Introduction
Among the
many soil-borne species worldwide, Fusarium is considered a major group
of plant pathogens (Dean et al. 2012). Specifically, F. oxysporum, which causes vascular wilt
disease in many important cash crops including tomato, cucumber and banana, is
one of the most common and highly pathogenic Fusarium species (Michielse
and Rep 2009). Banana (Musa spp.) Fusarium wilt (Panama disease) caused by
F. oxysporum f. spp. cubense (Foc) is a serious threat to banana
production globally (Hwang and Ko 2004).
According to the sensitivity
of different banana varieties in the field, banana Foc is divided into three races, Foc1,
Foc2 and Foc4 (Ploetz 2015). Foc1 can infect banana cultivars “Gros Michel”
(AAA) and “Silk” (AAB), while Foc2 infects only “Bluggoe” (ABB). In contrast,
Foc4 readily attacks not only Cavendish (AAA) but also all other Foc1- and
Foc2-sensitive cultivars (Ploetz 2015). Therefore, Foc4 is thought to be the
most highly infectious race of the pathogen (Ploetz 2006). Indeed, Grimm (2008)
pointed out that Latin America bananas might be destroyed if Foc4 invaded the
region. To date, there are no 100% Foc4 resistant varieties or any suitable
management strategy for Fusarium wilt
in plants infected with Foc4 (Butler 2013; Ordonez et al. 2015).
Banana wilt disease has been reported in all the main banana producing
areas in China and already the national banana industry, has been seriously
affected, as many plantations have been completely devastated by this disease,
causing great economic losses. Therefore, understanding the molecular basis of
the pathogenicity of banana wilt might provide a method to counter the disease;
however, its pathogenesis has not been fully elucidated, as to date, only some
of the pathogenic genes associated with Foc4 have been reported (Ploetz and
Randy 2015; Guo et al. 2016; Ding et al. 2020); thus pathogenesis
of Foc4 warrants further study.
Plant cell walls, the first chemical and physical barriers against
pathogen invasion, are mainly composed of three polysaccharides. Although the
fungal cell wall per se cannot sense an external stimulus, a large
number of glycoproteins surrounding the cell wall may participate in the
interaction between pathogenic fungi and the host plant (Geoghegan et al. 2017). Some of the cell wall
degrading enzymes (CWDEs) secreted by pathogenic fungi, including Foc4, have
been proved to be important pathogenicity factors during infection (Dong and
Wang 2011, 2015). Thus, for example, Guo et
al. (2014) found that Foc1 and Foc4 genomes contained a large number of
carbohydrate-active enzymes (CAZymes). Furthermore, there were more specific
CAZymes expressed in Foc4 than Foc1 when grown in proximity to the host cell
wall, implying that CAZymes may play an irreplaceable role in Foc
pathogenicity, while there might still be some special CAZymes in Foc4 contributing
to make it more infective (Guo et al.
2014). In our previous studies, differences among polygalacturonases (PGs)
produced by Foc1 and Foc4 and two other forma of F. oxysporum were compared (Dong et al. 2010). Now, in this
study, a novel endoPG isozyme named as PGC1, was purified from F. oxysporum f. spp. cubense race 4 (Foc4) in vitro
cultures. The cloned gene and heterologous expression of PGC1 were then tested
for tissue maceration and necrosis. Our findings provide evidence that PGC1 may
play a role in Foc4 pathogenicity to plants and that it can be produced as a
fully functional PG in the P. pastoris
protein expression system.
Materials and Methods
Fungal strain and culture conditions
The pathogenic strain Foc4
was preserved at South China Agriculture University, where it was cultured on
SM medium with 1% [w/v] citrus pectin (Sigma) for pectinolytic enzyme
production and RNA extraction (Pietro and Roncero
1996).
PG activity and protein assays
PG activity was assayed in a mixture (1 mL of total volume) containing
0.5% polygalacturonate (PGA) (w/v), 50 mM sodium acetate buffer (pH 4.5)
and various amounts of enzyme solution at 50°C for 30 min. The
number of reducing groups, expressed as galacturonic acid (GA) released by
enzymatic action, was determined. One unit of enzyme activity (U) was defined
as the amount of enzyme that releases 1 μmol
of GA per minute under the assay conditions. The molecular weight of the
purified enzyme determined by SDS-PAGE was performed using 12% acrylamide.
