Proteomics of rice and Cochliobolus miyabeanus fungal interaction: Insight into proteins at intracellular and extracellular spaces

Necrotrophic fungal pathogen Cochliobolus miyabeanus causes brown spot disease in rice leaves upon infection, resulting in critical rice yield loss. To better understand the rice–C. miyabeanus interaction, we employed proteomic approaches to establish differential proteomes of total and secreted proteins from the inoculated leaves. The 2DE approach after PEG‐fractionation of total proteins coupled with MS (MALDI‐TOF/TOF and nESI‐LC‐MS/MS) analyses led to identification of 49 unique proteins out of 63 differential spots. SDS‐PAGE in combination with nESI‐LC‐MS/MS shotgun approach was applied to identify secreted proteins in the leaf apoplast upon infection and resulted in cataloging of 501 unique proteins, of which 470 and 31 proteins were secreted from rice and C. miyabeanus, respectively. Proteins mapped onto metabolic pathways implied their reprogramming upon infection. The enzymes involved in Calvin cycle and glycolysis decreased in their protein abundance, whereas enzymes in the TCA cycle, amino acids, and ethylene biosynthesis increased. Differential proteomes also generated distribution of identified proteins in the intracellular and extracellular spaces, providing a better insight into defense responses of proteins in rice against C. miyabeanus. Established proteome of the rice–C. miyabeanus interaction serves not only as a good resource for the scientific community but also highlights its significance from biological aspects.     

Induced phenylamide accumulation in response to pathogen infection and hormone treatment in rice (Oryza sativa)

Rice plants accumulate various specialized metabolites, including phenylamides, in response to pathogen attack. We prepared 25 phenylamides, and developed a method of analyzing them by multiple reaction monitoring with liquid chromatography coupled with tandem mass spectrometry. We analyzed phenylamides in rice leaves infected with Cochliobolus miyabeanus and Xanthomonas oryzae. The phenylamides induced included benzoyltryptamine, cinnamoyl-, p-coumaroyl-, feruloyl-, and benzoylserotonins, cinnamoyl and benzoyltyramines, feruloylagmatine, and feruloylputrescine. Some of the phenylamides exhibited antimicrobial activity against C. miyabeanus and X. oryzae, indicating that they are phytoalexins. Treatment with jasmonic acid, salicylic acid, 6-benzylaminopurine, and ethephone also induced phenylamide accumulation. The compositions of the induced amides varied depending on the plant hormone used, and cinnamoyltryptamine, cinnamoylserotonin, and cinnamoyltyramine were not induced by the plant hormones. These findings suggest that several plant hormones and additional factors are involved in phenylamide accumulation in response to pathogen infection in rice.     

A comparative screening of hybrid,modern varieties and local rice cultivar for brown leaf spot disease susceptibility and yield performance

Five rice cultivars, one hybrid (WR96), three modern (BR16, BR26, and BRRI Dhan27) and one local (Pari) were screened for reaction to brown leaf spot disease caused by Cochliobolus miyabeanus and performance of yield-related characters. The severity of brown leaf spot varied with growth stages of rice plant as well as different cultivars tested under field condition. Low disease severity was observed at maximum tillering stage compared to moderate to high at dough stage, with hybrid cultivar WR96 showing highest disease, while local cultivar Pari had the lowest. Brown spot disease severity in different cultivars under induced epiphytic condition also followed the similar trend. The results also revealed that most of the yield-contributing characters examined showed wide variations among the cultivars. Modern cultivar BR16 produced the highest panicle length, number of grain per panicle and grain yield per hectare. At the same time as local cultivar Pari generated the lowest number of tiller per plant, panicle length, grain number per panicle and grain yield per hectare. Moreover, hybrid cultivar WR96 produced the highest percentage of spotted grain per panicle and seed yielding C. miyabeanus, and also the lower percentage of seed germination, while the reverse was observed in local cultivar Pari. These findings may allow producers and breeders to select rice cultivar, resistant or tolerant to brown leaf spot disease and to avoid significant reductions in grain yields.     

