Transcriptome-Wide N6-Methyladenosine (m6A) Methylation Analyses in a Compatible Wheat-Puccinia striiformis f. sp. tritici Interaction.

N6-methyladenosine (m6A) is a prevalent internal modification in eukaryotic mRNA, tRNA, miRNA, and long non-coding RNA. It is also known for its role in plant responses to biotic and abiotic stresses. However, a comprehensive m6A transcriptome-wide map for Puccinia striiformis f. sp. tritici (Pst) infections in wheat (Triticum aestivum) is currently unavailable. Our study is the first to profile m6A modifications in wheat infected with a virulent Pst race. Analysis of RNA-seq and MeRIP-seq data revealed that the majority of differentially expressed genes are up-regulated and hyper-methylated. Some of these genes are enriched in the plant-pathogen interaction pathway. Notably, genes related to photosynthesis showed significant down-regulation and hypo-methylation, suggesting a potential mechanism facilitating successful Pst invasion by impairing photosynthetic function. The crucial genes, epitomizing the core molecular constituents that fortify plants against pathogenic assaults, were detected with varying expression and methylation levels, together with a newly identified methylation motif. Additionally, m6A regulator genes were also influenced by m6A modification, and their expression patterns varied at different time points of post-inoculation, with lower expression at early stages of infection. This study provides insights into the role of m6A modification regulation in wheat's response to Pst infection, establishing a foundation for understanding the potential function of m6A RNA methylation in plant resistance or susceptibility to pathogens.

Puccinia striiformis f. sp. tritici effectors in wheat immune responses.

The obligate biotrophic fungus Puccinia striiformis f. sp. tritici, which causes yellow (stripe) rust disease, is among the leading biological agents resulting in tremendous yield losses on global wheat productions per annum. The combatting strategies include, but are not limited to, fungicide applications and the development of resistant cultivars. However, evolutionary pressure drives rapid changes, especially in its "effectorome" repertoire, thus allowing pathogens to evade and breach resistance. The extracellular and intracellular effectors, predominantly secreted proteins, are tactical arsenals aiming for many defense processes of plants. Hence, the identity of the effectors and the molecular mechanisms of the interactions between the effectors and the plant immune system have long been targeted in research. The obligate biotrophic nature of P. striiformis f. sp. tritici and the challenging nature of its host, the wheat, impede research on this topic. Next-generation sequencing and novel prediction algorithms in bioinformatics, which are accompanied by in vitro and in vivo validation approaches, offer a speedy pace for the discovery of new effectors and investigations of their biological functions. Here, we briefly review recent findings exploring the roles of P. striiformis f. sp. tritici effectors together with their cellular/subcellular localizations, host responses, and interactors. The current status and the challenges will be discussed. We hope that the overall work will provide a broader view of where we stand and a reference point to compare and evaluate new findings.

Silyl-naphthalene endoperoxides as switchable sources of singlet oxygen for bactericidal activity.

Singlet oxygen is a short half-life cytotoxic agent which can be generated by chemical and photochemical methods. In order to make use of its antibacterial action at a selected location, it is desirable to have singlet oxygen in a relatively stable, "caged" structure, in the form of an endoperoxide. Here, the trimethylsilyl (TMS) group supplies the steric bulk, inhibiting the cycloreversion reaction to produce very little singlet oxygen under ambient conditions. However, when fluoride ions are added as tetrabutylammonium fluoride, very rapid removal of the TMS group takes place, followed by the unhindered cycloreversion, releasing singlet oxygen much faster. The bactericidal action on surfaces was demonstrated using E. coli, and imaged under fluorescence microscopy. Considering the issues related to emergence of antibiotic resistant bacterial strains, "on demand singlet oxygen" appears to be an exciting alternative.

In-depth secretome analysis of Puccinia striiformis f. sp. tritici in infected wheat uncovers effector functions.

