Chitin Deacetylases Are Required for Epichlo€e festucae
Endophytic Cell Wall Remodeling During Establishment
of a Mutualistic Symbiotic Interaction with Lolium perenne

Nazanin Noorifar,1 Matthew S. Savoian,1 Arvina Ram,1 Yonathan Lukito,1 Berit Hassing,1,2

Tobias W. Weikert,3 Bruno M. Moerschbacher,3 and Barry Scott1,2,†

1 School of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand
2Bioprotection Research Centre, Massey University, Palmerston North 4442, New Zealand
3 Institute for Biology and Biotechnology of Plants, Westf€alische Wilhelms-Universit€at, M€unster, Germany

Accepted 5 May 2021.

Epichlo€e festucae forms a mutualistic symbiotic association
with Lolium perenne. This biotrophic fungus systemically colo-
nizes the intercellular spaces of aerial tissues to form an endo-
phytic hyphal network and also grows as an epiphyte.
However, little is known about the cell wall–remodeling mech-
anisms required to avoid host defense and maintain intercalary
growth within the host. Here, we use a suite of molecular
probes to show that the E. festucae cell wall is remodeled by
conversion of chitin to chitosan during infection of L. perenne
seedlings, as the hyphae switch from free-living to endophytic
growth. When hyphae transition from endophytic to epiphytic
growth, the cell wall is remodeled from predominantly chitosan
to chitin. This conversion from chitin to chitosan is catalyzed
by chitin deacetylase. The genome of E. festucae encodes three
putative chitin deacetylases, two of which (cdaA and cdaB) are
expressed in planta. Deletion of either of these genes results in
disruption of fungal intercalary growth in the intercellular
spaces of plants infected with these mutants. These results
establish that these two genes are required for maintenance
of the mutualistic symbiotic interaction between E. festucae
and L. perenne.

Keywords: chitin, chitosan, endophyte, Epichlo€efestucae, molecular
probes

Fungal species from the genus Epichlo€e (Ascomycota, Clavici-
pitaceae) are symbionts of cool-season grasses that form systemic
hyphal networks within the apoplastic spaces of host aerial tissues
(Schardl et al. 2004, 2009). These infections are usually asymp-
tomatic and are generally regarded as mutualistic because the

fungal endophyte synthesizes a range of bioactive secondary
metabolites that protect the host against biotic stress (Schardl et al.
2013; Tanaka et al. 2012). In return, the host provides a source of
nutrients for the fungus either directly from the apoplastic space or
by way of specialized transport processes between the hyphae and
the attached plant cells (Christensen et al. 2002; Hinton and Bacon
1985). The stable association between E. festucae and Lolium per-
enne (perennial ryegrass) is now established as a model experi-
mental system for studying this important above-ground fungal-
grass symbiotic interaction (Scott et al. 2012; Scott et al. 2018).
E. festucae establishes a hyphal network within the aerial tissue

of ryegrass by two distinct growth mechanisms (Scott et al. 2012).
In the true stem, hyphae grow between the tightly packed cells by
tip growth, lateral branching, and hyphal cell-to-cell fusion to form
a hyphal network within these tissues. From here, hyphae ramify
into the leaf primordia and axillary buds, which give rise to new
leaves and shoots, respectively (Christensen and Voissey 2007).
Once hyphae colonize leaf primordia, which comprise sheath
and blade zones of cell division, they become attached to the plant
cell wall. As the leaf cells expand from these cell division zones,
the hyphae are stretched, triggering a unique pattern of hyphal
growth known as intercalary growth (Christensen et al. 2008).
This ensures the hyphae avoid mechanical shear as the host cells,
to which they are firmly attached, expand rapidly (Voisey 2010).
However, we have little understanding of how the fungal cell wall
is remodeled to enable this pattern of growth in the plant or how E.
festucae avoids eliciting a host immune response from direct phys-
ical contact between the fungal and plant cell walls.
Fungal cell walls are comprised of a complex matrix of polysac-

charides and proteins, including b -glucans (b-1,3-glucan and
b-1,6-glucan), a-glucans (a-1,3-glucan and a-1,4-glucan), chitin
and mannan (P�erez and Ribas 2004; Latg�e 2007). However, the
composition and amount of each of these components vary consid-
erably among different fungal species, and between different fun-
gal structures, such as hyphae, conidia, or capsules (Erwig and
Gow 2016; Fujikawa et al. 2009; Gow et al. 2017; Hopke et al.
2018). Chitin, a linear homopolymer of b-1,4-linked N-acetyl-D-
glucosamine (GlcNAc) monomers, is a structurally important
component of the cell wall (Bowman and Free 2006; Latg�e
2007, 2010). It is also recognized as an important microbe-
associated molecular pattern that will elicit pattern-triggered
immunity (PTI), unless it is masked, modified, or sequestered to
prevent interaction with plant chitin receptors (Boller and Felix
2009; Boutrot and Zipfel 2017; Zipfel and Oldroyd 2017). Fungal
secretion of apoplastic effectors, which bind to chitin oligomers in
the apoplastic space or to the fungal cell wall itself is a

†Corresponding author: B. Scott: d.b.scott@massey.ac.nz

Funding: This research was supported by a grant from the Tertiary Edu-
cation Commission (number RM20918) to the Bio-Protection Research
Centre and by Massey University. N. Noorifar was supported by a Massey
University PhD scholarship and B. Scott by an Alexander von Humboldt
Research Award.

*The e-Xtra logo stands for “electronic extra” and indicates there are
supplementary materials published online.

The author(s) declare no conflict of interest.

Copyright © 2021 The Author(s). This is an open access article
distributed under the CC BY-NC-ND 4.0 International license.

