Abstract
Background
The eukaryotic unicellular protist Plasmodiophora brassicae is an
endocellular parasite of cruciferous plants. In host cortical cells,
this protist develops a unicellular structure that is termed the
plasmodium. The plasmodium is actually a multinucleated cell, which
subsequently splits and forms resting spores. The mechanism for the
growth of this endocellular parasite in host cell is unclear.
Results
Here, combining de novo genome sequence and transcriptome analysis of
strain ZJ-1, we identified top five significant enriched KEGG pathways
of differentially expressed genes (DEGs), namely translation, cell
growth and death, cell communication, cell motility and cancers. We
detected 171 proto-oncogenes from the genome of P. brassicae that were
implicated in cancer-related pathways, of which 46 were differential
expression genes. Three predicted proto-oncogenes (Pb-Raf1, Pb-Raf2,
and Pb-MYB), which showed homology to the human proto-oncogenes Raf and
MYB, were specifically activated during the plasmodial growth in host
cortical cells, demonstrating their involvement in the multinucleate
development stage of the unicellular protist organism. Gene networks
involved in the tumorigenic-related signaling transduction pathways and
the activation of 12 core genes were identified. Inhibition of
phosphoinositol-3-kinase relieved the clubroot symptom and
significantly suppressed the development process of plasmodia.
Conclusions
Proto-oncogene-related regulatory mechanisms play an important role in
the plasmodial growth of P. brassicae.
Electronic supplementary material
The online version of this article (10.1186/s12864-018-5307-4) contains
supplementary material, which is available to authorized users.
Keywords: Plasmodiophora brassicae, Proto-oncogenes, Clubroot, Cancer,
Tumor, Brassica napus
Background
Plasmodiophora brassicae Woron. is an obligate intracellular plant
parasite in the protist subgroup Rhizaria [[42]1]. It is one of the
most economically important pathogens of cruciferous plants [[43]2]. P.
brassicae induces galls on the infected roots of cruciferous plants,
such as oilseed rape and cabbage [[44]3]. It severely disrupts the host
root functions by inducing the formation of deformed galls, which
reduce the uptake of water and nutrients from the soil and the growth
of the roots [[45]4]. Clubroot causes huge economic losses to oilseed
rape and cruciferous vegetable crops, accounting for up to 10–15% loss
of cruciferous crops production globally [[46]5, [47]6].
When host signals (root exudates) are sensed, the resting spores of P.
brassicae germinate and release primary zoospores. The zoospores attach
to and invade the root hairs. P. brassicae forms primary plasmodia and
primary zoosporangia in root hairs. This process is termed the
asymptomatic root hair infection stage [[48]2, [49]7]. The secondary
zoospores are released from the broken root hairs and directly invade
host cortical cells. P. brassicae forms galls on host roots or rootlets
by the modification of hormone levels [[50]8–[51]11]. In the cortical
cells, P. brassicae form the secondary plasmodia [[52]12]. In each
plasmodium, the nuclei continuously divide, but the plasmodium does not
split. Thus, a plasmodium is a single cell with multiple nuclei. After
meiotic cleavage, the multinuclear plasmodium returns to the haploid
state [[53]13]. Finally, the internal space of plant cells become
filled with the mature resting spores [[54]12].
The genome sequences of the single spore isolate Pbe3, Pb3, and Pb6 of
P. brassicae have been determined. In addition, studies have shown that
the significant reduction in intergenic space and low repeat content
contribute to the compact genome of P. brassicae, and some genes
involved in the regulation of the plant growth hormones (cytokinin and
auxin) and ancestry of chitin synthases have been identified [[55]3,
[56]14]. These genome data have helped clarify the biological
properties of P. brassicae. However, the mechanisms involved in special
developmental stages of P. brassicae, especially the cell division
events of multinucleate secondary plasmodia during cortical infection,
remain unclear. This prompted us to investigate the molecular
regulation mechanisms of the pathogen in response to growth and
development characteristics by the combined de novo genome sequencing
and transcriptome analysis of cell type-specific stages.
Multicellular organisms have evolved more sophisticated, higher-level
functional capability by a division of labor among component cells with
complementary behaviors [[57]15]. However, dissolution and death of
multicellular individuals due to conditions like cancer occur when the
cooperation of component cells in multicellular species breaks down
[[58]16, [59]17]. When cells of multicellular organism fail to regulate
their growth within the normal program of development, they face the
challenge of cancer, which has often been described as a loss of
multicellularity.
Unlike the closely regulated and controlled growth of normal cells,
most malignant cells have some common features, including a strong
proliferative activity, self-sufficiency in growth signals, limitless
regulation of replicative potential (in which antigrowth signals are
ignored and replication continues in the presence of a growth signal),
and evasion of cell death by a variety of pathways [[60]18]. Cancer
occurs in almost all metazoans in which adult cells proliferate, which
suggests that the regulatory mechanism of tumorigenic cells is
deep-rooted in the evolutionary history of metazoans [[61]19]. The
c-myc proto-oncogene encodes a transcription factor (Myc) with
oncogenic potential. Genetic studies of an ancestral myc proto-oncogene
from Hydra have dated the human oncogene myc back at least 600 million
years [[62]20, [63]21]. It has been argued that oncogenes are ancient
and highly conserved, and that cancer cells are not newly evolved types
of cell, but rather are heirs to a basic mode of survival that is
deeply embedded in multicellular life. In this scenario, cancer is an
atavistic state of multicellular life [[64]19].