Isolation and characterization of PGC1 from FOC4 culture supernatant
A Foc4 sample culture was
centrifuged at 16,000 rpm for 20 min at 4°C. Then, the supernatant in the tube
was transferred onto a new tube and concentrated 100-fold with an Amicon 8400
ultrafiltration system containing a 10 kDa MWCO membrane. This concentrated
extract filtrate was added to a gel filtration column (Sephacryl S-100 16/60,
Pharmacia) and eluted with 50 mM sodium acetate buffer (pH4.5) at a flow
rate of 1 mL min-1. The active eluate fraction containing PG was
collected and added to a cation exchange column (Sepharose SP XL 16/10,
Pharmacia) equilibrated with 20 mM sodium acetate buffer (pH 4.5). The
column was eluted with an NaCl
gradient (0–0.7 M) at a flow rate of
4 mL min-1. The fractions were collected and added to another cation
exchange column (Sepharose FF CM Hitrap 1 mL, Pharmacia) equilibrated with 20 mM
sodium acetate buffer (pH 4.5). The column was then eluted with NaCl gradient (0–0.7 M)
at a flow rate of 2 mL min-1.
Purified PGC1
was run on a SDS-PAGE gel, then transferred onto a PVDF membrane and submitted
for N-terminal analysis by automated Edman degradation (Pulsed liquid sequencer
model 470A; Applied Biosystems).
Construction of plasmid vectors and transformation
Re-PGC1 expression vector pPICZαA-pgc1-Myc-His6
was constructed as follows: the pgc1
cDNA was amplified using the forward primer
(5΄-GCGCTCGAGAAAAGAGATCCCTGCAGCGTCACTGACT-3΄) and the reverse primer
(5΄-CGCGCGGCCGCGTTAGGGCAAGTGTT-3΄); XhoI and NotI restriction
sites are underlined and the yeast consensus sequence is shown in bold. The primers are based on the
sequence of pgc1 (accession no. FJ593631). The PCR product was then cloned into the pPICZαA vector
(Invitrogen) in fusion with a C-terminal
Myc and His6 tag giving pPICZαA-pgc1-Myc-His6.
The correct sequence was verified by sequencing. Yeast transformation was
performed. SacI was used to digest
the recombinant plasmid pPICZαA-pgc1-Myc-His6.
After that, the linearized part was transformed into P. pastoris strain SMD1168 by electroporation. Then, they were
patterned at 28°C for 48 h in a yeast extract peptone dextrose (YPD) plate
containing 1% yeast extract, 2% dextrose, 2% peptone, and 100 µg
mL-1 of Zeocin.
Expression and purification of re-PGC1
Transformants containing pPICZαA-pgc1-Myc-His6
were inoculated in 10 mL of BMGY medium (1% yeast extract, 2% peptone, 1.34%
yeast nitrogen base, 100 mM potassium phosphate, 4×10-5%
biotin and 1% glycerol) at 28°C for 24 h and then inoculated in 200 mL BMMY (1%
yeast extract, 2% peptone, 1.34% yeast nitrogen base, 100 mM potassium
phosphate, 4×10-5% biotin and 0.5% methanol). Samples were taken
every 12 h. One milliliter of 100% methanol was added into it every 24 h to
ensure a final methanol concentration of 0.5% assuming methanol was completely
utilized in 24 h. The culture supernatant was analyzed by 10% (w/v) SDS-PAGE
followed by silver staining.
The recombinant protein was extracted from the culture
supernatant and purified using a Ni-NTA His Bind resin column as per manufacturer
instructions (Novagen). All steps were carried out at 4°C. Protein
concentrations were determined by the Bradford assay and analyzed by 10% (w/v)
SDS-PAGE. Proteins were transferred onto a PVDF membrane for Western blot
analysis. Re-PGC1 was detected using an Anti-Myc-HRP Antibody (Invitrogen). The
membrane was developed using the chemiluminescent substrate HRP-DAB Kit
(TIANTEN), according to manufacturer instructions.
