<i>PUF3</i> and mRNA/RFP divergence for mitochondrial translation related genes.

(A) The PUF3 motif is enriched in 3’ UTRs of genes in mitochondrial translation. (B) The expression of mitochondrial translation genes and PUF3 from the 22 yeast strains. Data was from Skelly et al. [19]. (C) Categorizing the regulatory effects of PUF3 on mitochondrial translation genes. Genes were classified into four categories based on their significance (FDR ≤ 5%) in mRNA or translation efficiency (TE).</p

Transcriptome and translatome profiling for YJM789 and BY4742.

(A) Workflow for the experimental procedures. The two yeast strains were cultured in YPD to log-phase. Cells were collected and treated with cycloheximide (CHX) for two minutes, and then divided into two aliquots, one for RNA-Seq and the other for ribosomal footprint profiling. Raw sequencing reads were cleaned and mapped to the yeast genomes for further analyses. (B) Spearman correlation coefficient (ρ) among mRNA and RFP data between biological replicates (indicated by numbers) and strains (indicated by letter, B: BY4742 and Y: YJM789). (C) Improving the YJM789 annotation using both mRNA and RFP data. Representative cases are depicted for three types of modifications: missing ORF (YIR019C), ORF extension (YEL046C) and ORF trimming (YER050C).</p

Coordinated Evolution of Transcriptional and Post-Transcriptional Regulation for Mitochondrial Functions in Yeast Strains

<div><p>Evolution of gene regulation has been proposed to play an important role in environmental adaptation. Exploring mechanisms underlying coordinated evolutionary changes at various levels of gene regulation could shed new light on how organism adapt in nature. In this study, we focused on regulatory differences between a laboratory <i>Saccharomyces cerevisiae</i> strain BY4742 and a pathogenic <i>S</i>. <i>cerevisiae</i> strain, YJM789. The two strains diverge in many features, including growth rate, morphology, high temperature tolerance, and pathogenicity. Our RNA-Seq and ribosomal footprint profiling data showed that gene expression differences are pervasive, and genes functioning in mitochondria are mostly divergent between the two strains at both transcriptional and translational levels. Combining functional genomics data from other yeast strains, we further demonstrated that significant divergence of expression for genes functioning in the electron transport chain (ETC) was likely caused by differential expression of a transcriptional factor, <i>HAP4</i>, and that post-transcriptional regulation mediated by an RNA-binding protein, <i>PUF3</i>, likely led to expression divergence for genes involved in mitochondrial translation. We also explored mito-nuclear interactions via mitochondrial DNA replacement between strains. Although the two mitochondrial genomes harbor substantial sequence divergence, neither growth nor gene expression were affected by mitochondrial DNA replacement in both fermentative and respiratory growth media, indicating compatible mitochondrial and nuclear genomes between these two strains in the tested conditions. Collectively, we used mitochondrial functions as an example to demonstrate for the first time that evolution at both transcriptional and post-transcriptional levels could lead to coordinated regulatory changes underlying strain specific functional variations.</p></div

Strain differences in mRNA and RFP.

<p><b>(A)</b> Diagram describing the resampling statistics. As introduced by Artieri and Fraser [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153523#pone.0153523.ref013" target="_blank">13</a>], for a given ortholog pair, we calculated the marginal nucleotide frequencies (π_B = [π_B(A), π_B(T), π_B(G), π_B(C)] for BY4742 and π_Y = [π_Y(A), π_Y(T), π_Y(G), π_Y(C)] for YJM789) and mappable lengths (L_B for BY4741 and L_Y for YJM789) for both genes, We began to generate the null ortholog pairs by resampling, with replacement, the base level counts from either BY4742 or YJM789 ortholog using the same length and nucleotide frequency from the orthologs (L_B, π_B or L_Y, π_Y). The resampling was repeated 10,000 times to form the null distribution of log₂(YJM789/BY4742) either from the BY4742 ortholog (green boxplot) or from the YJM789 ortholog (blue boxplot). The <i>P</i> value was obtained by comparing the observed log₂ ratio to the null distributions, and the max <i>P</i> value from all four comparisons was assigned to that gene. Log₂-ratio of YJM789 to BY4742 for genes involved in mitochondrial translation <b>(B)</b> and glucose metabolism <b>(C)</b>. The log₂(Y/B) for genes with insignificant <i>P</i> values were manually set to zero in both (B) and (C). The dashed box shows the pathways involved in mitochondrial metabolisms.</p

5’-extension, 3’-readthrough and ribosome stalling for the two strains.