The possibility of the existence of cancer in other multicellular
organisms or even in unicellular protozoa is contentious
[[65]22–[66]24]. Despite the demonstration that differentiated plant
cells have the unique potential of reverting to a pluripotent state,
proliferating, and transdifferentiating, the current knowledge suggests
that plant cells are highly resistant to oncogenic transformation and
strikingly tolerant to altered levels of cell-cycle regulators and to
hyperplasia [[67]25]. So far, it is still unclear whether the
regulatory mechanism of tumorigenic cells is involved in the growth and
development of unicellular protozoa.
The current study is an initial multi-omics analysis aimed at providing
novel insights into the regulatory pattern of the critical
multinucleate cell division lifestyle of P. brassicae. The data suggest
that mechanisms of the proto-oncogenes are highly conserved and
deep-rooted in evolutionary history, and play important roles in the
specific developmental stage of the examined eukaryotic unicellular
organism.
Results
Cell type-specific transcriptome analysis of P. brassicae
Adopting a whole-genome shotgun sequencing strategy, a combined de novo
genome sequence data and cell type-specific transcriptome analysis of
P. brassicae, including multinucleate secondary plasmodia stage, we
constructed the final 24.1 Mb genome assembly with high-quality clean
reads.
During the cortical stage of its life cycle, P. brassicae can be
multinucleate in a cell termed the plasmodium (Fig. [68]1a). The
transition from the unicellular state to the multinucleate secondary
plasmodium with a rapidly proliferating cell type is an important
course of cell developmental. It leads to the formation of galls
containing mononucleate resting spores that occupy most of the cell
[[69]12, [70]26] (Fig. [71]1b, c). To investigate the transition from
unicellular cells to multinucleate secondary plasmodia, we conducted
transcriptome analysis of P. brassicae at three time points-the
unicellular resting spore (RS) stage, unicellular germinating resting
spore (GS) stage (Fig. [72]1d), and multinucleate early cortical
infection (IN) stage. Compared to the RS stage, in the IN stage 508
genes were up-regulated and 89 genes were down-regulated.
Fig. 1.
Fig. 1
[73]Open in a new tab
Plasmodia, resting spores, and germination of resting spores
(zoospores) of P. brassicae. a Multinucleate plasmodia in a root
cortical cell of B. napus; the left upper corner shows an enlarged
portion from the white square frame showed membranes of two plasmodia
(white arrows); N, nucleus. b Mature resting spores in cortical cells
of roots of B. napus. c Resting spores observed by transmission
electron microscopy. d Germinated resting spores (large) and zoospores
(small) stained with 4′,6-diamidino-2-phenylindole. The resting spores
are empty, with no nuclei present
Furthermore, compared with the GS stage, the germination of the resting
spores and release of primary zoospores in the IN stage were associated
with the up-regulation of 594 genes and the down-regulation of 86 genes
(Fig. [74]2a). The majority of the differentially expressed genes
(DEGs) were up-regulated during the IN stage with multinucleate
secondary plasmodia (Fig. [75]2a and Additional file [76]1: Figure S1),
suggesting that the transition from unicellular to the multinucleate
state is critical for the whole cell developmental biology of P.
brassicae.
Fig. 2.
[77]Fig. 2
[78]Open in a new tab
Transcriptome analysis of the entire infection process of P. brassicae.
a DEGs during the infection stages. The early cortical infection stage
(IN) was compared with the RS (resting spores) stage and GS
(germinating resting spores) stage. b Significantly enriched Gene
Ontology (GO) terms (P < 0.05) of DEGs from the comparison of IN-VS-RS
and IN-VS-GS. GO terms belong to biological processes (GOBP), cellular
components (GOCC), and molecular functions (GOMF) were shown,
respectively. GO terms were sorted based on P-values. c Validation of
GO enrichment of DEGs by qRT-PCR. Fifteen DEGs from significant GO
Classification Enrichment were chosen randomly for qRT-PCR validation.
The relative expression level of each gene was expressed as the
fold-change between three different samples (RS, GS, IN) in the RNA-Seq
data (red line chart) and qRT-PCR data (black histogram). Data from
qRT-PCR represent the means and standard deviations (three
replications). The actin gene was used as an internal control to
normalize the expression data. Pearson’s correlation coefficient
(R-value) was used to measure the consistency of the RNA-seq data and
qRT-PCR. DEGs from Biological Process: PlasB_07290, PlasB_01053,
PlasB_26969, PlasB_09671; DEGs from Cellular Component: PlasB_02130,
PlasB_09605, PlasB_02486, PlasB_01173, PlasB_03329, PlasB_07163; DEGs
from Molecular Function: PlasB_09800, PlasB_01922, PlasB_07507,
PlasB_01791, PlasB_03590. See Additional file [79]4: Table S2 for
putative functions of these genes. See Additional file [80]5: Table S3
for genes information
To confirm the RNA-Seq profiles, qRT-PCR was conducted on 12 randomly
selected DEGs. The data between RNA-Seq and qRT-PCR of the genes
displayed a high correlation, indicating the basic consistency between
the two approaches (Additional file [81]2: Figure S2 and and Additional
file [82]3: Table S1). To further understand the function of these
DEGs, gene ontology (GO) term enrichment analysis was performed. The GO
categories were ranked based on P-values. The significantly enriched
classes (P ≤ 0.05) are presented. For these DEGs during multinucleate
secondary plasmodia stage, the most significantly enriched GO terms
were GO: 0030154 cell differentiation, GO: 0040007 growth, GO: 0000003
reproduction, GO: 0006412 translation in biological processes level,
GO: 0005623 cell, GO: 0030312 external encapsulating structure, GO:
0005622 intracellular, GO: 0043226 organelle, and GO: 0005198
structural molecule activity in molecular functions level (Fig.
