Gain or Loss? Evidence for Legume Predisposition to Symbiotic Interactions with Rhizobia via Loss of Pathogen-Resistance-Related Gene Families.

Nodulation is a hallmark yet non-universal characteristic of legumes. It is unknown whether the mechanisms underlying nitrogen-fixing symbioses evolved within legumes and the broader nitrogen-fixing clade (NFC) repeatedly de novo or based on common ancestral pathways. Ten new transcriptomes representing members from the Cercidoideae and Caesalpinioideae subfamilies were supplemented with published omics data from 65 angiosperms, to investigate how gene content correlates with nodulation capacity within Fabaceae and the NFC. Orthogroup analysis categorized annotated genes into 64150 orthogroups, of which 19 were significantly differentially represented between nodulating versus non-nodulating NFC species and were most commonly absent in nodulating taxa. The distribution of six over-represented orthogroups within Viridiplantae representatives suggested that genomic evolution events causing gene family expansions, including whole-genome duplications (WGDs), were unlikely to have facilitated the development of stable symbioses within Fabaceae as a whole. Instead, an absence of representation of 13 orthogroups indicated that losses of genes involved in trichome development, defense and wounding responses were strongly associated with rhizobial symbiosis in legumes. This finding provides novel evidence of a lineage-specific predisposition for the evolution and/or stabilization of nodulation in Fabaceae, in which a loss of pathogen resistance genes may have allowed for stable mutualistic interactions with rhizobia.

A Tale of Two Families: Whole Genome and Segmental Duplications Underlie Glutamine Synthetase and Phosphoenolpyruvate Carboxylase Diversity in Narrow-Leafed Lupin (Lupinus angustifolius L.).

Narrow-leafed lupin (Lupinus angustifolius L.) has recently been supplied with advanced genomic resources and, as such, has become a well-known model for molecular evolutionary studies within the legume family-a group of plants able to fix nitrogen from the atmosphere. The phylogenetic position of lupins in Papilionoideae and their evolutionary distance to other higher plants facilitates the use of this model species to improve our knowledge on genes involved in nitrogen assimilation and primary metabolism, providing novel contributions to our understanding of the evolutionary history of legumes. In this study, we present a complex characterization of two narrow-leafed lupin gene families-glutamine synthetase (GS) and phosphoenolpyruvate carboxylase (PEPC). We combine a comparative analysis of gene structures and a synteny-based approach with phylogenetic reconstruction and reconciliation of the gene family and species history in order to examine events underlying the extant diversity of both families. Employing the available evidence, we show the impact of duplications on the initial complement of the analyzed gene families within the genistoid clade and posit that the function of duplicates has been largely retained. In terms of a broader perspective, our results concerning GS and PEPC gene families corroborate earlier findings pointing to key whole genome duplication/triplication event(s) affecting the genistoid lineage.

Zearalenone lactonohydrolase activity in Hypocreales and its evolutionary relationships within the epoxide hydrolase subset of a/b-hydrolases.

