Abstract
Background
Sporisorium scitamineum causes the sugarcane smut disease, one of the
most serious constraints to global sugarcane production. S. scitamineum
possesses a sexual mating system composed of two mating-type loci, a
and b locus. We previously identified and deleted the b locus in S.
scitamineum, and found that the resultant SsΔMAT-1b mutant was
defective in mating and pathogenicity.
Results
To further understand the function of b-mating locus, we carried out
transcriptome analysis by comparing the transcripts of the mutant
strain SsΔMAT-1b, from which the SsbE1 and SsbW1 homeodomain
transcription factors have previously been deleted, with those from the
wild-type MAT-1 strain. Also the transcripts from SsΔMAT-1b X MAT-2
were compared with those from wild-type MAT-1 X MAT-2 mating. A total
of 209 genes were up-regulated (p < 0.05) in the SsΔMAT-1b mutant,
compared to the wild-type MAT-1 strain, while 148 genes down-regulated
(p < 0.05). In the mixture, 120 genes were up-regulated (p < 0.05) in
SsΔMAT-1b X MAT-2, which failed to mate, compared to the wild-type
MAT-1 X MAT-2 mating, and 271 genes down-regulated (p < 0.05). By
comparing the up- and down-regulated genes in these two sets, it was
found that 15 up-regulated and 37 down-regulated genes were common in
non-mating haploid and mating mixture, which indeed could be genes
regulated by b-locus. Furthermore, GO and KEGG enrichment analysis
suggested that carbon metabolism pathway and stress response mediated
by Hog1 MAPK signaling pathway were altered in the non-mating sets.
Conclusions
Experimental validation results indicate that the bE/bW heterodimeric
transcriptional factor, encoded by the b-locus, could regulate S.
scitamineum sexual mating and/or filamentous growth via modulating
glucose metabolism and Hog1-mediating oxidative response.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2691-5)
contains supplementary material, which is available to authorized
users.
Keywords: Mating, Sugarcane smut, bE/bW heterodimeric transcriptional
factor, Glucose, Hog1
Background
Sugarcane smut is a devastating disease in sugarcane growing areas
globally. The characteristic symptom of the disease is a black or gray
growth that is referred to as a “smut whip” [[37]1]. Sugarcane smut is
caused by the fungus S. scitamineum, a bipolar species [[38]2, [39]3]
with two mating type strains MAT-1 and MAT-2 [[40]4] producing haploid
sporidia by budding. The compatible sporidia fuse to develop pathogenic
dikaryotic hyphae, which grow within the stalk of sugarcane and form
diploid teliospores to complete the pathogenic life cycle [[41]3]. The
teliospores are disseminated by wind or rain splashes and germinate to
form four sporidia, and initiate next round of life cycle by mating.
The sexual mating process of S. scitamineum is similar to the maize
pathogen Ustilago maydis, which is regulated by two unlinked mating
type loci, a locus and b locus [[42]5–[43]7]. The bi-allelic a loci
that encode a pheromone/pheromone receptor system that is responsible
for recognition of the opposite haploid sporidia and formation of
conjugation tubes [[44]8]. The b locus composed of the bE and bW genes,
encoding a heterodimeric transcription factor to maintain the
dikaryotic filament and promote subsequent penetration of the host
plant, after fusion of the sporidia [[45]8–[46]10].
It has been reported that in U. maydis, the bE/bW transcription factor
acts through a regulatory cascade to affect various pathways in
triggering pathogenic development, including cell cycle regulation,
mitosis and DNA replication [[47]11]. However, the physiology of S.
scitamineum mating is largely unknown, due to unavailability of genome
sequence and effective method of genetic manipulation, previously.
Recently, with the genome sequencing performed by Que et al. [[48]2]
and Taniguti et al. [[49]12], and optimizing of the ATMT transformation
procedure for S. scitamineum [[50]13], investigation on S. scitamineum
differentiation and pathogenesis on molecular level becomes feasible.
Recently, we identified and characterized a b-locus homolog in S.
scitamineum, and found that it is essential for sexual mating and
filamentous growth [[51]14], but the underlying mechanism remained
unclear. Given that b-locus encodes a homeodomain transcription
complex, comparative transcriptome analysis may provide useful clues to
possible b-locus target gene(s) and functional study of such candidate
gene(s) may reveal the molecular basis of b-locus regulating S.
scitamineum sexual mating and/or filamentous growth. Therefore, we
carried out transcriptome analysis with wild-type MAT-1 and SsΔMAT-1b
mutant, and with mating and non-mating mixtures of S. scitamineum
haploids. Our study identified several potential target genes of
b-locus encoding transcriptional factor, that are likely involved in S.
scitamineum sexual mating and/or filamentous growth, and further
reveals two critical endogenous/environmental cues: nutrient and redox
homeostasis, for mating and/or filementous growth in S. scitamineum.
