Journal of Integrative Agriculture 2019, 18(1): 96–107

Available online at www.sciencedirect.com

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RESEARCH ARTICLE

Comparative analysis of protein kinases and associated domains

between Ascomycota and Basidiomycota

PEI Guo-liang, GUO Jun, WANG Qin-hu, KANG Zhen-sheng

State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling
712100, P.R.China

Abstract
Protein kinases play an important role in every aspect of cellular life. In this study, we systemically identified protein
kinases from the predicted proteomes of 59 representative fungi from Ascomycota and Basidiomycota. Comparative
analysis revealed that fungi from Ascomycota and Basidiomycota differed in the number and variety of protein kinases.
Some groups of protein kinases, such as calmodulin/calcium regulated kinases (CMGC) and those with the highest group
percentages are the most prevalent protein kinases among all fungal species tested. In contrast, the STE group (homologs
of the yeast STE7, STE11 and STE20 genes), was more abundant in Basidiomycetes than in Ascomycetes. Importantly,
the distribution of some protein kinase families appeared to be subphylum-specific. The tyrosine kinase-like (TKL) group
had a higher protein kinase density in Agaricomycotina fungi. In addition, the distribution of accessory domains, which could
have functional implications, demonstrated that usage bias varied between the two phyla. Principal component analysis
revealed a divergence between the main functional domains and associated domains in fungi. This study provides novel
insights into the variety and expansion of fungal protein kinases between Ascomycota and Basidiomycota.

