BACKGROUND
sugarcane belongs to the saccharum genus of the poaceae family , an economically important seed plant family that includes maize, wheat, rice, sorghum, and many other forage crops.
sugarcane is the world’s largest crop.
in  <dig>  the food and agriculture organization estimates that it was cultivated in  <dig> countries, on approximately  <dig>  million hectares, and with a worldwide harvest of  <dig>  billion tons.
the 10-year trend for sugarcane production has been upward.
sugarcane is not only a cash crop, but it can also be used as livestock fodder.
the main product of sugarcane is sucrose, which accounts for 80% of all sugar produced in the world and 92% of that in china.
it is also used as raw material in human food industries  and is fermented to produce sugarcane ethanol.
sugarcane ethanol represents 40% of the world’s total ethanol fuel.
the increasing world demand for sugar and sugarcane ethanol keeps driving the development of sugarcane agriculture.
the average yield of cane stalk is 60- <dig> tons per hectare per year.
however, this figure can vary between  <dig> and  <dig> tons per hectare depending on knowledge and crop management approaches.
during the production period, a sugarcane crop is sensitive to several biotic and abiotic factors, including ‘sugarcane smut’ which is a disease caused by the fungus sporisorium scitamineum.
sugarcane smut has been an increasing large problem in almost all countries where sugarcane is grown.
it was first reported in  <dig> in the natal region of south africa
<cit> .
sugarcane smut can cause substantial losses in cane yield and sugar content in susceptible varieties.
losses can range from 30% to total crop failure, and the disease even leads to variety elimination due to susceptibility to this fungus
<cit> .
the smuts are multicellular fungi-in spite of the unicellular yeast phase, which are characterized by their large numbers of dark, thick-walled and dust-like teliospores.
ustilago, sporisorium and macalpinomyces are the three genera of smut fungi .
these three genera are comprised of approximately  <dig> described species that all infect grasses
<cit> .
compared to the ambiguous position of macalpinomyces, previous studies have demonstrated that ustilago and sporisorium together form a monophyletic group with the ustilaginomycotina
<cit> .
the close relationship between ustilago and sporisorium can also be evidenced by the misclassification of s. scitamineum as u. scitaminea.
generally, ustilago infects all aerial parts of the plant and rapidly forms galls or tumors filled with spores.
in contrast, sporisorium infects young seedlings, remains asymptomatic, and grows systemically until the emergence of mass of sooty spores.
s. scitamineum, previously known as u. scitaminea, is a basidiomycete and the causal agent of the sugarcane smut disease.
it is among the most important phytopathogenic fungi in sugarcane and has significant impact on sugarcane production.
this pathogen results in thin stalks, stunted plant growth, and an outgrowth of fungus of the stalk on the cane known as culmicolous
<cit>  .
the infected plants have increased tillering that results in more slender and much weaker leaves.
the most recognizable characteristic of this disease is a black or gray outgrowth of a whip-like structure that is referred to as a “smut whip”
<cit>  .
these structures emerge from the terminal bud or from the lateral shoots of infected stalks and are composed of both plant and fungal tissues .
within these whip-like structures are millions of teliospores that are responsible for the quick dissemination of the disease.
teliospores germinate and undergo meiosis to form four haploid basidiospores.
however, haploid cells are not infective and only the dikaryotic hyphae formed by fusion of compatible sporidia can infect the host.
it is assumed that developing varieties of sugarcane resistant to sugarcane smut is the only economical method for control of this disease and the best course of action for management.
during the selection process of sugarcane breeding, breeders have to abandon the development of a cross combination once smut disease appears in any of the progeny.figure  <dig>
infection process of
sporisorium scitamineum
on sugarcane.
<dig>  teliospore;  <dig>  soil or sugarcane plants contaminated with s. scitamineum;  <dig>  teliospore germination;  <dig>  the formation of infection hyphae;  <dig>  hypha infection in sugarcane meristem and growth point;  <dig>  the formation of disease panicle.
despite the high worldwide losses in cane yield and sucrose caused by s. scitamineum, a limited amount of information is available about its genome structure and pathogenic mechanisms.
a better understanding of the mechanisms of smut fungus-sugarcane interactions is necessary for the development and deployment of more effective and durable resistant cultivars.
however, sugarcane smut fungi are biotrophic pathogens that cannot be cultured on artificial media.
most of their life stages, including urediniospores that are commonly used for aetiological and evolutionary biology studies, are dikaryotic.
therefore, it is extremely difficult to conduct molecular studies and functional characterizations of genes in sugarcane smut fungus.
besides its biological features, the genome is especially interesting because this is the first fungus important to the sugarcane industry to be completely sequenced.
the insights from the three sequenced genomes of the biotrophic fungal plant pathogens, ustilago maydis
<cit>  and sporisorium reilianum
<cit>  in maize and ustilago hordei
<cit>  in barley, have highlighted the power of comparative genomics of closely related species for identification of virulence determinants and will no doubt facilitate the elucidation of the pathogenic mechanisms of sugarcane smut.
in the present study, we investigate the pathogenic mechanisms of sugarcane smut by sequencing, assembling, and annotating the genome of the smut fungus s. scitamineum.
a comparative genomics study with the three related fungi u. maydis, s. reilianum and u. hordei was also conducted.
this is the first high-quality genome sequence of s. scitamineum and is also the first reported genome of sugarcane fungi.
this study may serve as a model for studying the pathogenic mechanisms in sugarcane, and this study provides knowledge for improving the sugar yield in sugarcane agriculture.
RESULTS
de novogenome sequencing and assembly
the genome of s. scitamineum was sequenced using a whole-genome shotgun approach.
a total of  <dig>  gb of raw sequences were generated from the illumina hiseq <dig> platform  at bgi-shenzhen.
the total assembly size of the genome of s. scitamineum is  <dig>  mb which was assembled into  <dig> contigs and  <dig> scaffolds .
according to the k-mer analysis , the evaluated genome size of s. scitamineum is  <dig>  mb, larger than the assembled one, on account of repeat sequences in the genome.
in additional file
2: figure s1b, the gc content and depth is normal which illustrates that the genome of s. scitamineum has less heterozygosity.
the genome properties of s. scitamineum, including genome size, and gc content, is similar to the species closely related to smut fungi, especially u. maydis.table  <dig>
sporisorium scitamineum
genome statistics compared to that of other sequenced smut fungi
ss
uh
um
sr
n <dig> contig, size in kilobases of ≥50% of the assembled contigs.
ss, sporisorium scitamineum; uh, ustilago hordei; um, ustilago maydis; sr, sporisorium reilianum.
genome annotation
we predicted  <dig>  protein-coding genes with an average transcript size of  <dig>  bp via ab initio and homology-based analyses .
the gene density was  <dig>  kb per gene, which is lower than that in s. reilianum  and similar to the other two smut pathogens u. maydis  and u. hordei .
the absolute number of exons was largest in s. scitamineum, however, the average exon size was the shortest among these smut fungi.
interestingly, the whole genome character of s. scitamineum is most similar to u. maydis which belongs to a separate genus.
only  <dig>  ,  <dig>  ,  <dig>  ,  <dig>  , and  <dig>  of the predicted genes had homologies with known functions in the swissprot, gene ontology , clusters of orthologous groups , kyoto encyclopaedia of genes and genomes , and non-redundant proteins  databases, respectively.
there were  <dig>  genes common to all these classical protein databases .
with regard to non-coding genes, we identified,  <dig> trnas,  <dig> snrnas, and  <dig> rrna fragments from the assembly .
transposable elements play an important role in fungal pathogens
<cit> .
the s. scitamineum genome is comprised of  <dig> % repetitive elements and dna transposons are dominant .
comparative genome analyses
the homology between s. scitamineum and three other smut fungi was examined via gene families.
there were  <dig>  gene families shared among all four smut fungi, and there were  <dig> predicted genes in  <dig> gene families that appear to be unique to s. scitamineum .
among these unique genes, most have unknown functions.
twenty-seven genes did not receive hits in protein databases, and  <dig> genes annotated to nr even belong to hypothetical or uncharacterized protein encoding genes.
these unique genes could act as a potential mine to explore.
main gene families, which have large gene members, included atp-binding cassette  transporter, oligopeptide transporter, p-type atpase , helicase and integrase , and heat shock protein  <dig> family.figure  <dig>
overlap among gene families for four smut fungi species.
this is the venn diagram used to compare the gene families of four smut fungi, four colors  mean four gene family sets, and the overlap parts have the overlapping color.
numbers indicate the gene families in each comparison.
s means the gene families specific to s. scitamineum, s-u means common gene families of s. scitamineum and u. hordei, s-u-r means common gene families of s. scitamineum, u. hordei and s. reilianum, s-u-r-m means common gene families of four smut fungi.
a phylogenetic analysis revealed the evolutionary relationship among smut fungi and positioning of s. scitamineum.
it showed that s. scitamineum and s. reilianum are sister taxa and that u. maydis branches earlier in the evolutionary history .
