PAMP treatment or pathogen infection. Combest et al.RLK7, encoding the receptor-like kinase that binds AtPIP18, suggesting that StPIP1_1 is a functional homolog of AtPIP1.Similar to AtPIP1, exogenous application of a synthetic peptide covering the C-terminal 13 amino acids of StPIP1_1 to Arabidopsis seedlings induces FRK1 expression. Importantly, this induction does not take place in a mutant defective in 23 reported a ROS burst induced by StPIP1 only in nodes, but not in leaves. On the other hand, both StPIP1 and StPIP1_1 induced defense gene expression. In addition, we showed that defense metabolites, such as p-coumaroyl- and feruloyloctopamine, as well as p-coumaroyltyramine, accumulated in StPIP1_1-infiltrated plants. In potato, these compounds are synthesized in response to pathogen infection or PAMP treatment32. They are incorporated into the cell wall and are therefore postulated to contribute to pathogen resistance31. Indeed, overexpression of the tomato gene encoding the biosynthetic enzyme THT in transgenic plants resulted in enhanced levels of HCAAs and correlated with reduced growth of Pseudomonas syringae pv. tomato35.In response to treatment with StPIP1_1, we observed significant ROS production in potato leaf disks. In contrast, Combest et al.23. However, inoculation with potato virus Y resulted in the activation of a large number of genes in StPIP1 overexpressing plants, compared to the lower number of genes induced by PVY in control plants23, suggesting that StPIP1, once induced, amplifies the defense response. This is in accordance with Arabidopsis plants expressing AtPIP1, which displayed enhanced resistance to the root pathogen Fusarium oxysporum8.The StPIP1_1-induced production of ROS and the accumulation of defense transcripts and metabolites in potato leaves suggest that StPIP1_1 indeed acts as a phytocytokine. In transgenic plants overexpressing StPIP1, no difference in gene expression to wild type plants was observed2+-dependent metacaspases36. More specifically, the metacaspase MC4 cleaves the precursor of PEP1 (PROPEP1) after a conserved arginine, resulting in the release of the C-terminal 23 amino acids that make up PEP137. In contrast, AtPIP1-GFP fusion proteins were present in the apoplast of non-disrupted cells after transient expression in N. benthamiana leaves8. Here we show that the apoplastic localization of potato StPIP1_1 is dependent on the presence of the predicted N-terminal signal peptide, suggesting that potato StPIP is targeted to the extracellular compartment by the secretory pathway. Similarly, IDA, a peptide with sequence similarity to PIPs, was also shown to be secreted in a signal peptide-dependent manner38.In order to act as a phytocytokine, peptides have to be present in the apoplast. This can be achieved by secretion or by release into the apoplast after cellular damage. The proprotein of Arabidopsis AtPEP1 is released upon wounding and subsequently processed by CaNicotiana benthamiana cells transiently expressing StPIP1_1-mCh contain proteins with a lower molecular weight than those expressing ΔSP_StPIP1_1-mCh, suggesting extracellular processing of the proprotein in the apoplast by yet unidentified proteases. In vitro experiments with GST-tagged AtPIP1 and AtPIP2, incubated with Arabidopsis protein extracts, also revealed processing of the proproteins, however, the proteases responsible or the nature of the released peptides had not been confirmed by proteomics8. For only a few apoplastic peptides, processing by proteases has been shown. The IDA peptide, for example, is released by subtilisin-like proteases39, whereas the apoplastic immune signaling peptide ZIP1 from maize is released from its proprotein by the action of Papain-like cysteine proteases40. In this context it is interesting to note that exogenously applied synthetic peptides are rapidly degraded, possibly by apoplastic proteases that are part of the immune barrier at the plant pathogen interface.StSERK3A/B silenced plants. Loss of ROS production in StSERK3A/B-silenced plants had also been observed in response to Pep-13 and flg22, but not chitin, suggesting that StSERK3A/B are required for peptide perception27. In Arabidopsis, the ROS burst induced by AtPIP1 was shown to be partially dependent on BAK18. In addition, the induction of stomatal closure by AtPIP1 in Arabidopsis is also dependent on functional BAK141. Thus, PIP peptides are suggested to be perceived by a receptor complex involving BAK1 in both Arabidopsis and potato. Data obtained in Arabidopsis moreover imply that such a receptor complex would include receptor kinase RLK7(41). Since StPIP1_1 is a functional homolog of AtPIP1, we searched for a possible homolog of RLK7 in potato. Comparison of the amino acid sequence of RLK7 to the potato database revealed homology to StR7L1 and StR7L2. As reported by Combest et al.23, transcripts of StR7L1 did not accumulate in virus-inoculated StPIP1 overexpressing plants, which is in accordance with our observation that expression is not induced after Pep-13 treatment. In contrast, transcripts of StR7L2 accumulate in response to PAMP treatment and pathogen infection. Thus, the encoded RLK is a candidate for the RLK7 homolog of potato.ROS production induced in potato leaf disks by StPIP1_1 was dependent on the functional co-receptors StSERK3A/B, the potato homologs of BAK1 from Arabidopsis, since no hydrogen peroxide formation was detected in Our data suggest that the potato homolog of AtPIP1, StPIP1_1, is secreted to the apoplast, causes an oxidative burst in a BAK1-dependent manner and induces defense gene expression as well as defense metabolite accumulation.Solanum tuberosum cv. Désirée) were cultivated in sterile tissue culture in a phytochamber . Plants were transferred to steam-sterilized soil and grown for 4 weeks under long day conditions in a phytochamber . PAMP treatment was performed with leaf disks that were cut out from 4-week-old potato plants with a biopsy puncher (4 mm diameter) and placed with the abaxial side onto the surface of 250 µl of water in a 96-well plate. The plate was incubated overnight at 22 °C in the dark. Water from the wells was removed and 100 µl sterilized fresh water per well was added. The plate was incubated for 30 min in the phytochamber (20 °C). Elicitation was performed by adding 100 nM StPIP1_1 (RLPSGPSPRGPGH), Pep-13 (VWNQPVRGFKVYE) or the nearly inactive analog W2A (VANQPVRGFKVYE27). For whole plant assays, peptides were infiltrated as a 100 µM solution into the abaxial side of leaves of 3-week-old potato plants growing in a phytochamber. Phytophthora infestans infections were achieved by drop-inoculation of 10 µl of a zoospore suspension (1 × 105 zoospores/ml) onto the abaxial leaf surface. Inoculated leaves were kept at 100% humidity for the duration of the experiment. As controls, water was pipetted onto the leaves42. Nicotiana benthamiana was grown from seeds in the greenhouse. Transient expression was performed with Agrobacterium tumefaciens containing binary vectors. Eleven-day-old Arabidopsis seedlings (Col-0 and rlk7-2 (SALK_083114)) grown in microtiter plates were treated with 1µM StPIP1_1.Potato plants  and cDNA synthesis using Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) were performed according to the manufacturer´s instructions. For quantitative PCR, Maxima Probe qPCR MasterMix (Thermo Fischer Scientific) was used and the samples were run on an Mx3005P qPCR system (Agilent).RNA was isolated from potato leaves, leaf disks or Arabidopsis seedlings as describedStPIP1_1: 5′-GGAAATTCGTCTCGCAACTT-3′, 5′-CTGAAGGTAGCCTCTCAAGCA-3′ and Roche Universal Probe Library Probe #155, StPR1: 5′-TCAGTTCGACTAGGTTGTGGTC-3′, 5′-GTCCGACCCAGTTTCCAAC-3′ and probe #157, St4CL: 5′-TGCTGTTGTCCCAATGATAGA-3′, 