The importance of wheat yellow rust disease, caused by Puccinia striiformis f. sp. tritici (Pst), has increased substantially due to the emergence of aggressive new Pst races in the last couple of decades. In an era of escalating human populations and climate change, it is vital to understand the infection mechanism of Pst in order to develop better strategies to combat wheat yellow disease. The present study focuses on the identification of small secreted proteins (SSPs) and candidate-secreted effector proteins (CSEPs) that are used by the pathogen to support infection and control disease development. We generated de novo assembled transcriptomes of Pst collected from wheat fields in central Anatolia. We inoculated both susceptible and resistant seedlings with Pst and analyzed haustoria formation. At 10 days post-inoculation (dpi), we analyzed the transcriptomes and identified 10550 Differentially Expressed Unigenes (DEGs), of which 6072 were Pst-mapped. Among those Pst-related genes, 227 were predicted as PstSSPs. In silico characterization was performed using an approach combining the transcriptomic data and data mining results to provide a reliable list to narrow down the ever-expanding repertoire of predicted effectorome. The comprehensive analysis detected 14 Differentially Expressed Small-Secreted Proteins (DESSPs) that overlapped with the genes in available literature data to serve as the best CSEPs for experimental validation. One of the CSEPs was cloned and studied to test the reliability of the presented data. Biological assays show that the randomly selected CSEP, Unigene17495 (PSTG_10917), localizes in the chloroplast and is able to suppress cell death induced by INF1 in a Nicotiana benthamiana heterologous expression system.

Fusarium head blight in wheat: contemporary status and molecular approaches.

Fusarium head blight (FHB) disease that occurs in wheat is caused by Fusarium graminearum and is a major risk to wheat yield. Although several research efforts focusing on FHB have been conducted in the past several decades, conditions have become more critical due to the increase in its virulent forms. In such a scenario, conferring complete resistance in plants seems to be difficult for handling this issue. The phenotyping for FHB and finding a solution for it at the genetic level comprises a long-term process as FHB infection is largely affected by environmental conditions. Modern molecular strategies have played a crucial role in revealing the host-pathogen interaction in FHB. The integration of molecular biology-based methods such as genome-wide association studies and marker-based genomic selection has provided potential cultivars for breeding programs. In this review, we aim at outlining the contemporary status of the studies conducted on FHB in wheat. The influence of FHB in wheat on animals and human health is also discussed. In addition, a summary of the advancement in the molecular technologies for identifying and developing the FHB-resistant wheat genetic resources is provided. It also suggests the future measures that are required to reduce the world's vulnerability to FHB which was one of the main goals of the US Wheat and Barley Scab Initiative.

Biocontrol of Fusarium wilt disease in strawberries using bioorganic fertilizer fortified with Bacillus licheniformis X-1 and Bacillus methylotrophicus Z-1.

Fusarium wilt caused by Fusarium oxysporum f. sp. fragariae is one of the most serious indigenous soil-borne fungal disease of strawberry. In this study, we have identified and investigated two sets of bacterial samples: Bacillus licheniformis (X-1) and Bacillus methylotrophicus (Z-1). Both of them were isolated from the rhizosphere soil of healthy strawberries which showed a strong inhibitory effect on Fusarium wilt caused by Fusarium oxysporum f. sp. fragariae. Bioorganic fertilizer developed by our team exhibiting a strong inhibition ability against the pathogen in comparison with the chemical and organic fertilizers. It allowed 80% disease free strawberry production together with improved physical and biochemical indexes in the pot experiments. The enzyme activity analysis of SOD, PPO, POD, and CAT in the bioorganic fertilizer (BOF) group showed significant increase with values; 48.8%, 68.7%, 85.9%, and 41.1% than that of the control group, respectively. The results of bacterial diversity showed that Bacillus in group BOF was almost three times as large as in the healthy soil control group (CK). Besides, the results of microbial diversity showed that Fusarium and Fusicolla of BOF was nearly five times less than that in CK and chemical fertilizer groups, where the Bacillus content reached to three times as much of the CK. Moreover, the enzymes activity and the content of beneficial microorganisms in the rhizosphere increased significantly. In this study, the bioorganic fertilizer developed by the isolated strains had significant effects on the control of strawberry Fusarium wilt disease. Our results demonstrate that BOF is a promising approach to control this disease in strawberry production.

