BACKGROUND
verticillium dahliae is a soilborne, plant pathogenic fungus that causes wilt disease on over  <dig> plant species worldwide.
verticillium wilt diseases are also referred to as vascular wilts since the fungus invades the plant xylem, disrupting water transport and causing characteristic leaf wilt symptoms.
control of verticillium wilt diseases is complicated by the lack of genetic resistance in many plant hosts, and also by the persistence of v. dahliae in the soil  <cit> .
verticillium dahliae produces melanized survival structures, microsclerotia, that are able to survive in the soil for at least  <dig> years  <cit> .
root exudates provide a signal for microsclerotial germination  <cit> , and upon germination infectious hyphae emerge from the microsclerotia to initiate plant root infection.
following penetration through the plant root epidermis, and an extended period of colonization in which plants are asymptomatic, microsclerotia are produced in large quantities within the dying and necrotic plant tissues, and are subsequently returned to the soil with plant debris to initiate a new round of the disease cycle  <cit> .
wild-type microsclerotia of v. dahliae are characterized by the presence of dense black melanin deposits that appear as granules within and exterior to the cell wall  <cit> .
this melanin is proposed to act as an anti-desiccant and protect against temperature extremes and enzymatic lysis  <cit> , and is synthesized via a dihydroxynaphthalene  melanin biosynthesis pathway  <cit> .
the initial substrate molecule in the dhn pathway is  <dig> , <dig> -tetrahydroxynaphthalene , derived by cyclization of acetate molecules by polyketide synthase  activity.
dhn monomers are generated from the  <dig> , <dig> -thn through alternating reduction and dehydration reactions involving several intermediates, including scytalone,  <dig> ,8-thn , and vermelone  <cit> .
the dhn monomers are subsequently oxidized and polymerized into melanin by laccases  <cit> .
though the microsclerotia are typically darkly pigmented, the processes of melanin production and microsclerotial development in v. dahliae can be uncoupled  <cit> .
because of their central role in pathogen survival and initiating plant root infection, the microsclerotia of v. dahliae have been considered important targets for disease control  <cit> .
this suggestion is supported by the correlation between reduced microsclerotial and pigment production, and reduced survival of v. dahliae <cit> .
the results of the analyses of multiple agrobacterium tumefaciens-mediated transformation  insertional mutants of v. dahliae also indicate an important link between microsclerotial formation and developmental processes needed for virulence  <cit> .
for example, a strain of v. dahliae with mutation of the stress-responsive glutamic acid-rich protein encoding-gene  shows both decreased virulence on cotton, and severely reduced microsclerotia formation under nutrient limiting conditions  <cit> .
in contrast, however, vdpkac <dig> mutants exhibit both reduced virulence, and under certain growth conditions enhanced microsclerotia production  <cit> .
the vdh <dig> mutant, which exhibits an amicrosclerotial phenotype under both nutrient-rich and starvation growth conditions, is not compromised in virulence  <cit> .
taken together, these data indicate that microsclerotial development is not always coupled with virulence.
analyses of gene expression in verticillium dahliae during microsclerotia formation have been carried out previously by comparisons of expressed sequence tag  libraries generated from v. dahliae cultures in which microsclerotia were developing , and those generated from cultures grown in simulated xylem fluid medium , which does not stimulate microsclerotia development  <cit> .
those est analyses revealed in the dms library expression of pigment synthesis genes, and increased expression of transporter type proteins encoded by several sxm library genes.
with the availability of the genomic sequence and the transcriptome of strain vdls.
<dig> of v. dahliae via the broad institute  <cit> , more in-depth analysis of gene expression during microsclerotia formation in v. dahliae can be conducted using the high-throughput, deep coverage rna sequencing  technology.
as an additional resource, bioinformatic analyses have enabled categorization of gene sets in v. dahliae, strain vdls.
<dig>  <cit> .
these gene sets include sequences encoding predicted secreted proteins, secondary metabolite synthesis genes, cysteine-rich proteins, and a set of  <dig> proteins encoded in four ~ 300 kb lineage -specific “genome islands”, that showed a significantly increased rate of expression relative to the core genome sequence  <cit> .
all of these individually categorized gene sets can be useful for rapid data-mining in projects employing high throughput analyses of gene expression.
the aim of this study was to identify genes that are differentially expressed in the melanized microsclerotia  of v. dahliae.
to accomplish this aim, rna-seq libraries were generated from ms-producing  cultures of strain vdls.
<dig>  and from cultures of vdls.
<dig> not producing ms , and analyzed for differential gene expression.
additional comparative analyses of gene expression, under growth conditions that promote or preclude microsclerotial development, were conducted using a microarray approach, with rna derived from v. dahliae strain dvd-t <dig> and an amicrosclerotial mutant  derived from dvd-t <dig> by targeted mutagenesis  <cit> .
taken together, these results yielded additional insights into gene sets that are of likely importance to the processes involved in melanin production in v. dahliae, as well as in ms formation and maintenance, or inhibition of these processes.
RESULTS
rna-seq analyses
microsclerotia formation in verticillium dahliae is a default process that occurs as cultures age, and on the potato dextrose agar  used for the rna-seq analysis is typically initiated after 3–4 days growth.
however, during our studies of v. dahliae we have noted that on occasion cultures may spontaneously become amicrosclerotial .
we considered this phenomenon as an opportunity that could be exploited for the analysis of microsclerotial development.
two different rna-seq approaches were therefore used to assess differential gene expression between ms + and noms pda-grown cultures  of v. dahliae.
in the first, a genome-wide analysis  was performed using untrimmed rna-seq library sequences and fdr-corrected p values , and differences were evaluated in gene expression between ms + and noms libraries.
this method detected a total of  <dig> differentially expressed genes,  <dig> of which were up-regulated, and  <dig> down-regulated in the ms + versus noms cultures .
gwa was also carried out using trimmed rna sequence reads without applying fdr correction to p values, and selecting genes having > 10-fold up- or down-regulation .
this latter approach resulted in the detection of increased numbers of up-regulated genes involved in melanin synthesis , a hallmark feature of ms maturation  <cit> .
specifically, nine pigment synthesis genes related to fungal melanin biosynthesis were identified by data mining analysis  as up-regulated in the ms + library vs. noms library , whereas only two of these genes were detected with the first gwa .
the results of this second gwa  were therefore a focal point for this study and were combined into the dma  as described below.
the dma was conducted with  <dig> sets of genes of interest  .
one set of genes examined in this analysis was created by inclusion of the selected genes from gwa <dig> having > 10-fold up- or down-regulation .
the other  <dig> gene sets were obtained either from categories of v. dahliae genes identified in previous analyses  <cit> , or from multigene families  identified in this study as containing differentially expressed genes .
all genes in each set were analyzed for differential expression between the ms + and noms libraries, using 10-base end-trimmed rna sequence reads, and employing common significance criteria of fdr-corrected p values <  <dig> , and expression fold change of at least  <dig> -fold up- or down-regulation.
this analysis yielded a total of  <dig> differentially expressed genes,  <dig> of which were up-regulated  and  <dig> down-regulated .
those genes with > <dig> fold changes in differential expression are shown in table  <dig>
*differential expression between ms + and noms libraries.
