Identification of Cis-Regulatory Modules that Function in the Male Germline of Flowering Plants.

The male germline of flowering plants develops within the vegetative cell of the male gametophyte and displays a distinct transcriptional profile. Key to understanding the development of this unique cell lineage is determining how gene expression is regulated within germline cells. This knowledge impacts upon our understanding of cell specification, differentiation, and plant fertility. Here, we describe methods to identify cis-regulatory modules (CRMs) that act as key regulatory regions in the promoters of germline-expressed genes. We detail the complimentary techniques of phylogenetic footprinting and the use of fluorescent reporters in pollen for the identification and verification of CRMs

Core phasome analysis of a subset of <i>Neisseria</i> species.

<p>Core phasome analysis of a subset of <i>Neisseria</i> species.</p

Tract length distribution in different <i>Neisseria</i> species.

<p>Data are represented by heat maps. Colour intensity represents the percentage that a given tract length comprises of the total number of identified tracts of that type for each species. ‘-’ is indicative of no identified repeats of the given length. Information on the numbers of strains for each species, and numbers of tract lengths analysed can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196675#pone.0196675.t001" target="_blank">Table 1</a>.</p

Exemplar gene groupings associated with in frame and read through phase variation.

<p>Exemplar gene groupings associated with in frame and read through phase variation.</p

Variety of repeat types found in pathogenic and commensal <i>Neisseria ssp</i>.

<p>Variety of repeat types found in pathogenic and commensal <i>Neisseria ssp</i>.</p

Flowchart and visual output of Phasome<i>It</i>.

<p><b>(A)</b> Outputs of Phasome<i>It</i> can be viewed visually on the index page. Green bars indicate there is an homopolymeric tract within the open reading frame; orange bars indicate there is an SSR close to the gene of interest (for example in a promoter region); grey bars indicate there is a non-PV gene homologous to a PV gene in that same homology grouping; the remaining coloured bars are indicative of SSRs other than homopolymers which can be further derived from the dataset below the visual output. <b>(B)</b> Gene groupings corresponding to the visual output are found in a table below. From here, functions, PV status in each strain and tract entries can be obtained for the grouping of interest. The full dataset from which this figure is derived, containing further phasome information not discussed in this manuscript are available (<a href="https://figshare.com/s/d31b7b0b6ca4aeeb48df" target="_blank">https://figshare.com/s/d31b7b0b6ca4aeeb48df</a>). A red outline shows highlights both the graphical and interactive outputs for the <i>opa</i> loci as an example.</p

Range of phase variable genes identified in each species.

<p>Data shown the median, range, upper and lower quartile number of PV genes, as indicated by presence of a repeat tract. These data exclude gene groupings which contain dinucleotide repeat tracts, due to the insufficient evidence of phase variation associated with dinucleotide repeats in the literature, and the loci discussed herein. Statistical analysis were performed with a Kruskal-Wallis test with Dunn’s multiple comparisons. NS; not significant, ***; p-value of <0.0005.</p

Distribution of phase variable genes between phase variable modules.

<p>Distribution of phase variable genes between phase variable modules.</p

Phasome analysis of pathogenic and commensal Neisseria species expands the known repertoire of phase variable genes, and highlights common adaptive strategies

Pathogenic Neisseria are responsible for significantly higher levels of morbidity and mortality than their commensal relatives despite having similar genetic contents. Neisseria possess a disparate arsenal of surface determinants that facilitate host colonisation and evasion of the immune response during persistent carriage. Adaptation to rapid changes in these hostile host environments is enabled by phase variation (PV) involving high frequency, stochastic switches in expression of surface determinants. In this study, we analysed 89 complete and 79 partial genomes, from the NCBI and Neisseria PubMLST databases, representative of multiple pathogenic and commensal species of Neisseria using PhasomeIt, a new program that identifies putatively phase-variable genes and homology groups by the presence of simple sequence repeats (SSR). We detected a repertoire of 884 putative PV loci with maxima of 54 and 47 per genome in gonococcal and meningococcal isolates, respectively. Most commensal species encoded a lower number of PV genes (between 5 and 30) except N. lactamica wherein the potential for PV (36–82 loci) was higher, implying that PV is an adaptive mechanism for persistence in this species. We also characterised the repeat types and numbers in both pathogenic and commensal species. Conservation of SSR-mediated PV was frequently observed in outer membrane proteins or modifiers of outer membrane determinants. Intermittent and weak selection for evolution of SSR-mediated PV was suggested by poor conservation of tracts with novel PV genes often occurring in only one isolate. Finally, we describe core phasomes—the conserved repertoires of phase-variable genes—for each species that identify overlapping but distinctive adaptive strategies for the pathogenic and commensal members of the Neisseria genus

Scripts_for_Figure_Preparation

This folder contains the scripts used for preparation of the figures in the mBio paper