Proteomic-based investigations on the mode of action of the marine anticancer compound rhizochalinin.

Rhizochalinin (Rhiz) is a novel marine natural sphingolipid-like compound, which shows promising in vitro and in vivo activity in human castration-resistant prostate cancer. In the present study, a global proteome screening approach was applied to investigate molecular targets and biological processes affected by Rhiz in castration-resistant prostate cancer. Bioinformatical analysis of the data predicted an antimigratory effect of Rhiz on cancer cells. Validation of proteins involved in the cancer-associated processes, including cell migration and invasion, revealed downregulation of specific isoforms of stathmin and LASP1, as well as upregulation of Grp75, keratin 81, and precursor IL-1ß by Rhiz. Functional analyses confirmed an antimigratory effect of Rhiz in PC-3 cells. Additionally, predicted ERK1/2 activation was confirmed by Western blotting analysis, and revealed prosurvival effects in Rhiz-treated prostate cancer cells indicating a potential mechanism of resistance. A combination of Rhiz with MEK/ERK inhibitors PD98059 (non-ATP competitive MEK1 inhibitor) and FR180204 (ATP-competitive ERK1/2 inhibitor) resulted in synergistic effects. This work provides further insights into the molecular mechanisms underlying Rhiz bioactivity. Furthermore, our research is exemplary for the ability of proteomics to predict drug targets and mode of action of natural anticancer agents.

Transcriptional Responses of Creeping Bentgrass to 2,3-Butanediol, a Bacterial Volatile Compound (BVC) Analogue.

Bacterial volatile compounds (BVCs) have been reported to enhance plant growth and elicit plant defenses against fungal infection and insect damage. The objective of this study was to determine transcriptomic changes in response to synthetic BVC that could be associated with plant resistance to Rhizoctonia solani in creeping bentgrass. The 2,3-butanediol (BD) (250 µM) was sprayed on creeping bentgrass leaves grown in jam jars. The result showed that synthetic BD induced plant defense against R. solani for creeping bentgrass. Transcriptomic analysis demonstrated that more genes were repressed by BD while less showed up-regulation. BD suppressed the expression of some regular stress-related genes in creeping bentgrass, such as pheromone activity, calcium channel activity, photosystem II oxygen evolving complex, and hydrolase activity, while up-regulated defense related transcription factors (TFs), such as basic helix-loop-helix (bHLH) TFs, cysteine2-cysteine2-contans-like (C2C2-CO) and no apical meristem TFs (NAC). Other genes related to disease resistance, such as jasmonic acid (JA) signaling, leucine rich repeats (LRR)-transmembrane protein kinase, pathogen-related (PR) gene 5 receptor kinase and nucleotide binding site-leucine rich repeats (NBS-LRR) domain containing plant resistance gene (R-gene) were also significantly up-regulated. These results suggest that BD may induce changes to the plant transcriptome in induced systemic resistance (ISR) pathways.

Visualizing fungal metabolites during mycoparasitic interaction by MALDI mass spectrometry imaging.

Studying microbial interactions by MALDI mass spectrometry imaging (MSI) directly from growing media is a difficult task if high sensitivity is demanded. We present a quick and robust sample preparation strategy for growing fungi (Trichoderma atroviride, Rhizoctonia solani) on glass slides to establish a miniaturized confrontation assay. By this we were able to visualize metabolite distributions by MALDI MSI after matrix deposition with a home-built sublimation device and thorough recrystallization. We present for the first time MALDI MSI data for secondary metabolite release during active mycoparasitism.

New insights into membrane-active action in plasma membrane of fungal hyphae by the lipopeptide antibiotic bacillomycin L.

Bacillomycin L, a natural iturinic lipopeptide produced by Bacillus amyloliquefaciens, is characterized by strong antifungal activities against a variety of agronomically important filamentous fungi including Rhizoctonia solani Kühn. Prior to this study, the role of membrane permeabilization in the antimicrobial activity of bacillomycin L against plant pathogenic fungi had not been investigated. To shed light on the mechanism of this antifungal activity, the permeabilization of R. solani hyphae by bacillomycin L was investigated and compared with that by amphotericin B, a polyene antibiotic which is thought to act primarily through membrane disruption. Our results derived from electron microscopy, various fluorescent techniques and gel retardation experiments revealed that the antifungal activity of bacillomycin L may be not solely a consequence of fungal membrane permeabilization, but related to the interaction of it with intracellular targets. Our findings provide more insights into the mode of action of bacillomycin L and other iturins, which could in turn help to develop new or improved antifungal formulations or result in novel strategies to prevent fungal spoilage.