*fdr p value < <dig>  and fold change >  <dig> .
in many instances, the recorded magnitude of gene expression fold-change was greater in the dma than in the gwa .
in dma, the largest fold-change among the up-regulated and down-regulated genes in the ms + library was recorded for vdag_ <dig> , and vdag_ <dig> , respectively.
the dma also enabled detection of additional pigment synthesis genes expected to be identified in the ms + library, as indicated above.
the number of up- and down-regulated genes observed in dma of the various categories of v. dahliae genes is summarized in table  <dig>  among the  <dig> categories, predicted secretome, ls region protein-encoding genes, and small cysteine-rich protein-encoding genes represented the largest fractions of genes differentially expressed between the two libraries.
functional classification of the differentially expressed genes detected with dma revealed that over half encode hypothetical proteins of unknown function, with proteins involved in cell metabolism and transport functions representing the second and third largest category, respectively .
functional categories of differentially expressed genes
the lists of genes identified as differentially expressed during microsclerotia development were functionally annotated in-silico to identify genes encoding the different types of proteins that are potentially involved in microsclerotia biogenesis.
among these were genes coding for pigment biosynthesis and secondary metabolic enzymes, as well as those coding for cell growth, morphogenesis, signaling and transcription factors associated with pigment production and/or nutrient acquisition, and carbohydrate active enzymes and transport proteins.
below we describe in more detail some of the genes identified as differentially expressed in each one of these major categories.
pigment synthesis and secondary metabolism
a number of known melanogenesis-related enzyme-encoding genes were identified by the data mining analysis as up-regulated in ms + culture vs. the noms culture, including thn reductase , scytalone dehydratase , pigment biosynthesis protein ayg <dig> , conidial yellow pigment biosynthesis pks , two laccases , versicolorin reductase , polyketide synthase , .
among these, those encoding the pks , versicolorin reductase , laccase  and a second polyketide synthase  are found clustered in a region spanning  <dig>  kb on chromosome  <dig>  supercontig  <dig> of the v. dahliae strain vdls.
<dig> genome  .
microsclerotia formation in fungi involves dramatic morphological changes and activation of biosynthetic pathways involved in pigment synthesis  <cit> , processes that are expected to entail transcriptional activation of genes of various cellular functions.
consistent with this premise, several transcriptional regulators were observed as up-regulated in the dma, including some that are known to control melanin biosynthesis .
homologs of the pig <dig> and cmr <dig> transcription factors, which have been previously reported to be involved in fungal melanin synthesis  <cit> , were both up-regulated 12-fold in v. dahliae vdls.
<dig> .
these genes share sequence homology, and are located next to each other in the aforementioned  <dig>  kb gene cluster .
in addition, a novel transcription factor, encoded by vdag_ <dig> and located in the same cluster, was transcriptionally activated in the ms + culture.
the pig <dig> and cmr <dig> homologs were originally annotated in the verticillium genome sequence  as two separate genes , but are probably encoded by a single gene .
this possibility is supported by the finding in the current study of nearly identical rna-seq expression levels of vdag_ <dig> and vdag_ <dig> .
secondary metabolite biosynthesis in fungi is tightly linked with functions of non-ribosomal peptide synthetases , pkss, and activities of the cytochrome p <dig> enzymes .
of the eight nrpss identified in the v. dahliae genome , none were significantly differentially expressed in the ms + vs. noms rna-seq libraries, whereas of the  <dig> pks-encoding genes identified in this species, only the two above-mentioned, vdag_ <dig> and vdag_ <dig>  which share homology with fungal genes involved in the secondary metabolic pathway leading to pigment production  <cit> , and are part of the melanin biosynthesis cluster , were detected as differentially expressed.
also, 3/ <dig> of the cyps were up-regulated in the ms + library .
expression of the cyp gene vdag_ <dig> was up-regulated 247-fold in the ms + library , and that of vdag_ <dig> was up-regulated 5-fold .
though its function in v. dahliae is currently unknown, blast searches revealed that vdag_ <dig> shares homology with o-methylsterigmatocystin oxidoreductase proteins involved in the conversion of o-methylsterigmatocystin to aflatoxins in aspergillus spp.
<cit> .
cell growth, morphogenesis and cell death-related genes
a homolog of idi- <dig>  a cell death-associated gene of podospora anserina <cit> , was strongly up-regulated  in the ms + culture , as were cell cycle/cytoskeleton-related genes.
the keratin-associated protein- <dig> gene homolog  encodes a large  cysteine-rich protein, predicted to be extracellular  <cit>  and displaying weak homology with the developmentally regulated conidiospore surface protein cmp <dig>  from trichoderma sp.
<cit> .
genes encoding homologs of actin and other cytoskeletal elements that play critical roles in fungal morphogenesis  <cit>  were also differentially expressed in the ms + or noms cultures.
a gene encoding a homolog of the cortical actin regulatory protein, asp <dig>  important for polarized cell growth  <cit> , was up-regulated 4-fold in the ms + culture .
there was also down-regulation of other cytoskeletal-related protein genes , and of a homolog of sun <dig> , which in s. cerevisiae regulates cell wall morphogenesis and septation  <cit> .
catalases, whose products mediate the conversion of hydrogen peroxide to o- and h2o, have been implicated in oxidative stress protection  <cit> , and in mediating sclerotial differentiation in filamentous fungi  <cit> .
the expression of only one of three genes encoding predicted secreted catalases, vdag_ <dig>  was up-regulated 61-fold in the ms + culture , while the others  were neither significantly up- nor down-regulated .
we observed a 2-fold increase in the expression level of vdpkac <dig> , which encodes the catalytic subunit of a camp-dependent kinase that is involved in regulation of microsclerotial development  <cit> .
carbohydrate-active enzymes
the genome of v. dahliae encodes an abundance of carbohydrate-active enzymes  with diverse predicted activities  <cit> , including roles in fungal cell wall remodeling and plant cell wall degradation.
cazy-encoding sequences significantly up-regulated in the ms library included protein-encoding genes endochitinase vdag_ <dig> , pectate lyase vdag_ <dig> , alpha-amylase a vdag_ <dig> , glucan  <dig> -beta-glucosidase vdag_ <dig> , glucanase b vdag_ <dig> , and chitin deacetylase vdag_ <dig> .
cazy genes significantly down-regulated included those encoding an alpha-galactosidase a vdag_ <dig> , a glucan  <dig> -beta-glucosidase vdag_ <dig> , endoglucanase vdag_ <dig> , glucan endo- <dig> -alpha-glucosidase , and an endochitinase vdag_ <dig> .
the periplasmic trehalase vdag_ <dig> and the glucose-repressive grg <dig> <cit>  gene homolog vdag_ <dig> were also up-regulated 10- and 13-fold .
transport proteins
the data-mining analyses revealed differential expression in the ms + vs. noms libraries of genes encoding atp-binding cassette  and major facilitator  transport proteins, and other transporters.
in total, 4/ <dig> abc type proteins were differentially regulated in the ms library, while 15/ <dig> mfs proteins were differentially regulated .
of the differentially expressed abc transporter-encoding genes, the observed magnitudes of the differences in expression were relatively small, from  <dig> -fold up- to 2-fold down-regulation.
only one of the abc type and two of the predicted mfs genes were identified as down-regulated .
the sugar transport mfs protein-encoding gene vdag_ <dig> , was strongly induced, with a 17-fold change in expression.
of the additional transporter type genes identified, the rta1/lipid translocating exporter gene vdag_ <dig> , was up-regulated 31-fold.
the sphingolipid long-chain base-response protein  gene , involved in endocytic transport regulation  <cit> , was also up-regulated 12-fold .
hypothetical protein-encoding genes
approximately 50% of the differentially expressed genes in the ms + library encode hypothetical proteins for which cellular functions are not yet assigned .
some of these genes were strongly induced in the ms + library with expression fold changes >  <dig> .
vdag_ <dig>  up-regulated 252-fold in the ms + library, was also identified as an est  in the library derived from a developing microsclerotia  culture of v. dahliae  hypothetical protein-encoding genes in the ms library .
protein motif searches  were done for each of the hypothetical proteins in a quest for additional functional information.
most of the hypothetical protein encoding genes appeared to have no identifiable domain in protein motif searches, while others possess domains that function in diverse cellular activities, such as a fasciclin  extracellular cell adhesion domain , a camp-regulated phosphoprotein/endosulfine-conserved region  which might be involved in cell signaling, and fungal zn-cys binuclear cluster  or bzip domains  with potential roles in transcription regulation.
reverse-transcription quantitative pcr  analyses of gene expression in ms and noms cultures
real-time pcr with taqman assays were conducted to confirm differential expression of seven selected genes in ms + and noms cultures of v. dahliae .
these seven genes included known and hypothetical protein coding genes, as indicated in figure  <dig>  among these, rna-seq analyses had revealed that expression of the hypothetical protein encoding gene vdag_ <dig> was strongly down-regulated in the ms + culture as compared to the noms culture.
the six other genes in this set were up-regulated as determined by the rna-seq analyses .
