Deep mRNA sequencing reveals stage-specific transcriptome alterations during microsclerotia development in the smoke tree vascular wilt pathogen, Verticillium dahliae
- Dianguang Xiong†1,
- Yonglin Wang†1,
- Jie Ma2,
- Steven J Klosterman3,
- Shuxiao Xiao1 and
- Chengming Tian1Email author
© Xiong et al.; licensee BioMed Central Ltd. 2014
Received: 27 August 2013
Accepted: 22 April 2014
Published: 1 May 2014
Verticillium dahliae is a soil-borne fungus that causes vascular wilt diseases in a wide range of plant hosts. V. dahliae produces multicelled, melanized resting bodies, also known as microsclerotia (MS) that can survive for years in the soil. The MS are the primary source of infection of the Verticillium disease cycle. Thus, MS formation marks an important event in the disease cycle of V. dahliae.
In this study, next generation sequencing technology of RNA-Seq was employed to investigate the global transcriptomic dynamics of MS development to identify differential gene expression at several stages of MS formation in strain XS11 of V. dahliae, isolated from smoke tree. We observed large-scale changes in gene expression during MS formation, such as increased expression of genes involved in protein metabolism and carbohydrate metabolism. Genes involved in glycolytic pathway and melanin biosynthesis were dramatically up-regulated in MS. Cluster analyses revealed increased expression of genes encoding products involved in primary metabolism and stress responses throughout MS development. Differential expression of ubiquitin-dependent protein catabolism and cell death-associated genes during MS development were revealed. Homologs of genes located in the lineage-specific (LS) regions of V. dahliae strain VdLs.17, were either not expressed or showed low expression. Furthermore, alternative splicing (AS) events were analyzed, revealing that over 95.0% AS events involve retention of introns (RI).
These data reveal the dynamics of transcriptional regulation during MS formation and were used to construct a comprehensive high-resolution gene expression map. This map provides a key resource for understanding the biology and molecular basis of MS development of V. dahliae.
KeywordsVerticillium dahliae Microsclerotia development RNA-Seq Transcriptome Gene expression Alternative splicing
Verticillium dahliae Kleb. (Eukaryota, Fungi, Ascomycota) is a ubiquitous soil-borne fungus that penetrates plant roots, enters the plant vascular system, and causes vascular wilt diseases collectively known as Verticillium wilts [1, 2]. V. dahliae can infect more than 200 plant species, including important crops, flowers, vegetables, trees, and shrubs, causing economically significant losses each year [1, 2].
Verticillium wilt is a threat to smoke tree (Cotinus coggygria Scop.) stands in China. Smoke trees are of primary importance as ornamentals, highly valued for the brilliant red leaf scenery that these trees provide in the Beijing region during autumn, especially in Fragrant Hills Park, a 160 hectare forest park in Beijing. Vascular wilt in smoke tree was reported in Fragrant Hills Park as early as 1990, and subsequently the disease spread to other smoke tree growing areas in Beijing . Symptoms of vascular wilt in smoke trees include stunted growth of the stem, early senescence of leaves, and early mortality. Without adequate control, the famous “red leaf scenery” of Fragrant Hills and other areas is threatened by the detrimental effects of the Verticillium wilt. Currently available fungicides and other control measures are not effective in controlling the disease due in part to the ability of the fungus to survive for long periods in soil.
The survival of V. dahliae in soil depends on the production of melanized, multicellular structures known as microsclerotia (MS) . Melanin deposition and a thickened cell wall enable the MS of the pathogen to resist UV irradiation, temperature extremes, enzymatic lysis, and fungicidal activities . The MS can survive in soils in the absence of a host plant for as long as 10 years, and are the primary infectious propagules of the Verticillium wilt disease . The MS germinate to form hyphae in the soil, and penetrate the plant roots, where the fungus colonizes the xylem tissue of the plant vascular system. As wilt symptoms progress, V. dahliae produces MS in dying plant tissues, which are returned to the soil to initiate new primary infections. Thus, the production of MS represents a significant developmental event in the life cycle of V. dahliae.