[83]2B). Furthermore, qRT-PCR verified that 15 randomly selected genes
from the significant GO Classification Enrichment were dramatically
activated during the multinucleate secondary plasmodia stage (Fig.
[84]2c and Additional file [85]3: Table S1). Cell type-specific
transcriptome analysis revealed the conversion from unicellular to
multinucleate status during P. brassicae cell developmental.
Proto-oncogene mechanism involved in the specific developmental stage of
multinucleate plasmodium
Infected plant roots transform into galls, with multinucleate secondary
plasmodia cell division taking place rapidly. In the process, the
secondary zoospores first become myxamoebae and then invade internal
root tissues, where they become multinucleate secondary plasmodia
[[86]26]. The spherical or subspherical young plasmodia divide into
several small plasmodia and the small multinucleate plasmodia form a
cluster by repeated cell division. The plasmodia fuse with each other,
which is followed by the development of vegetative plasmodia. At the
end of cortical infection stage, the mature resting spores form
[[87]27] (Fig. [88]1b).
Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
analysis, we identified 171 proto-oncogenes that were implicated in the
“Cancers” (Human Diseases) related pathways from the P. brassicae ZJ-1
genome, of which 46 genes were DEGs (Additional file [89]4: Table S2).
Of these 171 proto-oncogenes, three predicted proto-oncogene proteins
from P. brassicae-PbRaf-1 (PlasB_06593), PbRaf-2 (PlasB_09434) and
Pbmyb (PlasB_01331)-were found to contain conserved functional domains
(S_TKc and myb) homologous with Raf and myb proto-oncogenes proteins
from Homo sapiens, respectively (Fig. [90]3a, b). Raf and myb
proto-oncogenes proteins regulate fundamental cellular processes such
as growth, proliferation, differentiation, metabolism, and apoptosis,
and the deregulation of Raf and myb proto-oncogenes is frequently
observed in tumorigenesis [[91]28–[92]30]. Presently, these three
predicted proto-oncogenes from P. brassicae were specifically activated
with the rapidly proliferating multinucleate plasmodium cell type (Fig.
[93]3c). This indicated that the proto-oncogene-related cancer cell
development pathway may be highly conserved and deeply embedded in
multicellular life and in unicellular protists.
Fig. 3.
[94]Fig. 3
[95]Open in a new tab
Multiple alignment of conserved domains of proto-oncogenes proteins and
cell type-specific expression patterns of these proto-oncogenes. a
Alignment of S_TKc domain of P. brassicae Raf proto-oncogenes with
selected homologs. (GenBank accession nos.: Blastocystis sp.,
[96]OAO16337.1; Malus domestica, [97]XP_008390530.1; Prunus mume,
[98]XP_008223365.1; Camelina sativa, [99]XP_019092831.1; Malus
domestica, [100]XP_008390530.1; Nicotiana sylvestris,
[101]XP_009791599.1; Solanum tuberosum, [102]XP_015169630.1;
Acanthamoeba castellanii, [103]XP_004334709.1; Polysphondylium
pallidum, [104]EFA76341.1; Homo sapiens B-raf proto-oncogene protein,
[105]AAA96495.1; Homo sapiens RAF1 protein, [106]AAA60247.1). b
Alignment of myb domain of P. brassicae proto-oncogene protein Pbmyb
with its homologs. (GenBank accession nos.: Rhagoletis zephyria,
[107]XP_017478845.1; Bactrocera latifrons, [108]XP_018796786.1;
Alligator mississippiensis, [109]XP_019340180.1; Camelina sativa,
[110]XP_019092831.1; Homo sapiens MYB proto-oncogene protein,
[111]AAA52031.1; Dictyostelium fasciculatum, [112]XP_004355527.1;
Physcomitrella patens, [113]AAF78887.1). Identical residues are shaded
in black and gray, and gaps are indicated by dashed lines. Alignments
were generated by using the ClustalW algorithm with additional manual
adjustments. c The cell type-specific expression patterns of
proto-oncogenes from P. brassicae were measured by RNA-seq data (red
line chart) and qRT-PCR data (black histogram). The P. brassicae actin
gene was used as an internal control to normalize the expression data.
The histograms and error bars represent means and standard deviations
of qRT-PCR, respectively. Pearson’s correlation coefficient (R-value)
was used to measure the consistency of the RNA-seq data and qRT-PCR.
