Comparative Genome Structure, Secondary Metabolite, and Effector Coding Capacity across Cochliobolus Pathogens

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25x higher than those between inbred lines and 50x lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP-encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence

Comparative genome structure, secondary metabolite, and effector coding capacity across Cochliobolus pathogens.

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP-encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence

<i>S. turcica</i> has an ortholog of the <i>C. carbonum</i> NRPS HTS1 responsible for HC-toxin biosynthesis.

<p>A. Gene annotation and comparisons of the <i>S. turcica</i>, <i>P. tritici-repentis</i>, and <i>F. semitectum</i> regions carrying orthologs of the HC-toxin locus genes in <i>C. carbonum</i> strain SB111. Protein designations (color coded) correspond to <i>C. carbonum</i> and <i>F. semitectum</i> (APS) nomenclature. HTS1 is an NRPS, ToxA, E, F correspond to efflux pump, DNA-binding, and branched chain amino acid transaminase, proteins, respectively, and FAS α is a fatty acid synthase alpha subunit. Tox C (FAS beta subunit), ToxD (dehydrogenase), and ToxG (alanine racemase) in the cluster in <i>C. carbonum</i>, are not clustered in the other species but map to different scaffolds in the <i>S. turcica</i> and <i>P. tritici-repentis</i> assemblies. In <i>C. carbonum</i>, all of the known genes required for HC-toxin production are multicopy, in two linked, but separated clusters in a 600 kb region in isolate SB111; the genes are absent from toxin non-producing <i>C. carbonum</i> isolates that have been examined <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen.1003233-Ahn3" target="_blank">[122]</a>. B. Portions of the full phylogenetic tree (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen.1003233.s005" target="_blank">Figure S5A</a>) showing placement of the HTS1 AMPs, extracted from tree to the left. HTS1 has four AMP domains cartooned bottom left. Each <i>C. carbonum</i> AMP domain (red), groups, with high bootstrap support, with <i>S. turcica</i> protein 29755, a four AMP domain NRPS, (red), except for AMP4. In each of these matches, <i>C. carbonum</i> is represented twice, once by the SB111 AMP domain of the deposited sequence #AAA33023, and once as extracted by Augustus from our Illumina Velvet assembly of strain 26-R-13. Note all HTS1 AMP domains group separately one from another and AMP2 is distant from the others.</p

Cartoon of cross-species phylogenomic analyses of individual ketosynthase domains from PKS proteins.

<p>The ketosynthase (KS) domains were extracted from all five <i>C. heterostrophus</i> and from the <i>C. victoriae</i>, <i>C. carbonum</i>, <i>C. miyabeanus</i>, <i>C. sativus</i> and <i>S. turcica</i> genomes. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen-1003233-g003" target="_blank">Figure 3</a> for species designations, color codes and format. KS domains colored black and therefore absent in analyzed genomes include outgroups and KS domains in animal fatty acid synthases (FAS). KS domains not grouping with the previously annotated <i>C. heterostrophus</i> set are labeled as ‘New _1 through _10’. Gene/KS nomenclature and bootstrap values as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen-1003233-g003" target="_blank">Figure 3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen-1003233-g004" target="_blank">Figure 4</a> for AMP domains.</p

NPS1, NPS3, and NPS13 are examples of NRPS proteins encoded by highly recombinogenic and expanded <i>NPS</i> genes.

<p>The reference NPS1, NPS3 and NPS13 proteins are cartooned bottom left and AMP domains are color coded as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen-1003233-g005" target="_blank">Figure 5</a>. AMP domains corresponding to these proteins are completely conserved in the five strains of <i>C. heterostrophus</i>, but show discontinuous presence in all other <i>Cochliobolus</i> species (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen-1003233-g005" target="_blank">Figure 5</a>) and <i>Setosphaeria</i>. Thumbnail of full AMP tree (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen.1003233.s005" target="_blank">Figure S5A</a>) is shown at left. Note some AMP domains from NPS1 and NPS3 group at the top of the tree (AMPs 2 and 4, green), while the rest group at the bottom of the tree (AMPs 1 and 3, red); NPS13 AMP1 (blue) also groups at the bottom of the tree. Branches correspond to individual AMP domains which group together and the particular corresponding AMP domain is colored coded on the right of the diagram. Branches not in the original reference set of <i>C. heterostrophus</i> AMPs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen.1003233-Bushley1" target="_blank">[57]</a> are labeled as ‘expanded’ (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen-1003233-g005" target="_blank">Figure 5</a>). Gene/AMP nomenclature and bootstrap values as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen-1003233-g004" target="_blank">Figure 4</a>. Double red asterisk indicates <i>C. sativus</i> protein ID 115356 discussed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen-1003233-g007" target="_blank">Figure 7</a>).</p

<i>C. heterostrophus</i> race O strain C5, race T strain C4, <i>C. sativus</i> and <i>S. turcica</i> genome statistics.

<p><i>C. heterostrophus</i> race O strain C5, race T strain C4, <i>C. sativus</i> and <i>S. turcica</i> genome statistics.</p

Comparative small secreted protein candidate effector inventories.

a<p>Total SSPs for all strains examined.</p>b<p>Total for each strain (e.g., <i>Ch</i> C5).</p

NPS2 is an example of a highly conserved Dothideomycete NRPS.