5′-TGATCTAACAACAAAAGCCACTG-3′ and probe #7, StTHT: 5′-CCTCCTTAGAGGGCTTGCTT-3′, 5′-AGTACGGATGGCCCGTAGA-3′ and probe #144, StRLK7: 5′-TGGAAAGCCTAGAGAATGGTACA-3′, 5′-CAAATGAAACGAGCACCACA-3′ and probe #83, StEF1α: 5′-CACTGCCCAGGTCATCATC-3′, 5′-GTCGAGCACTGGTGCATATC-3′ and Roche Universal Probe Library Probe #163, AtFRK1: 5′-GAGACTATTTGGCAGGTAAAAGGT-3′, 5′-AGGAGGCTTACAACCATTGTG-3′ and probe #33.The following primers and real time probes were used: 43 with the following modifications: Each well contained 200 µl of water supplied with 5 µM luminol L-012 , 2 µg horseradish peroxidase (Fluka) and 100 nM peptide.ROS analyses were performed as described45 with the following modifications of the binary gradient with 0.150 µl/min: 0–1 min isocratic 95% A  and 5% B ; 1–10 min linear from 5 to 60% B; 10–10.2 min linear to 95% B; 10.2–12 min isocratic 95% B; 12–14 min isocratic 5% B. Eluting compounds were detected from m/z 50 to 1000. LC–MS profiling data were analysed as described previously46.Leaf disks from 4-week-old infiltrated potato plants were cut out at defined time points after infiltration and frozen in liquid nitrogen. Chromatographic separations of methanolic extracts were performed as described previouslyInfiltrated peptides of StPIP1_1 and Pep-13, which both had a carboxy-terminal amidation, were annotated by comparison of consistent retention time, accurate mass and MS/MS identity to the applied authentic standards. Deamidated peptides and peptide fragments were annotated by accurate mass as calculated from possible peptide structures generated in silico and by MS/MS sequence interpretation.47. As plasma membrane marker, Arabidopsis SULTR1;2 was included33. The coding sequence of SULTR1;2 without the stop codon was amplified from Arabidopsis cDNA using the primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCGTCAAGAGCTCACCCTGTGG-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGACCTCGTTGGAGAGTTTTGG-3, cloned into pDONR221 via BP Ligation, and mobilized into pB7FWG248 to generate ProCaMV35:SULTR1;2-GFP. Agrobacterium tumefaciens GV3101 containing StPIP1_1-mCh, SULTR1;2 or the silencing suppressor p19 in a 1:1:2 ratio were co-infiltrated for transient expression in N. benthamiana. Three days after infiltration, microscopy was performed using a Zeiss LSM 780 inverted microscope using a 40× water-immersion objective. The excitation wavelengths for GFP and mCherry were 488 nm and 561 nm, respectively, and emission was detected between 495 and 550 nm (GFP) and 570 and 620 nm (RFP). Bright field images were captured with the TPMT detector. Plasmolysis was induced by application of 150 mM NaCl to excised leaf discs mounted on object slides.A gene encompassing the coding region of StPIP1_1 fused to mCherry containing an intron was synthesized by GeneArt (Thermofisher). The gene was cloned into a binary vector under the control of the 35S promoter by Golden Gate CloningN. benthamiana leaves expressing StPIP1_1-mCherry or ΔSP-StPIP1_1-mCherry 3 days after Agrobacterium infiltration. Leaves were ground in liquid nitrogen and the leaf material was mixed with 2× Laemmli buffer in a 1:1 ratio. For each sample, 20 µl were loaded on a 10% SDS gel and blotted for 1 h. Blots were blocked in 1× TBS with 2% [w/v] milk powder and 2% [w/v] BSA for 1 h at RT. For protein detection, blots were incubated over night at 4 °C with a primary mouse α-RFP antibody  and subsequently for 2 h at RT with a secondary α-mouse antibody coupled to horseradish peroxidase . Western blot images were acquired with a FluorChem system detection using ECL-Prime (Amersham/Cytiva) for detection.Proteins were analyzed from The authors confirm that all methods were carried out in accordance with the relevant guidelines.Supplementary Figures.


The isolation of pure LMs from a single preparation enables direct transcriptomic and proteomic analyses, as well as functional studies on freshly isolated LMs, which can be considered the proper counterparts of LMs in vivo and have potential applications in tissue engineering.Limbal melanocytes (LMs) are found in the corneoscleral limbus basal epithelial layer and interact with neighboring limbal epithelial progenitor cells. The difficulty of isolating and cultivating LMs is due to the small fraction of LMs in the overall limbal population and the frequent contamination of primary cultures by other cell types. This has limited the research on freshly isolated LMs and the investigation of their biological significance in the maintenance of the limbal stem cell niche. Here, we describe an optimized protocol for the efficient isolation and expansion of LMs from cadaveric corneal limbal tissue using CD90 and CD117 as selective markers in fluorescence-activated cell sorting to obtain a pure population of LMs (CD90 Limbal epithelial stem/progenitor cells (LEPCs), present in the basal layers of limbal epithelium, are responsible for the homeostasis of corneal epithelium. The self-renewal, quiescence, and differentiation status of LEPCs are regulated by the limbal niche, a specialized microenvironment proposed to be composed of a distinct extracellular matrix (ECM), limbal vasculature, and neighboring limbal niche cells (LNCs) ,2. The EFew reports have been published on obtaining pure melanocyte cultures from limbal tissue using G418 (geneticin), a cytotoxic agent that affects rapidly growing cells ,10. Desp− CD117+; 1.2 ± 0.3% cells) with self-renewal capacity and sustained melanin production [The CD90 molecule, also known as Thy-1, is a cell surface glycoprotein that is commonly expressed on stromal cells ,17,18. Coduction  and FACSoduction , these m− CD117+  cells were sorted and cultured. An overview of the stages of the protocol is presented in Limbal melanocytes were isolated from organ-cultured corneoscleral tissues through a combination of enzymatic digestion and cell sorting. The limbal segments were digested with collagenase A to obtain limbal clusters, which consisted of epithelial–mesenchymal melanocytes and single cells. The obtained limbal clusters were dissociated into single cells using trypsin and ethylenediaminetetraacetic acid (EDTA). The obtained single-cell suspension was stained with anti-CD90 and anti-CD117 antibodies, and FACS sorting was performed. The CD90The following glassware and plasticware were used in this protocol. However, you may test any other available commercial source of the same requirements.➢Micropipette tips  ;➢®, Kaiserslautern, Germany, catalog number: 3513);A 12-well plate ;A 60 mm cell culture dish ;A 100 mm cell culture dish ;➢®, catalog number: 201000010);Disposable scalpel blades, No. 10  ;➢Conical tubes (15 mL) ;➢Conical tubes (50 mL) ;➢T75 flasks ;➢Reversible cell strainers ;➢Cell filter 20 µm ;➢FACS tubes .Cell-specific antibodies and their corresponding isotype controls were required for cell sorting and characterization of cells. Anti-CD117 antibody was used as a selective marker for limbal melanocytes, whereas anti-CD90 was used to identify limbal mesenchymal stromal cells. For characterization, epithelial marker pan-cytokeratin; stromal marker vimentin; and melanocyte markers Melan-A, SRY-box-10 (Sox10), human melanoma black (HMB)-45, and tyrosinase-related protein (TRP)1 were used.➢PE mouse anti-human CD117 ;➢APC mouse anti-human CD90 ;➢Mouse IgG2a, k isotype-APC ;➢Mouse IgG2a, k isotype-PE ;➢Mouse anti-human cytokeratin pan AE1/AE3 ;➢Rat anti-human vimentin ;➢Rabbit anti-human Melan-A ;➢Rabbit anti-human Sox10 ;➢Mouse anti-human HMB-45 ;➢Mouse anti-human TRP1 .You may test any other manufacturers’ antibodies of the same type.