Keywords: protein kinases, associated domains, Ascomycota, Basidiomycota

et al. 2009). Including molds, mushrooms, rusts, smuts

1. Introduction
Dikarya is a subkingdom of fungi that contains the two
largest fungal phyla, Ascomycota and Basidiomycota,
accounting for the vast majority of terrestrial fungi (Stajich
Received 27 April, 2018 Accepted 21 June, 2018
PEI Guo-liang, E-mail: peiguoliang@nwafu.edu.cn; Correspondence
KANG Zhen-sheng, Tel/Fax: +86-29-87080061, E-mail: kangzs@
nwsuaf.edu.cn; GUO Jun, Tel/Fax: +86-29-87082439, E-mail:
guojunwgq@nwsuaf.edu.cn
© 2019 CAAS. Published by Elsevier Ltd. This is an open
access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/)
doi: 10.1016/S2095-3119(18)62022-2
and yeasts, Ascomycota and Basidiomycota make
essential contributions to the biosphere, bio-product
industry, medicine, and as animal, human and plant
pathogens. Although separated by almost two billion years,
and comprising of a huge number of family members,
Ascomycota and Basidiomycota have retained the common
feature of dikaryotic hyphae, and thus belong to the
Dikarya (Mclaughlin et al. 2009). In addition to similarities
in mating, in which nuclear fusion does not follow directly
to gamete fusion (Taylor and Berbee 2006), Ascomycota
and Basidiomycota share several general characteristics,
as well as some specialties (Wang et al. 2010). Thus, it is
important to study functional protein families in the Dikarya
in order to understand the molecular characteristics of
these organisms. Such studies may provide fundamental
 PEI Guo-liang et al. Journal of Integrative Agriculture 2019, 18(1): 96–107
information for understanding how Ascomycota and
Basidiomycota fungi evolved.
Protein kinases (PKs) constitute a key class of enzymes
and regulate numerous cellular processes, including
mitosis, communication, differentiation, metabolism, and
transcription (Hanks and Hunter 1995; Cohen 2000).
By adding a phosphate group from ATP/GTP to the side
chains of target proteins, PKs often profoundly alter the
biological activity of the target molecules (LaRonde-LeBlanc
and Wlodawer 2005; Wuichet et al. 2010). All PKs can
be classified into two super-families: 1) eukaryotic or
conventional protein kinases (ePKs), which are the most
prevalent and have a common ancestry; and 2) atypical
protein kinases (aPKs) (Hanks and Hunter 1995). ePKs
have two functional domains, a catalytic domain which binds
and phosphorylates target proteins, and a regulatory region.
The catalytic domain of ePKs contains a core domain of
250–300 amino acid residues that is divided further into 12
sub-domains that have highly conserved individual amino
acid residues and motifs (Hanks 2003). aPKs lack sequence
similarity to the ePK catalytic domain, but do have kinase
activity (Hanks and Hunter 1995). Based on the catalytic
domain, the ePK super-family can be classified into several
major groups (Hanks and Hunter 1995; Becker and Joost
1998; Manning et al. 2002b), and members of these groups
are grouped according to broad functional categories
with distinct sequences and structural features. Several
classification schemes for PKs have been proposed in the
literatures. In their original classification work, Hanks and
Hunter (1995) took into consideration conservation and
phylogenetic analyses of catalytic domains. This has been
followed by several research groups for PK classification
(Manning et al. 2002a; Krupa et al. 2004; Scheeff and
Bourne 2005; Martin et al. 2009). The database KinBase
(http://www.kinase.com/kinbase) has been developed for
PK classification (Manning et al. 2002b; Martin et al. 2009).
Currently, over 3 000 PK genes divided into 10 groups can
be found in KinBase. Goldberg et al. (2013) designed a
new software, Kinannote, which can identify and classify
members of the PK super-families. Kinannote significantly
improved the average sensitivity and precision for full
classification of conserved PKs and had been implementes
in several PK annotation programs.
Several hundreds of fungal genomes have been
published (Zhao et al. 2013), including Ascomycota and
Basidiomycota, allowing for representative fungal data to
be utilized. Kosti et al. (2010) compared and analyzed the
PKs from 30 fungi, allowing some meaningful discoveries,
although their study had several weaknesses. First, they
were unable to develop a precise classification of PKs that
could be correlated to specific functions. Second, the PKs of
the 30 fungi were not compared on the basis of evolutionary
97
stage. Lastly, the important role of PKs in cell life was not
emphasized and the relationship of PKs to the life cycle of
fungi was not explored. As many fungal genomes have been
sequenced and new tools are available to classify PKs, it is
necessary to analyze fungal PKs with new methods.
In this study, we identified and compared the full
repertoires of PKs from 59 representative fungal species
from Ascomycota and Basidiomycota using Kinannote.
All 59 fungi can be classified into six sub-phyla, as
demonstrated by Hibbett et al. (2007). The six sub-phyla
are Pucciniomycetes, Ustilaginomycetes, Agaricomycotina,
Pezizomycetes, Saccharomycetes and Taphrinomycetes,
and the evolutionary stages of these sub-phyla are
mentioned in order. The 59 fungi PKs were classified into
10 groups and 119 families/sub-families using different
strategies. In addition, we identified all domains associated
with PKs according to the Pfam database (http://pfam.xfam.
org/) classification, followed by identifying the differences
between Ascomycota and Basidiomycota.
2. Materials and methods
The procedures used in this study can be classified into
three phases (Fig. 1). First, fungal proteomes were collected
from a genome database. Second, Kinannote was used
to annotate proteomes. Finally, the presence of each
domain within a specific fungal group was determined and
