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Frontiers in Microbiology 01 frontiersin.org
Global characterization of GH11
family xylanases genes in
Neostagonosporella sichuanensis
and functional analysis of Nsxyn1
and Nsxyn2
LijuanLiu
1,2, ChengsongLi
1,2, FangLiang
1,2, ShanHan
1,2,
ShujiangLi
1,2, ChunlinYang
1,2* and YinggaoLiu
1,2*
1 College of Forestry, Sichuan Agricultural University, Chengdu, China, 2 National Forestry and
Grassland Administration Key Laboratory of Forest Resources Conservation and Ecological Safety on
the Upper Reaches of the Yangtze River and Forestry Ecological Engineering in the Upper Reaches of
the Yangtze River Key Laboratory of Sichuan Province, College of Forestry, Sichuan Agricultural
University, Chengdu, China
Rhombic-spot disease, caused mainly by Neostagonosporella sichuanensis,
significantly impacts the yield and quality of fishscale bamboo (Phyllostachys
heteroclada). Xylanases are essential for pathogenic fungi infection, yet their
specific functions in the physiology and pathogenicity of N. sichuanensis remain
unclear. Here, wecharacterized three xylanase proteins with glycosyl hydrolase
11 domains from the N. sichuanensis SICAUCC 16–0001 genome and examined
the function of Nsxyn1 and Nsxyn2. Purified Nsxyn1 and Nsxyn2 proteins displayed
specific xylanase activity in vitro and induced cell death in Nicotiana benthamiana,
independent of their enzymatic function. Both proteins possessed signal peptides
and were confirmed as secreted proteins using a yeast secretion system. Subcellular
localization revealed that Nsxyn1 and Nsxyn2 localized in both the cytoplasm and
nucleus and can trigger cell death in N. benthamiana through Agrobacterium
tumefaciens-mediated transient transformation. qRT-PCR results showed notable
upregulation of Nsxyn1 and Nsxyn2 during infection, with Nsxyn1 exhibiting an
80-fold increase at 15 days post-inoculation. Deletion of Nsxyn1 and Nsxyn2 in
N. sichuanensis impaired xylan degradation, adaptation to osmotic and oxidative
stress, and pathogenic full virulence. Deletion of Nsxyn1 notably slowed fungal
growth and reduced spore production, whereas only a reduction in microconidial
production was observed in Nsxyn2 mutants. Complementation of Nsxyn1 and
Nsxyn2 only partially restored these phenotypic defects in the ∆Nsxyn1 and
∆Nsxyn2 mutants. These findings suggest that Nsxyn1 and Nsxyn2 contribute
to N. sichuanensis virulence and induced plant defense responses, providing
new insights into the function of xylanases in the interaction between fishscale
bamboo and N. sichuanensis.
KEYWORDS
Neostagonosporella sichuanensis, xylanase, prokaryotic expression, transient
expression, gene knockout
OPEN ACCESS
EDITED BY
Nazia Manzar,
National Bureau of Agriculturally Important
Microorganisms (ICAR), India
REVIEWED BY
Shakil Ahmad,
Hainan Normal University, China
Yuqiang Zhao,
Jiangsu Province and Chinese Academy of
Sciences, China
Devanshu Dev,
Bihar Agricultural University, India
*CORRESPONDENCE
Chunlin Yang
yangcl0121@163.com
Yinggao Liu
11468@sicau.edu.cn
RECEIVED 08 October 2024
ACCEPTED 11 November 2024
PUBLISHED 21 November 2024
CITATION
Liu L, Li C, Liang F, Han S, Li S, Yang C and
Liu Y (2024) Global characterization of GH11
family xylanases genes in Neostagonosporella
sichuanensis and functional analysis of
Nsxyn1 and Nsxyn2.
Front. Microbiol. 15:1507998.
doi: 10.3389/fmicb.2024.1507998
COPYRIGHT
© 2024 Liu, Li, Liang, Han, Li, Yang and Liu.
This is an open-access article distributed
under the terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in other forums is
permitted, provided the original author(s) and
the copyright owner(s) are credited and that
the original publication in this journal is cited,
in accordance with accepted academic
practice. No use, distribution or reproduction
is permitted which does not comply with
these terms.
TYPE Original Research
PUBLISHED 21 November 2024
DOI 10.3389/fmicb.2024.1507998
Liu et al. 10.3389/fmicb.2024.1507998
Frontiers in Microbiology 02 frontiersin.org
1 Introduction
Known for its rapid growth and sustainability, bamboo is an
essential resource worldwide (especially in Asia). Phyllostachys
heteroclada Oliver, known as shscale bamboo, is a crucial
waterlogging-tolerant and industrial bamboo species native to China.
It plays an important role in riparian zone restoration, bamboo shoots,
and handicra production and serves as a vital food source for wild
giant pandas (Jing etal., 2022; Liang etal., 2024; Shi and Chen, 2022).
e culms are the primary economic component of bamboo; however,
culm diseases signicantly impact bamboo wood quality, leading to
considerable ecological and nancial losses (Huang L. etal., 2023).
Although numerous culm diseases have been reported, there is a lack
of in-depth studies (Huang L. etal., 2023; Yang etal., 2024). Notably,
major culm diseases such as bamboo blight and bamboo witches’
broom are receiving increased attention, including investigations into
their pathogenic molecular mechanisms (Li etal., 2020; Gu etal.,
2024). For example, the whole genome of the bamboo blight pathogen,
Arthrinium phaeospermum, has been published, identifying key
pathogenic factors such as the ApCE12 and ApCE22 eectors and
Ctf1β cutinase (Li etal., 2020; Fang etal., 2021; Fang etal., 2022).
Among these diseases, rhombic-spot disease is recognized as one of
the most signicant threats to shscale bamboo, with
Neostagonosporella sichuanensis being the most commonly reported
pathogen (Yang, 2019). N. sichuanensis primarily infects shscale
bamboo branches and culms through the stomatal apparatus and
various wounds (Yang, 2019). Numerous pycnidia and ascostromata
can beobserved at infection sites, with ascostromata being particularly
abundant, continuously released from November through April of the
following year (Liu etal., 2022). During the disease spread period
from May to October, N. sichuanensis mainly persists in the branches
and culms of the host in the form of mycelia and conidia (Liu
etal., 2022).
Plant cell walls, consisting of complex polysaccharides, which
primarily include cellulose, hemicellulose, and pectin, form an
eective physical barrier against fungal invasion (Underwood, 2012).
Xylan, a hemicellulose characterized by a β-1,4-linked xylose
backbone, is the predominant hemicellulose that exists in both
monocots and dicots. It is integral to plant cell wall structure by
providing exibility and strength through its interactions with
cellulose and lignin (Rennie and Scheller, 2014). Xylan-degrading
enzymes, known as xylanases, are necessary for the full degradation
of hemicellulose (Bhardwaj etal., 2019). Xylanases mainly belong to
glycoside hydrolase families 10 and 11 according to their molecular
weight and catalytic domains (Bhardwaj etal., 2019). GH10 xylanases
generally have higher molecular weights and broader substrate
specicities, whereas GH11 xylanases are smaller and exhibit better
catalytic eectiveness on linear xylan chains (Liu etal., 2021).