➢For washing tissues and cells, Dulbecco’s Phosphate Buffered Saline (DPBS)   was used;➢Dulbecco’s Modified Eagle Medium (DMEM) high glucose  and fetal bovine serum (FBS)  were used to inhibit trypsin action;➢To propagate LMs, we recommend using CnT-40 melanocyte proliferation medium ;➢For successful expansion of LMs, we used iMATRIX 511  to coat the cultureware;➢Penicillin–streptomycin 100× ; ➢EDTA 0.5 M  was used in FACS buffer to reduce cell clump formation;➢As a disinfectant, 70% isopropyl alcohol was used;➢4′,6-diamidino-2-phenylindole (DAPI)  was used during FACS to differentiate live and dead cells. You may experiment with any viability indicator that works well with the conjugated antibodies used in sorting.➢Collagenase A  was used to digest the limbal segments;➢®, catalog number: 25200056) was used to dissociate limbal clusters into single cells as well as passaging of limbal melanocytes.Trypsin–EDTA 0.25%  were required;Pipettes: micropipettes of all sizes  and pipette aids . You may use any kind of cell counter to count the cell numbers;➢®-H) were required;Surgical instruments: to dissect the tissues, forceps  was required to create an aseptic environment for cell isolation and culturing. All the steps need to be performed under a biosafety cabinet;➢2 incubator: for optimal cell culturing, CO2 incubator  was required to maintain 37 °C and 5% CO2;CO➢Microscope: to visualize cell phases, contrast-inverted microscope with a camera  was required;➢Refrigerator and freezer: a refrigerator at 2–8 °C and freezer at −20 °C were required for storage of media and reagents;➢Flow sorter: In this study, we used FACS Aria II sorter (BD Biosciences) to sort the cells. You may use any other cell sorter to sort the cells using this protocol;➢®, Karlsbad, Germany, catalog number: 1013) was required to warm the reagents.Water bath: a water bath ;➢FACSDiva v8.0.1 software ;➢FlowJo v10.2 software .Collagenase solution: to dissect corneolimbal tissue, collagenase solution was required. Collagenase solution (2 mg/mL) was prepared and stored at −20 °C. To prepare collagenase solution, dissolve 500 mg of collagenase A in 220 mL of DMEM high glucose containing 25 mL of fetal calf serum and 5 mL of penicillin–streptomycin 100×. Mix well by inverting until it dissolves completely. Filter the solution using 0.2 µm filter, aliquot as desired , and store at −20 °C. Avoid repeated freeze–thawing.CnT-40 medium: thaw the CNT-40 medium at 2–8 °C overnight. Then, add 5 mL of penicillin–streptomycin 100× to 500 mL of CnT-40 medium. Mix by inverting, and prepare aliquots if needed. Store at 2–8 °C, and use within one month.FACS buffer: to prepare FACS buffer, add 1 mL of FBS and 25 µL of EDTA (0.5 mM EDTA) to 24 mL of DPBS. Mix well by inverting. Always prepare a fresh solution.2 to coat the cultureware. For coating of the T75 culture flask, dilute 75 µL of iMatrix-511 in 8 mL of 1× DPBS. Coat the T75 flask with 8 mL of dilute iMatrix-511 (0.5 µg/cm2) solution for 1 h at 37 °C, 5% CO2 in the incubator, or at 4 °C overnight. After incubation, remove the iMatrix-511 solution, and add 10 mL of CnT-40 medium until use.Laminin-coated plates: coat the culture plates with iMatrix-511 according to the manufacturer’s instructions. We used 0.5 µg/cmThe dissection of the limbus and preparation of limbal clusters is similar to the previously published procedure ,22.A. 1.2) or the presence of neurological disorders or malignancies in the donors can be used. Donor cornea remnants after corneal endothelial transplant preparation are also a valuable source if appropriate research consent has been obtained.Organ-cultured corneoscleral tissues  were obtained from the cornea bank with appropriate research consent and ethical approval . TissuesTissueB. 2.Place the organ-cultured corneoscleral tissue in a 60 mm culture dish, and wash twice with DPBS. Using a scalpel blade and forceps, scrape the posterior side  of the tissue to remove unwanted tissue, such as trabecular meshwork and iris/ciliary tissue;3.Place the tissue in a new 60 mm culture dish containing 1× DPBS, and cut the tissue into four equal quarters A of cornNote: to obtain adequate LMs for downstream applications, use 4–6 corneoscleral tissues.4.Make incisions of corneoscleral segments at 1 mm before and beyond the anatomical limbus to obtain limbal segments C.Dissection of limbusC. 5.2 to digest the stromal collagen and obtain limbal cell clusters;Place the limbal segments in a 60 mm dish with 5 mL of collagenase A (2 mg/mL), and cut them into smaller pieces  with a scalpel blade. Incubate overnight at 37 °C with 5% CO6.After overnight incubation (~16–18 h), triturate the suspension with an up-and-down motion using a 1 mL pipette (P1000), and examine for the presence of cell clusters and single cells under the microscope D;Note: If incomplete digestion of limbal segments occurs after overnight incubation trituration, re-incubate for an additional 2 h in the same solution at 37 °C with 5% to ensure complete digestion. In contrast, prolonged digestion of tissue (more than 20 h) may have a negative impact on cell viability and quality.7.To separate limbal cell clusters from single cells, filter the tissue digest using 20 µm cell filters that allow single cells to flow through while retaining the clusters;8.To remove any remaining single cells, wash the filters twice with DPBS;9.Place the strainer in the opposite direction on a 60 mm dish. Add 5 mL of 0.25% trypsin–EDTA to flush clusters E into a Note: In place of 20 µm cell filters, 37 µm reversible cell strainers can be used. Do not allow the filter to dry as this might cause cell death. The filtered single-cell suspension can be discarded or used for other purposes, such as obtaining limbal fibroblasts.10.After incubation, triturate the cell suspension with an up-and-down motion using a 1 mL pipette. Observe the cell suspension under the microscope to ensure the complete dissociation of clusters into single cells F. Add 5 2 to achieve complete dissociation. Prolonged incubation of clusters in trypsin–EDTA might adversely affect cell viability and quality.Note: If clusters are not completely dissociated after 15 min of incubation and trituration, re-incubate for an additional 5 min in the same solution at 37 °C with 5% CO11.Filter the cell suspension using a 40 µm cell strainer into a 15 mL Falcon tube to remove any cell clumps. Wash the cell strainer with DPBS twice;12.g for 5 min at room temperature;Centrifuge at 200× 13.After centrifugation, remove the supernatant carefully, and resuspend the cell pellet in 200 µL of ice-cold FACS buffer see  by pipetPreparation of limbal cell suspensionD. 14.Transfer the cell suspension to FACS tubes (100 µL/tube). Add a mouse APC-conjugated anti-human CD90 antibody (5 µL/106 cells) and PE-conjugated anti-human CD117 antibody (5 µL/106 cells) to one tube and an IgG2a-Isotype APC and an IgG2a-Isotype PE to another tube at 4 °C;7 cells/mL. If the cell number is high, adjust the volumes and concentration of antibodies accordingly.Note: The cell density should be <1 × 1015.Incubate the cells on ice for 45 min, agitating every 15 min16.g at 4 °C. Repeat the washing process twice;After incubation, add 1 mL of FACS buffer to each FACS tube, and centrifuge for 5 min at 400× 17.After washing, add 500 µL of FACS buffer containing DAPI (1:5000) to the cell pellet to exclude dead cells, and proceed to flow sorting using a FACS Aria II sorter ;18.Set up the FACS Aria II machine according to manufacturer’s instructions;19.Analyze the limbal cell suspensions. Set the gate on forward scatter (FSC-A) and side scatter (SSC-A) to select cells of interest based on size and granularity A. To rem20.− CD117+  when stained with the corresponding isotype control antibody.21.− CD117+ cells into 2 mL of FACS buffer in FACS tubes.Sort the CD90− CD117− populations mainly contain LEPCs and can be used to enrich LEPCs using cell type-specific media, whereas the CD90+ population containing LMSCs can be used for the expansion of LMSCs in cell type-specific media [Note: the CD90Fluorescence-activated cell sorting (FACS)E. 22.