Proteomic response of Rhizoctonia solani GD118 suppressed by Paenibacillus kribbensis PS04.

Rice sheath blight, caused by Rhizoctonia solani, is considered a worldwide destructive rice disease and leads to considerable yield losses. A bio-control agent, Paenibacillus kribbensis PS04, was screened to resist against the pathogen. The inhibitory effects were investigated (>80 %) by the growth of the hyphae. Microscopic observation of the hypha structure manifested that the morphology of the pathogenic mycelium was strongly affected by P. kribbensis PS04. To explore essentially inhibitory mechanisms, proteomic approach was adopted to identify differentially expressed proteins from R. solani GD118 in response to P. kribbensis PS04 using two-dimensional gel electrophoresis. Protein profiling was used to identify 13 differential proteins: 10 proteins were found to be down-regulated while 3 proteins were up-regulated. These proteins were involved in material and energy metabolism, antioxidant activity, protein folding and degradation, and cytoskeleton regulation. Among them, material and energy metabolism was differentially regulated by P. kribbensis PS04. Protein expression was separately inhibited by the bio-control agent in oxidation resistance, protein folding and degradation, and cytoskeleton regulation. Proteome changes of the mycelium assist in understanding how the pathogen was directly suppressed by P. kribbensis PS04.

D-Glucosamine-derived synthons for assembly of L-threo-sphingoid bases. Total synthesis of rhizochalinin C.

A five-step transformation of D-glucosamine, commencing with indium-mediated Barbier reaction without isolation of intermediates, into (R,R)-2-aminohex-5-ene-1,3-diol in 45-51% is described. The latter is a useful synthon for assembly of L-threo-sphingoid bases: long-chain aminoalkanols and aminoalkanediols with configurations antipodal to that found in mammalian D-erythro-sphingosine but prevalent among invertebrate-derived sphingolipids. The utility of the method is demonstrated by the first total synthesis of rhizochalinin C, the long-chain, "two-headed" sphingoid base aglycon of the natural product rhizochalin C from the marine sponge Rhizochalina incrustata.

Design, Synthesis, and Evaluation of the Antifungal Activity of Novel Pyrazole-Thiazole Carboxamides as Succinate Dehydrogenase Inhibitors.

Succinate dehydrogenase (SDH) is regarded as a promising target for fungicide discovery. To continue our ongoing studies on the discovery of novel SDH inhibitors as fungicides, novel pyrazole-thiazole carboxamides were designed, synthesized, and evaluated for their antifungal activity. The results indicated that compounds 9ac, 9bf, and 9cb showed excellent in vitro activities against Rhizoctonia cerealis with EC50 values from 1.1 to 4.9 mg/L, superior to that of the commercial fungicide thifluzamide (EC50 = 23.1 mg/L). Compound 9cd (EC50 = 0.8 mg/L) was far more active than thifluzamide (EC50 = 4.9 mg/L) against Sclerotinia sclerotiorum. Compound 9ac exhibited promising in vivo activity against Rhizoctonia solani (90% at 10 mg/L), which was better than that of thifluzamide (80% at 10 mg/L). The field experiment showed that compound 9ac had 74.4% efficacy against Rhizoctonia solani on the 15th day after two consecutive sprayings at an application rate of 4.80 g a.i./667 m2, which was close to that of thifluzamide (83.3%). Furthermore, molecular docking explained the possible binding mode of compound 9ac in the RcSDH active site. Our studies indicated that the pyrazole-thiazole carboxamide hybrid is a new scaffold of SDH inhibitors.

Septal pore cap protein SPC18, isolated from the basidiomycetous fungus Rhizoctonia solani, also resides in pore plugs.