The rice/maize pathogen Cochliobolus spp. infect and reproduce on Arabidopsis revealing differences in defensive phytohormone function between monocots and dicots

The fungal genus Cochliobolus describes necrotrophic pathogens that give rise to significant losses on rice, wheat, and maize. Revealing plant mechanisms of non‐host resistance (NHR) against Cochliobolus will help to uncover strategies that can be exploited in engineered cereals. Therefore, we developed a heterogeneous pathosystem and studied the ability of Cochliobolus to infect dicotyledons. We report here that C. miyabeanus and C. heterostrophus infect Arabidopsis accessions and produce functional conidia, thereby demonstrating the ability to accept Brassica spp. as host plants. Some ecotypes exhibited a high susceptibility, whereas others hindered the necrotrophic disease progression of the Cochliobolus strains. Natural variation in NHR among the tested Arabidopsis accessions can advance the identification of genetic loci that prime the plant’s defence repertoire. We found that applied phytotoxin‐containing conidial fluid extracts of C. miyabeanus caused necrotic lesions on rice leaves but provoked only minor irritations on Arabidopsis. This result implies that C. miyabeanus phytotoxins are insufficiently adapted to promote dicot colonization, which corresponds to a retarded infection progression. Previous studies on rice demonstrated that ethylene (ET) promotes C. miyabeanus infection, whereas salicylic acid (SA) and jasmonic acid (JA) exert a minor function. However, in Arabidopsis, we revealed that the genetic disruption of the ET and JA signalling pathways compromises basal resistance against Cochliobolus, whereas SA biosynthesis mutants showed a reduced susceptibility. Our results refer to the synergistic action of ET/JA and indicate distinct defence systems between Arabidopsis and rice to confine Cochliobolus propagation. Moreover, this heterogeneous pathosystem may help to reveal mechanisms of NHR and associated defensive genes against Cochliobolus infection.     

First Extensive Polyphenolic Profile of Erodium cicutarium with Novel Insights to Elemental Composition and Antioxidant Activity

Erodium cicutarium is known for its total polyphenolic content, but this work reveals the first highly detailed profile of E. cicutarium, obtained with UHPLC‐LTQ OrbiTrap MS⁴ and UHPLC‐QqQ‐MS/MS techniques. A total of 85 phenolic compounds were identified and 17 constituents were quantified. Overall, 25 new compounds were found, which have not yet been reported for the Erodium genera, or the family Geraniaceae. Along with methanolic extracts, the so far poorly investigated water extracts exhibited in vitro antioxidant activity according to all performed assays, including the ferric reducing/antioxidant power assay (FRAP), 2,2‐diphenyl‐1‐picrylhydrazyl assay (DPPH), 2,2′‐azinobis(3‐ethylbenzthiazoline‐6‐sulfonic acid) assay (ABTS) and cupric ion reducing antioxidant capacity assay (CUPRAC). Elemental composition analysis performed with inductively coupled plasma atomic emission spectrometry (ICP‐AES) and, additionally, hydride generation atomic absorption spectrometry (HydrEA‐ETAAS) showed six most abundant elements to be decreasing as follows: Mg>Ca>K>S>P>Na, and gave first data regarding inorganic arsenic content (109.3–248.4 ng g^?1). These results suggest E. cicutarium to be a valuable source of various phenolic compounds with substantial potential for further bioactivity testing.     

Quantitative Analysis of Ingenol in Euphorbia species via Validated Isotope Dilution Ultra‐high Performance Liquid Chromatography Tandem Mass Spectrometry

Introduction

Various species of the Euphorbia genus contain diterpene ingenol and ingenol mebutate (ingenol‐3‐angelate), a substance found in the sap of the plant Euphorbia peplus and an inducer of cell death. A gel formulation of the drug has been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the topical treatment of actinic keratosis.

Objective

To develop a rapid and reliable method for quantification of ingenol in various plant extracts.

Methodology

Methanolic extracts of 38 species of the Euphorbia genus were analysed via ultra‐high performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) after methanolysis and solid‐phase extraction (SPE) purification. The ¹⁸O–labelled ingenol analogue was prepared and used as an internal standard for ingenol content determination and method validation.

Results

The highest ingenol concentration (547 mg/kg of dry weight) was found in the lower leafless stems of E. myrsinites. The screening confirms a substantial amount of ingenol in species studied previously and furthermore, reveals some new promising candidates.

Conclusion

The newly established UHPLC–MS/MS method shows to be an appropriate tool for screening of the Euphorbia genus for ingenol content and allows selection of species suitable for raw material production and/or in vitro culture initiation. Copyright © 2017 John Wiley & Sons, Ltd.     