− CD117+ LMs into a well of a 12-well plate coated with i-Matrix-511  to expand LMs. Change media every 2 days. Visualize the morphology of LMs using phase-contrast microscopy.Cultivate the LMs at 37 °C with 5% COExpansion of limbal melanocytesF. 24.Remove the media from the culture vessel at 70 to 80% confluency;25.2 for 5 min;Wash the cells using DPBS, and add 1 mL (per well 12-well plate) of trypsin–EDTA . Incubate at 37 °C with 5% CO26.After incubation, add 1 mL of DMEM containing 10% FBS to inhibit trypsin action, and mix well;27.g for 5 min. Remove the supernatant carefully, resuspend the cell pellet in CnT-40 medium and count the total cell number using a hemocytometer;Transfer the cell suspension to a 15 mL tube, and centrifuge at 200× Note: after 10 to 15 days of culture (roughly 70 to 80% confluency), the number of cells obtained from the well of a 12-well culture plate ranges from 40 to 60,000 cells.28.3cells/cm2, i.e., 50,000 cells/T25 flask) coated with i-Matrix-511 in CnT-40 medium, and change the media every other day;Seed the cells in T25 flask  and prolonged trypsin digestion (more than 5 min) adversely affect cell viability and the quality of cells during sub-culturing. Always passage cells at 70 to 80% confluence. Avoid prolonged incubations in trypsin. We recommend the seeding density of 2 × 1029.2 for 5 min;After 10 to 14 days, repeat the steps from 24 to 27. Use the cells for the application of choice or sub-culturing. For subculturing, seed all the cells in T75 flask coated with i-Matrix-511 see  in CnT-430.3 cells/cm2) until the desired number of LMs is achieved for application of choice. Melanocytes take 10 to 14 days to reach confluence (with seeding density of 2 × 103 cells/cm2). The culturing and passaging of LMs can be carried out for 24 months without any significant changes in cell proliferation or behavior;Passage the cells  per limbus varied between samples. The cultured CD90− CD117+ cells appeared large and flattened, with smooth bodies and multiple dendrites under phase-contrast microscopy  . Briefly7+ cells D. Limbal7+ cells E. The nucroscopy A and staMelan-A+ B, which − CD117+ cells, immunostaining confirmed the expression of human melanoma black-45 (HMB-45), SRY-box transcription factor 10 (Sox10), and tyrosinase-related protein 1 (TRP1) (green) (2) . In all (green) C. The seeen) (2) D. The coeen) (2) E. These During the isolation of LMs from donor limbal tissue, reagent preparations, or the culture of LMs, some problems described in In conclusion, we described a step-by-step protocol for obtaining pure and functional LMs from organ-cultured corneoscleral tissues in less than a day. The unpassaged LM populations obtained enable direct transcriptomic analysis, proteomic profiling, stem cell-based tissue engineering approaches, and functional studies to further explore melanocytes’ role in the limbal stem cell niche.


Class I is divided into three subtypes (types I-III) that have diverse mechanisms of sensing preQ1, which is involved in queuosine biosynthesis. To provide insight into translation control, we determined a 2.30 Å-resolution cocrystal structure of a class I type III preQ1-sensing riboswitch identified in Escherichia coli (Eco) by bioinformatic searches. The Eco riboswitch structure differs from previous preQ1 riboswitch structures because it has the smallest naturally occurring aptamer and the SDS directly contacts the preQ1 metabolite. We validated structural observations using surface plasmon resonance and in vivo gene-expression assays, which showed strong switching in live E. coli. Our results demonstrate that the Eco riboswitch is relatively sensitive to mutations that disrupt noncanonical interactions that form the pseudoknot. In contrast to type II preQ1 riboswitches, a kinetic analysis showed that the type III Eco riboswitch strongly prefers preQ1 over the chemically similar metabolic precursor preQ0. Our results reveal the importance of noncanonical interactions in riboswitch-driven gene regulation and the versatility of the class I preQ1 riboswitch pseudoknot as a metabolite-sensing platform that supports SDS sequestration.Riboswitches are small noncoding RNAs found primarily in the 5′ leader regions of bacterial messenger RNAs where they regulate expression of downstream genes in response to binding one or more cellular metabolites. Such noncoding RNAs are often regulated at the translation level, which is thought to be mediated by the accessibility of the Shine-Dalgarno sequence (SDS) ribosome-binding site. Three classes (I-III) of prequeuosine Riboswitches are small noncoding RNAs found primarily in the 5′-leader regions of bacterial messenger RNAs , 2. Thes1 (preQ1)-sensing riboswitch family was one of the first to be identified (1 family can be divided into three classes. All three sense preQ1 (A). Lack of queuosine can diminish bacterial virulence  riboswitch is the smallest known naturally occurring ligand-binding aptamer and the most abundant of the three preQ1 classes as indicated by its broad distribution across numerous bacterial phyla (B). High-resolution crystal structures of class I type II preQ1-I (preQ1-III) riboswitches from Thermoanaerobacter tengcongenesis (Tte) (C) and Bacillus subtilis  , 14  C and Bacsubtilis  revealedigations , 18, 19,igations  and biopigations  that proigations , 19, genigations , 18, andigations , 21. In function , 23.1  riboswitches sense two equivalents of preQ1 through interactions in which metabolites hydrogen bond and π-stack with one other in the same binding pocket . This work demonstrated how structural studies on different sequence cohorts within the same riboswitch class can reveal new modes of RNA-small molecule recognition, providing insight into the chemical diversity of RNA, and its potential for ligand recognition and catalysis in an RNA world  riboswitch subclass was identified in a bioinformatic search carried out after the type I and II subgroups were identified (1-IIII riboswitch. The type III covariation model predicted the presence of an H-type pseudoknot fold similar to types I and II (B). However, the type III riboswitch has a shorter pyrimidine-rich hairpin loop and a longer 3′-tail having a variable length that comprises A-rich sequences that link the P1 helix to the expression platform (B). The ability of the preQ1-IIII riboswitch to bind preQ1 was validated previously using in-line probing with representative sequences derived from Shigella dysenteriae  (The class I type III preQentified . CurrentI and II B. Howeveplatform B. The abenteriae  and isot cloacae . Each teene yhhQ . This obnd preQ0 A 13)..1  sequestration that represses gene expression, we determined the cocrystal structure of a preQ1-IIII riboswitch from Escherichia coli, termed Eco, to 2.30 Å resolution. Phasing by single-wavelength anomalous diffraction (SAD) used Mn2+ ions that mimic naturally occurring divalent ion binding sites. The structure revealed an H-type pseudoknot fold that recognizes preQ1. The type III Eco riboswitch sequesters its SDS through predicted  and a reporter-gene assay in live E. coli to detect riboswitch function. Mutations that disrupt the noncanonical interactions in the binding pocket were tolerated poorly compared to other class I riboswitches. Our results also revealed a high level of kinetic discrimination for preQ0 relative to preQ1. Overall, our findings detail an economical aptamer that directly utilizes its expression platform for metabolite sensing, underscoring the diverse strategies by which riboswitches control translation.To investigate the mode of metabolite recognition by preQredicted  