WRKY transcription factors, which play critical roles in stress responses, have not been characterized in eggplant or its wild relative, turkey berry. The recent availability of RNA-sequencing data provides the opportunity to examine WRKY genes from a global perspective. We identified 50 and 62 WRKY genes in eggplant (SmelWRKYs) and turkey berry (StorWRKYs), respectively, all of which could be classified into three groups (I–III) based on the WRKY protein structure. The SmelWRKYs and StorWRKYs contain ~76% and ~95% of the number of WRKYs found in other sequenced asterid species, respectively. Positive selection analysis revealed that different selection constraints could have affected the evolution of these groups. Positively-selected sites were found in Groups IIc and III. Branch-specific selection pressure analysis indicated that most WRKY domains from SmelWRKYs and StorWRKYs are conserved and have evolved at low rates since their divergence. Comparison to homologous WRKY genes in Arabidopsis revealed several potential pathogen resistance-related SmelWRKYs and StorWRKYs, providing possible candidate genetic resources for improving stress tolerance in eggplant and probably other Solanaceae plants. To our knowledge, this is the first report of a genome-wide analyses of the SmelWRKYs and StorWRKYs. Solanum melongena L.) is the third most agriculturally important crop from the genus Solanum after potato (S. tuberosum) [S. lycopersicum) [http://faostat.fao.org). Eggplant is susceptible to many bacterial and fungal pathogens and insects, such as the Verticillium dahlia fungus and nematodes [S. torvum Sw., commonly known as turkey berry, is a wild relative of eggplant and is resistant to root-knot nematodes and the most serious soil-borne diseases, such as those caused by Ralstonia solanacearum, V. dahlia Klebahn and Fusarium oxysporum f. sp. Melongenae [Eggplant (berosum)  and tomaersicum) . In 2011ematodes , which clongenae . Thus, tlongenae ,6,7,8,9.de novo transcriptome assemblies of eggplant and turkey berry from short-read RNA-sequencing data [Oryza sativa) [In spite of their economic and experimental importance, genome-wide research resources are limited for eggplant and turkey berry, because of the lack of whole-genome sequences. The recent availability of comprehensive and high-quality ing data  has alloing data . WRKY pring data . Severaling data , seed deing data , embryoging data ,16 and ling data . WRKY pring data , such asing data ,20,21 an sativa) , and are sativa) ,24,25,26N terminus of the WRKY domain is characterized by a highly-conserved WRKYGQK motif, whereas the C-terminal region of the domain contains a metal-chelating zinc finger motif, either C–X4-5–C–X22-23–H–X–H (C2H2) or C–X5–8–C–X25–28–H–X1-2–C (C2HXC) [N-terminal and C-terminal) with distinct functions and include a C2H2 motif, whereas Group II and III WRKY TFs contain one WRKY domain with a C2H2 zinc finger motif and C2HXC zinc finger motif, respectively [The WRKY genes are primarily restricted to plants with the exception of several examples in protozoa . Most WR (C2HXC) . The WRK (C2HXC) ,30. WRKY (C2HXC) . Group Iectively . Based oectively .s of these TFs based on Arabidopsis orthologs. Our results may provide candidate SmelWRKYs and StorWRKYs for genetic improvement of stress tolerance in eggplant and, probably, other Solanaceae plants.In this study, we performed a genome-wide identification of WRKY genes in eggplant (SmelWRKYs) and turkey berry (StorWRKYs) using transcriptome data. Phylogenetic trees were constructed to classify WRKY genes and to evaluate their evolutionary relationships in a wide range of plant species. Detailed analyses of SmelWRKYs and StorWRKYs were performed, including identification of variants of the highly-conserved WRKY domain, analyses of evolutionary selective pressure and inference with the biological functionde novo transcriptome assemblies of eggplant and turkey berry [Amborella trichopoda, greater duckweed (Spirodela polyrhiza), Arabidopsis, Populus trichocarpa, grape (Vitis vinifera), tomato, potato and hot pepper (Capsicum annuum L. Zunla-1), and the results are listed in http://plntfdb.bio.uni-potsdam.de/v3.0/fam_mem.php?family_id=WRKY&sp_id=ATH) and by PlantTFDB . We also identified 81 SlWRKYs (WRKY TFs in tomato), which are identical to those identified by Huang et al. [As the first step in identifying important WRKY regulators that function in response to abiotic and biotic stresses , we sougey berry . To ideney berry . With they berry . All of g et al. , confirmA. trichopoda, and of greater duckweed (Physcomitrella patens [Selaginella moellendorffii [Ricinus communis) [Cucumis sativus [Carica papaya [Based on our results, the eggplant WRKY family may be the smallest reported Solanaceae WRKY family to date, but it is larger than the WRKY families of the basal angiosperm, duckweed . In addia patens , the lycndorffii , and casommunis)  have few sativus  and Caria papaya  with 55 Solanum relatives, tomato and potato  is presented in N- and C-terminal domains of Group I and the domains of Group III are monophyletic; however, those from Group II were not monophyletic and were grouped into three distinct clades, IIa + IIb, IIc and IId + IIe. Group IIc is closely related to Group IC and clustered with Group IIa + IIb, whereas Group IId + IIe clustered with Group III.To determine the phylogenetic relationships and groupings among WRKY TFs identified in this study, an unrooted phylogenetic tree was constructed based on the amino acid alignment of 712 complete WRKY domains (from 606 WRKY TFs) identified by NCBI CDD from ey berry . The twoA. trichopoda, and of a wide range of species not only reduced potential long-branch attractions during phylogenetic tree construction, but also provided valuable information about the evolutionary history and classification of WRKY TFs since the emergence of the basal angiosperm. Interestingly, all of the WRKY TFs were more similar to those in the same groups/subgroups in divergent species than they were to other WRKY proteins in the same species  with distinct functions. However, in this study, several WRKY TFs with only one WRKY domain were phylogenetically grouped into Group IC or IN   .N-terminal and C-terminal WRKY domains of AtWRKY1 , AtWRKY40 , AtWRKY61 , AtWRKY57 , AtWRKY21 , AtWRKY65  and AtWRKY46 .The WRKY domains from eggplant and turkey berry WRKY TFs and several homologous Arabidopsis proteins were aligned in MEGA 6.0  Figure . For eacN-terminus followed by a zinc finger motif. Previous reports indicate that some plants contain variants of the WRKY domain, such as WRKYGKK, WRKYGEK and WRKYGSK [Glycine max) [cis-elements.The WRKY domain contains the highly-conserved heptapeptide stretch, WRKYGQK, at its  WRKYGSK . In toma WRKYGSK . In turk WRKYGSK , tomato  WRKYGSK  and largine max) . Variatiine max) . In contine max) . TherefoL. japonicus and M. truncatula [Because they are involved in responses to abiotic and biotic stresses , the WRKuncatula . TherefodN/dS) is a measure of natural selection acting on a protein. Generally, values for ω of 1, >1 and <1 indicate neutral, positive and purifying selection, respectively. The branch-site model, which allows ω ratios to vary among sites and lineages simultaneously, appeared to be most suitable for describing evolutionary processes of the WRKY gene family [et al. [L. japonicus and M. truncatula appear to be under purifying selection. Taken together, these results demonstrate that, in WRKY TFs, the natural selective pressures are likely to vary across different plant species and that subgroups have different evolutionary rates in particular species.The ω ratio , which assumes an independent ω ratio for each branch. As shown in dS values. Higher dN values in these branches relative to those in branches within each group also demonstrated that there is higher divergence at the amino acid level among groups than within each group. dS is recognized as an indicator of evolutionary rates. The high dS values  and in giant cells induced by the root-knot nematode (Meloidogyne incognita) [H. schachtii, tomato is susceptible and eggplant is less susceptible [dS value  with genes from turkey berry, but without genes from eggplant . These Scognita) . Eggplancognita) ; and witceptible . The pree ~1.21,  was obseet al. [Solanum. Interestingly, we did not find an ortholog of WRKY72 in eggplant, turkey berry or three other Solanaceae species, although this TF is proposed to play a partially conserved role in basal defense in tomato and Arabidopsis [Jasmonate and its derivatives are widely distributed in plants and affect a variety of processes, including responses to wounding and abiotic stress and defenses against insects and pathogens . With puet al.  identifiet al. , has twobidopsis .Belonolaimus longicaudatus) and root-knot nematodes (Meloidogyne spp.). As noted above, eggplant is susceptible to root-knot nematodes, whereas turkey berry is tolerant. Recently, Bagnaresi et al. [M. incognita infection. To investigate whether these root-knot nematode-responsive genes include WRKY TFs, we performed a BLASTN [et al. [et al. [et al. [et al. [Sclerotina sclerotiorum, which causes Sclerotinia blight in eggplant [et al. [S. sclerotiorum.Plant parasitic nematode infections generally occur as a result of root dysfunction and contribute to yield reductions. The most widespread and economically damaging nematode species include the sting nematode . The eg [et al. . Oxalic  [et al. , such aseggplant . Chen et [et al.  found thS. lycopersicum and S. tuberosum were downloaded from Sol Genomics Network . The annotated genome sequences of Arabidopsis, greater duckweed, A. trichopoda and hot pepper were downloaded from the Arabidopsis Information Resource , Mockler Lab (http://spirodelagenome.org/), the Amborella Genome Database (http://amborella.huck.psu.edu/) and the Pepper Genome Database (http://peppersequence.genomics.cn/page/species/download.jsp), respectively. The annotated genome sequences of V. vinifera and P. trichocarpa were downloaded from JGI phytozome v9.0 (ftp://ftp.jgi-psf.org/pub/compgen/phytozome/v9.0/Vvinifera and ftp://ftp.jgi-psf.org/pub/compgen/phytozome/v9.0/Ptrichocarpa/). To obtain high-quality representative gene sets for the above genomes, the longest translation form was chosen to represent each gene, and gene models with open reading frames shorter than 150 bp in the genomes were removed. Eggplant and turkey berry transcripts were downloaded from the NCBI Transcriptome Shotgun Assembly database under Accession Numbers GBEF00000000 (eggplant) and GBEG00000000 (turkey berry) [The annotated genome sequences of −5 was performed against public protein databases, including the NCBI non-redundant database, SwissProt [Solanum (tomato and potato) protein datasets and the NCBI database if conflicting results were obtained. ESTSCAN software [A BLASTX  search wwissProt  and the software  was alsosoftware . Again, A. trichopoda, Arabidopsis, hot pepper, P. trichocarpa, tomato, greater duckweed, potato, V. vinifera, eggplant and turkey berry.InterProScan version 4.5  was usedhttp://tree.bio.ed.ac.uk/software/figtree/). The WRKY genes were classified into different groups and subgroups based on the Arabidopsis WRKY classifications.The protein sequences of the complete WRKY domains identified by the NCBI CDD were collected using an in-house Perl script. Multiple sequence alignment of these domain sequences from all ten plant species was performed using MUSCLE v3.8.31  with defdS and dN and to detect positive selection using CODEML in the PAML 4.8 package in a maximum likelihood framework. The branch-specific dN, dS and ω ratios were calculated using the free-ratios model and an F3×4 codon frequency model.The amino acid sequences of complete WRKY domains from eggplant and turkey berry WRKY TFs were used