for the selected genes rt-qpcr analyses of relative expression was compared as follows: expression in the darkly pigmented 12 day ms + cultures  vs. that in the 12 day pure white noms  culture, and that in seven day-old cultures that showed an intermediate level of pigmentation .
expression of the selected genes in darkly pigmented 6 month-old cultures  was also quantified and compared with expression in the above mentioned conditions .
culture types subjected to this study where pigment was either present or absent , were analyzed microscopically at each of the selected time points.
microscopic observations confirmed absence of structures resembling microsclerotia in those cultures lacking dark pigmentation .
the results of the rt-qpcr analyses in  <dig> d ms + vs.  <dig> d noms cultures revealed significant up-regulation of vdag_ <dig>  vdag_ <dig>  vdag_ <dig>  vdag_ <dig>  and vdag_ <dig> in the  <dig> d ms + culture, but showed no significant up- or down-regulation of vdag_ <dig> or vdag_ <dig>  .
however, all of the genes tested that were significantly up-regulated in the ms culture, as determined by rna-seq analyses, were also shown by rt-qpcr to be significantly upregulated in the 6 month ms + culture compared to that in the  <dig> d noms culture.
comparison of relative expression between the 6mo ms + culture and the  <dig> d noms culture types also revealed significant down-regulation of vdag_ <dig>  in the former .
with exceptions of vdag_ <dig> or vdag_ <dig> in the  <dig> d ms + vs.  <dig> d noms comparison, fold-changes in gene expression were similar as assessed by both rna-seq and rt-qpcr .
as may be expected in the comparison of the expression levels in two  <dig> d and  <dig> d ms + cultures, differences in rt-qpcr-assessed relative gene expression were reduced for vdag_ <dig>  vdag_ <dig>  vdag_ <dig>  and vdag_ <dig> .
as mentioned, the  <dig> d ms + cultures exhibited an intermediate level of melanization .
gene expression was markedly reduced for several of the genes when comparing the darkly pigmented 6 month ms + and  <dig> d ms + cultures.
in particular, vdag_ <dig>  vdag_ <dig>  and the known pigment biosynthesis gene vdag_ <dig> were significantly down-regulated in the 6 month ms + culture, relative to the expression seen for the  <dig> d ms + culture .
microarray analyses
in addition to the rna-seq analyses done for ms + and noms cultures of v. dahliae strain vdls.
<dig>  a small-scale microarray analysis was conducted in a second laboratory.
v. dahliae strains and culture conditions used in the microarray and rna-seq experiments is shown in additional file  <dig>  this microarray analysis used a second wild type strain, dvd-t <dig>  and an amiscrosclerotial  strain, with cultures grown on either a complete medium agar , or a basal medium agar  that induces enhanced production of microsclerotia .
in this analysis genes were identified that were induced during growth on bma, and/or repressed in vdh <dig> cultures, or dvd-t <dig> cultures grown in a liquid cm, i.e.
in the absence of microsclerotial development.
genes that exhibited such an expression profile belong to the aforementioned categories of genes found by rna-seq analysis to be induced during microsclerotial development  – namely, pigment biosynthesis and secondary metabolism genes, transcriptional regulators, and hypothetical genes.
normalized hybridization ratios of specific genes significantly induced during growth on bma and/or repressed in the vdh <dig> strain and during growth in liquid are shown in table  <dig>  many of these genes are involved in secondary metabolism, including the following five pigment-biosynthesis protein-encoding genes that were also shown by the rna-seq analyses to be up-regulated during microsclerotial development: scytalone dehydratase , versicolorin reductase , thn reductase , laccase , and ayg <dig>  .
also among the secondary metabolism genes that were up-regulated during growth in microsclerotia-promoting conditions in both the arrays and the rna-seq analysis was the cytochrome p <dig> gene vdag_ <dig> .
acontrol: wild-type strain dvd-t <dig> grown on complete medium ,agar; bvdh1: vdh <dig> strain  grown on ,agar; cwild-type strain  grown in liquid cm ; dwild-type strain grown on basal medium  agar; ** p ≤  <dig> ; *p ≤  <dig> .
among hypothetical genes, two that were up-regulated in the rna-seq analysis, vdag_ <dig> and vdag_ <dig>  were also represented among the differentially expressed genes identified by the array analysis, with both shown to be induced during growth on bma.
notably, the microarray analysis showed up-regulation of hypothetical gene vdag_ <dig>  which was, conversely, down-regulated in the rna-seq analysis .
the microarray analysis also identified several differentially expressed genes in v. dahliae dvd-t <dig> that were not identified in the rna-seq analysis .
among these was the transcriptional regulator gene ace <dig> , which in dvd-t <dig> was repressed in cultures that were not producing ms, and induced during ms development.
a cellulose growth specific protein encoding gene , was also repressed under non- microsclerotial producing conditions, as were two genes with possible roles in lipid metabolism, an acetyl coa synthetase gene , and a fatty acid binding protein-encoding gene .
northern blot validation of microarray and rna-seq data
the expression of four genes, identified by rna-seq as up-regulated in the ms + cultures of strain vdls.
<dig>  and by microarray analyses in microsclerotial-producing cultures of strain dvd-t <dig>  was validated by northern blot analyses .
these genes, which included two hypothetical protein encoding genes  and two pigment biosynthesis genes  were highly expressed at four days post-inoculation in wild-type cultures of v. dahliae grown on bma, but suppressed in a vdh <dig> mutant strain of v. dahliae grown on cma , and during growth of strain dvd-t <dig> in liquid cm.
northern blot analysis also verified that the hypothetical gene vdag_ <dig>  which was down-regulated in the rna-seq analysis, was up-regulated in four-day old bma-grown cultures .
the expression profiles of two genes, identified as differentially expressed in the microarray analysis but not in the vdls.
<dig> rna-seq analysis, verified by northern analysis; both the ace <dig> transcriptional regulator gene vdag_ <dig> and the fatty acid binding protein vdag_ <dig> were induced in dvd-t <dig>  and repressed in both the vdh <dig> mutant and in liquid-grown cultures .
ten genes were shown by microarray analysis to be strongly induced in strain dvd-t <dig> during microsclerotia development.
to evaluate inter-experimental results the expression of the genes as determined by microarray analysis was compared to that as assessed by northern blot analysis, rna-seq and rt-qpcr .
of the ten genes, eight were also up-regulated in the ms + pda cultures of strain vdls.
<dig> as compared to those in the rna-seq analysis that were not producing ms on pda .
the exceptions were vdag_ <dig>  which was shown as down-regulated by both rna-seq and rt-qpcr, but up-regulated by both microarray and northern blot analyses, and vdag_ <dig> which was, as mentioned above, not detected as differentially expressed by rna-seq  in strain vdls.
<dig> under the conditions tested .
discussion
microsclerotia are critically important in the disease cycle of v. dahliae, and their development has been the subject of intense research for decades  <cit> .
though significant advances have been made in understanding the morphological and biochemical aspects of microsclerotia biogenesis, the molecular mechanisms underpinning the formation of these structures are still not well understood  <cit> .
the aim of the current study was to identify genes that are differentially expressed in microsclerotia-producing  and noms cultures of v. dahliae, to shed further light on these molecular mechanisms.
to conduct this work, we took advantage of genome-wide gene expression technologies , and also of the recently sequenced genome of v. dahliae strain vdls.