Methods
Growth conditions and strains used in this study
Teliospores of sugarcane smut were collected from the fields in
Guangdong province of China (21°12′ 36′′ N; 101°10′ 12′′ E), and no
specific permissions were required for sampling diseased plants in this
location. Haploid colonies of MAT-1 and MAT-2 were isolated from these
teliospores by serial dilution and plating on YePSA medium, as
previously described [[52]15]. Synthetic complete dextrose (SCD) medium
is consisted of 0.7 % (wt/vol) yeast nitrogen base without amino acids,
0.17 % complete amino acids powder, and 2 % (wt/vol) glucose [[53]16].
Synthetic complete (SC) medium was formulated as SCD medium without
addition of glucose [[54]16].
RNA extraction and sequencing strategies
TRIzol Reagent (Life Technologies, UK) was used for Total RNA
extraction from haploid MAT-1 and SsΔMAT-1b mutant. MAT-1 and MAT-2
haploids were mixed and plated on YePSA medium for 24 h before total
RNA extraction with TRIzol Reagent. Similarly, SsΔMAT-1b and MAT-2
haploids were mixed and inoculated on YePSA medium for 24 h before
total RNA extraction.
Libraries were constructed following Illumina manufacturer’s protocol
of the “TruSeq RNA Sample Prep v2 Low Throughput (LT)” kit. Paired-end
sequencing was performed on the Illumina HiSeq™2000. Reads were
analyzed by FASTQC
([55]http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and low
quality bases (phred ≥20), Illumina adapters and poly-A tails were
removed using the NGS QC Toolkit v2.3.3
([56]http://59.163.192.90:8080/ngsqctoolkit/) [[57]17].
Transcriptome assembly and annotations
De novo short read assembly was performed using tophat and cufflinks
softwares [[58]18]. The assembled reads were mapped to the complete
genome of S. scitamineum SSC39B strain
([59]ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank/fungi/Sporisorium_scita
mineum/latest_assembly_versions/GCA_000772675.1_Sporisorium_scitamineum
_v1) using Tophat and Bowtie2 [[60]19].
Unigene generated by De novo short read assembly was aligned to NCBI NR
Database ([61]ftp://ftp.ncbi.nih.gov/blast/db), SWISSPROT Database
([62]http://www.uniprot.org/downloads), and KOG Database (Clusters of
orthologous groups for eukaryotic complete genomes,
[63]ftp://ftp.ncbi.nih.gov/pub/COG/KOG/kyva), respectively. Unigene
encoding proteins with high similarity (e <1e-5) to the known proteins
in aforementioned databases were used to annotate the corresponding
Unigene. GO annotation was performed by Blast2GO software [[64]20] and
the database [65]http://www.geneontology.org/. KEGG annotation was
performed with the database [66]http://www.genome.jp/kegg/pathway.html
[[67]21].
Transcriptome analysis
Differential transcript accumulation among treatments (SsΔMAT-1b vs
MAT1, SsΔMAT-1b X MAT-2 vs MAT-1 X MAT-2) was observed using bowtie2
([68]http://bowtie-bio.sourceforge. net/bowtie2/manual.shtml) [[69]19]
and eXpress [[70]22]. The gene expression level is calculated by using
FPKM method (fragments Per kb per Million reads) [[71]22]. Baggerley’s
test and the false discovery rate (FDR) with a significance level of
≤0.05 and the absolute value of Log2Ratio ≥1 was set as the threshold
to judge the significance of gene expression difference.
GO enrichment analysis was performed as firstly mapping all DEGs
(Differential Expressed Genes) to GO terms in the database
([72]http://www.geneontology.org/), calculating gene numbers for every
term, then using hypergeometric test to find significantly enriched GO
terms in the input list of DEGs, based on GO::TermFinder
([73]http://smd.stanford.edu/help/GOTermFinder/GO_
TermFinder_help.shtml/). P value was calculated using the following
formula:
[MATH: P=1−∑i=0
m−1MiN−Mn−iNn<
/mrow> :MATH]
Where N is the number of all genes with GO annotation; n is the number
of DEGs in N; M is the number of all genes that are annotated to
certain GO terms; m is the number of DEGs in M. The calculated p-value
goes through Bonferroni Correction [[74]23], taking corrected
p-value ≤ 0.05 as a threshold. GO score was calculated as follows:
[MATH: Enrichmentscore=mnMN :MATH]
.
KEGG database is used to perform pathway enrichment analysis of DEGs.
The calculating formula is the same as that in GO analysis. Here N is
the number of all genes that with KEGG annotation, n is the number of
DEGs in N, M is the number of all genes annotated to specific pathways,
and m is the number of DEGs in M.
