nematology -applied molecular biology2012
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Nematology, 2011
*Corresponding author, email: mprivilege@hotmail.com
Cloning and Sequence analysis of cellulase genes from Bursaphelenchus
xylophilus isolates
Privilege T. MAKUNDE
Universidade de Évora, 7002-554 Évora Lab. Nematologia/ICAM, Pólo da Mitra, Portugal
Summary- Bursaphelenchus xylophilus is inimitable in that, besides feeding on fungi, they
parasitizes live pine trees and subsequently cause pine wilt disease. The power behind this
kind of plant parasitism by nematodes is their ability to secrete and inject effector proteins
like cellulase, a protein which degrades the plant cell wall mainly composed of cellulose. We
cloned B. xylophilus cellulase gene, sequenced it and subjected the sequence to orthologs
search using Basic Local Alignment Search Tool in the public databases and we found that
the cellulase gene is of B. xylophilus belongs to glycoside hydrolase family (GHF) 45. We
also found that the cloned and sequenced gene is more related to GHF45 endoglucanases of
fungi since orthologs retrieved from the databases were of B. xylophilus and fungi species. A
phylogenetic analysis revealed that B. xylophilus isolates from Portugal, HF and 7C are
closely related to the isolates from Asian countries and less related to the isolates from USA.
Searching for co-transcribed genes with cellulase revealed that there are no genes in close
proximity of B. xylophilus gene and this finding together with the relatedness of the cellulase
gene of fungi evidently support the hypothesis that independent horizontal gene transfer
events have helped in shaping the evolution of B. xylophilus.
Keywords-Bursaphelenchus xylophilus; cellulase; secretions; glycoside hydrolase family 45,
Lateral Gene Transfer.
Nematology, 2011
Cellulose is a major structural component
of the plant cell wall which provides its
rigidity in concert with xyloglucans,
hemicelluloses, pectin and proteins. It is a
homopolymer composed of β-D-
glucopyranosyl units linked in a β-1, 4
fashion (Pembroke, 1998). The complexity
nature of cellulose microfibrils support the
plant cells as well as the plant itself, and
protects the cell from the environment
constrains. Plant cell wall is therefore the
main formidable barrier to the entrance,
propagation and development of plant
parasitic nematodes and other plant
pathogens; it keeps the nematodes and
other pathogens at bay. To circumvent this
cell wall barrier plant parasitic nematodes
in addition to the sclerotized, protrusible
stylet which mechanically breach the host
plant cell wall, have evolved in such a way
that they secrete cell-wall-degrading
enzymes to weaken it. The cell wall degrading enzymes
are produced from the sub ventral glands
and delivered into plant tissues and cells
through the hollow stylet. These stylet
secretions have been proved to play an
important role in breaking down cellulose
from the onset of penetration, migration
within the host cells and the rest phase of
parasitism (Jaubert et al., 2005). Plant
parasitic nematodes’ cellulase genes were
found to be exclusively expressed in the
sub ventral gland cells and were first
identified in Heterodera glycines and
Globodera rostochiensis (Smant, et al.,
1998; Yan et al., 1998) and following this,
cellulase genes were found in several plant
parasitic genera; Globodera, Meloidogyne,
Heterodera, Ditylenchus, Pratylenchus,
Radopholus, Aphelenchus and
Rotylenchulus (Wang et al., 1999; de Boer
et al., 1999; Rosso et al., 1999; Yan et al.,
1998, 2001 Goellner et al., 2000, 2001; De
Meutter et al., 2001; Gao et al., 2003,
2004b; Kikuchi et al., 2004; Ledger et al.,
2006; Bellafiore et al., 2008; Opperman et
al., 2008; Roze et al., 2008; Karim et al.,
2009; Rehman et al., 2009a; Haegeman et
al., 2008, 2011a). These characterised
endogenous cellulases belong to glycosyl
hydrolase family (GHF) 5 and resembles
more to cellulases of bacteria than of
eukaryotes, which gives a blue print that
the cellulase genes possessed by these
nematodes have been probably acquired
from bacteria through horizontal gene
transfer (Smant et al., 1998; Yan, et al.,
1998; Jones et al., 2005; Ledger et al.,
2006; Danchin et al., 2010). Contrary to
this, the catalytic module of
Bursaphelenchus xylophilus (Kikuchi et
al., 2004) cellulase is classified into GH45
family according to sequence based
homology (Henrissat, et al., 1993), which
resembles much to fungi cellulases.
