Comparative Genome Structure, Secondary Metabolite, and Effector Coding Capacity across Cochliobolus Pathogens
[['The filamentous ascomycete genus Cochliobolus ( anamorph Bipolaris/Curvularia ) is comprised of more than forty closely related , often highly aggressive , pathogenic species with particular specificity to their host plants .', 'All members of the genus known to cause serious crop diseases fall in a tight phylogenetic group suggesting that a progenitor within the genus gave rise , over a relatively short period of time ( <20 MYA , Ohm et al . , [1] ) to the series of distinct species [2] , each distinguished by unique pathogenic capability to individual types of cereal ( Table 1 ) .', 'Aggressive members include the necrotrophic corn pathogens Cochliobolus heterostrophus and Cochliobolus carbonum , the oat pathogen , Cochliobolus victoriae , the rice pathogen , Cochliobolus miyabeanus , the sorghum pathogen , Bipolaris sorghicola , the sugarcane pathogen , Bipolaris sacchari , and the hemibiotrophic generalized cereal and grass pathogen , Cochliobolus sativus .', 'All of the known Cochliobolus pathogens are classified as necrotrophs , except for C . sativus , which , although previously classified as such , has more recently been described as a hemibiotroph [3] .', 'Many necrotrophic Cochliobolus spp .', 'and related taxa ( e . g . , Pyrenophora tritici-repentis , Stagonospora nodorum ) are notorious for their ability to evolve novel , highly virulent , races producing Host Selective Toxins ( HSTs ) and their concomitant capacity to cause diseases on cereal crops that were bred , inadvertently , for susceptibility to the HST-producing pathogen [4] , [5] .', 'For example , in 1970 , race T , a previously unseen race of C . heterostrophus ( Bipolaris maydis ) caused a major epidemic [Southern Corn Leaf Blight ( SCLB ) ] , destroying more than 15% of the maize crop [6] .', 'Before 1970 , C . heterostrophus was known as an endemic pathogen ( race O ) of minor economic importance , first described in 1925 [7] .', 'Subsequent research over the ensuing four decades since 1970 has demonstrated that the epidemic was triggered by the unfortunate confluence of complex DNA recombination events in both the fungal pathogen and the plant host .', 'Race T is genetically distinct from race O in that it possesses an extra 1 . 2 Mb of DNA [8] , [9] encoding genes for biosynthesis of the polyketide secondary metabolite , T-toxin , an HST essential for high virulence [4] .', 'These genes are missing in race O . On the plant side , the presence in Texas male sterile cytoplasm ( Tcms ) maize , of a hybrid mitochondrial gene called T-urf13 , composed of segments of two mitochondrial and one chloroplast gene , is essential for susceptibility .', 'Tcms corn does not need to be detasseled to prevent self-crossing because it is male sterile , a desirable trait for breeders producing hybrid seed .', 'The resulting popularity of Tcms maize was disastrous from the perspective of pathogen attack , however , as it served as a monoculture of susceptible germplasm [10] , [11] .', 'Similarly , C . victoriae ( Bipolaris victoriae ) , causal agent of Victoria Blight , produces the chlorinated cyclic pentapeptide HST , victorin , rendering it highly virulent to oats carrying the dominant Vb allele [12] .', "The fungus caused widespread destruction ( 20 states ) in the 1940's on oat varieties containing the recently introduced Pc-2 gene for resistance to crown rust caused by Puccinia coronata [13] .", 'Like the C . heterostrophus T-toxin/Tcms case , the monoculture of Victoria oats carrying Pc-2 was the perfect milieu for attack by C . victoriae producing victorin , which elicits Pc-2-dependent Programmed Cell Death ( PCD ) .', 'Recent work with Arabidopsis identified a resistance-like protein responsible for susceptibility to C . victoriae and victorin [14] , [15] .', 'This work is seminal in demonstrating fungal HSTs can target resistance proteins to promote disease .', 'In contrast to the dominant plant host genes required for susceptibility to C . heterostrophus and C . victoriae , susceptibility to Northern Corn Leaf Spot caused by C . carbonum ( Bipolaris zeicola ) is conferred by a homozygous recessive maize gene ( s ) [16] , [17] .', 'C . carbonum race 1 produces the