the genomes of u. maydis
<cit> , s. reilianum
<cit> , and u. hordei
<cit>  are all organized in  <dig> chromosomes.
they could be used as references to map the genome of s. scitamineum.
our results exhibited a remarkably higher degree of synteny between s. scitamineum and s. reilianum than that between s. scitamineum and u. maydis or u. hordei .
meanwhile, the fact that s. scitamineum and s. reilianum share most common gene families could support the relationship of these two species.
we conclude that the chromosome number of s. scitamineum is possibly twenty-three based on the high synteny between s. scitamineum and s. reilianum and the chromosome numbers of the three smut fungi.figure  <dig>
taxonomic placement of
sporisorium scitamineum
and genome synteny with
s. reilianum
,
ustilago maydis,
and
u. hordei
.
evolutionary placing of s. scitamineum in relation to other smut fungi and a. nidulans.
numbers on the branch indicate branch length.
numbers for the internal node indicate bootstrap value;  dot plot of the synteny occurring between the assembled chromosomes of s. reilianum and the assembled scaffolds of s. scitamineum;  dot plot of the synteny occurring between the assembled chromosomes of u. maydis and the assembled scaffolds of s. scitamineum;  dot plot of the synteny occurring between the assembled chromosomes of u. hordei and the assembled scaffolds of s. scitamineum.
characterization of mating type loci in s. scitamineum
for mating to occur, two haploid cells of different mating-type need to recognize each other and fuse to form the infectious dikaryon.
mating is regulated by two loci, a and b, which harbor conserved genes.
at the a locus, these genes encode pheromones and pheromone receptors while at the b locus two subunits of a heterodimeric transcription factor are encoded
<cit> .
the bipolar species s. scitamineum and u. hordei as well as the tetrapolar species u. maydis and s. reilianum possess one divergently transcribed gene pair that encode the homeodomain proteins be  and bw .
the mat- <dig> locus, gene order, orientation, as well as the genomic context are conserved in the b mating-type genes except for the u. hordei mat- <dig> locus .
interestingly, both be and bw mating-type genes are present in the genomes of ustilaginaceae including the two genera of ustilago and sporisorium.figure  <dig>
genetic organization of the mating-type loci of smut fungi.
genes are indicated by arrows with the arrow denoting the direction of transcription.
related genes are denoted by the same color and respective gene functions are explained in the lower part of the figure.
*indicates that the relative order and orientation of these genes have not been determined.
in the tetrapolar species, u. maydis and s. reilianum, the a and b specific sequences reside on different chromosomes, while they are linked by spacer regions  in the bipolar species u. hordei and s. scitamineum.
the black bars on top of the figure indicate the regions of the b locus, which covers the two homeodomain protein genes be and bw, and the a locus  from the lba gene to the rba gene.
sequence information was obtained from the following accession numbers: af <dig>  am <dig>  af <dig>  af <dig>  z <dig>  aj <dig>  aj <dig>  aj <dig>  aj <dig>  aj <dig>  aj <dig>  u <dig>  m <dig>  aacp <dig> and aacp <dig>
in addition to the b mating-type complexes, smut fungi contain genes necessary for cell–cell recognition which are located in the a mating type loci.
the detailed structure of these loci has been determined for both the mat- <dig> alleles of s. scitamineum and u. hordei, an allele of u. maydis, and for all three alleles of s. reilianum .
both u. maydis and u. hordei have two alleles of an a mating system with one pheromone receptor  and one functional pheromone gene  per locus.
in s. scitamineum and u. hordei which have a bipolar mating system, the a and b loci are linked and the mating-type locus  segregates as one locus.
however, in tetrapolar species such as s. reilianum and u. maydis, these genetic loci segregate independently
<cit> .
in s. scitamineum, the a locus encodes a lipopeptide with pheromone and pheromone membrane receptor functions responsible for cell recognition and compatible hyphal fusion, whereas the b locus encodes transcription factors that control the expression of genes responsible for the maintenance of the dikaryotic hyphal growth in plants .
during their life cycle, s. scitamineum presents two distinct monokaryotic and dikaryotic stages.
the monokaryotic stage is marked by haploid cells that grow saprophytically and are not able to cause disease, while in the second phase, dikaryotic hyphae are formed by mating  and are able to infect the host.
the induction of the pathogenic program in s. scitamineum implies not only strong morphological changes  but also genetic changes .
evolution of bipolar mating in s. scitamineum may have been beneficial for the fungus because it promoted inbreeding and stabilization of the genome.
the same process has been proved to be beneficial for the transposon elements .
a study concluded that inbreeding helped fix tes within a population in u. hordei
<cit> .
in tetrapolar species, such as u. maydis and s. reilianum, outcrossing increases heterozygosity
<cit> .
overall, sequence analysis and comparison of the mating-type regions of tetrapolar and bipolar smut fungi revealed that they are not fundamentally different.
bipolar and tetrapolar smuts as well as related species contain the genes for these a and b mating-type complexes.
in the bipolar species s. scitamineum and u. hordei, these mating-type complexes are encoded on the same chromosome and in a recombination-suppressed region ensuring genetic linkage.
g-protein–coupled receptors
to successfully infect a host plant, plant pathogenic fungi and oomycetes must make appropriate responses to a variety of environmental cues, including the chemical and physical characteristics of the host plant surface.
this is achieved via cell surface receptors that respond to external stimuli and relay that information into the cell.
g protein-coupled receptors , which are characterized by having seven transmembrane helices, represent the largest family of cell surface receptors and are responsible for transducing extracellular signals into intracellular responses that involve complex intracellular-signaling networks
.
the genomes of animals contain large numbers of gpcrs, while the genomes of the model ascomycete yeast species saccharomyces cerevisiae and schizosaccharomyces pombe contain only three and four gpcrs
<cit> , respectively.
however, a significant number of putative gpcrs have been demonstrated in the analysis of the genomes of filamentous phytopathogenic ascomycete fungi, as many as  <dig> have been reported in the rice blast fungus magnaporthe grisea and  <dig> have been demonstrated in the head blight pathogen fusarium graminearum
<cit> .
previous researches also revealed that gpcrs are extremely diverse in sequence and function.
constraints in structure prediction impaired research on these proteins for a long time
, highlighting the necessity of identifying and characterizing far more gpcrs.
here we present an exploration of the genomes of  <dig> fungi, the s. scitamineum plus other  <dig> fungi, and identify members of the g protein-coupled receptor family from the entire deduced proteomes.
it demonstrated that s. scitamineum possesses only  <dig> gpcrs, which are grouped into  <dig> classes .
this total set of analyses also resulted in the identification of  <dig> and  <dig> putative gpcrs in s. reilianum and u. maydis respectively, compared to the  <dig> predicted gpcrs in the u. hordei .
this suggests that the number of gpcrs in s. scitamineum is closer to those of s. reilianum and u. maydis than that of u. hordei.
this is in accordance to the phylogeny of these fungi based on comparative genome analyses showing in figure
<dig>  pth <dig>  a cell-surface integral membrane protein required for pathogenicity
<cit> , has seven transmembrane regions and an amino-terminal extracellular cysteine-rich egf-like domain .
the pth11-like gpcr is a phi protein shown to regulate m. grisea appressorium differentiation in response to the plant surface
<cit> .
we demonstrated that no pth11-like gpcrs were found in the genome of all the four sequenced smut fungi, including s. scitamineum, s. reilianum, u. maydis and u. hordei .table  <dig>
classification of putative gpcrs identified among various plant pathogen fungi
ss
uh
um
sr
mlp
pgt
mo
fg
vd
fo
fv
nh
ss, sporisorium scitamineum; uh, ustilago hordei; um, ustilago maydis; sr, sporisorium reilianum; mlp, melampsora laricis-populina; pgt, puccinia graminis f. sp.
tritici; mo, magnaporthe oryzae; fg, fusarium graminearum; vd, verticillium dahliae; fo, fusarium oxysporum; fv, fusarium verticillioides; nh, nectria haematococca.
s. scitamineum may acquire the ability to respond to a variety of environmental cues using different gpcrs that activate conserved intracellular signaling pathways and give new triggers to response systems.
the identification and characterization of gpcrs will provide insights into how s. scitamineum communicates with its environment and senses the presence of intracellular signaling.
carbohydrate degrading enzymes
phytopathogenic fungi secrete a cocktail of hydrolytic enzymes  for degrading the plant cell wall and penetrating into the host tissue.
the s. scitamineum genome encoded  <dig> putative cazymes including  <dig> glycoside hydrolases ,  <dig> glycosyltransferases ,  <dig> carbohydrate esterases ,  <dig> carbohydrate binding modules , and  <dig> polysaccharide lyases  comprising more than  <dig> distinct families .
there were  <dig> genes that could be annotated by the five databases utilized , among which  <dig> genes are putative plant cell wall hydrolytic enzymes
<cit> .
most of these enzymes belong to gh <dig> family  and are involved in pectin degradation.
interestingly, pectin is not the main part of the sugarcane cell wall
<cit> .