Xylanases are crucial in the pathogenicity and infection
processes of various fungal pathogens. However, reports on the
contribution of GH10 family xylanases to fungal pathogenicity
remain limited. For instance, the GH10 xylanase RcXYN1 from
Rhizoctonia cerealis has been found to exacerbate disease severity
during wheat infections (Lu etal., 2020). Additionally, two GH10
xylanases have been identified from the genome of Botrytis cinerea
B05.10, but relevant experiments have not yet determined the
contributions of these xylanases to the pathogenicity of this
fungus (García etal., 2017). In contrast, there is considerable
literature on the involvement of GH11 xylanases in fungal
pathogenicity. GH11 family xylanases enable fungi to penetrate
the plant cell wall by degrading xylan, which facilitates tissue
colonization and nutrient acquisition for fungal growth (Wang
etal., 2021). Beyond their role in cell wall degradation, GH11
family xylanases contribute to fungal development, reproduction,
and virulence. For instance, in Magnaporthe oryzae, the rice blast
fungus, the GH11 family xylanase MoXYL1A is crucial for
conidiation, appressorium maturation, and virulence,
underscoring its importance in effective host colonization
(Shabbir etal., 2022). Furthermore, some xylanases function as
protein elicitors activating PAMPs-triggered immunity (PTI)
during host-pathogen interactions, triggering host defense
responses that lead to cell necrosis (Enkerli etal., 1999). For
example, the xylanase BcXyn11A from Botrytis cinerea can
berecognized by plants as PAMPs and elicits plant defenses (Frías
etal., 2019). This necrosis-inducing effect not only disrupts local
cell integrity, providing an entry point for fungal invasion, but also
suppresses nearby immune responses, creating an environment
conducive to nutrient acquisition and sustained infection
(Derbyshire and Raffaele, 2023). These findings emphasize that
GH11 family xylanases are important components of the
sophisticated strategies employed by fungal pathogens to penetrate
and develop infections in their plant hosts.
Screening for xylanase genes within fungal genomes enables the
systematic identication of xylanase genes potentially implicated in
plant cell wall degradation, facilitating further studies through gene
knockout or overexpression techniques to validate their roles in
pathogenicity. Moreover, this method helps elucidate the diversity and
evolutionary history of xylanase gene families, providing insights into
their contribution to fungal adaptability and virulence. For example,
identifying and annotating carbohydrate-active enzymes(CAZy)
enhanced the comprehension of the genomic structure of plant cell
wall-degrading enzymes (PCWDEs) in Fusarium virguliforme.
Sequence and structural investigations of FvXyn11A and FvXyn11B
revealed conserved residues facilitating XIP-I inhibition, with both
xylanases expressed during soybean root infection (Chang etal.,
2016). Comparative genomics investigations indicated that the
Verticillium dahliae genome contains a family of six xylanases, with
VdXyn4 facilitating the degradation of the plant cell wall and
contributing to the pathogenicity of V. dahliae (Wang etal., 2021).
e genome of Neostagonosporella sichuanensis has been released,
detailing the quantity and distribution of plant cell wall-degrading
enzymes (PCWDEs) within it (Liu et al., 2024). Prior research
demonstrated that N. sichuanensis exhibits strong xylan degradation
activity, suggesting that the pathogen may employ hemicellulose
degradation to penetrate plant cell walls during infection (Liu etal.,
2024). Despite the well-established importance of xylanases in fungal
pathogenicity, their specic role in the virulence of N. sichuanensis
remains unknown. In this study, wecomprehensively examined the
involvement of GH11 family xylanases in the pathogenicity of
N. sichuanensis in plant systems. e principal aims were to (1)
determine whether these xylanases are secreted proteins and examine
their location in plant cells; (2) evaluate the relationship between the
enzymatic activity and cell necrosis-inducing activity; (3) determine
whether N. sichuanensis xylanases are up-regulated during pathogen
infection; and (4) investigate the contribution of xylanases to
N. sichuanensis virulence.
Liu et al. 10.3389/fmicb.2024.1507998
Frontiers in Microbiology 03 frontiersin.org
2 Materials and methods
2.1 Plants, strains, and culture conditions
e wild-type strain Neostagonosporella sichuanensis SICAUCC
16–0001 was acquired from the Culture Collection at Sichuan
Agricultural University (SICAUCC) and activated on PDA medium
at 25°C for 7 days and then subcultured on fresh PDA medium at 25°C
for 30 days. Nicotiana benthamiana was cultivated at 25°C under a
14-h light and 10-h dark cycle in an articial climate incubator.
Healthy one-year shscale bamboo seedlings were cultivated at 25°C
under a 12-h light and 12-h dark cycle in an articial climate incubator
aer transplanted from Zhougongshan Town (103°2′59.87″E,
29°50′8.56″N) in Ya’an City, Sichuan Province, China, to the Chengdu
Academy of Agriculture and Forestry Sciences (103°85′73.17″E,
30°70′32.61″N) in Chengdu City, Sichuan Province, China.
2.2 Identification and bioinformatics
analysis of GH11 xylanase family proteins in
Neostagonosporella sichuanensis SICAUCC
16–0001 genome
Xylanase family proteins were identied by querying the HMM
prole of the Glyco_hydro_11 (GH11) domain (Pfam ID: PF00457)
against the Neostagonosporella sichuanensis SICAUCC 16–0001
genome and nine other phaeosphaeriacous species (Ampelomyces
quisqualis, Leptosphaeria microscopica, Ophiobolus disseminans,
Paraphoma chrysanthemicola, Parastagonospora nodorum,
Phaeosphaeria poagena, Stagonospora sp., Setomelanomma holmii, and
Setophoma terrestris) using HMMER v3.4 (Potter etal., 2018) with an
E-value threshold of ≤1 × 10−5. e returned hits with scores > 30 were
selected. e genome sequences used for the xylanase proteins
screening of four strains, Leptosphaeria microscopica, Paraphoma
chrysanthemicola, Phaeosphaeria poagena, and Setophoma terrestris
were downloaded from the JGI Genome Portal (Nordberg etal.,
2014), and the genome sequences of the remaining ve species were
downloaded from the NCBI Genome (Sayers et al., 2022).
Subsequently, these candidate proteins were searched for the Glutamic
acid enzymatic activity sites and conserved glycoside hydrolase
families 11 motifs using the ProSITE webserver (Sigrist etal., 2012).
ese sequences without enzyme active sites were discarded. e
remaining sequences were submitted to SignalP v5.0 (Almagro
Armenteros etal., 2019), TMHMM v2.0 (Krogh etal., 2001), BIG-PI
Fungal Predictor (Eisenhaber etal., 2004), and WoLF PSORT (Horton
et al., 2007) for secretory protein prediction. Protein sequences
possessing a signal peptide, devoid of the transmembrane region and
anchor location, and extracellular subcellular localization were
identied as xylanase proteins and analyzed further. e phylogenetic
tree was constructed with these selected xylanase proteins using
MEGA v11 (Tamura etal., 2021) with the neighbor-joining method
and visualized through Evolview v3.0 webserver (Subramanian etal.,
2019). ClustalX v2.0 (Larkin etal., 2007) and ESpriPT3.0 webserver
(Robert and Gouet, 2014) performed multiple sequence alignment.
SOMPA (Geourjon and Deléage, 1995) and SWISS-MODEL web tools
(Waterhouse et al., 2024) were used to predict the secondary and
tertiary structures with the AlphaFold v2 method, respectively. e
quality of the predicted models was assessed by SAVES v6.1 (Bowie
etal., 1991; Lüthy etal., 1992; Colovos and Yeates, 1993; Laskowski
etal., 1996) and Qmean (Benkert etal., 2011).
2.3 Total RNA extraction, reverse
transcription, and cloning of CDS
full-length of Nsxyn1 and Nsxyn2 genes
Fresh Neostagonosporella sichuanensis mycelia were collected for
total RNA extraction utilizing the Quick RNA Isolation Kit
(Huayueyang, Beijing, China). e concentration and purity of RNA
were assessed using a NanoDrop2000 spectrophotometer (ermo
Fisher Scientic, Waltham, MA, USA). e isolated RNA was
preserved at-80°C for subsequent study. High-quality total RNA was
reverse transcribed into cDNA with the PrimeScript™ RT Reagent Kit
(Perfect Real Time; Takara, Beijing, China). Only two candidate
xylanase genes (Nsxyn1 and Nsxyn2) were successfully cloned from
the cDNA of Neostagonosporella sichuanensis SICAUCC 16–0001.