cyclic-tetrapeptide HST , HC-toxin , which is specifically active against corn with the genotype hmhm , as is the fungus itself [4] , [18] , [19] .', 'Hm1 and Hm2 encode carbonyl reductases which inactivate the toxin [16]; hmhm lines , cannot inactivate the toxin , and are therefore sensitive .', 'The site of action of HC-toxin in susceptible corn is histone deacetylase; it is hypothesized that HC-toxin acts to promote infection of maize of genotype hm1hm1 by inhibiting this enzyme , resulting in accumulation of hyperacetylated core ( nucleosomal ) histones .', 'This then alters expression of genes encoding regulatory proteins involved in plant defense [20] , [21] .', 'C . carbonum races 2 and 3 do not produce the toxin .', 'C . miyabeanus ( Bipolaris oryzae ) is the causal agent of Brown Spot of rice which contributed , along with a cyclone and tidal waves , to the Bengal rice famine of 1942/1943 that resulted in starvation of more than two million people [22] .', 'To date , no HST has been associated with virulence , although C . miyabeanus culture filtrates can suppress plant phenol production [23] .', 'C . sativus ( Bipolaris sorokiniana ) causes diseases of roots ( Common Root Rot ) , leaves ( Spot Blotch ) , and spikes ( known as black point or kernel blight ) of cereals ( mainly barley and wheat ) [24] , [25] , but also attacks many grasses , including switch grass ( Panicum virgatum L . ) [3] , [26] , [27] and Brachypodium distachyon ( S . Zhong , unpublished ) .', 'Three C . sativus pathotypes ( 0 , 1 and 2 ) have been described [28] , based on differential virulence patterns on three barley genotypes ( ND5883 , Bowman , and NDB112 ) .', 'Pathotype 0 isolates show low virulence on all three barley genotypes .', 'Pathotype 1 isolates show high virulence on ND5883 but low virulence on other barley genotypes .', 'Pathotype 2 isolates show high virulence on Bowman but low virulence on ND5883 and NDB112 .', 'Genetic analysis and molecular mapping indicates that a single locus , VHv1 , controls high virulence of the pathotype 2 isolate ND90Pr on Bowman [29] , [30] , however , the exact nature of the gene ( s ) was unknown before this study ( see Results ) .', 'More recently , a new pathotype , highly virulent on NDB112 , the most durable spot blotch resistance source in barley [31] , has been found in North Dakota [32] and Canada [33] .', 'Setosphaeria turcica ( Exserohlium turcicum , Helminthosporium turcicum ) , a hemibiotrophic vascular leaf pathogen , is a member of the closest genus to Cochliobolus ( see Figure 1 in Ohm et al . , [1] ) , and causes Northern Leaf Blight ( NLB ) , a major disease of maize and sorghum in the US and internationally [34] .', 'To date , at least four races of S . turcica have been identified based on their differential virulence performance on maize carrying resistance genes known as Ht [35] , [36] .', 'In recent work , Martin et al . [34] , identified new resistance genes ( named St ) in both maize and sorghum .', 'The connections between the Ht and St resistance genes are unclear at this point .', 'Until recently , it was assumed that necrotrophs use brute force methods ( e . g . , arsenals of cell wall degrading enzymes , HSTs ) to invade and kill host tissues and do not subtly manipulate the host during infection , as do biotrophs with their arsenal of effectors [37] .', 'Several lines of evidence , from studies of the Dothideomycete , necrotrophic wheat pathogens , Pyrenophora tritici-repentis [38] and Stagonospora nodorum , [39] indicate that , like biotrophs , these pathogens secrete protein effectors ( in this case also called HSTs ) that interact with host targets in a gene-for-gene manner .', 'Unlike biotrophs , however , interaction of the fungal effector and host protein results in susceptibility , rather than resistance .', 'The above-mentioned research on C . victoriae , further indicates mechanistic overlap of pathogenic lifestyle and has major implications regarding the challenge plants face in defending themselves against both necrotrophs and biotrophs [37] , [40] .', 'Recent studies involving Arabidopsis have revealed that victorin-induced PCD requires a host NB-LRR-type resistance protein [15] , [41] .', 'Thus a protein with canonical resistance protein structure is required for susceptibility .', 'These observations point toward victorin , subverting effector triggered defenses against biotrophs , such as P . coronata ( see above ) , to promote susceptibility to a necrotroph .', 'Here we provide a comparative analysis of genome similarities and differences among Cochliobolus and Setosphaeria pathogens , with particular emphasis on strain and species-unique sequences , secondary metabolism genes , and genes encoding small secreted proteins .', 'Identification of these key structural genomic and molecular differences is the first step in understanding species-specificity and how closely related necrotrophic and hemibiotrophic pathogens cause disease .', 'As proof of concept , we offer several examples of how comparative approaches pinpoint virulence associated , species-specific regions of interest .', 'The Joint Genome Institute ( JGI ) has sequenced five strains of C . heterostrophus ( three race T and two race O strains ) and four additional species of Cochliobolus , including C . carbonum , C . victoriae , C . miyabeanus , and C . sativus and one strain of S . turcica .', 'Add to this that host genome sequences ( corn , rice , barley and B . distachyon ) for six of these pathogens are available and one has the information base for dissecting both sides of the interaction mechanism going forward .'], ['Five strains of C . heterostrophus , one strain of C . sativus , and one strain each of C . victoriae , C . carbonum , C . miyabeanus , and S . turcica were sequenced by JGI ( Table 2 , Table 3 ) .', 'Two C . heterostrophus strains and one strain each of C . sativus and S . turcica were fully sequenced as described in Materials and Methods , while the remaining genomes were sequenced using Illumina and assembled de novo using Velvet , as described in Materials and Methods .', 'The highly inbred race O lab strain C5 was used as the reference sequence for all comparisons , as it is the most complete , consisting of only 68 scaffolds .', 'Overall sequence assembly and annotation statistics are presented in Table 2 and Table 3 .', 'All Cochliobolus genomes are in the 32–38 Mb range with an estimated gene content of 11 , 700–13 , 200 .', 'The S . turcica genome is ∼43 Mb with ∼11 , 700 genes .', 'Overall gene content and genome organization are highly similar within this group of fungi .', 'In contrast , comparative analysis of C . heterotrophus , C . sativus and S . turcica in the context of 14 more distantly related Dothideomycetes genomes described elsewhere ( Ohm et . al . , [1] ) revealed significant variation .', 'C . heterostrophus: A genetic map with 125 RFLP markers was constructed previously [8] using C . heterostrophus race O field strain Hm540 ( sequenced herein ) and race T C-strain B30 . A3 . R . 45 ( same backcross series as strains C5 and C4 , sequenced herein [42] , [43] ) as parents .', 'RFLP sequences were used to refine the C . heterostrophus race O strain C5 physical assembly and link it to the genetic RFLP map ( Figure 1 , Text S1 ) .', 'The interconnected genetic and physical maps allowed comparisons of physical and genetic distance , which was found to be ∼13 kb/cM on average ( Figure S1 ) .', 'Correlations were also made between previously estimated chromosome sizes based on CHEF gel analysis [9] , and physical size based on sequence assemblies ( Tables S1 , S2 ) .', 'Based on RFLP map data , parental field isolate Hm540 was reported to lack the dispensable chromosome present in the other parental strain ( B30 . A3 . R . 45 ) used to build the map [8] .', 'Comparisons of all five sequenced C . heterostrophus strains , using the Mauve alignment tool [44] , supported this observation and revealed that all sequenced strains carried the previously recognized ‘B’ or dispensable chromosome ( which corresponds to strain C5 scaffold 16 ) ( Figure S2 ) .', 'Race O chromosomes 6 and 12 are of interest because counterparts are reciprocally translocated in race T and the high-virulence conferring Tox1 locus , encoding genes for biosynthesis of T-toxin , maps genetically to both breakpoints [9] .', 'Comparison of the race O and race T assemblies provided some clues as to the physical locations of these breakpoints , but the exact positions remain elusive , due to structural complexity associated with these regions [45] .', 'Additional details regarding linkage of the C . heterostrophus physical assembly to the genetic map are available in Text S1 .', 'C . sativus: Before mapping sequenced scaffolds to the previously constructed C . sativus genetic map [30] 121 polymorphic simple sequence repeat ( SSR ) markers were identified in the assembly sequences of the ND90Pr and ND93-1 parents , as described in Materials and Methods .', 'Then , sequences of the SSRs and other markers were used to assign sequenced scaffolds to the updated map .', 'Thirty of these linkage groups contained SSR markers and were found to be associated with 16 scaffolds , summing to 29 . 