Vol. 34, No. 10, 2021 / 1181

MPMI Vol. 34, No. 10, 2021, pp. 1181–1192, https://doi.org/10.1094/MPMI-12-20-0347-R

mailto:d.b.scott@massey.ac.nz
http://creativecommons.org/licenses/by-nc-nd/4.0/
http://creativecommons.org/licenses/by-nc-nd/4.0/


well-characterized mechanism for preventing elicitation of a PTI
response (de Jonge et al. 2010; van Esse et al. 2007). Other strat-
egies used by fungi to evade plant immunity include masking of
chitin with a-1,3-glucan (Fujikawa et al. 2012) or conversion of
chitin to chitosan by chitin deacetylases (Cord-Landwehr et al.
2016; El Gueddari et al. 2002; Gao et al. 2019; Nampally et al.
2012; Xu et al. 2020). While chitin appears to be an important
component of the cell wall of E. festucae grown in axenic culture,
it is not detected in the cell wall of endophytic hyphae, although it
is clearly present in septa, as seen following infiltration of leaf tis-
sue with the chitin-specific wheat germ agglutinin-AlexaFluor 488
(WGA-AF488) probe (Becker et al. 2015, 2016; Eaton et al.
2015).
Besides growing as an endophyte, E. festucae is also capable of

epiphytic growth across the surface of a grass leaf (Christensen
et al. 1997; Moy et al. 2000). These epiphytic hyphae arise from
and remain connected to endophytic hyphae within the leaf.
Endophytic hyphae colonize the epidermal cells beneath the
cuticle in this leaf zone, where they differentiate to form an
appressorium-like structure called an expressorium that facilitates
cuticle penetration and exit of the hyphae onto the leaf surface
(Becker et al. 2016). Dramatically, the entire cell wall of these epi-
phytic hyphae becomes labeled with WGA-AF488 within a few
cell divisions following emergence, suggesting chitin becomes a
dominant component of the cell wall.
These observations suggest that the chitin/chitosan component

of the E. festucae cell wall is modified during the transition
from free-living to endophytic hyphae and from endophytic to epi-
phytic hyphae. To test this hypothesis, we employ a range of
molecular probes to identify whether chitin or chitosan is present
in the cell wall of E. festucae at these different developmental
stages and test genetically whether chitin deacetylases are required
for establishment and maintenance of this mutualistic symbiotic
interaction.

RESULTS

Endophytic and epiphytic hyphal cell walls have distinct
chitin staining profiles.
Tissue from L. perenne seedlings at an early stage of infection

with E. festucae Fl1 were stained with WGA-AF488 and aniline
blue, which stain chitin and hyaline fungal structures, respectively.
Confocal laser scanningmicroscopy (CLSM) showed that only the
septa of endophytic hyphae were labeled with WGA-AF488,
while the hyphal cell wall was labeled with aniline blue (Fig.
1A through D). In contrast, both the cell wall and septa of epiphyl-
lous hyphae around the site of infection were labeled with WGA-
AF488 (Fig. 1A through C). Similarly, labeling of infected plant
sheath sampleswith chitin-binding protein (CBP) (Fuenzalida et al.
2014) showed that only fungal septa of endophytic hyphae labeled
with this chitin-specific probe (Fig. 1E through G). This suggests
that chitin is either masked or absent from the cell walls of endo-
phytic hyphae.
Strikingly, labeling of leaf sheath samples with WGA-AF488

and aniline blue showed that epiphytic hyphae near the expresso-
rium emergence point on the leaf surface had a combination of
both staining patterns. A transition was observed from septa-
only WGA-AF488 labeling at the emergence point to a progres-
sively more homogeneous labeling of the hyphal cell walls outside
of the plant (Fig. 2). This suggests that the cell wall of E. festucae
is remodeled during the transition from endophytic to epiphytic
growth.

Endophytic hyphal cell walls can be labeled with a
chitosan-specific probe.
Given that conversion of chitin to chitosan in the cell walls of

pathogenic fungi is a known mechanism for evading a host

defense response (El Gueddari et al. 2002; Nampally et al.
2012), we hypothesized that a similar change occurs in the cell
wall of E. festucae endophytic hyphae. To test this hypothesis,
we probed fungal endophytic hyphae with chitosan affinity protein
(CAP), which selectively recognizes chitosan (Nampally et al.
2012). CLSM analysis of the plant cell division zone and leaf pri-
mordia revealed that CAP exclusively labeled the endophyte cell
wall in this zon, and did not label septa (Fig. 3A and B). A similar
labeling pattern was observed for endophytic hyphae within the
leaf sheath, with the hyphal cell wall labeled along its full visible
length with this chitosan-specific probe and no signal observed in
septa (Fig. 3C and D).

The E. festucae Fl1 genome encodes
three chitin deacetylases.
We analyzed the genome of E. festucae for genes encoding

chitin deacetylases, enzymes that catalyze the conversion of chitin
to chitosan (Christodoulidou et al. 1996). A tBLASTn search of
the E. festucae Fl1 genome sequence using aMagnaporthe oryzae
chitin deacetylase (CDA) protein sequence queries (MGG_12939,
MGG_14966, MGG_09159, MGG_04172, MGG_08774,
MGG_01868, MGG_08356, MGG_05023, MGG_04704, and
MGG_03461) (Geoghegan and Gurr 2016) identified three chitin
deacetylase genes, EfM3.035615, EfM3.030650, and
EfM3.042050 (Schardl et al. 2013), which were designated cdaA,
cdaB, and cdaC, respectively. These share 24 to 56% identity
with M. oryzae chitin deacetylase proteins (Supplementary Table
S1). The domain structure of the encoded CdaA, CdaB, and
CdaC proteins was determined using InterProScan (Blum et al.
2021), showing that all three proteins contain polysaccharide chitin
deacetylase (Pfam 01522) domains but no chitin binding (Pfam
00187) domains (Supplementary Fig. S1). In addition, CdaA and
CdaB contain an N-terminal signal peptide that targets proteins
toward the classical protein secretion pathway, although CdaB is
also predicted to contain a transmembrane helix, as found in the
M.oryzae (Geoghegan andGurr 2016) andTrichodermaatroviride
(Kappel et al. 2020) homologs CDA8 and CDA2, respectively. An
alignment of the three putative E. festucae chitin deacetylases with
the structurally characterized Streptococcus pneumoniae peptido-
glycan deacetylase and Colletotrichum lindemuthianum chitin
deacetylase confirmed that the S. pneumoniae catalytic residues
D275 and R364 as well as H417 and D391, which comprise the
two charged relay pairs, and the zinc (Zn)-binding triad of D276,
H326, and H330 are conserved in the E. festucae sequences (Sup-
plementary Fig. S2) (Blair et al. 2005, 2006).
To gain insight into which of the three chitin deacetylase genes