<dig>  <cit>  for in silico functional annotation of the genes we identified as differentially expressed during ms development.
the rna-seq analyses revealed a number of differentially expressed genes including those that encoded enzymes previously known to be involved in the fungal dhn-melanin biosynthetic pathway, including polyketide synthases, thn reductases, scytalone dehydratases, and laccases,  <cit> .
since ms maturation culminates with the synthesis and deposition of melanin granules in cell walls and intercellular spaces  <cit> , the identification of the highly expressed genes encoding melanin biosynthetic-like enzymes indicated that the rna-seq libraries were appropriate for evaluation of gene expression during ms maturation.
in the microarray analysis pigment biosynthesis-encoding genes likewise formed the largest group of genes up-regulated during microsclerotial production, and showing high levels of expression, an expression pattern that was confirmed by northern blot analyses of two of the putative melanin biosynthesis gene orthologs, vdag_ <dig> and vdag_ <dig>  moreover, pigment biosynthesis gene expression was similarly evident in an earlier est study of v. dahliae cultures undergoing microsclerotia development  <cit> .
collectively, the overall consistency of these results signals the soundness of the experimental designs used herein.
although the general biochemical scheme/pathway of fungal dhn melanin biosynthesis is well defined based on experimental evidence gleaned from genetic and biochemical studies  <cit> , the exact identities, functions and multiplicity of all genes/enzymes involved in melanin biosynthesis, and the possible variation of function among species or strains has yet to be determined.
the melanin synthesis pathway is complex, involving many regulatory, secretory and structural genes, a complexity that was made apparent by the identification of nine melanin synthesis-related genes that were detected in the rna-seq study as highly expressed in the ms + culture.
some of these synthetic genes and putative regulators of melanin biosynthesis are clustered together in a  <dig>  kb gene region of the v. dahliae genome.
similar melanin biosynthetic gene clusters are present in other fungi  <cit> , and such clusters often include one or more transcription factors, as found in this study.
the rt-qpcr analyses provided confirmation of differential gene expression between ms + and noms cultures, and further enabled analyses of gene expression at several culture time points.
for example the known pigment biosynthesis gene vdag_ <dig>  encoding a protein highly similar to the melanin biosynthesis protein ayg <dig>  <cit> , the cytochrome p <dig> gene vdag_ <dig>  and the hypothetical protein-encoding gene vdag_ <dig>  were significantly down-regulated in the 6 month ms + culture, relative to the expression seen for the 12 day ms + culture.
the down-regulation of vdag_ <dig> in the six month 4°c culture may be indicative of completion of pigment synthesis.
similarly, the function of the cytochrome p <dig>  may not be required at this late stage.
specific genes, such as vdag_ <dig> and vdag_ <dig>  were identified by rt-qpcr analyses as being expressed in the  <dig> d ms + cultures at higher levels compared to that in the  <dig> d ms + cultures.
that this enhanced expression is suggestive of their roles in ms  biogenesis is confirmed by the corresponding genes having previously been identified as ests  expressed in dms cultures  <cit> , and also observed by microarray analysis to be highly expressed during microsclerotia development  <cit> .
morphological changes that occur during microsclerotia formation are well documented  <cit> .
in the initial stage of ms formation, the hyphae aggregate, swell and form numerous septa.
the septate cells enlarge and become spherical, forming clusters of cells by lateral budding, and during this process of ms maturation some cells undergo autolysis, resulting in a mixture, in individual ms, of dead and live cells  <cit> .
the transition from vegetative  growth to survival structure formation is expected to involve external and internal signals , as well as developmental reprogramming of molecular pathways that direct cell division, growth, and death.
intriguingly, the v. dahliae gene vdag_ <dig>  up-regulated 72-fold in the ms + culture, is highly similar to the podospora anserina cell death-related gene idi- <dig>  the expression of which is induced during the vegetative incompatibility reaction  <cit> .
likewise, vdag_ <dig> was up-regulated  in the 10 day ms + culture, and is highly similar to the p. anserina gene grg <dig>  which exhibits increased expression in senescent cultures and has been implicated in control of lifespan  <cit> .
signaling and effector molecules that orchestrate the death of some cells during v. dahliae ms biogenesis may thus involve mechanisms similar to those that in other fungi control senescence and cell death during fungal vegetative incompatibility  <cit> , with genes such as vdag_ <dig> and vdag_ <dig> involved in the developmental reprogramming of cells during ms maturation.
however, the absence in the verticillium group database of homologs of genes encoding other idi proteins  <cit> , as determined by blastp and tblastn , suggests divergence between p. anserina and v. dahliae in the molecular mechanisms operating in stress responses and/or cell death control.
the process of sclerotia formation and maturation in filamentous fungi involves active translocation of molecules from hyphae to developing sclerotia, and exudation/excretion, from sclerotia to the cell environment, of various molecules such as water, amino acids, proteins, soluble carbohydrates, fatty acids, and enzymes  <cit> .
some of these molecules may be required for synthesis of storage compounds that act as energy reserves for sclerotia germination  <cit> , and the observed increased expression in this study of certain transport protein genes, including those of abc, mfs, and lipid transport systems, might reflect such a scenario.
secreted carbohydrate-active enzymes  may function not only in the disease process, but also in remodeling of fungal cell walls during ms maturation.
the beta- <dig> -glucanase encoded by vdag_ <dig>  which is up-regulated in the ms + library, may, for example, contribute to cell wall modifications that accompany morphological transitions in ms development, much as beta- <dig> -glucanase is involved in morphogenesis in s. cerevisiae <cit> .
in addition, fungal periplasmic trehalases, which convert trehalose to glucose, are up-regulated under nutrient limiting or starvation conditions  <cit> , and the observed enhanced expression, in the ms + library, of the putative periplasmic trehalase vdag_ <dig> may therefore reflect depletion of nutrient levels in the actively growing 10-day v. dahliae cultures.
it is conceivable too that reduced expression of genes encoding specific cell wall-degrading activities may also be important for ms maturation.
the endochitinase gene vdag_ <dig> and the alpha-glucanase sequence vdag_ <dig>  were both down-regulated in the ms library.
while the s. pombe protein degrades the septum, allowing cell separation  <cit> , such separation may not be necessary in v. dahliae microsclerotia, which comprise both chains and clusters of spherical cells  <cit> .
the chitin deacetylase encoded by vdag_ <dig>  which is up-regulated in the ms + library, may on the other hand protect the fungus by converting chitin to a de-n-acetylated product like that which has previously been shown to be unsuitable as a chitinase substrate  <cit> .
a decrease in chitinolytic enzyme production during ms maturation, with a concomitant increase in cell wall protection, might be expected for production of the long-lived ms.
in addition to the new candidate genes identified by rna-seq as differentially expressed in the ms library in this study, there was also a pattern of overlap with genes previously identified in the dms est library  <cit> , and by the microarray analysis presented here .
as described above, pigment biosynthesis genes formed one such clearly identifiable group, as did certain hypothetical/unknown genes.
notably, however, one of the hypothetical genes identified in both analyses  showed expression patterns that seem to be culture age dependent.
this gene was shown by both microarray and northern blot analyses to be up-regulated in 4-day old microsclerotia-producing cultures, but by rna-seq and rt-qpcr analysis was down-regulated in 10-day and 6 month microsclerotial cultures of vdls.
<dig>  likewise, the acetyl coa synthase gene  was induced in the 4-day old cultures , but not in the 10-day old cultures used to produce the rna-seq libraries.
also, fluxes in the expression levels of certain other genes, like vdh <dig> <cit> , could have occurred at a developmental stage different than that sampled in the rna-seq study.
such variation in results obtained through rna-seq vs. microarray and previous est analyses may also reflect the difference in strains used for the studies , and/or the different culture conditions under which the fungi were grown prior to harvest .
while enhanced expression of cellulolytic enzymes was not expected to be observed in rna-seq libraries prepared from cultures grown on nitrocellulose membranes, it is possible that starvation of the fungus in the presence of the cellulose may induce expression of genes encoding cell wall degrading enzymes, such as was observed for vdag_ <dig> in the est analysis  <cit> , and in the present microarray analysis.
CONCLUSIONS
information on the molecular control of microsclerotia development in v. dahliae is currently still sparse  <cit> .
the developmental reprogramming from the normal growth state to survival structure  formation is expected to involve external and internal signals  as well as unique cell signaling pathway, all of which are yet to be identified and elucidated.
we envision that future functional characterization of the melanin synthesis and regulatory genes identified in this study, in conjunction with biochemical analyses, will help refine our understanding of the molecular basis of melanogenesis and ms morphogenesis in v. dahliae.
some of the genes identified as up- or down-regulated in the ms + vs. noms libraries may be involved in the morphogenetic processes that regulate different aspects of microsclerotial development.
in addition, similar to the results obtained in previous est analyses  <cit> , approximately 50% of the genes that we identified by rna-seq lacked significant similarity to any known genes.
functional analyses of some of these differentially expressed, unknown genes will undoubtedly advance our understanding of verticillium development.
the microsclerotia of v. dahliae have been considered important targets for disease control  <cit> , and gene products identified in this study could potentially represent new targets for disease control through prevention of survival structure development.

