Novel Tyrosine Kinase-Mediated Phosphorylation With Dual Specificity Plays a Key Role in the Modulation of Streptococcus pyogenes Physiology and Virulence

Streptococcus pyogenes (Group A Streptococcus, GAS) genomes do not contain a gene encoding a typical bacterial-type tyrosine kinase (BY-kinase) but contain an orphan gene-encoding protein Tyr-phosphatase (SP-PTP). Hence, the importance of Tyr-phosphorylation is underappreciated and not recognized for its role in GAS pathophysiology and pathogenesis. The fact that SP-PTP dephosphorylates Abl-tyrosine kinase-phosphorylated myelin basic protein (MBP), and SP-STK (S. pyogenes Ser/Thr kinase) also autophosphorylates its Tyr101-residue prompted us to identify a putative tyrosine kinase and Tyr-phosphorylation in GAS. Upon a genome-wide search of kinases possessing a classical Walker motif, we identified a non-canonical tyrosine kinase M5005_Spy_1476, a ∼17 kDa protein (153 aa) (SP-TyK). The purified recombinant SP-TyK autophosphorylated in the presence of ATP. In vitro and in vivo phosphoproteomic analyses revealed two key phosphorylated tyrosine residues located within the catalytic domain of SP-TyK. An isogenic mutant lacking SP-TyK derived from the M1T1 strain showed a retarded growth pattern. It displayed defective cell division and long chains with multiple parallel septa, often resulting in aggregates. Transcriptomic analysis of the mutant revealed 287 differentially expressed genes responsible for GAS pathophysiology and pathogenesis. SP-TyK also phosphorylated GAS CovR, WalR, SP-STP, and SDH/GAPDH proteins with dual specificity targeting their Tyr/Ser/Thr residues as revealed by biochemical and mass-spectrometric-based phosphoproteomic analyses. SP-TyK-phosphorylated CovR bound to PcovR efficiently. The mutant displayed sustained release of IL-6 compared to TNF-α during co-culturing with A549 lung cell lines, attenuation in mice sepsis model, and significantly reduced ability to adhere to and invade A549 lung cells and form biofilms on abiotic surfaces. SP-TyK, thus, plays a critical role in fine-tuning the regulation of key cellular functions essential for GAS pathophysiology and pathogenesis through post-translational modifications and hence, may serve as a promising target for future therapeutic developments.

The ancient Salicoid genome duplication event: A platform for reconstruction of de Novo gene evolution in Populus trichocarpa

Orphan genes are characteristic genomic features that have no detectable homology to genes in any other species and represent an important attribute of genome evolution as sources of novel genetic functions. Here, we identified 445 genes specific to Populus trichocarpa. Of these, we performed deeper reconstruction of 13 orphan genes to provide evidence of de novo gene evolution. Populus and its sister genera Salix are particularly well suited for the study of orphan gene evolution because of the Salicoid whole-genome duplication event (WGD) which resulted in highly syntenic sister chromosomal segments across the Salicaceae. We leveraged this genomic feature to reconstruct de novo gene evolution from inter-genera, inter-species, and intra-genomic perspectives by comparing the syntenic regions within the P. trichocarpa reference, then P. deltoides, and finally Salix purpurea. Furthermore, we demonstrated that 86.5% of the putative orphan genes had evidence of transcription. Additionally, we also utilized the Populus genome-wide association mapping panel (GWAS), a collection of 1,084 undomesticated P. trichocarpa genotypes to further determine putative regulatory networks of orphan genes using expression quantitative trait loci (eQTL) mapping. Functional enrichment of these eQTL subnetworks identified common biological themes associated with orphan genes such as response to stress and defense response. We also identify a putative cis-element for a de novo gene and leverage conserved synteny to describe evolution of a putative transcription factor binding site. Overall, 45% of orphan genes were captured in trans-eQTL networks.