to estimate a phylogenetic tree as described above. The amino acid sequence alignments were then converted into the corresponding CDS alignments, which were, together with the tree, used to estimate p-values calculated based on this mixture distribution were used to guide against violations of model assumptions. Posterior probabilities were calculated using the BEB method. The nodes were considered to have undergone positive selection if they showed a statistically-significant LRT, and positively selected sites were identified in the BEB analysis.The recommended branch-site test of positive selection was applied to detect positive selection affecting a few particular sites along selected lineages. We compared the null hypothesis, in which sites may evolve either neutrally (ω = 1) or under purifying selection (ω < 1), with the alternative hypothesis, which allows sites to be under positive selection (ω > 1). We then conducted the LRT analysis. The null distribution was a 50:50 mixture of chi-squared distributions with 1 degree of freedom and a point mass at zero; therefore, the A. trichopoda, greater duckweed, Arabidopsis, P. trichocarpa, V. vinifera, tomato, potato and hot pepper [E-value of 1 × 10−5, and hits with identity <30% and coverage <30% were removed. Orthologous groups were constructed from the BLASTP results with OrthoMCL v2.0.9 [The translated eggplant and turkey berry WRKY TF amino acid sequences were pooled into a WRKY TF protein database with sequences from another eight plant species: t pepper . Only set pepper  was condL v2.0.9  using deIn this study, we identified 50 SmelWRKYs and 62 StorWRKYs, and all of them could be classified into three groups (I–III). The SmelWRKY and StorWRKY families contain ~76% and ~95% of the number of WRKYs found in other sequenced asterid species, respectively. Different selection constraints could have affected the evolution of these groups. Sites with high probabilities of having been under positive selection were found in a subgroup of Group II (Group IIc) and Group III. Most WRKY domains from eggplant and turkey berry WRKY proteins are conserved and have evolved at low rates since their divergence. We also identified several pathogen resistance-related SmelWRKYs and StorWRKYs, providing possible candidate genetic resources for improving stress tolerance in eggplant and, probably, other Solanaceae plants. Overall, our results not only further our understanding of the evolutionary processes of eggplant and turkey berry WRKY genes, but also facilitate future functional genomics studies in these economically and genetically important crops.


Background and Aim. Helicobacter pylori (HP) and diet are both risk factors for gastric cancer. The aim of this study was to evaluate the Helicobacter pylori infection and dietary habits common in Khuzestan province. Methods. This cross-sectional study was conducted in 2011–2013 on 374 patients. Participants were interviewed using a food frequency questionnaire and tissue sample of the antrum was sent for pathology lab. The histopathological major variables were graded on a scale of 3  and data analyzed using nonparametric tests. Results. In this study, of 160 patients (43%) that were determined, 8.1 percent had severe contamination. Among dietary patterns, relationship between energy intake and carbohydrate with H. pylori was significant. A direct association was found between mean daily intakes of sausage (P = 0.001) and burgers (P < 0.05) with HP infection. Low intake of fresh vegetables and fruits was the most significant risk factors (P < 0.05). Conclusion. There is a possibility that some dietary factors such as consumption of fast foods and low intake of fresh vegetables may increase the chance of HP and severity of this infection. Helicobacter pylori is a spiral, gram negative, acid tolerant, microaerophilic bacterium that lives in the stomach and duodenum [ H. pylori infection is present in nearly 90% of adult population [ Helicobacter pylori infection [duodenum , 2. In Ipulation  and appepulation . Despitepulation . Epideminfection . H. pylori was introduced as a class I carcinogen [ H. pylori infection but also varieties of environmental factors are important risk factors for GC [ H. pylori infection [ H. pylori infection [ H. pylori gastric disorders [Besides the fact thatrcinogen , the infrcinogen . Previous for GC –12. It infection . Adequatnfection . Knoops isorders , 16. H. pylori has rarely been investigated [ H. pylori-infected subjects. The aim of this study was to evaluate whether there are differences in dietary habits between patients who were infected and subjects who were not infected.Furthermore, which environmental factor is involved in the development of GC among persons infected withstigated . TherefoThe study was carried out in 2011–2013 in a group of patients from the Gastroenterological Unit of the Emam Khomeini Hospital in Ahvaz who had functional dyspepsia and had been referred for endoscopic examination of the upper digestive tract. To minimize any known confounding effects, the subjects with the following conditions have been excluded from the study: subjects with habits such as smoking, tobacco chewing, and alcohol consumption; on corticosteroid therapy at pharmacological levels for duration of more than 6 months; diabetes mellitus .For all patients, energy and nutrient intakes were measured using a 3-day 24 h recall (two week days and one weekend day). Three-day food diary form contains foods at every meal, snack, and the amount consumed per person. A validated food frequency questionnaire  was alsoValues of each food were converted to grams by using household measures. An interview on dietary habits was performed by a dietician. Nutrients were analyzed by Nutritionist IV software , which was modified for Iranian foods.P < 0.05 was the significance threshold.After upper GI endoscopy a tissue sample from antrum, body, and fundus in plate containing formalin buffer was sent to the pathology lab. All samples were examined by a pathologist. Contamination detection was performed with hematoxylin eosin (H&E). Semiquantitative method of scoring according to the Updated Sydney Classification System was undertaken. Statistical analysis was done using SPSS software (version 13). To correlate variables with normal and nonnormal distribution, Pearson's and Spearman's correlations, respectively, were used. All tests were two-tailed, and  H. pylori infection, 214 healthy subjects (57%) and 160 patients (43%) were diagnosed that 8% of them had severe contamination and 37.5% and 54.4% had moderate and mild contamination, respectively.In this study, 374 patients were evaluated, 182 patients (48%) of them were females and 192 (52%) were males. Based on pathological studies on H. pylori infection.  There was also a negative and significant correlation between daily intake of fish (P = 0.001), olive oil (P = 0.002) and honey (P = 0.002), and peas and beans (P = 0.03) with H. pylori infection.As it is shown in P = 0.001), hamburgers (P = 0.002), fat mayonnaise (P = 0.002), and soft drinks (P = 0.001) with H. pylori infection.P = 0.001), onions (P = 0.002), green pepper (P = 0.01), apple (P = 0.002), and citrus fruits (P = 0.001) was significantly lower than in healthy individuals .Subjects with H. pylori infection.As it is shown in  H. pylori is probably one of the most common bacterial infections throughout the world, involving 30% of the population living in developed countries and up to 80%–90% of the population in developing countries [Epidemiological studies have shown thatountries . H. pylori is difficult, requires a two-week application of at least three medicines (proton pump inhibitors and two antibiotics) simultaneously, proves successful in only 80%–90% of cases, and is connected with the risk of adverse effects of therapy with antibiotics (15%–30% of the treated) [The treatment oftreated) , 21. H. pylori infection and diet has been suggested [ H. pylori infection (group I) and in the control not-infected group (group II). We demonstrated a significantly higher consumption of fruit, vegetables, and vitamin C among persons who were not infected. Vitamin C is highly concentrated in stomach mucosa and gastric juice and probably lowers the risk of gastric cancer and influences the course of H. pylori infection through a number of mechanisms [ H. pylori is now being examined. Jarosz et al. [ H. pylori infected patients with chronic gastritis with a high dose of vitamin C caused H. pylori eradication in 30% of cases. In those patients, a highly significant rise in gastric juice total vitamin C concentration was demonstrated, which persisted for at least four weeks after treatment. Ruiz et al. [ H. pylori infection and low ascorbic acid levels in the gastric juice. Their findings supported two hypotheses that explain this phenomenon: increased oxidation and decreased secretion of ascorbic acid.A synergistic interaction betweenuggested . In thischanisms , 24. It z et al.  showed tz et al.  found a  H. pylori pathogenicity or protective agents that hamper its activity as related to the appearance of cancer diseases. Production of nitrosylating species plays a fundamental role in this regard. Observational cohort studies should ideally provide much more reliable evidence. Analysis of the data substantially confirmed the significant increased risk of developing GC due to high intake of total carbohydrates, processed meat, refined grains, and saturated fat [Also diet may provide noxious agents that contribute toated fat , 27. Proated fat . H. pylori infection.Previous studies have reported the protection role of allium vegetables (onion) in gastric cancer –29. In tP = 0.001), hamburgers (P = 0.002), fat mayonnaise (P = 0.002), and soft drinks intake (P = 0.001) with H. pylori infection.In this study there was positive relation between carbohydrate, sausages  or regarding specific types of unsaturated fatty acids and gastric cancer is very limited, but, in some studies, protective effect of vegetable oil consumption in gastric cancer has been reported . H. pylori infection [Some studies of healthy adults demonstrated subclinical deficiency of some B vitamins and high prevalence ofnfection .H. pylori stimulates the macrophage system through the l-arginine/nitric oxide (NO) pathway [ H. pylori infection of the human stomach may increase endogenous NO formation, yielding, after oxidation, the nitrosating agents N2O3 and N2O4, which can produce nitrosamines or cause other types of DNA damage [ H. pylori-induced atrophic gastritis. This may result from bacteria within the stomach generating nitrite from dietary nitrate [ pathway . As a coA damage . NitrosaA damage . Nitrite nitrate .In present study among the micronutrients reported, there were significant differences in intake of folate between the healthy subjects and patient groups. H. pylori infection causes atrophic gastritis and decreased absorption of vitamin folic acid in patients with this condition. Because methylation of homocysteine to methionine requires folate, an increased plasma homocysteine level is found in H. pylori-infected patients [ H. pylori infection. Third limitation was that level of education, socioeconomic, and race of subjects are not considered.It has been established that chronicpatients . Our stu H. pylori-infection displayed that a diet rich in fruit and vegetables and poor in meat, fat, and salt has a good prophylactic potential for cancer. Fruits and vegetables are sources (among other protective agents) of ascorbic acid, which were associated with a high effect against the progression of gastric carcinogenesis.In summary, the present study on food interaction andThus, “diet for cancer prevention” can be recommended as a general role of well-being and can represent the basis for a rational health policy.