Biochemical characterization of PGC1 and re-PGC1
To analyze
the hydrolysis products, the samples (0.02 U enzyme in 0.5 mL water) were added
to 1 mL of 0.5% (w/v) PGA in 50 mM sodium acetate buffer (pH 4.5) and
incubated at 50°C, for 10, 20, 30, 40, 50, 60 min and then used for PG
activity assay. The
3% of the substrate being hydrolyzed by endo-PGs can lead to 50% reduction of
viscosity, while exo-PGs need 20% of the substrate being hydrolyzed.
The Michaelis constant (Km) and Vmax values were
determined from Lineweaver–Burk plots of enzyme activity measured with the PGA
as substrates, at concentrations between 0.25 and 1.25% at optimum pH and
temperature, and then plotted the results.
To determine the
optimal pH, the PG activity was assayed using 100 mM potassium
phosphate buffer for pH values between 3 and 10 at 50°C and 0.5% (w/v) PGA as
substrates. The consequence of temperature on PG activity was determined in 100
mM potassium phosphate buffer at pH
4.5, between 10 and 90°C. Three replicates were tested per treatment as well as
the negative control.
Tissue maceration and necrosis assayed with PGC1 and re-PGC1
Table
1: Purification of the PGC1 from FOC4 cultured with synthetic medium (SM)
medium supplemented with 1% citrus
pectin
|
Protein (mg) |
Activity (Unit) |
Yield (%) |
Specific
activity (Unit/mg) |
Crude |
36.6 |
131.2476 |
100 |
3.586 |
Ultrafiltration |
8.21 |
70.2604 |
53.53 |
8.5579 |
Sephacryl S-100 16/60 |
1.85 |
20.35 |
15.51 |
11 |
Sepharose SP XL 16/10 |
0.66 |
12.50 |
9.52 |
18.9394 |
Sepharose FF CM Hitrap |
0.15 |
3.22 |
2.45 |
21.4667 |
To evaluate banana tissue
maceration, the cultivars tested comprised
Musa AAA ‘Cavendish’ cv. ‘Baxi’, resistant to FOC1 and susceptible to FOC4;
Musa AAB cv. ‘Guangfen-1’,
susceptible to FOC1 and FOC4. 1 cm lengths of tissue (0.5 g) were taken from
the healthy stems of the four-leaf stage banana and placed in test tubes. A
mixture of 1 unit of purified enzyme with 1 ml of 50 mM sodium acetate
buffer, pH 5.0, was inoculated with the sterilized banana tissue and maceration
was evaluated after 48 h at 45°C. Control tubes contained the same buffer
without enzyme. Released reducing sugar was calculated from standards of GalA
after incubation 48 h at 45°C.
For the tissue necrosis assay, 1 unit of enzyme was
applied to the stems of healthy banana plant by injection. For each treatment,
stems were cut (vertical-sectioned) 5 day later to
observe vascular necrosis, ten replicates. Sterile double distilled water and
50 mM sodium acetate buffer pH 5.0 were used as controls.
Results
Purification
of PGC1
Polygalacturonase
PGC1 was finally purified from Foc4 through several steps including three steps
of ultra-filtration, gel filtration chromatography, and cation exchange
chromatography. The enzyme activity increased from 3.57 to 21.46 units mg protein-1 min-1 after the
purification process (Table 1). Concentrated crude PG from the shaking culture
showed one unclear single PG-activity peak when applied onto a Sephacryl S-100
16/60 gel filtration column. Subsequently, the fraction with PG activity was
collected and applied onto a cation exchange (Sepharose SP XL 16/10)
chromatography column that yielded a significantly enriched single PG peak.
This concentrated PG sample was loaded onto another cation exchange column
(Sepharose FF CM Hitrap 1 mL) that yielded a single PG and protein peak.
SDS-PAGE showed a single protein band, thus proving that PGC1 was totally
purified to homogeneity (Fig. 1) with molecular weight of a 42.3 kDa.