 verticillium dahliae is a soilborne, plant pathogenic fungus that causes wilt disease on over plant species worldwide. verticillium wilt diseases are also referred to as vascular wilts since the fungus invades the plant xylem, disrupting water transport and causing characteristic leaf wilt symptoms. control of verticillium wilt diseases is complicated by the lack of genetic resistance in many plant hosts, and also by the persistence of v. dahliae in the soil. verticillium dahliae produces melanized survival structures, microsclerotia, that are able to survive in the soil for at least years. root exudates provide a signal for microsclerotial germination, and upon germination infectious hyphae emerge from the microsclerotia to initiate plant root infection. following penetration through the plant root epidermis, and an extended period of colonization in which plants are asymptomatic, microsclerotia are produced in large quantities within the dying and necrotic plant tissues, and are subsequently returned to the soil with plant debris to initiate a new round of the disease cycle. wild-type microsclerotia of v. dahliae are characterized by the presence of dense black melanin deposits that appear as granules within and exterior to the cell wall. this melanin is proposed to act as an anti-desiccant and protect against temperature extremes and enzymatic lysis, and is synthesized via a dihydroxynaphthalene melanin biosynthesis pathway. the initial substrate molecule in the dhn pathway is, -tetrahydroxynaphthalene, derived by cyclization of acetate molecules by polyketide synthase activity. dhn monomers are generated from the, -thn through alternating reduction and dehydration reactions involving several intermediates, including scytalone,,8-thn, and vermelone. the dhn monomers are subsequently oxidized and polymerized into melanin by laccases. though the microsclerotia are typically darkly pigmented, the processes of melanin production and microsclerotial development in v. dahliae can be uncoupled. because of their central role in pathogen survival and initiating plant root infection, the microsclerotia of v. dahliae have been considered important targets for disease control. this suggestion is supported by the correlation between reduced microsclerotial and pigment production, and reduced survival of v. dahliae. the results of the analyses of multiple agrobacterium tumefaciens-mediated transformation insertional mutants of v. dahliae also indicate an important link between microsclerotial formation and developmental processes needed for virulence. for example, a strain of v. dahliae with mutation of the stress-responsive glutamic acid-rich protein encoding-gene shows both decreased virulence on cotton, and severely reduced microsclerotia formation under nutrient limiting conditions. in contrast, however, vdpkac mutants exhibit both reduced virulence, and under certain growth conditions enhanced microsclerotia production. the vdh mutant, which exhibits an amicrosclerotial phenotype under both nutrient-rich and starvation growth conditions, is not compromised in virulence. taken together, these data indicate that microsclerotial development is not always coupled with virulence. analyses of gene expression in verticillium dahliae during microsclerotia formation have been carried out previously by comparisons of expressed sequence tag libraries generated from v. dahliae cultures in which microsclerotia were developing, and those generated from cultures grown in simulated xylem fluid medium, which does not stimulate microsclerotia development. those est analyses revealed in the dms library expression of pigment synthesis genes, and increased expression of transporter type proteins encoded by several sxm library genes. with the availability of the genomic sequence and the transcriptome of strain vdls. of v. dahliae via the broad institute, more in-depth analysis of gene expression during microsclerotia formation in v. dahliae can be conducted using the high-throughput, deep coverage rna sequencing technology. as an additional resource, bioinformatic analyses have enabled categorization of gene sets in v. dahliae, strain vdls.. these gene sets include sequences encoding predicted secreted proteins, secondary metabolite synthesis genes, cysteine-rich proteins, and a set of proteins encoded in four ~ 300 kb lineage -specific “genome islands”, that showed a significantly increased rate of expression relative to the core genome sequence. all of these individually categorized gene sets can be useful for rapid data-mining in projects employing high throughput analyses of gene expression. the aim of this study was to identify genes that are differentially expressed in the melanized microsclerotia of v. dahliae. to accomplish this aim, rna-seq libraries were generated from ms-producing cultures of strain vdls. and from cultures of vdls. not producing ms, and analyzed for differential gene expression. additional comparative analyses of gene expression, under growth conditions that promote or preclude microsclerotial development, were conducted using a microarray approach, with rna derived from v. dahliae strain dvd-t and an amicrosclerotial mutant derived from dvd-t by targeted mutagenesis. taken together, these results yielded additional insights into gene sets that are of likely importance to the processes involved in melanin production in v. dahliae, as well as in ms formation and maintenance, or inhibition of these processes.  rna-seq analyses microsclerotia formation in verticillium dahliae is a default process that occurs as cultures age, and on the potato dextrose agar used for the rna-seq analysis is typically initiated after 3–4 days growth. however, during our studies of v. dahliae we have noted that on occasion cultures may spontaneously become amicrosclerotial. we considered this phenomenon as an opportunity that could be exploited for the analysis of microsclerotial development. two different rna-seq approaches were therefore used to assess differential gene expression between ms + and noms pda-grown cultures of v. dahliae. in the first, a genome-wide analysis was performed using untrimmed rna-seq library sequences and fdr-corrected p values, and differences were evaluated in gene expression between ms + and noms libraries. this method detected a total of differentially expressed genes, of which were up-regulated, and down-regulated in the ms + versus noms cultures. gwa was also carried out using trimmed rna sequence reads without applying fdr correction to p values, and selecting genes having > 10-fold up- or down-regulation. this latter approach resulted in the detection of increased numbers of up-regulated genes involved in melanin synthesis, a hallmark feature of ms maturation. specifically, nine pigment synthesis genes related to fungal melanin biosynthesis were identified by data mining analysis as up-regulated in the ms + library vs. noms library, whereas only two of these genes were detected with the first gwa. the results of this second gwa were therefore a focal point for this study and were combined into the dma as described below. the dma was conducted with sets of genes of interest. one set of genes examined in this analysis was created by inclusion of the selected genes from gwa having > 10-fold up- or down-regulation. the other gene sets were obtained either from categories of v. dahliae genes identified in previous analyses, or from multigene families identified in this study as containing differentially expressed genes. all genes in each set were analyzed for differential expression between the ms + and noms libraries, using 10-base end-trimmed rna sequence reads, and employing common significance criteria of fdr-corrected p values < , and expression fold change of at least -fold up- or down-regulation. this analysis yielded a total of differentially expressed genes, of which were up-regulated and down-regulated. those genes with > fold changes in differential expression are shown in table  *differential expression between ms + and noms libraries. *fdr p value < and fold change > . in many instances, the recorded magnitude of gene expression fold-change was greater in the dma than in the gwa. in dma, the largest fold-change among the up-regulated and down-regulated genes in the ms + library was recorded for vdag_, and vdag_, respectively. the dma also enabled detection of additional pigment synthesis genes expected to be identified in the ms + library, as indicated above. the number of up- and down-regulated genes observed in dma of the various categories of v. dahliae genes is summarized in table  among the categories, predicted secretome, ls region protein-encoding genes, and small cysteine-rich protein-encoding genes represented the largest fractions of genes differentially expressed between the two libraries. functional classification of the differentially expressed genes detected with dma revealed that over half encode hypothetical proteins of unknown function, with proteins involved in cell metabolism and transport functions representing the second and third largest category, respectively. functional categories of differentially expressed genes the lists of genes identified as differentially expressed during microsclerotia development were functionally annotated in-silico to identify genes encoding the different types of proteins that are potentially involved in microsclerotia biogenesis. among these were genes coding for pigment biosynthesis and secondary metabolic enzymes, as well as those coding for cell growth, morphogenesis, signaling and transcription factors associated with pigment production and/or nutrient acquisition, and carbohydrate active enzymes and transport proteins. below we describe in more detail some of the genes identified as differentially expressed in each one of these major categories. pigment synthesis and secondary metabolism a number of known melanogenesis-related enzyme-encoding