in terms of carbohydrates, the larger amount of hemicelluloses that comprise up to 50% of sugarcane cell wall
<cit>  could be broken down by the gh <dig> family  coding xyloglucan:xyloglucosyltransferase and beta;-xylosidase as well as the gh <dig> family  coding alpha;-l-arabinofuranosidase and alpha-l-arabinofuranosidase.
the minimal set of hemicellulases found in s. scitamineum seems perfectly in line with its biotrophic lifestyle, in which damage to the host is minimized and the release of cell wall fragments, which often trigger plant defense responses, is avoided
<cit> .table  <dig>
comparative analysis of cazyme family among various plant pathogen fungi
biotroph
ss
uh
um
sr
obligate_biotroph
mlp
pgt
hemibiotroph
mo
fg
necrotroph
vd
fo
fv
nh
ss, sporisorium scitamineum; uh, ustilago hordei; um, ustilago maydis; sr, sporisorium reilianum; mlp, melampsora laricis-populina; pgt, puccinia graminis f. sp.
tritici; mo, magnaporthe oryzae; fg, fusarium graminearum; vd, verticillium dahliae; fo, fusarium oxysporum; fv, fusarium verticillioides; nh, nectria haematococca.
cbm, carbohydrate binding module; ce, carbohydrate esterase; gh, glycoside hydrolases; gt, glycosyltransferase; pl, polysaccharide lyase; pcw, plant cell wall degradation related enzyme.
real-time quantitative pcr  analysis was performed to examine the expression of these  <dig> genes during the course of infection ).
the result suggests that there are only ten genes of the gh <dig> family were expressed and half of them expressed in the initial stage of infection .
one gene  encoding alpha;-l-rhamnosidase , expressed in all stages and reached the highest expression at  <dig> hpi.
it is quite a surprise that no expression was detected for the gh <dig>  gh <dig> and pl <dig> families, while the gene expression profiles of the gh <dig> family are distinct at different infection time points.
the regulated expression of ten members of the gh <dig> family suggests that these key enzymes have a higher activity and play an important role during host infection.
however, the expression of a specific gene encoding degradation-associated enzymes should cause damage to the sugarcane plant during infection only at a certain point.figure  <dig>
the expression pattern of candidate genes for encoding pant cell wall degradation enzymes of
sporisorium scitamineum
at six stages in the infection process.
these infection stages includes six time points during the course of infection .
the number on the left side, such as ss  <dig>  means gene id.
the color scale indicates relative rt-qpcr expression.
biotrophic properties and utilization of carbon and nitrogen substrates
in order to grow and propagate, phytopathogens need to take up nutrients, mainly ammonium and sulfate, from their parasitic host.
obligate biotrophic pathogens which include powdery mildews, rust fungi, downy mildews and albugo spp., lack genes encoding for particular classes of plant cell wall hydrolases and for various metabolic processes of products, such as nitrates and proteins involved in sulfate assimilation
<cit> .
these losses are interpreted as convergent adaptations.
we anticipated that growth profiles could illuminate correlations between a pathogen and its respective gene complements.
here, we present the comparison of growth on eight carbon and  <dig> nitrogen substrates.
as shown in figure
<dig>  s. scitamineum can utilize all the eight carbon substrates, which is similar to the assimilation of cladosporium fulvum and dothistroma septosporum on carbon substrates
<cit> .
in general, s. scitamineum grows more slowly on the media with carbon substrates of mannitol, lactose, and galactose than on the media with carbon substrates of glucose, fructose, mannose or maltose.
the pattern was reversed when the minimal control medium czapek was also present.
the pronounced growth of s. scitamineum on the medium with sucrose is particularly striking and suggests that the fungus can utilize sucrose available in apoplastic fluid during its early biotrophic colonization phase.
the result is in accordance with previous reports on c. fulvum
<cit> .
the present study also showed that s. scitamineum can uptake a broad range of nitrogen sources, including sodium nitrate, ammonium sulfate, ammonium acetate, and all the twenty kinds of amino acids .
the same phenomenon of extensive use of nitrogen sources has also been witnessed in aspergillus
<cit>  and this emphasizes the impact of nutritional flexibility on fungal pathogenicity.
the above-mentioned carbohydrate and nitrogen metabolism pathways including glycolysis, fructose and mannose metabolism, sucrose metabolism, amino acid metabolism, as well as nitrate and sulphate assimilation, are present in the genome of s. scitamineum.
however, due to the non-uniform growth habit of s. scitamineum on these media, further information is needed for conclusions about its ability to utilize carbon and nitrogen substrates.
in addition, more experiments must be conducted to support the hypothesis that transporter genes are responsible for the uptake of carbon and nitrogen from plant cells and to confirm the assumption that the broad range of utilization in carbon and nitrogen substrates should result in the wide adaptability of s. scitamineum to various environmental conditions.figure  <dig>
comparative growth profiling of
sporisorium scitamineum
on various carbon substrates.
this figure is used to explain that s. scitamineum can utilize all the eight carbon substrates without significant differences.
comparative growth profiling of
sporisorium scitamineum
on various nitrogen substrates.
this figure is used to explain that s. scitamineum can uptake a broad range of nitrogen sources, including sodium nitrate, ammonium sulfate, ammonium acetate, and all the twenty kinds of amino acids, without significant differences.
the smut fungus u. maydis has a reduced set of genes encoding plant cell wall hydrolytic enzymes, presumably as an adaptation to its biotrophic lifestyle
<cit> .
a similar schematic representation of cazyme is found in the s. scitamineum with the approximate types and sum  as typical biotrophic fungi.
biotrophic fungi tend to have fewer plant cell wall degrading enzymes  than necrotrophic and hemibiotrophic fungi, and the necrotrophic fungi have the most abundant numbers of pcws.
virulence associated genes
as a biotrophic pathogenic fungus, s. scitamineum is expected to possess a number of pathogen-host interaction genes.
the genome was searched using the pathogen-host interaction database
<cit>  and  <dig>  putative phi genes were identified.
after filtering the genes from bacteria plant pathogens and animal or human pathogens, there were  <dig> genes left that could be commonly annotated by five databases  as the candidate virulent factor.
these  <dig> genes play diverse roles in redox of short-chain compounds, cell wall breakdown energy metabolism and transport .
rt-qpcr analysis was performed to examine the expression profiles of these  <dig> genes during the course of infection  and 52% of them expressed in the process of infection .
the result suggests that  <dig> genes were expressed in all the stages and mainly encoded energy metabolism and redox of short-chain compound related enzymes .
the gene expression of smutadna4_glean_ <dig> which encodes peptidyl-prolyl cis-trans isomerase b  continued to increase at all stages and reached its maximum at  <dig> hpi .
cyclophilin b possesses cis-trans peptidyl-prolyl isomerase activity and probably is an important determinant of the virulence of pathogenesis
<cit> .
the function of these virulence associated genes could be elucidated by gene disruption or complementation.figure  <dig>
the expression pattern of candidate virulence associated genes of
sporisorium scitamineum
at six stages in the infection process.
these infection stages includes six time points during the course of infection .
the number on the left side, such as ss  <dig>  means gene id.
the color scale indicates relative rt-qpcr expression.
cseps and their expression
recent researches have revealed the extensive repertoires of pathogen effectors and their various functions.
the pathogenicity of s. scitamineum has been studied.
however, no effector has been reported prior to our analysis.
effectors have been screened via de novo analysis from the gene set according to the description of olof emanuelsson et al.
in the nature protocol
<cit> .
there are  <dig> proteins  that comprise the candidates for secreted effector proteins  of s. scitamineum which are clustered into  <dig> families  in table
<dig>  most of cseps in these  <dig> families have more than  <dig> % cysteine content and longer amino acids.
interestingly, there were just two cseps un-annotated to the nr database, however,  <dig> of  <dig> annotated proteins are conserved hypothetical proteins or uncharacterized proteins.
many of the functions of cseps are unknown in biotrophic phytopathogenic fungi, such as u. maydis
<cit>  and blumeria graminis
<cit> .
rt-qpcr analysis revealed that 47% of cseps expressed in the infection process.
among them,  <dig> expressed at all stages while  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> cseps started to express at  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> hpi, respectively .
four of the above-mentioned seven cseps belong to conserved hypothetical proteins with unknown function and did not receive hits from any protein database.
the remaining three cseps have a clan gh-d domain , a catalytic domain , and a barwin-related endoglucanase domain  as well as sections encoding glycoside hydrolase and lytic transglycosylase-like protein.
these cseps are mainly involved in carbohydrate degradation.
it is unclear how these cseps facilitate infection and/or trigger defense responses, how they are secreted and whether there is specific invasion structure to help the secretion.
these conundrums require additional endeavors and deeper research.figure  <dig>
the expression pattern of genes coding for secreted effector proteins of
sporisorium scitamineum
at six stages in the infection process.
these infection stages includes six time points during the course of infection .
the number on the left side, such as ss  <dig>  means gene id.
the color scale indicates relative rt-qpcr expression.