statistically analyzed.
2.1. Data collection
The proteomes of 17 fungi were downloaded from the Fungal
Genome Initiative site of the Broad Institute (http://www.
broadinstitute.org/), 19 were obtained from GenBank of the
National Center of Biological Information (NCBI) (http://www.
ncbi.nlm.nih.gov/), and the remaining 23 were downloaded
from the United States Department of Energy (DOE) Joint
Predicted proteome
Kinannote
HMMER search
Motif scoring
BLAST against local database and
classification to 10 groups
Pfam analysis Data analysis
Fig. 1 Flow chart of the protein kinase (PK) analysis pipeline.
98 PEI Guo-liang et al. Journal of Integrative Agriculture 2019, 18(1): 96–107
Genome Institute (JGI) site (http://genome.jgi-psf.org/).
2.2. Kinase annotation and statistical analysis
The Kinannote Program, downloaded from Sourceforge
(http://sourceforge.net/projects/kinannote/), was used to
extract and classify putative PKs from all fungal proteomes.
Kinannote identified and classified PKs in three phases.
First, the proteomes were searched with a PK hidden
Markov models (HMMs) and a relaxed cutoff was used for
PK identification. Candidates were searched using position-
specific scoring matrices and against a local version of
KinBase using BLAST, and BLAST results were then parsed.
Second, BLAST results were used to identify conserved PKs
with poor HMM scores. Finally, BLAST search results were
applied to classify PKs.
The PK groups are divided as follows: The CMGC group
was named after a set of families (CDK, MAPK, GSK3 and
CLK) and has a diverse set of functions, which include cell
cycle control, MAPK (mitogen-activated protein kinase)
signaling, splicing, and other unknown functions. The other
group consists of several families and some unique PKs that
are clearly ePKs but do not fit into other ePK groups. The
AGC group was named after the protein kinase A, G, and
C families (PKA, PKC and PKG) and contains many core
intracellular signaling PKs which are modulated by cyclic
nucleotides, phospholipids and calcium. The STE group
includes homologs of the yeast STE7, STE11 and STE20
proteins, which form the MAPK cascade transducing signals
from the cell surface to the nucleus. The Ser/Thr group
consists of only one family. The CK1 group is a small but
ancient family, originally known as Casein Kinase 1 (from a
biochemically assay with a non-physiological substrate), and
has been renamed Cell Kinase 1. The TKL group is most
similar to tyrosine kinases. The PAB group was named after
the PAB-dependent poly(A)-specific ribonuclease subunit,
a catalytic subunit of the poly(A)-nuclease de-adenylation
complex, which have PK activity. There are two large
groups, RGC and TK, which are not found in fungi and are
not discussed in this article. It should be noted that the
classifications determined using the Kinannote database,
based on KinBase, is slightly different from the newer version
of KinBase. We annotated all the families of PKs using
Saccharomyces cerevisiae S290C genes, and genes which
cannot be found in S. cerevisiae were identified by other
fungal genes (Appendix A). In order to avoid unnecessary
misunderstanding in different classification, we do not
discuss the functions of PKs from any special fungus.
All data were extracted using Kinannote Software.
OriginPro 8.5 (http://originlab.com/index.aspx?go=
PRODUCTS/OriginPro) was used to generate statistical
box charts. Independent sample t-tests were performed
using SPSS Statistic 20.0 Software (http://www.01.ibm.com/
software/analytics/spss/).
2.3. Domains searches
InterProScan 5.20 was used to identify domain sequences
from each putative PKs by searching sequences against the
Pfam A (V30) HMMs library (Jones et al. 2014). A minimum
score value of e-value of 1×10–4 was used.
2.4. Principal component analysis (PCA)
The 59 fungi were classified according to evolutionary
stages, limiting the data to those domains present in at
least half of the fungi (at least 30). The PCA procedure was
applied PKs and the domains identified as PK sequences,
using the GNU R Software in the ade4 Package (Dray and
Dufour 2007).
3. Results and discussion
3.1. Distribution of PKs
The predicted proteomes of 59 Basidiomycetes
and Ascomycetes fungi representing six sub-phyla
(Pucciniomycetes, Ustilaginomycetes, Agaricomycotina,
Pezizomycetes, Saccharomycetes and Taphrinomycetes)
(Appendix B) were systematically screened for different
families of kinomes using Kinannote Software. These
fungi also represent the five types of nutritional modes:
saprophytic, facultative parasitic, hemi-biotrophic, biotrophic
and mutualistic symbiotic fungi, including pathogens of
plants, human, and insects as well as non-pathogenic fungi.
The focus of this study was on the fungal evolutionary status,
also taking into account other features of fungi, such as
nutritional modes and pathogenicity.
A total of 7 557 putative PK sequences were obtained.
They were classified into 119 families/sub-families and 10
PK groups (RGC and TK groups were not represented). All
putative PKs were identified in fungal predicted proteomes.
As shown in Fig. 2, the distribution of the 10 PK groups was
classified using Kinannote Software. All PK groups in fungi
can be classified into three classes by group percentage,
the number of genes in a PK group compared to the total
number of all PKs, which were then adjusted by a median
value. The most populated PK groups were “CMGC” and
“Other”, averaging >20% from all fungi. The AGC, CAMK,
STE and Ser/Thr groups comprised approximately 10% of
all fungal PKs, while the CK1, PAB and TKL groups were
the least populated groups (Appendix C). Most PK groups
were stably distributed in all fungi studied, especially the
CK1 and PAB groups. However, in the TKL and the Ser/Thr
 PEI Guo-liang et al. Journal of Integrative Agriculture 2019, 18(1): 96–107 99