The morphological events of MS formation have been well studied by both light and electron microscopy [8–11]. In the early stages of MS development, hyphae become swollen, vacuolated, and form numerous septa. Subsequently, clusters of hyphal cells form in the swollen hyphae that resemble the microsclerotial initial [11, 12]. In the final phase of MS formation, melanin particles are extruded into the interhyphal spaces of the microsclerotium, and peripheral microsclerotial cells are killed by autolysis . The genes involved in melanin biosynthesis have been identified and their functions have been characterized [10, 13–15]. The results indicate that melanin is necessary for the formation of fully functional MS . However, little is known about the molecular pathways involved in MS formation.
Genome-wide identification of genes expressed during MS formation is a first step in elucidating the pathways and molecular mechanisms underlying MS formation in V. dahliae. Methods for genome-wide expression analyses include expressed sequence tag (EST) analysis [16, 17], suppression subtractive hybridization (SSH) [18, 19], serial analysis of gene expression (SAGE) , massive parallel signature sequencing (MPSS) [21–23] and RNA-Sequencing (RNA-Seq) [24–26]. Next-generation sequencing (NGS) technologies have provided new platforms for comprehensive transcriptional studies [27–31]. Transcriptome sequencing is an efficient means to generate transcriptomic data, and RNA-Seq is one approach transcriptome profiling that provides highly accurate measurements of gene expression by counting the number of sequencing reads, which map to a genome or annotated transcripts [28, 32], and further enable genome-wide identification of coding sequences, gene structure, alternative splicing . Transcriptomic data produced by RNA-Seq methods have increased our understanding of gene expression involved in growth and development of pathogenic fungi [30, 34–39]. For example, transcriptional analysis of appressorium formation in the rice blast fungus Magnaporthe oryzae revealed the role of autophagy, lipid metabolism and melanin biosynthesis, and a Pmk1 MAPK kinase as a key global regulator in appressorium differentiation .
Resources available to facilitate transcriptome analyses of MS formation include transcript data for 10,535 genes and the 33.8 MB genome sequence for V. dahliae, strain VdLs.17 (Broad Institute Verticillium Group Database) . Subsequent characterizations of this sequence resource also provide useful information to place transcriptomic studies in context. For example, comparison of the genome structure of V. dahliae strain VdLs.17 to that of the Verticillium alfalfae strain VaMs.102 revealed four lineage-specific (LS) regions of about 350 kb in length present in VdLs.17 but absent in the VaMs.102 strain. The VdLs.17 LS regions encoded 354 predicted genes, some of which had been associated with virulence and host range specificity. Only about 7.0% the LS genes encode predicted secreted proteins, and the LS regions were nearly devoid of “housekeeping” type genes . Additional comparative genomics analyses of multiple V. dahliae strains indicated that the LS regions are diverse in length and gene content, enriched for in planta-expressed genes, and that chromosomal rearrangements associated with LS regions in V. dahliae are common . An additional valuable resource includes expressed sequence tag (EST) libraries previously used to identify expressed genes in V. dahliae during pathogenic growth and MS development in V. dahliae. Neumann and Dobinson  obtained about 1000 ESTs, many of which corresponded to melanin biosynthetic enzymes, exclusive to the developing MS culture type. The analysis of genes associated with specific ESTs has accelerated molecular characterization of MS formation. Recently, Duressa et al. performed a RNA-Seq analysis between MS producing cultures and those not producing MS in V. dahliae, revealing over 200 significantly expressed genes involved in melanin synthesis and other processes . While this work focused on up- or down-regulation of gene expression between these two culture types, the focus was not on assessing gene expression associated with gradual developmental changes during MS formation.
In addition to those resources, several individual genes involved in MS formation have been characterized in V. dahliae[12, 44–47]. VDH1, encoding a class II hydrophobin, is one of the many genes significantly differentially expressed during MS formation, and VDH1 is required for MS formation of V. dahliae[12, 45]. VMK1, encoding a mitogen-activated protein kinase, modulates MS formation ; mutation of VdGARP1, encoding a glutamic acid-rich protein, significantly delays development of melanized MS . The non-LS copy of VdHOG1, a homolog of the high osmolarity glycerol response protein kinase, positively regulates MS formation (Xiao, et al., unpublished data), and the G protein β subunit negatively controls MS formation . Functional characterizations of these individual genes have provided valuable insight into the genetic control of MS formation, yet there remain major gaps in our understanding of the molecular determinants that trigger and regulate MS formation in V. dahliae.