32 Mb .', 'Seven linkage groups were unassigned ( Figure S3 ) .', 'The two AFLP marker sequences ( E-AG/M-CG-121 and E-AG/M-CA-207 ) , cosegregating with the VHv1-associated high virulence of C . sativus pathotype 2 on cultivar Bowman [30] , were used as blast queries against the ND90Pr genome assembly .', 'E-AG/M-CG-121 mapped to scaffold 5 , while E-AG/M-CA-207 mapped to scaffold 40 ( Figure S3 ) .', 'Details of the construction , linking , and analysis of the C . sativus map are available in Text S1 .', 'To begin to identify regions in the C . heterostrophus C5 assembly not represented in other strains , we first mapped gaps in the reference assembly ( thick vertical black bars , Figure 2 ) .', 'A single gap was present in the assembly of 9 of 16 chromosomes and we speculate that these gaps correspond to centromeric regions .', 'We then mapped sequence reads of all Cochliobolus genomes in this study to the C . heterostrophus C5 reference , identifying regions in the C5 reference that were not present in the query genome ( Table S5 ) .', 'The sets of C5 genomic regions that were absent in a given query were combined to determine genomic regions unique and/or conserved at different taxonomic levels .', 'Individual reference-unique region counts were recorded for each of the C . heterostrophus strains .', 'There were 609 areas of the C5 assembly unique ( C4 reads did not map there ) to the C5 genome when C4 reads were mapped to it .', 'For C . heterostrophus strains Hm540 , PR1x412 , and Hm338 , there were 3 , 279 , 4 , 383 and 1 , 480 such regions , respectively .', 'When only regions greater than 5 , 000 bp were considered , there were 19 when C4 was used as the query , and 33 , 75 , and 30 , when PR1x412 , Hm540 , and Hm338 were used as the queries , respectively .', 'Many of the gaps associated with Hm540 mapped to reference C5 scaffold 16 corresponding to the dispensable B chromosome , which is absent in Hm540 ( Figure S2 ) .', 'The C5 reference-unique regions were then combined and filtered to identify conserved genomic regions at the strain or species level , where regions were unique in one type of comparison , but not in others ( Table S5 ) .', 'We designated “inbred C strain”-specific regions as gaps found when all field strains were aligned to C5 but not when C4 was aligned to C5 , race O specific regions as gaps found when all race T strains were aligned to C5 but not when race O strain Hm540 was aligned to race O strain C5 , and C . heterostrophus-specific regions as gaps found when all Cochliobolus species were aligned to C5 , but not when any C . heterostrophus strain was aligned .', 'A total of 28 , 556 bp is missing from all C . heterostrophus field strains , yet present in the C4 assembly ( Table S5 ) .', 'This ∼30 kb of DNA represents sequence uniquely conserved in the inbred C strains .', 'There are at least six fungal-specific Zn2Cys6 transcription factors ( ID# 1019013 , 1020538 , 1021058 , 1021066 , 1100349 , 1100899 ) present in this C strain unique cache that may signify “early action” strain diversification .', 'Zn2Cys6 transcription factors were among the most abundant predicted domains in the C . heterostrophus gene catalogue [1] .', 'There were almost no race O specific regions identified ( sequence found only in C5 and Hm540 ) ; only 11 regions , summing to 4 , 309 bp , were identified ( Table S5 ) .', 'Our working hypothesis is that the essential difference between race O and race T is the 1 . 2 Mb of Tox1 race T DNA ( not in race O C5 and therefore not able to be aligned ) .', 'Both race O only regions ( scaffold 12 , 732532–734880 , and scaffold 19 , 441888–443521 ) contain a single protein each ( ID# 59063 , 34937 ) with no conserved domains or predicted function .', 'Most significantly , at the species level , a total of 11 . 