Primers (Supplementary Table S1) were designed by Primer Premier
5.0 (Premier Bioso International, Palo Alto, CA, USA). e target
fragments were amplied using 2 × TransTaq® High Fidelity (HiFi)
PCR SuperMix II (−dye) (TRANS, Beijing, China) and puried using
a Universal DNA Purication Kit (TIANGEN, Beijing, China). en,
they were connected to pMD™-19 T vectors (Takara, Beijing, China),
respectively, by the manufacturer’s guidelines. e ligation mixture
was transformed into Escherichia coli DH5α cells, which were then
spread onto antibiotic-containing Luria-Bertani (LB) agar. Positive
clones were screened by colony PCR and veried by sequencing.
2.4 Recombinant protein expression
optimization, purification, and enzyme
activity assays of Nsxyn1 and Nsxyn2
Nsxyn1 and Nsxyn2 fragments (Amplied primers were in
Supplementary Table S1), devoid of the predicted signal peptides and
stop codons, were introduced into the pET-32a plasmid at the EcoR
Iand Xho Isites, respectively, and subsequently transformed into
Escherichia coli BL21(DE3) for expression. To get adequate soluble
proteins for subsequent analysis, expression conditions were optimized
by inducing with IPTG at various concentrations and temperatures,
followed by an analysis of expression levels by SDS-PAGE (Zhao etal.,
2022). Large-scale protein production was carried out under optimal
conditions, and the His6-tagged protein was puried utilizing a
His-tag protein purication kit (Reductant&Chelator-resistant;
Beyotime, Shanghai, China). Two Glu residues of the Nsxyn1rec/
Nsxyn2rec mutant were replaced with Lys residues utilizing the Hie
Mut™ Site-Directed Mutagenesis Kit (Yeasen, Shanghai, China) to
eliminate xylanase activity of Nsxyn1 and Nsxyn2. Following
verication through sequence alignment, the Nsxyn1rec and
Nsxyn2rec mutants were expressed under optimal conditions and
puried as described above. Protein concentrations were then
determined using a BCA protein assay kit (Beyotime, Shanghai, China).
For qualitative analysis, recombinant proteins were introduced to
wells containing a medium with hardwood xylan (Beyotime, Shanghai,
China) as the exclusive carbon source (Meddeb-Mouelhi etal., 2014).
Aer 2 h of incubation, the medium was treated with 1% Congo red
solution for 1 h and then eluted with 1 mol/L NaCl solution for 3 h.
Liu et al. 10.3389/fmicb.2024.1507998
Frontiers in Microbiology 04 frontiersin.org
Hydrolytic zones were then examined using sterile eluents as negative
controls. Xylanase activity was quantied by a modied DNS
(dinitrosalicylic acid) assay (Fu etal., 2019) with some modications.
A mixture of 90 μL of 1% hardwood xylan solution (pH 6.5) and 10 μL
of recombinant xylanase solution was incubated in PCR tubes at 45°C
for 30 min. Subsequent to the reaction, 100 μL of DNS solution was
immediately added, and the mixture was heated in a boiling water
bath for 10 min before cooling to ambient temperature. e negative
controls comprised 90 μL of 1% xylan solution (pH 6.5) and 10 μL of
heat-inactivated recombinant xylanase. Absorbance was measured at
540 nm. All experiments were performed in triplicate.
2.5 Assessment of necrotic activity of
purified Nsxyn1 and Nsxyn2 xylanase
proteins in Nicotiana benthamiana
To assess the potential necrotic activity of the puried proteins,
leaves of Nicotiana benthamiana were inltrated with 10 μmol/L of each
puried xylanase protein using a needleless syringe. e inltration sites
were marked, and the plants were incubated under controlled conditions
at 25°C under a 14-h light and 10-h dark cycle. Leaf tissue was observed
for the development of necrosis throughout 5 days post inltration, with
leaves inltrated with buer only used as negative controls.
2.6 qRT-PCR analysis
e transcript levels of Nsxyn1 and Nsxyn2 at dierent infection
times in shscale bamboo were quantied by qRT-PCR on a CFX96™
real-time system (Bio-Rad, Hercules, CA, USA). Specic primers for
amplication were designed according to the coding regions of
Nsxyn1 and Nsxyn2, with the Neostagonosporella sichuanensis
elongation factor 1 alpha gene as an internal control (the coding
sequence is detailed in Supplementary Table S2). e qRT-PCR
reaction setup and conditions followed those described by Liu etal.
(2019). A no-template control (ddH2O) was included. e infection
timepoint exhibiting the lowest expression served as the baseline
(assigned a value of 1.0). Relative expression levels were calculated
utilizing the 2-∆∆Ct method (Ritter and Schulz, 2004; Zhu etal., 2015).
Each qRT-PCR experiment was performed in triplicate. Data were
analyzed and plotted using GraphPad version 8.4.2, with statistical
signicance assessed by unpaired Student’s t-test.
2.7 Signal peptides secretion function
detection of Nsxyn1 and Nsxyn2
To detect whether the signaling peptides of Nsxyn1 and Nsxyn2
proteins possess secretory functions, the relevant signaling peptide
sequences were inserted into the pSUC2 vector and transformed into
Saccharomyces cerevisiae YTK12 competent cells, which are lacking
invertase secretion. Transformants were selected on CMD-W medium
and cultured at 30°C for 48 h. Positive colonies were transferred to the
YPRAA medium to evaluate the secretory activity and incubated at 30°C
for 48 h. e CMD-W and YPRAA mediums were prepared according
to Huang’s method (Huang C. P. etal., 2023). e secretory function was
further conrmed by staining with 2,3,5-triphenyl tetrazolium chloride
(TTC), as described by Yin et al. (2018). e appearance of a red
precipitate indicated successful secretion of the expressed proteins.
2.8 Subcellular localization assays and
transient expression in Nicotiana
benthamiana of Nsxyn1 and Nsxyn2
To investigate the subcellular localization of Nsxyn1 and Nsxyn2
proteins in planta and assess their necrosis-inducing activity, the coding
sequences of Nsxyn1 and Nsxyn2 (without stop codons) were ligated to
the 5′ end of the enhanced green uorescent protein (EGFP) gene, driven
by the MAS promoter (Amplied primers were in
Supplementary Table S1). ese constructs were then inserted into the
pCAMBIAsuper1300-EGFP vector at the KpnI and XbaI restriction sites
to generate recombinant expression vectors, pCAMBIAsuper1300:Nsxyn1-
EGFP and pCAMBIAsuper1300:Nsxyn2-EGFP.
Onion epidermal cells and Arabidopsis thaliana protoplasts were
used for subcellular localization analysis. Disinfected onion inner
epidermal slices (~1 cm
2
) were incubated in Agrobacterium suspensions
(GV3101) containing either Nsxyn1-EGFP, Nsxyn2-EGFP, or the empty
pCAMBIAsuper1300-EGFP vector as a control, for 30 min. e
epidermal slices were then placed on MS medium and incubated at
25°C under a 14-h light and 10-h dark cycle. Fluorescence was observed
24 h later using an Ultra-High Resolution Confocal Microscope (Leica,
Wetzlar, Germany), with excitation at 488 nm. Moreover, protoplasts
were prepared from the leaves of 3–4 week-old A. thaliana seedlings for
protoplast localization. e Nsxyn1 and Nsxyn2 plasmids were
co-transformed with an SV40 NLS plasmid into the protoplasts via
PEG4000-mediated transformation. Aer overnight incubation at 23°C
under low light conditions, the protoplasts were centrifuged at 400 rpm
for 5 min, and the supernatant was discarded. Fluorescent signals were
visualized using a confocal laser microscope (FV1000). Fluorescence
detection parameters were GFP excitation at 488 nm, mCherry
excitation at 561 nm, and chloroplast autouorescence at 640 nm.