genes were identified by the data mining analysis as up-regulated in ms + culture vs. the noms culture, including thn reductase, scytalone dehydratase, pigment biosynthesis protein ayg, conidial yellow pigment biosynthesis pks, two laccases, versicolorin reductase, polyketide synthase,. among these, those encoding the pks, versicolorin reductase, laccase and a second polyketide synthase are found clustered in a region spanning  kb on chromosome supercontig of the v. dahliae strain vdls. genome. microsclerotia formation in fungi involves dramatic morphological changes and activation of biosynthetic pathways involved in pigment synthesis, processes that are expected to entail transcriptional activation of genes of various cellular functions. consistent with this premise, several transcriptional regulators were observed as up-regulated in the dma, including some that are known to control melanin biosynthesis. homologs of the pig and cmr transcription factors, which have been previously reported to be involved in fungal melanin synthesis, were both up-regulated 12-fold in v. dahliae vdls.. these genes share sequence homology, and are located next to each other in the aforementioned  kb gene cluster. in addition, a novel transcription factor, encoded by vdag_ and located in the same cluster, was transcriptionally activated in the ms + culture. the pig and cmr homologs were originally annotated in the verticillium genome sequence as two separate genes, but are probably encoded by a single gene. this possibility is supported by the finding in the current study of nearly identical rna-seq expression levels of vdag_ and vdag_. secondary metabolite biosynthesis in fungi is tightly linked with functions of non-ribosomal peptide synthetases, pkss, and activities of the cytochrome p enzymes. of the eight nrpss identified in the v. dahliae genome, none were significantly differentially expressed in the ms + vs. noms rna-seq libraries, whereas of the pks-encoding genes identified in this species, only the two above-mentioned, vdag_ and vdag_ which share homology with fungal genes involved in the secondary metabolic pathway leading to pigment production, and are part of the melanin biosynthesis cluster, were detected as differentially expressed. also, 3/ of the cyps were up-regulated in the ms + library. expression of the cyp gene vdag_ was up-regulated 247-fold in the ms + library, and that of vdag_ was up-regulated 5-fold. though its function in v. dahliae is currently unknown, blast searches revealed that vdag_ shares homology with o-methylsterigmatocystin oxidoreductase proteins involved in the conversion of o-methylsterigmatocystin to aflatoxins in aspergillus spp.. cell growth, morphogenesis and cell death-related genes a homolog of idi- a cell death-associated gene of podospora anserina, was strongly up-regulated in the ms + culture, as were cell cycle/cytoskeleton-related genes. the keratin-associated protein- gene homolog encodes a large cysteine-rich protein, predicted to be extracellular and displaying weak homology with the developmentally regulated conidiospore surface protein cmp from trichoderma sp.. genes encoding homologs of actin and other cytoskeletal elements that play critical roles in fungal morphogenesis were also differentially expressed in the ms + or noms cultures. a gene encoding a homolog of the cortical actin regulatory protein, asp important for polarized cell growth, was up-regulated 4-fold in the ms + culture. there was also down-regulation of other cytoskeletal-related protein genes, and of a homolog of sun, which in s. cerevisiae regulates cell wall morphogenesis and septation. catalases, whose products mediate the conversion of hydrogen peroxide to o- and h2o, have been implicated in oxidative stress protection, and in mediating sclerotial differentiation in filamentous fungi. the expression of only one of three genes encoding predicted secreted catalases, vdag_ was up-regulated 61-fold in the ms + culture, while the others were neither significantly up- nor down-regulated. we observed a 2-fold increase in the expression level of vdpkac, which encodes the catalytic subunit of a camp-dependent kinase that is involved in regulation of microsclerotial development. carbohydrate-active enzymes the genome of v. dahliae encodes an abundance of carbohydrate-active enzymes with diverse predicted activities, including roles in fungal cell wall remodeling and plant cell wall degradation. cazy-encoding sequences significantly up-regulated in the ms library included protein-encoding genes endochitinase vdag_, pectate lyase vdag_, alpha-amylase a vdag_, glucan -beta-glucosidase vdag_, glucanase b vdag_, and chitin deacetylase vdag_. cazy genes significantly down-regulated included those encoding an alpha-galactosidase a vdag_, a glucan -beta-glucosidase vdag_, endoglucanase vdag_, glucan endo- -alpha-glucosidase, and an endochitinase vdag_. the periplasmic trehalase vdag_ and the glucose-repressive grg gene homolog vdag_ were also up-regulated 10- and 13-fold. transport proteins the data-mining analyses revealed differential expression in the ms + vs. noms libraries of genes encoding atp-binding cassette and major facilitator transport proteins, and other transporters. in total, 4/ abc type proteins were differentially regulated in the ms library, while 15/ mfs proteins were differentially regulated. of the differentially expressed abc transporter-encoding genes, the observed magnitudes of the differences in expression were relatively small, from -fold up- to 2-fold down-regulation. only one of the abc type and two of the predicted mfs genes were identified as down-regulated. the sugar transport mfs protein-encoding gene vdag_, was strongly induced, with a 17-fold change in expression. of the additional transporter type genes identified, the rta1/lipid translocating exporter gene vdag_, was up-regulated 31-fold. the sphingolipid long-chain base-response protein gene, involved in endocytic transport regulation, was also up-regulated 12-fold. hypothetical protein-encoding genes approximately 50% of the differentially expressed genes in the ms + library encode hypothetical proteins for which cellular functions are not yet assigned. some of these genes were strongly induced in the ms + library with expression fold changes > . vdag_ up-regulated 252-fold in the ms + library, was also identified as an est in the library derived from a developing microsclerotia culture of v. dahliae hypothetical protein-encoding genes in the ms library. protein motif searches were done for each of the hypothetical proteins in a quest for additional functional information. most of the hypothetical protein encoding genes appeared to have no identifiable domain in protein motif searches, while others possess domains that function in diverse cellular activities, such as a fasciclin extracellular cell adhesion domain, a camp-regulated phosphoprotein/endosulfine-conserved region which might be involved in cell signaling, and fungal zn-cys binuclear cluster or bzip domains with potential roles in transcription regulation. reverse-transcription quantitative pcr analyses of gene expression in ms and noms cultures real-time pcr with taqman assays were conducted to confirm differential expression of seven selected genes in ms + and noms cultures of v. dahliae. these seven genes included known and hypothetical protein coding genes, as indicated in figure  among these, rna-seq analyses had revealed that expression of the hypothetical protein encoding gene vdag_ was strongly down-regulated in the ms + culture as compared to the noms culture. the six other genes in this set were up-regulated as determined by the rna-seq analyses. for the selected genes rt-qpcr analyses of relative expression was compared as follows: expression in the darkly pigmented 12 day ms + cultures vs. that in the 12 day pure white noms culture, and that in seven day-old cultures that showed an intermediate level of pigmentation. expression of the selected genes in darkly pigmented 6 month-old cultures was also quantified and compared with expression in the above mentioned conditions. culture types subjected to this study where pigment was either present or absent, were analyzed microscopically at each of the selected time points. microscopic observations confirmed absence of structures resembling microsclerotia in those cultures lacking dark pigmentation. the results of the rt-qpcr analyses in d ms + vs. d noms cultures revealed significant up-regulation of vdag_ vdag_ vdag_ vdag_ and vdag_ in the d ms + culture, but showed no significant up- or down-regulation of vdag_ or vdag_. however, all of the genes tested that were significantly up-regulated in the ms culture, as determined by rna-seq analyses, were also shown by rt-qpcr to be significantly upregulated in the 6 month ms + culture compared to that in the d noms culture. comparison of relative expression between the 6mo ms + culture and the d noms culture types also revealed significant down-regulation of vdag_ in the former. with exceptions of vdag_ or vdag_ in the d ms + vs. d noms comparison, fold-changes in gene expression were similar as assessed by both rna-seq and rt-qpcr. as may be expected in the comparison of the expression levels in two d and d ms + cultures, differences in rt-qpcr-assessed relative gene expression were reduced for vdag_ vdag_ vdag_ and vdag_. as mentioned, the d ms + cultures exhibited an intermediate level of melanization. gene expression was markedly reduced for several of the genes when comparing the darkly pigmented 6 month ms + and d ms + cultures. in particular, vdag_ vdag_ and the known pigment biosynthesis gene vdag_ were significantly down-regulated in the 6 month ms + culture, relative to the expression seen for the d ms + culture. microarray analyses in addition to the rna-seq analyses done for ms + and noms cultures of v. dahliae strain vdls. a small-scale microarray