BACKGROUND: Zearalenone is a mycotoxin produced by several species of Fusarium genus, most notably Fusarium graminearum and Fusarium culmorum. This resorcylic acid lactone is one of the most important toxins causing serious animal and human diseases. For over two decades it has been known that the mycoparasitic fungus Clonostachys rosea (synonym: Gliocladium roseum, teleomorph: Bionectria ochroleuca) can detoxify zearalenone, however no such attributes have been described within the Trichoderma genus. RESULTS: We screened for the presence of zearalenone lactonohydrolase homologs in isolates of Clonostachys and Trichoderma genera. We report first finding of expressed zearalenone lactonohydrolase in Trichoderma aggressivum. For three isolates (T. aggressivum, C. rosea and Clonostachys catenulatum isolates), we were able to reconstruct full coding sequence and verify the biotransformation ability potential. Additionally, we assessed progression of the detoxification process (in terms of transcript accumulation and mycotoxin decomposition in vitro).In silico, search for origins of zearalenone lactonohydrolase activity in model fungal and bacterial genomes has shown that zearalenone lactonohydrolase homologs form a monophyletic fungal clade among the a/b hydrolase superfamily representatives. We corroborated the finding of functional enzyme homologs by investigating the functional sites (active site pocket with postulated, noncanonical Ser-Glu-His catalytic triad) conserved in both multiple sequence alignment and in homology-based structural models. CONCLUSIONS: Our research shows the first finding of a functional zearalenone lactonohydrolase in mycoparasitic Trichoderma aggressivum (an activity earlier characterised in the Clonostachys rosea strains). The supporting evidence for presence and activity of functional enzyme homologs is based on the chemical analyses, gene expression patterns, homology models showing conservation of key structural features and marked reduction of zearalenone content in cultured samples (containing both medium and mycelium). Our findings also show divergent strategies of zearalenone biotransformation ability (rapid induced expression and detoxification vs. gradual detoxification) present in several members of Hypocreales order (Trichoderma and Clonostachys genera). The potential for lactonhydrolase activity directed towards zearalenone and/or similar compounds is likely ancient, with homologs present in several divergent filamentous fungi among both Sordariomycetes (Bionectria sp., Trichoderma sp., Apiospora montagnei) and Leotiomycetes (Marssonina brunnea f. sp. 'multigermtubi').

New evidence of ancestral polyploidy in the Genistoid legume Lupinus angustifolius L. (narrow-leafed lupin).

KEY MESSAGE: This is the first clear evidence of duplication and/or triplication of large chromosomal regions in a genome of a Genistoid legume, the most basal clade of Papilionoid legumes. Lupinus angustifolius L. (narrow-leafed lupin) is the most widely cultivated species of Genistoid legume, grown for its high-protein grain. As a member of this most basal clade of Papilionoid legumes, L. angustifolius serves as a useful model for exploring legume genome evolution. Here, we report an improved reference genetic map of L. angustifolius comprising 1207 loci, including 299 newly developed Diversity Arrays Technology markers and 54 new gene-based PCR markers. A comparison between the L. angustifolius and Medicago truncatula genomes was performed using 394 sequence-tagged site markers acting as bridging points between the two genomes. The improved L. angustifolius genetic map, the updated M. truncatula genome assembly and the increased number of bridging points between the genomes together substantially enhanced the resolution of synteny and chromosomal colinearity between these genomes compared to previous reports. While a high degree of syntenic fragmentation was observed that was consistent with the large evolutionary distance between the L. angustifolius and M. truncatula genomes, there were striking examples of conserved colinearity of loci between these genomes. Compelling evidence was found of large-scale duplication and/or triplication in the L. angustifolius genome, consistent with one or more ancestral polyploidy events.

Molecular Re-Diagnosis with Whole-Exome Sequencing Increases the Diagnostic Yield in Patients with Non-Syndromic Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of disorders with progressive loss of photoreceptor and pigment epithelial function. Nineteen unrelated Polish probands clinically diagnosed with nonsyndromic RP were recruited to this study. We used whole-exome sequencing (WES) to identify potential pathogenic gene variants in molecularly undiagnosed RP patients, as a molecular re-diagnosis after having performed targeted NGS in the past. Targeted NGS allowed for identification of the molecular background in only 5 out of 19 patients. Fourteen patients who remained unsolved despite the targeted NGS were subjected to WES. WES revealed potentially causative variants in RP-related genes in another 12 patients. Together, NGS methods revealed the coexistence of causal variants affecting distinct RP genes in 17 out of 19 RP families, with a very high efficiency of 89%. With the improvement of NGS methods, including higher sequencing depth, broader target enrichment, and better bioinformatic analysis capabilities, the ratio of identified causal gene variants has significantly increased. Therefore, it is important to consider repeating high-throughput sequencing analysis in those patients in whom the previously performed NGS did not reveal any pathogenic variants. The study confirmed the efficiency and clinical utility of re-diagnosis with WES in molecularly undiagnosed RP patients.