For necrosis induction assays, Agrobacterium tumefaciens
GV3101 (pSoup-19) harboring the Nsxyn1 or Nsxyn2 constructs was
inltrated into the leaves of Nicotiana benthamiana. Empty vector
pCAMBIAsuper1300-EGFP-MGS served as a negative control. Aer
3 days of incubation at 25°C with a 14-h light and 10-h dark cycle, the
inltrated leaves were collected, and the development of necrosis
was assessed.
2.9 Generation of gene deletion mutants
and complementation
Nsxyn1 and Nsxyn2 mutants were created via targeted gene
disruption using homologous recombination. Gene-specic le, and
right anking regions (1,000–1,500 bp) along with the hygromycin
resistance anking region were amplied and inserted into the
pCE-Zero vector (detailed primers are in Supplementary Table S1).
Using the method described by Liang etal. (2024), the knockout
construct was introduced into Neostagonosporella sichuanensis
SICAUCC 16–0001 via protoplast transformation. Successful mutant
generation was veried by PCR analysis.
For the complementation of mutants, the full-length Nsxyn1 and
Nsxyn2 genes, including their native promoter regions, were inserted
Liu et al. 10.3389/fmicb.2024.1507998
Frontiers in Microbiology 05 frontiersin.org
into the pEASY-NeoR vector, which contains a neomycin resistance
(NeoR) marker (detailed primers are in Supplementary Table S1).
ese constructs were reintroduced into the respective mutant strains.
Complemented strains were selected on G418-containing mediums,
and successful integration was veried by PCR.
2.10 Phenotypic and virulence analysis of
Nsxyn1 and Nsxyn2 gene deletion mutants
and complemented strains
To assess the impact of Nsxyn1 and Nsxyn2 deletions on fungal
development and stress adaptation, a comprehensive phenotypic
analysis was conducted on the gene deletion mutants, complemented
strains, and wild-type controls. Growth rates were determined by
measuring colony diameters on a PDA medium incubated at 25°C
over a 30-day incubation period. Sporulation was quantied by
counting conidia produced on the PDA plates aer 30 days using a
hemocytometer. Spore morphology was examined under a light
microscope. At the same time, conidial germination rates were
assessed by incubating conidia in sterile distilled water for 24 h at
25°C, followed by counting germinated spores under the microscope.
Oxidative and osmotic stress assays were performed to evaluate stress
tolerance. Strains were grown on a PDA medium supplemented with
10 μmol/L Congo red for oxidative stress and either 1 mol/L NaCl or
0.02% SDS for osmotic stress. Colony growth was monitored by
measuring the diameters aer a 30-day incubation period. To evaluate the
capacity of these strains to degrade primary cell wall components, they
were cultured on the PDA mediums containing 1% (w/v) cellulose, xylan,
or pectin as the sole carbon source. Enzyme activity was quantied by
measuring the diameter of clear zones aer Congo red staining, providing
insight into the ability to degrade cell wall polysaccharides of these strains.
For virulence assessment, Nsxyn1 and Nsxyn2 gene deletion
mutants, complemented strains, and the wild-type strains were
cultured on PDA at 25°C for 30 days. Single conidia were isolated and
transferred to fresh PDA medium for 15-day incubation. Conidia
were harvested and diluted to a concentration of 1 × 10
6
conidia/mL
in sterile distilled water. Surface-sterilized host plant leaves were
wounded with a sterile needle, and 10 μL of the conidial suspension
was applied to each wound. Inoculated plants grow at 25°C with 80%
relative humidity. Disease symptoms were monitored daily. Lesion
sizes were recorded at 30 days post-inoculation to determine virulence.
e assays were repeated in triplicate in each treatment.
Data analysis was performed using One-way ANOVA followed
by Duncan’s multiple range test in SPSS version 27.0 (SPSS Inc.,
Shanghai, China). Graphical data visualization was conducted using
GraphPad Prism 8.4.2 to illustrate signicant dierences in disease
indices among strains.
3 Results
3.1 Identification of GH11
domain-containing xylanases in
Neostagonosporella sichuanensis
ree xylanases containing GH11 domain proteins (Nsxyn1-
Nsxyn3) were identied in the genome of Neostagonosporella
sichuanensis SICAUCC 16–0001 through bioinformatics analysis.
ese xylanases were identied as secreted proteins with signaling
peptides, lacking transmembrane regions and GPI-anchor signals
(Supplementary Table S3). e same screening method predicted 51
xylanase proteins from nine other phaeosphaeriacous species
(Supplementary Table S3). ese xylanase proteins and Nsxyn1-
Nsxyn3 were subjected to phylogenetic analysis with the neighbor-
joining method. e generated phylogenetic tree clustered the proteins
into six distinct clades, designated Branch1 to Branch6, widely
represented among the analyzed phaeosphaeriacous species. Most
xylanases exhibited relatively higher sequence similarity within the
same branches, indicating a degree of conservation within the
Phaeosphaeriaceae family (Figure1). However, sequence alignment
analysis revealed low homology between the GH11 domains of the
three xylanases from N. sichuanensis, with pairwise identities of
44.04% between Nsxyn1 and Nsxyn2, 29.44% between Nsxyn1 and
Nsxyn3, and 47.67% between Nsxyn2 and Nsxyn3 (Figure2). Despite
the low sequence homology, all three xylanases shared two conserved
catalytic sites associated with glutamic acid residues (Figure2).
3.2 Structural model prediction and quality
assessment of Nsxyn1-Nsxyn3 proteins of
Neostagonosporella sichuanensis
e number and locations of alpha helices and extended strands
were similar between Nsxyn1, Nsxyn2, and Nsxyn3 proteins according
to the analysis of their secondary structures (Figures 3A–C) and
tertiary structures (Figures3D–F), leading us to hypothesize that all
three proteins have similar functions. e tertiary structure quality of
the three xylanase proteins was assessed using the SAVES pipeline and
Qmean. e overall quality factor of the tertiary structure of Nsxyn1,
Nsxyn2, and Nsxyn3 was 90.45, 89.11, and 88.89, respectively, as
determined by the ERRAT program and Nsxyn1 and Nsxyn3 passed
the quality test of the Verify3D program, in contrast to Nsxyn2 where
less than 80% (77.97%) of the residues had an averaged 3D-1D
score > = 0.1. Nevertheless, the Ramachandran plots generated by
PROCHECK showed that over 90% of the amino acid residues of all
three proteins were in acceptable regions (Figures3G–I). On the other
hand, the Nsxyn1, Nsxyn2, and Nsxyn3 models evaluated by Qmean
had Z-scores of-1.27, −1.49, and-1.40, respectively (Figures3J–L). e
SAVES pipeline and the Qmean evaluation results revealed that the
tertiary structures of all three protein models predicted by the SWISS-
MODEL using the Alphafold v2 method were consistently of good
quality and suitable for further experimental study.
3.3 Cloning two candidate xylanases from
Neostagonosporella sichuanensis
Two candidate xylanase genes (Nsxyn1 and Nsxyn2) were cloned
from the cDNA of Neostagonosporella sichuanensis SICAUCC 16–0001
(Supplementary Figure S1); however, Nsxyn3 was not obtained despite
numerous attempts. e sequences of two genes obtained by cloning,
Nsxyn1 and Nsxyn2, were identical to the sequences from the genome
annotation of Neostagonosporella sichuanensis SICAUCC 16–0001,
reecting the accuracy of the genome assembly and annotation results.
e functions of Nsxyn1 and Nsxyn2 genes were then further investigated.