might be important for symbiosis, the expression of each of these
genes was compared between hyphae growing in planta and in
axenic culture (Hassing et al. 2019; Winter et al. 2018). This anal-
ysis revealed that cdaA was significantly upregulated in planta
compared with axenic culture, whereas cdaB had comparable lev-
els of expression under the two physiological conditions. cdaC
was not expressed under either condition (Supplementary Table
S2). We therefore generated a deletion mutant of cdaA and ana-
lyzed the axenic culture and plant interaction phenotypes of three
independently isolated mutants, DcdaA #28, #41, and #49
(Supplementary Fig. S3).

Phenotype analysis of DcdaA mutant.
Radial growth and morphology of DcdaA strains on potato dex-

trose agar (PDA) media was indistinguishable from the wild-type
(WT) strain. Widefield epifluorescence light microscopy analysis
of cultures grown on water agar and stained with Calcofluor white
showed that the morphology and frequency of hyphal bundles,
hyphal cell-to-cell fusions, and hyphal coils was similar between
DcdaA and WT (Supplementary Fig. S4). We next examined
the role of cdaA in the symbiotic interaction between E. festucae

1182 / Molecular Plant-Microbe Interactions



and L. perenne. Cultures of WT and DcdaA were inoculated into
L. perenne seedlings and the interaction phenotype was examined
at 10 weeks postinoculation (wpi). No significant differences in til-
ler height or tiller number were observed between plants infected
withWT and theDcdaAmutants (Fig. 4A). However, CLSM anal-
ysis revealed that DcdaA hyphae were less abundant and exhibited
a dramatically different cellular phenotype compared with WT
(Fig. 5). In contrast to WT, which had a highly restricted form
of growth, with each intercellular space containing a single hypha
running parallel to the leaf axis, with occasional fusion of lateral
hyphal branches, the hyphae of the DcdaA mutants were swollen
and occasionally formed dense convoluted structures (Fig. 5; Sup-
plementary Fig. S5). Analysis of relative endophyte biomass was
performed by quantitative PCR (qPCR), using the ratio of the

single-copy E. festucae pacC gene (Lukito et al. 2015) to the
single-copy L. perenne LpCCR1 gene (McInnes et al. 2002).
This showed no significant difference in the degree of host leaf
sheath and blade colonization between the DcdaA mutant and
WT (Supplementary Fig. S6). Introduction of the WT cdaA allele
into DcdaA complemented these in planta cellular phenotypes
(Fig. 6), confirming that the defects were specific to the deletion
of cdaA.

Phenotype analysis of DcdaB single and DcdaADcdaB
double mutants.
Given the relatively mild whole-plant interaction phenotype

observed following infection with DcdaA, we generated DcdaB
single (#17 and #22) and DcdaADcdaB double (#18 and #30)

Fig. 1. Labeling of Epichlo€e festucae hyphae at the site of inoculation and in mature leaf sheath tissue of Lolium perenne plants, using chitin-specific molec-
ular probes. A through D, Plant tissue was incubated with either wheat germ agglutinin-AlexaFluor 488 (WGA-AF488) or E through G, with chitin binding
protein (cyan), propidium iodide (yellow) and aniline blue (red). A, L. perenne seedling infected with E. festucae wild-type Fl1 (WT) 2 weeks postinoculation
showing epiphyllous hyphae (asterisks) stained with WGA-AF488 (blue pseudocolor). The rectangle labeled I outlines endophytic hyphae colonizing the
seedling meristem zone stained with aniline blue (red pseudocolor) and chitin in cell-wall septa stained with WGA-AF488 (blue pseudocolor). B, Higher
magnification of I in panel A, showing plant meristem colonization. C and D, Higher magnification of II and III in panel B, indicating epiphyllous (asterisk)
and endophytic hyphae respectively. E, Longitudinal section of L. perenne leaf sheath tissue infected with WT 10 weeks postinoculation, stained with chitin
binding protein (cyan), propidium iodide (yellow), and aniline blue (red). F, Higher magnification of I in panel E. G, Higher magnification of II in panel E.
Arrows identify fungal septa. Images were generated by maximum intensity projection of confocal z-stacks. Bar = 20 lm.

Vol. 34, No. 10, 2021 / 1183



mutants to assess how the loss of both deacetylase genes might
affect axenic culture growth and plant interaction phenotypes
(Supplementary Fig. S7). Radial growth and morphology of
DcdaB and DcdaADcdaB mutants on PDA media were indistin-
guishable from the WT strain. Epifluorescence light microscopy
analysis of cultures grown on water agar and stained with Calco-
fluor white showed that DcdaB and DcdaADcdaB mutants form
hyphal bundles, undergo hyphal cell-to-cell fusion, and form
hyphal coils with the same morphology and frequency observed
for theWT (Supplementary Fig. S8). However, the cell-wall integ-
rity of the DcdaADcdaB double mutant appears to be compro-
mised, as they frequently formed intrahyphal hyphae (Fig. 7).
In contrast to the DcdaAmutant, the DcdaBmutant had a strong