summary of the  <dig> csep families in the
sporisorium scitamineum
genome
protein essential during penetration  <dig> , representing a novel effector for establishing a biotrophic interaction, has been identified and characterized in u. maydis
<cit> .
pep <dig> is also found to be highly conserved in related pathogens such as s. reilianum and u. hordei
<cit> .
however, all the above  <dig> cseps of s. scitamineum have no ortholog to pep <dig>  accordingly, we expanded the search to all proteins and found that one protein encoded by smutadna4_glean_ <dig>  has  <dig> amino acids with 65% identity to the u. maydis pep <dig>  it was demonstrated that all pep <dig> proteins from the  <dig> smut fungi have an n-terminal secretion signal and four cysteine residues whose spacing is conserved as well as glycine-rich c-terminal regions .
in u. maydis, a glycine-rich domain of  <dig> aa at the c-terminus was deleted without affecting biological activity, however, cysteine residues are necessary for secretion of pep1
<cit> .
the molecular function of these two regions in s. scitamineum may be similar to that in u. maydis.
interestingly, phylogenetic tree based on pep <dig>  is in accordance with that based on the whole genome , implying that pep <dig> can probably be used as a molecular marker for phylogenetic analysis or even for race identification in smut fungi.
secondary metabolic pathways
plant pathogenic fungi produce diverse secondary metabolites that aid in pathogenicity, such as host selective toxins.
we identified  <dig> putative secondary metabolite genes and  <dig> gene clusters in the s. scitamineum genome.
there are  <dig>   <dig> and  <dig> genes predicted to encode polyketide synthases , non-ribosomal peptide synthetases , and terpenes, respectively.
two nrps gene clusters may participate in iron-chelating siderophores ferrichrome and ferrichrome a biosynthesis.
one terpene gene cluster may involve the conversion of gibberellins.
iron is an important element for many essential processes in living organisms.
to acquire iron, s. scitamineum uses two iron-chelating siderophores to uptake iron from host sugarcane cells.
in figure
10a, one nrps gene cluster including  <dig> genes, is involved in ferrichrome generation.
the core gene smutadna4_glean_ <dig> encodes ferrichrome siderophore peptide synthetase, and encodes  <dig>  amino acids with 76% identity to the nrps sid <dig> of u. maydis 521
<cit> .
the gene has three amp-dependent synthetase/ligase domains, four acyl carrier domains  to activate the cognate amino acid, four condensation domains to catalyze peptide bond formation between the amino acyl and peptidyl substrates of adjacent domains and one amp-binding enzyme c-terminal domain
<cit> .
the other nrps gene cluster consists of  <dig> genes, responsible for ferrichrome a biosynthesis .
the skeleton gene smutadna4_glean_ <dig> is located in the fifth gene cluster and encodes siderophore peptide synthetase.
it encodes  <dig>  amino acids with 75% identity to the nrps fer <dig>  of s. reilianum srz <dig>  which is composed of three amp-dependent synthetase/ligase domains, five acyl carrier domains, four condensation domains and one amp-binding enzyme c-terminal domain.figure  <dig>
three gene clusters in the genome of
sporisorium scitamineum
.
physical map of ferrichrome gene cluster .
coding regions are indicated with arrows and are putatively involved in ferrichrome biosynthesis , siderophore transport , or unknown functions .
location of interpro domains in nrps smutadna4_glean_ <dig> is as bottom.
ad, amp-dependent synthetase/ligase domain , ipr00873; ac, acyl carrier domain , ipr009081; cd, condensation domain , ipr001242; ab, amp-binding enzyme c-terminal domain , ipr <dig>   physical map of ferrichrome a gene cluster .
coding regions are indicated with arrows and are putatively involved in ferrichrome a biosynthesis , siderophore transport , or unknown functions .
location of interpro domains in nrps smutadna4_glean_ <dig> is as bottom.
ad, amp-dependent synthetase/ligase domain , ipr00873; ac, acyl carrier domain , ipr009081;cd, condensation domain , ipr001242; ab, amp-binding enzyme c-terminal domain , ipr <dig>   physical map of terpene gene cluster .
coding regions are indicated with arrows and are putatively involved in terpene biosynthesis  or unknown functions .
two nrpss involved in siderophore biosynthesis have also been identified in the ascomycete cochliobolus heterostrophus and the ferricrocin biosynthesis enzyme is conserved in other ascomycetes
<cit> .
a. fumigatus is one of the most common aspergillus species to cause disease in individuals with an immunodeficiency a. fumigatus produces four siderophores, fusarinine c, triacetylfusarinine c, ferricrocin and hydroxyferricrocin
<cit> .
a. fumigatus has been recognized as a model species to study fungal siderophore biosynthetic pathways.
these two nrps gene clusters require further study to describe the function of gene members in this cluster by metabolite detection methods such as high performance liquid chromatography , gas chromatography  and mass spectrometry.
molecular biology experiments also need to be conducted such as knock-out, knock-in, and heterogeneous expression.
in figure
10c, the terpenes gene cluster is composed of three unknown function genes and seven biosynthesis related genes.
the gene smutadna4_glean_ <dig>  which encodes gibberellin 20-oxidase, is known to encode  <dig> amino acids that have 90% identity with sr <dig>  of s. reilianum srz <dig>  gibberellin 20-oxidase is a key oxidase in the biosynthesis of gibberellin .
gibberellin 20-oxidase catalyzes the conversions of ga <dig> and ga <dig> to ga <dig> and ga <dig> respectively, via a three-step oxidation at c- <dig> of the ga skeleton.
ga <dig> probably plays an important role in s. reilianum resistance in infected sorghum bicolor, however, ga <dig> has no effect
<cit> .
this implies that the gene cluster is involved in weakening the resistance of sugarcane to smut disease and contributes to the successful infection of s. scitamineum.
with gc-ms detection, s. scitamineum can biosynthesize  <dig> compounds and secrete  <dig> extracellularly .
the intracellular metabolites are mainly esters and the distribution of molecular weight  is from  <dig> to  <dig> amu.
however, the extracellular metabolites are alcohol , aldoketones , alkanes , and carboxylic acids  and no esters.
the extracellular metabolites exhibited a smaller mw distribution .
neither intracellular nor extracellular metabolites had strong infection to sugarcane in the inoculation experiments by supernatant  and precipitation of fungal elicitor.
the questions of whether this was due to the application of mixed metabolites, and if only the single or some secreted metabolites function in the infection process, require further investigation through additional inoculation validation.
determining the function of each metabolite in this process could elucidate the corresponding mechanism causing sugarcane smut.
cytochrome p450s play an important role in various hydroxylation and oxidation processes including those associated with secondary metabolites as well as the breakdown of toxins and other xenobiotic compounds
<cit> .
plant pathogenic fungi use a wide range of strategies to gain access to the carbon sources and nitrogen sources of their host plants and to counter the plant defense response.
transporters are involved in toxin and effector secretion as well as nutrition uptake.
there are  <dig> p450s and  <dig> transporter genes  in the genome of s. scitamineum.
a large proportion of transporters belong to the major facilitator superfamily  , but the abc superfamily , p-type atpase  superfamily , and sugar/inositol transporter  are all well represented.
these four smut fungi have similar composition of p450s and transporters.
compared to the typical pathogens m. grisea or fusarium graminearum
<cit> , the mfs number of s. scitamineum is small.
it is unsurprising that few metabolites could be secreted extracellularly.table  <dig>
comparative analysis of p450s and transporter genes among various plant pathogen fungi
ss
uh
um
sr
mlp
pgt
mo
fg
vd
fo
fv
nh
ss, sporisorium scitamineum; uh, ustilago hordei; um, ustilago maydis; sr, sporisorium reilianum; mlp, melampsora laricis-populina;
pgt, puccinia graminis f. sp.
tritici; mo, magnaporthe oryzae; fg, fusarium graminearum; vd, verticillium dahliae; fo, fusarium oxysporum;
fv, fusarium verticillioides; nh, nectria haematococca.
CONCLUSIONS
although sugarcane smut was first reported in  <dig>  little is known about its pathogenic mechanisms.
the s. scitamineum genome sequence and our comparative analysis with the genome sequences of u. maydis, s. reilianum and u. hordei have shed new light on the pathogenic mechanisms of this fungus and have provided insights into aspects of genome evolution, biotrophy features, carbohydrate degrading enzymes and secondary metabolic pathways likely to be common to all smut fungi.
these results represent the initial step in realizing the full potential of these genomes.
as a result of the genome analysis, efforts are underway to validate the function of all these potential pathogenicity determinants, especially the virulence associated genes.
however, the genome sequence of s. scitamineum provides only a first glimpse into the genomic basis of pathogenic mechanisms of sugarcane smut.
these efforts and ongoing sequencing projects for additional sugarcane fungi will provide extraordinary opportunities for comparative analyses that will expand our understanding of the interaction between sugarcane and fungi in this agriculturally and industrially important crop.
further sequencing of s. scitamineum isolates from different geographical locations or different host varieties will lead to comprehensive population genetic analysis.