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3.4 Optimization of expression conditions
and purification of Nsxyn1, Nsxyn2,
Nsxyn1SM113–205, and Nsxyn2SM121–212
recombinant proteins
Recombinant plasmids pET-32a-Nsxyn1 and pET-32a-Nsxyn2
were successfully constructed and transformed into Escherichia coli
BL21 (DE3) competent cells (Supplementary Figure S2). Expression
parameters, including IPTG concentration, induction duration, and
temperature, were carefully optimized to improve Nsxyn1 and
Nsxyn2 protein yield and enhance protein solubility. e ndings
indicated that IPTG concentration did not impact Nsxyn1 and
Nsxyn2 protein expression, so the lowest IPTG concentration of
this experiment, 0.2 mM, was chosen to continue optimizing other
expression conditions (Supplementary Figures S3A,B). Similarly,
the expression of Nsxyn1 was not aected by the duration of
induction, so 2 h was optimal (Supplementary Figure S3C).
Conversely, 5 h was chosen for pET-Nsxyn2 induction due to the
signicantly increased amount of induced protein compared to the
previous hours (Supplementary Figure S3D). e expression of
Nsxyn1 and Nsxyn2 proteins was highest at 30°C
(Supplementary Figures S3E,F). e optimal screening conditions
induced the expression of Nsxyn1 and Nsxyn2 proteins
before purication.
To determine whether the two Glu sites in Nsxyn1 and Nsxyn2
proteins are essential for the enzyme activity, we conducted
site-directed mutagenesis genes, Nsxyn1
SM113–205
and Nsxyn2
SM121–212
.
Subsequently, they were transferred to E. coli BL21 (DE3) for
overexpression using the previously optimized conditions.
Solubility detection experiments showed that Nsxyn1, Nsxyn2,
Nsxyn1
SM113–205
, and Nsxyn2
SM121–212
were present as soluble proteins
(Supplementary Figures S3G,H). e Nsxyn1, Nsxyn2, Nsxyn1
SM113–
205
, and Nsxyn2
SM121–212
proteins were puried before detection of
enzyme activity.
e Nsxyn1 and Nsxyn2 recombinant proteins have xylanase
activity and can induce the necrosis phenotype of Nicotiana
benthamiana independently of xylanase activity.
e puried Nsxyn1 and Nsxyn2 proteins exhibited the ability to
hydrolyze xylan to form hydrolytic rings when they were added to the
small holes of the medium with xylan as the sole carbon source
(Figures4A,B). On the other hand, Nsxyn1
SM113–205
and Nsxyn2
SM121–212
lost the ability to degrade xylan (Figure4C). e above ndings align
with the quantitative enzyme activity analysis of the puried protein
using xylan as substrate (Figures4D,E).
Injection of puried Nsxyn1 and Nsxyn2 proteins into Nicotiana
benthamiana leaves resulted in necrosis approximately 5 days aer
treatment (Figures4F,G). To determine whether this necrosis was
related to the enzymatic activity of these proteins, site-directed
mutagenesis was used to generate catalytic site mutants (Nsxyn1SM113–
205
and Nsxyn2
SM121–212
) in both proteins. Despite the loss of enzymatic
activity, the mutant proteins still induced necrosis in N. benthamiana
leaves, comparable to the unmutated proteins (Figures4H,I). ese
FIGURE1
Phylogenetic analysis of GH11 xylanase proteins between Neostagonosporella sichuanensis and nine other phaeosphaeriacous species constructed
using the neighbor-joining method. Weselect the first sequence in each branch in a counterclockwise direction for sequence similarity comparison
with other sequences within the branch. Nsxyn1, Nsxyn2, and Nsxyn3 are highlighted in bold red. In the phylogenetic tree, nodes with support values
below 50% were not displayed.
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ndings suggested that the necrosis-inducing activity of these xylanase
proteins is independent of their enzymatic function.
3.5 Quantitative analysis of Nsxyn1 and
Nsxyn2 expression
To assess the involvement of Nsxyn1 and Nsxyn2 in the infection
process of Neostagonosporella sichuanensis, their expression levels
at dierent periods of infection with shscale bamboo were
determined using qRT-PCR (Figure 5). Under non-inductive
conditions, the expression levels of both genes were at their lowest,
normalized to 1. Expression analysis revealed signicant
up-regulation of Nsxyn1 and Nsxyn2 during the infection process,
with Nsxyn1 showing a particularly strong increase, reaching an
80-fold up-regulation at 15 days post-inoculation. ese results
suggest that Nsxyn1 and Nsxyn2 are strongly induced
during infection.
3.6 Signal peptides of Nsxyn1 and Nsxyn2
proteins have secretory functions
e Nsxyn1 and Nsxyn2 genes were inserted into the pSUC2
vector and subsequently transformed into the YTK12 yeast strain.
Both transformants grew on CMD-W and YPRAA selective media,
indicating successful gene expression (Figure6). Similarly, the positive
control strain harboring the pSUC2-Avr1bSP construct also exhibited
normal growth, whereas the negative control showed no growth,
conrming the validity of the experimental system. Further TTC
staining analysis revealed that YTK12 strains expressing Nsxyn1 and
Nsxyn2 produced a red precipitate. ese results strongly suggest that
the proteins encoded by these genes contain functional signal peptides
that facilitate secretion.
3.7 Subcellular localization of Nsxyn1 and
Nsxyn2 proteins and their role in inducing
necrosis in Nicotiana benthamiana
e plasmid maps and electrophoretic analysis during vector
construction are presented in Supplementary Figure S4. All uorescence
signals were eectively recorded using confocal laser scanning
microscopy in the onion epidermal cells (Figure7A) and Arabidopsis
thaliana protoplasts (Figure7B) expressing the Nsxyn1 and Nsxyn2.
Results from the transient expression system showed that onion
epidermal cells with empty PCAMBIAsuper1300-GFP carriers emitted
green uorescence throughout the cell structure. However, EGFP green
uorescence was mainly distributed in the nuclear onion cells with
pCAMBIAsuper1300-Nsxyn1-EGFP or pCAMBIAsuper1300-Nsxyn2-
EGFP overexpression carriers. Furthermore, when examined in
A. thaliana protoplasts, the Nsxyn1 and Nsxyn2 xylanases displayed a
dual localization pattern in both the nucleus and the cytoplasm.
Together, Nsxyn1 and Nsxyn2 proteins were localized in the nucleus
and cytoplasm.
Transient expression of Nsxyn1-EGFP and Nsxyn2-EGFP in
Nicotiana benthamiana leaves resulted inlocalized cell necrosis, as
evidenced by visible necrotic lesions at the inltration sites
(Figures7C,D). In contrast, the EGFP control did not induce any signs
of necrosis, and the leaves remained healthy. is suggested that
Nsxyn1 and Nsxyn2 possessed necrosis-inducing activity in plant
cells, independent of the uorescent tag.
FIGURE2
Sequence alignments were performed for Nsxyn1, Nsxyn2 and Nsxyn3. Numbers denote the positions of the amino acid residues. Bidirectional green
arrows mark signal peptides, while black lines represent GH11 domains. Green triangles highlight the two conserved glutamate enzymatic activity sites.
The alignment sequences of each site are highlighted with red characters on a white background with blue frames when the amino acid similarity in
the same column is greater than 0.7 and highlighted with white characters on a red background with blue frames when they are strictly conserved in
the column. Amino acids with a similarity of less than 0.7in the same column are highlighted with black characters on a white background.
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3.8 Obtaining gene knockout and
knockout complementation
Deletion and complementation strains for both genes were generated
to investigate the role of Nsxyn1 and Nsxyn2in cell wall degradation
(Supplementary Figures S5, S6). It was observed that Neostagonosporella
sichuanensis exhibited minimal growth on plates containing either 90 μg/
mL hygromycin B or 200 μg/mL G418 (Supplementary Figure S7),
leading to the selection of these concentrations for transformant
screening. e deletion strains of Nsxyn1 and Nsxyn2 were veried by
PCR analysis (Supplementary Table S1), and positive transformants were
chosen for further study (Supplementary Figures S8, S9).