Results
Unigenes identification and gene annotation
Our RNAseq analysis produced a total length of 17.8344 Mb (Table [75]1)
for all the transcripts, out of 2G clean sequencing data, representing
about 100 X coverage of the transcriptome. Compared to previous
published genomic sequence of S. scitamineum [[76]2, [77]12], the total
length of sequence is slightly low, likely due to the fact that only
transcripts (with poly-A tails) were anchored and sequenced in this
study. De novo assembly of transcripts was performed as described in
Methods. We identified 7341 unigenes in total, with length from 145 bp
to 16628 bp (Table [78]1). Most of the identified unigenes are of
200–2000 bp (Fig. [79]1a), and GC content is within the range of
50–60 % (Fig. [80]1b). The unigenes were mapped to NR, SWISSPROT, and
KOG Database for annotation, as listed in Additional file [81]1: Table
S1.
Table 1.
Unigene statistics
All > = 200 bp > = 500 bp > = 1000 bp Total Length (Mb) Max Length Min
Length Avg Length
PRJNA240344 - - - - 19.7235 - - -
PRJEB5169 7711 - - - 19.4279 - - -
PRJNA275631 6677 - - - 20.0676 - - -
Unigene 7341 7338 7131 6123 17.8344 16628 145 2429.42
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Fig. 1.
Fig. 1
[83]Open in a new tab
Length and GC-content of All-Unigene. a Bar chart depicting length
distribution of All-Unigene identified in this study. b GC content
frequency distribution of All-Unigene of this study
Transcriptome analysis identified differentially expressed genes between
mating and non-mating strains/conditions
In this study, we compared two sets of non-mating vs mating
strain/condition, with an aim to identify the genes related to S.
scitamineum mating and likely regulated by b-locus. Differentially
Expressed Genes (DEGs) were identified in the SsΔMAT-1b mutant compared
to the wild-type MAT-1 strain, as well as in the non-mating mixture,
SsΔMAT-1b X MAT-2, compared to the mating mixture of MAT-1 X MAT-2.
DEGs of significance (p ≤ 0.05) in the haploid and mating set were
listed in Additional file [84]2: Table S2 and Additional file [85]3:
Table S3 respectively. In total, there are 357 DEGs identified in the
SsΔMAT-1b mutant, among which 209 genes were up-regulated and 148
down-regulated (Table [86]2). Under mating condition, a total of 391
genes were differentially expressed in the non-mating mixture, with 120
up-regulated and 271 down-regulated (Table [87]2). By comparing the up-
and down-regulated genes in these two conditions, we found that 15
up-regulated and 37 down-regulated genes were common in non-mating
haploid and mating mixture. We listed in Table [88]3 for those with
annotation in SWISSPROT Database.
Table 2.
DEGs statistics
Control Case Up_diff Down_diff Total_diff
MAT-1 b-deletion 209 148 357
MAT-1 b-deletion + MAT-2 MAT-1 + MAT-2 120 271 391
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Table 3.
List of Up- and Down- regulated genes common in SsΔMAT-1b vs wild-type
MAT-1 and SsΔMAT-1b X MAT-2 vs wild-type MAT-1 X MAT-2 comparing sets
Swiss ID Length (bp) Fold change p value Fold change p value
swiss.Description
SsΔMAT-1b vs MAT-1 SsΔMAT-1b X MAT-2 vs MAT-1 X MAT-2
sp|[90]P38938|CEK1_SCHPO 13208 13.42056 1.68E-06 3.44586 0.01867
Serine/threonine-protein kinase cek1 OS = Schizosaccharomyces pombe
(strain 972/ATCC 24843) GN = cek1 PE = 1 SV = 3
sp|[91]P39960|BEM2_YEAST 8330 1.796835 0.023621 1.718937 0.023855
GTPase-activating protein BEM2/IPL2 OS = Saccharomyces cerevisiae
(strain ATCC 204508/S288c) GN = BEM2 PE = 1 SV = 1
sp|[92]Q6R3K9|YSL2_ARATH 2834 1.946333 0.003032 1.913564 0.010979
Metal-nicotianamine transporter YSL2 OS = Arabidopsis thaliana