76 Mb DNA was missing from all non-C .', 'heterostrophus Cochliobolus genomes analyzed , yet found in all field strains of C . heterostrophus .', 'Only 1 . 6 Mb of this was found in pieces larger than 5 kb .', 'Most of the sequence that separates C . heterostrophus from other species , therefore , is not the result of large wholesale insertions or deletions of DNA , but from a more piecewise gain and loss .', 'Nonribosomal peptide synthetases ( NRPSs ) , found in fungi and bacteria , are multimodular megasynthases that catalyze biosynthesis of small bioactive peptides ( NRPs ) , including virulence determinants , such as HSTs , via a thiotemplate mechanism independent of ribosomes [50] , [51] , [52] , [53] , [54] .', 'NRPSs synthesize peptides using sets of core domains , known as modules , which consists of three domains:', '1 ) an adenylation ( AMP ) domain which recognizes and activates the substrate via adenylation with ATP ,', '2 ) a thiolation ( T ) or peptidyl carrier protein ( PCP ) domain which binds the activated substrate to a 4′- phosphopantetheine ( PP ) cofactor via a thioester bond and transfers the substrate to', '3 ) a condensation ( C ) domain which catalyzes peptide bond formation between adjacent substrates on the megasynthase complex .', 'NRPSs can be mono- , bi- , or multi-modular and core domains in any particular multimodular enzyme may be most closely related to one another or to a domain from a different NRPS .', 'The suites of NRPS encoding genes ( NPS ) in the C . heterostrophus C4 and C5 genomes were identified and annotated previously [55] , [56] , [57] .', 'To address degree of conservation and evolutionary relationships of NRPS proteins in our subject species in order to make inferences about function , we used the fungal AMP-binding ( AMP ) domain Hidden Markov Model ( HMM ) developed by Bushley and Turgeon [57] to identify individual AMP domains in the additional strains of C . heterostrophus and other Cochliobolus species , plus S . turcica .', 'Phylogenetic trees were built to develop a comparative NRPS AMP domain inventory and included the known C . heterostrophus C5 AMP domains as a reference ( Table S6 ) .', 'Polyketide synthases ( PKSs ) , like NRPSs , are large multidomain enzymes that produce small molecules ( polyketides ) with functions that include HSTs .', 'The suites of PKS encoding genes ( PKS ) in the C . heterostrophus C4 and C5 genomes were identified and annotated previously [65] .', 'To address degree of conservation and evolutionary relationships of PKSs in our subject species in order to make inferences about function , we used the PFAM ketosynthase domain ( KS ) HMM as a query to search for orthologs in the additional strains of C . heterostrophus and other species , and the related maize pathogen , S . turcica .', 'Several publications demonstrate that species/strain unique sequences tend to reside in variable regions of the genome such as in subtelomeric locations [68] , [69] and dispensable chromosomes [70] .', 'All C . heterostrophus reference strain C5 NPS and PKS genes were mapped to the assembled linkage groups ( Figure 2 ) .', 'For NPSs , 13 of the 14 total could be mapped to one of the 16 linkage groups/chromosomes , and 6 of the 13 were <200 kb from the end of the linkage group .', 'Two ( NPS 5 , 9 ) of the six are unique to C . heterostrophus and two ( NPS1 , 11 ) have limited distribution in Cochliobolus spp .', '.', 'For PKSs , 22 of the 25 total could be mapped to one of the 16 linkage groups , 9 of the 22 were <200 kb from the end of the corresponding linkage group and one ( PKS25 ) mapped to the B chromosome .', 'Five ( PKS 13 , 16 , 17 , 20 , 25 ) of the ten are unique to C . heterostrophus and one more ( PKS11 ) has limited distribution in Cochliobolus spp .', '.', 'In sum , approximately half of the NPSs and PKSs map to scaffold ends , in some cases with mapped telomeres ( Figure 1 ) .', 'As chromosome ends are notoriously variable , this placement could indicate a mechanism for patchy phylogenetic distribution of these genes .', 'Note that the two PKSs involved in T-toxin production by race T are absent in race O strain C5 , but