analysis was conducted in a second laboratory. v. dahliae strains and culture conditions used in the microarray and rna-seq experiments is shown in additional file this microarray analysis used a second wild type strain, dvd-t and an amiscrosclerotial strain, with cultures grown on either a complete medium agar, or a basal medium agar that induces enhanced production of microsclerotia. in this analysis genes were identified that were induced during growth on bma, and/or repressed in vdh cultures, or dvd-t cultures grown in a liquid cm, i.e. in the absence of microsclerotial development. genes that exhibited such an expression profile belong to the aforementioned categories of genes found by rna-seq analysis to be induced during microsclerotial development – namely, pigment biosynthesis and secondary metabolism genes, transcriptional regulators, and hypothetical genes. normalized hybridization ratios of specific genes significantly induced during growth on bma and/or repressed in the vdh strain and during growth in liquid are shown in table  many of these genes are involved in secondary metabolism, including the following five pigment-biosynthesis protein-encoding genes that were also shown by the rna-seq analyses to be up-regulated during microsclerotial development: scytalone dehydratase, versicolorin reductase, thn reductase, laccase, and ayg. also among the secondary metabolism genes that were up-regulated during growth in microsclerotia-promoting conditions in both the arrays and the rna-seq analysis was the cytochrome p gene vdag_. acontrol: wild-type strain dvd-t grown on complete medium,agar; bvdh1: vdh strain grown on,agar; cwild-type strain grown in liquid cm; dwild-type strain grown on basal medium agar; ** p ≤ ; *p ≤ . among hypothetical genes, two that were up-regulated in the rna-seq analysis, vdag_ and vdag_ were also represented among the differentially expressed genes identified by the array analysis, with both shown to be induced during growth on bma. notably, the microarray analysis showed up-regulation of hypothetical gene vdag_ which was, conversely, down-regulated in the rna-seq analysis. the microarray analysis also identified several differentially expressed genes in v. dahliae dvd-t that were not identified in the rna-seq analysis. among these was the transcriptional regulator gene ace, which in dvd-t was repressed in cultures that were not producing ms, and induced during ms development. a cellulose growth specific protein encoding gene, was also repressed under non- microsclerotial producing conditions, as were two genes with possible roles in lipid metabolism, an acetyl coa synthetase gene, and a fatty acid binding protein-encoding gene. northern blot validation of microarray and rna-seq data the expression of four genes, identified by rna-seq as up-regulated in the ms + cultures of strain vdls. and by microarray analyses in microsclerotial-producing cultures of strain dvd-t was validated by northern blot analyses. these genes, which included two hypothetical protein encoding genes and two pigment biosynthesis genes were highly expressed at four days post-inoculation in wild-type cultures of v. dahliae grown on bma, but suppressed in a vdh mutant strain of v. dahliae grown on cma, and during growth of strain dvd-t in liquid cm. northern blot analysis also verified that the hypothetical gene vdag_ which was down-regulated in the rna-seq analysis, was up-regulated in four-day old bma-grown cultures. the expression profiles of two genes, identified as differentially expressed in the microarray analysis but not in the vdls. rna-seq analysis, verified by northern analysis; both the ace transcriptional regulator gene vdag_ and the fatty acid binding protein vdag_ were induced in dvd-t and repressed in both the vdh mutant and in liquid-grown cultures. ten genes were shown by microarray analysis to be strongly induced in strain dvd-t during microsclerotia development. to evaluate inter-experimental results the expression of the genes as determined by microarray analysis was compared to that as assessed by northern blot analysis, rna-seq and rt-qpcr. of the ten genes, eight were also up-regulated in the ms + pda cultures of strain vdls. as compared to those in the rna-seq analysis that were not producing ms on pda. the exceptions were vdag_ which was shown as down-regulated by both rna-seq and rt-qpcr, but up-regulated by both microarray and northern blot analyses, and vdag_ which was, as mentioned above, not detected as differentially expressed by rna-seq in strain vdls. under the conditions tested. discussion microsclerotia are critically important in the disease cycle of v. dahliae, and their development has been the subject of intense research for decades. though significant advances have been made in understanding the morphological and biochemical aspects of microsclerotia biogenesis, the molecular mechanisms underpinning the formation of these structures are still not well understood. the aim of the current study was to identify genes that are differentially expressed in microsclerotia-producing and noms cultures of v. dahliae, to shed further light on these molecular mechanisms. to conduct this work, we took advantage of genome-wide gene expression technologies, and also of the recently sequenced genome of v. dahliae strain vdls. for in silico functional annotation of the genes we identified as differentially expressed during ms development. the rna-seq analyses revealed a number of differentially expressed genes including those that encoded enzymes previously known to be involved in the fungal dhn-melanin biosynthetic pathway, including polyketide synthases, thn reductases, scytalone dehydratases, and laccases,. since ms maturation culminates with the synthesis and deposition of melanin granules in cell walls and intercellular spaces, the identification of the highly expressed genes encoding melanin biosynthetic-like enzymes indicated that the rna-seq libraries were appropriate for evaluation of gene expression during ms maturation. in the microarray analysis pigment biosynthesis-encoding genes likewise formed the largest group of genes up-regulated during microsclerotial production, and showing high levels of expression, an expression pattern that was confirmed by northern blot analyses of two of the putative melanin biosynthesis gene orthologs, vdag_ and vdag_ moreover, pigment biosynthesis gene expression was similarly evident in an earlier est study of v. dahliae cultures undergoing microsclerotia development. collectively, the overall consistency of these results signals the soundness of the experimental designs used herein. although the general biochemical scheme/pathway of fungal dhn melanin biosynthesis is well defined based on experimental evidence gleaned from genetic and biochemical studies, the exact identities, functions and multiplicity of all genes/enzymes involved in melanin biosynthesis, and the possible variation of function among species or strains has yet to be determined. the melanin synthesis pathway is complex, involving many regulatory, secretory and structural genes, a complexity that was made apparent by the identification of nine melanin synthesis-related genes that were detected in the rna-seq study as highly expressed in the ms + culture. some of these synthetic genes and putative regulators of melanin biosynthesis are clustered together in a  kb gene region of the v. dahliae genome. similar melanin biosynthetic gene clusters are present in other fungi, and such clusters often include one or more transcription factors, as found in this study. the rt-qpcr analyses provided confirmation of differential gene expression between ms + and noms cultures, and further enabled analyses of gene expression at several culture time points. for example the known pigment biosynthesis gene vdag_ encoding a protein highly similar to the melanin biosynthesis protein ayg, the cytochrome p gene vdag_ and the hypothetical protein-encoding gene vdag_ were significantly down-regulated in the 6 month ms + culture, relative to the expression seen for the 12 day ms + culture. the down-regulation of vdag_ in the six month 4°c culture may be indicative of completion of pigment synthesis. similarly, the function of the cytochrome p may not be required at this late stage. specific genes, such as vdag_ and vdag_ were identified by rt-qpcr analyses as being expressed in the d ms + cultures at higher levels compared to that in the d ms + cultures. that this enhanced expression is suggestive of their roles in ms biogenesis is confirmed by the corresponding genes having previously been identified as ests expressed in dms cultures, and also observed by microarray analysis to be highly expressed during microsclerotia development. morphological changes that occur during microsclerotia formation are well documented. in the initial stage of ms formation, the hyphae aggregate, swell and form numerous septa. the septate cells enlarge and become spherical, forming clusters of cells by lateral budding, and during this process of ms maturation some cells undergo autolysis, resulting in a mixture, in individual ms, of dead and live cells. the transition from vegetative growth to survival structure formation is expected to involve external and internal signals, as well as developmental reprogramming of molecular pathways that direct cell division, growth, and death. intriguingly, the v. dahliae gene vdag_ up-regulated 72-fold in the ms + culture, is highly similar to the podospora anserina cell death-related gene idi- the expression of which is induced during the vegetative incompatibility reaction. likewise, vdag_ was up-regulated in the 10 day ms + culture, and is highly similar to the p. anserina gene grg which exhibits increased expression in senescent cultures and has been implicated in control of lifespan. signaling and effector molecules that orchestrate the death of some cells during v. dahliae ms