Use of plant extracts and biocontrol agents for the management of brown spot disease in rice

Fifty plant extracts, four oil cakes and eight antagonistic organisms were tested against Bipolaris oryzae (Cochliobolus miyabeanus), the causal agent of brown spot disease of rice. In vitro studies indicated that two leaf extracts, Nerium oleander and Pithecolobium dulce exerted the higher percent inhibition to mycelial growth (77.4, 75.1%) and spore germination (80.3, 80.0%) of B. oryzae. Among the four oil cake extracts tested in vitro against B. oryzae, neem cake extract showed the maximum inhibition percent to mycelial growth (80.18%) and spore germination (81.13%) of the pathogen followed by mahua cake extract, castor and gingelly cake extract. Trichoderma viride (Tv2) was significantly effective in inhibiting the mycelial growth (62.92%) and spore germination (77.03%) of the pathogen followed by Trichoderma harzianum (Th5) and Trichoderma reesei (Tr3). The promising leaf extracts, oil cake extracts and antagonistic microorganisms were further evaluated for their efficacies in disease management under glasshouse and field conditions. In glasshouse studies, post-infectional spraying of rice plants with neem cake extract, N. oleander leaf extract and T. viride (Tv2) was significantly effective in reducing the incidence of brown spot of rice by 66, 52 and 45 percent respectively. Two rounds of spraying of rice plants with neem cake extract, N. oleander leaf extract and T. viride (Tv2) in the field at initial appearance of disease and 15 days later reduced the incidence of brown spot (70, 53 and 48% disease reduction respectively) and increased the yield by 23, 18 and 15 percent respectively.     

Involvement of Gluconeogenic Pathway in Virulence of Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases of rice. A phosphoenolpyruvate synthase (ppsA)‐disrupted mutant OSPAM was generated by homologous suicide plasmid integration. The mutant was unable to grow in medium with pyruvate or C₄‐dicarboxylates as the sole carbon source, compared with the wild‐type, indicating a disruption in ppsA function. The mutant showed a reduction in virulence on rice but still induced a hypersensitive response in tobacco. When the mutant was complemented, the response was recovered to wild‐type. These results suggested that X. oryzae pv. oryzae possesses only PPSA route in gluconeogenesis, which is necessary for virulence.     

The identification and deletion of the polyketide synthase-nonribosomal peptide synthase gene responsible for the production of the phytotoxic triticone A/B in the wheat fungal pathogen Pyrenophora tritici-repentis

The economically important necrotrophic fungal pathogen, Pyrenophora tritici-repentis (Ptr), causes tan spot of wheat, a disease typified by foliar necrosis and chlorosis. The culture filtrate of an Australian Ptr isolate, M4, possesses phytotoxic activity and plant bioassay guided discovery led to the purification of necrosis inducing toxins called triticone A and B. High-resolution LC–MS/MS analysis of the culture filtrate identified an additional 37 triticone-like compounds. The biosynthetic gene cluster responsible for triticone production (the Ttc cluster) was identified and deletion of TtcA, a hybrid polyketide synthase (PKS)-nonribosomal peptide synthase (NRPS), abolished production of all triticones. The pathogenicity of mutant (ttcA) strains was not visibly affected in our assays. We hypothesize that triticones possess general antimicrobial activity important for competition in multi-microbial environments.     

Metabolomics reveals herbivore‐induced metabolites of resistance and susceptibility in maize leaves and roots

Plants respond to herbivory by reprogramming their metabolism. Most research in this context has focused on locally induced compounds that function as toxins or feeding deterrents. We developed an ultra‐high‐pressure liquid chromatography time‐of‐flight mass spectrometry (UHPLC‐TOF‐MS)‐based metabolomics approach to evaluate local and systemic herbivore‐induced changes in maize leaves, sap, roots and root exudates without any prior assumptions about their function. Thirty‐two differentially regulated compounds were identified from Spodoptera littoralis‐infested maize seedlings and isolated for structure assignment by microflow nuclear magnetic resonance (CapNMR). Nine compounds were quantified by a high throughput direct nano‐infusion tandem mass spectrometry/mass spectrometry (MS/MS) method. Leaf infestation led to a marked local increase of 1,3‐benzoxazin‐4‐ones, phospholipids, N‐hydroxycinnamoyltyramines, azealic acid and tryptophan. Only few changes were found in the root metabolome, but 1,3‐benzoxazin‐4‐ones increased in the vascular sap and root exudates. The role of N‐hydroxycinnamoyltyramines in plant–herbivore interactions is unknown, and we therefore tested the effect of the dominating p‐coumaroyltyramine on S. littoralis. Unexpectedly, p‐coumaroyltyramine was metabolized by the larvae and increased larval growth, possibly by providing additional nitrogen to the insect. Taken together, this study illustrates that herbivore attack leads to the induction of metabolites that can have contrasting effects on herbivore resistance in the leaves and roots.     

Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes

Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.     

Detection of flavonoids in microalgae from different evolutionary lineages

Flavonoids are important secondary plant metabolites believed to be present mainly in land plants. As phenolics were detected previously in microalgae using photometric assays, we wanted to investigate the nature of these phenolics and verify whether flavonoids are present. Therefore, in this study, we used state‐of‐the‐art ultra‐high performance liquid chromatography‐two‐dimensional mass spectrometry (UHPLC‐MS/MS) technology to investigate whether microalgae also contain flavonoids. For this, representative microalgal biomass samples from divergent evolutionary lineages (Cyanobacteria, Rhodophyta, Chlorophyta, Haptophyta, Ochrophyta) were screened for a set of carefully selected precursors, intermediates, and end products of the flavonoid biosynthesis pathways. Our data unequivocally showed that microalgae contain a wide range of flavonoids and thus must possess the enzyme pool required for their biosynthesis. Further, some of the microalgae displayed an intricate flavonoid pattern that is compatible with the established basic flavonoid pathway as observed in higher plants. This implies that the flavonoid biosynthesis pathway arose much earlier in evolution compared to what is generally accepted.     

UHPLC‐HRMS/MS Based Profiling of Algerian Lichens and Their Antimicrobial Activities

Lichens are complex symbiotic organisms able to produce a vast array of compounds. The Algerian lichen diversity has only prompted little interest even given the 1085 species listed. Herein, the chemodiversity of four Algerian lichens including Cladonia rangiformis, Ramalina farinaceae, R. fastigiata, and Roccella phycopsis was investigated. A dereplication strategy, using ultra high performance liquid chromatography‐high resolution‐electrospray ionization‐mass spectrometry (UHPLC‐HRMS/MS), was carried out for a comprehensive characterization of their substances including phenolics, depsides, depsidones, depsones, dibenzofurans, and aliphatic acids. Some known compounds were identified for the first time in some species. Additionally, the lichenic extracts were evaluated for their antifungal and antimicrobial activities on human pathogenic strains (Candida albicans, C. glabrata, Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli). Cyclohexane extracts were found particularly active against human pathogenic fungi with MIC₈₀ values ranging from 8 to 62.5 μg/mL, without cytotoxicity. This study highlights the therapeutic and prophylactic potential of lichenic extracts as antibacterial and antifungal agents.     

A secreted chitinase‐like protein (OsCLP) supports root growth through calcium signaling in Oryza sativa

Chitinases belong to a conserved protein family and play multiple roles in defense, development and growth regulation in plants. Here, we identified a secreted chitinase‐like protein, OsCLP, which functions in rice growth. A T‐DNA insertion mutant of OsCLP (osclp) showed significant retardation of root and shoot growth. A comparative proteomic analysis was carried out using root tissue of wild‐type and the osclp mutant to understand the OsCLP‐mediated rice growth retardation. Results obtained revealed that proteins related to glycolysis (phosphoglycerate kinase), stress adaption (chaperonin) and calcium signaling (calreticulin and CDPK1) were differentially regulated in osclp roots. Fura‐2 molecular probe staining, which is an intracellular calcium indicator, and inductively coupled plasma‐mass spectrometry (ICP‐MS) analysis suggested that the intracellular calcium content was significantly lower in roots of osclp as compared with the wild‐type. Exogenous application of Ca²⁺ resulted in successful recovery of both primary and lateral root growth in osclp. Moreover, overexpression of OsCLP resulted in improved growth with modified seed shape and starch structure; however, the overall yield remained unaffected. Taken together, our results highlight the involvement of OsCLP in rice growth by regulating the intracellular calcium concentrations.     