map ( genetically ) to the breakpoints of race T chromosomes 12;6 and 6;12 which are reciprocally translocated with respect to chromosomes 6 and 12 in race O C5 [9] .', 'Note also that PKS3 , which has a phylogenetic relationship , but without bootstrap support , to PKS2 ( Figure 10 , Figure 11B ) , maps internally to race O chromosome 6 ( Figure 2 ) , and that PKS7 , which is the closest ( with bootstrap support ) C . heterostrophus PKS to PKS1 ( Figure 11B ) , maps to the end of unplaced scaffold 20 ( Figure 2 ) .', 'To identify candidate effector proteins , we searched the gene catalog of each species for proteins that were cysteine rich ( ≥2% cysteine ) , small ( <200 amino acids ) , predicted to be secreted ( using Phobius [71] ) , and without transmembrane domains .', 'Between 141 and 289 SSPs per genome ( Table 10 ) were identified with C . sativus ND90Pr containing the most and C . heterostrophus Hm338 the fewest .', 'We next conducted an all versus all blast analysis to determine if SSPs were strain or species-unique , using an 80% blast cutoff .', 'Very few C . heterostrophus SSPs were unique to any particular strain as most could be found in at least one other C . heterostrophus field or lab strain .', 'Using this approach , we identified between one and 21 unique SSPs ( Table 10 , master inventory Table S11 ) .', 'We found more strain-unique SSPs in the other Cochliobolus genomes , as our analysis included five C . heterostrophus strains .', 'S . turcica and C . sativus had the most isolate-unique SSPs , containing 191 and 167 candidates , respectively .', 'As these are the two strains thought to act as hemibiotrophs , it is interesting that they contain more SSPs , and more unique SSPs , than the necrotrophic isolates , although this is only a correlation at this point .', 'The C . heterostrophus C5 assembly has 180 predicted secreted proteins matching the criteria .', 'We examined each of these in the JGI browser with respect to EST support , SNPs , and predicted functional domains .', 'Seventy-two of these calls had absolutely no EST support , while 24 calls had incomplete EST support ( ESTs matching some portion , but not all , of the gene call ) , leaving 84 with complete ( spanning the entire gene model ) EST support ( Table 11 ) .', 'Genes in the no-EST support category are of special interest , as they may be specifically expressed in planta and thus not expressed under conditions used for preparing our EST libraries ( fungus grown in vitro on a variety of complete ( CM ) and minimal ( MM ) media , and mixed into CM or MM pools ) .', 'Lack of strong EST support may also suggest an erroneous gene call .', 'As is typical with candidate effectors , functional domain predictions were lacking , with only 37 candidates having some predicted function , generally involved in cell wall or extracellular matrix function ( Table 11 , Table S11 ) .', 'An additional 23 candidates were conserved in other fungi outside of the Dothideomycetes .', 'The remaining 120 calls were featureless and seemingly unique to the Dothideomycetes .', 'C . heterostrophus strain C5 SSP calls were rich in SNP calls to other Cochliobolus genomes: 101 candidate SSPs had SNPs with at least one other Cochliobolus genome .', 'In our all versus all blast analysis , only 6 of the 180 C . heterostrophus C5 SSPs were found in all 10 strains examined and 14 were unique to strain C5 ( Table 11 ) .', 'The presence or absence of most SSPs did not fall into easily categorized bins such as C . heterostrophus-specific , or maize-pathogens only .', 'Instead , SSPs were present and absent in no particular pattern across the genomes .', '115 SSPs were present in at least one other species ( C . victoriae , C . miyabeanus , C . carbonum , or S . turcica ) , with seven found in all species , and 27 in all Cochliobolus species .', 'SSPs mapped to all scaffolds larger than S26 ( Figure 2 ) .', 'Unlike those in some phytopathogens , such as Leptosphaeria maculans [72] , SSP encoding genes did not occur in clusters; candidates seldom were located within 10 kb of each other ( Figure 2 ) .', 'These genes were , however , often located in or near regions we identified as C . heterostrophus species unique ( Figure 2 ) .'], ['Our whole-genome alignment data support graduated degrees of