plant interaction phenotype, with the tiller length of infected plants
significantly reduced compared with plants infected withWT (Fig.
4). Surprisingly, the phenotype of plants infected with the
DcdaADcdaB double mutants were not noticeably different from
WT; while some plants had shorter tillers than WT, there was
no significant overall difference. This difference in whole-plant
interaction phenotype is most likely due to differences in host col-
onization, as the endophyte biomass in both leaf sheath and blade
was significantly greater than that of WT for the DcdaB single
mutant but not for the DcdaADcdaB double mutant (Supplemen-
tary Fig. S6). Analysis of the cellular phenotype by CSLM
revealed a striking cellular phenotype for both mutants, with
hyphae showing an irregular pattern of growth compared with
WT and frequently forming very complex convoluted structures
(Fig. 5; Supplementary Fig. S5). These hyphal growth defects
were particularly prominent for the DcdaB single mutant. Intro-
duction of the WT cdaB allele into DcdaB complemented these
in planta cellular phenotypes (Fig. 6). These hyphal morpholo-
gies were never observed in the WT, indicating that deletion of
these genes results in disruption of intercalary growth within
the host leaf tissue. However, even with deletion of both genes,
the cell walls of endophytic hyphae were still depleted of chitin
(Fig. 5; Supplementary Fig. S5). This absence of chitin is
unlikely to be due to depletion of the third chitin deacetylase,
i.e., CdaC, as the level of expression of cdaC was beyond the
limit of detection (Cp values of >35) in WT and the DcdaA,
DcdaB, or DcdaADcdaB mutant backgrounds (Supplementary
Fig. S9).

DISCUSSION

For phytopathogenic and symbiotic fungi to establish a compat-
ible interaction with their host plant, it is crucial that the cell-wall
components of the fungus that elicit a host defense response, such
as chitin and b-glucans, do not interact with the corresponding
host receptors (Fesel and Zuccaro 2016; S�anchez-Vallet et al.
2015). Secretion of fungal effectors into the apoplastic space is
a well-characterized mechanism for preventing elicitation of a
pathogen-associated molecular pattern (PAMP) PTI response
(de Jonge et al. 2010; Ma et al. 2018; van Esse et al. 2007; Wawra
et al. 2016). Other strategies used by fungi to evade plant immu-
nity include conversion of one or both cell-wall chitin and cell
wall–derived chitin oligomers to chitosan by chitin deacetylases
(Cord-Landwehr et al. 2016; El Gueddari et al. 2002; Gao et al.
2019; Nampally et al. 2012) and the masking of chitin with
a-1,3-glucan (Fujikawa et al. 2012). Here, we show that infection
of L. perenne seedlings with E. festucae results in conversion of
chitin to chitosan as the hyphae switch from free-living to endo-
phytic growth. Similarly, chitin becomes predominant in the fun-
gal cell wall as hyphae transition from endophytic to epiphytic
growth.
By probing the structure of the E. festucae cell wall in infected

plant tissues using the chitin-specific probe WGA-AF488, we
found this probe stains only the septa of endophytic hyphae but
decorates the entire cell wall of epiphytic hyphae (Becker et al.
2016). The absence of this probe from the endophytic cell wall
is unlikely to be due to inaccessibility due to chitin masked by
a-1,3 glucan (Fujikawa et al. 2012), as there is no gene encoding
an a-1,3 glucan synthase in the E. festucae genome (Becker et al.
2015). However, there are other cell-wall appositions that can
mask chitin (Freytag and Mendgen 1991). Also, chitin sources
internal to the fungal cell were uniformly labeled with WGA-
AF488, as observed for septa and the cell walls of intrahyphal
hyphae in some symbiotic mutants (Becker et al. 2015, 2016;
Eaton et al. 2015). To better understand the changes in cell-wall
chitin during the developmental transitions that accompany entry
and exit of E. festucae into leaf tissue, we employed chitosan-
and chitosan-specific molecular probes (Fuenzalida et al. 2014;
Nampally et al. 2012). Infiltration of leaf tissue with the chitosan
probe CAP resulted in the labeling of E. festucae cell walls but not

Fig. 2. Wheat germ agglutinin-AlexaFluor 488 (WGA-AF488) labeling of endophytic and epiphyllous hyphae of Epichlo€e festucae at the site of expressorium-
mediated exit from a leaf of Lolium perenne. A, E. festucae Fl1 endophytic hyphae stained with aniline blue (red), chitin in cell-wall septa with WGA-AF488
(blue), and fungal and plant nuclei with propidium iodide (yellow). B, E. festucae Fl1 expressorium (ex). The epiphyllous hyphae (red asterisk) stained with
WGA-AF488 (blue). C and D, Higher magnification of (I) and (II) showing the expressorium (ex), emergence point (yellow asterisk) and epiphyllous hyphae
(red asterisk). White arrows identify fungal septa. Images were generated by maximum intensity projections of confocal z-stacks. Bar = 20 lm.

1184 / Molecular Plant-Microbe Interactions



septa of hyphae growing between the tightly packed meristematic
cells at the base of the leaves and in the intercellular spaces of
mature leaf sheath tissue. This indicates that chitosan rather than
chitin predominates in the cell wall of endophytic hyphae, regard-
less of the host aerial tissue type. In contrast, infiltration of leaf tis-
sue with a second chitin binding probe, CBP, only labeled the
septa of endophytic hyphae, as previously observed for the
WGA-AF488 probe (Becker et al. 2015; Eaton et al. 2015). Sim-
ilarly, WGA-AF488 stained the entire cell wall of epiphyllous
hyphae, suggesting that chitin predominates in the cell wall fol-
lowing expressorium-mediated exit of endophytic hyphae onto
the leaf surface. Interestingly, complete restoration of chitin pres-
ence in the cell wall takes several cell divisions following exit of
the endophytic hyphae from the leaf cuticle (Becker et al. 2016). A
number of phytopathogenic fungi have also been shown to
undergo remodeling of the cell wall from chitin to chitosan during
host infection (El Gueddari et al. 2002; Nampally et al. 2012). The
presence of chitosan rather than chitin in the cell wall of endo-
phytic hyphae is a potential mechanism for E. festucae to avoid
a host immune response, given that chitosan with a sufficiently
low fraction of acetylation is a much weaker PAMP than chitin
and also a poorer substrate for chitinases, resulting in reduced lev-
els of putatively PAMP-active cell-wall polymer fragments (Cord-
Landwehr et al. 2016; Gubaeva et al. 2018; Vander et al. 1998).
Conversion of chitin to chitosan is catalyzed by chitin deacety-