A Puccinia striiformis f. sp. tritici secreted protein activates plant immunity at the cell surface.

Pathogens secrete effector proteins to suppress host immunity, mediate nutrient uptake and subsequently enable parasitism. However, on non-adapted hosts, effectors can be detected as non-self by host immune receptors and activate non-host immunity. Nevertheless, the molecular mechanisms of effector triggered non-host resistance remain unknown. Here, we report that a small cysteine-rich protein PstSCR1 from the wheat rust pathogen Puccinia striiformis f. sp. tritici (Pst) activates immunity in the non-host solanaceous model plant Nicotiana benthamiana. PstSCR1 homologs were found to be conserved in Pst, and in its closest relatives, Puccinia graminis f. sp. tritici and Puccinia triticina. When PstSCR1 was expressed in N. benthamiana with its signal peptide, it provoked the plant immune system, whereas no stimulation was observed when it was expressed without its signal peptide. PstSCR1 expression in N. benthamiana significantly reduced infection capacity of the oomycete pathogens. Moreover, apoplast-targeted PstSCR1 triggered plant cell death in a dose dependent manner. However, in Brassinosteroid insensitive 1-Associated Kinase 1 (SERK3/BAK1) silenced N. benthamiana, cell death was remarkably decreased. Finally, purified PstSCR1 protein activated defence related gene expression in N. benthamiana. Our results show that a Pst-secreted protein, PstSCR1 can activate surface mediated immunity in non-adapted hosts and contribute to non-host resistance.

Assessment of genetic variability for grain nutrients from diverse regions: potential for wheat improvement.

BACKGROUND: A total of 150 bread wheat genotypes representing 121 Indian and 29 Turkish origin were screened for nutrient concentrations and grain protein content. Elemental and grain protein composition were studied by Inductively Coupled Plasma-Atomic Emission Spectrophotometer and LECO analyser, respectively. The study was performed to determine the variability in nutrient concentrations present in the collected wheat genetic material from two countries. RESULTS: Several fold variations among genotypes existed for almost all the elements. Three major components of principal component analysis (PCA) revealed 60.8% variation among the genotypes. Nutrient variables segregated into two groups, one group containing all the macroelements except sulphur; and another cluster containing proteins and all the microelements except Zn and Mn. Pearson correlation analysis and heat-map were in accordance with each other determining strong positive association between P-K, Mn-Zn, Mg-S and Cu-protein content. Also, PCA and hierarchical grouping divided all the Indian and Turkish genotypes in two main clusters. CONCLUSIONS: Nutritional profile differentiated the genotypes from two countries into separate groups. However, some of the varieties were closely associated and indicated the success of global wheat exchange programs. While most of the correlations were in agreement with the previous studies, non-association of zinc with grain protein content directed towards its control by some other genetic factors. Some of the experimental wheat varieties with promising nutrient content have been suggested for future wheat advancement programs. Results obtained will be supportive for breeders involved in wheat biofortification programs, food industries and people relying on whole grain wheat products.

Ancient DNA from 8400 Year-Old Çatalhöyük Wheat: Implications for the Origin of Neolithic Agriculture.

Human history was transformed with the advent of agriculture in the Fertile Crescent with wheat as one of the founding crops. Although the Fertile Crescent is renowned as the center of wheat domestication, archaeological studies have shown the crucial involvement of Çatalhöyük in this process. This site first gained attention during the 1961-65 excavations due to the recovery of primitive hexaploid wheat. However, despite the seeds being well preserved, a detailed archaeobotanical description of the samples is missing. In this article, we report on the DNA isolation, amplification and sequencing of ancient DNA of charred wheat grains from Çatalhöyük and other Turkish archaeological sites and the comparison of these wheat grains with contemporary wheat species including T. monococcum, T. dicoccum, T. dicoccoides, T. durum and T. aestivum at HMW glutenin protein loci. These ancient samples represent the oldest wheat sample sequenced to date and the first ancient wheat sample from the Middle East. Remarkably, the sequence analysis of the short DNA fragments preserved in seeds that are approximately 8400 years old showed that the Çatalhöyük wheat stock contained hexaploid wheat, which is similar to contemporary hexaploid wheat species including both naked (T. aestivum) and hulled (T. spelta) wheat. This suggests an early transitory state of hexaploid wheat agriculture from the Fertile Crescent towards Europe spanning present-day Turkey.