 sugarcane belongs to the saccharum genus of the poaceae family, an economically important seed plant family that includes maize, wheat, rice, sorghum, and many other forage crops. sugarcane is the world’s largest crop. in the food and agriculture organization estimates that it was cultivated in countries, on approximately million hectares, and with a worldwide harvest of billion tons. the 10-year trend for sugarcane production has been upward. sugarcane is not only a cash crop, but it can also be used as livestock fodder. the main product of sugarcane is sucrose, which accounts for 80% of all sugar produced in the world and 92% of that in china. it is also used as raw material in human food industries and is fermented to produce sugarcane ethanol. sugarcane ethanol represents 40% of the world’s total ethanol fuel. the increasing world demand for sugar and sugarcane ethanol keeps driving the development of sugarcane agriculture. the average yield of cane stalk is 60- tons per hectare per year. however, this figure can vary between and tons per hectare depending on knowledge and crop management approaches. during the production period, a sugarcane crop is sensitive to several biotic and abiotic factors, including ‘sugarcane smut’ which is a disease caused by the fungus sporisorium scitamineum. sugarcane smut has been an increasing large problem in almost all countries where sugarcane is grown. it was first reported in in the natal region of south africa. sugarcane smut can cause substantial losses in cane yield and sugar content in susceptible varieties. losses can range from 30% to total crop failure, and the disease even leads to variety elimination due to susceptibility to this fungus. the smuts are multicellular fungi-in spite of the unicellular yeast phase, which are characterized by their large numbers of dark, thick-walled and dust-like teliospores. ustilago, sporisorium and macalpinomyces are the three genera of smut fungi. these three genera are comprised of approximately described species that all infect grasses. compared to the ambiguous position of macalpinomyces, previous studies have demonstrated that ustilago and sporisorium together form a monophyletic group with the ustilaginomycotina. the close relationship between ustilago and sporisorium can also be evidenced by the misclassification of s. scitamineum as u. scitaminea. generally, ustilago infects all aerial parts of the plant and rapidly forms galls or tumors filled with spores. in contrast, sporisorium infects young seedlings, remains asymptomatic, and grows systemically until the emergence of mass of sooty spores. s. scitamineum, previously known as u. scitaminea, is a basidiomycete and the causal agent of the sugarcane smut disease. it is among the most important phytopathogenic fungi in sugarcane and has significant impact on sugarcane production. this pathogen results in thin stalks, stunted plant growth, and an outgrowth of fungus of the stalk on the cane known as culmicolous. the infected plants have increased tillering that results in more slender and much weaker leaves. the most recognizable characteristic of this disease is a black or gray outgrowth of a whip-like structure that is referred to as a “smut whip”. these structures emerge from the terminal bud or from the lateral shoots of infected stalks and are composed of both plant and fungal tissues. within these whip-like structures are millions of teliospores that are responsible for the quick dissemination of the disease. teliospores germinate and undergo meiosis to form four haploid basidiospores. however, haploid cells are not infective and only the dikaryotic hyphae formed by fusion of compatible sporidia can infect the host. it is assumed that developing varieties of sugarcane resistant to sugarcane smut is the only economical method for control of this disease and the best course of action for management. during the selection process of sugarcane breeding, breeders have to abandon the development of a cross combination once smut disease appears in any of the progeny.figure infection process of sporisorium scitamineum on sugarcane. teliospore; soil or sugarcane plants contaminated with s. scitamineum; teliospore germination; the formation of infection hyphae; hypha infection in sugarcane meristem and growth point; the formation of disease panicle. despite the high worldwide losses in cane yield and sucrose caused by s. scitamineum, a limited amount of information is available about its genome structure and pathogenic mechanisms. a better understanding of the mechanisms of smut fungus-sugarcane interactions is necessary for the development and deployment of more effective and durable resistant cultivars. however, sugarcane smut fungi are biotrophic pathogens that cannot be cultured on artificial media. most of their life stages, including urediniospores that are commonly used for aetiological and evolutionary biology studies, are dikaryotic. therefore, it is extremely difficult to conduct molecular studies and functional characterizations of genes in sugarcane smut fungus. besides its biological features, the genome is especially interesting because this is the first fungus important to the sugarcane industry to be completely sequenced. the insights from the three sequenced genomes of the biotrophic fungal plant pathogens, ustilago maydis and sporisorium reilianum in maize and ustilago hordei in barley, have highlighted the power of comparative genomics of closely related species for identification of virulence determinants and will no doubt facilitate the elucidation of the pathogenic mechanisms of sugarcane smut. in the present study, we investigate the pathogenic mechanisms of sugarcane smut by sequencing, assembling, and annotating the genome of the smut fungus s. scitamineum. a comparative genomics study with the three related fungi u. maydis, s. reilianum and u. hordei was also conducted. this is the first high-quality genome sequence of s. scitamineum and is also the first reported genome of sugarcane fungi. this study may serve as a model for studying the pathogenic mechanisms in sugarcane, and this study provides knowledge for improving the sugar yield in sugarcane agriculture.  de novogenome sequencing and assembly the genome of s. scitamineum was sequenced using a whole-genome shotgun approach. a total of gb of raw sequences were generated from the illumina hiseq platform at bgi-shenzhen. the total assembly size of the genome of s. scitamineum is mb which was assembled into contigs and scaffolds. according to the k-mer analysis, the evaluated genome size of s. scitamineum is mb, larger than the assembled one, on account of repeat sequences in the genome. in additional file 2: figure s1b, the gc content and depth is normal which illustrates that the genome of s. scitamineum has less heterozygosity. the genome properties of s. scitamineum, including genome size, and gc content, is similar to the species closely related to smut fungi, especially u. maydis.table sporisorium scitamineum genome statistics compared to that of other sequenced smut fungi ss uh um sr n contig, size in kilobases of ≥50% of the assembled contigs. ss, sporisorium scitamineum; uh, ustilago hordei; um, ustilago maydis; sr, sporisorium reilianum. genome annotation we predicted protein-coding genes with an average transcript size of bp via ab initio and homology-based analyses. the gene density was kb per gene, which is lower than that in s. reilianum and similar to the other two smut pathogens u. maydis and u. hordei. the absolute number of exons was largest in s. scitamineum, however, the average exon size was the shortest among these smut fungi. interestingly, the whole genome character of s. scitamineum is most similar to u. maydis which belongs to a separate genus. only,,,, and of the predicted genes had homologies with known functions in the swissprot, gene ontology, clusters of orthologous groups, kyoto encyclopaedia of genes and genomes, and non-redundant proteins databases, respectively. there were genes common to all these classical protein databases. with regard to non-coding genes, we identified, trnas, snrnas, and rrna fragments from the assembly. transposable elements play an important role in fungal pathogens. the s. scitamineum genome is comprised of % repetitive elements and dna transposons are dominant. comparative genome analyses the homology between s. scitamineum and three other smut fungi was examined via gene families. there were gene families shared among all four smut fungi, and there were predicted genes in gene families that appear to be unique to s. scitamineum. among these unique genes, most have unknown functions. twenty-seven genes did not receive hits in protein databases, and genes annotated to nr even belong to hypothetical or uncharacterized protein encoding genes. these unique genes could act as a potential mine to explore. main gene families, which have large gene members, included atp-binding cassette transporter, oligopeptide transporter, p-type atpase, helicase and integrase, and heat shock protein family.figure overlap among gene families for four smut fungi species. this is the venn diagram used to compare the gene families of four smut fungi, four colors mean four gene family sets, and the overlap parts have the overlapping color. numbers indicate the gene families in each comparison. s means the gene families specific to s. scitamineum, s-u means common gene families of s. scitamineum and u. hordei, s-u-r means common gene families of s. scitamineum, u. hordei and s. reilianum, s-u-r-m means common gene families of four smut fungi. a phylogenetic analysis revealed the evolutionary relationship among smut fungi and positioning of s. scitamineum. it showed that s. scitamineum and s. reilianum are sister taxa and that u. maydis branches earlier in the evolutionary history. the genomes of u. maydis, s. reilianum, and u. hordei are all organized in chromosomes. they could be used as references to map the genome of s. scitamineum. our results exhibited a remarkably higher degree of synteny between s. scitamineum and s. reilianum than that between s. scitamineum and u. maydis or u. hordei. meanwhile, the fact that s. scitamineum and s. reilianum share most common gene families could support the relationship of these two species. we conclude that the chromosome number of s. scitamineum is possibly twenty-three based on the high synteny between s. scitamineum and s. reilianum and the chromosome numbers of the three smut fungi.figure taxonomic placement of sporisorium scitamineum and genome synteny with s. reilianum, ustilago maydis, and u. hordei. evolutionary placing of s. scitamineum in relation to other smut fungi and a. nidulans. numbers on the branch indicate branch length. numbers for the internal node indicate bootstrap value; dot plot