lase, which was first biochemically characterized from Mucor

rouxii (Kafetzopoulos et al. 1993) and was then genetically char-
acterized in Saccharomyces cerevisiae, in which there are two
functionally redundant copies of the gene (Christodoulidou et al.
1996, 1999). Analysis of the genome of E. festucae identified three
genes, cdaA, cdaB, and cdaC, encoding proteins with domains
consistent with chitin deacetylases, including a Zn-binding motif
and catalytic-site amino acid residues known to be required for
peptidoglycan and chitin deacetylase activities in Streptococcus
pneumoniae and Colletotrichum lindemuthianum, respectively
(Blair et al. 2005, 2006). CdaA and CdaB also have predicted
N-terminal signal peptides, as found in their homologs from M.
oryzae and T. atroviride (Geoghegan and Gurr 2016; Kappel et al.
2020), suggesting they are both directed into the secretory path-
way. However, CdaB also has a putative transmembrane domain,
suggesting that it is localized to the outer membrane of the cell.
None of these putative chitin deacetylases are predicted to have
a C-terminal glycosylphosphatidylinositol anchor, which is a char-
acteristic feature of the three Cryptococcus neoformans (Baker
et al. 2007) and two Saccharomyces cerevisiae chitin deacetylases
(Christodoulidou et al. 1996, 1999), which all group in a separate
clade than the M. oryzae and T. atroviride chitin deacetylases
(Geoghegan and Gurr 2016; Kappel et al. 2020). Given that there
was a significant difference in cdaA expression in planta compared
with axenic culture (Hassing et al. 2019; Winter et al. 2018), we
first generated a targeted deletion of this gene and analyzed both
the axenic culture and plant-interaction phenotypes. While plants
infected with the DcdaA mutants had no obvious whole-plant
interaction phenotype, there was a distinct cellular phenotype.
CLSM analysis revealed that the endophytic hyphae of this mutant
were defective in intercalary growth within the leaves. Instead of
the restrictive pattern of growth observed for WT (Christensen
et al. 2008; Tanaka et al. 2006), hyphae of the DcdaA mutants
had an irregular pattern of growth, were frequently branched,
and occasionally formed dense convoluted structures. Interest-
ingly, while patches of WGA-AF488 labeling indicative of
increased chitin deposition were occasionally observed in endo-
phytic DcdaA hyphae, most DcdaA hyphae did not exhibit this
defect, suggesting deletion of this gene alone did not result in res-
toration of chitin to the endophytic cell wall.
To determine if the residual chitin deacetylating activity was

due to redundancy with CdaB, additional targeted gene deletions
were performed to generate DcdaB mutants and DcdaADcdaB
double mutants. While cdaB is not differentially expressed in
planta compared with axenic culture (Hassing et al. 2019; Winter
et al. 2018), the plant interaction phenotype for DcdaB was much
more severe than for both theDcdaA single and theDcdaADcdaB
double mutant. Plants infected with the DcdaB mutant were fre-
quently stunted, suggesting a severe antagonistic interaction
between this mutant and the host grass, as previously observed
for other symbiotic mutants (Becker et al. 2015; Scott et al.
2018; Tanaka et al. 2006). The absence of a whole-plant growth
phenotype for the DcdaA and DcdaADcdaBmutants is consistent
with their relative biomass being similar to the WT, whereas that
of the DcdaB mutant is increased. The results also suggest that
cdaA is dominant to cdaB. However, at the cellular level, the
hyphal growth phenotype was similar for all three mutants.
The E. festucae Dcdc42 mutant also lacks a whole-plant growth
phenotype but does have a cellular phenotype of reduced host
colonization and disrupted intercalary growth (Kayano et al.
2018). Even with deletion of both genes, chitin was still absent
in most of the endophytic hyphal cell walls. The lack of detect-
able expression of cdaC in these mutant backgrounds rules this
out as an explanation for the phenotype observed. One possible
explanation as to why chitin is still not detected in the cell walls
of the DcdaADcdaB double mutant is deletion of these two genes
somehow interferes with chitin as well as chitosan biosynthesis
(Christodoulidou et al. 1999).

Fig. 3. Epichlo€e festucae endophytic hyphae in leaf tissue of Lolium perenne
probed with chitosan affinity protein. Fungal cell wall labeled with chitosan
affinity protein (CAP) (green) and fungal nuclei stained with propidium
iodide (red). A and B, Hyphal labeling with CAP in leaf tissue at base of
tiller and C and D, in leaf sheath tissue. B and D show a higher magnifica-
tion image of I from panels A and C, respectively. Images were generated by
maximum intensity projection of confocal z-stacks. Bar = 10 lm.

Vol. 34, No. 10, 2021 / 1185



Fig. 4. Plant phenotype of Lolium perenne infected with Epichlo€e festucae wild type (WT), DcdaA, DcdaB, and DcdaADcdaB mutants. A through C, Whole
plant interaction phenotype of L. perenne plants infected with WT (Fl1), DcdaA, DcdaB, and DcdaADcdaB mutants 10 weeks after inoculation. Box plots
show tiller number and tiller height of L. perenne plants infected with WT or cda mutant strains. Significant differences (P < 0.05 as determined by a two-
tailed Student’s t test, n ³ 10) are indicated by a red asterisk (*). Outliers are indicated by circles.