Xa7, a Small Orphan Gene Harboring Promoter Trap for AvrXa7, Leads to the Durable Resistance to Xanthomonas oryzae Pv. oryzae

Background The rice (Oryza sativa) gene Xa7 has been hypothesized to be a typical executor resistance gene against Xanthomonas oryzae pv. oryzae (Xoo), and has conferred durable resistance in the field for decades. Its identity and the molecular mechanisms underlying this resistance remain elusive. Results Here, we filled in gaps of genome in Xa7 mapping locus via BAC library construction, revealing the presence of a 100-kb non-collinear sequence in the line IRBB7 compared with Nipponbare reference genomes. Complementary transformation with sequentially overlapping subclones of the BACs demonstrated that Xa7 is an orphan gene, encoding a small novel protein distinct from any other resistance proteins reported. A 27-bp effector binding element (EBE) in the Xa7 promoter is essential for AvrXa7-inducing expression model. XA7 is anchored in the endoplasmic reticulum membrane and triggers programmed cell death in rice and tobacco (Nicotiana benthamiana). The Xa7 gene is absent in most cultivars, landraces, and wild rice accessions, but highly homologs of XA7 were identified in Leersia perrieri, the nearest outgroup of the genus Oryza. Conclusions Xa7 acts as a trap to perceive AvrXa7 via EBEAvrXa7 in its promoter, leading to the initiation of resistant reaction. Since EBEAvrXa7 is ubiquitous in promoter of rice susceptible gene SWEET14, the elevated expression of which is conducive to the proliferation of Xoo, that lends a great benefit for the Xoo strains retaining AvrXa7. As a result, varieties harboring Xa7 would show more durable resistance in the field. Xa7 alleles analysis suggests that the discovery of new resistance genes could be extended beyond wild rice, to include wild grasses such as Leersia species.

Analysis of the chromosomal clustering of Fusarium-responsive wheat genes uncovers new players in the defence against head blight disease

There is increasing evidence that some functionally related, co-expressed genes cluster within eukaryotic genomes. We present a novel pipeline that delineates such eukaryotic gene clusters. Using this tool for bread wheat, we uncovered 44 clusters of genes that are responsive to the fungal pathogen Fusarium graminearum. As expected, these Fusarium-responsive gene clusters (FRGCs) included metabolic gene clusters, many of which are associated with disease resistance, but hitherto not described for wheat. However, the majority of the FRGCs are non-metabolic, many of which contain clusters of paralogues, including those implicated in plant disease responses, such as glutathione transferases, MAP kinases, and germin-like proteins. 20 of the FRGCs encode nonhomologous, non-metabolic genes (including defence-related genes). One of these clusters includes the characterised Fusarium resistance orphan gene, TaFROG. Eight of the FRGCs map within 6 FHB resistance loci. One small QTL on chromosome 7D (4.7 Mb) encodes eight Fusarium-responsive genes, five of which are within a FRGC. This study provides a new tool to identify genomic regions enriched in genes responsive to specific traits of interest and applied herein it highlighted gene families, genetic loci and biological pathways of importance in the response of wheat to disease.

Xa7 , A Small Orphan Gene Harboring Promoter Trap for AvrXa7, Leads to the Durable Resistance to Xanthomonasoryzae pv. Oryzae.

BackgroundThe rice (Oryza sativa) gene Xa7 has been hypothesized to be a typical executor resistance gene against Xanthomonas oryzae pv. oryzae (Xoo), and has conferred durable resistance in the field for decades. Its identity and the molecular mechanisms underlying this resistance remain elusive.Results Here, we filled in gaps of genome in Xa7 mapping locus via BAC library construction, revealing the presence of a 100-kb non-collinear sequence in the line IRBB7 compared with Nipponbare reference genomes. Complementary transformation with sequentially overlapping subclones of the BACs demonstrated that Xa7 is an orphan gene, encoding a small novel protein distinct from any other resistance proteins reported. A 27-bp effector binding element (EBE) in the Xa7 promoter is essential for AvrXa7-inducing expression model. XA7 is anchored in the endoplasmic reticulum membrane and triggers programmed cell death in rice and tobacco (Nicotiana benthamiana). The Xa7 gene is absent in most cultivars, landraces and wild rice accessions, but highly homologs of XA7 were identified in Leersia perrieri, the nearest outgroup of the genus Oryza.ConclusionsXa7 acts as a trap to perceive AvrXa7 via EBEAvrXa7 in its promoter, leading to the initiation of resistant reaction. Since EBEAvrXa7 is ubiquitous in promoter of rice susceptible gene SWEET14, the elevated expression of which is conducive to the proliferation of Xoo, that lends a great benefit for the Xoo strains retaining AvrXa7. As a result, varieties harboring Xa7 would show more durable resistance in the field. Xa7 alleles analysis suggests that the discovery of new resistance genes need not be limited to wild rice, but could instead be extended to wild grass such as Leersia genus.