Thefore, plant parasitic nematodes
cellulases are mostly likely secreted for
hydrolysis and breaking down of cellulose
to facilitate penetration and migration in
the plant cells during parasitism (de Boer
et al., 1999).
Bursaphelenchus is a large group
of nematodes that has a global distribution.
Most Bursaphelenchus species are
exclusively fungal feeders and some
species utilize fungi at some stage of their
life cycle. The pinewood nematode
(PWN), Bursaphelenchus xylophilus (Steiner & Buhrer, 1934), an endoparasitic
migratory nematode is the causal agent of
pine wilt disease, which cause great
wreckages to many conifer trees in China,
Japan, Korea and Portugal (Mamiya,
1988; Lee et al., 1990; Yang, 2003).The
pathogenic mechanisms of B. xylophilus
are still complex and not completely
understood. Three main theories therefore
exist; the enzyme theory, cavitation theory
and the toxin theory (Odani et al., 1985;
Kuroda, 1989). B. xylophilus is vectored
from wilt-killed pine tree to a healthy tree
by the beetle Monochamus
galloprovincialis (Mamiya & Enda, 1972;
Mamiya, 1983). The nematodes feed on
cells in the tree and migrate through the
tissues, spreading through the tree. This
causes cell destruction of cells, leading to
wilt symptoms that result in the death of
the tree within one year of infection.
Nematology, 2011
However the origin of pine wilt nematode
found in Portugal remains elusive (Mota
et., al 2006).
Here, we cloned; sequenced
cellulase gene from different
Bursaphelenchus xylophilus isolates and
systematically investigated the
evolutionary history of cellulase in plant
parasitic nematodes and traced back the
origin of the isolates. We show that these
proteins most likely originate from
horizontal gene transfer from fungi.
Material and methods BIOGICAL MATERIALS
The Bursaphelenchus isolates
which were grown on the fungus Botritys
cinerea at 25-28°C were provided.
Approximately 2000-10000 nematodes
including the growth media were washed
onto a filter paper in a Baermann funnel
with sterile physiological salt solution
(0.9% NaCl) and incubated for 24 hours.
There Baermann funnel facilitated the
removal of dead nematodes and growth
media. 10 ml of the nematodes suspension
were pipetted into microtubes tubes and
centrifuged at 17g for 6 minutes. The
supernatant was discarded and a sundry of
microorganism were partially removed by
an equivalent volume of 3% hydrogen
peroxide for 5 minutes. The suspension
was then centrifuged at 17g for 6 minutes,
the supernatant were discarded and 500µl
of distilled water was added and the
procedure was repeated three times. The
nematodes were then kept at -20 °C.
NEMATODE DNA EXTRACTION AND
ANALYSIS
DNA isolation from nematodes was
performed in accordance with GENOMED
JETQUICK Tissue DNA Spin Kit following
the manufacturer’s instructions. Nematode
remains were transferred into an
Eppendorf tube and nuclei lysis and
protein denaturation were done by 200µl
of Buffer T1 supplied with the kit and to
the lysed nematodes, 20 µl of proteinase K
was added to digest the denatured proteins.
The lysates in the tubes were mixed
thoroughly and incubated at 56°C (1h). In
between the incubation, the lysates were
repeatedly inverted to enhance the
efficiency of poteinase K, until the
observation of a clear solution. Preceding
the incubation, 20 µl RNase A was added
and the tubes were incubated at 37°C (5
min) for the removal of cellular RNA. To
the lysate 200 µl of Buffer T1 was added
and thoroughly mixed followed by
incubation 70°C (10 min). Following this,
200 µl of absolute ethanol was added and
DNA precipitation was avoided by
shaking. The lysates were transferred into
a JETQUICK column, placed into a suitable
receiver tube and centrifuged at 10.000 x g
(1 min). The flow through was discarded
and 500 µl of reconstituted buffer TX was
pipetted into the micro-spin column and
centrifuged at 10.000 x g (1 min). The
same procedure was done using
reconstituted buffer T3 except that,
centrifugation was at 13.000 rpm (1min).