See Additional file [114]3: Table S1 and Additional file [115]6: Table
S4 for detail of genes
We conducted KEGG pathway classification enrichment analysis on
significantly enriched DEGs (P ≤ 0.05). During the IN stage, the top
five significantly enriched DEGs identified in the KEGG pathway
classifications were “Translation” (Genetic information processing),
“Cell growth and death”, “Cell communication”, “Cell motility”
(Cellular Processes), and “Cancers” (Human Diseases). However, the
tendency of this significant enrichment of pathways was no longer
evident at the GS stage (Fig. [116]4a). Furthermore, qRT-PCR analysis
verified 18 randomly chosen genes from those significantly enriched in
the KEGG pathway. “Translation” and “DNA replication” (Genetic
information processing), “Cell growth and death”, “Cell communication”,
“Cell motility” (Cellular processes), and “Cancer” (Human diseases)
were dramatically activated during the multinucleate plasmodium
developmental stage (Fig. [117]4b and Additional file [118]3: Table
S1). Our results suggest that the “Translation” and “DNA replication”,
“Cell growth and death”, “Cell communication”, “Cell motility” and
“Cancer” KEGG pathways are distinctively activated and are important in
the transition from the unicellular to multinucleate plasmodium states
with the rapidly proliferating cell type during cortical infection. The
findings reveal the sophisticated mechanism of the
proto-oncogene-related pathways by which the pathogen can switch from
the normal unicellular state to the continuously dividing tumorigenic
state.
Fig. 4.
[119]Fig. 4
[120]Open in a new tab
KEGG pathway classification enrichment of DGEs of P. brassicae in three
developmental stages. a Enriched pathways. X-axis, the significance
(−Log P-value) of KEGG Pathway Enrichment was calculated by the method
of hypergeometric distribution; Y-axis, the category of KEGG Pathway
(a, Metabolism; b, Genetic Information Processing; c, Environmental
Information Processing; d, Cellular Processes; e, Organismal Systems;
f, Human Diseases). Vertical dotted lines: P = 0.01. b Eighteen DEGs
from significant KEGG Pathway Classification Enrichment were randomly
selected for qRT-PCR validation. The relative expression level of each
gene was expressed as the fold change between three different samples
(RS, GS, IN) in the RNA-Seq data (red line chart) and qRT-PCR data
(black histogram). The P. brassicae actin gene was used as an internal
control to normalize the expression. Data from qRT-PCR represent the
means and standard deviations. Pearson’s correlation coefficient
(R-value) was used to measure the consistency of the RNA-seq data and
qRT-PCR. Up, middle and down panes, DEGs from Translation and DNA
replication KEGG Pathway, Cell growth and death KEGG Pathway and
Cancers KEGG Pathway, respectively. See Additional file [121]4: Table
S2 for the information of the putative genes
Phosphoinositol-3-kinase (PI3K) inhibitor treatment relieves the severity of
clubroot symptom
Extensive research over the past decade has revealed the critical role
for the signaling transduction pathways in the regulation of the
dynamic process of tumorigenesis. The dysregulation of these pathways,
for example the Ras/PI3K-Akt and mammalian target of rapamycin (mTOR)
signaling pathways, lead to massive overgrowth of tissue
[[122]31–[123]33]. The present organization analysis identified 17
gene-encoded proteins containing conserved domains, which were
predicted to be involved in cancer-related signaling pathways in the
genome of P. brassicae (Fig. [124]5a, Additional file [125]5: Table S3,
and Additional file [126]6: Table S4). qRT-PCR confirmed the expression
pattern of the selected core components of the Ras/PI3K-Akt and mTOR
signaling pathways during the developmental stage of the multinucleate
plasmodium. The findings indicate the specific activation of the
Ras/PI3K-Akt and mTOR signaling pathways during the cortical infection.
Compared with other developmental stages, the core genes involved in
the Ras/PI3K-Akt signaling pathway (Regulation of actin cytoskeleton),
Ras/MAPK signaling pathway (Cell proliferation), PI3K-Akt signaling
pathway (Survival signal, Growth and proliferation, Cell cycle
progression, Cell survival, Protein synthesis), and mTOR signaling
pathway (Translation, Cell growth) were significantly activated in the
rapidly proliferating cell stage (Fig. [127]5b and Additional file
[128]3: Table S1). The transcriptome analysis of P. brassicae genes and
gene networks involved in tumorigenic-related signaling transduction
pathways documented the activation of the core components of the
Ras/PI3K-Akt and mTOR signaling pathways, providing insights into the
mechanisms by which the parasite can change the normal growth and
development into a tumorigenic life-style.
Fig. 5.
[129]Fig. 5
[130]Open in a new tab
Proteins involved in cancer-related signaling pathways in P. brassicae
and qRT-PCR validation of the expression pattern. a Schematic diagram
of proteins encoded by genes of cancer-related signaling pathways in P.
brassicae. The black frames represented conserved domains in the genes
encoded proteins. The information of conserved domain, e-value, and
length was obtained from NCBI database. b Twelve core genes of
cancer-related signaling pathways (marked with black solid triangle in
(a) were chosen for qRT-PCR validation. Expression levels of these 12
genes from the three different samples (RS, GS and IN) were measured by
RNA-seq data (Red line chart) and qRT-PCR data (black histogram). The
actin gene of P. brassicae was used as an internal control to normalize
the expression level. Data from qRT-PCR represent the means and
standard deviations (three replications). R-value of Pearson’s
correlation coefficient was used to measure the consistency of the
RNA-seq data and qRT-PCR. See Additional file [131]3: Table S1 and
Additional file [132]4: Table S2 for genes information
Due to the feature of obligate biotrophy, there is still no successful
and stable genetic transformation system for P. brassicae. Thus, we
conducted inhibitor treatment experiments to explore the requirement of
tumorigenesis-related signaling transduction pathways during the
tumorigenic proliferating process of the multinucleate plasmodium
stage. GDC-0032 is a potent, next-generation β isoform-sparing PI3K
inhibitor targeting PI3Kα/δ/γ [[133]34, [134]35]. GDC-0032 treatment
affects the normal growth and root formation and plant development of
oilseed rape plants (Brassica napus). The expression of Cyclin
([135]XM_013809141.1) from B. napus between the mock-treated and
GDC-0032-treated groups did not differ significantly (Additional file
[136]7: Figure S3 a, b). Host plants were inoculated with
GDC-0032-pretreated resting spores and then grown in nutrient solution
that contained the inhibitor. GDC-0032 had no effect on the germination
rate of the resting spores or the root hair infection rate of P.