biogenesis may thus involve mechanisms similar to those that in other fungi control senescence and cell death during fungal vegetative incompatibility, with genes such as vdag_ and vdag_ involved in the developmental reprogramming of cells during ms maturation. however, the absence in the verticillium group database of homologs of genes encoding other idi proteins, as determined by blastp and tblastn, suggests divergence between p. anserina and v. dahliae in the molecular mechanisms operating in stress responses and/or cell death control. the process of sclerotia formation and maturation in filamentous fungi involves active translocation of molecules from hyphae to developing sclerotia, and exudation/excretion, from sclerotia to the cell environment, of various molecules such as water, amino acids, proteins, soluble carbohydrates, fatty acids, and enzymes. some of these molecules may be required for synthesis of storage compounds that act as energy reserves for sclerotia germination, and the observed increased expression in this study of certain transport protein genes, including those of abc, mfs, and lipid transport systems, might reflect such a scenario. secreted carbohydrate-active enzymes may function not only in the disease process, but also in remodeling of fungal cell walls during ms maturation. the beta- -glucanase encoded by vdag_ which is up-regulated in the ms + library, may, for example, contribute to cell wall modifications that accompany morphological transitions in ms development, much as beta- -glucanase is involved in morphogenesis in s. cerevisiae. in addition, fungal periplasmic trehalases, which convert trehalose to glucose, are up-regulated under nutrient limiting or starvation conditions, and the observed enhanced expression, in the ms + library, of the putative periplasmic trehalase vdag_ may therefore reflect depletion of nutrient levels in the actively growing 10-day v. dahliae cultures. it is conceivable too that reduced expression of genes encoding specific cell wall-degrading activities may also be important for ms maturation. the endochitinase gene vdag_ and the alpha-glucanase sequence vdag_ were both down-regulated in the ms library. while the s. pombe protein degrades the septum, allowing cell separation, such separation may not be necessary in v. dahliae microsclerotia, which comprise both chains and clusters of spherical cells. the chitin deacetylase encoded by vdag_ which is up-regulated in the ms + library, may on the other hand protect the fungus by converting chitin to a de-n-acetylated product like that which has previously been shown to be unsuitable as a chitinase substrate. a decrease in chitinolytic enzyme production during ms maturation, with a concomitant increase in cell wall protection, might be expected for production of the long-lived ms. in addition to the new candidate genes identified by rna-seq as differentially expressed in the ms library in this study, there was also a pattern of overlap with genes previously identified in the dms est library, and by the microarray analysis presented here. as described above, pigment biosynthesis genes formed one such clearly identifiable group, as did certain hypothetical/unknown genes. notably, however, one of the hypothetical genes identified in both analyses showed expression patterns that seem to be culture age dependent. this gene was shown by both microarray and northern blot analyses to be up-regulated in 4-day old microsclerotia-producing cultures, but by rna-seq and rt-qpcr analysis was down-regulated in 10-day and 6 month microsclerotial cultures of vdls. likewise, the acetyl coa synthase gene was induced in the 4-day old cultures, but not in the 10-day old cultures used to produce the rna-seq libraries. also, fluxes in the expression levels of certain other genes, like vdh, could have occurred at a developmental stage different than that sampled in the rna-seq study. such variation in results obtained through rna-seq vs. microarray and previous est analyses may also reflect the difference in strains used for the studies, and/or the different culture conditions under which the fungi were grown prior to harvest. while enhanced expression of cellulolytic enzymes was not expected to be observed in rna-seq libraries prepared from cultures grown on nitrocellulose membranes, it is possible that starvation of the fungus in the presence of the cellulose may induce expression of genes encoding cell wall degrading enzymes, such as was observed for vdag_ in the est analysis, and in the present microarray analysis.  information on the molecular control of microsclerotia development in v. dahliae is currently still sparse. the developmental reprogramming from the normal growth state to survival structure formation is expected to involve external and internal signals as well as unique cell signaling pathway, all of which are yet to be identified and elucidated. we envision that future functional characterization of the melanin synthesis and regulatory genes identified in this study, in conjunction with biochemical analyses, will help refine our understanding of the molecular basis of melanogenesis and ms morphogenesis in v. dahliae. some of the genes identified as up- or down-regulated in the ms + vs. noms libraries may be involved in the morphogenetic processes that regulate different aspects of microsclerotial development. in addition, similar to the results obtained in previous est analyses, approximately 50% of the genes that we identified by rna-seq lacked significant similarity to any known genes. functional analyses of some of these differentially expressed, unknown genes will undoubtedly advance our understanding of verticillium development. the microsclerotia of v. dahliae have been considered important targets for disease control, and gene products identified in this study could potentially represent new targets for disease control through prevention of survival structure development. 
verticillium wilt is difficult to control since v. dahliae is capable of persisting in the soil for to years as melanized microsclerotia, rendering crop rotation strategies for disease control ineffective. the identified gene products could therefore potentially represent new targets for disease control through prevention of survival structure development. collectively, the data acquired from these investigations provide additional insight into gene expression and molecular processes that occur during ms biogenesis and maturation in v. dahliae. differential expression of selected genes observed by rna-seq or microarray analysis was confirmed using rt-qpcr or northern hybridizations. microsclerotia of v. dahliae overwinter and germinate to produce infectious hyphae that give rise to primary infections. consequently, microsclerotia formation, maintenance, and germination are critically important processes in the disease cycle of v. dahliae. to shed additional light on the molecular processes that contribute to microsclerotia biogenesis and melanin synthesis in v. dahliae, three replicate rna-seq libraries were prepared from 10 day-old microsclerotia -producing cultures of v. dahliae, strain vdls. additional comparative analyses of gene expression in v. dahliae, under growth conditions that promote or preclude microsclerotial development, were conducted using a microarray approach with rna derived from v. dahliae strain dvd-t and from the amicrosclerotial vdh strain. analyses of these libraries for differential gene expression revealed over differentially expressed genes, including up-regulation of melanogenesis-associated genes tetrahydroxynaphthalene reductase and scytalone dehydratase, and additional genes located in a kilobase melanin biosynthetic gene cluster of strain vdls. the soilborne fungus, verticillium dahliae, causes verticillium wilt disease in plants.
 the soilborne fungus, verticillium dahliae, causes verticillium wilt disease in plants. verticillium wilt is difficult to control since v. dahliae is capable of persisting in the soil for to years as melanized microsclerotia, rendering crop rotation strategies for disease control ineffective. microsclerotia of v. dahliae overwinter and germinate to produce infectious hyphae that give rise to primary infections. consequently, microsclerotia formation, maintenance, and germination are critically important processes in the disease cycle of v. dahliae.  to shed additional light on the molecular processes that contribute to microsclerotia biogenesis and melanin synthesis in v. dahliae, three replicate rna-seq libraries were prepared from 10 day-old microsclerotia -producing cultures of v. dahliae, strain vdls., and those not producing microsclerotia. analyses of these libraries for differential gene expression revealed over differentially expressed genes, including up-regulation of melanogenesis-associated genes tetrahydroxynaphthalene reductase and scytalone dehydratase, and additional genes located in a kilobase melanin biosynthetic gene cluster of strain vdls. nearly 50% of the genes identified as differentially expressed in the ms library encode hypothetical proteins. additional comparative analyses of gene expression in v. dahliae, under growth conditions that promote or preclude microsclerotial development, were conducted using a microarray approach with rna derived from v. dahliae strain dvd-t and from the amicrosclerotial vdh strain. differential expression of selected genes observed by rna-seq or microarray analysis was confirmed using rt-qpcr or northern hybridizations.  collectively, the data acquired from these investigations provide additional insight into gene expression and molecular processes that occur during ms biogenesis and maturation in v. dahliae. the identified gene products could therefore potentially represent new targets for disease control through prevention of survival structure development. verticillium dahliaemorphogenesismicrosclerotiarna-seqgene expression 