The eINTACT system dissects bacterial exploitation of plant osmosignalling to enhance virulence.

Bacteria inject effector proteins into host cells to manipulate cellular processes that promote disease. Since bacteria deliver minuscule amounts of effectors only into targeted host cells, it is technically challenging to capture effector-dependent cellular changes from bulk-infected host tissues. Here, we report a new technique called effector-inducible isolation of nuclei tagged in specific cell types (eINTACT), which facilitates affinity-based purification of nuclei from Arabidopsis plant cells that have received Xanthomonas bacterial effectors. Analysis of purified nuclei reveals that the Xanthomonas effector XopD manipulates the expression of Arabidopsis abscisic acid signalling-related genes and activates OSCA1.1, a gene encoding a calcium-permeable channel required for stomatal closure in response to osmotic stress. The loss of OSCA1.1 causes leaf wilting and reduced bacterial growth in infected leaves, suggesting that OSCA1.1 promotes host susceptibility. eINTACT allows us to uncover that XopD exploits host OSCA1.1/abscisic acid osmosignalling-mediated stomatal closure to create a humid habitat that favours bacterial growth and opens up a new avenue for accurately elucidating functions of effectors from numerous gram-negative plant bacteria in native infection contexts.

Results from Genetic Studies in Patients Affected with Craniosynostosis: Clinical and Molecular Aspects.

Background: Craniosynostosis (CS) represents a highly heterogeneous genetic condition whose genetic background has not been yet revealed. The abnormality occurs either in isolated form or syndromic, as an element of hundreds of different inborn syndromes. Consequently, CS may often represent a challenging diagnostic issue. Methods: We investigated a three-tiered approach (karyotyping, Sanger sequencing, followed by custom gene panel/chromosomal microarray analysis, and exome sequencing), coupled with prioritization of variants based on dysmorphological assessment and description in terms of human phenotype ontology. In addition, we have also performed a statistical analysis of the obtained clinical data using the nonparametric test χ2. Results: We achieved a 43% diagnostic success rate and have demonstrated the complexity of mutations' type harbored by the patients, which were either chromosomal aberrations, copy number variations, or point mutations. The majority of pathogenic variants were found in the well-known CS genes, however, variants found in genes associated with chromatinopathies or RASopathies are of particular interest. Conclusion: We have critically summarized and then optimised a cost-effective diagnostic algorithm, which may be helpful in a daily diagnostic routine and future clinical research of various CS types. Moreover, we have pinpointed the possible underestimated co-occurrence of CS and intellectual disability, suggesting it may be overlooked when intellectual disability constitutes a primary clinical complaint. On the other hand, in any case of already detected syndromic CS and intellectual disability, the possible occurrence of clinical features suggestive for chromatinopathies or RASopathies should also be considered.

Genome sequence comparison of Col and Ler lines reveals the dynamic nature of Arabidopsis chromosomes.

Large differences in plant genome sizes are mainly due to numerous events of insertions or deletions (indels). The balance between these events determines the evolutionary direction of genome changes. To address the question of what phenomena trigger these alterations, we compared the genomic sequences of two Arabidopsis thaliana lines, Columbia (Col) and Landsberg erecta (Ler). Based on the resulting alignments large indels (>100 bp) within these two genomes were analysed. There are approximately 8500 large indels accounting for the differences between the two genomes. The genetic basis of their origin was distinguished as three main categories: unequal recombination (Urec)-derived, illegitimate recombination (Illrec)-derived and transposable elements (TE)-derived. A detailed study of their distribution and size variation along chromosomes, together with a correlation analyses, allowed us to demonstrate the impact of particular recombination-based mechanisms on the plant genome evolution. The results show that unequal recombination is not efficient in the removal of TEs within the pericentromeric regions. Moreover, we discovered an unexpectedly high influence of large indels on gene evolution pointing out significant differences between the various gene families. For the first time, we present convincing evidence that somatic events do play an important role in plant genome evolution.