pseudoknswitches , 30, 31.1 recognition for class I type I (preQ1-II) riboswitches that went undiscovered for 15 years  that offsets an unfavorable entropy  (A). The equilibrium binding constant is consistent with our previous ITC assays for a preQ1-IIII riboswitch from E. cloacae, which had a KD of 72 nM (We next used ITC to characterize ligand binding by the  binding . The WT l mol−1) A. The eqof 72 nM .Table 1IEco riboswitch and other type III riboswitch sequences were refractory to crystallization (data not shown). The covariation model suggests that three positions of the SDS, 5′-GGA-3′, form the pseudoknot helix P2  connected to the base of the P1 helix via a linker having variable length and sequence (B). Based on available structures for class I type I and II riboswitches from Can . A single G deletion  enhanced the KD to 33.4 ± 1.9 nM (B). A ΔGG double deletion mutant  had a markedly improved affinity with an average KD of 14.0 ± 1.6 nM (C). Finally, the triple deletion mutant ΔGGU  had higher affinity relative to WT with an average KD of 30.2 ± 1.3 nM, which was comparable to the 35-mer ΔG single mutant (D). Thus, shortening the poorly conserved linker indeed improved the affinity of preQ1-IIII riboswitches for preQ1.To test this hypothesis, we generated three mutants with successively longer nucleotide deletions in the linker and tested the preQUAAUC-3′ A. A sing± 1.9 nM B. A ΔGG ± 1.6 nM C. Finalle mutant D. Thus, 1, only the 34-mer double mutant readily crystallized. However, crystals were sensitive to cryoprotection even after dehydration . Placement of adenines in the proper register is known to be important for minor-groove stabilization at P1. Moreover, the binding-pocket floor of type I and II riboswitches adopts a quintuple-base transition motif .Importantly, our 30-mer gulation B.Eco riboswitch sequence showed marginally better preQ1 binding compared to WT with an average KD of 51.9 ± 1.8 nM (E). This binding constant suggests that the length and sequence of the linker region allows metabolite binding similar to WT but is not as ideal as the double-deletion 34-mer (A and C), which does not appear to occur naturally because binding could be too tight to allow effective gene regulation. Nonetheless, under low-salt conditions, the Eco 30-mer produced well-diffracting crystals.Consistent with this possibility, the 30-mer ± 1.8 nM E. This bn 34-mer , A and CEco riboswitch would adopt the same H-type pseudoknot fold as other class I preQ1 riboswitches (1 riboswitches (B). To circumvent this issue, we crystallized the 30-mer Eco crystal with MnCl2 for SAD phasing. We previously demonstrated for the Tte riboswitch that Mn2+ ions can act as a proxy for Mg2+ through coordination at magnesium-binding sites . To our knowledge, this is this first report of an RNA structure solved using Mn2+ SAD phasing.Despite the prediction that the switches , moleculswitches B. To cirng sites . Indeed,geometry , althoug to Mg2+ . In the 1-IIIIEco riboswitch was refined to 2.30 Å resolution. The Rwork/Rfree values were 20.5%/25.2% with bond and angle RMS deviations from ideality of 0.003 Å and 0.524˚ (A); chain B also lacked density for the C12 base (B). In the P1 helix, position U1 does not form the expected canonical base pair with A19. Instead, U1 is disordered, causing A19 to engage in a crystal contact with the Hoogsteen edge of A19 from a symmetry-related molecule (C).The structure of the 30-mer preQd 0.524˚ . Two RNAd 0.524˚ A; chain C12 base B. In themolecule C.Table 2Eco preQ1-IIII riboswitch folds as an extraordinarily compact H-type pseudoknot characterized by numerous noncanonical base interactions (A and B). The fold involves a short, conserved P1 helix that transitions through a single-nucleotide L1 loop at U6 into the P2 (pseudoknot) helix, which comprises canonical and noncanonical base pairs. At only three nucleotides, the L2 loop is especially short and the nucleobase of each loop points toward the bound preQ1 metabolite. Following the 3′-end of helix P1, the backbone makes a sharp bend into loop L3, which exhibits a series of contacts including A-amino kissing interactions (A and B) that are functionally equivalent to those seen in type I and type II preQ1-I riboswitches (A).The ractions , A and Bractions , A and Bswitches , 26. Posswitches  that linswitches A.Figure Eco riboswitch molecules in the asymmetric unit contained four site-bound Mn2+ ions at different locations (A). In anomalous difference Fourier maps, all Mn2+ ions exhibited strong anomalous scattering with peak heights of 6.5σ (site I), 8.5σ (site II), 8.5σ (site III), and 6.0σ (site IV). At site I, the ion coordinates N7 of G4 in helix P1 (D), consistent with findings that Mn2+ strongly coordinates N7 of guanine nucleotides in the major groove . These observations suggest that Mg2+ binds at this position and appears to stabilize the top of the stem to facilitate interaction between the preQ1 exocyclic amine and the G5 O6 keto group (C–E).Each of the two ocations A. In anohelix P1 D, consisr groove , 37. Theg pocket B and 3C.to group , C–E.2+ ions at sites II, III, and IV do not appear to contribute to the stabilization of the overall fold. Instead, these ions engage in crystal contacts. Mn2+ at sites II and III coordinate N7 of G30 in chain A and a nonbridging oxygen of the phosphate backbone at C9, which is donated by the crystallographically related chain B (B). Intermolecular Mn2+ coordination connects the flush-end stack between the C9-G30 Watson-Crick base pairs that cap the P2 stems of chains A and B. The Mn2+ ion at site IV also mediates a crystal contact between nonbridging oxygens of A18 and A19 in chains A and chain B, which form inner-sphere ion contacts (C). Taken together, these results explain the requirement of Mg2+ or Mn2+ ions for crystallization of the Eco preQ1-IIII riboswitch.In contrast to site I, Mn chain B B. Intermcontacts C. Taken Eco riboswitch superimposes with representative chains from the Can preQ1-II and Tte preQ1-III riboswitch cocrystal structures with average rmsd values of 1.51 Å and 1.69 Å for all paired atoms (A). The largest conformational differences localize to helix P2 where the type I Can riboswitch has three additional nucleotides at positions 9 to 11 that expand the backbone of the pseudoknot loop  to accommodate a second preQ1 metabolite . By contrast, the type III Eco riboswitch adopts a very tight pseudoknot loop that allows binding of only a single metabolite . The type II Tte riboswitch also binds one metabolite but has a loop of intermediate size relative to Eco and Can .The ed atoms A. The lanot loop B, yellowtabolite B, purple and Can B, pink.FC). Here, the Eco riboswitch backbone conformation most closely resembles that of the Can riboswitch, which shows a one-to-one correspondence for each nucleotide  although the sequences are different . In general, the Eco riboswitch makes tighter turns with fewer nucleotides than its type I and type II subclass counterparts.The P1-to-L3 turn also exhibits different conformations among the riboswitches C. Here, Eco-binding pocket to other class I riboswitches reveals a striking similarity with the α-site–binding pocket of the type I Can and type II Tte riboswitches (D). Each riboswitch has a highly conserved cytidine in L2 acting as a specificity base that reads out the Watson-Crick-face of preQ1 through canonical hydrogen bonding. Meanwhile, highly conserved adenines from L3 and uridines from L1 recognize the metabolite via its minor-groove edge equivalent (D). The only difference among the three binding sites is the orientation of the flexible methylamine group (D), which is likely caused by differences in the local charge environment. In particular, the Eco riboswitch forms salt-bridge interactions