brassicae (Additional file [137]7: Figure S3 c, d). Clubroot symptom
was then quantified at 21 and 28 days post-infection (dpi). Clubroot
symptom was less frequent in the inhibitor-treated plants with a lower
Disease Index (DI) than the mock-treated group (Fig. [138]6a, b). qPCR
was used to evaluate the relative accumulated amount of pathogen in the
root tissues of host plants. P. brassicae was 37-fold more prevalent in
the infected roots of mock-treated group than the inhibitor-treated
group (Fig. [139]6c). Furthermore, the expression levels of the
components from PI3K-related signaling transduction pathways were
significantly reduced in the inhibitor-treated group (Fig. [140]6d).
These results indicated that GDC-0032 treatment could relieve clubroot
symptom of infected roots by inhibiting the core components of PI3K
signaling transduction.
Fig. 6.
[141]Fig. 6
[142]Open in a new tab
Effect of PI3K inhibitor treatment on the clubroot symptom of B. napus.
a PI3K-treated and P. brassicae-inoculated plants showed less symptom
and rich rootlets; non-inoculated plants are shown as control (CK), and
plants inoculated P. brassicae showed heavy symptoms (mock).
Photographs were taken at 28 dpi. Bar denotes 1 cm. b Clubroot symptoms
of infected roots from three biological replicates were evaluated using
the percentage of plants in individual disease classes and disease
index (DI) at 21 dpi and 28 dpi. Asterisks indicate statistically
significant differences at the level of P = 0.05. M: Mock-treated P:
PI3K inhibitor-treated. c At 28 dpi, the infected roots of mock-treated
plants and inhibitor-treated plants were harvested. The relative
accumulated amount of pathogen DNA was quantified by qPCR. The actin
gene of B. napus was set as the control to normalize the accumulation
amount of the pathogen. M, mock treated; P, PI3K inhibitor-treated. d
The expression levels of genes from PI3K-related pathways between
mock-treated and inhibitor-treated groups. The P. brassicae actin gene
was used as an internal control to normalize the expression level. The
expression level of inhibitor-treated groups was set as 1.0. Data
represent the means and standard deviations
Discussion
Conversion from unicellular to multinucleate state is the key stage of P.
brassicae cell development
The conversion from the unicellular state to multinucleate secondary
plasmodium is critical in P. brassicae cell developmental biology, as
the cell division of secondary plasmodia provokes the rapidly cleavage
into resting spores, which enable survival during harsh environmental
conditions and the initial infection source of the next cycle
[[143]12]. The rapidly proliferating cell types of multinucleate
plasmodia are part of this conversion. The formation of secondary
plasmodia is accompanied by hypertrophy and hyperplasia of the infected
host cells, leading to the development of galls that obstruct nutrient
and water transport, with resting spores released back into the soil
when the galls decompose [[144]14]. Although the transition from the
unicellular to the multinucleate state has been the focus of P.
brassicae infection biology research for a long time [[145]12,
[146]26], there is still a poor understanding of this phase change, in
particular, the details of the transcriptional regulatory network of
multinucleate secondary plasmodium. Understanding the mechanisms
involved in cell division of multinucleate secondary plasmodia could
help reveal the universal regulation pattern of cell division and
development of multinucleate cells in this type of protist.
Presently, GO enrichment analysis of cell type-specific transcriptome
suggested that the activation of cell differentiation, growth,
reproduction, and translation related biological processes are required
in multinucleate cell division. Based on the functional study of the
significantly enriched KEGG pathways, we speculate that many pathways,
including “Cancer” (Human Diseases) maybe vital for the conversion from
a normal unicellular state to the rapidly proliferating multinucleate
cell state.
Proto-oncogenes of P. brassicae are specifically activated during the
multinucleate cell division stage
The 171 proto-oncogenes implicated in the “Cancer” (Human Disease)
pathway were mined from the genome of strain ZJ-1 by in silico
analysis. Proto-oncogenes may be ancient and highly conserved
[[147]36]. Such conservation indicates that proto-oncogenes are normal
cellular genes homologous to oncogenes, but they have served vital and
indispensable functions in normal cellular and organismic physiology,
and that their role in carcinogenesis represents only an unusual and
aberrant diversion from their usual functions [[148]19, [149]36]. For
example, the deregulation of the c-myc proto-oncogene leads to
tumorigenesis and is a hallmark of approximately 30% of all human
cancers [[150]37, [151]38]. The ancestral forms of myc and max genes
have been identified and extensively characterized in the early
diploblastic cnidarians, Hydra magnipapillata [[152]20]. The myc gene
was observed to be specifically activated in all rapidly proliferating
cells, such as the interstitial stem cell system and gland cells.
However, the expression of myc is not detectable in terminally
differentiated nerve cells, nematocytes, or epithelial cells. The
results reveal that the stem cell-specific activation of the ancestral
myc protooncogene is indispensable for the regenerative ability of the
early metazoan Hydra, which confirms that the principal functions of
the Myc master regulator arose very early in metazoan evolutionary
history [[153]20].