Terpenoid Biosynthesis Dominates among Secondary Metabolite Clusters in Mucoromycotina Genomes.

Early-diverging fungi harbour unprecedented diversity in terms of living forms, biological traits and genome architecture. Before the sequencing era, non-Dikarya fungi were considered unable to produce secondary metabolites (SM); however, this perspective is changing. The main classes of secondary metabolites in fungi include polyketides, nonribosomal peptides, terpenoids and siderophores that serve different biological roles, including iron chelation and plant growth promotion. The same classes of SM are reported for representatives of early-diverging fungal lineages. Encouraged by the advancement in the field, we carried out a systematic survey of SM in Mucoromycotina and corroborated the presence of various SM clusters (SMCs) within the phylum. Among the core findings, considerable representation of terpene and nonribosomal peptide synthetase (NRPS)-like candidate SMCs was found. Terpene clusters with diverse domain composition and potentially highly variable products dominated the landscape of candidate SMCs. A uniform low-copy distribution of siderophore clusters was observed among most assemblies. Mortierellomycotina are highlighted as the most potent SMC producers among the Mucoromycota and as a source of novel peptide products. SMC identification is dependent on gene model quality and can be successfully performed on a batch scale with genomes of different quality and completeness.

Geographical Origin Does Not Modulate Pathogenicity or Response to Climatic Variables of Fusarium oxysporum Associated with Vascular Wilt on Asparagus.

Asparagus crop is distributed worldwide, covering very different climatic regions. Among the different diseases that affect asparagus, vascular Fusarium wilt, caused by Fusarium oxysporum f. sp. aparagi (Foa), stands out. It is not only the cause of large economic losses due to a decrease in yield and shortened longevity of the plantation, but also prevents replanting. This work aimed to determine if F. oxysporum isolates associated with vascular wilt on asparagus have adapted differentially to the different agro-environmental conditions. The potential correlation between origin and mycelial growth under different temperatures and humidity conditions was analysed for isolates from asparagus fields cultivated in northern and southern Europe. The genetic and pathogenic variability were also analysed. While a clear effect of water activity on mycelial growth was observed, all isolates responded in a similar way to changes in water activity in the medium, regardless of their geographical origin. The results revealed a low genetic variability of F. oxysporum isolates associated with vascular wilt on asparagus without signs of differentiation correlated to geographical origin. The southernmost isolates of the two cultivated varieties inoculated did not express more pathogenicity than those isolated from the colder region.

Domain Hierarchy and closed Loops (DHcL): a server for exploring hierarchy of protein domain structure.

Domain hierarchy and closed loops (DHcL) (http://sitron.bccs.uib.no/dhcl/) is a web server that delineates energy hierarchy of protein domain structure and detects domains at different levels of this hierarchy. The server also identifies closed loops and van der Waals locks, which constitute a structural basis for the protein domain hierarchy. The DHcL can be a useful tool for an express analysis of protein structures and their alternative domain decompositions. The user submits a PDB identifier(s) or uploads a 3D protein structure in a PDB format. The results of the analysis are the location of domains at different levels of hierarchy, closed loops, van der Waals locks and their interactive visualization. The server maintains a regularly updated database of domains, closed loop and van der Waals locks for all X-ray structures in PDB. DHcL server is available at: http://sitron.bccs.uib.no/dhcl.

Cyclodepsipeptide Biosynthesis in Hypocreales Fungi and Sequence Divergence of The Non-Ribosomal Peptide Synthase Genes.