FIGURE3
Structural analysis and model quality assessment of Nsxyn1, Nsxyn2 and Nsxyn3. (A–C) Secondary structures. The red helices, blue arrows, and black
lines indicate alpha helices, extended strands, and random coils. (D–F) 3D structures with “confidence (gradient)” color schemes of Nsxyn1, Nsxyn2,
and Nsxyn3 were constructed from AlphaFold DB models of 4ZRR9_LEPMJ and W6YRM9_COCMI, respectively. The bluer the models are, the higher
the confidence level, while the redder the models are, the lower the confidence level. The coverage and sequence identity of Nsxyn1 (D) were 100 and
82.35%, respectively. The coverage and sequence identity of Nsxyn2 (E) were 100 and 87.22%, respectively. The coverage and sequence identity of
Nsxyn3 (F) were 99 and 84.19%, respectively. (G–I) Ramachandran plots of the protein conformations generated by PROCHECK. There are 88.6% core
region, 10.3% allow region, 0.5% general allow region, and 0.5% disall region of all amino acid residues of Nsxyn1 (G), respectively, while 88.0% core
region, 10.4% allow region, 1.0% general allow region, and 0.5% disall region of all amino acid residues of Nsxyn2 (H). In comparison, 90.7% core region,
8.8% allow region, 0.5% general allow region, and 0.0% disall region of the amino acid residues of Nsxyn3 (I). (J–L) Comparison with the non-
redundant set of PDB structures using QMEAN.
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3.9 Nsxyn1 and Nsxyn2 proteins display cell
wall-degradation activity
e capacity of the deletion mutants (ΔNsxyn1 and ΔNsxyn2) and
the complementation strains (ctNsxyn1 and ctNsxyn2) to degrade cell
wall components was evaluated on the C’zapek media that was devoid
of carbon sources but supplied with xylan, cellulose, or pectin. On the
C’zapek medium with xylan as the sole carbon source, colony
diameters of ΔNsxyn1 and ΔNsxyn2 mutants were signicantly
smaller than those of the wild type (Figure8). Additionally, the colony
diameter of the ΔNsxyn1 mutants was unexpectedly decreased on
C’zapek media with cellulose or pectin, while the ΔNsxyn2 mutants
showed a similar reduction only when grown on C’zapek medium
with pectin. e complementation strains, ctNsxyn1, and ctNsxyn2,
completely restored cellulose and pectin degradation capacity and
partially restored the potential for xylan degradation.
FIGURE4
Qualitative and quantitative determination of the enzyme activity of purified recombinant Nsxyn1 and Nsxyn2 proteins. Determination of the ability of
purified Nsxyn1 (A) and Nsxyn2 (B) proteins to degrade xylan, respectively. 1–4: Recombinant Nsxyn1/Nsxyn2 protein. 5: Control eluent. The hydrolysis
circle reflects the ability of Nsxyn1/Nsxyn2 to degrade xylan, and the larger the hydrolysis circle, the greater the ability of the recombinant Nsxyn1/
Nsxyn2 protein to degrade xylan. (C) Determination of the xylanase activity of purified Nsxyn1SM113–205 and Nsxyn2SM121–212 proteins to degrade xylan. 1–2:
Nsxyn1SM113–205 recombinant protein. 3–4: Nsxyn2SM121–212 recombinant protein. 5: Eluent as control. (D,E) Determination of xylanase activity of Nsxyn1/
Nsxyn2 proteins and two site-directed mutants. Xylan was used as the only carbon source for induction in each case. Error bars indicate the standard
errors (SE) of the mean. Asterisks *** indicates statistical significance at p < 0.001 based on unpaired Student’s t-tests. Nsxyn1 (F), Nsxyn2 (G),
Nsxyn1SM113–205 (H), and Nsxyn2SM121–212 (I) inducing cell necrosis in Nicotiana benthamiana leaves. The white ring on the left side of the leaf indicates the
blank control for the eluent, while the white circle on the right indicates the location of the purified proteins.
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FIGURE6
Functional assessment of the Nsxyn1 and Nsxyn2 signal peptides.
YTK12 strains carrying pSUC2-Nsxyn1SP, pSUC2-Nsxyn2SP together
with pSUC2-Avr1bSP (positive control) are capable of growth on
CMD-W medium and YPRAA media, whereas the YTK12 strain devoid
of vector and possessing pSUC2 empty vector (negative controls) do
not. Additionally, the YTK12 strains containing pSUC2-Nsxyn1SP,
pSUC2-Nsxyn2SP, and the positive control change the color of the
reaction mixture from colorless to bright and dark red in the TTC
assay, whereas the negative controls show no color change.
3.10 Analysis of phenotypic, sporulation,
and spore germination of transformation
To examine the impact of Nsxyn1 and Nsxyn2 gene deletion on
the growth of Neostagonosporella sichuanesis, mycelial plugs of wild
type, ΔNsxyn1 and ΔNsxyn2 mutants, and the ctNsxyn1 and ctNsxyn2
complement strains were inoculated on PDA medium under light/
dark (12 h/12 h) conditions at 25°C for 30 days. A signicant decrease
in colony diameter and spore production was observed in the
ΔNsxyn1 mutant relative to the wild type (Figures9A–C). In contrast,
no strong negative eect on the growth rate was observed in the
ΔNsxyn2 mutant (Figures 9A,B). However, it was found that the
colony morphology of the ΔNsxyn2 mutant changed, shiing from a
smooth surface to a cotton-like appearance, with a corresponding
reduction in microconidia production compared to the wild type
(Figures9A,C). Spore production only statistically partially recovered,
and the growth rate was restored aer complementation. Additionally,
the conidial morphology of all transformant strains did not dier
signicantly from that of the wild type, and all strains were able to
germinate normally (Figure9D).
3.11 Stress response of transformants
e integrity of the fungal cell wall is crucial for successful host
cell infection, as it maintains the shape of the fungal cell and regulates
interactions with the external environment (Cabib et al., 2001).
Continuous cell wall remodeling is required for proper fungal growth
and development (Bowman and Free, 2006). Weassessed the eects
of cell wall-disrupting agents on the growth of the ΔNsxyn1 and
ΔNsxyn2 mutant strains (Figure10). Colony size measurements were
employed to quantify the growth inhibition rate, revealing that the
ΔNsxyn1 and ΔNsxyn2 strains exhibited signicant sensitivity to cell
wall stressors, including Congo red, SDS, and NaCl. Notably, the
ΔNsxyn2 strain displayed greater sensitivity than the ΔNsxyn1 strain.
3.12 Pathogenicity test of transformants
Pathogenicity assays were conducted to determine the roles of
Nsxyn1 and Nsxyn2 in rhombic-spot disease on living shscale
bamboo culms. e wild-type strain caused typical necrotic symptoms.
In contrast, while the ΔNsxyn1 and ΔNsxyn2 mutants displayed similar
symptoms, their virulence was signicantly reduced (Figure 11).
Complementation strains ctNsxyn1 and ctNsxyn2 restored most of the
virulence phenotype. ese ndings underscore the critical roles of
Nsxyn1 and Nsxyn2in the pathogenicity of N. sichuanensis.
4 Discussion
Bamboos are widely used in construction, furniture, and
ecological restoration (Ndavaro etal., 2022). However, bamboo culm
FIGURE5
Relative expression levels of Nsxyn1 (A) and Nsxyn2 (B) at dierent infection times of Neostagonosporella sichuanensis. Error bars represent standard
errors (SE). Asterisks *, **, and *** on the SE line are statistically significant dierences at p < 0.05, p < 0.01, and p < 0.001, respectively, as determined by
unpaired Student’s t-tests.