1186 / Molecular Plant-Microbe Interactions



Endophytic hyphae of all mutants had a very irregular pattern of
growth marked by the presence of convoluted structures, particu-
larly in the DcdaB mutant. Additionally, septa appeared markedly
closer together than in the WT, indicating that individual hyphal
cells were smaller. Unlike a number of previously isolated symbi-
otic mutants that showed a host-stunting phenotype (Charlton et al.
2012; Green et al. 2017; Tanaka et al. 2006, 2008, 2013; Take-
moto et al. 2006), all cda mutants were still able to undergo
cell-to-cell fusion of lateral branches, which is crucial for develop-
ment of a hyphal network within the plant (Scott et al. 2018). The
apparent reduction in hyphal cell length suggests there is a disrup-
tion to intercalary growth within the leaf tissue when chitosan syn-
thesis is reduced. Chitosan may therefore have a key role in the
remodeling of the cell wall of endophytic hyphae during interca-
lary growth, as hyphae are stretched and undergo septation in
response to expansion of the leaf cells (Christensen et al. 2008;
Voisey 2010). The positive charge of the chitosan could confer
some affinity for the negative charge of the glucuronoarabinoxylan
and pectin in the host cell walls (Geoghegan and Gurr 2016). The
severe cellular phenotype observed with the chitin deacetylase
mutants is consistent with these hypotheses and identifies a vital
role for cell-wall remodeling in both fungal morphology and plant
symbiosis.

Despite the strong whole-plant and cellular interaction pheno-
types observed for the E. festucae cdamutants, no obvious pheno-
type difference to WT was observed when the DcdaA and DcdaB
single mutants were grown in axenic culture. The radial growth
and morphology of DcdaA and DcdaB mutants were also similar
to WT. These mutants formed hyphal bundles, were able to
undergo hyphal tip-to-side cell fusion, and formed characteristic
coil-like structures from which conidiophores arise, with a mor-
phology and frequency similar to WT (Becker et al. 2015; Scott
et al. 2012). While the whole-colony morphology and radial
growth phenotype of the DcdaADcdaB double mutant also
appeared normal, microscopic examination of the cellular pheno-
type revealed that the cell-wall integrity of these mutants was com-
promised, as they frequently formed intrahyphal hyphae. This
phenotype has been observed for a number of other symbiotic
E. festucaemutants, including those involved in cell-wall integrity
mitogen-activated protein kinase and calcineurin signaling
(Becker et al. 2015; Green et al. 2017; Mitic et al. 2018), and
for T. atroviride Dcda1 and Dchs1 mutants (Kappel et al. 2020).
In conclusion, this study highlights the very dynamic state of the

chitin component of the E. festucae cell wall and how it rapidly
changes during the developmental transitions that accompany
the free-living to endophytic and the endophytic to epiphytic

Fig. 5. In planta cellular phenotype of Lolium perenne infected with Epichlo€e festucae wild type (WT), DcdaA, DcdaB, and DcdaADcdaB mutants. A,
Maximum intensity projections from longitudinal sections of L. perenne leaf sheath tissue infected with WT (Fl1), DcdaA, DcdaB, and DcdaADcdaB mutant
strains 10 weeks postinoculation. Hyphae are stained with aniline blue (red), chitin in cell-wall septa are stained with wheat germ agglutinin-AlexaFluor 488
(blue), and fungal and plant nuclei are stained with propidium iodide (yellow). Bar = 50 lm. B, and C, Higher magnification of sections I and II in panel A,
showing normal hyphal compartment in WT, abnormal growth pattern of fungal hyphae, and increased hyphal branching in cda mutant strains, respectively.
Images were generated by maximum intensity projection. Bar = 50 lm.

Vol. 34, No. 10, 2021 / 1187



lifestyle changes. Chitin deacetylase-catalyzed conversion of cell-
wall chitin to chitosan is crucial for maintaining a mutualistic sym-
biotic association between E. festucae and its grass host. It will be
of considerable interest to determine how widespread this particu-
lar mechanism is among other symbiotic fungi for maintaining
compatible interactions with their plant hosts.

MATERIALS AND METHODS

Strains and growth condition.
Cultures of Escherichia coli were grown overnight in LB

(lysogeny broth) or on 1.5% LB agar containing 100 lg ampicillin

per milliliter, as previously described (Miller 1972). Cultures of E.
festucae (Supplementary Table S3) were grown on 2.4% (wt/vol)
PDA (1.5% water) plates or in PD broth as previously described
(Moon et al. 1999, 2000).

Plant growth and endophyte inoculation conditions.
Endophyte-free seedlings of perennial ryegrass (Lolium perenne

cv. Samson) were inoculated with E. festucae as previously
described (Latch and Christensen 1985). Plants were grown in
root trainers in an environmentally controlled growth room at
22�C with a photoperiod of 16 h of light (about 100 lE per square

Fig. 6. Complementation of in planta cellular phenotype of Lolium perenne infected with Epichlo€e festucae DcdaA and DcdaB mutants. A, Maximum inten-
sity projections from longitudinal sections of L. perenne leaf sheath tissue infected with wild-type Fl1 (WT), DcdaA, DcdaB, DcdaA/cdaA, and DcdaB/cdaB
mutant strains 10 weeks postinoculation. Hyphae are stained with aniline blue (red), chitin in cell-wall septa are stained with wheat germ agglutinin-
AlexaFluor 488 (blue), and fungal and plant nuclei are stained with propidium iodide (yellow). Bar = 50 lm. B and C, Higher magnification of I and II,
respectively, in the images in A showing normal hyphal growth in WT, abnormal growth pattern of fungal hyphae in cda mutant strains, and restoration
of WT-like growth in the complementation mutants. Images were generated by maximum intensity projection. Bar = 50 lm.

Fig. 7. Intrahyphal hyphae phenotype of the DcdaADcdaB mutant strains. A, Differential interference contrast images of wild type Fl1 and DcdaADcdaB
mutant strains showing intrahyphal hyphae. B, Higher magnification of I sections in A, with white arrow heads pointing to intrahyphal hyphae. Bar = 20 lm.