Fungi from the Hypocreales order synthesize a range of toxic non-ribosomal cyclic peptides with antimicrobial, insecticidal and cytotoxic activities. Entomopathogenic Beauveria, Isaria and Cordyceps as well as phytopathogenic Fusarium spp. are known producers of beauvericins (BEAs), beauvenniatins (BEAEs) or enniatins (ENNs). The compounds are synthesized by beauvericin/enniatin synthase (BEAS/ESYN1), which shows significant sequence divergence among Hypocreales members. We investigated ENN, BEA and BEAE production among entomopathogenic (Beauveria, Cordyceps, Isaria) and phytopathogenic (Fusarium) fungi; BEA and ENNs were quantified using an LC-MS/MS method. Phylogenetic analysis of partial sequences of putative BEAS/ESYN1 amplicons was also made. Nineteen fungal strains were identified based on sequence analysis of amplified ITS and tef-1α regions. BEA was produced by all investigated fungi, with F. proliferatum and F. concentricum being the most efficient producers. ENNs were synthesized mostly by F. acuminatum, F. avenaceum and C. confragosa. The phylogeny reconstruction suggests that ancestral BEA biosynthesis independently diverged into biosynthesis of other compounds. The divergent positioning of three Fusarium isolates raises the possibility of parallel acquisition of cyclic depsipeptide synthases in ancient complexes within Fusarium genus. Different fungi have independently evolved NRPS genes involved in depsipeptide biosynthesis, with functional adaptation towards biosynthesis of overlapping yet diversified metabolite profiles.

Divergence of Beauvericin Synthase Gene among Fusarium and Trichoderma Species.

Beauvericin (BEA) is a cyclodepsipeptide mycotoxin, showing insecticidal, antibiotic and antimicrobial activities, as well as inducing apoptosis of cancer cell lines. BEA can be produced by multiple fungal species, including saprotrophs, plant, insect and human pathogens, particularly belonging to Fusarium, Beauveria and Isaria genera. The ability of Trichoderma species to produce BEA was until now uncertain. Biosynthesis of BEA is governed by a non-ribosomal peptide synthase (NRPS), known as beauvericin synthase (BEAS), which appears to present considerable divergence among different fungal species. In the present study we compared the production of beauvericin among Fusarium and Trichoderma strains using UPLC methods. BEAS fragments were sequenced and analyzed to examine the level of the gene's divergence between these two genera and confirm the presence of active BEAS copy in Trichoderma. Seventeen strains of twelve species were studied and phylogenetic analysis showed distinctive grouping of Fusarium and Trichoderma strains. The highest producers of beauvericin were F. proliferatum and F. nygamai. Trichoderma strains of three species (T. atroviride, T. viride, T. koningiopsis) were minor BEA producers. The study showed beauvericin production by Fusarium and Trichoderma species and high variance of the non-ribosomal peptide synthase gene among fungal species from the Hypocreales order.

Targeted Next-Generation Sequencing in the Diagnosis of Facial Dysostoses.

BACKGROUND: Defects in the development of the first and second pharyngeal arches and their derivatives result in abnormal formation of the craniofacial complex, consequently giving rise to facial dysostoses (FDs). FDs represent a group of rare and highly heterogeneous disease entities that encompass mandibulofacial dysostoses (MFDs) with normal extremities and acrofacial dysostoses (AFDs) with limb anomalies in addition to craniofacial defects. METHODS: We examined 11 families with variable clinical symptoms of FDs, in most of which only one member was affected. We applied two custom gene panels-first comprising 37 genes related to the genetic disorders of craniofacial development such as FDs (On-Demand AmpliSeq Thermo Fisher Scientific gene panel with two primer pools) and second composed of 61 genes and 11 single nucleotide variants (SNVs) known to be involved in the development of skull malformations, mainly in the form of craniosynostoses (SureSelect Agilent Technologies). Targeted next-generation sequencing (NGS) was performed using the Ion Torrent S5 platform. To confirm the presence of each detected variant, we have analyzed a genomic region of interest using Sanger sequencing. RESULTS: In this paper, we summarized the results of custom targeted gene panel sequencing in the cohort of sixteen patients from 11 consecutive families affected by distinct forms of FDs. We have found three novel pathogenic variants in the TCOF1 gene-c.2145_2148dupAAAG p.(Ser717Lysfs ∗42), c.4370delA p.(Lys1457Argfs ∗118), c.83G>C p.(Arg28Pro) causing Treacher Collins syndrome type 1, two novel missense variants in the EFTUD2 gene-c.491A>G p.(Asp164Gly) and c.779T>A p.(Ile260Asn) in two female patients affected by acrofacial dysostosis Guion-Almeida type, one previously reported-c.403C>T (p.Arg135Cys), as well as one novel missense variant-c.128C>T p.(Pro43Leu) in the DHODH gene in the male patient with Miller syndrome and finally one known pathogenic variant c.574G>T p.(Glu192∗) in the SF3B4 gene in the patient with Nager syndrome. CONCLUSION: Our study confirms the efficiency and clinical utility of the targeted gene panel sequencing and shows that this strategy is suitable and efficient in the molecular screening of variable forms of FDs.