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diseases have signicantly challenged bamboo production and
quality. Despite the increasing prevalence of reported bamboo culm
diseases (Huang L. etal., 2023; Sonali etal., 2023; Yang etal., 2024),
the pathogenic mechanisms of bamboo culm pathogens, including
Neostagonosporella sichuanensis (the primary pathogen of rhombic-
spot disease), remain few investigated. Xylanases have been identied
as key virulence factors in numerous previous studies (Nguyen etal.,
2011; Yu etal., 2016; Yu etal., 2018; Wang etal., 2021). Our early
experiments have demonstrated that N. sichuanensis exhibited good
xylan degradation capabilities (Liu et al., 2024), but what role
xylanases play in the rhombic-spot disease remains uninvestigated.
e high-quality whole genome sequences of N. sichuanensis
SICAUCC 16–0001 (GenBank accession number:
JAUGWR000000000) have been published in the NCBI database,
which provides a valuable resource for gene identication and
functional validation. Here, weidentied the xylanases belonging to
the GH11 family in the N. sichuanensis SICAUCC 16–0001 genome,
in which two xylanases (Nsxyn1 and Nsxyn2) were deeply studied for
their roles in pathogenesis. Despite numerous attempts, wecould not
obtain the Nsxyn3 gene; therefore, synthesizing the Nsxyn3 gene
fragment for future functional studies would be a promising
approach. Weidentied Nsxyn1 and Nsxyn2 as secretory proteins
possessing conserved functional domains and enzyme activity sites
through multiple sequence alignments. e secretion potential and
enzyme activity of Nsxyn1 and Nsxyn2 proteins were veried
through signal peptide secretion assays and enzyme activity
detection, suggesting their potential roles in host cell wall
degradation. Expression analysis via qRT-PCR showed that Nsxyn1
and Nsxyn2 were up-regulated to varying degrees during the
infection of shscale bamboo, with Nsxyn1 showing the most
signicant upregulation. e upregulation of xylanase genes has been
demonstrated to bea critical factor in the pathogenicity of fungi,
particularly during interaction with host plants (Brito etal., 2006; Lai
and Liou, 2018). e signicant expression levels of Nsxyn1 and
Nsxyn2 further indicate that they play a vital role in degrading the
cell wall of shscale bamboos and potentially function as eectors
that modulate plant immune responses. Moreover, the signicant
decrease in pathogenicity observed in the Nsxyn1 and Nsxyn2
mutants provides convincing evidence of its functional involvement.
However, xylanases are not always essential for pathogenicity, as
FIGURE7
Subcellular localization of the Nsxyn1 and Nsxyn2 proteins is examined in onion epidermal cells (A) and Arabidopsis thaliana protoplasts (B). The
pCAMBIA1300-EGFP vector is used as a control. In Arabidopsis thaliana protoplasts, red fluorescence indicates nuclear markers, while purple
fluorescence is due to chloroplast autofluorescence, and the green fluorescence emitted by the pCAMBIA1300-GFP vector is visible. Membrane and
nuclear markers exhibit red fluorescence. Transient expression of Nsxyn1 (C) and Nsxyn2 (D) are both observed to induce necrosis in N. benthamiana
leaves. The white ring on the left side of the leaf denotes the negative control, where only EGFP-expressing Agrobacterium suspensions are infiltrated.
The white circles on the right indicate the regions infiltrated with Nsxyn1-EGFP (C) and Nsxyn2-EGFP (D) Agrobacterium suspensions, respectively.
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FIGURE8
Growth observations of the Nsxyn1 and Nsxyn2 knockout mutants and complement strains on basic C’zapek medium, utilizing xylan, pectin, or
microcrystalline cellulose (MCC) as the sole carbon source. (A) Photographs of the growth phenotypes were taken after incubation at 25°C for 30 days.
(B) Comparison of colony diameters. Error bars represent the standard errors of the mean. Distinct lowercase letters indicate significant dierences in
colony diameter among the various strains on the same type of culture media at p < 0.05, as determined by one-way ANOVA.
demonstrated with other fungi, including Cochliobolus carbonum,
Fusarium oxysporum f. sp. Lycopersici, and Fusarium graminearum
(Apel, 1993; Gómez-Gómez etal., 2002; Sella etal., 2013).
Accumulating evidence suggests that plant cell wall-degrading
enzymes (PCWDEs), including xylanases, can induce host defense
responses and promote cell death independently of their enzymatic
activity (Enkerli etal., 1999; Noda etal., 2010; Gui etal., 2017). For
example, the strong cell death and PAMP-triggered immunity (PTI)
induced by BcXyl1 were observed in several plants and independent of
its enzyme activity (Yang etal., 2018). ese PCWDEs were dened as
a class of necrosis-inducing proteins (NIPs). Although Nsxyn1 and
Nsxyn2 are xylanase proteins, they can cause cell necrosis in Nicotiana
benthamiana independently of xylanase activity, indicating that a
specic motif or protein domain mediates cell death-inducing activities
(Zhu etal., 2017). e PCWDEs inducing cell necrosis have been
reported to act as pathogen-associated molecular patterns (PAMP) and
possibly recognized by plant leucine-rich repeat receptor-like proteins
(LRR-RLPs) (Sabnam etal., 2023). PAMP usually triggers early plant
immune responses, such as oxidation bursts, MAPK phosphorylation,
ROS production, and callose accumulation (Nicaise etal., 2009; Peng
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etal., 2018). Furthermore, the RLP-SOBIR1-BAK1 complex is essential
for the downstream signaling and cell death activity of PCWDEs (Frías
etal., 2019; Wang etal., 2022). e potential of Nsxyn1 and Nsxyn2 to
induce plant immune responses, particularly in shscale bamboo, and
whether the RLP-SOBIR1-BAK1 complex mediates their cell death-
inducing activity requires further investigation. e mechanism by
which plant cells receive the xylanase elicitor remains unclear. Previous
studies suggest that xylanase recognition may bedirectly detected by
plant cells through a receptor for this protein or indirectly through
fragments of plant cell walls generated by its enzymatic activity (Bucheli
etal., 1990; Hanania and Avni, 1997). Our study observed that the
xylanases Nsxyn1 and Nsxyn2 localized to cytoplasm and nuclei in
Arabidopsis thaliana protoplasts. Localization within the cytoplasm may
enable these xylanases to interact directly with host cellular components,
FIGURE9
Colony phenotypes (A), mycelial growth rates (B), conidial sporulation statistics (C), and germination morphology of megaspores and microspores
(D) from dierent strains (wild-type strain, Nsxyn1 and Nsxyn2 deletion strains, and their corresponding complementation strains) grown under light/
dark (12 h/12 h) conditions at 25°C for 30 days. Error bars represent the standard error of the mean. Dierent lowercase letters indicate significant
dierences in megaspore/microspore sporulation among the various strains at p < 0.05, as determined by one-way ANOVA. The unit for counting
megaspores is 1 × 106 spores, while the unit for counting microspores is 1 × 107 spores.