The objectives of this work were to (1) examine developmental stage-specific gene expression during MS formation in V. dahliae and (2) to identify genes differentially expressed in this developmental process. To accomplish these aims, extensive microscopy analyses were carried out to initially characterize four different stages of MS development in a smoke tree strain of V. dahliae. RNA-Seq analyses were employed to analyze transcript profiles of 10,158 genes (96.4% of the total predicted reference genes of the VdLs.17 strain) and global patterns of gene expression in V. dahliae using four identified MS developmental stages as reference points. This enabled identification genes significantly differentially expressed during stages of MS development, revealing major metabolic processes and signal pathways associated with MS development in V. dahliae. Elucidation of the molecular mechanisms that govern MS formation in V. dahliae may be useful in designing novel strategies to control Verticillium wilt, not only for the smoke tree pathogen, but also related pathogenic strains and other Verticillium spp. that produce MS .
Microscopic analyses of MS development
Overview of the V. dahliaetranscriptome
Insert size (bp)a
Perfect match readsd
Reads mapped to genesf
Of the mapped reads, 114,769,683 (72.4% of total) reads were perfectly mapped to the V. dahliae genome without mismatch. Furthermore, 56.9% of the total reads could be mapped to the annotated genes with less than 2 bp mismatches, indicating that almost 30.0% of the total reads mapped to non-annotated regions, including the intergenic regions or other non-coding regions. The numbers of differentially expressed perfectly matched reads and reads mapped to the annotated genes in each stage were not significantly different (Table 1, Additional file 1: Figure S1). The unmapped percentages of reads were found to be lower (between 12.6% and 15.9%).
Distribution of gene expression values among developmental stages examined
FPKMa > 0
0 < FPKM < =1
1 < FPKM < =10
10 < FPKM < =100
FPKM > 100
Clustering analysis reveals enrichment of particular gene categories expressed during MS formation
According to the gene expression pattern described earlier, and the cluster analysis results, a large variety of genes were differentially expressed during MS formation. To ascribe gene functions to those genes displaying differential expression patterns, gene ontology (GO) enrichment analysis was performed for genes from the top 18 clusters shown in Figure 3B by Blast2GO with Fisher’s Exact Test, and compared with the whole genome background filtered with false discovery rate (FDR) correction (≤0.01). The results of enriched GO terms revealed an overrepresentation of different gene functions in certain clusters. For example, genes encoding protein metabolic processes, such as ribonucleoprotein complex biogenesis, primary metabolic processes, and stress responses, were enriched in cluster 1, which was expressed at increased levels at each stage compared to the CO stage (Figure 3B). The genes in cluster 5 function mainly in signaling pathways, and increased in expression until MS3, at which point there were declining levels of expression (Figure 3B). Genes of cluster 7 were functionally enriched in transport. The functional enrichment of cluster 8 revealed cofactor metabolic processes, oxidation-reduction processes, and chromosome segregation. However, analyses did not reveal functional enrichment in other clusters, such as clusters 2, 4, 9, 10, 12, 13, 16 and 18. The full list of enriched GO terms and corresponding genes are provided in Additional file 3: Table S1.
Gene expression profile during MS development
RNA-Seq provides digital readings of gene expression levels . To determine which genes were expressed at each developmental stage, we examined the dynamics of gene expression throughout MS development in the genome-wide transcriptomic data. The distribution of gene expression values was different among the six stages studied. Nearly 12.0% of the expressed genes were detected with low expression values (0 < FPKM ≤ 1) in stages CO and GC, while only 8.0% (average) were detected in stages MS1-MS4, suggesting that many genes expressed with 0 < FPKM ≤1 in CO or GC stages were elevated transcript levels during the MS formation. However, the number of genes with moderate expression values (1 < FPKM ≤100) or high expression values (FPKM >100) in stages MS1-MS4 were increased relative to stages CO or GC, suggesting up-regulation of genes to meet the requirements of MS formation (Table 2). The number of expressed genes with FPKM >1 were gradually increased, from 8,818 (MS1) to 9,267 (MS4) (Table 2).