0 100 200 300 400

Puccinia striiformis
Melampsora laricis-populina
Pucciniomycotina Puccinia graminis
Microbotryum violaceum
Rhodotorula graminis
Mixia osmundae
Sporisorium reilianum
Tilletiaria anomala

Basidiomycota
Ustilaginomycotina Ustilago maydis
Ceraceosorus bombacis
Malassezia globosa
Botryobasidium botryosum
Paxillus involutus
Laccaria bicolor
Hebeloma cylindrosporum
Calocera viscosa
Agaricomycotina
Calocera cornea
Coniophora puteana
Cryptococcus neoformans
Cryptococcus gattii
Moniliophthora perniciosa
Nectria haematococca
Cordyceps militaris AGC
Metarhizium acridum
Neurospora crassa Atypica
Gaeumannomyces graminis var. tritici
Metarhizium anisopliae CAMK
Arthroderma benhamiae
CK1
Cladosporium fulvum
Aspergillus clavatus CMGC
Mycosphaerella graminicola
Pyrenophora teres f. sp. teres Other
Ajellomyces capsulatus
PAB
Trichophyton rubrum
Pezizomycotina
Magnaporthe oryzae STE
Cochliobolus sativus
Penicillium marneffei TKL

Ascomycota
Coccidioides immitis
Arthrobotrys oligospora
Ser/Thr
Phaeosphaeria nodorum
Dothistroma septosporum
Botryotinia fuckeliana
Sclerotinia sclerotiorum
Fusarium graminearum
Tuber melanosporum
Chaetomium thermophilum
Blumeria graminis f. sp. hordei
Saccharomyces cerevisiae
Debaryomyces hansenii
Candida tropicalis
Saccharomycotina
Candida lusitaniae
Candida albicans
Lodderomyces elongisporus
Schizosaccharomyces japonicus
Schizosaccharomyces cryophilus
Schizosaccharomyces octosporus
Schizosaccharomyces pombe
Pneumocystis jirovecii
Taphrinomycotina Taphrina deformans

Fig. 2 Comparative analysis of fungal protein kinases (PKs). The number of kinase genes is represented as horizontal bars.
Colors indicate the major kinase groups: AGC, Atypica, CAMK, CK1, CMGC, other, PAB, STE, TKL and Ser/Thr protein kinase.

groups, the number of PKs highly varied among different
fungi (Appendix D). The species with the most PKs (389)
was Botryobasidium botryosum, whereas Moniliophthora
perniciosa only had 48 PKs.
The differences in PKs among fungi were not limited
to the number but also in types of kinases. Kosti et al.
(2010) reported a large number of TKL protein kinase
genes in Laccaria bicolor. In the present study, TKL gene
duplication was found not only in L. bicolor, but also in most
Agaricomycotina. Duplicated genes mostly belonged to
TKL/TKL-CCIN group. In fact, the TKL/TKL-CCIN group
was only found in Agaricomycetes, whereas Cryptococcus
100 PEI Guo-liang et al. Journal of Integrative Agriculture 2019, 18(1): 96–107

gattii and Calocera cornea had no genes encoding TKL/
TKL-CCIN, suggesting that duplicated TKL/TKL-CCIN genes
could be a potential classifier for Agaricomycetes species.
The data show that PK gene duplication is a common
phenomenon in fungi. Besides the TKL/TKL-CCIN family,
which was largely observed in Agaricomycetes, and the
Ser/Thr group, which was identified in all fungi, there are
11 families/sub-families that were represented by more than
two copies in each fungus. For example, the families/sub-
families ratio of other/AGAK1 was 5.5, indicating that the
fungi containing these families/sub-families of PKs have at
least five copies of other/AGAK1 genes. Duplicate genes
are believed to be a major mechanism for establishing new
gene functions. These results suggest that the function of
some PK families/sub-families may change and play an
increasingly important role in the fungal cellular lifecycle.
The 119 families/sub-families vary greatly in the
distribution and abundance among fungi (Fig. 3). For
example, different members of the families Other/SCY1,
CMGC/CK2, STE/STE20/PAKA and STE/STE20/YSK are
present in the examined fungi. For the other 13 families/
sub-families, such as CMGC/CDKL, AGC/RSK/RSKP90 and
STE/STE20/KHS, only a single member was identified in one
predicted proteome, similar to the distribution in Arabidopsis
thaliana, whereas many copies were identified in human and
mouse genomes (Manning et al. 2002b; Caenepeel et al.