between the charged methylamine and the phosphate backbone at position 12 (E), and these interactions are absent in the Can and Tte riboswitch structures . In E. coli bacteria, the SDS consensus is 5'-(A/C)(A/U)GGA(A/G)AA (B) and the Eco riboswitch here. This observation provides a direct link between effector binding and gene regulation that is not a feature of the other preQ1-I subtypes AA , which cA(A/G)AA B and thesubtypes , 26. Simswitches , 31, alttabolite .Eco riboswitch, the SDS extends into the pocket ceiling, such that the second position of the SDS, A28, is sequestered in a base quartet that stacks atop preQ1 (A). This feature is reminiscent of the Tte riboswitch (B) but differs from the base-triple ceiling of the Can riboswitch, which sits above the β-site preQ1 (C). The core of the Eco riboswitch ceiling is a base triple that involves C8 and U11 from the anti-SDS  and A28 of the SDS (A). Additionally, the Watson-Crick face of A13 from L2 contacts the minor groove of U11 (A). In the Tte riboswitch, the core of the ceiling includes a G11-C30 canonical pair that forms a base quadruple in which C7(+) pairs with the Hoogsteen edge of G11, and the WC face of A14 of loop L2 interacts with the minor-groove edge of G11 (B). The latter interaction resembles U11 and A13 of the Eco riboswitch (A). Notably, the Tte riboswitch structure determined from crystals grown in the absence of preQ1 . The first two positions of the SDS, A27 and A28, engage in base triple interactions, while the final two guanines, G29 and G30, engage in cis Watson Crick interactions with C10 and C9 (D). Watson Crick pairing between U11-A28, C10-G29, and C9-G30 was inferred from the covariation model (B) built during the initial classification of this subtype (1 and C8•A28-U11 were only apparent in the structure shown here (D). Collectively, these and other similar interactions explain the unusual pyrimidine-rich composition of the aptamer loop that extends from nucleotides 6 through 12 of the Eco riboswitch (A).The terparts , D–F. Th0 and C9 D. Watsonon model B built down here D. Collecboswitch A.Eco riboswitch is quite strong compared to other preQ1-I riboswitch subtypes, in which only the first two SDS nucleotides are integrated into pseudoknot folds (E and F). Indeed, the Tte preQ1-III riboswitch strongly sequesters G33 via a Watson-Crick interaction with C9, whereas the first position, A32, has only a single hydrogen bond to A10 of the anti-SDS (E). Likewise, the Can preQ1-II riboswitch firmly sequesters the second SDS position, G34, via a strong Watson-Crick pair with C10. However, A33 in the first position of the SDS engages its Watson-Crick face with the sugar edge of G9 and the Watson-Crick face of A11 through three hydrogen bonds (F) ..Eco riboEco riboswitch differs from the type II Tte and type I Can riboswitches. Relative to the Eco riboswitch, both Tte and Can riboswitches have a less severe bend of the P2-helix hairpin due to the presence of a guanine (G8 in Tte and G9 in Can) that lies directly above the pocket ceiling where the N1 imino group can interact with a nonbridging phosphate oxygen of the backbone in P2 . The minor-groove of this guanine also interacts with the Watson-Crick face of a cross-strand adenine (A31 in Tte and A33 in Can) (E and F). Another adenine (A10 in Tte and A11 in Can) stacks between this noncanonical G•A pair and the terminal G-C pair and donates a single hydrogen bond to the first position of the SDS (A32 in Tte and A33 in Can) (E and F). By contrast, the Eco riboswitch, like all preQ1-IIII sequences (B) that cannot form these purine-mediated contacts (D) and thus has a more compact pseudoknot with more canonical pairing that together are predicted to render the Eco riboswitch more resistant to ribosome helicase activity.In addition to differences in SDS sequestration, the overall architecture of the P2 helix in the  in Can) , E and F in Can) , E and Fequences B that cacontacts D and thu1-IIII riboswitches for preQ0—the metabolic precursor of preQ1 (A)—would be lower than that for preQ1 due to geometric and electronic differences between the nitrile and the methylamine groups at the C7 position of the pyrrole ring (A and B). This possibility is intriguing since proteobacteria like E. coli lack queT transporter genes and instead utilize yhhQ genes to salvage preQ1-related molecules from the environment. Notably, a recent study demonstrated that yhhQ genes encode a protein that imports preQ0 with preference over preQ1 , was shown to have relatively high affinity for preQ0   . ConsideEco sequence for preQ1. The results revealed that the WT Eco riboswitch binds preQ1 with an apparent KD of 120 ± 6 nM, which is consistent with ITC measurements (A and 2A). The WT Eco riboswitch kon and koff for preQ1 is 66.1 × 103 M–1 s–1 and 7.9 × 10-3 s–1, corresponding to t1/2 of 88.4 s. The Eco riboswitch kon is similar to that for the Tte riboswitch (kon 77.7 × 103 M–1 s–1) (t1/2 is shorter due to its 50-fold difference in koff. This difference in dissociation rate is likely associated with the more stable fold of the thermophilic Tte riboswitch than that of Eco.We first used SPR to measure binding kinetics of the WT urements A and 2A.M–1 s–1) , but its0 binding to the Eco riboswitch showed lower affinity for preQ0 than preQ1 as demonstrated by a slower kon (9.8 × 103 M–1 s–1) and faster koff (155 × 10–3 s–1) for preQ0 that corresponds to a t1/2 of 4.5 s (B). Accordingly, the apparent KD of the Eco riboswitch for preQ0 was 15.9 ± 2.3 μM, which represents nearly a 133-fold loss in affinity compared to preQ1 (Tte riboswitch had a comparable kon for preQ0 (9.8 × 103 M–1 s–1versus 6.5 103 M–1 s–1), the Eco koff value was 44-fold slower (2.22 × 10–4 s–1), resulting in a t1/2 of 54 min. These results demonstrate that the type III Eco riboswitch strongly discriminates between preQ0 and preQ1 largely through the relatively fast dissociation of the preQ0 metabolite (B).The binding kinetics for preQof 4.5 s B. Accordto preQ1 . Althougtabolite B.0 discrimination, we next considered how the preQ0 metabolite would fit into the binding pocket in the Eco cocrystal structure. The flexible 7-aminomethyl group of preQ1 lies in a cleft between L2 and P1 where it acts as a hydrogen bond donor to O6 of G5 in the binding pocket floor and a nonbridging phosphate group of C12 in loop L2 (C). Although our experiments suggest that preQ0 can be accommodated in the binding pocket, the nitrile group at the 7-position of preQ0 is unprotonated (B), which precludes the hydrogen bond formation required for favorable binding to and specificity for preQ1 (C). The linear sp geometry of the preQ0 nitrile moiety likely causes a steric clash with the backbone based on modeling (D). These factors plausibly contribute to the 6.7-fold slower binding and faster off-rate for preQ0 than to preQ1 , we generated a series of homologous mutations that were probed by SPR to examine how conserved metabolite-binding nucleotides in the Eco riboswitch affect preQ1 recognition.Based on the structural similarity between the binding pockets of the switches , which bswitches D, we genEco sequence . This canonical pair contributes to the binding pocket floor (E and 4D) and is invariant across all preQ1-I subtypes (B) , or with N7 of G5 as observed in previous preQ1-I riboswitch structures  (1 affinity by around 645-fold compared to WT (KD of 77.4 ± 5.9 μM versus 0.12 μM for WT) , but this substantially reduced affinity highlights the role of the O6 keto group of G5 as an important determinant of preQ1 specificity and affinity that likely discriminates against other purine-like metabolites.We first analyzed the importance of the G6-C16 base pair in the WT et floor E and 4D subtypes B 9). Th. ThEco structure E, or witructures , 33. To ase pair D 13). A. AEco se and A26 C, but thA), which would disrupt the single hydrogen bond with the minor-groove edge equivalent of preQ1 (E). The U7C mutant indeed reduced affinity for preQ1, with a 35,000-fold change compared to WT . The analogous mutation in the Tte riboswitch also showed poor affinity with an estimated KD >274 μM, representing an approximate 134,000-fold decrease in binding , which would be disrupted by a U-to-C mutation. Together, these observations suggest that the quintuple-base transition motif involves U6 to form a stable floor of the binding pocket and plays a key role in connecting the aptamer to the P1 helix, which is likely essential to stabilize the pseudoknot fold.We next mutated U7 to cytosine , which is numbered A27 in the cocrystal structure (used here for convenience). Specifically, N1 and N6 groups of A27 hydrogen bond to N2 and N3 of preQ1 (E). The A27 Hoogsteen edge also interacts with the Watson-Crick face of U7 (D), similarly to the U6•A26 interaction directly below it . To ameliorate the loss of affinity caused by disruption of Hoogsteen edge interactions in the U6C mutant, we generated an A27Pur (purine) mutant to partially maintain the U7•A27 interaction while weakening preQ1 recognition by removing only the A27 exocyclic amine. The A27Pur mutant had a 39,000-fold reduction in affinity relative to WT  that represents the specificity base for preQ1 (E). The consensus model for type III riboswitches indicates 97% conservation at C15, which is hypothesized to recognize preQ1 through Watson-Crick readout of the ligand’s guanine-like face, akin to the Tte and Bsu type II riboswitches  , we placed the aptamer upstream of a GFPuv reporter gene to assess its function in live E. coli. The WT Eco riboswitch produced a preQ1-dependent dose response in which GFPuv emission decreased in the presence of the metabolite (B). The WT EC50 was 291.4 ± 1.6 nM with a 6.1-fold overall repression by preQ1 , but the Eco riboswitch had a 2.4-fold reduction in overall gene repression  and produces its highest repression at preQ1 levels over the EC50,2 of 7.1 ± 0.4 μM  each showed significantly higher GFPuv fluorescence than WT but neither showed gene regulation, even at saturating levels of preQ1 , whereas C16U disrupts the P1 stem in the pocket floor . Notably, a previous study observed a similar effect for the preQ1-II riboswitch when the anti-SDS sequence was mutated to inhibit pairing with the SDS  showed no gene regulation under saturating preQ1, as indicated by its flat dose response (B). Here, GFPuv fluorescence remained comparable to the WT gene-on state across all tested preQ1 doses. In the SPR analysis, we replaced A33 with a non-natural purine nucleobase, but for in vivo assays, we made the A33C mutation since cytosine preserves the SDS consensus 5'-(A/C)(A/U)GGA(A/G)AA  and ablates Hoogsteen edge interactions with U8 .The A33C mutant A showed response B. Here, A(A/G)AA  and can A and B). The C15 mutant had an estimated EC50 of 90.6 ± 0.4 μM, which is approximately 311-fold weaker than WT (D). Full repression was not achieved even at saturating preQ1 levels of 1 mM, and thus the EC50 value could be considerably higher. As with the other mutants, the fold-repression was extremely weak (C). C-to-U mutations in the specificity bases of the type I Can riboswitch also increased EC50 values, but only by ∼60- (C17U) and ∼210-fold (C31U) over WT, corresponding to 6-fold and 2-fold repression  but also promotes a new cross-strand interaction with A33  that competes with preQ1 binding. This possibility is consistent with the substantially weakened ligand-binding properties of this mutant measured by SPR (E).Interestingly, mutation of the specificity base, C15, to uracil was the only mutant that showed a dose response , A and B than WT D. Full rely weak C. C-to-Upression . Our Ecod by SPR E.Eco riboswitch in complex with preQ1, the first cocrystal structure of a preQ1-IIII (class I type III) riboswitch bound to preQ1. The structure and function analysis is the first major characterization of a type III preQ1 riboswitch since type III (preQ1-IIII) was first described as a distinct class I subgroup . The P1 helix is fortified in the minor groove by multiple A-amino kissing interactions from the A-rich L3 loop (B) that culminate in a quintuple-base transition motif that forms the binding pocket floor of the riboswitch . Perhaps most remarkably, all three riboswitch types use common nucleobase determinants to recognize at least one equivalent of preQ1 in a homologous-binding pocket designated as the α site (D)  that appears to stabilize the binding pocket floor to facilitate preQ1 sensing (D). An equivalent Mn2+ site was observed in the Tte preQ1-III riboswitch  , 21, 33. sensing D. An equEco riboswitch lacks several key interactions found in type I and II class I preQ1 riboswitches, suggesting that it may function as a ‘bare bones’ aptamer. This moniker is supported by the lower tolerance of the Eco riboswitch to mutations than representative type I Can and type II Tte riboswitches. As noted for preQ1 binding, U6 and A27  recognize the minor groove-edge equivalent of the metabolite but each position also engages in noncanonical interactions (E and 5D). In the Eco riboswitch, U6 pairs with the Hoogsteen edge of A26 in the binding pocket floor in a manner similar to U6 and A29 in the Can riboswitch, while the Hoogsteen edge of Eco A27 pairs with the Watson-Crick face of U6, similar to U7 and A30 in the Can riboswitch. However, the Eco riboswitch has poorer stacking upon the metabolite, wherein U11 sits above preQ1 and makes a canonical pair to A28, as well as a sugar-edge contact with A13 (A). By contrast, the type II Tte and type I Can riboswitch ceilings each stack a purine upon preQ1, which is held in place by base quadruple or triple interactions (B and C). Moreover, the Can riboswitch evolved to bind two stacked metabolites, adding another tier of interactions that are absent in type II or III aptamers (C and D and 3B). Hence, the dual metabolite recognition and RNA-folding roles of nucleobases U6 and A27 in the Eco riboswitch appear to render them less tolerant of mutations, as observed by the substantial loss of affinity for the U6C and A27Pur mutations (C and D). Similarly, the equivalent U7C and A33C mutants were each unresponsive to preQ1 in bacterial reporter-gene assays and had unregulated GFPuv expression, even in the presence of saturating amounts of preQ1 (B).Despite similarities with other riboswitches, the type III ractions E and 5D.with A13 A. By conractions , B and Cutations , C and Dof preQ1 B.1-III riboswitch gene regulation is widely assumed to be representative of the entire class . Some riboswitches, such as the cobalamin riboswitch . The mode of 5′-SDS sequestration differs between the two folds, but each has the first position of the SDS, an adenine, engaged in a noncanonical interaction and the second position, a guanine, engaged in a canonical Watson-Crick base pair (E and F). However, the Eco preQ1-IIII riboswitch integrates the SDS directly into the binding pocket, where the 5′-most adenine contacts the preQ1 metabolite via two hydrogen bonds (E). Thereafter, the SDS extends into the ceiling to participate in a base quartet (A) and is sequestered strongly by canonical Watson-Crick interactions that compose the remainder of P2 (D). Direct metabolite sensing by the SDS is rare but has been observed in the SAM/SAH riboswitch (+-II riboswitch (Eco riboswitch is somewhat distinct because the Watson-Crick face of the 5′-SDS adenine (A27) directly reads the preQ1 edge to confer specificity and affinity (E).Although the mode of preQre class , 21, 33,switches , D–F. Soboswitch , SAM-IIIboswitch , NAD+-IIboswitch , and preboswitch , use theboswitch , the THFboswitch , the TPPboswitch , and SAMboswitch , place te ligand , 47  . In the context of the Eco-binding pocket, the preQ0 nitrile group faces the O6 group of G5 and the negatively charged backbone at C12 (D), which are receptive to hydrogen bond donors but not to hydrogen bond acceptors. The noncomplementary charge between the nitrile group and the oxygen-rich RNA environment provides a plausible basis for the 20-fold increase in koff of preQ0 compared to preQ1  and exhibits no equivalent phosphate backbone interaction with the preQ1 methylamine. Accordingly, the nonbridging oxygen of C12 in the Eco riboswitch likely serves as a major specificity determinant. These results lead us to hypothesize that the yhhQ transport gene may have a preQ1 import preference in a cellular context.When this study was initiated, there was some ambiguity concerning type III riboswitch specificity, in part because the preQor preQ0 . Moreoveer preQ0 . These fanalysis . Our anaFig. 