Utricularia attract attention not only owing to their carnivorous lifestyle, but also due to an elevated substitution rate and a dynamic evolution of genome size leading to its dramatic reduction. To better understand the evolutionary dynamics of genome size and content as well as the great physiological plasticity in this mostly aquatic carnivorous genus, we analyzed the transcriptome of Utricularia vulgaris, a temperate species with well characterized physiology and ecology. We compared its transcriptome, namely gene content and overall transcript profile, with a previously described transcriptome of Utricularia gibba, a congener possessing one of the smallest angiosperm genomes.The species of U. vulgaris including leaves and traps, cultivated under sterile or outdoor conditions. 454 pyrosequencing resulted in more than 1,400,000 reads which were assembled into 41,407 isotigs in 19,522 isogroups. We observed high transcript variation in several isogroups explained by multiple loci and/or alternative splicing. The comparison of U. vulgaris and U. gibba transcriptomes revealed a similar distribution of GO categories among expressed genes, despite the differences in transcriptome preparation. We also found a strong correspondence in the presence or absence of root-associated genes between the U. vulgaris transcriptome and U. gibba genome, which indicated that the loss of some root-specific genes had occurred before the divergence of the two rootless species.We sequenced a normalized cDNA library prepared from total RNA extracted from shoots of Utricularia offers a unique opportunity to study adaptations related to the environment and carnivorous habit and also evolutionary processes responsible for considerable genome reduction. We show that a transcriptome may approximate the genome for gene content or gene duplication estimation. Our study is the first comparison of two global sequence data sets in Utricularia.The species-rich genus The online version of this article (doi:10.1186/s12870-015-0467-8) contains supplementary material, which is available to authorized users. Utricularia (Lentibulariaceae) are the most versatile and cosmopolitan among carnivorous plants, exhibiting great morphological and ecophysiological plasticity [Utricularia are aquatic or amphibious, growing in standing, nutrient-poor humic waters. While their ecology and carnivorous habit have been researched previously [Utricularia genomes - miniature size in many species within the family [Utricularia gibba possesses one of the smallest haploid angiosperm genomes known, approximately one-half that of Arabidopsis thaliana, with chromosomes of bacterial size [U. gibba was the subject of the first broad survey of nuclear gene transcripts in Utricularia species [U. gibba genome [Members of the rootless genus asticity -3. Approe family ,5, highle family -9, and te family ,10. Utriial size ,5. U. gi species ,12, revea genome  additionUtricularia spp. have since been identified as prime candidates for further research on the complexities of plant ecophysiology associated with carnivory, metagenomic surveys of trap microbial communities, novel plant nitrogen/nutrient utilization pathways, the ecology of prey attraction, whole-plant and trap comparative development, and the evolution of a minimalist angiosperm genome [Utricularia gibba, however, is not a good candidate species for many ecological and physiological experiments due to its minute size and extremely small traps. We have therefore chosen the ecologically well-characterized temperate Utricularia vulgaris [U. gibba, offering the possibility for a comprehensive comparison of genome-wide expression patterns between the two species.m genome ,5,14-20.vulgaris ,16-18 asU. vulgaris, derived from a pooled sample of multiple tissue types, including functional annotation of expressed gene content. We compared this transcriptome to the U. gibba transcriptome [U. vulgaris transcriptome, including a gene for which this post-transcriptional process has not been investigated in any plant species.In this study, we report the results of 454 GS-FLX Titanium sequencing of a polyA-selected and normalized cDNA library from criptome  and showU. vulgaris, provided that it is prepared from multiple tissues and various environmental conditions [U. vulgaris transcriptome for the identification of gene losses and duplications during the course of evolution of the genus Utricularia.Although any transcriptome should be viewed as incomplete, it may serve as an acceptable proxy for the genome in a species without complete genomic information, such as nditions ,22. We dU. vulgaris normalized cDNA library, 1,389,835 of them passed built-in quality filtering. 91.5% of the initial, raw reads were assembled by Newbler 2.7 and produced 19,522 isogroups containing 41,407 isotigs, roughly corresponding to the individual transcripts. In addition, 64,188 singletons longer than 100 nt were obtained. Isotigs and singletons were combined together into a unique transcript (UT_U.vulgaris) data set representing the U. vulgaris transcriptome. To facilitate the comparison between our data and the U. gibba transcriptome published by [U. gibba were downloaded from DNA Data Bank of Japan (DDBJ) under the submission SRA029151 and assembled by Newbler 2.7 using the same parameters as adopted for the U. vulgaris transcriptome (UT_U.gibba). Table U. vulgaris data set consisted of nearly twice as many raw reads, a higher proportion of which assembled into contigs, than the U. gibba dataset. The U. vulgaris assembly also resulted in a higher number of isogroups and much higher (about three fold) number of isotigs. Furthermore, our U. vulgaris assembly produced only 64,188 singletons compared to the 99,900 singletons remaining after de novo U. gibba assembly. The U. vulgaris data set contained about 2.1 isotigs per isogroup, whereas only 1.2 isotigs per isogroup, on average, were found in the U. gibba assembly. The much higher number of isotigs in the U. vulgaris transcriptome, both relative (per isogroup) and absolute, was at least partly caused by the method of cDNA library preparation. Our U. vulgaris cDNA library was normalized, increasing the number of rare transcripts represented by isotigs.In total, 1,405,703 reads were generated by 454 pyrosequencing of the ished by , raw reaU. vulgaris isotigs (96%) gave significant BLAST hit against the NCBI nr protein database . These sequences were further annotated using the BLAST2GO annotation pipeline. 30,392 isotigs  were successfully annotated. 9,794 isotigs (33% of annotated isotigs) were assigned with enzyme codes (E.C.). The average level of annotations in GO hierarchy was 5,868. The total number of assigned Gene Ontology terms was 212,122  gave a significant BLAST hit against the NCBI nr protein database under the same parameters as used for isotigs. 14,536 singletons  were successfully annotated and 4,121 singletons (28% of annotated singletons) were assigned with E.C. The average level of annotations in GO hierarchy was 5,791. The total number of assigned Gene Ontology terms was 90,735. The much lower proportion of U. vulgaris singletons yielding significant BLAST hits, compared with the isotigs, may be due to their short sizes and also due to the presence of transcripts derived from microbes without any NCBI record.Of the total 58,363 U. gibba than in U. vulgaris. This difference results from a higher amount of unassembled reads in UT_U.gibba relative to UT_U.vulgaris. The proportion of isotigs with an assigned E.C. was also higher in U. vulgaris than in U. gibba.The results of the GO annotations of the UT_U.gibba transcriptome are given in Table Utricularia species  which can be easily searched by BLAST or annotation.We summarized the results of our U. vulgaris and U. gibba. All remaining isotigs (38 in U. vulgaris and 87 in U. gibba) belonged to Fungi, Metazoa, unicellular eukaryotes, and prokaryotes  in both s Figure . The taxU. vulgaris transcriptome contained six isogroups with > 100 isotigs, 23 isogroups with > 45 isotigs, and 332 isogroups with > 10 isotigs. The largest, isogroup 00018 in U.vulgaris, included 480 isotigs derived from various members of a large BETA GLUCOSIDASE gene family.The isogroups containing numerous isotigs may include transcripts derived from several or many loci, e.g. retroposons, or from transcripts undergoing AS . The U. U. gibba assembly contained zero isogroups with > 100 isotigs, only two isogroups with > 45 isotigs, and 17 isogroups with > 10 isotigs  [U. gibba genome draft (CoGe-id36222).In contrast, the  family)  or the iU. vulgaris and in U. gibba. These isotigs contain contigs corresponding to Arabidopsis exons and also numerous contigs which may be assigned to introns based on their position between two exons. Additional file U. vulgaris and