Development and Use of Gene Therapy Orphan Drugs—Ethical Needs for a Broader Cooperation Between the Pharmaceutical Industry and Society

Gene therapy orphan medicinal products constitute a unique group of new drugs which in case of hereditary diseases are usually administered only once at an early age, in the hope to provide sufficient gene product to last for the entire life of the patients. The combination of an exceptionally large single payment and the life-long clinical follow-up needed for understanding the long-term benefits and safety of gene therapy, represent new types of scientific, financial, social and ethical challenges for the pharmaceutical industry, regulators and society. With special consideration of the uniqueness and importance of gene therapy, the authors propose a three points plan for a close cooperation between the pharmaceutical industry and society to develop orphan gene therapy. (1) In fully transparent health technology negotiations a close and long-lasting, contractually fixed cooperation should be established between the manufacturers and local health-care stakeholders for sharing the medical and scientific benefits, the financial risks as well as the burdens of the post-authorization clinical and regulatory development. (2) The parties should agree on a fair, locally affordable drug price without the usually very high premium price calculated to compensate for the low number of patients. In case of high manufacturing costs, the companies should offer prolonged, 15–20 years long payment by installment with risk-sharing, especially considering that the late outcome of the treatment is unknown. Society should assist scientifically and financially organizing a specific patient registry, treatment in specialized hospitals and adequate long-term follow-up of patients, the coordinated management of financial transactions related to the risk sharing program. (3) The post-authorization treatment and prolonged observation of additional new cases coordinated by society should provide real world data needed for the modern complex regulatory evaluation of gene therapy products by the competent authorities. We assume that fair sharing of the benefits and risks as well as a well-organized cooperation of society with the industry in collecting real world evidence might result in better drug evaluation and improved accessibility due to lower prices. The outlined concept might support gene therapy more efficiently than the presently requested outstandingly high prices.

Mitochondrial Fostering: The Mitochondrial Genome May Play a Role in Plant Orphan Gene Evolution

Plant mitochondrial genomes exhibit unique evolutionary patterns. They have a high rearrangement but low mutation rate, and a large size. Based on massive mitochondrial DNA transfers to the nucleus as well as the mitochondrial unique evolutionary traits, we propose a “Mitochondrial Fostering” theory where the organelle genome plays an integral role in the arrival and development of orphan genes (genes with no homologs in other lineages). Two approaches were used to test this theory: (1) bioinformatic analysis of nuclear mitochondrial DNA (Numts: mitochondrial originating DNA that migrated to the nucleus) at the genome level, and (2) bioinformatic analysis of particular orphan sequences present in both the mitochondrial genome and the nuclear genome of Arabidopsis thaliana. One study example is given about one orphan sequence that codes for two unique orphan genes: one in the mitochondrial genome and another one in the nuclear genome. DNA alignments show regions of this A. thaliana orphan sequence exist scattered throughout other land plant mitochondrial genomes. This is consistent with the high recombination rates of mitochondrial genomes in land plants. This may also enable the creation of novel coding sequences within the orphan loci, which can then be transferred to the nuclear genome and become exposed to new evolutionary pressures. Our study also reveals a high correlation between the amount of mitochondrial DNA transferred to the nuclear genome and the number of orphan genes in land plants. All the data suggests the mitochondrial genome may play a role in nuclear orphan gene evolution in land plants.