<p>NPS2 consists of four AMP domains (cartoon above partial tree) that produce the hexapeptide intracellular siderophore, ferricrocin, responsible for iron storage within cells. NPS2 is present in all five strains of <i>C. heterostrophus</i>, all other <i>Cochliobolus</i> species and <i>Setosphaeria</i>. Note four branches, which cluster together on the thumbnail of the full tree (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen.1003233.s005" target="_blank">Figure S5A</a>), each corresponding to one of the four NPS2 AMP domains (gray AMP in NRPS cartoon to the right). Each fungal race or strain is color coded as indicated. Reference C5 AMP indicated as e.g., ChC5 77609 NPS2 AMP1 4; 77607 is the Genbank protein ID, NPS2 is the NRPS designation, AMP1 4 denotes the 1^st AMP domain of a total of 4 AMPs in the NRPS. JGI protein IDs are given for <i>S. turcica</i>, <i>C. sativus</i>, and <i>C. heterostrophus</i> strain C5. All other AMPs indicated by NODE number and Augustus gene call number (e.g., Cvict NODE 1785 g7786 t1 1 is a <i>C. victoriae</i> AMP on NODE 1785 in the Velvet assembly carrying Augustus called gene 7786; t1 1 indicates the order in which the AMP domain was predicted by HMMER). <i>C. heterostrophus</i> strain C4 AMP domains are indicated by scaffold and gene call. Bootstrap values 80% and above are indicated on the branches.</p

<i>C. heterostrophus</i> RFLP sequences anchor sequenced scaffolds to the genetic map.

<p>Genetic linkage groups determined by Tzeng <i>et al. </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen.1003233-Tzeng1" target="_blank">[8]</a>, are in light blue (linkage group numbers on the left). Assembled scaffolds from the reference C5 strain that could be anchored to each linkage group are in light green; numbers above are internal JGI identifiers (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen.1003233.s010" target="_blank">Table S2</a>). Black bars indicate the relative locations of RFLPs Tzeng et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen.1003233-Tzeng1" target="_blank">[8]</a> or feature, linked by dotted lines. The relative scale of genetic/physical distance was determined by calculating the average genetic/physical distance between consecutively placed RFLP markers (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen.1003233.s001" target="_blank">Figure S1</a>). Scaffold ends marked with a T contain telomeric sequence. Unplaced scaffolds are shown in pink. Maps are to scale and shown as centiMorgans (cM) or kilobases (kb).</p

Genomic organization of the scaffold associated with the <i>VHv1</i> locus conferring high virulence of pathotype 2 isolate ND90Pr to barley cv. Bowman compared to the corresponding region in pathotype 0, isolate ND93-1.

<p>A. Mauve alignment <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen.1003233-Darling1" target="_blank">[44]</a> of ND93-1 scaffolds to ND90Pr. Colored blocks [Locally Collinear Blocks (LCB)] indicate matches between the two genomes, and vertical block shading corresponds with % similarity. Note very few colored blocks at right end of top row. The ∼133 kb <i>VHv1</i> locus, which maps to distal end of scaffold 5 (2.18 Mb), is unique to isolate ND90Pr, as indicated by absence of colored blocks at right end of the top row and the second row which is the same region at higher resolution. Below this Mauve alignment segment of the <i>VHv1</i> region on scaffold 5 (from position 2,044,422 to 2,177,878) is the JGI browser view of the same region, displaying gene models and repeats. There are forty-three predicted genes (blue) in this region, only a fraction of which have KOG or GO descriptions (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen.1003233.s015" target="_blank">Table S7</a>). Two NRPSs (ID # 115356 and 140513, shown in red) map to this region and are unique to the ND90Pr isolate and also not found in any of the genomes examined in this manuscript (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen-1003233-g003" target="_blank">Figure 3</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen-1003233-g005" target="_blank">Figure 5</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen-1003233-t008" target="_blank">Table 8</a>). E-AG/M-CG-121 is one of two AFLP markers (the other is E-AG/M-CA-207 on scaffold 40) are linked to the virulence locus, <i>VHv1</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen.1003233.s003" target="_blank">Figure S3</a>). B. Quantitative real-time PCR analysis of five <i>NPS</i> genes (protein ID 49884, 350779, 130053, 115356, 140513) during infection of barley cv. Bowman. Gene expression was normalized based on the expression of the β-Actin gene, and the values are the relative expression levels in comparison with M96, a mixture of mycelia harvested, at 96 hours after culture set up, from different media including PDA, MM, V8PDA, and water agar. B6, B12, B24, B48, B72, and B96 are samples collected at 6, 12, 24, 48, 72, and 96 hours after inoculation. Primers are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen.1003233.s017" target="_blank">Table S9</a>. The error bars indicate the minimum and maximum values of relative expression of the gene. C. Inoculation of barley cv. Bowman with wild type (ND90Pr) and a mutant lacking the gene corresponding to protein ID 115356. Images taken 7 days after inoculation. Virulence on Bowman is significantly reduced compared to that of plants inoculated with the wild-type strain (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003233#pgen.1003233.s007" target="_blank">Figure S7</a>).</p