U. gibba, respectively. They represent various genes or gene families belonging to similar structure and function categories.Alternative splicing appears to be the main reason for the transcript abundance and diversity in many of the largest isogroups in Utricularia species. It encodes a family of ATP dependent RNA helicases. Its Arabidopsis homologs  are involved in a wide range of RNA metabolism including pre-mRNA splicing, mRNA transport, turnover, translation initiation etc. [U. vulgaris match Arabidopsis exons, suggesting extensive AS of transcripts derived from at least two related genes. The more than four fold higher isotig count of the 00008 isogroup in U. vulgaris than in U. gibba may again reflect a significant enrichment in rare transcripts due to cDNA normalization of U. vulgaris transcriptome, or reflect a lower extent of AS in U. gibba. Three other isogroups of U. vulgaris could participate in the control of AS, including the isogroup 00013, encoding a homolog of AFC2 protein kinase, which underwent extreme AS (producing multiple splice variants from the same primary transcript) in Arabidopsis [U. vulgaris transcriptome. No large isogroup corresponding to transposons or retroposons was found in the U. gibba transcriptome, however three single isotigs were.Only one large isogroup (00008) appears to be the same in both ion etc. ,26. Theyion etc. . Five cobidopsis . The remU. vulgaris with very high isotig counts for more detailed analysis. After aligning all 277 isotigs of the isogroup 00007, we found that all of them were derived from the same locus, because only one sequence variant (contig) corresponded to each exon of the homologous Arabidopsis gene, At1g27980, coding for sphingoid long-chain base 1-phosphate lyase (LCB-1-P lyase)  and exon 15 (reverse) and ran PCR  in Arabidopsis is an example of the mixture of alternatively spliced transcripts derived from at least two loci. We identified contigs corresponding to the exons and introns of the EIN2 gene. Several putative exons existed in two sequence variants and occurred in chimeric isotigs. We confirmed the occurrence of two EIN2 gene copies by direct sequencing of U. vulgaris genomic DNA. We designed two primer pairs UV304_F1, R1 and UV308_F1, R1  of U. vulgaris sequences with EIN2 homologs across angiosperms was used in phylogenetic analysis to generate MP and ML trees .To achieve the correct assembly of alternative transcripts in a species without reference genome is very difficult. It becomes even more challenging if multiple similar paralogous genes are transcribed and alternatively spliced. In such cases, chimeric misassembled contigs are frequently generated . The isoEIN2-1 and EIN2-2 of U. vulgaris was 0.41, between EIN2 of U. vulgaris and EIN2 of U. gibba was 0.44 . The data suggest no variation in evolutionary constraints.The ratio of non-synonymous and synonymous substitutions (Ka/Ks) in the pairwise comparison between U. gibba, which represents an in silico transcriptome of this species. We chose the U. gibba transcriptome derived from a genomic draft, because it supposedly represented more complete set of transcripts than the experimental transcriptome UT_U. gibba.We performed a reciprocal BLAST hit search to identify putative orthologs between the UT_U.vulgaris transcriptome and a 19475-mRNA database derived from the genomic draft of U. vulgaris isotigs and the remaining 1,667 pairs contained U. vulgaris singletons. The orthologs represented about 42.9% of all genes annotated in the U. gibba genomic draft. We ordered putative orthologous pairs between U. gibba and U. vulgaris according to the sequence similarity of the regions aligned by BLAST. The distribution of orthologs assigned to individual similarity classes is shown in Figure U. vulgaris isotigs or singletons. Singletons are much shorter than an average isotig , we investigated which GO categories were enriched among the most conserved orthologous pairs. GO enrichment (AgriGo) [U. gibba and U. vulgaris revealed genes which were less similar to their Arabidopsis counterparts than the rest of the highly conserved orthologs, namely MYOSIN XI B (homolog of At1g04160) and TIP GROWTH DEFECTIVE 1 (TIP1) (a homolog of At5g20350). Interestingly, both genes play a role in root hair development in Arabidopsis. As neither U. gibba nor U. vulgaris produce roots, it is probable that the two genes have gained a novel or modified functions in Utricularia, explaining why their sequences are highly similar between both Utricularia species, but less similar to Arabidopsis homologs.Because the overall sequence similarity of putative (AgriGo)  analysisUtricularia vulgaris does not form roots, some of the genes involved in root development and function might have been lost. Ibarra-Laclette et al. [A. thaliana, but not found in the genome of U. gibba. They include MYB transcription factors, MADS box genes, cell-wall-associated kinases, nitrate transporter etc.  and the UT_U.vulgaris transcriptome. We found a strong correspondence between the absence of particular genes in the U. gibba genome [U. vulgaris transcriptome  or root cap development . These genes were also missing in the U. gibba genome (CoGe-id36222). On the other hand, some genes such as AUXIN RESPONSE FACTOR 4 (ARF4) or INDOLE-3-ACETIC ACID INDUCIBLE (IAA) genes found putative orthologs in both the U. vulgaris transcriptome and the U. gibba genome  under study contained complete ORFs, the rest of isotigs represented partial sequences, which reflected an overall incompleteness of experimental transcriptomes.As e et al.  publishee et al. ). We sela genome  and theiU. vulgaris transcriptome was caused by its low or missing transcription. However, the coincident absence of 48 root-associated genes and concordant presence of 11 root-associated genes in the U. vulgaris transcriptome and the U. gibba genome suggest that, at least in the case of root genes, the UT_U.vulgaris transcriptome reflects the gene content of the U. vulgaris genome.Because the transcriptomes are incomplete, the absence of an ortholog in the transcriptome, by itself, is not the evidence of its absence in the corresponding genome. Thus, we may not exclude the possibility that the absence of any respective gene in the AUXIN RESPONSE FACTOR 16 (ARF16), duplicated in the U. gibba genome [U. vulgaris transcriptome. The full agreement between the sets of root-associated genes lost in U. gibba and U. vulgaris and the concordance of the genes duplicated in both species support the notion that deletions and duplications of genes involved in root-associated genes occurred before the divergence of the two Utricularia species.Interestingly, two copies of the gene a genome , were alhttps://sites.google.com/a/ualberta.ca/onekp) is just one example of current efforts. The genomic draft of U. gibba [U. gibba generated by 454 pyrosequencing from various organs [U. gibba a suitable species for comparative transcriptomics in Utricularia. We chose a temperate congener U. vulgaris as a counterpart for comparison. Both species share an aquatic carnivorous life style, lack of roots, and display rapid apical shoot growth, but exhibit partly distinct ecophysiology  initiative . Alternatively, the impact of very different data sets cannot be excluded. In this case, it would affect only the two GO categories, which appears unlikely.The distribution of GO categories in rracenia . This stArabidopsis [U. vulgaris and Arabidopsis, because missing genomic information in U. vulgaris prevents the accurate assignment of splice variants. However, 23 largest isogroups in U. vulgaris  was shown to be alternatively spliced not only in bidopsis ,26 and iU. vulgaris homologous to the gene for LCB-1-P lyase in Arabidopsis. The function of LCB-1-P lyase or sphingosine-1-phosphate (SPH-1-P) lyase in plants is not fully understood. Ng et al. [AtDPL1, At1g27980), which was located in the endoplasmic reticulum. LCB-1-P lyase regulates LCB-1-P content and through this activity participates in stomata closure and dehydration stress response in Arabidopsis [We also documented extreme AS in the isogroup 00007 in g et al. , Coursolg et al.  describeg et al.  showed tbidopsis ,34.U. vulgaris transcriptome was generated from submersed plant organs which did not develop stomata. However, we have verified that above-water flower stems of U. vulgaris contain stomata . It is therefore possible that transcripts with retained introns represent a pool from which functional transcripts may be readily formed by additional splicing when LCB-1-P lyase becomes necessary. This protein may be needed when the submersed plant body continues to grow above water. A similar regulatory role of intron retention was observed, for example, in the fern Marsilea vestita, where transcripts with retained introns were stored in spores and spliced after germination [Our mination .Utricularia, not associated with stomata. As SPH-1-P affects ion channels in guard cell protoplasts [Utricularia traps, which is associated with potassium channels in trap bifid glands [U. gibba, because non-normalized transcriptomes have only limited potential to detect AS.The expression of the gene for LCB-1-P lyase in submersed plant organs lacking stomata may also suggest that it fulfills a distinct function in toplasts , we specd glands . FinallyEIN2 gene in U. vulgaris transcriptome. This gene is essential for ethylene signaling and occurs in a single copy in many plant species and its duplication is rare among angiosperms [EIN2 paralogs undergoing accelerated evolution were recently identified in Lotus japonicus [EIN2 paralogs in Utricularia vulgaris may be important for the interaction between plants and microbes, similar to the role of the two EIN2 genes in the symbiosis between leguminous plants and bacteria [EIN2 gene occurred early in the evolution of the genus Utricularia and might have been associated with the transition to a carnivorous life-style. Subsequently, one copy was lost in U. gibba. Similar Ka/Ks ratios in pairwise comparisons of Utricularia EIN2 genes do not indicate any shift in function. However, it should be emphasized, that only parts of the U. vulgaris EIN2 genes, confirmed by Sanger sequencing, were analyzed. The examination of additional species of Lentibulariaceae regarding EIN2 multiplication will shed light on the evolution and function of this important gene.We found a duplication of iosperms ,38. Two aponicus . They rebacteria . We specUtricularia species was only 87% - [U. vulgaris and U. gibba are classified in the same generic section Utricularia [U. gibba and U. vulgaris appears to be a high substitution rate, described in Lentibulariaceae as one of the characteristics of the plant carnivorous syndrome [Utricularia species.The median value of sequence similarity among 12,267 putative orthologous pairs  between the two % Figure , much los (98%)-  or betwes (98%)- ) which bicularia , althougsyndrome ,42. HoweU. gibba genome [U. vulgaris transcriptome. The correspondence between both Utricularia species was also observed in additional root-associated genes, not specifically analyzed by [U. vulgaris genome. It is probable that the loss of root-associated genes had occurred already in the ancestor of U. gibba and U. vulgaris. The comparison of the presence or absence of root-associated genes in additional Utricularia species will be very useful for understanding the adaptation to an aquatic rootless carnivorous life-style.We found a perfect coincidence between the absence of root-associated genes in the a genome  and the lyzed by   sterile plants from a meristem tissue culture , growing in a half-strength Gamborg B5 liquid medium with 500 mg l−1 KNO3, microelements, vitamins, and 2.5% sucrose, but without other organic substances [Carex spp.) used as a substrate [We utilized bstances ; (2) stebstances , with rebstances , with thubstrate  and partTissue for RNA extraction was harvested in the middle of the dark period to reduce the amount of Rubisco-coding mRNA in samples, frozen immediately in liquid nitrogen, and stored at-70°C until further processing.Total RNA was extracted from entire shoots, including leaves and traps, from all three sources described above. Inflorescences were not used in this study. Approximately 800 mg of fresh weight  of whole shoots in three replicates from each source were ground in liquid nitrogen using a mortar and pestle and RNA was extracted using RNeasy Plant Midi Kit . The results of the parallel extractions were pooled in equal proportions into a single sample and the quality and quantity of the extracted RNA were verified by gel electrophoresis and by spectrophotometer, measuring the 230/260 ratio with Biophotometer .A pooled RNA sample was provided to GATC Biotech  for the construction of a cDNA library and subsequent sequencing. Library construction involved DNAselection for polyadenylated (polyA+) transcripts to enrich for protein-coding mRNAs, DNase I treatment and normalization through denaturation/reassociation of cDNA, to improve the representation of low-copy transcripts and thereby maximize gene discovery. The resulting library was sequenced with a full picotiter plate on a 454 GS-FLX sequencer with Titanium reagents , using standard 454 protocols.http://www.ebi.ac.uk/ena/data/view/PRJEB8057).Raw reads obtained by 454 pyrosequencing were submitted to European Nucleotide Archive under the study accession number PRJEB8057  and singletons (transcripts represented by single reads) was done using the BLAST2GO pipeline  with theMEGAN v 5.7.0  with defU. vulgaris and U. gibba were identified by means of reciprocal BLAST search [U. vulgaris containing both isotigs and the singletons which passed quality filtering . As the U. gibba transcriptome published by Ibarra-Laclette et al. [U. gibba, from CoGe website (https://genomevolution.org/CoGe/-id36222). We generated local databases and used BLASTN, threshold e–10, to retrieve the best hits in reciprocal BLAST searches. Reciprocal BLAST outputs were compared by RBH  script and putative orthologs which appeared as the same pair in both BLAST outputs were identified. The similarity of orthologs in % was based on high-scoring segment pair (HSP) of BLAST alignments.The orthologous transcripts between T search . We usede et al.  might haU.vulgaris and Arabidopsis (TAIR10_prot_20101214) were searched in a similar way, with BLASTX (threshold e–10) and TBLASTN (threshold e–10) instead of BLASTN, which means that predicted protein sequences were aligned.The orthologs between U. vulgaris and more than 10 isotigs in U. gibba and found their Arabidopsis counterparts using the BLASTX search in TAIR (www.arabidopsis.org). We assigned their functions based on TAIR annotations. We aligned all isotigs from two selected isogroups of U. vulgaris, each displaying an extremely high number of isotigs, by MUSCLE implemented in Geneious 7.1.5. We identified introns retained in the transcripts with the help of Layout output from Newbler 2.7 and by comparison with gene structures in Arabidopsis (TAIR).We selected the isogroups containing more than 45 isotigs in U. gibba–U. vulgaris orthologs by means of AgriGO (http://bioinfo.cau.edu.cn/agriGO) [customized annotated reference and the list of orthologs with specific similarity as a customized annotation. We applied Yekutieli multi-test adjustment, significance level 0.05 and a minimum number of mapping entries of 4.We performed the analysis of enrichment of particular GO categories among putative /agriGO) . We inpuU. vulgaris plants grown outdoor and isolated total RNA by RNeasy Plant Mini Kit (Qiagen). RNA was treated with DNase I  to eliminate a DNA contamination. The treatment was repeated until no amplification was observed in PCR reaction with RNA as a template. One μg of RNA and oligo dT primers (500 ng) were heated 5 min at 65°C, chilled on ice and mixed with Transcriptor buffer , 0.5 μl of Protector RNase Inhibitor (Roche Scientific), 2 μl of 10 mM dNTPs and 10 units of Transcriptor Reverse Transcriptase (Roche Scientific). The first strand of cDNA was synthesized at 55°C for 30 min and then amplified with the primers , Genlisea aurea (EPS70151), Solanum tuberosum (XM006354273), Petunia hybrida (AY353249), Populus trichocarpa (XM002326149), Lotus japonicus (chr5.CM1729.230 and chr1. CM0012.1100), Dianthus caryophyllus (HQ441183), Arabidopsis thaliana (NM12040), Zea mays (AY359584), and Sorghum bicolor (XM002457067). We aligned these angiosperm EIN2 sequences with partial protein sequences EIN2-1 and EIN2-2 from U. vulgaris and created a 1,361 amino acid long gapped-alignment in Geneious 7.1.5. We then adopted a maximum parsimony (MP) approach with stepwise addition to the starting tree, no topology constraints, tree bisection-reconnection (TBR) swap, and 1000 bootstrap pseudoreplicates to construct a 60% consensus tree in PAUP*4 v10 [EIN2 paralogs in Utricularia we performed Maximum Likelihood (ML) analysis in PAUP*4 v10 [We downloaded UP*4 v10 . Gaps weUP*4 v10  of the sU. vulgaris transcriptome assembly and annotation and created a web-accessible database (http://utricularia.prf.jcu.cz/index.php) which can be easily searched by BLAST or annotation. Raw reads obtained by 454 pyrosequencing were submitted to European Nucleotide Archive under the study accession number PRJEB8057 (http://www.ebi.ac.uk/ena/data/view/PRJEB8057). The sequences of EIN2 paralogs were deposited in GenBank under the numbers KP279653 and KP279654. The data sets undelying phylogenetic EIN2 studies was submitted to TreeBase under the ID 17158. The sequences of U. gibba were obtained from the study of Ibarra-Laclette et al. [We summarized the results of our e et al.  (doi:10.