GO enrichment terms of significantly regulated genes during MS1-MS4 stages vs stage CO (p value <0.05)
Ribonucleoprotein complex biogenesis (GO:0022613)
Cellular carbohydrate metabolic process (GO:0044262)
Cellular component biogenesis (GO:0044085)
Cellular protein metabolic process(GO:0044267)
Generation of precursor metabolites and energy (GO:0006091)
Gene expression (GO:0010467)
Structural molecule activity (GO:0005198)
Peptidase activity (GO:0008233)
Motor activity (GO:0003774)
Oxidoreductase activity (GO:0016491)
Protein kinase activity (GO:0004672)
Hydrolase activity (GO:0016787)
Transporter activity (GO:0005215)
Oxidoreductase activity (GO:0016491)
Differential expression of ubiquitin-dependent protein catabolism and cell death-associated genes during MS development
Some of the MS cells undergo autolysis or death during MS formation in V. dahliae. According to the GO categories and enrichment analysis, there were more than twenty genes involved in protein metabolic processes, most of which participated in proteolysis. Among the genes involved in protein metabolic processes, five were involved in proteasome formation, and included VDAG_00111 (proteasome subunit alpha type 6), VDAG_08991 (proteasome component pup2), VDAG_02924 (proteasome component pre3 precursor), VDAG_03131 (proteasome component pup3), VDAG_04256 (proteasome subunit beta type 7 precursor) (Additional file 9: Figure S6A). Genes encoding products involved in protein modification processes were also enhanced during MS formation, such as ubiquitination, important for proteasome-mediated degradation of proteins .
Other genes involved in autophagy and cell death in V. dahliae were identified. In total, 23 autophagy genes were identified in V. dahliae, which were then divided into two groups, nonselective autophagy and selective autophagy . Expression profiles of these genes showed stable expression patterns across developmental stages examined, except VDAG_03183 (nonselective) and VDAG_01208 (selective), which were up-regulated during MS formation (Additional file 9: Figure S6B). In addition, some genes encoding the Het domain were up-regulated during MS formation in V. dahliae, although over 90.0% of the Het-related genes were neither up- nor down-regulated during MS formation (Figure 6C). Vegetative incompatibility is an apoptotic-type heterokaryon incompatibility, and proteins containing a Het domain mediate the apoptotic-type pathway [55–59]. Potentially, cell death observed during MS formation in V. dahliae is regulated through mechanisms that also mediate vegetative incompatibility.
Analyses of lineage-specific gene expression during MS development
Transcription factors encoded within the LS regions potentially regulate gene expression of not only LS-associated genes, but also other core genes in the genome of V. dahliae that have roles in MS development. We followed the expression patterns of bZIP transcription factors to ascertain a potential role of these genes in regulating development in V. dahliae. Among 20 bZIP transcription factor-encoding genes of V. dahliae, strain VdLs.17, five genes (VDAG_02348, VDAG_02408, VDAG_02411, VDAG_02415 and VDAG_09148) are located within the LS regions and were separated in a distinct clade with other bZIP transcription factors in a phylogenetic analysis (Additional file 10: Figure S7), similar to those results previously reported  All five homologs of these LS-encoded genes, with the exception of VDAG_09148, were not differentially expressed during MS formation in the smoke tree strain of V. dahliae. Furthermore these genes are Verticillium-specific (Additional file 11: Figure S8).
Expression profiles of genes involved in carbohydrate metabolism and melanin biosynthesis during MS formation
Genes involved in carbohydrate metabolic process were significantly up-regulated during the MS formation based on GO annotation analysis. To further study the dynamics of hexose metabolism during MS formation, glycolysis/gluconeogenesis, tricarboxylic acid, pentose phosphate and glyoxylate pathways were examined (Figure 4A, Additional file 6: Table S2). Expression profiles showed that genes controlling hexose metabolism pathways were differentially expressed during MS formation (Additional file 12: Figure S9). For example, two key enzyme encoding genes VDAG_04087 (hexokinase) and VDAG_01206 (pyruvate kinase) in glycolysis were significantly up-regulated during MS1-MS4 stages, indicative of an up-regulation of glycolysis during MS formation. Conversely, two genes that are critical for gluconeogensis (VADG_07446 and VDAG_10101) were down-regulated.