The present findings corroborate the requirement of 171 proto-oncogenes
in the multinucleate cell division stage to regulate proliferation and
self-renewal and to perturb or inhibit terminal differentiation, as has
been proposed in metazoans. For the S_TKc and myb conserved functional
domains, three predicted proto-oncogenes proteins from P. brassicae
displayed homology with Raf and myb from H. sapiens, respectively.
These proto-oncogenes were specifically activated during the cell
type-specific multinucleate cell division course of P. brassicae.
Considering the indispensable role of proto-oncogenes from the protist
subgroup Rhizaria in cell developmental biology, similar to the rapid
cell proliferation of multinucleate plasmodia, we suggest that the
proto-oncogenes are a group of genes that have been strongly
functionally conserved in metazoans and protists.
PI3K-related signaling transduction pathways have key roles during
multinucleate cell division stage
PI3K-Akt signaling, including downstream signaling pathways, such as
the mitogen-activated protein kinase kinase (MEK), extracellular
signal-regulated kinase (ERK), mitogen-activated protein kinase (MAPK)
pathways and the mTOR pathways, are activated by many types of cellular
stimuli or toxic insults. This activation regulates the fundamental
cellular functions of transcription, translation, proliferation,
growth, and survival [[154]32, [155]39]. The development and
progression of cancer are the result of a disturbance in the balance
between cell proliferation and apoptosis [[156]40]. PI3K-Akt signaling
is associated with cell proliferation and apoptosis. Notably, many
studies have demonstrated that the constitutive activation of the
PI3K-Akt pathway is frequently associated with human cancers [[157]32,
[158]41, [159]42]. These findings indicate that the PI3K-Akt pathway
plays a pivotal role in tumor progression. Genes associated with
tumorigenic-related signaling transduction pathways were identified
from P. brassicae. Our transcriptome analysis also revealed the
significant up-regulation of core compounds from the Ras/PI3K-Akt and
mTOR signaling pathways during the cell division course of
multinucleate secondary plasmodia.
The recent studies show that the PI3K pathway and the downstream
pathways are often deregulated in human cancer cells. As the key
component of these signaling cascades, PI3K an important target for
therapeutic interventions [[160]32, [161]41]. To date, several
compounds that directly inhibit PI3K-Akt activity have been developed.
In preclinical, phase I or II clinical trials, they have shown good
anti-tumor efficacy, such as inducing cell cycle arrest or apoptosis in
human cancer cells in vitro and in vivo [[162]43–[163]45]. GDC-0032 is
a potent and selective inhibitor of Class I PI3Kα, δ, and γ isoforms.
Preclinical data show that the combination of GDC-0032 enhances the
activity of other inhibitor medication resulting in tumor regression
and tumor growth delay [[164]34, [165]35]. Intriguingly, the present
results show that GDC-0032 can block cell proliferation course of
multinucleate secondary plasmodia and relieve the clubroot symptom of
the host plants. Our findings indicate that the PI3K related signaling
transduction pathways were specially required by P. brassicae during
the multinucleate plasmodium stage of cortical infection, but not in
the root hair infection stage. This is consistent with the distinct
activation of proto-oncogenes and related pathways during the
development of multinucleate plasmodium with a rapidly proliferating
cell type.
Our research highlights the highly concordant mechanism of cell
development regulated by the PI3K-Akt signaling pathway between
protists and metazoans, and provides insight into the convergent
evolution of this regulatory mechanism. Thus, PI3K-Akt-mediated
regulation of the transition from normal cell development to rapid
proliferation (such as the cell division of multinucleate secondary
plasmodia of P. brassicae and tumorigenesis of mammalian cells) shares
a common developmental mechanism. In the future, the plasmodiophorid
material of P. brassicae could be a potential model system for studies
of the PI3K-Akt signaling pathway mechanisms involved in mammalian
cancer.
Conclusions
Using a multi-omics analysis strategy, we provide an important
comprehensive insight into the critical multinucleate cell division
life-style of the obligate uncultivable protist pathogen, P. brassicae.
Our finding that the special activation of proto-oncogenes is important
in the development of P. brassicae will undoubtedly inform future
research on novel regulation mechanisms involved in the growth and
development of unicellular organisms.
Methods
Collection of field populations and isolation of single spores
P. brassicae strain ZJ-1 resting spores were maintained at − 20 °C in
sterilized double-distilled water containing 50 μg.mL^− 1 cefotaxime
sodium. The details of the strain and the inoculation methods have been
previously published [[166]46].