0 10 20 30 40 50 59 0 10 20 30 40 50 59
AGC AGC Other Other/SCY1
AGC/NDR/NDR Other/HAL
AGC/PDK1 Other/BUB
AGC/AKT Other/CDC7
AGC/RSK/RSK-UNCLASSIFIED Other/IRE
AGC/NDR/NDR-UNCLASSIFIED Other/ULK/ULK
AGC/PKA Other/AUR
AGC/PKC Other/NEK
AGC/YANK Other/RAN
AGC/RSK/RSKP70 Other/VPS15
AGC/NDR Other/NAK
AGC/DMPK Other/TTK
AGC/RSK Other/BUD32
AGC/DMPK/GEK Other/PEK/GCN2
AGC/DMPK/ROCK Other/IKS
AGC/RSK/RSKP90 Other/HASPIN
AGC/SGK Other/PLK
Atypical Other/CAMKK/CAMKK-META
Atypical/ABC1/ABC1-C Other/WEE/WEE-UNCLASSIFIED
Atypical/ABC1/ABC1-B Other/CAMKK
Atypical/RIO/RIO2 Other/NAK/BIKE
Atypical/RIO/RIO1 Other/CAMKK/ELM
Atypical/ABC1 Other/PEK
Atypical/ABC1/ABC1-A
Other/WEE
Atypical/PI4K
Other/PLK/PLK1
Atypical/PIKK/FRAP
Other/AGAK1
Atypical/HISK
Other/ULK
Atypical/PIKK
Other/CILIATE-A1
Atypical/BCR
Other/NAK/MPSK
Atypical/RIO
Other/NEK/NEK2
CAMK CAMK
Other/PLK/SAK

CAMK/CAMK1/CAMK1-RCK
CAMK/CAMKL/AMPK
CMGC CMGC/CK2
CMGC/CDK/CRK7
CAMK/CAMKL/KIN1
CMGC/SRPK
CAMK/CAMKL/KIN4
CMGC/CDK
CAMK/CAMK1
CMGC/CDK/CDC2
CAMK/CAMKL/PASK
CMGC/CDK/CDK8
CAMK/CAMK1/CAMK1-CMK
CMGC/CLK
CAMK/CAMKL/GIN4
CAMK/CAMKL/CHK1 CMGC/MAPK
CAMK/CAMKL CMGC/MAPK/P38
CAMK/RAD53 CMGC/CDK/CDK5
CAMK/CAMKL/MARK CMGC/CDK/CDK7
CAMK/CAMKL/BRSK CMGC/DYRK/YAK
CMGC/GSK
STE STE/STE20/PAKA CMGC/DYRK/DYRK2
STE/STE20/YSK CMGC/RCK/MAK
STE/STE11/BCK1 CMGC/DYRK/PRP4
STE/STE11/SSK CMGC/MAPK/ERK1
STE/STE11 CMGC
STE/STE7 CMGC/CDK/CDK9
STE/STE7/MEK1 CMGC/RCK
STE/STE11/CDC15 CMGC/CDK/PITSLRE
STE/STE7/MKK CMGC/DYRK
STE/STE20/FRAY CMGC/CDKL
STE/STE20
STE TKL TKL
STE/STE20/KHS TKL/LISK/LISK-DD1
TKL/TKL-CCIN
CK1 CK1/CK1/CK1-D
CK1/CK1/CK1-G PAB PAB-dependent
CK1/CK1
CK1 Ser/Thr Serine/Threonine

Fig. 3 Distribution of each protein kinase (PK) group among the 59 fungi. The colums showed the numbers of fungi that have
the members of the specific kinase group.
 PEI Guo-liang et al. Journal of Integrative Agriculture 2019, 18(1): 96–107 101

2004; Champion et al. 2004). comparison to the kinome size. At the sub-phylum level,
only Agaricomycetes had remarkable gene duplication in
3.2. Differences between Basidiomycetes and As- PKs (Appendix E).
comycetes In addition to those already mentioned above, several
PK families/sub-families were only observed in either
The studied fungi, which belong to Basidiomycetes and Basidiomycetes or Ascomycetes, but not both. Ten and
Ascomycetes, had different types and numbers of PKs. 13 PK families/sub-families are only found in Ascomycetes
Although these fungi of two phyla were separated long or in Basidiomycetes (Table 1). It should be noted that
ago, their PKs in some groups are still highly analogous, some families/sub-families, such as TKL/TKL-CCIN, Other/
however, the high homology was not found across all PK AGAK1 and TKL/LISK/LISK-DD1 in Basidiomycetes,
groups. Interestingly, the number of PKs in the STE group have similar numbers of duplicated genes, such as
is more abundant in Basidiomycetes than in Ascomycetes Other/NAK/BIKE, AGC/RSK/RSKP70 and Atypical/HISK
(Fig. 4-A, P<0.01). In contrast, the percentage of TKL in Ascomycetes. Interestingly, both TKL/TKL-CCIN and
in Basidiomycetes was higher than in Ascomycetes, Other/AGAK1 are only found in Agaricomycetes at high
whereas the group percentages of CMGC and other PKs
numbers. In contrast, TKL/LISK/LISK-DD1, STE/STE20/
in Basidiomycetes is lower than in Ascomycetes (Fig. 4-B,
FRAY and AGC/DMPK, which have more than 10 copies,
P<0.01). This result may indicate that Basidiomycetes
can be found in most Basidiomycetes. Other/NAK/BIKE
depend less on CMGC and other PKs during the cell cycle.
and AGC/RSK/RSKP70, which also have more than 10
PK gene duplication in Agaricomycetes may partially
copies, are found in most Ascomycetes and have no
explain why the STE group is more abundant in
obvious sub-phylum features.
Basidiomycetes than in Ascomycetes. In addition, the
The kinase percentage (the ratio of kinase number
decreased group percentage for CMGC and Other PKs
to gene number) differed between 0.5 and 2.5% among
in Basidiomycetes compared to Ascomycetes could be
different sub-phyla (Fig. 5). For most Taphrinomycetes,
partially due to gene amplification of TKL/TKL-CCIN. The
the kinase percentage was >2%, greater than all other
analysis indicates that, although Basidiomycetes fungi
fungi. Only Taphrina deformans has a kinase percentage
have bigger genomes than Ascomycetes, the genome
differences between these two phyla are not significant in of only 1.4%. As few fungal kinase percentages reach the
level of Taphrinomycetes, this may serve as a marker for
Taphrinomycetes species.