The receiver tubes were discarded and the
column was inserted into microtubes.
DNA was then elutated by 200 µl of
prewarmed (65-70°C) elution buffer (10
mM Tris-HCl [pH 9,0] which was placed
directly onto the surface of the silica
membrane and incubated at 22°C (5 min)
and centrifuged at 13,000 rpm (2 min).
The eluted DNA was then stored at -20°C.
For qualitative test, 5µl of DNA
samples were resolved by gel
electrophoresis in a 0.8% agarose gel with
ethidium bromide in TBE (0.5×) buffer at
80V (45 min). 1 kb Plus DNA Ladder
(Invitrogen) was run in parallel with the
samples. DNA fragments were visualized
under UV light using UV transiluminator
system.
GENE AMPLIFICATION BY PCR AND
ANALYSIS
Extracted DNA (5 µl) was transferred into
a 0.2 ml Eppendorf tube containing: 5µl 1
X Taq incubation buffer (NH4)2SO4; 1µl
(0.2 mM each) dNTPs mixture; 2 µl (10
Nematology, 2011
pmol) of each primer (1 and 2); 0, 2µl
(1.25 u / 50 µl) of Taq DNA polymerase,
4µl (2 mM) of MgCl2 and 31µl sterilized
water to a final volume of 50 µl. The
forward primer 1, ENG00s (5’
CTAAAATGAAGTCTCTTGTG 3’) and reverse
primer 2, ENG 00a (5’
AGTCCTCTAAGCATCGTC3’) were used in
the PCR. The PCR conditions were as
follows: pre-denaturation for 3 minutes at
95°C, 30 cycles denaturation of 1minute at
94°C, annealing at 50°C for 1 minute, and
polymerization at 72°C for 5 minutes, with
a final incubation at 10°C hold.
After completion of PCR, a mix of 5µl of
each PCR product with 5µl of distilled
water and 2µl of sample buffer x 6 and
were resolved in a 1% TBE (0.5×)
buffered agarose mini-gel at 80 V (60min).
The PCR fragments were visualized under
UV transiluminator after exposure of the
gel in Ethidium Bromide (0.5µg/mL) for
20 minutes.
PRODUCT PURIFICATION AND
QUANTIFICATION
The PCR products were purified
using the GFX column. The GFX columns
were placed in collection tubes and to each
500 µl of capture buffer was added. 100 µl
of the PCR product were transferred into
each of the GFX columns and pipetted up
and down 5 times. The mix was
centrifuged at full speed for 30 seconds
and the flow through was discarded. The
above procedure was repeated and the
collection tube was discarded. The GFX
columns were placed onto a new 1.5ml
micro centrifuge tubes and 40 µl elution
buffer (10mM Tris-HCl Ph 8.0. TE Ph 8.0)
was pipetted onto the glass fiber matrix in
the GFX column and incubated at room
temperature for 1 min. Centrifugation of
the samples at full speed was then done for
1 min to recover the purified DNA.
PCR product quantification was
done using Quant-iT ™ dsDNA High-
Sensitivity Assay Kit from Invitrogen.
During the routine the samples were
handled carefully to avoid warming. For
quantitative test, 1µl of the PCR product
was mixed with 199µl working solution
which composed of the buffer and
fluorescent dye. The mixture was vortexed
and incubated at room temperature for 3
minutes.
LIGATION WITH PLASMID VECTOR
The PCR products were cloned into
pGEM®-T Easy Vector following
Promega pGEM®-T protocol and
pGEM®-T Easy Vector Systems. The
vector was centrifuged before use. The
ligation mixture was composed of 5µl of
2x buffer, 1 µl pGEM®-T Easy Vector
(50ng), 1 µl of T4 DNA ligase and 3µl of
the PCR product (insert). Reaction
mixtures were mixed thoroughly by
pipetting; incubated at room temperature
for 1 hour and overnight at 4°C for
maximum transformants.