similarity at the highly inbred strain , field strain , species and genus levels .', 'C . heterostrophus strains C4 and C5 , offspring of successive backcrosses [42] , were highly similar to one another , with 20 fold fewer SNPs than pairwise comparisons of reference strain C5 to C . heterostrophus field strains .', 'This remarkably low number of SNPs highlights the power of selective inbreeding in establishing uniformity across the genome .', 'The two C . sativus field strains , when aligned to each other , had a comparable number of SNPs to those of C . heterostrophus field strains aligned to reference C . heterostrophus strain C5 .', 'Other Cochliobolus genomes had roughly 50 fold more SNPs than C . heterostrophus field strains when aligned to the C . heterostrophus C5 reference .', 'This level of similarity was seen when comparing any two Cochliobolus species to one another , with the exception of comparing C . victoriae to C . carbonum .', 'These two species are capable of successfully mating , although progeny of crosses are unable to cross to each other or to their parents ( Turgeon lab , unpublished ) .', 'We have hypothesized that C . victoriae may have evolved from a C . carbonum strain [49] .', 'This similarity is seen at the whole genome level , as C . victoriae and C . carbonum share an intermediary number of SNPs compared to C . heterostrophus inter- and intra-species comparisons .', 'Our SNP data show that approximately one quarter of the genome differs between Cochliobolus species and that only about one tenth of this is found in segments larger than 5 kb .', 'We and others [72] , [73] , [74] have recently introduced the term mesosyteny [1] to describe organizational conservation between species .', 'Genetic content is conserved across chromosomes , but not co-linearly .', 'It seems possible that our findings here , showing that many small , scattered differences summing to significant quantitative differences ( i . e . , 25% dissimilar ) , could be the product of the same mechanisms that result in mesosyntenic patterns .', 'Pathogens of the same host ( e . g . , C . carbonum and C . heterostrophus on maize ) or lifestyle were not more similar to each other than those of different hosts; instead overarching genetic patterns followed phylogenetic lines .', 'As it is estimated that the Pleosporaceae arose as a group less than 20 MYA ( see Figure 1 in Ohm et al . , [1] ) and the genus Cochliobolus is young in the group , genome comparisons provide us with an overall picture of a timeline of how genome diversity varies with speciation .', 'Our intra-species SNP tallies are comparable to SNP tallies found when strains of other species are examined .', 'For example , there were 10 , 495 SNPs called between two Fusarium graminearum strains [75] , and a range of 13 , 274–188 , 346 SNPs called for 18 Neurospora crassa classical genetic mutants [76] .', 'Both dataset tallies are in the same range as our C . heterostrophus field strain comparisons .', 'With respect to SNPs in different species of the same genus , it is unusual that we were able to perform a whole-genome SNP analysis at all , without limiting our scope to coding sequence .', 'We owe this to the very close phylogenetic relationship of these species .', 'Although individual functional domains of NPS/PKS proteins can be identified bioinformatically , attempting to predict their corresponding metabolite product is challenging .', 'The genes encoding these proteins evolve rapidly and through complex mechanisms [57] , [65] and whole gene alignment methods provide misleading or unclear results when determining presence or absence of a particular NPS or PKS gene .', 'Here , we extracted individual conserved signature catalytic domains , i . e . , the AMP-binding domains from mono- or multi-modular NRPSs or the ketosynthase ( KS ) domain from multidomain PKSs using customized HMM models , then built alignments and phylogenetic trees with these individual units to determine the presence or absence of whole or partial NRPS and PKS proteins , and their evolutionary relationships .', 'In our opinion this is a necessary first step towards understanding evolutionary history of the corresponding genes and the possible small molecules produced by these highly diverse proteins .', 'We found that