Developmental stage-specific sample preparation
To prepare resting spore stage samples, the resting spores were
extracted from single spore isolated from plant root clubroot as
previously described [[167]46]. For the collection of germinating
resting spore stage samples, the purified resting spores were thawed at
4 °C for 48 h followed by 24 h at room temperature in a root exudate
solution that allow germination in a dark environment. The root tissues
of the prepared seeds were immersed in 2.0 mL Eppendorf tubes with the
treated germinating spores (10^7 spores.mL^− 1). Samples were harvested
after 24 h when microscopy examination revealed appreciable aggregation
and adsorption in root tissues of the germinating resting spores. To
prepare the IN root samples for RNA extraction, seeds of oilseed rape
(Brassica napus) were surface sterilized in 1% NaClO for 5 min, washed
with distilled water, and germinated on moistened filter paper for
6 days in a growth chamber maintained at 22 °C/20 °C (day/night) with a
14-h photoperiod and 80% relative humidity. Some of the seeds were
transplanted to autoclaved potting mix in 60 cell
(4 cm × 3.5 cm × 6 cm) plastic pot trays in a controlled environment
growth chamber (HP300GS-C; Ruihua Instrument and Equipment, Wuhan,
China) at a constant 20 °C with a 14-h photoperiod and 80% relative
humidity. Samples were watered once daily. Nineteen days after sowing,
the plants were inoculated with resting spores derived from single
spore ZJ-1 strain (10^7 spores.mL^− 1). After inoculation, the plants
were grown in the glasshouse conditions described above. Root masses
were collected from at least 50 plants at 21 dpi, when signs of
secondary infection were visible. Roots were extensively washed in tap
water and inspected microscopically to ensure the absence of successful
infection of pathogens. Fine root tissues were trimmed away.
DNA and RNA extraction
DNA was extracted from resting spores of the strain ZJ-1 using a
modified CTAB method [[168]47] and stored at − 20 °C. Total RNA was
extracted with RNAiso Plus (Takara, Dalian, China) according to the
manufacturer’s protocols from the developmental stage-specific samples
described above. The pellet was resuspended in 20–30 μL of diethyl
pyrocarbonate-treated, RNase-free water and homogenized by pipetting
20–30 times. Each sample was stored at − 80 °C. RNA quantity and
quality was assessed using a spectrophotometer (NanoDrop Technologies,
Inc. Wilmington, DE USA) and by 1.5% (w/v) agarose gel electrophoresis.
Transcriptome expression analysis
We collected samples from three specific infection stages: resting
spore stage, primary zoospore stage, and early cortical infection
stage. Total RNA samples of three biological replicates for each stage
were prepared. According to the manufacturer’s instructions (Illumina,
San Diego, CA), an RNA-seq library was prepared, followed by sequencing
on an Illumina NextSeq 500 platform for paired-end 2 × 150 bp
sequencing, which was performed at Shanghai Personal Biotechnology Co.,
Ltd. (Shanghai, China). The RNA-Seq raw reads were processed to obtain
high quality reads by removing the adapter sequences and low-quality
bases at the 3′ end and trimming low-quality bases (Q < 20) from the 3′
to 5′ ends of the remaining reads. Reads containing ‘N’ and greater
than 50 bp were filtered out. The resulting reads were considered for
analysis. The filtered reads were mapped to the P. brassicae genome
using Tophat v2.0.9 ([169]http://tophat.cbcb.umd.edu/) [[170]48]. The
analysis of transcriptome differential expression was conducted with
HTSeq ([171]http://www-huber.embl.de/users/anders/HTSeq) [[172]49] and
DESeq ([173]http://www-huber.embl.de/users/anders/DESeq) [[174]50].
DESeq based on the theory of negative binomial distribution was used to
identify the DEGs and their corresponding P-values [[175]51]. The DEGs
were selected with the standard: P-value≤0.05 and the absolute value of
log[2] Fold Change≥1. GO term enrichment analysis first mapped all DEGs
to GO terms in the database ([176]http://www.geneontology.org/),
calculating gene numbers for every term, then used the hypergeometric
test to identify significantly enriched GO terms in DEGs compared to
the genome background. The KEGG pathway enrichment analysis identified
significantly enriched pathways of DEGs compared with the entire genome
background. To analyze gene expression data and genes with similar
functions, we exploited hierarchical clustering methods based on
transcriptome expression data. To compare the expression pattern of
each gene between samples, the abundance of each transcript was
normalized by reads per kilobase of transcript per million mapped reads
(RPKM) [[177]52]. The heat map of the clustered genes and samples was
generated by the MultiExperiment Viewer v4.9 software package
[[178]53]. An average-linkage hierarchical clustering method was used,
and Pearson’s correlation coefficient (the distance metric is the
default) was employed to measure the similarity of the expressed genes.
Quantitative RT-PCR validation
qRT-PCR primers were designed to generate amplicons to validate the
RNA-seq data (Additional file [179]3: Table S1). For RT-PCR and
qRT-PCR, 5 μg of total RNA was reverse transcribed into first-strand
cDNA using the oligo (dT) primer and M-MLV Reverse Transcriptase
according to the manufacturer’s instructions (TransScript, Beijing,
China). All qRT-PCR experiments were run using SYBR Green Real-Time PCR
Master Mix (Bio-Rad, Hercules, CA, USA) in 20 μL reactions with the
CFX96™ real-time PCR detection system (Bio-Rad). The P. brassicae actin
gene was used as an internal control to normalize the expression data.
Data was acquired and analyzed using the Bio-Rad CFX Manager™ Software
(version 2.0). The relative expression level of each target gene was
quantified by the comparative CT method (2 ^-△△Ct) [[180]54]. The
relative expression levels of each gene were validated for the RNA-seq
data.
