Conserved Motifs and Domains in Members of Pospiviroidae

In 1985, Keese and Symons proposed a hypothesis on the sequence and secondary structure of viroids from the family : their secondary structure can be subdivided into five structural and functional domains and “viroids have evolved by rearrangement of domains between different viroids infecting the same cell and subsequent mutations within each domain”; this article is one of the most cited in the field of viroids. Employing the pairwise alignment method used by Keese and Symons and in addition to more recent methods, we tried to reproduce the original results and extent them to further members of which were unknown in 1985. Indeed, individual members of consist of a patchwork of sequence fragments from the family but the lengths of fragments do not point to consistent points of rearrangement, which is in conflict with the original hypothesis of fixed domain borders.

Structural basis for effector recognition by an antibacterial type IV secretion system

Many soil-, water-, and plant-associated bacterial species from the orders Xanthomonadales, Burkholderales, and Neisseriales carry a type IV secretion system (T4SS) specialized in translocating effector proteins into other gram-negative species, leading to target cell death. These effectors, known as X-Tfes, carry a carboxyl-terminal domain of ∼120 residues, termed XVIPCD, characterized by several conserved motifs and a glutamine-rich tail. Previous studies showed that the XVIPCD is required for interaction with the T4SS coupling protein VirD4 and for T4SS-dependent translocation. However, the structural basis of the XVIPCD–VirD4 interaction is unknown. Here, we show that the XVIPCD interacts with the central all-alpha domain of VirD4 (VirD4AAD). We used solution NMR spectroscopy to solve the structure of the XVIPCD of X-TfeXAC2609 from Xanthomonas citri and to map its interaction surface with VirD4AAD. Isothermal titration calorimetry and in vivo Xanthomonas citri versus Escherichia coli competition assays using wild-type and mutant X-TfeXAC2609 and X-TfeXAC3634 indicate that XVIPCDs can be divided into two regions with distinct functions: the well-folded N-terminal region contains specific conserved motifs that are responsible for interactions with VirD4AAD, while both N- and carboxyl-terminal regions are required for effective X-Tfe translocation into the target cell. The conformational stability of the N-terminal region is reduced at and below pH 7.0, a property that may facilitate X-Tfe unfolding and translocation through the more acidic environment of the periplasm.

Evolutionary analysis of angiosperm dehydrin gene family reveals three orthologues groups associated to specific protein domains

Dehydrins (DHNs) are a family of plant proteins that play important roles on abiotic stress tolerance and seed development. They are classified into five structural subgroups: K-, SK-, YK-, YSK-, and KS-DHNs, according to the presence of conserved motifs named K-, Y- and S- segments. We carried out a comparative structural and phylogenetic analysis of these proteins, focusing on the less-studied KS-type DHNs. A search for conserved motifs in DHNs from 56 plant genomes revealed that KS-DHNs possess a unique and highly conserved N-terminal, 15-residue amino acid motif, not previously described. This novel motif, that we named H-segment, is present in DHNs of angiosperms, gymnosperms and lycophytes, suggesting that HKS-DHNs were present in the first vascular plants. Phylogenetic and microsynteny analyses indicate that the five structural subgroups of angiosperm DHNs can be assigned to three groups of orthologue genes, characterized by the presence of the H-, F- or Y- segments. Importantly, the hydrophilin character of DHNs correlate with the phylogenetic origin of the DHNs rather than to the traditional structural subgroups. We propose that angiosperm DHNs can be ultimately subdivided into three orthologous groups, a phylogenetic framework that should help future studies on the evolution and function of this protein family.

Mechanistic Investigations on Microbial Type I Terpene Synthases through Site-directed Mutagenesis

During the past three decades many terpene synthases have been characterised from all kingdoms of life. The type I of these enzymes from bacteria, fungi and protists commonly exhibit several highly conserved motifs and single residues, and the available crystal structures show a shared -helical fold, while the overall sequence identity is generally low. Several enzymes have been studied by site-directed mutagenesis, giving valuable insights into terpene synthase catalysis and the intriguing mechanisms of terpene synthases. Some mutants are also preparatively useful and give higher yields than the wildtype or a different product that is otherwise difficult to access. The accumulated knowledge obtained from these studies is presented and discussed in this review.

Structural analysis of the role of the two conserved motifs of the ECF41 family sigma factor in the autoregulation of its own promoter in Azospirillum brasilense Sp245

In Azospirillum brasilense, an extra-cytoplasmic function sigma factor (RpoE10) shows the characteristic 119 amino acid long C-terminal extension found in ECF41-type sigma factors, which possesses three conserved motifs (WLPEP, DGGGR, and NPDKV), one in the linker region between the sigma and sigma , and the other two in the SnoaL_2 domain of the C-terminal extension. Here, we have described the role of the two conserved motifs in the SnoaL_2 domain of RpoE10 in the inhibition and activation of its activity, respectively. Truncation of the distal part of the C-terminal sequence of the RpoE10 (including NPDKV but excluding the DGGGR motif) results in its promoter’s activation suggesting autoregulation. Further truncation of the C-terminal sequence up to its proximal part, including NPDKV and DGGGR motif, abolished promoter activation. Replacement of NPDKV motif with NAAAV in RpoE10 increased its ability to activate its promoter, whereas replacement of DGGGR motif led to reduced promoter activation. We have explored the dynamic modulation of sigma2 – sigma4 domains and the relevant molecular interactions mediated by the two conserved motifs of the SnoaL2 domain using molecular dynamics simulation. The analysis enabled us to explain that the NPDKV motif located distally in the C-terminus negatively impacts transcriptional activation. In contrast, the DGGGR motif found proximally of the C-terminal extension is required to activate RpoE1