of the synteny occurring between the assembled chromosomes of s. reilianum and the assembled scaffolds of s. scitamineum; dot plot of the synteny occurring between the assembled chromosomes of u. maydis and the assembled scaffolds of s. scitamineum; dot plot of the synteny occurring between the assembled chromosomes of u. hordei and the assembled scaffolds of s. scitamineum. characterization of mating type loci in s. scitamineum for mating to occur, two haploid cells of different mating-type need to recognize each other and fuse to form the infectious dikaryon. mating is regulated by two loci, a and b, which harbor conserved genes. at the a locus, these genes encode pheromones and pheromone receptors while at the b locus two subunits of a heterodimeric transcription factor are encoded. the bipolar species s. scitamineum and u. hordei as well as the tetrapolar species u. maydis and s. reilianum possess one divergently transcribed gene pair that encode the homeodomain proteins be and bw. the mat- locus, gene order, orientation, as well as the genomic context are conserved in the b mating-type genes except for the u. hordei mat- locus. interestingly, both be and bw mating-type genes are present in the genomes of ustilaginaceae including the two genera of ustilago and sporisorium.figure genetic organization of the mating-type loci of smut fungi. genes are indicated by arrows with the arrow denoting the direction of transcription. related genes are denoted by the same color and respective gene functions are explained in the lower part of the figure. *indicates that the relative order and orientation of these genes have not been determined. in the tetrapolar species, u. maydis and s. reilianum, the a and b specific sequences reside on different chromosomes, while they are linked by spacer regions in the bipolar species u. hordei and s. scitamineum. the black bars on top of the figure indicate the regions of the b locus, which covers the two homeodomain protein genes be and bw, and the a locus from the lba gene to the rba gene. sequence information was obtained from the following accession numbers: af am af af z aj aj aj aj aj aj u m aacp and aacp in addition to the b mating-type complexes, smut fungi contain genes necessary for cell–cell recognition which are located in the a mating type loci. the detailed structure of these loci has been determined for both the mat- alleles of s. scitamineum and u. hordei, an allele of u. maydis, and for all three alleles of s. reilianum. both u. maydis and u. hordei have two alleles of an a mating system with one pheromone receptor and one functional pheromone gene per locus. in s. scitamineum and u. hordei which have a bipolar mating system, the a and b loci are linked and the mating-type locus segregates as one locus. however, in tetrapolar species such as s. reilianum and u. maydis, these genetic loci segregate independently. in s. scitamineum, the a locus encodes a lipopeptide with pheromone and pheromone membrane receptor functions responsible for cell recognition and compatible hyphal fusion, whereas the b locus encodes transcription factors that control the expression of genes responsible for the maintenance of the dikaryotic hyphal growth in plants. during their life cycle, s. scitamineum presents two distinct monokaryotic and dikaryotic stages. the monokaryotic stage is marked by haploid cells that grow saprophytically and are not able to cause disease, while in the second phase, dikaryotic hyphae are formed by mating and are able to infect the host. the induction of the pathogenic program in s. scitamineum implies not only strong morphological changes but also genetic changes. evolution of bipolar mating in s. scitamineum may have been beneficial for the fungus because it promoted inbreeding and stabilization of the genome. the same process has been proved to be beneficial for the transposon elements. a study concluded that inbreeding helped fix tes within a population in u. hordei. in tetrapolar species, such as u. maydis and s. reilianum, outcrossing increases heterozygosity. overall, sequence analysis and comparison of the mating-type regions of tetrapolar and bipolar smut fungi revealed that they are not fundamentally different. bipolar and tetrapolar smuts as well as related species contain the genes for these a and b mating-type complexes. in the bipolar species s. scitamineum and u. hordei, these mating-type complexes are encoded on the same chromosome and in a recombination-suppressed region ensuring genetic linkage. g-protein–coupled receptors to successfully infect a host plant, plant pathogenic fungi and oomycetes must make appropriate responses to a variety of environmental cues, including the chemical and physical characteristics of the host plant surface. this is achieved via cell surface receptors that respond to external stimuli and relay that information into the cell. g protein-coupled receptors, which are characterized by having seven transmembrane helices, represent the largest family of cell surface receptors and are responsible for transducing extracellular signals into intracellular responses that involve complex intracellular-signaling networks. the genomes of animals contain large numbers of gpcrs, while the genomes of the model ascomycete yeast species saccharomyces cerevisiae and schizosaccharomyces pombe contain only three and four gpcrs, respectively. however, a significant number of putative gpcrs have been demonstrated in the analysis of the genomes of filamentous phytopathogenic ascomycete fungi, as many as have been reported in the rice blast fungus magnaporthe grisea and have been demonstrated in the head blight pathogen fusarium graminearum. previous researches also revealed that gpcrs are extremely diverse in sequence and function. constraints in structure prediction impaired research on these proteins for a long time, highlighting the necessity of identifying and characterizing far more gpcrs. here we present an exploration of the genomes of fungi, the s. scitamineum plus other fungi, and identify members of the g protein-coupled receptor family from the entire deduced proteomes. it demonstrated that s. scitamineum possesses only gpcrs, which are grouped into classes. this total set of analyses also resulted in the identification of and putative gpcrs in s. reilianum and u. maydis respectively, compared to the predicted gpcrs in the u. hordei. this suggests that the number of gpcrs in s. scitamineum is closer to those of s. reilianum and u. maydis than that of u. hordei. this is in accordance to the phylogeny of these fungi based on comparative genome analyses showing in figure  pth a cell-surface integral membrane protein required for pathogenicity, has seven transmembrane regions and an amino-terminal extracellular cysteine-rich egf-like domain. the pth11-like gpcr is a phi protein shown to regulate m. grisea appressorium differentiation in response to the plant surface. we demonstrated that no pth11-like gpcrs were found in the genome of all the four sequenced smut fungi, including s. scitamineum, s. reilianum, u. maydis and u. hordei.table classification of putative gpcrs identified among various plant pathogen fungi ss uh um sr mlp pgt mo fg vd fo fv nh ss, sporisorium scitamineum; uh, ustilago hordei; um, ustilago maydis; sr, sporisorium reilianum; mlp, melampsora laricis-populina; pgt, puccinia graminis f. sp. tritici; mo, magnaporthe oryzae; fg, fusarium graminearum; vd, verticillium dahliae; fo, fusarium oxysporum; fv, fusarium verticillioides; nh, nectria haematococca. s. scitamineum may acquire the ability to respond to a variety of environmental cues using different gpcrs that activate conserved intracellular signaling pathways and give new triggers to response systems. the identification and characterization of gpcrs will provide insights into how s. scitamineum communicates with its environment and senses the presence of intracellular signaling. carbohydrate degrading enzymes phytopathogenic fungi secrete a cocktail of hydrolytic enzymes for degrading the plant cell wall and penetrating into the host tissue. the s. scitamineum genome encoded putative cazymes including glycoside hydrolases, glycosyltransferases, carbohydrate esterases, carbohydrate binding modules, and polysaccharide lyases comprising more than distinct families. there were genes that could be annotated by the five databases utilized, among which genes are putative plant cell wall hydrolytic enzymes. most of these enzymes belong to gh family and are involved in pectin degradation. interestingly, pectin is not the main part of the sugarcane cell wall. in terms of carbohydrates, the larger amount of hemicelluloses that comprise up to 50% of sugarcane cell wall could be broken down by the gh family coding xyloglucan:xyloglucosyltransferase and beta;-xylosidase as well as the gh family coding alpha;-l-arabinofuranosidase and alpha-l-arabinofuranosidase. the minimal set of hemicellulases found in s. scitamineum seems perfectly in line with its biotrophic lifestyle, in which damage to the host is minimized and the release of cell wall fragments, which often trigger plant defense responses, is avoided.table comparative analysis of cazyme family among various plant pathogen fungi biotroph ss uh um sr obligate_biotroph mlp pgt hemibiotroph mo fg necrotroph vd fo fv nh ss, sporisorium scitamineum; uh, ustilago hordei; um, ustilago maydis; sr, sporisorium reilianum; mlp, melampsora laricis-populina; pgt, puccinia graminis f. sp. tritici; mo, magnaporthe oryzae; fg, fusarium graminearum; vd, verticillium dahliae; fo, fusarium oxysporum; fv, fusarium verticillioides; nh, nectria haematococca. cbm, carbohydrate binding module; ce, carbohydrate esterase; gh, glycoside hydrolases; gt, glycosyltransferase; pl, polysaccharide lyase; pcw, plant cell wall degradation related enzyme. real-time quantitative pcr analysis was performed to examine the expression of these genes during the course of infection ). the result suggests that there are only ten genes of the gh family were expressed and half of them expressed in the initial stage of infection. one gene encoding alpha;-l-rhamnosidase, expressed in all stages and reached the highest expression at hpi. it is quite a surprise that no expression was detected for the gh gh and pl families, while the gene expression profiles of the gh family are distinct at different infection time points. the regulated expression of ten members of the gh family suggests that these key enzymes have a higher activity and play an important role during host infection. however, the expression of a specific gene encoding degradation-associated enzymes should cause damage to the sugarcane plant during infection only at a certain point.figure the expression pattern of candidate genes for encoding pant cell wall degradation enzymes