Genetic diversity and population structure of wheat in India and Turkey.

Genetic diversity among plant species offers prospects for improving the plant characteristics. Its assessment is necessary to help tackle the threats of environmental fluctuations and for the effective exploitation of genetic resources in breeding programmes. Although wheat is one of the most thoroughly studied crops in terms of genetic polymorphism studies, phylogenetic affinities of Indian and Turkish Triticum species have not been assessed to date. In this study, genetic association of 95 tetraploid and hexaploid wheat genotypes originating from India and Turkey was determined for the first time. Combined analysis of random amplified polymorphic DNA and inter-simple sequence repeat markers disclosed 177 polymorphic bands, and both the dendrogram and two-dimensional scatterplot showed similar groupings of the wheat genotypes. Turkish hexaploid varieties were basically divided into two clusters, one group showed its close association with Indian hexaploid varieties and the other with Indian tetraploid varieties. Analysis of molecular variance revealed high (77 %) genetic variation within Indian and Turkish populations. Population structure analysis elucidated distinct clustering of wheat genotypes on the basis of both geographical origin and ploidy. The results revealed in this study will support worldwide wheat breeding programmes and assist in achieving the target of sustainable wheat production.

TRAINER: a general-purpose trainable short biosequence classifer.

Classifying sequences is one of the central problems in computational biosciences. Several tools have been released to map an unknown molecular entity to one of the known classes using solely its sequence data. However, all of the existing tools are problem-specific and restricted to an alphabet constrained by relevant biological structure. Here, we introduce TRAINER, a new online tool designed to serve as a generic sequence classification platform to enable users provide their own training data with any alphabet therein defined. TRAINER allows users to select among several feature representation schemes and supervised machine learning methods with relevant parameters. Trained models can be saved for future use without retraining by other users. Two case studies are reported for effective use of the system for DNA and protein sequences; candidate effector prediction and nucleolar localization signal prediction. Biological relevance of the results is discussed.

A probabilistic approach to microRNA-target binding.

Elucidation of microRNA activity is a crucial step in understanding gene regulation. One key problem in this effort is how to model the pairwise interactions of microRNAs with their targets. As this interaction is strongly mediated by their sequences, it is desired to set-up a probabilistic model to explain the binding preferences between a microRNA sequence and the sequence of a putative target. To this end, we introduce a new model of microRNA-target binding, which transforms an aligned duplex to a new sequence and defines the likelihood of this sequence using a Variable Length Markov Chain. It offers a complementary representation of microRNA-mRNA pairs for microRNA target prediction tools or other probabilistic frameworks of integrative gene regulation analysis. The performance of present model is evaluated by its ability to predict microRNA-target mRNA interaction given a mature microRNA sequence and a putative mRNA binding site. In regard to classification accuracy, it outperforms two recent methods based on thermodynamic stability and sequence complementarity. The experiments can also unveil the effects of base pairing types and non-seed region in duplex formation.

Cellular and transcriptional responses of wheat during compatible and incompatible race-specific interactions with Puccinia striiformis f. sp. tritici.