GN = YSL2 PE = 2 SV = 1
sp|[93]Q9JME5|AP3B2_MOUSE 9407 12.37208 9.87E-06 4.222222 2.16E-05 AP-3
complex subunit beta-2 OS = Mus musculus GN = Ap3b2 PE = 1 SV = 2
sp|[94]Q12019|MDN1_YEAST 16628 1.87115 0.005704 2.001882 0.002886
Midasin OS = Saccharomyces cerevisiae (strain ATCC 204508/S288c)
GN = MDN1 PE = 1 SV = 1
sp|[95]Q54YH4|DHKB_DICDI 6828 Inf 0.007605 10.75 0.005969 Hybrid signal
transduction histidine kinase B OS = Dictyostelium discoideum GN = dhkB
PE = 1 SV = 1
sp|[96]A2BGA0|RFX4_DANRE 4929 6.911589 1.76E-06 1.937224 0.015736
Transcription factor RFX4 OS = Danio rerio GN = rfx4 PE = 2 SV = 1
sp|[97]Q4P3W3|DBP10_USTMA 3384 12.41402 0.025351 4.851852 0.046387
ATP-dependent RNA helicase DBP10 OS = Ustilago maydis (strain 521/FGSC
9021) GN = DBP10 PE = 3 SV = 1
sp|[98]P56584|SID1_USTMA 2399 1.764062 0.017173 2.524876 0.016416
L-ornithine 5-monooxygenase OS = Ustilago maydis (strain 521/FGSC 9021)
GN = SID1 PE = 2 SV = 2
sp|[99]P36619|PMD1_SCHPO 7057 2.328467 0.01718 2.990596 0.006957
Leptomycin B resistance protein pmd1 OS = Schizosaccharomyces pombe
(strain 972/ATCC 24843) GN = pmd1 PE = 3 SV = 2
sp|[100]Q4PC06|HOG1_USTMA 5538 3.954272 0.001332 2.294118 0.037876
Mitogen-activated protein kinase HOG1 OS = Ustilago maydis (strain
521/FGSC 9021) GN = HOG1 PE = 3 SV = 1
sp|[101]P0CJ65|ATB54_ARATH 2265 0.02171 6.20E-19 0.178523 3.40E-06
Homeobox-leucine zipper protein ATHB-54 OS = Arabidopsis thaliana
GN = ATHB-54 PE = 2 SV = 1
sp|[102]P22943|HSP12_YEAST 1053 0.168388 9.19E-15 0.552964 0.01578
12 kDa heat shock protein OS = Saccharomyces cerevisiae (strain ATCC
204508/S288c) GN = HSP12 PE = 1 SV = 1
sp|[103]O14094|PPX1_SCHPO 2702 0.36517 0.002301 0.144718 0.000202
Putative exopolyphosphatase OS = Schizosaccharomyces pombe (strain
972/ATCC 24843) GN = SPAC2F3.11 PE = 3 SV = 1
sp|[104]Q6CHP9|CCM1_YARLI 2835 0.451759 0.010845 0.049724 1.68E-07
Mitochondrial group I intron splicing factor CCM1 OS = Yarrowia
lipolytica (strain CLIB 122/E 150) GN = CCM1 PE = 3 SV = 1
sp|[105]P22018|B4_USTMD 1539 0 9.33E-23 0.325834 0.001866 Mating-type
locus allele B4 protein OS = Ustilago maydis PE = 3 SV = 1
sp|[106]Q8VZ80|PLT5_ARATH 2427 0.452944 0.022087 0.493914 0.003544
Polyol transporter 5 OS = Arabidopsis thaliana GN = PLT5 PE = 1 SV = 2
sp|[107]Q4WFX9|LAP2_ASPFU 2169 0.053682 0.00954 0.337114 4.19E-05
Probable leucine aminopeptidase 2 OS = Neosartorya fumigata (strain
ATCC MYA-4609/Af293/CBS 101355/FGSC A1100) GN = lap2 PE = 3 SV = 2
sp|[108]Q5UP73|YR614_MIMIV 1884 0.353454 2.79E-06 0.340428 1.15E-05
Putative band 7 family protein R614 OS = Acanthamoeba polyphaga
mimivirus GN = MIMI_R614 PE = 3 SV = 1
sp|[109]Q8K4J6|MKL1_MOUSE 736 0.600119 0.019664 0.330712 5.01E-06
MKL/myocardin-like protein 1 OS = Mus musculus GN = Mkl1 PE = 1 SV = 2
sp|[110]Q9RPT1|RHLG_PSEAE 1073 0.337766 0.035915 0.16568 0.019132
Rhamnolipids biosynthesis 3-oxoacyl-[acyl-carrier-protein] reductase
OS = Pseudomonas aeruginosa (strain ATCC 15692/PAO1/1C/PRS 101/LMG
12228) GN = rhlG PE = 1 SV = 1
sp|[111]P80299|HYES_RAT 1160 0.467807 0.005301 0.425344 0.024971
Bifunctional epoxide hydrolase 2 OS = Rattus norvegicus GN = Ephx2
PE = 1 SV = 1
sp|[112]P36914|AMYG_ASPOR 2852 0.569668 0.010942 0.419058 0.010065
Glucoamylase OS = Aspergillus oryzae (strain ATCC 42149/RIB 40)
GN = glaA PE = 2 SV = 2
sp|[113]P34211|YUAR_ECOLI 2743 0.226761 3.58E-06 0.318024 3.27E-05
Putative hydrolase YuaR OS = Escherichia coli (strain K12) GN = yuaR
PE = 3 SV = 3
sp|[114]O35750|SHOX2_RAT 3616 0.490521 0.005405 0.55 0.023074 Short
stature homeobox protein 2 (Fragment) OS = Rattus norvegicus GN = Shox2
PE = 2 SV = 2
sp|[115]Q767C8|IH5GT_IRIHO 2482 0.263235 3.53E-09 0.630828 0.048192