Screening of Aptamers That Bind to the Multivalent Aminoglycoside Amikacin

Increased awareness of the multiple roles of RNA molecules has led to the realization that, in addition to their structural and functional roles, RNAs can be drug targets for small molecular therapy. The aim of this study was to identify multivalent amikacin specific RNA aptamers that can be a new target sites for aminoglycoside antibiotics, including amikacin using the systemic evolution of ligands by exponential enrichment (SELEX) method. Amikacin, a member of the aminoglycoside group of antibiotics, binds to specific sites in bacterial 16S ribosomal RNAs (rRNAs) and interferes with protein synthesis, leading to cell death. Here, we used the SELEX method to isolate high affinity RNA fragments (aptamers) that bind to amikacin. After five rounds of SELEX selection, in which a linear N25 DNA template was used for the first selection cycle, the resulting RNA was cloned and sequenced. Among the 38 clones generated, five groups of sequences (groups A through E) containing nine conserved motifs were identified. The sequences of groups A and B were almost identical, indicating that the selected RNA was enriched. Subsequently, the Basic Local Alignment Search Tool program was used to search for the conserved motifs in bacterial 16S rRNA sequences. Strikingly, no sequence homology was observed, suggesting that the conserved sequences (motifs) identified in this study may be novel target sites for amikacin.

Comparative secretomics identifies conserved WAxR motif-containing effectors in rust fungi that suppress cell death in plants

Identification of novel effectors with conserved features has always remained a challenge in plant-pathogen interaction studies. The introduction of the genomics era in plant-pathogen studies has led to the identification of significant candidate effectors with novel motifs such as RxLR and dEER motifs. However, in the case of fungal pathogens, limited conserved motifs associated with effectors have been discovered yet. In the present study, we have performed comparative secretome analysis for major plant pathogens of diverse nutrition mechanisms with the aim of dissecting the features underlying their corresponding secretome and conserved motifs. We showed that rust fungi possess the lowest Cell wall degrading enzymes (CWDEs) consortium lower than other biotrophic pathogens. We also showed rust fungi possess the highest secretory superoxide dismutase (SOD) than other studied plant pathogens. Further, we prioritized the candidate secretory effectors proteins (CSEPs) of all the studied pathogens by combining various effector mining parameters to highlight the candidates with potential effector features. A novel WAxR motif in conjugation with the Y/F/WxC (FGC) motif was identified in the effectors of various P. striiformis races present globally. The WAxR/WAxR like motifs ( WxxR, WAxx, xAxR) containing effectors were also found in the secretome of other rust fungi. Further, the functional validation of two candidate effectors with WAxR motif from P. striiformis Yr9 showed that these effectors localize to the nucleus as well as cytoplasm, and are able to suppress BAX induced cell death in Nicotiana benthamiana. The mutation analysis of individual residues of the WAxR motif (W, A, R ) however did not affect the cell death suppression nor subcellular localization of these effectors. Overall, the current study reports the presence of novel motifs in large numbers of effectors of rust fungi with cell death suppression features.

MetaLogo: a generator and aligner for multiple sequence logos

Sequence logos are used to visually display sequence conservations and variations. They can indicate the fixed patterns or conserved motifs in a batch of DNA or protein sequences. However, most of the popular sequence logo generators can only draw logos for sequences of the same length, let alone for groups of sequences with different characteristics besides lengths. To solve these problems, we developed MetaLogo, which can draw sequence logos for sequences of different lengths or from different groups in one single plot and align multiple logos to highlight the sequence pattern dynamics across groups, thus allowing users to investigate functional motifs in a more delicate and dynamic perspective. We provide users a public MetaLogo web server (http://metalogo.omicsnet.org), a standalone Python package (https://github.com/labomics/MetaLogo), and also a built-in web server available for local deployment. Using MetaLogo, users can draw informative, customized, aesthetic, and publishable sequence logos without any programming experience.

Genome-wide analysis of BBX gene family in Tartary buckwheat (Fagopyrum tataricum)

BBX (B-box), a zinc finger transcription factor with one or two B-box domains, plays an important role in plant photomorphogenesis, growth, and development as well as response to environmental changes. In this study, 28 Tartary buckwheat BBX (FtBBX) genes were identified and screened using a comparison program. Their physicochemical properties, gene structures, conserved motifs, distribution in chromosomal, and phylogeny of the coding proteins, as well as their expression patterns, were analyzed. In addition, multiple collinearity analysis in three monocots and three dicot species illustrated that the BBX proteins identified from monocots clustered separately from those of dicots. Moreover, the expression of 11 candidate BBX genes with probable involvement in the regulation of anthocyanin biosynthesis was analyzed in the sprouts of Tartary buckwheat during light treatment. The results of gene structure analysis showed that all the 28 BBX genes contained B-box domain, three genes lacked introns, and these genes were unevenly distributed on the other seven chromosomes except for chromosome 6. The 28 proteins contained 10 conserved motifs and could be divided into five subfamilies. BBX genes of Tartary buckwheat showed varying expression under different conditions demonstrating that FtBBXs might play important roles in Tartary buckwheat growth and development. This study lays a foundation for further understanding of Tartary buckwheat BBX genes and their functions in growth and development as well as regulation of pigmentation in Tartary buckwheat.