1188 / Molecular Plant-Microbe Interactions



meter per second) and were tested by immunoblot at 8 wpi for the
presence of the endophyte (Tanaka et al. 2005).

DNA isolation, PCR, qPCR, reverse transcription qPCR
(RT-qPCR) and sequencing.
Plasmid DNA from Escherichia coli was extracted, using the

High Pure plasmid isolation kit (Roche), according to manufac-
turer instructions. Fungal genomic DNA used for Southern digests
was extracted from freeze-dried mycelium as previously described
(Byrd et al. 1990). Standard PCR amplifications were performed
with Taq DNA polymerase (Roche) as per manufacturer instruc-
tions. When high fidelity PCR products were required, proofread-
ing polymerases such as Phusion (ThermoFisher Scientific) or Q5
(New England Biolabs, Inc.) were used as per manufacturer
instructions. DNA sequencing of plasmids and PCR products
were conducted at the Massey Genome Sequence Service (Palm-
erston North, New Zealand), using a 3730 DNA analyzer (Applied
Biosystems) with BigDye Terminator Version 3.1 chemistry
(Applied Biosystems).
For expression analysis by RT-qPCR, pseudostem tissues from

8-wpi plants were homogenized in liquid nitrogen with a mortar
and pestle and RNA was isolated using TRIzol (Invitrogen)
according to manufacturer instructions. One microgram of RNA
was used for complementary DNA (cDNA) synthesis, using the
QuantiTect RT kit (Qiagen), and cDNA was diluted threefold
with Tris-EDTA (TE) buffer. qPCR was performed using the Sso-
Fast EvaGreen supermix (Bio-Rad) on a LightCycler 480 System
(Roche), according to manufacturer instructions. Standard curves
were generated using the purified PCR product amplified by
each primer pair. Absolute quantification RT-qPCR was per-
formed using three biological replicates and two technical repli-
cates for all samples. Transcript levels were normalized using
the reference gene encoding elongation factor 2 (EfM3.021210).
Fungal biomass in infected plants was quantified at 8 wpi, using

at least three biological replicates (eight for WT) from either 5-cm
pseudostem sections taken from the base of each tiller or 10-cm
blade sections starting from the ligule. DNA was isolated as pre-
viously described (Liu et al. 2000) and was diluted 100-fold with
TE buffer. Two microliters of this DNA was used as template for
qPCR (SsoFast EvaGreen). Relative biomass was determined by
qPCR analysis using the ratio of pacC, a single-copy endophyte
gene encoding a transcription factor (Lukito et al. 2015), to
LpCCR1, a single-copy plant gene encoding cinnamoyl-CoA
reductase (McInnes et al. 2002). Standard curves were generated
for each primer pair using purified PCR products of the primers,
and absolute quantification qPCR was performed with two techni-
cal replicates per sample. Primers used in this study are listed in
Supplementary Table S4.

Generation of the DcdaA, DcdaB, and
DcdaADcdaB mutants.
The cdaA replacement construct was prepared by Gibson

Assembly (Gibson et al. 2009). The 59 1,091-bp and 39 1,084-bp
sequences flanking cdaA were amplified from an E. festucae Fl1
genomic DNA template, using PCR with primer pairs NN92/
NN93 and NN94/NN95, respectively. These DNA fragments
were purified and assembled along with a 2,181-bp hygromycin
resistance cassette (primers hph-F, pDB33_7) amplified from
plasmid pDB48 and a linearized pRS426 vector to generate
pNN05. The in vitro recombined DNA mixture was trans-
formed into chemically competent Escherichia coliDH5a cells,
and ampicillin-resistant transformants were screened using Clo-
nechecker (Life Technologies) for plasmids with restriction
enzyme digest patterns predicted from in silico construction
of pNN05. The sequence fidelity of the plasmid extracted
from one of these clones was verified by DNA sequencing.
The cdaA replacement fragment contained within pNN05 was

then excised by XmaI/HpaI digestion, was gel-purified, and
was transformed into E. festucae protoplasts as described
below, using hygromycin B for selection.
The cdaB replacement construct was also prepared by Gibson

Assembly (Gibson et al. 2009), using 2,359-bp cdaB 59 and
1,931-bp cdaB 39 fragments amplified from E. festucae Fl1 geno-
mic DNA, using primer pairs NN151/NN152 and NN155/NN156,
respectively, a 1,741-bp geneticin-resistance cassette amplified
from pSF17.1 plasmid DNA, using primers NN153 and NN154,
and a linearized pUC19 vector, with the resulting plasmid
(pNN15) being screened and isolated as described above. The
cdaB replacement fragment contained within pNN15 was PCR-
amplified using primer pairs NN151/NN156, was gel-purified,
and was transformed into E. festucae WT and DcdaA#41 proto-
plasts as described below, using geneticin for selection to generate
the DcdaB and DcdaADcdaB mutants.

Fungal transformation.
E. festucae protoplasts were prepared as previously described

(Yelton et al. 1984) and were transformed with 2 to 5 lg of circu-
lar or linearized plasmid DNA (Itoh et al. 1994). Transformants
were selected on 1.5% RG media containing either hygromycin
(150 lg/ml) or geneticin (250 lg/ml) and were nuclear-purified
by three rounds of subculturing on selection medium (Young et al.
2005).

DNA hybridization.
High-quality E. festucae genomic DNA (1 to 1.5 lg) was

digested overnight, using the appropriate restriction enzyme,
was separated by agarose gel electrophoresis, and was transferred
to a positively charged nylon membrane (Roche) (Southern 1975).
DNA was crosslinked to the nylon membrane by UV irradiation
for 2 min, using a CEX-800 UV cross-linker (120 mJ/cm2, 254
nm [Ultralum, Inc.]). DNA probes were labeled using the DIG
High Prime dna labeling and detection starter kit (Roche), as per
manufacturer instructions. Hybridizations were performed accord-
ing to manufacturer instructions.