within a Dothideomycete genus , in this case Cochliobolus , approximately half of the NPS and a third of the PKS genes are well conserved ( present in all strains ) .', 'When related S . turcica was considered these numbers dropped to a third and a fifth , respectively .', 'The rest were found to be poorly conserved or species-unique when the highly curated C . heterostrophus gene sets were used as reference .', 'The small molecules produced by the corresponding non-conserved proteins are largely uncharacterized , but the differences between strains and species imply that the potential for production of biochemically unique molecules is large and considerably beyond that expected for closely related strains ( housekeeping genes share ∼95% identity ) .', 'These findings refine our understanding of NPS and PKS genes , as very few are conserved .', 'For example , only two NPS genes and one PKS gene were found when 18 Dothideomycete genomes were analyzed [1] .', 'Broadly conserved secondary metabolism genes , where they have been characterized , produce small molecules that serve basic cellular functions ( ferricrocin , melanin [58] , [77] , [78] , [79] ) .', 'Poorly conserved NPS and PKS genes , while still largely uncharacterized , can include those involved in host-specific high virulence .', 'NPS1 and NPS3 AMP domains are discontinuously distributed and expanded across the Cochliobolus and Setosphaeria isolates sequenced and are sources of much of the NRPS diversity ( Figure 5 , Figure 6 and [57] ) .', 'The individual AMP modules do not cluster by protein , but instead , NPS1 , NPS3 and NPS13 AMP domains occur in two distinct , and mixed , groups ( Figure 6 ) .', 'Strikingly , each Cochliobolus species possesses either a complete C . heterostrophus NPS1 or NPS3 ortholog , but never both .', 'Furthermore , all species have one or more additional NRPS proteins consisting of NPS1/NPS3 related domains , and a NPS13 related domain that is absent from all C . heterostrophus genomes except Hm540 ( Figure 5 ) .', 'The C . heterostrophus Hm540 genome includes all four corresponding genes: NPS1 , NPS3 , NPS13 , and the additional NPS1/NPS3/NPS13 gene ( Figure 5 ) .', 'The pattern of duplication and loss appears to have been very rapid to account for this distribution , and is further complicated by the presence of additional bi- , mono- , tri- , and tetra-modular proteins , particularly in C . sativus , whose AMP domains group with NPS1/NPS3/NPS13 proteins , ( Figure 5 , Figure 7 , Figure S5 ) .', 'The origin of strain or species unique secondary metabolism 
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['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 .']
['The filamentous ascomycete genus Cochliobolus includes highly aggressive necrotrophic and hemibiotrophic pathogens with particular specificity to their host plants , often associated with production of host selective toxins ( HST ) that allow necrotrophs to trigger host cell death .', 'Hemibiotrophs must keep their hosts alive during initial infection stages and rely on subverting host defenses by secreting small protein effectors .', 'Many Cochliobolus species have emerged rapidly as devastating pathogens due to HSTs .', 'The genomes of Cochliobolus and related pathogens that differ in host preference , host specificity , and virulence strategies have been sequenced .', 'Our comparative results , at the whole-genome level , and with a spotlight on core genes for secondary metabolism and small secreted proteins , touch on how pathogens develop and hone these tools , according to host or lifestyle .', 'We suggest that , while necrotrophs and hemibiotrophs employ fundamentally contrasting mechanisms of promoting disease , the tools they utilize ( HSTs and protein effectors ) overlap .', 'The suites of secondary metabolite and SSP genes that each possesses reflect astounding diversity among species , hinting that gene products , particularly those associated with unique genomic regions , are candidates for pathogenic lifestyle differences .', 'Manipulations of strain-unique secondary metabolite genes associated with host-specific virulence provide tangible examples .']
['genomics', 'plant biology', 'crops', 'genetics', 'biology', 'computational biology', 'genetics and genomics', 'agriculture']