Additional file 6: of Genome-wide association study of Gossypium arboreum resistance to reniform nematode

The 18 genes out of the 146 genes that were significantly differentially expressed in Gossypium hirsutum in response to reniform nematode infestation. (XLSX 13 kb

Additional file 3: of Genome-wide association study of Gossypium arboreum resistance to reniform nematode

Bayesian information criterion (BIC) values of compressed mixed linear model with different numbers of principle components used for association analysis in GAPIT. (XLSX 8 kb

Additional file 1: of Genome-wide association study of Gossypium arboreum resistance to reniform nematode

The 246 Gossypium arboreum germplasm accessions selected for genome-wide association study including the reniform nematode female index value for each accession. (XLSX 23 kb

Additional file 5 of Genome-wide association study of Gossypium arboreum resistance to reniform nematode

The 146 annotated genes within 72 kb of the 15 significantly associated SNPs. (XLSX 19 kb

Additional file 4: of Genome-wide association study of Gossypium arboreum resistance to reniform nematode

Genome-wide association results for the 7220 SNPs included in the study. (XLSX 564 kb

Detection of two shunt products in the mutant strain.

<p>A: HPLC spectra of metabolites by the wild type strain (WT/pIJ8600), mutant strain (MS/pIJ8600) and control strain (CH999/pRM5), indicated as UV absorption at 434 nm. Control: CH999/pRM5 was a recombinant strain of <i>S</i>. <i>coeliololar</i> able to accumulate two shunt products DMAC <b>5</b> (Mr: 298, 16.7 min) and aloesaponarin II <b>6</b> (Mr. 254, 22.6 min), due to the presence of 6 genes on pRM5, encoding KS, CLF, ACP, KR, ARO and CYC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132431#pone.0132431.g001" target="_blank">Fig 1B</a>) from the earlier stages of ACT <b>1</b> pathway. These 6 genes control the formation of the bicyclic intermediate <b>4</b>. Then the bicyclic intermediate was spontaneously converted into two shunt products in CH999/pRM5 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132431#pone.0132431.g001" target="_blank">Fig 1B</a>). B: Mass spectra of peaks for authentic DMAC <b>5</b> and aloesaponarin II <b>6</b> by CH999/pRM5 and for the compounds at 16.9 min and 22.6 min by the <i>med</i>-ORF12- deficient mutant strain.</p

Functional Characterization of a Ketoreductase-Encoding Gene <i>med</i>-ORF12 Involved in the Formation of a Stereospecific Pyran Ring during the Biosynthesis of an Antitumor Antibiotic Medermycin

<div><p>Medermycin, a polyketide antibiotic, possesses strong bioactivity against a variety of tumors through a novel mechanism and is structurally featured with a pyran ring containing two chiral centers (3<i>S </i>and 15<i>R</i>). By far the biosynthetic origin of such enantiomerical conformations still remains obscure. In the present study, we reported the functional characterization of a proposed ketoreductase Med-ORF12 encoded by medermycin biosynthetic cluster and revealed its involvement in the stereochemical control at C3 center of medermycin. Firstly, bioinformatics analysis of Med-ORF12 suggested that it belongs to a group of stereospecific ketoreductases. Next, a Med-ORF12-deficient mutant was obtained and LC/MS measurements demonstrated that medermycin production was completely abolished in this mutant. Meanwhile, it was found that two shunt products were accumulated at the absence of Med-ORF12. Finally, the reintroduction of Med-ORF12 into this mutant could restore the production of medermycin. In a conclusion, these data supported that Med-ORF12 is essential for the biosynthesis of medermycin and performs its role as a stereospecifc ketoreductase in the tailoring steps of medermycin biosynthetic pathway.</p></div

Inactivation and complementation of <i>med</i>-ORF12.