Few studies have tested whether mutualisms may affect species distributions by altering the niches of partner species. We show that a fungal endophyte is associated with a shift in the soil moisture niche of its host plant relative to a co-occurring, endophyte-free congener. The endophyte appeared to initially restrict its host's distribution to wetter microsites before positively affecting its growth, suggesting the value of considering symbiont effects at different partner life stages. Our study identifies a symbiotic relationship as a potential mechanism facilitating the coexistence of two species, suggesting that symbiont effects on host niche may have community-level consequences. Epichloë) may shift the niche of its partner plant, marsh bluegrass (Poa leptocoma) relative to a closely related but endophyte-free grass species, nodding bluegrass (Poa reflexa). Using observations and a 3-year field experiment, we tested two questions: (i) Do P. leptocoma and P. reflexa occupy different ecological niches? and (ii) Does endophyte presence affect the relative fitness of P. leptocoma versus P. reflexa in the putative niches of these grass species? The two species were less likely to co-occur than expected by chance. Specifically, P. leptocoma grew closer to water sources and in wetter soils than P. reflexa, and also had higher root colonization by mycorrhizal fungi. Endophyte-symbiotic P. leptocoma seeds germinated with greater frequency in P. leptocoma niches relative to P. reflexa niches, whereas neither endophyte-free  P. leptocoma seeds nor P. reflexa seeds showed differential germination between the two niche types. Thus, endophyte presence constrained the germination and early survival of host plants to microsites occupied by P. leptocoma. However, endophyte-symbiotic P. leptocoma ultimately showed greater growth than endophyte-free plants across all microsites, indicating a net benefit of the symbiosis at this life history stage. Differential effects of endophyte symbiosis on different host life history stages may thus contribute to niche partitioning between the two congeneric plant species. Our study therefore identifies a symbiotic relationship as a potential mechanism facilitating the coexistence of two species, suggesting that symbiont effects on host niche may have community-level consequences.Mutualisms can play important roles in influencing species coexistence and determining community composition. However, few studies have tested whether such interactions can affect species distributions by altering the niches of partner species. In subalpine meadows of the Rocky Mountains, USA, we explored whether the presence of a fungal endophyte (genus  Mutualisms can play important roles in influencing species coexistence and determining community composition  and affect its co-occurrence with a similar but naturally endophyte-free grass species, Poa reflexa (nodding bluegrass) in subalpine meadows of the Rocky Mountains, USA. Although it has been suggested that P. leptocoma prefers wetter habitats (P. leptocoma and P. reflexa populations to document endophyte symbiosis and carried out endophyte species characterization via DNA sequencing. We then addressed two questions to explore the effect of endophyte symbiosis on multiple stages of host life history: (i) Do P. leptocoma and P. reflexa occupy different ecological niches? and (ii) Does endophyte presence affect the relative fitness of P. leptocoma versus P. reflexa in their putative niches? We hypothesized that (i) the two species occupy different ecological niches in the habitats examined, and that (ii) endophyte symbiosis alters the relative fitness of P. leptocoma versus P. reflexa in the two species' putative niches. We predicted that endophyte-symbiotic P. leptocoma seeds would perform best in P. leptocoma microsites (niches), and that P. reflexa seeds would perform best in P. reflexa microsites. If the endophyte were to shift the niche dimensions of its host, then endophyte-free P. leptocoma individuals would have niche requirements more similar to those of P. reflexa; thus, we predicted that P. leptocoma endophyte-free seeds would perform better in P. reflexa microsites than in P. leptocoma microsites.We evaluated whether the presence of a fungal endophyte may alter the niche of habitats . We survP. leptocoma (sect. Oreinos) and P. reflexa  grow in western North America in subalpine meadow and forest understory environments near streams  and hybrid and non-hybrid asexual species, formerly called Neotyphodium spp.   south to Gunnison County [seeSupporting Information]. Thin sections from the inner leaf sheaths of collected samples were stained with aniline blue lactic acid dye following the methods in To evaluate endophyte symbiosis in the two Epichloë species diversity present in P. leptocoma using plant material collected from six sites in Gunnison County, CO [seeSupporting Information]. Total DNA was extracted from P. leptocoma plant material using a QIAGEN MagAttract 96 DNA Plant Core Kit . Multiplex PCR was carried out using 19 established genus-specific primers representing housekeeping, mating type and key alkaloid biosynthesis genes  and T1.2 for tubB . The nearest individual of each species was marked approximately every 2 m along transects placed in three regions of the watershed: directly in and along a shady streambed (where only P. leptocoma was present), next to a sunny streambed (both species) and in a wet meadow between the two streams (both species). Then, during August 2011, we used the same transect method to mark a minimum of 10 individuals per species at each of two sites near Schofield Pass  and at one site on Snodgrass Mountain  [seeSupporting Informationfor sample sizes].To document the current distributions of the bluegrass species, we marked naturally occurring plants at four sites. First, during August 2008, we marked 77 P. leptocoma and P. reflexa densities in randomly placed quadrats versus quadrats placed around target plants of each species (24–25 July 2012). For the random samples, we placed 30 × 30 cm quadrats at 2 m intervals along eight transects in the Virginia Basin study area and in nearby Copper Creek . We counted the number of flowering P. leptocoma and P. reflexa individuals per quadrat to determine an expected density per square metre for reproductive adults of each species, evaluating a total of 58 quadrats. At each transect interval, we also located the nearest bluegrass individual (P. leptocoma or P. reflexa), placed the quadrat around the centre of the identified target plant and counted the number of flowering individuals of each species in the quadrat to quantify plant density. We evaluated a total of 24 P. leptocoma targeted quadrats and 25 P. reflexa targeted quadrats. We used a one-tailed, unequal variance t-test to compare the density of each species in the randomly located quadrats against their densities in the targeted quadrats. These analyses tested (i) whether targeted quadrats had higher densities of conspecifics than expected by chance, indicative of a clumped species distribution and (ii) whether the two species were less likely to co-occur than expected by chance, suggestive of different niches or competitive exclusion.To examine natural patterns of co-occurrence, we compared P. leptocoma prefers wetter environments relative to P. reflexa . A significant species × site interaction would indicate inconsistency in species niche characteristics across sites. When such interactions were significant, we used P. leptocoma and P. reflexa microhabitats and compared germination, seedling survival and plant growth.We used the marked plants at the Virginia Basin site as replicates of each species' specific microhabitat, i.e. its putative niche. This approach eliminated the need to replicate the precise abiotic and biotic conditions characteristic of each species' microhabitat. To test whether plant performance differed between species and/or with endophyte symbiosis, we planted seeds into natural P. reflexa seeds collected from field plants; two naturally endophyte-symbiotic (E+) P. leptocoma seeds collected from field plants ; three endophyte-free (E−) P. leptocoma seeds collected from greenhouse-grown plants from which the endophyte was experimentally removed; and one P. leptocoma endophyte-symbiotic (E+) ‘control’ seed collected from a greenhouse-grown plant. This approach controlled for maternal effects by decoupling endophyte presence from effects associated with plant genotype or maternal environment.Surrounding each marked, naturally occurring plant, we planted a total of nine seeds: three naturally endophyte-free (E−) P. leptocoma E+ natural and P. reflexa seed treatments, we collected seeds from the Virginia Basin site during late August 2009 and stored them at 4 °C until planting. Poa leptocoma E− seeds and E+ control seeds came from plants grown in a common greenhouse environment from the Virginia Basin seed stock. To create endophyte-free (E−) P. leptocoma seeds, we experimentally removed the endophyte by germinating seeds for 5 weeks on wet filter paper in standard plastic Petri plates containing the fungicide benomyl (2 g/L). Seeds for the P. leptocoma E+ control treatment were germinated in Petri plates that contained water only. Following germination, P. leptocoma E+ control and E− seedlings were transplanted into 2.5 × 4 cm plastic pots and grown for 2–6 weeks on a light bench. Once seedlings had established a root system, they were transplanted into 10 cm2 pots with a 1 : 1 mixture of Pro-mix BX  and sterile sand and grown until reproductive. Endophyte status of each plant was confirmed microscopically following the methods in For the N = 920) were planted on 9 July 2011 in randomized order, forming a circle with a radius of ∼5 cm around each original, naturally occurring plant [seeSupporting Information]. This spacing mimicked realistic field seedling densities.Prior to planting, we lightly glued seeds to plastic toothpicks  and counted the number of leaves and tillers on each plant twice in 2012 (14 June and 20 August) and twice in 2013 (20 June and 2 September). On each of these dates, for seeds that had previously germinated, we also recorded plant survival. On 2 September 2013, we harvested all remaining plants and counted the total number of leaves and the number of live leaves on each plant. We rinsed the roots of harvested plants to remove soil, divided each plant at the soil interface into aboveground and belowground biomass and dried the samples at 60 °C. Following drying, we weighed samples using a microbalance .P. leptocoma E+ seeds would perform best in P. leptocoma microsites, P. reflexa seeds would perform best in P. reflexa microsites and endophyte removal from P. leptocoma would shift the species' niche toward P. reflexa microsites. All models included the fixed effects of seed identity, microsite (P. leptocoma or P. reflexa niche) and their interaction, as well as the random effect of plant identity (nested within microsite) to account for the non-independence of seeds that were placed around the same naturally occurring plant. To test a priori hypotheses, we constructed contrasts to compare performance between the P. leptocoma and P. reflexa niches within each seed type. We also specifically compared the responses of greenhouse-grown seeds (P. leptocoma E+ control versus P. leptocoma E−) within each microsite.Analyses were designed to test our predictions that P. leptocoma or P. reflexa niche) and their