Cyanidin 3-O-rutinoside 5-O-glucosyltransferase OS = Iris hollandica
GN = 5GT PE = 1 SV = 1
sp|[116]O42922|YBIH_SCHPO 4584 0.240982 5.71E-10 0.61912 0.037253
Uncharacterized MFS-type transporter C16A3.17c OS = Schizosaccharomyces
pombe (strain 972 / ATCC 24843) GN = SPBC16A3.17c PE = 3 SV = 1
[117]Open in a new tab
Among the 12 up-regulated and 16 down-regulated genes listed in
Table [118]3, we noticed that genes encoding components of signaling
pathway, e.g. MAPK Cek1 (involved in mitosis in yeast [[119]24] and
fungicidal activity in Candida albicans [[120]25]) and Hog1 (oxidative
or osmotic stress response [[121]26–[122]28]), GTPase-activating
protein BEM2/IPL2 (for cellular morphogenesis and interacting with
mitosis regulator in yeast [[123]29]), or histidine kinase (possibly
involved in two-component signal pathway [[124]30]) were up-regulated
with deletion of b-locus. Also, proteins involved vesicular trafficking
(AP-3 complex subunit) or metal-nicotianamine transporter YSL2 were
possibly repressed by b-locus transcriptional factor (Table [125]3).
Another transcriptional factor, RFX4, and an RNA helicase were
potentially repressed by b-locus too (Table [126]3). This result
indicates that b-locus may negatively regulate some signaling pathway
and repressed transcription of a set of downstream genes, directly or
indirectly, after sexual mating induced and during filamentous growth.
On the other hand, genes induced, directly or indirectly, by b-locus
include several other transcriptional factors, e.g. ATHB-54 [[127]31],
MKL/myocardin-like protein [[128]32], Short stature homeobox protein 2
(Shox2; related to growth and development in human [[129]33];
Table [130]3). b-locus may also induce regulators involved in
biosynthesis, including polyol transporter 5, Rhamnolipids biosynthesis
3-oxoacyl-[acyl-carrier-protein] reductase, MFS-type transporter, and
several hydrolases or Glucoamylase, during mating and/or filamentous
growth (Table [131]3). PKA and MAPK signaling pathway were found to be
involved in b-locus regulating sexual mating and/or filamentous growth
in U. maydis [[132]11]. Here in our study, we also identified component
of MAPK pathway, Cek1 and Hog1, that was potentially regulated by S.
scitamineum b-locus, but not among those identified in U. maydis. Our
finding indicates that S. scitamineum b-locus may regulate small
molecular (e.g. metal-nicotianamine, polyol) transport, vesicular
trafficking, biosynthesis, stress-response mediated by MAPK signaling
(Hog1), and a cascade of transcriptional network, during mating and/or
filamentous growth. The candidate genes listed in Table [133]3 are of
great interest in our future investigation, in terms of elucidating
physiology and molecular mechanism of S. scitamineum differentiation
and pathogenesis.
Identification of starch/sucrose metabolism and Hog1 MAPK pathway in fungal
mating
As an international standard gene functional classification system,
Gene Ontology (GO), offers a dynamic-updated controlled vocabulary, as
well as a strictly defined concept to comprehensively describe
properties of genes and their products in any organism [[134]34].
Therefore GO enrichment analysis of the aforementioned DEGs may further
reveal the functional relevance of b-locus regulating genes and S.
scitamineum mating. Enriched GO (for both up- and down- regulated) in
the haploid and mating sets were listed in Additional file [135]4:
Table S4 and Additional file [136]5: Table S5 respectively, and
schematically represented following three ontologies (molecular
function, cellular component and biological process) as in Fig. [137]2.