The initial stages of Puccinia striiformis f. sp. tritici (the causal agent of yellow rust in wheat) infection triggered a hypersensitive cell death (HCD) response in both compatible and Yr1-mediated incompatible interactions, although the response was earlier and more extensive in the incompatible interaction. Later stages of fungal development were only associated with an HCD response in the incompatible interaction, the HCD response being effectively suppressed in the compatible interaction. Cell autofluorescence was seen in mesophyll cells in direct contact with fungal infection hyphae (primary HCD) and in adjacent mesophyll cells (secondary HCD), indicating the activation of cell-to-cell signalling. Microarray analysis identified a number of defence-related transcripts implicated in Yr1-mediated resistance, including classical pathogenesis-related (PR) transcripts and genes involved in plant cell defence responses, such as the oxidative burst and cell wall fortification. A quantitative reverse transcriptase-polymerase chain reaction time course analysis identified a number of defence-related genes, including PR2, PR4, PR9, PR10 and WIR1 transcripts, associated with the latter stages of Yr1-mediated resistance. A meta-analysis comparison of the Yr1-regulated transcriptome with the resistance transcriptomes of the race-specific resistance gene Yr5 and the race-nonspecific adult plant resistance gene Yr39 indicated limited transcript commonality. Common transcripts were largely confined to classic PR and defence-related genes.

Identification of differentially expressed transcripts from leaves of the boron tolerant plant Gypsophila perfoliata L.

Very recently some of the species of Gypsophila genus collected from the boron rich soils in Turkey were shown to be remarkably tolerant to high levels of boron. A limited amount of boron is necessary for the normal development of plants; however, a high level of boron in soil is generally toxic. Nevertheless, the adaptability of plant species allows them to withstand the presence of extreme amounts of metal ion by various strategies. This study is conducted on highly boron tolerant Gypsophila perfoliata L. collected from a location in the boron mining area. The plant samples were transferred into plant nutritional medium in the presence high; approximately 500 (35 mg/kg), 1,000, and 30 microM (considered normal) boron concentrations. We compared the transcriptome of the plant sample treated with the excess levels of boron to that of the samples grown under normal concentration using differential display PCR (DDRT-PCR) method. Thirty bands showing differential expression levels (presence or absence of bands or varying intensities) in either of approximately 500 or 30 microM B concentrations at varying time points were excised, cloned, and sequenced. Among which, 18 of them were confirmed via quantitative reverse transcription real time PCR (qRT-PCR). We are reporting the first preliminary molecular level study of boron tolerance on this organism by attempting to identify putative genes related in the tolerance mechanism. The gene fragments are consistent with the literature data obtained from a proteomics study and a metabolomics study performed in barley under varying boron concentrations.

Dexamethasone and basic-fibroblast growth factor regulate markers of mineralization in cementoblasts in vitro.

BACKGROUND: The aim of this study was to determine the effects of basic-fibroblast growth factor (b-FGF) and/or dexamethasone (Dex) on cementoblasts in vitro. METHODS: Murine cementoblasts were treated as follows: 1) 5% FBS (fetal bovine serum) + ascorbic acid (AA, 50 microg/ml, control); 2) 5% FBS + Dex (10(7)M) + AA; 3) 5% FBS + b-FGF (50 ng/ml)+AA; or 4) 5% FBS + Dex (10(7) M) + b-FGF (50 ng/ml)+AA and then evaluated by Northern analysis for changes in specific genes and by von Kossa stain for changes in mineral nodule formation. RESULTS: Mitotic activity: b-FGF stimulated DNA synthesis significantly versus negative control. Gene expression: osteocalcin (OCN): Dex or b-FGF or the combination resulted in a decrease in expression versus control. Bone sialoprotein (BSP): Dex increased expression of BSP mRNA levels, b-FGF decreased transcript for BSP at 6 and 24 hours. Long-term (8 days) Dex, b-FGF, or Dex plus b-FGF caused a decrease in BSP expression versus control; osteopontin (OPN): both Dex and b-FGF increased transcripts for OPN seen by 6 hours, with a greater increase noted with b-FGF versus Dex. No apparent additive effect of Dex with b-FGF was noted; matrix gamma-carboxyglutamic acid protein (MGP): b-FGF induced transcripts for MGP and addition of Dex increased this effect, while Dex alone had no effect on expression. Biomineralization: Dex increased cementoblast- mediated biomineralization, while b-FGF blocked this activity, and addition of Dex to b-FGF did not alter FGF associated inhibition. CONCLUSION: Dex and FGF alone and in combination alter cementoblast behavior, but additional studies are required to determine whether these factors have beneficial effects at the clinical level.