A B
60 Basidiomycota
Ascomycota Table 1 Phylum-specific protein kinase families/sub-families
25 Max.
99% Families Basidiomycetes Ascomycetes
95%
50 75% TKL/TKL-CCIN 552 0
Mean
Media Other/AGAK1 33 0
20 25%
TKL/LISK/LISK-DD1 18 0
Groups percentage (%)

40 5%
STE/STE20/FRAY 17 0
Number of kinases

1%
Min.
AGC/DMPK 12 0
15 30 AGC/DMPK/GEK 3 0
STE/STE20/KHS 1 0
CK1 1 0
10 20 CAMK/CAMKL/BRSK 1 0
Other/NEK/NEK2 1 0
AGC/RSK/RSKP90 1 0
10
5 AGC/DMPK/ROCK 1 0
Atypical/BCR 1 0
0 Other/NAK/BIKE 0 45
0 AGC/RSK/RSKP70 0 32
STE TKL CMGC Other Atypical/HISK 0 5
CMGC/DYRK 0 3
Other/CILIATE-A1 0 1
Fig. 4 Difference in protein kinases (PKs) between Ascomycetes
CMGC/CDKL 0 1
and Basidiomycetes. A, the number of PKs in group STE was
plotted for Ascomycetes and Basidiomycetes. B, the group AGC/SGK 0 1
percentage of PKs in groups TKL, CMGC and other were Other/PLK/SAK 0 1
plotted for Ascomycetes and Basidiomycetes respectively Atypical/RIO 0 1
(t-test, P<0.01). Other/NAK/MPSK 0 1
102 PEI Guo-liang et al. Journal of Integrative Agriculture 2019, 18(1): 96–107
3.0
Max.
99%
95%
75%
Mean
Media
25%
2.5 5%
1%
Min.
2.0
Kinase percentage (%)
1.5
1.0
0.5
0 identified in this study, representing 68.7% of all catalytic
A B C D E F
Fig. 5 Different percentages of protein kinases (PKs) for
sub-classification in Ascomycetes and Basidiomycetes. The
percentage of PKs was plotted for sub-classifications in
Ascomycetes and Basidiomycetes. A, Pucciniomycotina; B,
Ustilaginomycotina; C, Agaricomycotina; D, Pezizomycotina;
E, Saccharomycotina; F, Taphrinomycotina. The ratio of the
kinome in Saccharomycotina was more abundant than other
fungi studied.
3.3. Domain distribution of PKs
Protein kinases play an important role in the cell cycle and
function in protein interaction networks. In order to establish
contact with other proteins and initiate different functions,
most protein kinases have other domains in addition to the
catalytic domain. In terms of protein kinase sequence,
the catalytic domain is frequently tethered to one or more
non-kinase domains that are responsible for regulation,
substrate specificity, scaffolding, or other functions. In
previous studies, researchers focused on both the catalytic
and non-kinase domains (Manning et al. 2002a; Kosti et al.
2010), therefore providing a rationale for the present study to
search for the putative kinases against the Pfam database.
Kinases were annotated to determine the identity and
number of domains flanking the catalytic domain.
Among the 7 557 PKs identified from the studied 59
fungal species, 7 419 significantly matched 10 429 Pfam
domains belonging to 181 different domain types. These
results expand the study conducted by Kosti et al. (2010),
with more kinase sequences in more groups, increasing
from an average of 99.2 PK sequences per fungal species
to 128 sequences, although the average of 1.4 domain per
sequence did not change. Interestingly, the type of domains
and distribution of some domains changed greatly. These
differences between the two studies could partly be due to
the total number of kinase domains examined and changes
in the version of Pfam. In the present study, the following
17 domain types had kinase activity: protein kinase domain
(7 162 domains), protein tyrosine kinase (581), protein kinase
C terminal domain (210), C1 domain (123), RIO1 family
(103), kinase associated domain 1 (66), polydenylate sensor
of SNF1-like protein kinase (61), Ras-binding domain of
Byr2 (52), fungal kinase associated domain (44), His kinase
A (phospho-acceptor) domain (35), histidine kinase, DNA
gyrase B, and HSP90-like ATPase (31), lipopolysaccharide
kinase (Kdo/WaaP) family (26), lipopolysaccharide kinase
(Kdo/WaaP) family (26), phosphatidylinositol 3- and 4-kinase
(25), yeast phosphatidylinositol-4-OH kinase Pik1 (8), PIK
domain (2) and ecdysteroid kinase (1). The protein kinase
domain was the most common type of kinase domain
domains. Fig. 6-A shows 30 of the most common domain
types found in PKs, including 11 of 17 domains that have
kinase activity.
Interestingly, the distribution of domains between
Basidiomycetes and Ascomycetes is different. There are
3 088 PK sequences with 4 190 domains in Basidiomycetes,
while there are 4 469 protein kinase sequences with 6 239
domains in Ascomycetes. Among 181 different domain
types, 67 of 181 kinds of domains were only observed in
Basidiomycetes, in contrast to 59 of 181 kinds of domains
observed in Ascomycetes only. Only 55 common elements
were found in both Basidiomycetes and Ascomycetes.
The 16 most common types of kinase domains for each
phylum are shown in Fig. 6-B and C. Tyrosine kinase
domains are more abundant in Basidiomycetes than in
Ascomycetes. In contrast, the protein kinase C terminal
domain and ABC1 family (which lack kinase activity) are
more abundant in Ascomycetes than in Basidiomycetes.
Interestingly, like the prevalence of TKL/TKL-CCIN in
Agaricomycetes, tyrosine kinase domains are highly
represented in Agaricomycetes and the TKL/TKL-CCIN
family. In most cases, the frequency of domains lacking
kinase activity found in kinase sequences was very low, and
91 of 181 domain types were only observed once among the
7 557 protein kinase sequences. In contrast, some domain
types were found at high frequencies in the present study.
Fifteen copies of HET (accession number PF06985) were
identified in Ascomycetes, while the alpha/beta hydrolase
fold (accession number PF07859) was observed at five
copies in Basidiomycetes, the highest number found for
 PEI Guo-liang et al. Journal of Integrative Agriculture 2019, 18(1): 96–107
0 100 200
A Protein kinase domain
Protein tyrosine kinase
ABC1 family
Protein kinase C
FHA domain
C1 domain
P21-Rho-binding domain
Hr1 repeat
RIO1 family
Response regulator
C2 domain
POLO box
Kinase associated domain 1
SNF1-like protein kinase
Ribonuclease 2-5A
Binding domain of tRNAs
Ankyrin repeats
DUF3543
Mad3/BUB1
Histidyl-tRNA synthetase
RWD domain
PH domain
Byr2
Rio2, N-terminal
SAM domain
DUF3635
Ubiquitin associated domain
Kinase associated-1 domain
WD domain, G-beta repeat
His kinase A
Fig. 6 Distribution of domains in protein kinases (PKs). A, distribution of the number of domains found in all fungal kinases over
the 10 groups of PKs. B and C, distribution of the number of domains found in Ascomycetes and Basidiomycetes. Domains with
kinase catalytic activity are colored deep blue, while the others in green.
any domain in this phylum.
The distribution of kinase associated domains among
the 10 PK groups is different. As shown in Fig. 7-A, the
CAMK group contains 38 types of domains, making it the
most complicated group of all PKs. This indicates that
kinase proteins in the CAMK group have a richness of
functional domains and a flexibility/expansibility in function.
The CK1 group contains two types of domains, containing
only a protein kinase domain and a casein kinase 1 gamma
C terminus which lacks kinase activity. In contrast, the
distribution of 10 common types of kinase domains among
the 10 PK groups is different. As shown in Fig. 7-B, in most
cases, the kinase proteins in the “Other” PK group contain a
protein kinase domain and a small domain which has kinase
activity. In contrast, kinase proteins in the TKL group contain
both a protein kinase domain and a protein tyrosine kinase,
which takes up almost half of the entire sequence. This is
similar to the CAMK group, which contains more domain
types, reducing the protein kinase domain percentage
(Fig. 7-B). For the AGC and Atypical groups, domains
are found exclusively in Basidiomycetes or Ascomycetes,
103
300 400 0 100 200 300 400
7 192 B Protein kinase domain 2 714
581 Protein tyrosine kinase 581
ABC1 family
Protein kinase C
C1 domain
P21-Rho-binding domain
POLO box duplicated region
FHA domain
Hr1 repeat
RIO1 family
WD domain, G-beta repeat
Ankyrin repeats (3 copies)
C2 domain
Kinase associated domain 1
Response regulator receiver
PH domain
C Protein kinase domain 4 478
ABC1 family
Protein kinase C
FHA domain
P21-Rho-binding domain
Hr1 repeat
C1 domain
RIO1 family
Response regulator receiver
Protein tyrosine kinase
C2 domain
SNF1-like protein kinase
Fungal kinase associated-1
Anticodon binding domain
Ribonuclease 2-5A
DUF3543
and these two groups are equally represented. In the
CAMK group, there were more domains exclusive to
Basidiomycetes than Ascomycetes, but this was reversed
in the CMGC and Ser/Thr groups. For the TKL and CK1
groups, no such domains were found in Ascomycetes.
The PAB group only had common domains found in both
Basidiomycetes and Ascomycetes.
3.4. PKs in different nutrition modes and pathoge-
nicity of fungi
Different fungi live in very particular conditions, and PKs are
utilized to best adapt to their specific environment. Thus,
it is important to uncover differences in fungal life styles,
such as methods for obtaining nutrients or invading a host.
In the present study, the evolutionary status of each fungal
species is inextricably tied to survival. Except Pneumocystis
jirovecii and T. deformans, most Saccharomycotina and
Taphrinomycotina fungal species are yeasts, which have
the same nutritional mode and do not invade hosts. This
suggests that the nutritional modes within the same sub-
104 PEI Guo-liang et al. Journal of Integrative Agriculture 2019, 18(1): 96–107

phylum can be compared to determine significant differences
based on the same evolutionary status. Thus, nutritional
modes and pathogenicity affect the evolutionary status of
fungi.
As shown in Fig. 8-A, pathogenic fungi with different
hosts (including plant, insect and human pathogens) and
yeasts were compared. Interestingly, all fungi with different
hosts and yeasts have particular families of kinases. The
particular PK families of human pathogenic fungi are Other/
CILIATE-A1 and Other/PLK/SAK. The AGC/SGK family is
only found in insect pathogenic fungi, including Ajellomyces
capsulatus, Cordyceps militaris and Metarhizium anisopliae.
Seven PK families, which form the largest group among
all studied groups, only appear in plant pathogenic fungi,
suggesting that these fungi may have different strategies
in adapting to kinds of plants, such as monocotyledons and
dicotyledons. Some fungi from the Saccharomycotina and
Agaricomycotina were used as a negative control group
to determine the differences between pathogenic and
non-pathogenic fungi in the five PK families found in this
group.
The differences in PKs between fungi with different
nutritional modes were compared (Fig. 8-B). Common
PK families were the most prevalent and 84 families were