Acetyl-CoA is an important intermediate product of metabolism and plays a very important role in the cell’s energy requirement, metabolic pathways, and appressorium formation in rice blast disease . RNA-Seq analyses of genes expressed in MS formation revealed up-regulation of four genes in strain XS11 of V. dahliae (corresponding to VDAG_08164, VDAG_09433, VDAG_06356 and VDAG_01642) that are candidate genes involved in catalyzing the pyruvate to generate acetyl-CoA, suggesting an increase in the amount of acetyl-CoA during MS formation.
Discovery of alternative splicing
The formation of MS in V. dahliae is critically important for survival and propagation of this fungus, representing a significant developmental event in the disease cycle. The long term survival of the MS in soil poses a major obstacle for effective control of Verticillium wilts through cultural practices such as crop rotation. The structure of the MS, including a thickened cell wall and heavy melanin deposition, protects the pathogen from various environmental assaults and even fungicidal activity . This study was undertaken to begin elucidating of the genetics and biochemical processes underpinning MS development, which may yield insight into control strategies to combat vascular wilt disease of smoke trees, and other plant hosts of economic or aesthetic importance.
In this work, RNA-Seq was performed on samples from six developmentally distinct stages, ranging from conidia (CO) and germinating conidia (GC) through four stages of MS formation and maturation (MS1 to MS4). Due to the actual difficulties of obtaining MS from real condition such as in plant or soil, especially during MS development, we obtained MS in vitro. The rationale for this approach was to define MS-associated gene expression during MS formation. Analyses of MS formation on an artificial surface ensured that gene expression data were exclusively from the fungus, rather than the smoke tree host. It is also technically difficult to sample different developmental stages of MS in plant tissues and soil. Likewise, MJ Neumann and KF Dobinson  employed a method to collect MS samples for studies of gene expression during MS development in V. dahliae using EST analyses from in vitro cultures. Given the limitations of EST-based analyses and advancements in NGS technologies, the dynamics of transcriptome expression during MS formation were studied using the relatively new NGS technology of RNA-Seq. This technology enabled identification of metabolic pathways of interest, as well as alternative splicing. Analyses of these transcriptome data revealed that this was an effective approach, providing an extensive catalog of expression values over the range of six distinct developmental stages. Moreover, of the 158.5 million sequenced reads, 114.8 million reads uniquely mapped to the genome without mismatch, and 90.1 million reads mapped perfectly to the annotated genes of the V. dahliae strain VdLs.17 sequence.
The increased expression of particular genes observed in this study was expected; including those involved in melanin biosynthesis, or selected genes that were previously identified as important in MS formation. Melanin biosynthetic genes in V. dahliae were expressed at relatively high levels during MS formation consistent with the culture phenotype (increased pigmentation) observed. In addition, VDH1 (VDAG_02273), a hydrophobin protein-encoding gene, is involved in MS formation, and VDH1 is specifically expressed in developing microsclerotia [12, 45]. In concurrence with the results of that study, the expression level of VDH1 was significantly increased in the stages of MS1-MS3, especially in MS1 and MS2 (Additional file 16: Table S6). The finding of the developmentally regulated expression of VDH1 in this study may also explain the lack of differential expression of VDH1 observed in RNA-Seq analyses of gene expression in which only 10 day MS-producing cultures and 10 day cultures not forming MS were compared . In the study of Duressa et al.  stages equivalent to MS1 or MS2 were not analyzed.
Analyses of GO functional category enrichment indicated that carbohydrate and protein metabolic processes, and ribonucleoprotein biosynthetic processes were significantly enriched among up-regulated genes, while transport processes were significantly enriched among down-regulated genes. Functional classification revealed that peptidase, protein kinase, and hydrolase activities were significantly up-regulated during MS1-MS4 stages. Among the carbohydrate metabolic processes, several genes involved in energy production were enriched; such as VDAG_04087 (hexokinase), VDAG_01206 (pyruvate kinase), and VDAG_03029 (enolase). These results suggested that these differentially expressed genes are involved in a broad range of physiological functions, especially in proteolysis, protein modification, during MS formation. Enrichment of peptidase activity may also accompany processes associated with cell death or autolysis in MS formation . On the other hand, most genes encoding plant cell wall-degrading enzymes, such as the polysaccharide lyase and carbohydrate esterase gene families, showed low expression levels during MS formation. The carbohydrate-binding module 1 gene family, which is significantly enriched in V. dahliae relative to most other fungi , also showed low expression at the MS developmental stages analyzed in this study.