Among them, we noticed that the genes involved in membrane transport,
oxidation-reduction process and ATP-binding were overall differentially
regulated in non-mating haploid (SsΔMAT-1b mutant), as well as in
non-mating mixture (SsΔMAT-1b X MAT-2, Fig. [138]2). However, some
particular genes associated with the membrane transport process (GO:
0055085) were up-regulated, while some others, enriched in the same GO
term, were down-regulated, in both non-mating haploid and non-mating
mixture (Additional file [139]4: Table S4 and Additional file [140]5:
Table S5). Similar situation occurred for oxidation-reduction process
(GO: 0055114; Additional file [141]4: Table S4 and Additional file
[142]5: Table S5) as well as ATP-binding (GO: 0005524; Additional file
[143]4: Table S4 and Additional file [144]5: Table S5). On the other
hand, ATP catabolic process (GO: 0006200) was up-regulated in both
SsΔMAT-1b mutant and SsΔMAT-1b X MAT-2 mixture (Additional file [145]4:
Table S4 and Additional file [146]5: Table S5), indicating that S.
scitamineum mating may repress ATP catabolism. In summary, GO terms
enrichment analysis further verifies that metabolism, biosynthsis,
transmembrane transport and redox homeostasis would be tightly
regulated by b-locus during S. scitamineum mating and/or filamentous
growth.
Fig. 2.
Fig. 2
[147]Open in a new tab
GO-enrichment of DEGs. Left panal: SsΔMAT-1b mutant vs wild-type MAT-1;
right panal: SsΔMAT-1b X MAT-2 mixture vs MAT-1 X MAT-2. GO terms were
catalogued as Biological Process, Cellular Component, and Molecular
Function
Genes usually interact with each other to play roles in certain
biological functions. Pathway-based analysis helps to further
understand the biological functions of unigenes. KEGG-enrichment
analysis thus was carried out to identify significantly enriched
metabolic pathways or signal transduction pathways in DEGs comparing
with the whole genome background [[148]21]. Enriched KEGG terms were
listed in Additional file [149]6: Table S6 and Additional file [150]7:
Table S7, for SsΔMAT-1b vs wild-type MAT-1 and the non-mating mixture
of SsΔMAT-1b X MAT-2 vs wild-tyype MAT-1 X MAT-2, respectively. Among
the enriched pathways, we observed that starch and sucrose metabolism
pathway (ko00500; Additional file [151]8: Figure S1) was commonly found
in both haploid and mating sets. The predicted outcome of
differentially regulation of this pathway was that glucose production
would be reduced, while accumulation of 1,3-β-glucan would be increased
(Additional file [152]8: Figure S1), in SsΔMAT-1b or non-mating
mixture. Another commonly up-regulated gene, Hog1 (p38), was also found
in enriched KEGG pathway (ko04010, MAPK signaling) in both SsΔMAT-1b or
non-mating mixture (Additional file [153]9: Figure S2). Hog1 mediates
osmo- and oxidative stress response in yeast and fungi
[[154]26–[155]28], and is important for mating capacity in Candida
albicans [[156]16]. We infer that carbohydrate metabolism as well as
redox homeostasis may play important roles in S. scitamineum mating,
and be subjective to regulation (directly or indirectly) by the
b-locus.
Starch/sucrose metabolism and Hog1 MAPK pathway may regulate S. scitamineum
mating
To verify the involvement of starch/sucrose metabolism and Hog1 MAPK
pathway in S. scitamineum mating, we tested the growth of the wild-type
MAT-1, MAT-2 and SsΔMAT-1b mutant, as well as mating MAT-1 X MAT-2
mixtures, under osmotic and oxidative stresses. The results showed that
SsΔMAT-1b was more resistant to oxidative stress, compared to the
wild-type MAT-1 as well as mating mixture (Fig. [157]3a middle panel).
However, wild-type MAT-2 also showed higher resistance to H2O2 when
cultured alone but not in mating condition (Fig. [158]3a middle panel).
Osmotic stress imposed by 500 mM NaCl repressed the filamentous growth
in the mating mixture of MAT-1 X MAT-2 (Table [159]3A right panel).
However, the colonial growth was indistinguishable between the
wild-type MAT-1 and SsΔMAT-1b mutant strain, under the same osmotic
stress (Fig. [160]3a right panel). On the other hand, the YePSA medium
supplemented with high concentration (10 %, wt/vol) of glucose
repressed filamentous growth in the mating mixture of MAT-1 X MAT-2
(Fig. [161]3b). In contrast, glucose-depleted medium (SC) was more
favorable for filamentous growth in mating mixture of MAT-1 X MAT-2,
compared to the SCD medium containing 2 % glucose (Fig. [162]3c). As
1,3-β-glucan is an effective anti-oxidant, the significant enhancement
of Hog1 transcripts in non-mating haploid/mixture may be an indirect
consequence of elevated intracellular oxidative level in non-mating S.