Expression and purification
of re-PGC1 in P. pastoris
The pgc1 gene (GenBank accession no. FJ593631)
fragment encoding the mature PGC1 was amplified by RT-PCR and cloned into
pPICZαA to construct the recombinant pPICZαA-pgc1 plasmid, which was then transformed
into the sensitive cells of strain SMD1168 by electroporation. A mut+
SMD1168 recombinant strain was inoculated in 200 mL BMMY and post induction
samples from the cultures at different sampling time points were run on
SDS-PAGE and analyzed by silver staining. These showed protein expression as
early as 24 h after induction, which continued to increase from 24 to 72 h.
Re-PGC1 was purified from 100 mL of crude extract by using a Ni-NTA His Bind
resin column. SDS-PAGE of the purified re-PGC1 showed only one band on a 10%
polyacrylamide gel. Together with our Western blot results, this indicated that
the protein was probably a native protein of the SMD1168 transformant induced
by methanol (Fig. 2).
Biochemical characterization
of PGC1 and re-PGC1
The Kms of purified PGC1 was
0.462 mg mL-1, while the Vmax
was 256.41 units mg protein-1 min-1 (Fig. 3). The final
products of enzymatic hydrolysis of PGA were analyzed using paper
chromatography. The intermediate products that appeared during hydrolysis
suggest an endoPG activity for PGC1. The optimum pH and temperature for PGC1
and re-PGC1 activity were 5.0 and 50°C, respectively (Fig. 4). Both natural
PGC1 and re-PGC1 showed the same variation trend as a function of temperature
and pH.
Active PGC1 and re-PGC1
cause tissue maceration and necrosis
Fig. 1: Purification of PGC1 from FOC4 cultured with synthetic medium
supplemented with 1% citrus pectin
Lane M: protein marker. Line 1:
Concentrated culture. Line 2: PG after gel filtration.
Line 3: PGC1 after sepharose SP cation exchange. Line 4-5: PGC1 after sepharose FF CM
Fig. 2: The
maceration activity of PGC1 to banana tissue
Musa AAB: Musa
AAB cv. Guangfen-1; Cavendish Musa AAA: Musa
AAA Cavendish cv. Baxi)
Fig. 3: Enzymatic activity of PGC1
A:
Optimal pH. B:
Optimal temperature
Fig. 4: The Michaelis-Menten curve of PGC1
Fig. 5: SDS-PAGE and Western
blot analysis of the purified re-PGC1
Lane 1: Markers, Lane 2: Purified re-PGC1 with Coomassie blue R-250, Lane 3: Western blot analysis of the
purified re-PGC1 after SDS-PAGE with the Anti-Myc-HRP
antibody
Fig. 6:
Tissue necrosis analysis of Baxi banana tissue
A:
Banana plant was injected with PGC1 of non-activity. B: Banana plant was injected with sterile double distilled water. C: Banana plant was injected with
purified PGC1. D: Banana plant was
injected with re-PGC1
Either one unit of PGC1 or
of r-PGC1, mixed with one mL of 50 mM sodium acetate buffer (pH 5.0),
were inoculated to the sterilized banana tissues to check whether they can macerate the tissue; observations
were made after 48 h.
The results showed that the
maceration activity of PGC1 on tissues
of banana cultivar ‘Guangfen-1’ was much more extensive than that of re-PGC1,
while the maceration activity of PGC1 on ‘Baxi’, a ‘Cavendish’ banana, was
slightly lower than that of re-PGC1. The maceration activities of PGC1 and
re-PGC1 on ‘Guangfen-1’ were higher than those on ‘Baxi’ (Fig. 5); PG showed
differences in maceration ability on ‘Guangfen-1’ and ‘Baxi’ banana cultivars.
Stem
vascular tissues of ‘Cavendish’
cultivar ‘Baxi’ showed partial necrosis after inoculation with PGC1 or re-PGC1.In
contrast, ‘Cavendish’ cultivar ‘Baxi’ inoculated with non-active PGC1
orsterile double-distilled water developed no necrotic spots (Fig. 6).
Discussion
Purification of the PG
protein does not seem to be highly effective due to low activity. In order to
collect PG more effectively, it should be increased and concentrated. The
Sephacryl S-100 column for coarse separation can help to remove many
contaminating proteins. For most PG isozymes usually produced under acidic
conditions, weak cation columns are used to separate them from middle and
advanced ones. Obtaining protein purified to a greater degree can be achieved
by regulating the collected volume from collection pipes. Similar methods were
successfully used to purify other PGs from Foc4 (Dong and Wang 2011, 2015).