<p>A: Schematic representation of knock-out <i>med</i>-ORF12. A 2.8 kb genomic fragment is composed of left arm (L-arm) and right arm (R-arm) referring to the upstream and downstream regions flanking <i>med</i>-ORF12 respectively. These two DNA regions are linked together and then inserted into a suicide vector pYH7, giving a suicide plasmid pHSL124. After double- crossover between chromosome of the wild type strain and homologous regions on pHSL124, <i>med</i>-ORF12-deficient mutant strain was acquired. On the chromosome of the mutant, an artificially-designed sequence (<b>ATG</b><u>TCTAGA</u><b>TGA</b>) containing <i>Xba</i>I site in underline and start codon/stop codon in bold to replace in-frame <i>m</i>ed-ORF12 in the full-length. B: PCR confirmation of <i>med</i>-ORF12-deficient mutant strain (MS) and complementary strain (CS). The amplification using wild-type strain genomic DNA (lane 6) as template and a primer set (med12-qc1/ med12-qc1, showed in arrow in A) produced a 1 488 bp PCR product, while the genomic DNA of the <i>med</i>-ORF12- deficient mutant strains (lane 1–5) gave a 513 bp band as expected. The 975 bp genomic fragment of <i>med</i>-ORF12 was amplified by PCR (lane 8) and inserted into an integrative vector pIJ8600 (8.1 kb, lane 9: digestion with <i>Nde</i>I and <i>Bam</i>HI), generating an expression plasmid pHSAYT19 (lane 7: double digestion with <i>Nde</i>I and <i>Bam</i>HI). Genomic DNA isolated from the complementary strains (lane 10) and a primer set (med 12-A/med 12-B shown in arrow in A) were used for PCR amplification, giving a 975 bp band indicating the presence of <i>med</i>-ORF12, as same as for wild type strain genomic DNA as template (lane 11). On the contrary, the <i>med</i>-ORF12-deficient mutant strain (lane 12) could not produce an expected a 975 bp PCR product. M is 1 kb ladder as DNA marker.</p

Comparison between two biosynthetic gene clusters for medermycin and actinorhodin and phylogenetic analysis of Med-ORF12 and its homologies.

<p>A: Predicted function of genes in the clusters for ACT <b>1</b> and MED <b>2</b> respectively is indicated as bars with different filling styles. B: Phylogenetic tree was established using the amino acid sequences of Med-ORF12 and Med-ORF6 (accession number: BAC79036 and BAC79042, for MED <b>2</b>) from <i>Streptomyces sp</i> AM-7161, ActVI-ORF1 and ActIII (NP_629223 and NP_629236, for ACT <b>1</b>) from <i>S</i>. <i>coelicolor</i> A3(2), DauB (AAA87616 for aklaviketone) from <i>Streptomyces sp</i>., Gra-ORF5 and Gra-ORF6 (P16542 and P16543 for GRA <b>3</b>) from <i>Streptomyces violaceoruber</i>, AveF (NP_822111 predicated as ketoreductase for C5 of avermectin) from <i>Streptomyces avermitilis</i>, DnrU(Q_9ZAU1, marked as daunorubicin C-13 ketoreductase) from <i>Streptomyces peucetius</i>, Sa10 (ACK77759 for indigoidine/auricin) from <i>Streptomyces aureofaciens</i>, 3HAD (3HAD_B) from human heart and a hypothetical protein (WP_019763579) from <i>Streptomyces sp</i> Wigar10. The bar indicated the evolutionary distance. The numbers on branch nodes were percentages of 1000 sets of bootstrap supports. 3HAD: 3-hydroxyacyl-CoA dehydrogenase protein family; SDR: short-chain alcohol dehydrogenases family. C: Comparison between the reactions catalyzed by 3HAD and Med-ORF12 respectively.</p

Primers used in this study.

<p>Note:</p><p>^a: Sequences underlined in the primers were recognition sites for restriction enzymes indicated in parentheses. Start/stop codons (ATG/TGA) were shown in bold.</p><p>Primers used in this study.</p