The hyphae of filamentous fungi are compartmentalized by septa that have a central pore. The fungal septa and septum-associated structures play an important role in maintaining cellular and intrahyphal homeostasis. The dolipore septa in the higher Basidiomycota (i.e., Agaricomycotina) are associated with septal pore caps. Although the ultrastructure of the septal pore caps has been studied extensively, neither the biochemical composition nor the function of these organelles is known. Here, we report the identification of the glycoprotein SPC18 that was found in the septal pore cap-enriched fraction of the basidiomycetous fungus Rhizoctonia solani. Based on its N-terminal sequence, the SPC18 gene was isolated. SPC18 encodes a protein of 158 amino acid residues, which contains a hydrophobic signal peptide for targeting to the endoplasmic reticulum and has an N-glycosylation motif. Immunolocalization showed that SPC18 is present in the septal pore caps. Surprisingly, we also observed SPC18 being localized in some plugs. The data reported here strongly support the hypothesis that septal pore caps are derived from endoplasmic reticulum and are involved in dolipore plugging and, thus, contribute to hyphal homeostasis in basidiomycetous fungi.

Chelation of specific metal ions imparts coplanarity and fluorescence in two imidazo[1,2-a]pyridine derivatives: Potential chemosensors for detection of metal ions in aqueous and biosamples.

Synthesis and chelation induced fluorescence emission from two imidazo[1,2-a]pyridine derivatives are described. The nonfluorescent molecule 1 containing N and O donor atoms, achieves coplanarity upon interactions with trivalent cations Al3+, Fe3+ and Cr3+, that favors fluorescence emission. Molecule 2 containing two N donor atoms attains coplanarity upon interaction with the only Zn2+ and becomes fluorescent. Both molecules 1 and 2 form a 1:1 complex with interacting metal ions. Other trivalent metal ions (including Bi3+ and In3+) and common divalent metal ions (including Hg2+ and Cd2+) fail to form any complex with 1 or 2, and they do not interfere in the detection of Zn2+, Al3+, Fe3+ or Cr3+ ions. Noninterference of other metal ions renders 1 and 2 suitable for the detection of fungal cells contaminated with Zn2+, Al3+, Fe3+ or Cr3+ ions.

Synthesis and Biological Activity of Novel Succinate Dehydrogenase Inhibitor Derivatives as Potent Fungicide Candidates.

In searching for novel fungicidal leads, the novel bioactive succinate dehydrogenase inhibitor (SDHI) derivatives were designed and synthesized by the inversion of carbonyl and amide groups. Bioassay indicated that compound 5i stood out with a broad spectrum of in vitro activity against five fungi. Its EC50 value (0.73 µg/mL) was comparable to that of boscalid (EC50 of 0.51 µg/mL) and fluxapyroxad (EC50 of 0.19 µg/mL) against Sclerotinia sclerotiorum. For Rhizoctonia cerealis, 5i and 5p with EC50 values of 4.61 and 6.48 µg/mL, respectively, showed significantly higher activity than fluxapyroxad with the EC50 value of 16.99 µg/mL. In vivo fungicidal activity of 5i exhibited an excellent inhibitory rate (100%) against Puccinia sorghi at 50 µg/mL, while the positive control boscalid showed only a 70% inhibitory rate. Moreover, 5i showed promising fungicidal activity with a 60% inhibitory rate against Rhizoctonia solani at 1 µg/mL, which was better than that of boscalid (30%). Compound 5i possessed better in vivo efficacy against P. sorghi and R. solani than boscalid. Molecular docking showed that even the carbonyl oxygen atom of 5i was far from the pyrazole ring. It could also form hydrogen bonds toward the hydroxyl hydrogen and amino hydrogen of TYR58 and TRP173 on SDH, respectively, which consisted of the positive control fluxapyroxad. Fluorescence quenching analysis and SDH enzymatic inhibition studies also validated its mode of action. Our studies showed that 5i was worthy of further investigation as a promising fungicide candidate.

Optimized protein extraction methods for proteomic analysis of Rhizoctonia solani.

