Chromatin structure 

modifications by histone
acetyltransferase are involved in multiple biological
processes in eukaryotes. In the present study, the GCN5
homologue FocGCN5 was identified in Fusarium
oxysporum f. sp. cubense tropical race 4 (Foc TR4). The
coding gene was then knocked out to investigate the
roles of FocGNC5. The mutant ΔFocGCN5 was found
significantly reduced in growth rate and conidiation, and
almost completely lost pathogenicity to banana plantlets.
The RNA-seq analysis provide an insight into the
underlying mechanism. Firstly, transcription of the
genes involved in carbohydrate metabolism and fungal
cell wall synthesis was reduced in ΔFocGCN5, leading to
the impairment of apical deposition of cell-wall material.
Secondly, FocabaA, one of the pivotal regulators of
conidiation, was significantly reduced in expression in
ΔFocGCN5, which might be the main cause of the
conidiation reduction. Thirdly, the pathogenicity-
associated factors, including effectors and plant cell wall
degrading enzymes, were almost all down-regulated in
ΔFocGCN5, which accounts for the decrease of
pathogenicity. In addition, the stress tolerance to salt,
heat, and cell wall inhibitors was slightly increased in
ΔFocGCN5. Taken together, our studies clarify the roles
of FocGCN5 in growth, conidiation, and pathogenicity of
Foc TR4, and explore the possible mechanism behind its
biological functions.

Banana (Musa spp.) are the second largest fruit crops in the world. A
variety of biotic and abiotic stresses, such as drought, heat, diseases,
could limit the yields and quality of banana in various degrees.
Among them, Fusarium wilt, caused by Fusarium oxysporum f.
sp. cubense (Foc), is the most destructive disease to banana plantation
worldwide [[1], [2], [3]]. Until now, there are 4 races of Foc have been
identified; among them, the race 4 shows strong pathogenicity to Cav-
endish banana, the world's most widely planted cultivar [2,4]. Foc race
4 is further classified into ‘tropical race 4’ (Foc TR4) and ‘subtropical
race 4’ (Foc ST4). Given the fact that Foc TR4 causes disease on Cav-
endish in both tropical and subtropical conditions, the pathogen is be-
lieved to pose a serious threat to the banana production globally at
present [5]. However, there is still no effective control method against
this soil borne pathogen.
In the past decades, studies have shown that epigenetic regulation
plays important roles in the regulation of biological processes, such as
growth, development, tissue differentiation, diseases, and interactions
between organisms and the environment [[6], [7], [8], [9]]. Histones
are the main components of nuclear chromosomes in eukaryotes, and
different histone modifications are generally associated with specific
transcriptional states by altering dynamic transitions of chromatin
structure. Histone acetylation is balanced by histone acetyltransferases
(HATs) and histone deacetylases (HDACs) [10,11]. HATs acetylate
histones and physically relax chromatin configuration by reducing the
affinity between DNA and histones, making it easier for the binding of
transcription factors, and thus active gene expression. While HDACs
remove acetyl groups from histones and lead to relatively condensed
chromatin conformation and therefore repress gene transcription [12].
According to the subcellular location, HATs are divided into two
classes. Type A HATs, including GNATs family (GCN5-related N-
acetyltransferases), MYST family, P300/CBP family, and TFIID sub-
unit TAF250 family, mainly exist in the nucleus and bind to chromatin
[[13], [14], [15]]; while Type B HATs mainly present in the cytoplasm
[16].
GCN5 (General Control Non-derepressible 5), one important member
of the GNATs family of Type A HATs, can effectively catalyze his-
tone acetylation. GCN5 protein was first identified in Saccharomyces
cerevisiae, and it is the core part of the histone acetylation complex
SAGA (SPT-Ada-GCN5 acetyltransferase), as well as a component of
other important protein complexes [17,18]. It mainly functions at spe-
cific lysine residues in histones H3 and H2B, and thus regulates global
gene transcription. In animal cells, the roles of GCN5 in the regulation
of gene transcription, DNA damage repair, nucleosome assembly, cell
differentiation, cell cycle regulation and tumorigenesis have been well
documented [19,20]. In plants, GCN5 is involved in physiological
processes such as cell differentiation, organogenesis of leaf and
flower, and also participates in the regulation of plant responses to bi-
otic and abiotic stresses, such as drought, salt stress, low temperature,
and phosphate starvation [[21], [22], [23]]. In Candida albicans,
CaGcn5 is required for the filamentous and invasive growth, as well
as stress tolerance [24]. In Fusarium graminearum, GCN5 plays an
important role in mycelial growth, sexual and asexual reproduction,
biosynthesis of deoxynivalenol (DON), stress tolerance, and pathogen-
icity [25,26]. GCN5 (GCNE) of Aspergillus nidulans controls
the conidiation by regulating the expression of several conidiation re-
lated factors and plays a central role in asexual development [27].
In Aspergillus flavus, AflGCNE plays multiple roles
in morphogenesis, aflatoxin synthesis, stress tolerance, and pathogeni-
city [28]. Moreover, in Magnaporthe oryzae, GCN5 can directly acet-
ylate nonhistone, cytosolic proteins, and mediates phototropic and
starvation-induced autophagy [29,30]. However, the roles of GCN5 in
the soil borne pathogen Foc TR4 are still unknown. Here, we charac-
terized the functions of FocGCN5 in growth, conidiation, and stress
tolerance, and we sought possible target genes regulated by FocGCN5
through transcriptome analysis.

2. Materials and methods

2.1. Fungal strains and culture conditions

F. oxysporum f.sp. cubense race 4 (Foc TR4, isolate B2) strain was
used as wild type (WT) for gene manipulation. Foc TR4 was cultured
on minimal medium (0.5 g KCl, 2 g, NaNO3, 1 g KH2PO4, 0.5 g
MgSO4·7H2O, 0.01 g FeSO4·7H2O, 30 gdextrose, pH 6.5, for 1 L),
complete medium (2 g petone, 6 g NaNO3, 10 g Gloucose, 0.52 g KCl,
0.52 g MgSO4·7H2O, 1.52 g KH2PO4, 1 g casamino acids, 1 g yeast
extract, 0.01 mg vitamins, 0.01 mg trace elements, pH 6.5, for 1 L),
and potato dextrose agar (PDA) medium (Difco, Thermo Fisher Sci-
entific) as indicated.