of sporisorium scitamineum at six stages in the infection process. these infection stages includes six time points during the course of infection. the number on the left side, such as ss means gene id. the color scale indicates relative rt-qpcr expression. biotrophic properties and utilization of carbon and nitrogen substrates in order to grow and propagate, phytopathogens need to take up nutrients, mainly ammonium and sulfate, from their parasitic host. obligate biotrophic pathogens which include powdery mildews, rust fungi, downy mildews and albugo spp., lack genes encoding for particular classes of plant cell wall hydrolases and for various metabolic processes of products, such as nitrates and proteins involved in sulfate assimilation. these losses are interpreted as convergent adaptations. we anticipated that growth profiles could illuminate correlations between a pathogen and its respective gene complements. here, we present the comparison of growth on eight carbon and nitrogen substrates. as shown in figure  s. scitamineum can utilize all the eight carbon substrates, which is similar to the assimilation of cladosporium fulvum and dothistroma septosporum on carbon substrates. in general, s. scitamineum grows more slowly on the media with carbon substrates of mannitol, lactose, and galactose than on the media with carbon substrates of glucose, fructose, mannose or maltose. the pattern was reversed when the minimal control medium czapek was also present. the pronounced growth of s. scitamineum on the medium with sucrose is particularly striking and suggests that the fungus can utilize sucrose available in apoplastic fluid during its early biotrophic colonization phase. the result is in accordance with previous reports on c. fulvum. the present study also showed that s. scitamineum can uptake a broad range of nitrogen sources, including sodium nitrate, ammonium sulfate, ammonium acetate, and all the twenty kinds of amino acids. the same phenomenon of extensive use of nitrogen sources has also been witnessed in aspergillus and this emphasizes the impact of nutritional flexibility on fungal pathogenicity. the above-mentioned carbohydrate and nitrogen metabolism pathways including glycolysis, fructose and mannose metabolism, sucrose metabolism, amino acid metabolism, as well as nitrate and sulphate assimilation, are present in the genome of s. scitamineum. however, due to the non-uniform growth habit of s. scitamineum on these media, further information is needed for conclusions about its ability to utilize carbon and nitrogen substrates. in addition, more experiments must be conducted to support the hypothesis that transporter genes are responsible for the uptake of carbon and nitrogen from plant cells and to confirm the assumption that the broad range of utilization in carbon and nitrogen substrates should result in the wide adaptability of s. scitamineum to various environmental conditions.figure comparative growth profiling of sporisorium scitamineum on various carbon substrates. this figure is used to explain that s. scitamineum can utilize all the eight carbon substrates without significant differences. comparative growth profiling of sporisorium scitamineum on various nitrogen substrates. this figure is used to explain that s. scitamineum can uptake a broad range of nitrogen sources, including sodium nitrate, ammonium sulfate, ammonium acetate, and all the twenty kinds of amino acids, without significant differences. the smut fungus u. maydis has a reduced set of genes encoding plant cell wall hydrolytic enzymes, presumably as an adaptation to its biotrophic lifestyle. a similar schematic representation of cazyme is found in the s. scitamineum with the approximate types and sum as typical biotrophic fungi. biotrophic fungi tend to have fewer plant cell wall degrading enzymes than necrotrophic and hemibiotrophic fungi, and the necrotrophic fungi have the most abundant numbers of pcws. virulence associated genes as a biotrophic pathogenic fungus, s. scitamineum is expected to possess a number of pathogen-host interaction genes. the genome was searched using the pathogen-host interaction database and putative phi genes were identified. after filtering the genes from bacteria plant pathogens and animal or human pathogens, there were genes left that could be commonly annotated by five databases as the candidate virulent factor. these genes play diverse roles in redox of short-chain compounds, cell wall breakdown energy metabolism and transport. rt-qpcr analysis was performed to examine the expression profiles of these genes during the course of infection and 52% of them expressed in the process of infection. the result suggests that genes were expressed in all the stages and mainly encoded energy metabolism and redox of short-chain compound related enzymes. the gene expression of smutadna4_glean_ which encodes peptidyl-prolyl cis-trans isomerase b continued to increase at all stages and reached its maximum at hpi. cyclophilin b possesses cis-trans peptidyl-prolyl isomerase activity and probably is an important determinant of the virulence of pathogenesis. the function of these virulence associated genes could be elucidated by gene disruption or complementation.figure the expression pattern of candidate virulence associated genes of sporisorium scitamineum at six stages in the infection process. these infection stages includes six time points during the course of infection. the number on the left side, such as ss means gene id. the color scale indicates relative rt-qpcr expression. cseps and their expression recent researches have revealed the extensive repertoires of pathogen effectors and their various functions. the pathogenicity of s. scitamineum has been studied. however, no effector has been reported prior to our analysis. effectors have been screened via de novo analysis from the gene set according to the description of olof emanuelsson et al. in the nature protocol. there are proteins that comprise the candidates for secreted effector proteins of s. scitamineum which are clustered into families in table  most of cseps in these families have more than % cysteine content and longer amino acids. interestingly, there were just two cseps un-annotated to the nr database, however, of annotated proteins are conserved hypothetical proteins or uncharacterized proteins. many of the functions of cseps are unknown in biotrophic phytopathogenic fungi, such as u. maydis and blumeria graminis. rt-qpcr analysis revealed that 47% of cseps expressed in the infection process. among them, expressed at all stages while and cseps started to express at and hpi, respectively. four of the above-mentioned seven cseps belong to conserved hypothetical proteins with unknown function and did not receive hits from any protein database. the remaining three cseps have a clan gh-d domain, a catalytic domain, and a barwin-related endoglucanase domain as well as sections encoding glycoside hydrolase and lytic transglycosylase-like protein. these cseps are mainly involved in carbohydrate degradation. it is unclear how these cseps facilitate infection and/or trigger defense responses, how they are secreted and whether there is specific invasion structure to help the secretion. these conundrums require additional endeavors and deeper research.figure the expression pattern of genes coding for secreted effector proteins of sporisorium scitamineum at six stages in the infection process. these infection stages includes six time points during the course of infection. the number on the left side, such as ss means gene id. the color scale indicates relative rt-qpcr expression. summary of the csep families in the sporisorium scitamineum genome protein essential during penetration, representing a novel effector for establishing a biotrophic interaction, has been identified and characterized in u. maydis. pep is also found to be highly conserved in related pathogens such as s. reilianum and u. hordei. however, all the above cseps of s. scitamineum have no ortholog to pep accordingly, we expanded the search to all proteins and found that one protein encoded by smutadna4_glean_ has amino acids with 65% identity to the u. maydis pep it was demonstrated that all pep proteins from the smut fungi have an n-terminal secretion signal and four cysteine residues whose spacing is conserved as well as glycine-rich c-terminal regions. in u. maydis, a glycine-rich domain of aa at the c-terminus was deleted without affecting biological activity, however, cysteine residues are necessary for secretion of pep1. the molecular function of these two regions in s. scitamineum may be similar to that in u. maydis. interestingly, phylogenetic tree based on pep is in accordance with that based on the whole genome, implying that pep can probably be used as a molecular marker for phylogenetic analysis or even for race identification in smut fungi. secondary metabolic pathways plant pathogenic fungi produce diverse secondary metabolites that aid in pathogenicity, such as host selective toxins. we identified putative secondary metabolite genes and gene clusters in the s. scitamineum genome. there are and genes predicted to encode polyketide synthases, non-ribosomal peptide synthetases, and terpenes, respectively. two nrps gene clusters may participate in iron-chelating siderophores ferrichrome and ferrichrome a biosynthesis. one terpene gene cluster may involve the conversion of gibberellins. iron is an important element for many essential processes in living organisms. to acquire iron, s. scitamineum uses two iron-chelating siderophores to uptake iron from host sugarcane cells. in figure  10a, one nrps gene cluster including genes, is involved in ferrichrome generation. the core gene smutadna4_glean_ encodes ferrichrome siderophore peptide synthetase, and encodes amino acids with 76% identity to the nrps sid of u. maydis 521. the gene has three amp-dependent synthetase/ligase domains, four acyl carrier domains to activate the cognate amino acid, four condensation domains to catalyze peptide bond formation between the amino acyl and peptidyl substrates of adjacent domains and one amp-binding enzyme c-terminal domain. the other nrps gene cluster consists of genes, responsible for ferrichrome a biosynthesis. the skeleton gene smutadna4_glean_ is located in the fifth gene cluster and encodes siderophore peptide synthetase. it encodes amino acids with 75% identity to the nrps fer of s. reilianum srz which is composed of three amp-dependent synthetase/ligase domains, five acyl carrier domains, four condensation domains and one amp-binding enzyme c-terminal domain.figure three gene clusters in the genome of sporisorium scitamineum. physical map of ferrichrome gene cluster. coding regions are indicated with arrows and are putatively involved in ferrichrome biosynthesis, siderophore