Rhizoctonia solani (Teleomorph: Thanatephorus cucumeris, T. praticola) is a basidiomycetous fungus and a major cause of root diseases of economically important plants. Various isolates of this fungus are also beneficially associated with orchids, may serve as biocontrol agents or remain as saprophytes with roles in decaying and recycling of soil organic matter. R. solani displays several hyphal anastomosis groups (AG) with distinct host and pathogenic specializations. Even though there are reports on the physiological and histological basis of Rhizoctonia-host interactions, very little is known about the molecular biology and control of gene expression early during infection by this pathogen. Proteamic technologies are powerful tools for examining alterations in protein profiles. To aid studies on its biology and host pathogen interactions, a two-dimensional (2-D) gel-based global proteomic study has been initiated. To develop an optimized protein extraction protocol for R. solani, we compared two previously reported protein extraction protocols for 2-D gel analysis of R. solani (AG-4) isolate Rs23. Both TCA-acetone precipitation and phosphate solubilization before TCA-acetone precipitation worked well for R. solani protein extraction, although selective enrichment of some proteins was noted with either method. About 450 spots could be detected with the densitiometric tracing of Coomassie blue-stained 2-D PAGE gels covering pH 4-7 and 6.5-205 kDa. Selected protein spots were subjected to mass spectrometric analysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Eleven protein spots were positively identified based on peptide mass fingerprinting match with fungal proteins in public databases with the Mascot search engine. These results testify to the suitability of the two optimized protein extraction protocols for 2-D proteomic studies of R. solani.

Biosynthesis of Oxylipins by Rhizoctonia solani with Allene Oxide and Oleate 8S,9S-Diol Synthase Activities.

Oxylipin biosynthesis by fungi is catalyzed by both the lipoxygenase (LOX) family and the linoleate diol synthase (LDS) family of the peroxidase-cyclooxygenase superfamily. Rhizoctonia solani, a pathogenic fungus, infects staple crops such as potato and rice. The genome predicts three genes with 9-13 introns, which code for tentative dioxygenase (DOX)-cytochrome P450 fusion enzymes of the LDS family, and one gene, which might code for a 13-LOX. The objective was to determine whether mycelia or nitrogen powder of mycelia oxidized unsaturated C18 fatty acids to LDS- or LOX-related metabolites. Mycelia converted 18:2n-6 to 8R-hydroxy-9Z,12Z-octadecadienoic acid and to an α-ketol, 9S-hydroxy-10-oxo-12Z-octadecenoic acid. In addition to these metabolites, nitrogen powder of mycelia oxidized 18:2n-6 to 9S-hydroperoxy-10E, 12Z-octadecadienoic, and 13S-hydroperoxy-9Z,11E-octadecadienoic acids; the latter was likely formed by the predicted 13-LOX. 18:1n-9 was transformed into 8S-hydroperoxy-9Z-octadecenoic and into 8S,9S-dihydroxy-10E-octadecenoic acids, indicating the expression of 8,9-diol synthase. The allene oxide, 9S(10)epoxy-10,12Z-octadecadienoic acid, is unstable and decomposes rapidly to the α-ketol above, indicating biosynthesis by 9S-DOX-allene oxide synthase. This allene oxide and α-ketol are also formed by potato stolons, which illustrates catalytic similarities between the plant host and fungal pathogen.

Differentiation of mixed soil-borne fungi in the genus level using infrared spectroscopy and multivariate analysis.

Early detection of soil-borne pathogens, which have a negative effect on almost all agricultural crops, is crucial for effective targeting with the most suitable antifungal agents and thus preventing and/or reducing their severity. They are responsible for severe diseases in various plants, leading in many cases to substantial economic losses. In this study, infrared (IR) spectroscopic method, which is known as sensitive, accurate and rapid, was used to discriminate between different fungi in a mixture was evaluated. Mixed and pure samples of Colletotrichum, Verticillium, Rhizoctonia, and Fusarium genera were measured using IR microscopy. Our spectral results showed that the best differentiation between pure and mixed fungi was obtained in the 675-1800 cm-1 wavenumber region. Principal components analysis (PCA), followed by linear discriminant analysis (LDA) as a linear classifier, was performed on the spectra of the measured classes. Our results showed that it is possible to differentiate between mixed-calculated categories of phytopathogens with high success rates (~100%) when the mixing percentage range is narrow (40-60) in the genus level; when the mixing percentage range is wide (10-90), the success rate exceeded 85%. Also, in the measured mixed categories of phytopathogens it is possible to differentiate between the different categories with ~100% success rate.

Rhizoctonia solani fucomannogalactan: Chemical characterization and antiproliferative activity.