Stage-specific differences were extensive in V. dahliae at the gene regulatory level during MS development. Among the genes annotated in V. dahliae, strain VdLs.17, dozens of genes that were differentially expressed during MS formation were identified (Additional file 7: Figure S5). This indicated that MS-stage-specific gene regulation occurs at the transcriptome level during MS formation. These data provided a basis for further assessing the roles of individual genes, especially those MS-specific genes potentially involved in cellular differentiation and MS formation.
The four major lineage-specific (LS) regions identified in the genome of strain VdLs.17 of V. dahliae are thought to contribute to adaptation to different host niches  and encode virulence factors . While the total LS region contents differ between strains of V. dahliae, a total of 354 genes are located in the four LS regions of strain VdLs.17; and some of these genes encode proteins involved in lipid metabolism, plant-fungal interactions, and transcriptional regulation . Interestingly, analyses of three EST libraries produced under low nutrient or complete medium revealed the LS genes of VdLs.17 were significantly up-regulated compared to genes in the genome core sequence. Furthermore, recent work indicates enrichment for in planta-expressed LS genes in the V. dahliae strain JR2 . In this study, the majority of genes in LS regions of V. dahliae were either not expressed or showed low expression in MS1 to MS4, suggesting that genes of the LS regions are suppressed at the developmental stages examined relative to those of the genome core, and also may be influenced by the differing gene content of LS regions between different strains examined. Consistent with this hypothesis, expression of VDAG_02354, encoding a high osmolarity glycerol response (HOG1) protein, and located in LS region 1, was not detected in all six developmental stages examined in this study. The other copy of the HOG1 gene (VDAG_08982 was identified outside of the LS regions in the genome of VdLs.17 and expressed at a higher level during MS formation. The deletion of the HOG1 gene (VDAG_08982) from the smoke tree strain resulted in significantly reduced and delayed production and developmental progress of MS in vitro (Xiao et al., unpublished data). Additional analyses revealed that genes encoding LS region-associated bZIP transcription factors were not expressed, or showed low expression in the stages of MS formation, while other bZIP transcription factors located in non-LS regions were expressed during MS formation.
The RNA-Seq analysis provided useful information on alternatively spliced transcripts in V. dahliae. These analyses are critical for studies that aim to assess gene regulation and gene function in MS development and in other developmental processes. The number of genes undergoing AS were mainly estimated based on overlapping introns, which does not take intron retention and splice junctions into account . Over 95.0% AS events were due to intron retention. As in other fungi, such as Aspergillus oryzae and Ustilago maydis, RI is the predominant form of AS [66, 67]. A total of 5,212 genes underwent AS events, while there was an average of 3,250 genes that underwent AS events in each stage. This suggests that stage-specific genes undergo AS at different times during MS development. Further screens of these genes are required to increase understanding the role of AS in the development and pathogenesis in the smoke tree wilt fungus. Assembly of transcription from short sequencing reads remains a computational challenge. Therefore, it is essential to validate the predicted AS events by laboratory and field experiments.
In summary, an RNA-Seq strategy was employed to gain insight on the biology and molecular basis of MS development of smoke tree vascular wilt fungus, V. dahliae. Functional categories of genes such as those involved in carbohydrate metabolism, proteolysis, and cell death were differentially regulated during MS formation. Comprehensive, high-resolution gene expression maps enabled detection of a large number of AS events that provide a key resource for further studies that aim to understand the molecular underpinnings of MS development and other developmental processes in this fungus. Further, de novo assembly of transcripts from RNA-Seq data represents a potential avenue for gene annotation [68–71]. The analysis of splicing events detected herein may shed light on alternate splicing in V. dahliae, and help to understand the roles of AS in MS formation and other developmental processes.