Adapting SureSelect enrichment protocol to the Ion Torrent S5 platform in molecular diagnostics of craniosynostosis.

Obtaining reliable and high fidelity next-generation sequencing (NGS) data requires to choose a suitable sequencing platform and a library preparation approach, which both have their inherent assay-specific limitations. Here, we present the results of successful adaptation of SureSelect hybridisation-based target enrichment protocol for the sequencing on the Ion Torrent S5 platform, which is designed to work preferably with amplicon-based panels. In our study, we applied a custom NGS panel to screen a cohort of 16 unrelated patients affected by premature fusion of the cranial sutures, i.e. craniosynostosis (CS). CS occurs either as an isolated malformation or in a syndromic form, representing a genetically heterogeneous and clinically variable group of disorders. The approach presented here allowed us to achieve high quality NGS data and confirmed molecular diagnosis in 19% of cases, reaching the diagnostic yield similar to some of the published research reports. In conclusion, we demonstrated that an alternative enrichment strategy for library preparations can be successfully applied prior to sequencing on the Ion Torrent S5 platform. Also, we proved that the custom NGS panel designed by us represents a useful and effective tool in the molecular diagnostics of patients with CS.

Comparative analysis of the Brassica oleracea genetic map and the Arabidopsis thaliana genome.

We further investigated genome macrosynteny for Brassica species and Arabidopsis thaliana. This work aimed at comparative map construction for B. oleracea and A. thaliana chromosomes based on 160 known A. thaliana probes: 147 expressed sequence tags (ESTs) and 13 full-length cDNA clones. Based on an in silico study of the A. thaliana genome, most of the selected ESTs (83%) represented unique or low-copy genes. We identified conserved segments by the visual inspection of comparative data with a priori assumptions, and established their significance with the LineUp algorithm. Evaluation of the number of B. oleracea gene copies per A. thaliana EST revealed a fixed upward trend. We established a segregation distortion pattern for all genetic loci, with particular consideration of the type of selection (gametic or zygotic), and discuss its possible impact on genetic map construction. Consistent with previous reports, we found evidence for numerous chromosome rearrangements and the genome fragment replication of B. oleracea that have taken place since the divergence of the two species. Also, we found that over 54% of the B. oleracea genome is covered by 24 segments conserved with the A. thaliana genome. The average conserved segment is composed of 5 loci covering 19.3 cM in the B. oleracea genetic map and 2.42 Mb in the A. thaliana physical map. We have also attempted to use a unified system of conserved blocks (previously described) to verify our results and perform a comprehensive comparison with other Brassica species.

Efflux pumps as an additional source of resistance to trichothecenes in Fusarium proliferatum and Fusarium oxysporum isolates.

Role of efflux-mediated toxin resistance to trichothecenes is known in trichothecene-producing species. However, the role of trichothecene efflux pump homologues in non-producing fusaria such as F. oxysporum and F. proliferatum was not investigated in detail. Analysis of the homologues of trichothecene efflux pump from multiple fungal species allowed us to uncover and catalogue functional gene copies of conserved structure. Putative Tri12 candidates in Fusarium oxysporum and F. proliferatum were characterised via expression profiling in response to different trigger compounds, providing supporting evidence for role of Tri12 homologues in the resistance to trichothecenes. Our analysis of Tri12 phylogeny also suggests that efflux-mediated trichothecene resistance is likely to predate the divergence of Trichoderma and Fusarium species. On the regulatory level, we posit that the increased tolerance of trichothecenes by F. oxysporum is possibly related to the decoupling of Tri12 homologue expression from pH, due to the deletion of PACC/RIM101 transcription factor binding site in its promoter region.