A fucomannogalactan from Rhizoctonia solani biomass was obtained after hot aqueous extraction and purified by freeze-thaw cycles and gel filtration chromatography on Sepharose CL-6B. The polysaccharide was homogeneous after HPSEC/RID analysis (Mw/Mn~1.1), displaying an average molecular weight of 15.4×103Da. Its chemical structure was determined by methylation analysis (GC/MS) and spectroscopy (FTIR, 1D and 2D NMR). The polysaccharide had a branched α-1,6-linked Galp backbone with 66% linear residues, a number of which were at O-3 methylated. Side chains (34%) were always linked at O-2 positions of the main chain and consisted of single, non-reducing ends of α-d-Manp (6%) and α-l-Fucp (28%). Analysis of its biological activity showed that the highly purified fucomannogalactan from R. solani inhibited the proliferation of colon cancer cells in vitro, but that it did not have the same activity against lung cancer cells.

Involvement of metabolic components, volatile compounds, PR proteins, and mechanical strengthening in multilayer protection of cucumber plants against Rhizoctonia solani activated by Trichoderma atroviride TRS25.

In the present study, the spread of Rhizoctonia solani-induced disease was limited when cucumber plants were pretreated with Trichoderma atroviride TRS25. The systemic disease suppression was related to TRS25-induced resistance (TISR) induction with simultaneous plant growth promotion. Protection of cucumber was related to enhanced activity of defense enzymes, e.g., guaiacol peroxidase (GPX), syringaldazine peroxidase (SPX), phenylalanine ammonia lyase (PAL), and polyphenol oxidase (PPO) as well as phenolic (PC) concentration increases in the conditions of hydrogen peroxide (H2O2) accumulation, resulting in thiobarbituric acid reactive substance (TBARS) decrease. Moreover, the obtained results indicated that TISR might depend on accumulation of salicylic acid derivatives, that is methyl salicylate (MeSA), ethylhexyl salicylate (EHS), salicylic acid glucosylated conjugates (SAGC), and ß-cyclocitral as well as volatile organic compounds (VOC) such as Z-3-hexanal, Z-3-hexenol, and E-2-hexenal. The results point to important, not previously documented, roles of these VOC in TISR signaling with up-regulation of PR1 and PR5 gene characteristic of systemic acquired resistance (SAR) and of PR4 gene, marker of induced systemic resistance (ISR). The study established that TRS25 enhanced deposition of callose and lignin in specialized plant cells, which protected vascular system in cucumber shoots and roots as well as assimilation cells and dermal tissues in shoots and leaves. These compounds protected cucumber organs against R. solani influence and made them more flexible and resilient, which contributed to better nutrition and hydration of plants. The growth promotion coupled with systemic mobilization of biochemical and mechanical strengthening might be involved in multilayer protection of cucumber against R. solani activated by TRS25.

Analysis of lectin concentrations in different Rhizoctonia solani strains.

Lectins are carbohydrate-binding proteins that contain at least one carbohydrate binding domain which can bind to a specific mono- or oligosaccharide. These proteins are widely distributed in plants. However, over the last decade evidence is accumulating that lectins occur also in numerous fungi belonging to both the Ascomycota and Basiodiomycota. Rhizoctonia solani is known to be an important pathogen to a wide range of host plants. In this study, isolates of R. solani from different anastomosis groups have been screened for the presence of lectin using agglutination assays to detect and quantitate lectin activity. The evaluation included determination of the lectin content in mycelium as well as in sclerotia. The amount of lectin in the sclerotia was higher than in the mycelium of the same strains. The R. solani strains with the highest amounts of lectin have been selected for cultivation, extraction and purification of the lectin.

Metabolites contributing to Rhizoctonia solani AG-1-IA maturation and sclerotial differentiation revealed by UPLC-QTOF-MS metabolomics.