In this study, we have conducted a RNA-Seq analysis of the MS developmental process in smoke tree wilt fungus V. dahliae XS11. A global view of gene expression profiles and a large-scale stage-specific transcriptome alterations during MS development are revealed. Further analysis show that genes involved in glycolytic pathway, melanin biosynthesis and protein catabolism are dramatically up-regulated in MS stages. In addition, a large number of AS events are detected among CO, GC and MS stages. Our results provides a key resource for understanding the biological and molecular basis of MS development of V. dahliae.
Fungal strain and growth conditions
Verticillium dahliae strain XS11, which was single spore-isolated from a smoke tree in Fragrant Hills Park, Beijing, was used in these experiments. Cultures were initially grown on PDA (potato dextrose agar). Conidia were harvested from cultures grown in liquid CM, as previously described . Conidia were collected by filtering through two layers of Miracloth (Calbiochem, USA), and the conidial suspension was sedimented by low speed (4000 rpm) centrifugation. The conidia were cultured at a concentration 105 conidia/ml for germination in the liquid basal medium (BM, 10 g/L glucose, 0.2 g/L sodium nitrate, 0.52 g/L KCl, 0.52 g/L MgSO4.7H2O, 1.52 g/L KH2PO4, 3 μM thiamine HCl, 0.1 μM biotin, 15 g/L agar, kindly provided by Dr. Katherine Dobinson, Agriculture and Agri-Food Canada, London, Canada) by shaking at 30 rpm for 12 hours at 24°C. Germinating conidia were harvested similarly.
Microsclerotial developmental stages
To observe the developmental process of MS formation of V. dahliae in stages MS1-MS4, a cellulose membrane (Ø =80 mm; pore size = 0.22 μm) was placed on BM agar and a suspension of 105 conidia/ml of V. dahliae strain XS11 was spread over the cellulose membrane, and incubated in the dark at 24°C. Developmental stages were observed under light microscopy (DM2500, Leica) at 12 hour intervals after incubation on the membrane until 7 days post incubation (dpi). After 7 dpi, the observations were conducted every 2 days for 1 week.
RNA extraction and validation of expression by RT-PCR and qRT-PCR
Total RNA was extracted from conidia, germinating conidia, and the MS1-MS4 stages of MS formation by using TRIzol Reagent (Invitrogen) and purified with the RNA Mini Kit (Ambion) according to the manufacturer’s instructions. All samples were ground to a fine powder with a mortar and pestle in liquid nitrogen. Total RNA was eluted in RNase-free water and stored at −80°C until further use. For each sample, two biological replicates were used for library preparations. The integrity and quantity of RNA was determined using a Qubit fluorometer (Invitrogen), agarose electrophoresis and the Agilent Bioanalyzer 2100 (Additional file 17: Table S7).
The RNA samples for RT-PCR were incubated at 37°C for 30 min with DNase I (RNase-free) (TaKaRa) to remove DNA contamination before reverse transcription. The mRNA were enriched using Oligo DT, then were transcribed to cDNA using SuperScript III Reverse Transcriptase (Invitrogen). The qRT-PCR was carried out using SYBR green (SuperReal Premix Plus; TIANGEN, China) methodology and the ABI 7500 real-time PCR system (Applied Biosystems, USA). The V. dahliae β-tublin gene was used as internal reference for all the qPCR analyses. Analyses of each gene were conducted in quadruplicate. Relative gene expression was calculated according to the ΔΔCT method. The primers used are described in Additional file 18: Table S8.
Library preparation for RNA-Seq
Libraries were prepared using RNA-Seq sample preparation kit from Illumina and poly(A) mRNA was enriched from total RNA using oligo (dT) beads. Fragmentation buffer was added for breaking mRNA to short fragments. Using these fragments as template, first-strand cDNA was synthesized by reverse transcription with a random hexamer primer. The second-strand cDNA was synthesized using buffer, dNTPs, RNase H and DNA polymerase I according to kit manufacturer instructions.. Short fragments were purified with QiaQuick PCR extraction kit (Qiagen) and resolved with EB buffer. Sequencing adaptors were added and amplified with PCR. Agarose gel electrophoresis was used to select the fragments with about 200 bp in size. Finally, the libraries were sequenced on Illumina HiSeq™ 2000 (Beijing Genomics Institute, Shenzhen) to produce 90 bp paired-end reads.