scitamineum haploid and mixture. Furthermore, we tested the effect of
anti-oxidant, Glutathione (GSH) on colonial and filamentous growth of
haploid and mating strains. All the strains were more resistant to GSH
on SC (glucose-deplete) medium compared to SCD (glucose-containing)
medium (Fig. [163]3c). This indicates that the glucose may indeed be
utilized for synthesis of anti-oxidant 1,3-β-glucan, therefore
depletion of glucose resulted in more resistance to GSH, another
anti-oxidant. Overall, these results indicate that glucose may play a
negative role in promoting S. scitamineum mating and/or filamentous
growth, and the b-locus encoding heterodimeric transcriptional factor
may regulate starch/sucrose metabolism on transcriptional level. We
further predicted, based on transcriptome analysis, that the b-locus
encoded heterodimeric transcriptional factor may regulate S.
scitamineum mating and/or filamentous growth by promoting synthesis of
1,3-β-glucan (probably from D-glucose) and meanwhile repressed the
stress response signaling pathway mediated by Hog1 MAPK. A working
model, adopted and modified from b-locus regulatory network proposed in
U. maydis [[164]11], is depicted in Fig. [165]4 .
Fig. 3.
Fig. 3
[166]Open in a new tab
Starch/sucrose metabolism and Hog1 MAPK pathway are likely involved in
S. scitamineum mating, and subject to regulation of the b-locus. a
Serially diluted cells of MAT-1, MAT-2, MAT-1 X MAT-2, and SsΔMAT-1b,
were spotted onto YePSA medium supplemented with 2.5 mM hydrogen
peroxide or 500 mM NaCl. b Cells of MAT-1, SsΔMAT-1b, MAT-1 X MAT-2,
and SsΔMAT-1b X MAT-2, were spotted onto YePSA medium with or without
10 % (wt/vol) of glucose. c Serially diluted cells of MAT-1, SsΔMAT-1b,
MAT-1 X MAT-2, and SsΔMAT-1b X MAT-2, were spotted onto SCD or SC
medium, with 1 mM or 5 mM GSH
Fig. 4.
Fig. 4
[167]Open in a new tab
Proposed Model. bE and bW proteins derived from opposite mating type
form functional transcriptional complex to activate or repress Class 1
(direct) target genes. Class 2, indirect targets, are in turn activated
or repressed by products encoded by Class 1 targets. Starch/sucrose
metabolism genes as well as HOG1 may be indirectly repressed by the
bE/bW transcriptional complex, during mating. b-repressed glucose
metabolism gene may promote production of glucose, which may repress
mating and/or filamentous growth. Meanwhile, glucose production may
lead to elevated intracellular oxidative level, and thus induce Hog1
MAPK pathway, which also negatively regulates mating and/or filamentous
growth. Overall, b-locus may act in shutting down mating/filamentous
growth inhibitors, including high level of glucose and Hog1 MAPK
signaling
Discussion
Investigation on molecular mechanism on S. scitamineum mating
and/pathogenicity was impeded due to lack of S. scitamineum genome
sequence, until 2014, when Que et al., published the first genome
sequence of the pathogen [[168]2]. More recently, a Brazil group
published a complete genome assembly of S. scitamineum, as well as the
fungal transcriptome profiles revealing the candidate genes unique to
interaction with sugarcane [[169]12]. Such genomic and transcriptome
analyses have provided enormous convenience for functional study of
mating and pathogenic genes in S. scitamineum. In current study, we
conducted transcriptome analysis and comparison between mating vs
non-mating haploid/mixture, which present useful information on the
b-regulated gene expression cascade during S. scitamineum mating and/or
filamentous growth. Our transcriptome analysis predicted 7341 unigenes
(transcripts), which is similar to the predicted genome sizes of the
three published S. scitamineum strains
([170]http://www.ncbi.nlm.nih.gov/assembly/organism/49012/all/;
Table [171]1). The GC-content of our identified unigenes is ranged from
50 to 60 %, peaking at 55 % (Fig. [172]1b), which is also consistent
with the GC-content of these three genome projects (54.9, 54.8 and
55.04 % respectively). These data suggest that our de novo assembly of
transcripts in S. scitamineum is valid for the identification of DEGs
as well as GO and KEGG enrichment.
Our transcriptome analyses identified 357 DEGs in SsΔMAT-1b mutant
compared to the wild-type MAT-1, and 391 DEGs in non-mating (SsΔMAT-1b
X MAT-2) mixture compared to mating (MAT-1 X MAT-2) mixture. Among
them, 28 annotated genes (12 up-regulated and 16 down-regulated,
Table [173]3) were common in these two sets of comparisons, thus are
most likely associated with mating/filamentous growth and subject to
regulation by bE/bW heterodimeric transcription factor.