Rhizoctonia solani is a causative agent of sheath blight, which results in huge economic losses every year. During its life cycle, the formation of sclerotia helps Rhizoctonia solani withstand a variety of unfavorable factors. Oxidative stress is a key factor that induces sclerotium formation. The differentiated and undifferentiated phenotypes of R. solani AG-1-IA were obtained by controlling aerial conditions. Metabolomics based on the mass spectrometry technique combined with multivariate and univariate analyses was used to investigate the metabolic variation in vegetative, differentiated and undifferentiated mycelia. Our results revealed that during maturation, the metabolic levels of N2-acetyl-L-ornithine, 3,1'-(OH)2-Gamma-carotene, (5Z,7E)-(1S,3R)-24,24-difluoro-24a-homo-9,10-seco-5,7,10(19)-cholestatrien-1,3,25-triol, stoloniferone O, PA(O-18:0/12:0), PA(P-16:0/14:0), PA(P-16:0/16:(19Z)) and PA(P-16:0/17:2(9Z,12Z)) were suppressed in both differentiated and undifferentiated mycelia. The concentrations of PE(20:1(11Z)/14:1(9Z)), PE(P-16:0/20:4(5Z,8Z,11Z,13E)(15OH[S])) and PS(12:0/18:1(9Z)) were increased in the differentiated group, while increased levels of N(gamma)-nitro-L-arginine, tenuazonic acid and 9S,10S,11R-trihydroxy-12Z,15Z-octadecadienoic acid were found in the undifferentiated group. Our results suggest that different levels of these metabolites may act as biomarkers for the developmental stages of R. solani AG-1-IA. Moreover, the mechanisms of sclerotium formation and mycelium differentiation were elucidated at the metabolic level.

Proteomic Investigation of Rhizoctonia solani AG 4 Identifies Secretome and Mycelial Proteins with Roles in Plant Cell Wall Degradation and Virulence.

Rhizoctonia solani AG 4 is a soilborne necrotrophic fungal plant pathogen that causes economically important diseases on agronomic crops worldwide. This study used a proteomics approach to characterize both intracellular proteins and the secretome of R. solani AG 4 isolate Rs23A under several growth conditions, the secretome being highly important in pathogenesis. From over 500 total secretome and soluble intracellular protein spots from 2-D gels, 457 protein spots were analyzed and 318 proteins positively matched with fungal proteins of known function by comparison with available R. solani genome databases specific for anastomosis groups 1-IA, 1-IB, and 3. These proteins were categorized to possible cellular locations and functional groups and for some proteins their putative roles in plant cell wall degradation and virulence. The majority of the secreted proteins were grouped to extracellular regions and contain hydrolase activity.

In vitro and in silico antifungal efficacy of nitrogen-doped carbon nanohorn (NCNH) against Rhizoctonia solani.

We have investigated in vitro antifungal efficiency of nitrogen-doped carbon nanohorn (NCNH) against Rhizoctonia solani (R. solani) plant pathogenic fungi. NCNH with size of 50-60 nm and concentrations of 10, 50, 100, and 150 µg mL(-1) were used. The results showed that growth of fungi in the presence of NCNH was significantly (p > .05) inhibited at 150 µg mL(-1) (85.13 ± .97) after 72 h. The results were validated through computational approaches. Molecular docking analysis of NCNH with endochitinase protein of R. solani was performed to validate the potential of antifungal activity of NCNH. Docking results showed different conformations of interaction of NCNH with endochitinase enzyme. The conformation with least binding energy -13.54 kcal/mol was considered further. It is likely that NCNH interacts with the pathogens by mechanically wrapping, which may be one of the major toxicity actions of NCNH against R. solani. The analysis showed that NCNH might interwinds to endochitinase of R. solani leading to the deactivation of the enzyme. To best of our knowledge, this is the first report of antifungal efficacy of NCNH against R. solani and provides useful information about the application of NCNH in resisting crop disease.

Comparison of two total cellular fatty acid analysis protocols to differentiate Rhizoctonia oryzae and R. oryzae-sativae.

Two fatty acid analysis protocols (the MIDI and a modified MIDI method) were investigated for their utility to characterize and differentiate Rhizoctonia oryzae and R. oryzae-sativae isolates from four countries. Only the modified MIDI method permitted a clear differentiation between the two species, regardless of the isolates' country of origin. The modified MIDI method gave the most consistent and reproducible fatty acid results. The failure of the MIDI method to differentiate between R. oryzae and R. oryzae-sativae isolates suggests that the 30 minutes saponification step is insufficient to completely break the cell wall of these two species. This study demonstrated that fatty acid profiles, obtained by the modified MIDI protocol, have the potential as a diagnostic tool for both R. oryzae and R. oryzae-sativae.