transport, or unknown functions. location of interpro domains in nrps smutadna4_glean_ is as bottom. ad, amp-dependent synthetase/ligase domain, ipr00873; ac, acyl carrier domain, ipr009081; cd, condensation domain, ipr001242; ab, amp-binding enzyme c-terminal domain, ipr physical map of ferrichrome a gene cluster. coding regions are indicated with arrows and are putatively involved in ferrichrome a biosynthesis, siderophore transport, or unknown functions. location of interpro domains in nrps smutadna4_glean_ is as bottom. ad, amp-dependent synthetase/ligase domain, ipr00873; ac, acyl carrier domain, ipr009081;cd, condensation domain, ipr001242; ab, amp-binding enzyme c-terminal domain, ipr physical map of terpene gene cluster. coding regions are indicated with arrows and are putatively involved in terpene biosynthesis or unknown functions. two nrpss involved in siderophore biosynthesis have also been identified in the ascomycete cochliobolus heterostrophus and the ferricrocin biosynthesis enzyme is conserved in other ascomycetes. a. fumigatus is one of the most common aspergillus species to cause disease in individuals with an immunodeficiency a. fumigatus produces four siderophores, fusarinine c, triacetylfusarinine c, ferricrocin and hydroxyferricrocin. a. fumigatus has been recognized as a model species to study fungal siderophore biosynthetic pathways. these two nrps gene clusters require further study to describe the function of gene members in this cluster by metabolite detection methods such as high performance liquid chromatography, gas chromatography and mass spectrometry. molecular biology experiments also need to be conducted such as knock-out, knock-in, and heterogeneous expression. in figure  10c, the terpenes gene cluster is composed of three unknown function genes and seven biosynthesis related genes. the gene smutadna4_glean_ which encodes gibberellin 20-oxidase, is known to encode amino acids that have 90% identity with sr of s. reilianum srz gibberellin 20-oxidase is a key oxidase in the biosynthesis of gibberellin. gibberellin 20-oxidase catalyzes the conversions of ga and ga to ga and ga respectively, via a three-step oxidation at c- of the ga skeleton. ga probably plays an important role in s. reilianum resistance in infected sorghum bicolor, however, ga has no effect. this implies that the gene cluster is involved in weakening the resistance of sugarcane to smut disease and contributes to the successful infection of s. scitamineum. with gc-ms detection, s. scitamineum can biosynthesize compounds and secrete extracellularly. the intracellular metabolites are mainly esters and the distribution of molecular weight is from to amu. however, the extracellular metabolites are alcohol, aldoketones, alkanes, and carboxylic acids and no esters. the extracellular metabolites exhibited a smaller mw distribution. neither intracellular nor extracellular metabolites had strong infection to sugarcane in the inoculation experiments by supernatant and precipitation of fungal elicitor. the questions of whether this was due to the application of mixed metabolites, and if only the single or some secreted metabolites function in the infection process, require further investigation through additional inoculation validation. determining the function of each metabolite in this process could elucidate the corresponding mechanism causing sugarcane smut. cytochrome p450s play an important role in various hydroxylation and oxidation processes including those associated with secondary metabolites as well as the breakdown of toxins and other xenobiotic compounds. plant pathogenic fungi use a wide range of strategies to gain access to the carbon sources and nitrogen sources of their host plants and to counter the plant defense response. transporters are involved in toxin and effector secretion as well as nutrition uptake. there are p450s and transporter genes in the genome of s. scitamineum. a large proportion of transporters belong to the major facilitator superfamily, but the abc superfamily, p-type atpase superfamily, and sugar/inositol transporter are all well represented. these four smut fungi have similar composition of p450s and transporters. compared to the typical pathogens m. grisea or fusarium graminearum, the mfs number of s. scitamineum is small. it is unsurprising that few metabolites could be secreted extracellularly.table comparative analysis of p450s and transporter genes among various plant pathogen fungi ss uh um sr mlp pgt mo fg vd fo fv nh ss, sporisorium scitamineum; uh, ustilago hordei; um, ustilago maydis; sr, sporisorium reilianum; mlp, melampsora laricis-populina; pgt, puccinia graminis f. sp. tritici; mo, magnaporthe oryzae; fg, fusarium graminearum; vd, verticillium dahliae; fo, fusarium oxysporum; fv, fusarium verticillioides; nh, nectria haematococca.  although sugarcane smut was first reported in little is known about its pathogenic mechanisms. the s. scitamineum genome sequence and our comparative analysis with the genome sequences of u. maydis, s. reilianum and u. hordei have shed new light on the pathogenic mechanisms of this fungus and have provided insights into aspects of genome evolution, biotrophy features, carbohydrate degrading enzymes and secondary metabolic pathways likely to be common to all smut fungi. these results represent the initial step in realizing the full potential of these genomes. as a result of the genome analysis, efforts are underway to validate the function of all these potential pathogenicity determinants, especially the virulence associated genes. however, the genome sequence of s. scitamineum provides only a first glimpse into the genomic basis of pathogenic mechanisms of sugarcane smut. these efforts and ongoing sequencing projects for additional sugarcane fungi will provide extraordinary opportunities for comparative analyses that will expand our understanding of the interaction between sugarcane and fungi in this agriculturally and industrially important crop. further sequencing of s. scitamineum isolates from different geographical locations or different host varieties will lead to comprehensive population genetic analysis. 
there are two non-ribosomal peptide synthetase gene clusters that are involved in the generation of ferrichrome and ferrichrome a, while the terpenes gene cluster is composed of three unknown function genes and seven biosynthesis related genes. there are virulence associated genes in the genome of s. scitamineum, among which expressed in all the stages, which mainly encode for energy metabolism and redox of short-chain compound related enzymes. this phytopathogen can utilize a wide range of carbon and nitrogen sources. as a bipolar mating fungus, a and b loci are linked and the mating-type locus segregates as a single locus. sugarcane smut can cause losses in cane yield and sugar content that range from 30% to total crop failure. however, it remains unclear how the fungus breaks through the cell walls of sugarcane and causes the formation of black or gray whip-like structures on the sugarcane plants. sporisorium scitamineum is the fungus that causes sugarcane smut. as a destructive pathogen to sugar industry, the s. scitamineum genome will facilitate future research on the genomic basis and the pathogenic mechanisms of sugarcane smut. sporisorium scitamineum, sugarcane smutpathogenic mechanisms, g-protein coupled receptors, carbohydrate degrading enzymes, biotrophic properties, candidates for secreted effector proteins, secondary metabolic pathwaysissue-copyright-statement© the author 2014 a reduced set of genes encoding plant cell wall hydrolytic enzymes leads to its biotrophic lifestyle, in which damage to the host should be minimized. the s. scitamineum genome has only g protein-coupled receptors grouped into five classes, which are responsible for transducing extracellular signals into intracellular responses, however, the genome is without any pth11-like gpcr. here, we report the first high-quality genome sequence of s. scitamineum assembled de novo with a contig n of kb, a scaffold n of kb and genome size mb, containing an estimated genes. sixty-eight candidates for secreted effector proteins were found in the genome of s. scitamineum, and of them expressed in the different stages of sugarcane infection, which are probably involved in infection and/or triggering defense responses.
 sugarcane smut can cause losses in cane yield and sugar content that range from 30% to total crop failure. losses tend to increase with the passage of years. sporisorium scitamineum is the fungus that causes sugarcane smut. this fungus has the potential to infect all sugarcane species unless a species is resistant to biotrophic fungal pathogens. however, it remains unclear how the fungus breaks through the cell walls of sugarcane and causes the formation of black or gray whip-like structures on the sugarcane plants.  here, we report the first high-quality genome sequence of s. scitamineum assembled de novo with a contig n of kb, a scaffold n of kb and genome size mb, containing an estimated genes. this phytopathogen can utilize a wide range of carbon and nitrogen sources. a reduced set of genes encoding plant cell wall hydrolytic enzymes leads to its biotrophic lifestyle, in which damage to the host should be minimized. as a bipolar mating fungus, a and b loci are linked and the mating-type locus segregates as a single locus. the s. scitamineum genome has only g protein-coupled receptors grouped into five classes, which are responsible for transducing extracellular signals into intracellular responses, however, the genome is without any pth11-like gpcr. there are virulence associated genes in the genome of s. scitamineum, among which expressed in all the stages, which mainly encode for energy metabolism and redox of short-chain compound related enzymes. sixty-eight candidates for secreted effector proteins were found in the genome of s. scitamineum, and of them expressed in the different stages of sugarcane infection, which are probably involved in infection and/or triggering defense responses. there are two non-ribosomal peptide synthetase gene clusters that are involved in the generation of ferrichrome and ferrichrome a, while the terpenes gene cluster is composed of three unknown function genes and seven biosynthesis related genes.  as a destructive pathogen to sugar industry, the s. scitamineum genome will facilitate future research on the genomic basis and the pathogenic mechanisms of sugarcane smut. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords sporisorium scitamineum, sugarcane smutpathogenic mechanisms, g-protein coupled receptors, carbohydrate degrading enzymes, biotrophic properties, candidates for secreted effector proteins, secondary metabolic pathwaysissue-copyright-statement© the author 2014 