In the enriched KEGG pathway, we noticed that sucrose/starch metabolism
pathway was altered in the SsΔMAT-1b mutant in a way that intracellular
glucose is predicted to be reduced and 1,3 β-glucan elevated. Also,
glucoamylase encoded gene was identified as potentially b-locus induced
(Table [174]3). Our results (Fig. [175]3b) showed in contrast to our
prediction, that elevated glucose level repressed, but not promoted,
filamentous growth and/or mating. We infer that the timing (24 h post
mating) for detecting glucoamylase transcription might not be suitable,
when at this time point the transcripts started translating into
proteins. Therefore, the apparent low level of glucoamylase in
non-mating sets would reflect active glucose production, and b-locus
may actually repress glucoamylase during mating and/or filamentous
growth. We further hypothesize that glucose may be channeled to
synthesis of 1,3-β-glucan during S. scitamineum filamentous growth
after mating and likely regulated by b-locus, through repression of
glucoamylase. As 1,3-β-glucan is an anti-oxidant, its production may
relief the cell from endogenous oxidative stress therefore Hog1 was not
induced in wild-type condition. In b-deletion condition, glucose level
may elevated and therefore repress filamentous growth; meanwhile the
reduced1,3-β-glucan level resulted in endogenous oxidative stress and
induction of Hog1 as a response. SsΔMAT-1b mutant was slightly more
resistant to H2O2, likely due to hyper-induced Hog1. Our hypothesis was
supported by the observation that glucose-depleted medium (SC) promoted
filamentous growth in the mating mixture of MAT-1 X MAT-2 spores
(Fig. [176]3c). It has been reported that glucose plays an important
role in asexual/sexual sporulation in other pathogenic/filamentous
fungi, including Magnaporthe oryzae [[177]35], U. maydis [[178]36], and
Fusarium graminearum [[179]37]. Also, glucose was reported to suppress
mating competency in Candida albicans [[180]16]. Our results fit well
with the established notion that glucose promotes unicellular
spore/cell production while represses filamentous growth, thus acting
as a switch between dimorphic transition.
Another interesting observation from common DEGs and KEGG enrichment is
that the stress-activating MAPK signaling pathway mediated by Hog1 was
significantly up-regulated, in both SsΔMAT-1b mutant and SsΔMAT-1b X
MAT-2 mixture. One possibility is that, elevated glucose production in
SsΔMAT-1b haploid resulted in reduced production of 1, 3-β-glucan,
which is also known as an anti-oxidant. As a result, HOG1 was
transcriptionally induced in response to elevated intracellular
oxidative level. Alternatively, HOG1 may be repressed by the bE/bW
transcriptional complex, directly or indirectly, during mating. Our
tests showed that SsΔMAT-1b is less sensitive to oxidative stress.
Meanwhile, repression on colonial growth caused by anti-oxidant GSH was
more prominent with presence of glucose. Overall, these results suggest
that Hog1 MAPK signaling may be repressed by the bE/bW transcriptional
complex. Such observation is consistent with the reported function of
the Hog1 ortholog in Candida albicans that negatively regulates its
mating capacity [[181]16]. However, we are not aware of whether SsHOG1
is one of the direct targets (class I) genes of the bE/bW
transcriptional complex, or among the indirect (class II) targets, as
no obvious b-locus binding site (bbs [[182]38, [183]39]) was predicted
in the promoter region of SsHOG1.
It has been reported in U. maydis that GO categories “Cell Cycle”,
“Chromosome” and “DNA metabolic process” were significantly enriched as
b-down-regulated genes [[184]40]. However, we observed that “DNA
replication” was enriched as up-regulated GO terms in non-mating
mixture (Fig. [185]2; Additional file [186]5: Table S5; GO: 0006260),
and mitosis regulator Cek1 [[187]24] and GAP Bem2 that related to
mitosis [[188]29] were up-regulated in non-mating sets, which may also
account for the failure of mating, with deletion of b-locus in MAT-1.
Conclusions
Overall, our transcriptome analysis contributes to prediction of
candidate genes of the regulatory cascade of S. scitamineum b-locus, in
terms of mating and/or filamentous growth after recognition of opposite
sex mediated by the a-locus. In future, further investigation on such
candidate genes would help elucidate molecular mechanism of S.
scitamineum mating, including but not limited to, b-locus regulating
cell fate decision, morphogenesis, carbon/nitrogen metabolism, mitosis,
stress (oxidative) response, etc. This would certainly enrich our
knowledge in fungal sexual differentiation and/or pathogenesis, and
likely of great potential towards development/design of anti-fungal
pathogen strategy.
Ethics and consent to participate
Not applicable.
Consent to publish
Not applicable.
Availability of data and materials
All the data supporting our findings is contained within the manuscript
and supplementary files.
Acknowledgements