<p><b>(A)</b> Two examples for 5’ extension and 3’ read-through in two strains. The blue and yellow lines indicate the positions of the original start and stop codons, respectively. The gray line shows the extended position of either the start or stop codons. Arrows indicate the ORF directions. <b>(B)</b> A cartoon diagram depicting the A-、P- and E- sites in the ribosome. The A-site is occupied by aminoacyl-tRNA, which functions as the acceptor for the growing protein during peptide bond formation. The P-site is occupied by the peptidyl-tRNA, the tRNA carrying the growing peptide chain. The E-site contains the deacylated tRNA on transit out from ribosome. <b>(C)</b> Ribosome residence time (RRT) for the 25 selected codons on A-, P- or E- sites of ribosome in two strains. Positions for those codons with insignificant <i>P</i> values were set to zero (RRT = 1). All other codons are not included because they do not show any significant stalling (defined as RRT ≥ 1.2 and <i>P</i> ≤ 0.0001) in either background. Codons were shown on the columns, with the respective amino acids indicated above. To compare the RRT for selected codons between strains, the ratio of RRT for two strains (B/Y) was calculated for each permutation. The observed RRT ratio was then compared with the permutated null ratios to obtain the <i>P</i> value. The criteria defining significance are the same as for ribosome stalling.</p

<i>HAP4</i> and mRNA divergence for ETC genes.

<p><b>(A)</b> The <i>HAP4</i> motif is enriched in the promoter regions of the ETC genes. <b>(B)</b> Log₂ (expression change) for genes involved in glucose metabolisms for the <i>HAP2</i>/<i>3</i>/<i>4</i>/<i>5</i> gene deletion mutants. Data was adopted from Kemmeren <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153523#pone.0153523.ref056" target="_blank">56</a>]. Genes with insignificant <i>P</i> values were manually set to zero. <b>(C)</b> Correlation coefficient of gene expression between each of the HAP complex components and the ETC genes among 22 yeast strains (upper panel: overall distribution of the correlation coefficients; lower panel: individual correlation coefficients). The RNA-Seq data was from Skelly <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153523#pone.0153523.ref019" target="_blank">19</a>].</p

Compatible mito-nuclear genomes in two yeast strains.

<p><b>(A)</b> Cytoduction was used to construct mtDNA replacement strains in the YJM789 genetic background [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153523#pone.0153523.ref047" target="_blank">47</a>]. Successful replacements underwent two rounds of mating. In the first round, an mtDNA donor strain was mated with an intermediate strain, the <i>KAR1-1</i> mutant, which is devoid of mtDNA. Cells with a nuclear background from <i>KAR1-1</i> and mtDNA from the donor strain were selected for the next round of mating with the recipient strain, which is also mtDNA deficient. Finally, cells with nuclear DNA from the recipient strain and mtDNA from the donor strain were selected and confirmed for their genotypes using several markers from different chromosomes. <b>(B)</b> Yeast cells were cultured in either glucose or ethanol growth medium starting at OD600 = 0.1. Three strains were used for the experiment, WT: YJM789 wild type, N_YMt_Y: YJM789 nuclear genome + YJM789 mtDNA; N_YMt_B: YJM789 nuclear genome + BY4742 mtDNA. The shown values were the average from three independent colonies for each strain. <b>(C)</b> Transcriptome profiling for each strain cultured in glucose or ethanol medium. Red dots show genes with significant expression changes, with the number of red dots indicated for each plot. The cutoff defining significance in these figures is FDR ≤ 10%.</p

Enrichment of up-regulated genes in YJM789 that are related with mitochondrial functions.

<p>Enrichment of up-regulated genes in YJM789 that are related with mitochondrial functions.</p

Additional file 1 of Combining BN-PAGE and microscopy techniques to investigate pigment-protein complexes and plastid transitions in citrus fruit

Additional file 1: Figure S1. Separation of plastids from exocarp of ‘Rong An’ kumquat and ‘Hong Anliu’ sweet orange on a discontinuous sucrose gradient, and light microscopy of isolated plastids from band 3. Figure S2. Normalized volume of ten protein complexes from BN-PAGE, MCPs, multiple protein complexes. Figure S3. 2D gel electrophoresis of plastid proteins in citrus fruit

Data_Sheet_1_Cell-Wall-Degrading Enzymes Required for Virulence in the Host Selective Toxin-Producing Necrotroph Alternaria alternata of Citrus.xlsx

The necrotrophic fungal pathogen Alternaria alternata attacks many citrus species, causing brown spot disease. Its pathogenic capability depends primarily on the production of host-selective ACT toxin. In the current study a Ste12 transcription factor was characterized to be required for conidial formation and the production of cell-wall-degrading enzymes (CWDEs) in the tangerine pathotype of A. alternata. The Ste12 deficiency strain (ΔSte12) retained wild-type growth, ACT toxin production, and sensitivity to oxidative and osmotic stress. However, pathogenicity tests assayed on detached Dancy leaves revealed a marked reduction in virulence of ΔSte12. Transcriptome and quantitative RT-PCR analyses revealed that many genes associated with Carbohydrate-Active Enzymes (CAZymes) were downregulated in ΔSte12. Two cutinase-coding genes (AaCut3 and AaCut7) regulated by Ste12 were individually and simultaneously inactivated. The AaCut3 or AaCut7 deficiency strain unchanged in cutinase activities and incited wild-type lesions on Dancy leaves. However, the strain carrying an AaCut3 AaCut7 double mutation produced and secreted significantly fewer cutinases and incited smaller necrotic lesions than wild type. Not only is the host-selective toxin (HST) produced by A. alternata required for fungal penetration and lesion formation, but so too are CWDEs required for full virulence. Overall, this study expands our understanding of how A. alternata overcomes citrus physical barriers to carry out successful penetration and colonization.</p