interaction, as well as the random effect of plant identity (nested within microsite) to account for the non-independence of seeds that were placed around the same naturally occurring plant. To test a priori hypotheses, we constructed contrasts to compare performance between the P. leptocoma and P. reflexa niches within each seed type. We also specifically compared the responses of greenhouse-grown seeds (P. leptocoma E+ control versus P. leptocoma E−) within each microsite. For growth responses, P. leptocoma E+ control and P. leptocoma E+ natural data were combined because few individuals were alive at the time of harvest.We analysed germination and survival data with a log-linear model, including the binomial response (0/1) with a logit link function . For the subset of seeds that germinated, we analysed the time to germinate (in weeks), and for the subset of seedlings that died, we analysed survival time (in weeks) using generalized linear models with a Gaussian distribution. Growth responses  were analysed using MANOVA followed by univariate analysis. Aboveground and belowground biomass data were log-transformed, and live leaf data were square-root-transformed. All models included the fixed effects of seed identity, microsite .In our field surveys, tubB and tefA genes, indicating the presence of a non-hybrid Epichloë species in P. leptocoma. Phylogenies revealed that all tubB and tefA sequence data grouped in the Epichloë typhina subspecies poae subclade [seeSupporting Information]. Sexual Epichloë spp. are heterothallic, requiring individuals of two mating types for successful reproduction; all examined samples were positive for mtBA markers, suggesting that the examined populations of P. leptocoma endophytes comprise a single mating type, MTB. While it is possible that the examined P. leptocoma endophytes are capable of sexual reproduction, we saw no evidence of both mating types within the populations we evaluated. In addition, stromata were not observed in field or greenhouse-grown plants, further suggesting that the examined endophytes likely represent asexual, non-hybrid populations of E. typhina subsp. poae.Examined samples contained single copies of endophyte E. typhina subsp. poae populations. All samples were positive for perA (PER locus required for peramine production) markers, indicating the potential to produce peramine, a known insect feeding deterrent. All samples from the Washington Gulch population and half of samples from the Slate River population also contained ergot alkaloid biosynthesis genes (EAS locus), indicating the potential to produce the ergot alkaloid intermediate chanoclavine. All samples examined from the Virginia Basin site contained identical endophyte genotypes and were positive for PER markers but negative for EAS markers.We also found genes linked to alkaloid production within the Poa species. Poa leptocoma individuals were absent from quadrats placed around target P. reflexa individuals, and in the randomly placed quadrats were significantly less likely to co-occur with P. reflexa than with a conspecific  = −2.77, P = 0.005) or than expected by chance  = −2.34, P = 0.014). Poa reflexa was significantly clumped relative to the randomly sampled quadrats  = −1.76, P = 0.045), and significantly more likely to co-occur with a conspecific target than with P. leptocoma  = −1.92, P = 0.034). However, P. reflexa was equally abundant in quadrats with a target P. leptocoma relative to the random expectation  = 1.05, P = 0.306).Our species distribution surveys indicated low local co-occurrence of the two Poa leptocoma plants typically occupied wetter microenvironments than P. reflexa. For instance, at the Virginia Basin site, P. leptocoma grew in environments an average of 8.3 m from streams, while P. reflexa grew in environments an average of 22.0 m from streams  contained AM hyphae, compared with 47 % of P. reflexa root intersections (±2 % s.e.) . Colonization by other types of root fungi did not differ significantly between the two Poa species or among sites .Colonization of roots by AM fungi was ∼15 % greater for P. leptocoma seeds germinated in P. leptocoma habitats relative to P. reflexa habitats  seeds had 85–187 % higher germination in P. reflexa microsites than did P. leptocoma E+ control seeds (P = 0.13) or E+ natural seeds (P = 0.0005), respectively. For the P. reflexa seeds, there was no significant difference in percentage germination between P. leptocoma and P. reflexa niches, contrary to our original prediction  took an additional 31 weeks, on average, to germinate compared with P. reflexa seeds . The length of time to germinate was not affected by microsite for any of the P. leptocoma seed types . For P. reflexa, the effects of microsite on survival were opposite to the effects of microsite on time to germination, with 133 % higher survival of P. reflexa seedlings in the P. leptocoma microsite relative to the P. reflexa microsite  generally survived longer in P. leptocoma microsites than in P. reflexa microsites . Mean aboveground biomass was significantly greater for P. leptocoma E+ individuals relative to P. leptocoma E− individuals, with E+ plants having ∼600 % more biomass than E− plants .Plant growth was affected only by seed identity and not by microsite , which consume both roots and shoots of grasses , significant differences were not observed between P. leptocoma and P. reflexa in mean distance from the closest water source or in soil water content. At Snodgrass Mountain, soil moisture was overall much lower than at the other sites, and differences between P. leptocoma and P. reflexa habitats were smaller, perhaps accounting for the results obtained. Overall densities of both species were also low at this site relative to the other surveyed locations, possibly indicative of marginal habitat for both species. Other, less closely related Poa species are common in the general region that we sampled, and future studies might incorporate their distributions in a community phylogenetics context  (grants DEB-1354972 and DEB-1145588), the Rocky Mountain Biological Laboratory  and the University of New Mexico Department of Biology.Our work was funded by the M.R.K. contributed to data collection and analysis and wrote the manuscript. C.L.D. and J.A.R. designed the experiment, carried out data collection and contributed to writing the manuscript. L.R. and Y.A.C. contributed to experimental design and collected data. W.Q.H. collected data and, with N.D.C. and C.A.Y., completed DNA sequencing and phylogenetic analysis. T.H.P. completed root fungal colonization data collection and analysis. All authors edited the manuscript.None declared.The following additional information is available in the online version of this article –Table S1. Locality information of sites at which P. leptocoma and P. reflexa plants were collected and/or marked, along with date of sampling, sample size and percentage of plants that were symbiotic with the fungal endophyte E. typhina subsp. poae.Image S1. Photograph showing placement of plastic toothpicks around a focal, naturally occurring Poa individual.Figure S1. Gene phylogenies placing the fungal endophyte of P. leptocoma within the E. typhina subsp. poae subclade.


Bacillus subtilis BAB-1, isolated from cotton rhizosphere soil, is an excellent biocontrol agent for tomato gray mold. The genome of B. subtilis strain BAB-1 was fully sequenced and annotated, genes encoding the antifungal active compound were identified, and multiple sets of regulatory systems were found in the genome. Bacillus subtilis has been widely studied and used for plant disease control. The contributing factors to the success of this bacterium include the production of various antimicrobial compounds  (http://soap.genomics.org.cn/soapdenovo.html) based on the genome sequence of type strain B. subtilis 168 (AL009126.3), and 98.6% of the reads were assembled into 23 large scaffolds. Most of the gaps within the scaffolds were filled by local assembly of Solexa reads. The remaining gaps between scaffolds were filled by sequencing of PCR amplification using an ABI 3730 sequencer. Protein-encoding genes, rRNA operons, and tRNAs were predicted by Glimmer v. 3.0  in China. Sequence reads were generated from a 500-bp paired-end library, giving 600-Mb sequences with approximately 120× genome sequence coverage. The paired reads were Botrytis cinerea, and when applied in combination with fengycin, the surfactin showed synergistic actions, which were confirmed by antifungal assay in vivo  antibiotics, polyketide synthase (PKS) antibiotics, and lantibiotics, as well as bacillibactin. Among those products, the lipopeptides surfactin and fengycin were identified from strain BAB-1 by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The fengycin was identified as a major antifungal active compound in inhibition against the growth of hoR/phoP , and degegU/degS . FinallyCP004405.The complete genome sequence has been deposited in GenBank under accession no.


Lactobacillus crispatus might not be the only commensal protective against BV: healthy women from different countries and ethnicities harbour alternative commensals. Microbiotas associated with BV have also shown global variation, further complicating effective diagnosis via culture-based assays as some species are difficult to grow. Antibiotics and probiotics have been suggested to be key in controlling BV infection, but the efficacy of this treatment might rely on reconstituting endogenous commensals while targeting a specific species of BV-associated bacteria (BVAB). Alternatively, therapy could inhibit essential BV bacterial growth factors e.g. sialidases or provide anti-microbial compounds e.g. lactic acid associated with a healthy cervicovaginal microbiome. But without global investigation into the mechanism of BV pathogenesis and its association with HIV, selection of such compounds could be limited to Caucasian women from certain regions. To confirm this suggestion and guide future therapy we require standardised diagnostic assays and research methodologies. This review will focus on research papers that describe the global variation of BV aetiology and how this influences the identification of determinants of BV pathogenesis and potential probiotic and antimicrobial therapy.Bacterial vaginosis (BV) is the most common vaginal disorder affecting women of reproductive age and is associated with increased risk of sexually transmitted infections such as human immunodeficiency syndrome (HIV-1). Sub-Saharan Africa has the highest BV and HIV-1 burden and yet very few studies have focused on understanding the aetiology of BV and its association with HIV in this region. It has been suggested that we need to accurately diagnose and treat BV to lower the risk of HIV infection globally. However, effective diagnosis requires knowledge of what constitutes a “healthy” cervicovaginal microbiome and current studies indicate that  Lactobacillus spp. within the genital tract [Bacterial vaginosis (BV) is associated with sexually transmitted infections (STIs) as well as pelvic inflammatory disease and pregnancy complications . The mosal tract . Given tLactobacilli spp. [L. crispatus, L. gasseri, L. jensenii and L. iners [L. crispatus was significantly associated with the absence of BV, and transition to BVAB occurred via outgrowth of L. iners, confirming a previous study that L. gasseri and/or L. iners are associated with BV-related microflora whereas L. crispatus protected against dysbiosis [Lactobacillus species varied both within and between countries. Three South African studies reported conflicting results: one indicated that L. crispatus was associated with normal cervicovaginal microflora (p = 0.024), supporting studies on Caucasian women, whereas another showed that BV- and HIV-negative women carried predominantly L. salivarius. Finally, the last study showed that most women carried both L. crispatus and L. jensenii and that L. jensenii and not L. crispatus was associated with lack of BV (p = 0.053) [L. iners with coincident anaerobic microbes [L. crispatus and L. vaginalis associated with low Nugent scores [L. iners and L. gasseri and Ugandan women carried primarily L. reuteri, L. crispatus, L. vaginalis and L. jensenii [Lactobacilli were found in women from different countries, the dominant species differed and some healthy women carried non-Lactobacilli anaerobic microflora.Seventy percent of healthy Caucasian females carry predominantly genital lli spp.  with theL. iners . Meta-anysbiosis , 9. Howe= 0.053) –12. The microbes . Anothert scores . Nigeriajensenii , 16. TheLeptotrichia amnionii, Atopobium vaginae and BVAB1 found in more African-American BV-negative women than Caucasians. Furthermore, the healthy microbiomes of African-American—women were dominated by L. iners and those of Caucasians, L. crispatus [Lactobacilli spp. and African-American and Hispanic individuals carried more non-Lactobacilli anaerobic bacteria (p < 0.0001) [Lactobacillus species, similar to African-American women. The dominant Lactobacillus spp. was also L. iners, suggesting that healthy women of African descent could be less likely to carry cervicovaginal L. crispatus [L. iners could play a role in BV pathology, this finding could in part explain the high incidence of BV amongst black women especially in Southern Africa [These results were confirmed when women from different ethnicities were compared from the same region. It is unknown why black women have a higher prevalence of BV than Caucasians , 18. Howrispatus . Healthy 0.0001) . Interesn Africa . Howevern Africa .Lactobacilli and gram-negative rods . Recently, BV-associated dysbiosis was shown not to correlate with three of the four Amsel criteria and the Amsel method was unlikely to identify BV-positive women if they lacked dominant Lactobacilli species [BV diagnosis is usually based on four physiological Amsel criteria or Nugent score—a gram stain that determines the relative amounts of gram-positive  species . This isLactobacilli commensals across countries and ethnicities could lead to varying levels of protection against BV and HIV-1. Comparative functional genomic studies have shown that Lactobacillus spp. have evolved in a species-specific manner to adjust to the cervicovaginal environment, each expressing alternative adaptive factors. Therefore, microbes could influence the health of the genital tract through multiple mechanisms [Lactobacilli produce strain-specific bacteriocins such as reuterin by L. reuteri and lactocepin by L. casei and L. paracase [Lactobacilli in the hypoxic environment of the genital tract would be too low to inhibit HIV [Variation in healthy genital tract chanisms  such as chanisms –27. Lactparacase . Despiteibit HIV .Lactobacillus spp. was associated with lower risk of HIV infection [L. crispatus was better associated with lower HIV RNA levels than L. iners [Overall, high pH correlated best with high Nugent scores  and low nfection  and lowenfection . HoweverL. iners . These sL. iners .Lactobacillus spp. produced either l- or d-lactic acid and only the l-isoform inhibited HIV-1 infection. l-lactic acid induces the IL-23/IL-17 T cell pathway, release of pro-inflammatory cytokines, lymphocyte activation and increase in metalloproteases responsible for disruption of the cervix. The release of different cytokines depended on the species of Lactobacillus present [L. crispatus,L. gasseri and L. iners do not have the same number of copies of the l- and d-lactate dehydrogenase (LDH) genes [Lactobacillus spp. dependent and as the commensals of healthy women differ globally, it is likely that microbes other than Lactobacilli could be protective via similar mechanisms (production of l-lactic acid) or novel ways, altering the definition of a “healthy” genital tract.Witkin et al.  reported present  and coulH) genes . TherefoVeillonella parvula, Bacteroides, Peptococcus asaccharolyticus, Gardnerella vaginalis, Mobiluncus spp., Mycoplasma hominis and Chlamydia trachomatis [Atopobium vaginae, newly identified BVAB strains (BVAB1-3), Megasphaera spp. and Leptotrichia spp. [G. vaginalis in Kenya but not in Uganda [Prevotella bivia or Lachnospiraceae were identified in Tanzania [Mycoplasma hominis infected 35 % of HIV-negative Nigerian women whereas G. vaginalis, Prevotella spp., Mobiluncus, Atopobium spp. and E. coli, which predominate in Caucasians, were not identified [Mycoplasma lacks a cell wall and thus cannot be identified using the Nugent scoring system. It is thus possible that this organism is underrepresented in Caucasian BV populations because of the type of diagnostic assay used in some studies [G. vaginalis and M. hominis in the genital tract was associated with increased CVL HIV RNA. When analysis compared these two organisms singly only Mycoplasma remained significantly associated with HIV levels (p = 0.0001) [Mycoplasma plays an important role in HIV acquisition and that correct screening and diagnostic assays should be used to confirm whether it is associated with BV and HIV globally.A number of studies have identified different bacteria associated with BV in Caucasian women such as chomatis –38. Unrahia spp. . Howevern Uganda  and PrevTanzania . Mycoplaentified . Mycopla studies , 41. The 0.0001) . This coG. vaginalis and A. vaginae [G. vaginalis first and its biofilm production facilitates the colonisation of secondary anaerobes [G. vaginalis has been isolated from healthy women and introduction of vaginal secretions and not inoculation with pure G. vaginalis culture resulted in BV, it was thought to be a component of the normal genital microbiome and thus not the causative agent for BV [G. vaginalis adherence displaced L. crispatus, grew threefold better in the presence of certain anaerobes and encouraged the biofilm growth of mainly P. bivia. The authors suggest an interdependent relationship between Lactobacilli and BVAB and that this association might be species-specific [The primary bacteria associated with BV biofilms are  vaginae , 43. It naerobes –46. Howet for BV –50. Macht for BV  showed tG. vaginalis in BV as nearly 100 % of women with BV carry this specific bacterium whereas other colonising anaerobes are highly heterogeneous. They also suggest that G. vaginalis diversity could result in both pathogenic and non-pathogenic strains [G. vaginalis strains- one from a BV-infected woman and the other from a BV-negative women- showed that the former strain was pathogenic with enhanced biofilm production [G. vaginalis strains, reiterating the importance of genetic variation between strains. What this means for BV in sub-Saharan Africa where BV microbiomes differ between countries remains unknown.Schwebke et al.  reviewed strains  with onl strains . Genomicoduction . In suppG. vaginalis facilitates bacterial growth [A heat stable factor found in CVLs of BV-infected women enhanced HIV replication, suggesting that BVAB could increase HIV acquisition directly –55. Vagil growth  and enhal growth .trans-infection of CD4+ T cells, thus facilitating HIV transmission [HIV-1 Envelope (Env) glycosylation may play a role in HIV transmission and thus glycosidases that alter viral glycans could help select specific transmitted founder variants. Dendritic cell DC-SIGN receptor binds to HIV via Env glycans and enables smission . One stusmission . Using lsmission . The autsmission –62.Bacteroides spp. and G. vaginalis help produce biofilms and both mucinases and sialidases are involved in STIs (reviewed by Wiggins) [Bacterial vaginosis was also associated with fewer CVL sialic acid residues as expected with an increase in sialidase levels associated with the onset of BV . SialidaWiggins)  by disruWiggins) . The negWiggins)  influencWiggins) –70, suggA. vaginae increased epithelial cell mucins and pro-inflammatory cytokines; L. iners activated pattern-recognition receptor-signaling activity [Prevotella bivia and L. crispatus seemed to have no effect. Therefore infection with A. vaginae could induce a pro-inflammatory immune response that disrupts barrier functions whereas other microbes could elicit different responses [It has been suggested that the genital tract immune response plays a very important role in the pathophysiological condition of BV  because activity  whereas esponses . This reLactobacilli spp. should be included [trans-infection of CD4 T cells without affecting the growth of commensal Lactobacilli [Treatment of BV with metronidazole did not prevent recurrent BV infections nor lower levels of viral RNA (shed virus) and viral DNA (cell associated) in CVLs . One reaincluded . One potobacilli .Lactobacilli population is rarely reconstituted [Lactobacilli have been shown to be protective against a number of bacterial infections and have been used in probiotics [Lactobacilli and the culture supernatant of Lactobacilli inhibited HIV infection. The most effective of the Lactobacilli tested was L. gasseri [Lactobacillus strains with varying effects [L. acidophilus, L. rhamnosus, and L. fermentum normalised cervicovaginal microbiome resulting in curing BV and preventing relapse. In contrast, L. fermentum and L. plantarum were shown by Vicariotto et al. [L. crispatus, L. reuteri, and L. iners, disrupted biofilms in another study [Metronidazole and clindamycin do not prevent recurrent BV infections as the stituted . Probiotobiotics , 87. Liv gasseri . As dysb gasseri , selecti effects . Homayou effects  review oo et al.  to reducer study .L. crispatus probiotic was able to colonise only in the absence of endogenous L. crispatus, lack of condom use and without recent sexual activity, suggesting that the choice of Lactobacillus probiotic, the identity of the natural microflora and sexual practices could affect the efficacy of probiotics [Despite this evidence, review and analysis of a number of studies in 2009 indicated that there was insufficient evidence to support the use of probiotics in the treatment of BV and that large randomised trials with standard methodology was still outstanding . Factorsobiotics . Due to obiotics , it is mLactobacilli spp., BVAB and host genetic differences within the context of social behaviour. This synergy is complicated by differences in Lactobacilli spp. and BVAB across race and nationality so that diagnosis and treatment within resource-poor settings such as sub-Saharan Africa requires new consideration. We need to know whether differences across regions and ethnicities reflect true diversity or are due to study design. Thus we need to first standardise a global methodology for BV screening and identification of commensals and BVAB before rigorous longitudinal comparisons between different races and countries are carried out. In conjunction with this, basic research needs to apply in vitro assays that circumvent CVL donor variation to identify novel markers of BV and potential targets for drug design. Without a global approach, controlling BV in sub-Saharan Africa is highly unlikely.Studies suggest that BV pathology is highly dependent on