Transcriptome-derived investigation of biosynthesis of quinolizidine alkaloids in narrow-leafed lupin (Lupinus angustifolius L.) highlights candidate genes linked to iucundus locus.

Unravelling the biosynthetic pathway of quinolizidine alkaloids (QAs), regarded as antinutritional compounds of narrow-leafed lupin (NLL) seeds, is fundamental to best exploit NLL as food or feed. We investigated 12 candidate genes connected to QA biosynthesis, selecting them by transcriptomic and genomic approaches, from the landscape of genes differentially expressed in leaves of the high- and low-alkaloid NLL accessions. Linkage analysis enabled the assessment of the location of the candidate genes in relation to iucundus, a major locus of unknown identity, that confers reduced QA content in seeds. The key finding was the identification of APETALA2/ethylene response transcription factor, RAP2-7, cosegregating with the iucundus locus and located within a region with highly significant QTLs that affect QA composition. We additionally identified a 4-hydroxy-tetrahydrodipicolinate synthase (DHDPS) gene involved in L-lysine biosynthesis as being closely linked to iucundus. The distributed location of other remaining candidates (including previously known QA genes) across different linkage groups, also indirectly supports the transcription factor as a possible regulator of lupin alkaloid biosynthesis. Our findings provide crucial insight into QA biosynthesis in NLL. Additionally, we evaluated and selected appropriate reference genes for qRT-PCRs to analyse the expression levels of QA genes in NLL.

Homologues of HSV-1 nuclear egress factor UL34 are potential phosphoinositide-binding proteins.

During the herpesvirus replication cycle, viral transcription, DNA replication, formation of capsids and DNA packaging occur in the nucleus. The subsequent nuclear egress of newly synthesized nucleocapsids is performed by budding of the inner leaflet of the nuclear membrane, which creates the primary envelope. Although products of two genes conserved throughout the Herpesviridae family (HSV-1 UL34 and UL31) have previously been shown to be involved in the execution of this process, the molecular basis of their activity is not clear. Here we present results of protein structure prediction for the conserved domain of UL34. The applied methodology suggests that this protein adopts a pleckstrin homology (PH) fold to perform its function. A detailed inspection of the ligand binding site strongly supports the hypothesis that UL34 orthologs can recognize phosphoinositides. Since previous works suggest that alterations of UL34 gene product result in a drastic impairment of primary envelopment of HSV-1 and trapping of capsids in the nucleus, the presented data may lead to the development of novel anti-herpetic therapeutic strategies where analogs of phosphoinositides are administered.

Wheat-infecting Fusarium species in Poland--their chemotypes and frequencies revealed by PCR assay.

Three Fusarium species: F. graminearum, F. culmorum and F. cerealis were identified in laboratory cultures and in sporodochia from spikelets of scabby wheat. SCAR (sequence characterized amplified region) primers were used to identify Fusarium species and nivalenol (NIV) and deoxynivalenol (DON) chemotypes within species in laboratory cultures and field collected heads harvested in 2006. Results from PCR analyses confirmed preliminary identifications of species on the basis of examination of macroconidia under a light microscope and identification of cultures on agar media. NIV and DON (3Ac-DON and 15Ac-DON) chemotypes were identified using PCR assay. Among samples and isolates of F. graminearum, the 15Ac-DON chemotype dominated, and among those where F. culmorum was identified, the 3Ac-DON chemotype prevailed. Only 5 of the 41 isolates of F. graminearum tested, displayed the NIV chemotype. An increase in the frequency of F. graminearum and a decrease in the frequency of F. culmorum were found during 1998 to 2006.