A Percentage (%) B
0 10 20 30 40
Atypical Protein kinase domain
CAMK
Protein tyrosine kinase
Other TKL
Protein kinase C terminal domain
Ser/Thr PAB
C1 domain
CMGC Ser/Thr RIO1 family

Atypical Other Kinase associated domain 1

STE Adenylate sensor of

TKL SNF1-like protein kinase
CAMK Ras-binding domain of Byr2
STE
CMGC Fungal kinase
AGC
associated-1 domain
CK1
PAB His kinase A domain
AGC
CK1
0 20 40 60 80 100
Percentage (%)

Fig. 7 Domain distribution in protein kinase (PK) groups. A, the number of domains found in PK groups. B, the distribution of
kinase catalytic domains between PK groups. The x-axis denotes the percentage of genes contained the domains in PK groups.

Facu
ltativ
A Insect Plant B e pa
rasit
2 ic
c

7
hi

1
p

N
ro

on 0 1
ot
an

0 e 1
Bi

5 3 1 0
Hemibio

0
um

2 0 2
H

0 1
1 0 3 0
2 5 0 3
trophic

84 0
2
0 1
3 85 0 1
0 0
0 1
1
0 1
9 0 2
Sy

gi
m

6
fun
bio

ic
1 hyt
tic

aprop
S

Fig. 8 Venn diagrams of shared protein kinase (PK) families and unique PK families in fungi. A, Venn diagram showing the
common and specific PKs among fungi. Three hosts of pathogenic fungi (human, insect, plant) and yeast were used to generate
the Venn diagram. In the graph were reported the number of species-specific and common PKs. B, Venn diagram showing the
common and specific PKs in five modes of nutrition in fungi. Five types of nutritional modes: saprophytic, facultative parasitic,
hemi-biotrophic, biotrophic, and symbiotic fungi were used to generate the Venn diagram. In the graph were reported the number
of species-specific and common PKs.
 PEI Guo-liang et al. Journal of Integrative Agriculture 2019, 18(1): 96–107 105

found. Dissimilar from other kinds of nutritional modes, stands for one or several PK families/sub-families, which
mutualistic symbiotic fungi show no particular PK families. were clustered together in small groups. Where circles
This result confirms that mutualistic symbiotic fungi have intersect with each other a bias for PK families/sub-families
lost many functional genes during the symbiosis and part within different groups of fungi is suggested. As illustrated
of the gene regulatory networks is dependent on the host in Fig. 9, the circle of Agaricomycotina covers the largest
regulation system. In contrast, saprophytic fungi have the area and gives a good description of PK gene amplification
largest amount of PK families. This suggested that this kind in this sub-phylum. The two sub-phyla Taphrinomycotina
of fungi must be more flexible in order to survive complex and Saccharomycotina cover an area close to one another,
environments. More details on the PK families/sub-families providing a good indication that most of the fungi in these
in this analysis are included in Appendix F. two sub-phyla are yeasts and have a close evolutionary
status and similar lifestyles. It should be noted that all of
3.5. Principal component analysis of fungal kinases the circles are consistent with evolutionary status, and the
discontinuous distribution suggested that evolution of the
PCA is one of the most useful statistical tools for analyzing PKs are complicated and associated with the lifestyle.
multivariate data and has been widely applied to analyze In parallel, 26 of the most common domains were
biological data. PCA transforms a number of correlated analyzed (Fig. 10). This is distinguished from the result of
variables into a smaller number of uncorrelated variables,
which are called principal components (PCs) with a minimal
loss of information. The reduced numbers of top ranked PCs
are calculated by projecting samples onto spaces spanned by
“eigenvectors” of a sample covariance matrix and selecting
the “eigenvectors” that comprise the largest contribution of
the sample variation. There are two approaches to perform
PCA, using “eigenvalue” decomposition (P-mode), or
singular value decomposition (Q-mode). The present study
utilized the ade4 package of R language to analyze the data
with the “eigenvalue” decomposition (P-mode) method,
which uses the covariance relationships between markers,
focusing on the differentiation of PK families/sub-families
with different evolutionary status. Discovering relationships
PC2 value