Mapping reads to the V. dahliaereference genome
Low quality (Q ≤5) reads containing adapters were removed from the raw reads, and Tophat software (version 2.0.0)  was used to align the filtered reads to the published reference genome of V. dahliae strain VdLs.17 (http://www.broadinstitute.org/annotation/genome/verticillium_dahliae/MultiHome.html) and to predict exon splice sites allowing less than two mismatches. The pipeline of RNA-Seq analysis is shown in Figure 1B. Total mapped reads were obtained with the parameters “-G (Vd) -r 20 --segment-length 30” provided by Tophat and perfect mapped reads were obtained using the parameters “-G (Vd) –r 20 --segment-length 30 –read-mismatches 0” provided by Tophat. In addition, reads only mapped to the annotated genes of V .dahliae were carried out with the parameters “-T -G (Vd) –r 20 --segment-length 30”.
Transcript assembly and genes expression analysis
Cufflinks software (version1.30)  was used to assemble the individual transcripts from RNA-Seq reads which had been aligned to the genome of V. dahliae, strain VdLs.17, with Tophat. Gene expression level was calculated using FPKM (fragments per kilobase of transcript per million mapped fragments) in Cufflinks. Due to their low reliability for assembly purposes, Cufflinks filtered low abundance transcripts using the default parameter. Cuffmerge, a component of Cufflinks, was used to merge the transcripts of several samples. Cuffdiff, a package of Cufflinks, was used to identify differentially expressed genes among samples (p value ≤0.05), and the CummeRbund R package  was used to visualize differentially expressed genes. Fold changes in gene expression were calculated with log2 FPKM compared with that of the control sample, CO. A MultiExperiment Viewer  was used to visualize changes in gene expression, cluster analyses, PCA analyses and CAST analyses with threshold affinity value 0.9 and other default parameters. The global views of gene expression patterns were visualized by Circos . Pearson correlation coefficient was calculated among the six samples according to genes’ expression profiles. Venn diagram was drawn through the interactive tool of VENNY .
Gene ontology and functional annotation
Gene Ontology was identified in the GO database through Blast2GO  software using the in silico translated sequence and default parameters. In addition, functional annotation, classification, and enrichment analysis were performed by Blast2GO software with the default parameters.
Alternative splicing analysis
Alternative splicing events were identified by SplicingViewer . First, reads were aligned to the reference genome by Bwa allowing less than two mismatches . Second, Samtool was used to obtain unmapped reads from the first step , to detect the splice junctions, and obtain the splice junction sequences. Unmapped reads were mapped to the splice junction sequences by Bwa with less than two mismatches. Seven types of alternative splicing events were identified by AlternativeSplicing.jar (http://bioinformatics.zj.cn/splicingviewer/index.php).
Amino acid sequences were aligned using Clustal X (version 1.83) without masking unreliable aligned positions . Phylogenetic trees were constructed using Mega 5.0 by Maximum Likelihood method with at least 1000 bootstrap replications .
Availability of supporting data
RNA-Seq data were submitted to the NCBI SRA database (http://www.ncbi.nlm.nih.gov/Traces/sra/) with the accession number: SRR1232601, SRR1232602, SRR1232630, SRR1232631, SRR1232632, SRR1232674, respectively.
Next generation sequencing
Microsclerotia formation 60 h
Microsclerotia formation 72 h
Microsclerotia formation 96 h
Microsclerotia formation 14d
days post incubation
Fragments per kilobase of transcript per million mapped fragments
Cell wall degrading enzymes
Expressed sequence tags
Open reading frame
Mitogen-activated protein kinase
High osmolarity glycerol
G protein–coupled receptor
Regulator of G-protein signaling
Mutually exclusive exon
Alternative 5′ splicing site
Alternative 3′ splicing site
Alternative first exon
Alternative last exon
We are grateful to the Center for Computational Biology, Beijing Forestry University for providing the Linux platform. The research was supported by National Natural Science Foundation of China (31370013 and 31000302), the Fundamental Research Funds for the Central Universities (NO. TD2011-06 and YX2013-10), and the Research Fund for the Doctoral Program of Higher Education (20100014120018) to YW.
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