4D) , 21. Theotonated B. In thee at C12 D, which to preQ1 . Notably1 pocket shared by type I, II, and III riboswitches, as revealed by this investigation (D). The preQ1-II riboswitch shows the highest number of riboswitch sequences per nucleotide (B), as well as greater phylogenetic diversity than all other preQ1-I subtypes  suggest that the β-binding site was lost more than once during bacterial evolution.We next considered the evolution of the α-site preQtigation D. The prcleotide B, as welsubtypes . Type I ia class , which iia class . By contia class . Given ting site , we specswitches , B and C1-I riboswitch types adopt the same fold (A), the structural data reveal that, despite their common α-site preQ1-binding pockets, these three preQ1 riboswitches have three different modes of SDS sequestration (D–F). Accordingly, our results demonstrate that riboswitch subgroups within the same class can produce substantially different modes of gene regulation and metabolite recognition  in the syringe. A total of 19 injections were made with a 0.5 μl technical injection and a 4 μl volume was used for all subsequent injections that were spaced 150 s apart. Best fits of thermograms were determined using a single-sites–binding model with the MicroCal analysis software for the PEAQ-ITC . All experiments were performed in duplicate. Thermodynamic parameters are listed in Lyophilized RNA was prepared for ITC experiments as described . A typicEco 30-mer RNA was dissolved in 20 μl 0.01 M Na-cacodylate pH 7.0. The RNA concentration was adjusted to 0.5 mM and the solution was heated at 65 °C for 3 min in an aluminum heat block. A folding mix comprising 0.01 M Na-cacodylate pH 7.0, 0.004 M MgCl2, and 0.1 M preQ1 was also heated at 65 °C for 3 min before an equal volume was added slowly to the RNA with gentle mixing. The RNA-folding mix solution was heated for an additional 3 min and slowly cooled to room temperature in the aluminum block.Lyophilized 2 tetrahydrate, 31% MPD, 0.05 M Na-cacodylate pH 6.0, 0.012 spermine–HCl, 0.1 M KCl, and 0.002 M MgCl2. Crystals grew in ≥2 weeks at 20 °C and were harvested using nylon loops attached to 18 mM copper pins. The crystals were serially transferred for 3 min into synthetic mother liquors containing 35% and 40% MPD for cryoprotection and then flash-frozen by plunging into N2 (l). Crystals were shipped to the Stanford Synchrotron Radiation Lightsource for data collection.Crystals were grown in VDX Plates (Hampton Research) using 2 μl of folded RNA at 250 μM combined with 1 μl well solution comprising 0.005 M MnClhttps://smb.slac.stanford.edu/facilities/software/xds/#autoxds_script), which uses XDS and the CCP4 programs POINTLESS, AIMLESS, and TRUNCATE  between half datasets dropped below a threshold of 0.15. Phenix.HySS  riboswitch was superimposed on chain A of the bound-state Can (type I) riboswitch (PDB code 7REX) (Can riboswitch. Chain A of the Eco riboswitch was superimposed onto chain A of the bound-state Tte (Type II) riboswitch (PDB code 6VUI) .Phasing efforts using molecular replacement were unsuccessful based on search models derived from preQswitches , 21, 26.nix.HySS  identifi of 0.30 . The Phe of 0.30  noise-fi of 0.30  produced of 0.30  produceding COOT , yieldining COOT  with TLSing COOT . Refineming COOT . The anox.Refine . Least-sx.Refine . In FiguEco riboswitches were dissolved in 200 μl 0.01 M Na-cacodylate pH 7.0. The RNA was folded by heating at 90 °C for 3 min and cooled quickly on ice for 3 min, during which an equal volume of ice-cold folding mix (0.01 M Na-cacodylate pH 7.0 and 0.006 M MgCl2) was added slowly. The RNA was then warmed to room temperature. The concentration of folded RNA was adjusted to 0.5 mM in SPR running buffer . Each folded riboswitch was injected over NeutrAvidin-conjugated flow cells to give a surface density of ∼ 3000 response units; the reference flow cell had only NeutrAvidin.NeutrAvidin-conjugated CM5 chips were prepared on a Biacore T200 instrument (Biacore Inc) using reagents from the manufacturer. Riboswitch samples with 5′-biotinylation were obtained in deprotected and desalted form (Horizon Discovery)  and the apparent equilibrium binding constant (KD). To evaluate the steady-state equilibrium binding data, the response during the equilibrium binding phase (Req) was calculated by averaging the response over 5 s just before the end of the metabolite injection. Equilibrium binding data were fit using nonlinear least-squares curve fitting in Prism version 9  to a one-site binding equation to determine KD. Kinetic and equilibrium binding constants are provided in All measurements were made in duplicate at 25 °C with a data collection rate of 10 Hz. Experimental data were processed using Biacore T200 Evaluation software version 3.2.0.5 . The double referencing method was used to remove instrumental and bulk shift effects . Buffer-Eco riboswitch was inserted into the pBR327-Lrh(WT)-GFPuv plasmid upstream of the GFPuv reporter gene as described (uv transcript. All constructs were verified by DNA sequencing. Sequences are provided in The WT E. coli strain JW0434-1 ΔqueC cells (Horizon Discovery), which cannot synthesize preQ0, were transformed with the desired plasmid and grown on chemically-defined salt broth (CSB) agar plates containing ampicillin (100 μg mL−1) and kanamycin (50 μg mL−1). Single colonies were isolated to inoculate overnight liquid cultures of 3 ml CSB-amp-kan media. These cultures were used to inoculate 2 ml of fresh CSB-amp-kan liquid media containing varying concentrations of preQ1: 0, 0.1 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, 500 nM, 1 μM, 5 μM, 10 μM, 100 μM, and 1 mM. A negative control was also evaluated at each preQ1 concentration wherein the WT SDS was replaced with the complementary sequence.The reporter assay was performed as described , 39 with1 concentration (Eco riboswitch and C15U curves showed single transitions described by 3 parameter fits of log(inhibitor) dose versus response.Six biological replicates were measured for each concentration. All measurements and analyses were performed as described  using Prntration . The WT p value for WT Eco versus U7C was 0.0001 . The p value for WT Eco versus C15U was 0.0009 . The p value for WT Eco versus C16U was 0.0008 . The p value for WT Eco versus A33C was 0.0012 .The Eco preQ1-IIII riboswitch structure were deposited into the Protein Data Bank under accession code 8fza.All data produced in this investigation are provided in the manuscript. Any additional information or reagents can be obtained by contacting the corresponding author. Coordinates and structure factors of the refined This article contains The authors declare that they have no conflicts of interest with the contents of this article.