The final products of enzymatic hydrolysis of PGA were analyzed; the
results suggested that PGC1 showed endoPG activity. Endopolygalacturonases
(endoPGs; poly-a-1, 4-galacturonide glycanohydrolase, EC
We found that PGC1 and re-PGC1 caused tissue maceration and necrosis on
banana plants. Many pectinases have been purified and found to macerate
vascular plant tissues. For example, Phytophthora capsici Pcipg5 was found to
increase leaves symptom development on pepper (Li et al. 2012), Purified
recombinant RsPG2 from Rhizoctonia solani degraded rice tissue 48 h
after inoculation (Chen et al. 2017). However, PG1 was purified and
cloned from F. oxysporum f. spp. lycopersici and then introduced into F. oxysporum f. spp. melonis without altering the virulence
pattern toward muskmelon, suggesting that PG1 could not macerate the vascular
tissues of muskmelon (Pietro and Roncero 1998). Here,
we found that banana inoculated with PG showed the same symptoms upon infection
by Fusarium wilt disease. Although
the maceration activity of re-PGC1 was lower than that of PGC1, its maceration
ability was high enough to allow detection of the activity.
It seems that future PGC1 mass production in Pichia pastoris is possible. The pectin of ‘Baxi’
banana varieties was a poor substrate for PGC1, compared with that from
‘Guangfen-1’, suggesting that the structure of pectin polymers in ‘Guangfen-1’
and ‘Baxi’ might differ from one another. Nonetheless, how PGs function and
whether a single gene can cause the disease or whether they work together,
needs to be further researched.
Optimum pH for PGC1 and re-PGC1 activity was 5.0, while optimum
temperature was 50°C. Further, both natural PGC1 and re-PGC1 showed the same
variation trend in when considered as a function of temperature and pH. In areas
where bananas are grown, ambient temperature is generally close to 30 degrees,
indicating that the enzyme is more active at a relatively high ambient
temperature, which can play a role in promoting pathogen infection. It also
shows that the enzyme has the ability to be industrialized, because it is
optimally active at 50°C and can be used for industrial purposes, such as plant
stem-pectin digestion.
In vitro expression
and preparation of active proteins is important to better research the function
of the gene involved. Although the prokaryotic protein-expression system is
widely used in various fields, the eukaryotic expression system displays more
advantages. The eukaryotic protein expression system in yeast has been
successfully used in different species (Chen et al. 2015; Meng et al.
2017). Here, PGC1 can be expressed in the yeast eukaryotic protein expression
system quite satisfactorily. Purified PGC1 and re-PGC1 have a similar function;
this implies that the activity of re-PGC1 is very similar to that of purified
PGC1 from Foc4 directly. Additionally, further successful purification of the
protein made functionality research more convenient. So considering maceration
activity level, high expression level, optimal temperature and pH, we propose
that re-PGC1 is a candidate endoPG for use in further researching fungal
pathogenicity and in developing the de-pectinization industry.
Conclusion
The present study provides the basic
characterization of a newly purified PGC1 protein. PGC1 can be produced as a fully
functional PG by using the P. pastoris
protein expression system. As purified PGC1 and re-PGC1 proteins both caused
maceration of banana tissue, PGC1 seemingly plays an important role in Foc4
pathogenicity. However, further studies are needed to elucidate the role of
PGC1 in pathogenicity role and its interactive target in host banana cultivars.
Functional analysis of pathogenic factors will provide new ideas for the
prevention and control of banana wilt.
Acknowledgements
This study
was supported by the Key projects of Guangdong Universities (Natural science)
[grant number: 2019KZDXM040) and “National and provincial fund” cultivation
project of Zhongkai College of Agricultural Engineering.
Author Contributions
ZY Dong
planned the basic research and conducted the experiments. ZY Dong and M Luo analyzed the data. ZY Dong, M Luo
prepared the manuscript. ZZ Wang revised the manuscript.
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