Symptom quantification during treatment with PI3K antagonist
At 28 days after PI3K inhibitor treatment, growth and development
status of oilseed rape plants were checked. A group treated with
dimethylsulfoxide (DMSO) was the control. The root tissues of treated
plants were harvested and the expression level of the PI3K gene
([181]XM_013869861.1) from B. napus was quantified by qRT-PCR as
described above. The effect of the PI3K inhibitor on resting spore
germination rate and root hair infection rate were analyzed as
previously described [[182]55, [183]56]. At 10 days after germination,
the prepared plants were inoculated with the pretreatment resting
spores diluted to 10^7 spores.mL^− 1 with modified 1/2 Hoagland
nutrient solution and inhibitor solution (GDC-0032: 100 nM,
MedChemExpress) in 10 mL EP tubes. As a parallel control, 1/2 Hoagland
nutrient solution and equivalent DMSO treatment was set as the
mock-treated group under the same condition. The CK group
(non-inoculated) was added to EP tubes contained equivalent volumes of
1/2 Hoagland nutrient solution and inhibitor solution. Solutions in the
EP tubes were refreshed every 7 days. The disease index (DI) was
calculated by categorizing the individual roots at 21 and 28 dpi into
five classes: 0 (no symptoms); 1 (very small clubs, mainly on lateral
roots that do not impair the main root); 2 (small clubs covering the
main root and few lateral roots); 3 (medium sized to bigger clubs, also
including the main root and hypocotyl, fine roots are partly
unaffected, plant growth may be impaired); 4 (severe clubs in lateral,
main root, hypocotyls or rosette, fine roots completely destroyed,
plant growth is affected) [[184]57], using the following formula:
[MATH: DI=1n1+2<
mi
mathvariant="normal">n2+3<
mi
mathvariant="normal">n3+4<
mi
mathvariant="normal">n4100/4Nt :MATH]
where n[1]–n[4] are the numbers of plant in the indicated classes and
N[t] is the total number of plant tested. At 28 days after infection,
all seedlings were harvested and the clubroot symptoms were
investigated by phytopathological analysis. The plants were cut at the
top of the hypocotyl into shoots and roots. For quantitative
estimation, genomic DNA was prepared from the pool of at least 30 root
samples. The abundance of the actin gene of P. brassicae, which
represents the accumulation amount of the pathogen in the root was
normalized to the actin gene of B. napus. A similar result was derived
from three independent biological experiments. For each biological
experiment, at least 30 plants were analyzed.
Additional files
[185]Additional file 1:^ (1.9MB, tif)
Figure S1. Heatmap of DEGs of P. brassicae at three stages. IN,
multinucleate secondary plasmodia stage in plant cortical cells; GS,
germinating resting spores stage when the resting spores germinating
and releasing primary zoospores; and RS, resting spores stage. (TIF
1974 kb)
[186]Additional file 2:^ (736.1KB, tif)
Figure S2. Validation of RNA-seq results of P. brassicae by qRT-PCR.
Comparison of expression levels for the randomly selected 12 genes from
the three different samples (RS, GS and IN) were measured by RNA-seq
data (gray line chart) and qRT-PCR data (black histogram). The P.
brassicae actin gene was used as an internal control to normalize the
expression level. Data from qRT-PCR represent the means and standard
deviations. Pearson’s correlation coefficient (R-value) was used to
measure the consistency of the RNA-seq data and qRT-PCR. See Additional
file [187]3: Table S1 for primer information. (TIF 736 kb)
[188]Additional file 3:^ (39KB, docx)
Table S1. Genes examined and primer pairs used in this study. (DOCX 38
kb)
[189]Additional file 4:^ (32.9KB, docx)
Table S2. The genes involve in Cancers related pathways on the genome
of P. brassicae and corresponding expression pattern. (DOCX 32 kb)
[190]Additional file 5:^ (26.1KB, docx)
Table S3. The amino acid sequences encoded by genes involved in
cancer-related signaling pathways in P. brassicae. (DOCX 26 kb)
[191]Additional file 6:^ (105.4KB, docx)
Table S4. Alignments of P. brasicae proteins associated with
cancer-related signaling pathways with their homologs. (DOCX 105 kb)
[192]Additional file 7:^ (642.9KB, tif)
Figure S3. Effect of PI3K inhibitor treatment on the growth,
development of oilseed rape plants, resting spores germination rate and
root hair infection rate of P. brassicae. a Growth and development
status of oilseed rape plants treated with PI3K inhibitor (right). MOCK
(DMSO) treatment served as control (left). The pictures of plants were
taken at 28 day after treatment. Bar = 1.5 cm. b At 28 day after
treatment, the roots of MOCK treated plants and inhibitor treated
plants were harvested. The expression level of Cyclin gene
(XM_013809141.1, downstream gene of PI3K signaling pathway) in B. napus
with MOCK and inhibitor treatment was quantified by qPCR. The actin
gene of B. napus was used as control to normalize the expression level.
Data represent the means and standard deviations. The expression level
of MOCK treated group was set as 1.0. Statistically significant
difference of data between MOCK and inhibitor treated groups was
compared, same letter in the graph indicates no significant differences
at the level of P = 0.05. c-d Resting spores germination rate and root
hair infection rate of P. brassicae were compared between H[2]O, MOCK
and PI3K-Inhibitor treated groups. At 6 day, the treated spores were
stained with orcein (Sigma-Aldrich Canada). The germination rate of
spores was counted under microscope. At 7 dpi, the roots of oilseed
rape plants were stained with Trypan Blue, then the root hair infection
rate was counted with microscopic examination. The graphic data
represent the means and standard deviations from three biological
replicates. At the level of P = 0.05, statistically significant
differences of data between H[2]O, MOCK and inhibitor treated groups
were compared, same letters in the graph indicate no significant
differences. (TIF 642 kb)
Acknowledgments