between fungi of different evolutionary status is a difficult

task, especially as the dissimilarities are based on individual
elements within the six sub-phyla, which includes fungi at the
same evolutionary status but with different lifestyles. This C B
is further complicated by difficulties in finding species that A D
from all six sub-phyla with similar lifestyles that also have
published and sequenced genomes. As a result, the PCA E
F
inevitably mirrors both the evolutionary aspects of a genome
and conservation of the functional genome.
The methodology for this research is not entirely
consistent with previous studies. First, all fungi were grouped
by evolutionary status, followed by listing the frequency
and type of the most common PK families/sub-families or
domains found among all fungal kinomes. Next, PCA was PC1 value
utilized to cluster fungi with PK families/sub-families or
domains based on grouping data. Fig. 9 shows the PCA Fig. 9 Principal component analysis (PCA) result for protein
clustering of the different fungi, based on the frequency kinases (PKs) from 59 fungi in six sub-phyla. The PCA results
from the 59 fungi in six sub-phyla. A total of 76 of 119 PKs
and type of the 76 most common PK families/sub-families families which could be found in at least half of these fungi were
found among all fungal kinomes. This figure composed of used to generate the PCA figure. Each dot represents one cluster
a round circle and some dots, and the circle area covered of PKs. The color for sub-phylum from Pucciniomycotina (A),
Ustilaginomycotina (B), Agaricomycotina (C), Pezizomycotina
depends on the site of the dots, suggesting frequency and (D), Saccharomycotina (E), and Taphrinomycotina (F) are
type of PK families/sub-families within the group. Each dot black, red, green, navy blue, light blue, and pink, respectively.
106 PEI Guo-liang et al. Journal of Integrative Agriculture 2019, 18(1): 96–107
the PK families/sub-families, as this figure was drawn using
common domains, showing that the areas of each circle
intersect each other more steadily and continuously, except
the areas of Taphrinomycotina and Saccharomycotina which
are irregularly distributed. This does not reflect a clear
correlation between domain distributions and taxonomic
classification, while also reflecting the divergence between
main functional domains and associated domains, which
have different roles in natural selection.
In contrast, when the PK families/sub-families for
fungi from different nutritional modes and pathogenicity
were analyzed by PCA, 48 of the 59 fungi were clearly
PC2 value
A
C than in Ascomycetes. Importantly, some of the PK family
B D
F E
PC1 value
Fig. 10 Principal component analysis (PCA) result for associated
domains from 59 fungi in six sub-phyla. The PCA result of the
59 fungi from six sub-phyla. A total of 26 associated domains
which can be found in at least half of these fungi were used
to generate the PCA figure. Each dot represents one cluster
of PKs. The color for sub-phylum from Pucciniomycotina (A),
Ustilaginomycotina (B), Agaricomycotina (C), Pezizomycotina
(D), Saccharomycotina (E), and Taphrinomycotina (F) are black,
red, green, navy blue, light blue and pink, respectively.
differentiated for the five nutritional modes: saprophytic,
facultative parasitic, hemi-biotrophic, biotrophic, and
mutualistic symbiotic fungi (Appendix G). As a result, the PK
composition for biotrophic fungi was remarkably consistent
with the symbiotic fungi, as most the areas coincided.
Although the hemi-biotrophic and biotrophic fungi have
similar stages in their lifecycles, there is a difference in the
PK composition between these two kinds of fungi. The
largest area covered by saprophytic fungi may suggest that
this kind of fungi have a complete PK composition compared
with the four other kinds of nutritional modes. Clustering the
PK families/sub-families of pathogenic fungi with different
hosts (plant, insect and human) and yeasts revealed that
the PK compositions of plant pathogenic fungi and insect
pathogenic fungi are almost identical as all of the areas
covered by insect pathogenic fungi were surrounded by plant
pathogenic fungi (Appendix H). Human pathogenic fungi are
very different from plant pathogenic and insect pathogenic
fungi. The greatest area covered by yeasts, which do not
have a host, suggests that there is a big difference in PK
composition between pathogenic and non-pathogenic fungi.
4. Conclusion
We systemically identified PKs from the predicted proteomes
of 59 representative fungi belonging to Ascomycota and
Basidiomycota. Comparative analysis revealed that fungi
between Ascomycota and Basidiomycota exhibited a high
diversity in the number and variety of PKs. Some PK groups,
CMGC and Other, were the most prevalent groups, followed
by AGC, CAMK, STE and Ser/Thr, and then CK1, PAB and
TKL were the least populated groups. On the other hand,
the STE group was more abundant in Basidiomycetes
distribution appeared to be phylum-specific. Ten families/
sub-families were found only in Ascomycetes, and 13
families/sub-families were only found in Basidiomycota.
More interestingly, the TKL/TKL-CCIN group had remarkable
gene duplication in many Agaricomycotina species and this
appearred to be subphylum-specific. Although there were
different kinase percentages among the six sub-phyla, this
is not an ideal method for classifying sub-phylum, but in
most cases should be a practical method for identifying
Taphrinomycetes. In addition, the distribution of accessory
domains, which may have functional implications, has a
usage bias that varies between the two phyla. The protein
kinase Tyr, which has kinase catalytic activity, is highly
represented in Agaricomycotina. The PCA demonstrates
that there is a divergence between the main functional
domains and associated domains, and that the two parts in
the PK sequences have different roles in natural selection,
allowing for more selection and choice with the associated
 PEI Guo-liang et al. Journal of Integrative Agriculture 2019, 18(1): 96–107
domains. Furthermore, the relationships between different
classifications of fungi, such as evolutionary status,
nutritional mode and pathogenicity, indicate that the
distribution of PK families is influenced by both evolutionary
status and by fungal lifestyle. Thus, the present study
provides insights into the variety and expansion of PK
families, which will be useful in understanding the differences
in regulatory mechanisms among different species of fungi.
Acknowledgements
This work was supported by the National Science &
Technology Pillar Program of China during the Twelfth
Five-Year Plan period (2012BAD19B04), the National
Natural Science Foundation of China (31371924), the 111
Project from the Ministry of Education of China (B07049),
and the National Basic Research Program of China
(2013CB127700). We thank Prof. Liu Huiquan, College
of Plant Protection, Northwest A&F University, China for
helpful discussion.
Appendices associated with this paper can be available on
http://www.ChinaAgriSci.com/V2/En/appendix.htm
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