Quantification of rice brown leaf spot through Taqman real-time PCR specific to the unigene encoding Cochliobolus miyabeanus SCYTALONE DEHYDRATASE1 involved in fungal melanin biosynthesis

Rice brown leaf spot is a major disease in the rice paddy field. The causal agent Cochliobolus miyabeanus is an ascomycete fungus and a representative necrotrophic pathogen in the investigation of rice-microbe interactions. The aims of this research were to identify a quantitative evaluation method to determine the amount of C. miyabeanus proliferation in planta and determine the method’s sensitivity. Real-time polymerase chain reaction (PCR) was employed in combination with the primer pair and Taqman probe specific to CmSCD1, a C. miyabeanus unigene encoding SCYTALONE DEHYDRATASE, which is involved in fungal melanin biosynthesis. Comparative analysis of the nucleotide sequences of CmSCD1 from Korean strains with those from the Japanese and Taiwanese strains revealed some sequence differences. Based on the crossing point (CP) values from Taqman real-time PCR containing a series of increasing concentrations of cloned amplicon or fungal genomic DNA, linear regressions with a high level of reliability (R²>0.997) were constructed. This system was able to estimate fungal genomic DNA at the picogram level. The reliability of this equation was further confirmed using DNA samples from both resistant and susceptible cultivars infected with C. miyabeanus. In summary, our quantitative system is a powerful alternative in brown leaf spot forecasting and in the consistent evaluation of disease progression.     

Volatile secretions and epicuticular hydrocarbons of the beetle Ulomoides dermestoides

Many species of tenebrionids produce and secrete a defensive volatile blend containing mainly benzoquinones and alkenes. In this study we characterized the volatile organic compounds (VOC) of the beetle Ulomoides dermestoides (Coleoptera: Tenebrionidae). Solid phase microextraction (SPME) coupled to capillary gas chromatography–mass spectrometry (CGC–MS) analysis was used to identify methyl-1,4-benzoquinone (MBQ), ethyl-1,4-benzoquinone (EBQ), 1-tridecene (C_13:1), and 1-pentadecene (C_15:1), representing more than 90% of the volatile blend. We also used CGC–MS to analyze the epicuticular hydrocarbons of U. dermestoides. Saturated, unsaturated, and branched structures with chain lengths ranging from 13 to 43 carbons were detected. n-pentacosane (C_25:0) and 9,11-pentacosadiene (9,11-C_25:2) were the most abundant components, representing more than 40% of the cuticular hydrocarbons.     

A systematic study of the polyphenolic composition of aqueous extracts deriving from several Cistus genus species: evolutionary relationship

Introduction – Cistaceae is a large family of shrubs widely spread over the Mediterranean area. It includes Helianthemum, Halimium and Cistus genus. Cistus genus contains approximately 20 species distributed in three subgenus. The essential oil of Cistus species has been thoroughly studied, but the polyphenolic composition of the aerial parts of the different Cistus species needs further characterisation. Objective – To perform a comparative analysis of the qualitative and quantitative polyphenolic composition of the aerial parts of the most commonly distributed Spanish Cistus species in order to find a relationship between chemotype and subgenus. Methodology – Thirteen aqueous extracts derived from 10 different Cistus species were analysed by using HPLC with diode array‐detection coupled to electrospray ion trap mass spectrometry technique (HPLC‐DAD‐ESI‐MS/MS). Their major compounds were identified and ellagitannins were quantified. Principal component analysis (PCA) was performed on the most relevant compounds to find out the statistical association between chemotype and variety. Results – Three main groups of compounds were found, i.e. ellagitannins, flavonoids and phenolic acids derivatives. The polyphenolic profile was specific for each species, although the abundance of some compounds also varied depending on the soil type. Whereas C. ladanifer, C. salviifolius, C. populifolius and C. libanotis were specially rich in ellagitannins, C. clusii, C. laurifolius and C. monspeliensis contained significant amounts of flavonoids and much less ellagitannins. In contrast, C. crispus, C. incanus and C. albidus showed a polyphenolic profile mostly based on flavonoids. PCA analysis showed a strong relationship between Cistus subgenus and its chemotype based on the most relevant water‐soluble polyphenolic compounds. Conclusions – Chemical composition of the leaves' aqueous extracts from plants belonging to the Cistus genus is strongly related to their subgenus, in agreement to previous taxonomical and phylogenetic divisions. In contrast, soil and climate are less influencing factors. Leucocistus and Halimioides subgenus showed a higher content in ellagitannins. However, Cistus subgenus had higher flavonoid content. Copyright © 2011 John Wiley & Sons, Ltd.