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FIGURE10
Sensitivity of Nsxyn1 and Nsxyn2 mutants to cell wall stress-inducing agents. Colony morphology (A) and colony diameter measurements (B–D) of the
wild-type strain (WT), Nsxyn1 and Nsxyn2 deletion mutants (ΔNsxyn1 and ΔNsxyn2), and their corresponding complement strains (ctNsxyn1 and
ctNsxyn2) were assessed after culturing on PDA media supplemented with various cell wall perturbing agents for 30 days. (B) 10 μmol/L Congo Red
(CR); (C) 0.02% sodium dodecyl sulfate (SDS); (D) 1 mol/L NaCl. Colony diameters were averaged from ten technical replicates, and error bars represent
standard errors. Asterisks (***) indicate a significant dierence (p < 0.001) based on unpaired Student’s t-tests. Dierent letters on the bar charts denote
statistically significant dierences at p < 0.05 (one-way ANOVA).
thereby modulating plant defense responses. For example, the
cytoplasmic eector Avr1b from Phytophthora sojae has been shown to
interfere with host immune signaling pathways by directly interacting
with host proteins, ultimately promoting disease development (Dou
etal., 2008). e dual localization of Nsxyn1 and Nsxyn2 suggests their
involvement in multiple stages of the infection process, with nuclear
localization possibly indicating a role in inuencing host transcriptional
responses (Kim etal., 2020; Harris etal., 2023). A 25-residue peptide
originating from xylanase BcXyn11A was reported to induce PTI
immune responses, including cell necrosis, ROS burst, and seedling
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growth inhibition (Frías etal., 2019). Notably, despite the low similarity
of Nsxyn1 (43.53% identity) and Nsxyn2 (59.39% identity) to
BcXyn11A, both proteins contained two similar conserved regions with
four consecutive amino acid residues to BcXyn11A, namely YGWT and
YYIV (with YYIQ in Nsxyn1 and YYVV in Nsxyn2). ese observations
indicate that Nsxyn1, Nsxyn2, and BcXyn11A potably share functional
commonalities. Moreover, the virulence role of BcXyn11A is linked to
its necrotizing activity (Frías et al., 2019); further investigation is
required to determine whether Nsxyn1 and Nsxyn2 contribute to
virulence through a similar mechanism.
e growth rate or sporulation rate of many xylanase mutants of
fungi decreased signicantly (Yu etal., 2016; Shabbir etal., 2022; Cui
etal., 2024). e deletion of Nsxyn1 had a notable adverse impact on
fungal growth rates and sporulation quantity, whereas only a reduction
in microconidial production was observed in Nsxyn2 mutants. ese
results suggest that Nsxyn1 may play a key role in fungal growth and
nutrient acquisition, whereas other genes may supplement the
function of Nsxyn2. To substantiate this hypothesis, the eect of both
deletions on fungal growth can befurther investigated through a
double knockout experiment. Conidia are critical for the pathogenicity
of Neostagonosporella sichuanensis as they serve as important primary
inocula for infecting stomatas or wounds of shscale bamboo through
wind or rain drops (Liu etal., 2022). e severity of rhombic-spot
disease correlates directly with the conidia count within rhombic-spot
lesions. e number of conidia in the Nsxyn1 mutant decreased
signicantly, suggesting its importance in asexual sporulation.
Xylan has a highly complex structure and is a signicant
component of the hemicellulose found in plant cell walls, whose
complete degradation requires the coordinated activity of multiple
enzymes (Sarangi and Tahatoi, 2024). In this study, the knockdown of
Nsxyn1 and Nsxyn2, respectively, signicantly reduced the ability of
Neostagonosporella sichuanensis to degrade xylanase, especially Nsxyn1,
but did not wholly prevent colony growth. ere are multiple xylanase
family genes in the N. sichuanensis genome, and other xylanase genes
may partially or wholly compensate for the function of the knocked-
down genes, helping to maintain a certain degree of xylan degradation
and colony growth. In addition, the knockdown of Nsxyn1 and Nsyxn2
not only made a dierence in the ability of N. sichuanensis to degrade
xylan but also aected pectin and cellulose degradation. One possibility
is that changes in xylanase production aect the overall secretion
system or metabolic pathways of the microorganism, indirectly
inuencing the production of other enzymes like pectinase. is is
supported by studies showing that enzyme secretion and growth are
tightly linked in lignocellulose-degrading microorganisms, and
altering one enzyme may impact others (ite and Nerurkar, 2018).
e polysaccharide network in fungal cell walls is crucial for
regulating the ow of chemical substances between the fungal cells
and their external environment (Díaz-Jiménez etal., 2012; Garcia-
Rubio etal., 2020). e heightened sensitivity of Nsxyn1 and Nsxyn2
mutants to cell wall-disrupting osmolytes implies that these genes are
crucial for maintaining cell wall integrity.
5 Conclusion
In summary, this study identied and cloned two xylanase genes,
Nsxyn1 and Nsxyn2, and demonstrated that the corresponding
proteins are secretory proteins localized in the nucleus and cytoplasm
of Arabidopsis thaliana. Both proteins exhibit signicant xylanase
enzyme activity but also induce cell necrosis in Nicotiana
benthamiana independently of this activity. Further research is
necessary to clarify the exact regions within these enzymes
recognized by plants as pathogen-associated molecular patterns
(PAMPs). Additionally, our results suggest that Nsxyn1 and Nsxyn2
are essential for xylan degradation, adaptation to osmotic and
oxidative stress, and full pathogenic virulence. Deletion of Nsxyn1
notably slowed fungal growth and reduced spore production, whereas
only a reduction in microconidial production was observed in
Nsxyn2 mutants. ese ndings lead us to hypothesize that Nsxyn1
plays a key role in fungal growth and nutrient acquisition, whereas
the function of Nsxyn2 may be compensated by other genes.
Exploring the eects of double knockout mutants of these xylanase
genes will beessential to elucidate their functional redundancy and
cooperative roles in pathogenicity. ese ndings will enhance our
understanding of the mechanisms by which xylanases contribute to
the pathogenicity of fungi.
FIGURE11
Photographs depicting the symptoms of infection on fishscale bamboo culms (A) and the corresponding statistics of lesion size (B) after 30 days of
inoculation with conidial suspension. From left to right, the treatments include sterile water, conidial suspensions of wild-type, and the Nsxyn1 and
Nsxyn2 detection mutants (ΔNsxyn1 and ΔNsxyn2), along with their corresponding complementary strains (ctNsxyn1 and ctNsxyn2). Wounds were
inoculated with sterile water as a control. Each treatment was conducted in triplicate. Dierent lowercase letters indicate statistically significant
dierences among the treatments under the same stress conditions, as determined by a one-way ANOVA at p < 0.05. Asterisks (**) indicate a significant
dierence (p < 0.01) based on unpaired Student’s t-tests.
Liu et al. 10.3389/fmicb.2024.1507998
Frontiers in Microbiology 16 frontiersin.org
Data availability statement
e datasets presented in this study can befound in online
repositories. e names of the repository/repositories and accession
number(s) can befound in the article/Supplementary material.
Author contributions
LL: Conceptualization, Data curation, Formal analysis,
Methodology, Soware, Visualization, Writing – original dra,
Writing – review & editing. CL: Conceptualization, Data curation,
Methodology, Soware, Visualization, Writing – review & editing.
FL: Methodology, Soware, Writing – review & editing. SH: Writing
– review & editing. SL: Writing – review & editing. CY: Supervision,
Writing – review & editing. YL: Data curation, Formal analysis,
Funding acquisition, Methodology, Project administration,
Resources, Supervision, Writing – review & editing.
Funding
e author(s) declare that no nancial support was received for
the research, authorship, and/or publication of this article.
Acknowledgments
We are grateful to the Chengdu Academy of Agriculture and
Forestry Sciences for oering us a location to plant shscale bamboo
to complete the pathogenicity test.
Conflict of interest
e authors declare that the study was conducted without any
nancial or commercial relationships that could beconsidered as
potential conict of interest.
Generative AI statement
e author(s) verify and take full responsibility for the use of
generative AI in the preparation of this manuscript. Generative AI was
used while preparing this work, the authors used ChatGPT 4o mini to
improve language. Aer using this tool, the authors reviewed and
edited the content as needed and take full responsibility for the
content of the publication.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their aliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may beevaluated in this article, or
claim that may bemade by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
e Supplementary material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/fmicb.2024.1507998/
full#supplementary-material
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