Microscopy.
Cultures to be analyzed by wide-field epifluorescence light

microscopy were inoculated at the edge of a thin layer of 1.5%
(wt/vol) water agar, were layered on top of a glass microscope
slide embedded in 1.5% water agar, and were grown for 7 days.
Square blocks were then extracted and placed onto new slides,
were covered with a cover slip, and were analyzed using an epi-
fluorescence microscope (Olympus IX83) with a 60× oil immer-
sion objective, numerical aperture (NA) = 1.42, outfitted with
differential interference contrast optics. Calcofluor white was visu-
alized using a U-MWU2 filter cube. All images were captured
with a Retiga 6000M (QImaging) camera using a Bin2×2 and
controlled by cellSens software (Olympus).
Growth and morphology of hyphae in planta was determined by

staining leaves with aniline blue diammonium salt (Sigma) and
WGA-AF488 (Molecular Probes/Invitrogen) as follows: infected
pseudostem tissue was incubated in 95% (vol/vol) ethanol over-
night at 4�C, were then treated with 10% potassium hydroxide
for 3 h or overnight at 4�C. The tissue was washed three times
in phosphate buffered saline (PBS) (pH 7.4) and was incubated
in staining solution (0.02% aniline blue, 10 lg of WGA-AF488
per milliliter, and 0.02% Tween-20 [Invitrogen] in PBS [pH
7.4]) for 10 min, followed by a 20-min vacuum infiltration step.
Hyphal growth and fungal cellular phenotypes were documented
by CLSM, using a Leica SP5 DM6000B (Leica Microsystems)
confocal microscope outfitted with a 40×, NA 1.3 or 63× NA
1.4 oil immersion objective lens. WGA-AF488 was excited at
488 nm to detect chitin, and aniline blue was excited at 561 nm
to detect b-1,3-glucan, with emissions collected at 498 to 551

Vol. 34, No. 10, 2021 / 1189



and 571 to 633 nm, respectively. While aniline blue itself is not
fluorescent, there is a minor fluorochrome component present,
Sirofluor, that does fluoresce (Stone et al. 1984).
Fungal cell-wall chitin and chitosan distribution was analyzed in

plant sheath samples using the enhanced green fluorescent
protein–fused molecular probes CBP (Fuenzalida et al. 2014)
and CAP (Nampally et al. 2012), respectively. The specificity of
these probes for chitin and chitosan have been experimentally ver-
ified by previous studies (Fuenzalida et al. 2014; Nampally et al.
2012). Plant samples fixed in 95% ethanol were washed as
described above and were incubated in staining solution contain-
ing 0.1 mg of CAP per milliliter (1 mg of stock solution per mil-
liliter in TEA buffer [20mM triethanolamine, 400mMNaCl, 10%
{vol/vol} glycerol, pH 8.0], 0.02% [vol/vol] Tween 20, and 0.02%
[wt/vol] propidium iodide in PBS [pH 7.4]). Samples were
vacuum-infiltrated for 30 min and were stored at 4�C overnight,
before analysis using CLSM. The WGA-AF488 chitin probe
was excited using 488 nm light and its emission collected from
493 to 555 nm. CBP and propidium iodide were excited at 488
and 561 nm, respectively, and their emissions were collected at
493 to 555 and 571 to 684 nm. The chitosan probe CAP was
excited at 488 nm and emitted light was collected from 493 to
555 nm. All imaging was performed with a 40× NA 1.3 oil objec-
tive lens and a step size of 1.2 lm. Images obtained from all
microscopy approaches were produced with ImageJ (National
Institutes of Health) software using the maximum intensity projec-
tions of five to 10 sections acquired at 1.2-lm intervals.

Bioinformatic analysis.
E. festucae genes encoding chitin deacetylases were obtained

from the Massey University Epichlo€e database (the E. festucae
E2368 genome) and the domain structures of the encoded products
were annotated using Pfam and InterProScan (v.5) (Quevillon et al.
2005; Zdobnov and Apweiler 2001). InterProScan lookup service
(v.43.1), Phobius (v.1.01) (K€all et al. 2004), SignalP (v.4.1), and
TMHMM (v.2.0c) were used, plus all software given by default
with InterProScan (BlastProDom, FprintScan, HMMPIR,
HMMPfam, HMMSmart, HMMTigr, ProfileScan, HAMAP, Pat-
ternScan, SuperFamily, Gene3D). Gene and protein sequences for
E. festucae (Fl1) as well as other species were obtained from the
genome databases curated by C. L. Schardl at the University of
Kentucky (Schardl et al. 2013). The proposed genemodels were val-
idated based on hiddenMarkovmodel (HMM)-based gene structure
prediction by FGENESH (Softberry). TheMagnaporthe oryzea chi-
tin deacetylase gene sequences (Geoghegan and Gurr 2016) used to
perform tBlastn against the Fl1 genome were obtained through Fun-
giDB, Fungal and Oomycete Genomics Resources.
Alignment of nucleotide and amino acid sequences were per-

formed with ClustalW (Thompson et al. 1994), as provided with
MacVector 14.5 (MacVector, Inc.) and using default parameters.
The DNA and amino acid sequence alignments utilized for syn-
teny analyses were performed using the MAFFT (v7.017) algo-
rithm (Katoh and Standley 2013; Katoh et al. 2002).

ACKNOWLEDGMENTS

The authors thank J. Taylor and N. Minards (Manawatu Microscopy and
Imaging Center) for technical advice.

AUTHOR-RECOMMENDED INTERNET RESOURCES

E. festucae Fl1 genome project database:
http://csbio-l.csr.uky.edu/endophyte/cpindex.php

FungiDB: https://fungidb.org/fungidb
Genome Projects at the University of Kentucky database:

http://csbio-l.csr.uky.edu/endophyte/cpindex.php
Softberry homepage: http://www.softberry.com

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