E. COLI TRANSFORMATION
The E. coli transformation was
done following the Protocol Promega
pGEM®-T Easy Vector Systems. Four
LB/ampicillin/IPTG plates were prepared
for the ligation reaction. 15g agar was
added to 1 litre of LB medium and
autoclaved. The medium was allowed to
cool to 50°C; before addition of ampicillin
to 100µg/ml and 0.5mM IPTG and
80µg/ml X-Gal. 35ml of medium was then
poured into 85mm Petri dishes and the
agar was left to harden.
The ligation products were
centrifuged and 5ul of each ligation
reaction was added to sterile 1.5ml micro
centrifuge tubes on ice. Frozen JM109
High Efficiency Competent Cells were just
thawed (about 5 minutes) and the cells
were mixed by gently flicking the tube.
The cells were heat-shocked for 45
seconds in a water bath exactly 42°C and
immediately returned to ice for 2 min.
Following this 950µl of SOC medium was
added to the tubes containing competent
cells and ligation mixture and incubated at
37°C (1.5 h) with shaking (~150rpm).
100µl of each transformation culture was
Nematology, 2011
poured into LB/ampicillin/
plates and glass pellets were used to spread
the innoculum and then incubate
(24 h). White colonies were picked
inoculated in 2 ml LB with ampicillin,
overnight at 37°C, and shak
rpm/min. Following this the
collected by centrifugation in
at 10.000 rpm for 5 min and the
removed and discarded.
PLASMID DNA EXTRACTION FROM
POSITIVE CLONES
The plasmid DNA from the
positive cell was extracted and purifie
following Fermentas Protocol
Plasmid Miniprep Kit. All steps
carried out at room temperature (20
The cells were resuspended in 250
resuspension solution, lysed by 250
Lysis Solution and neutralized using 350
of Neutralization Solution and the tubes
were inverted 6 times after each solution
and centrifuged for 5 minutes
supernatant was loaded to GeneJET™ spin
column and centrifuged for
centrifugations were done at
(~11000rpm). The columns w
centrifuged for 1 min after addition of
500µl of Wash Solution. The flowthrough
was discarded and the same procedure was
repeated. The empty column
centrifuged for 1 min and the
was done using 50µl of Elution Buffer
incubated for 2 min and centrifuged
min.
The presence of the insert
checked by digestion of the recombinant
plasmid with restriction enzyme
releases the insert .The product were
analyzed by resolving on
electrophoresis, together with linearized
vector.
The cloned genes were
sequencing.
SEQUENCING AND PHYLOGENY ANALYSIS
After sequencing, the sequences of
the three isolates were edited
BioEdit 7.13 and orthologs
LB/ampicillin/ IPTG/X-Gal
plates and glass pellets were used to spread
ncubated at 37°C
were picked and
2 ml LB with ampicillin,
, and shaken at 200
Following this the cell were
centrifugation in micro tubes,
at 10.000 rpm for 5 min and the medium
EXTRACTION FROM
The plasmid DNA from the
positive cell was extracted and purified
Fermentas Protocol GeneJET™
All steps were
carried out at room temperature (20°C).
The cells were resuspended in 250µl of
resuspension solution, lysed by 250µl of
Lysis Solution and neutralized using 350µl
of Neutralization Solution and the tubes
6 times after each solution
centrifuged for 5 minutes. The
supernatant was loaded to GeneJET™ spin
column and centrifuged for 1 min. All
centrifugations were done at ≥ 12000 x g
were washed,
centrifuged for 1 min after addition of
The flowthrough
was discarded and the same procedure was
The empty columns were then
and the DNA elution
l of Elution Buffer,
and centrifuged for 2
he presence of the insert was
by digestion of the recombinant
plasmid with restriction enzyme NotI, that
he product were
on agarose gel
with linearized
ere sent for
ENY ANALYSIS
After sequencing, the sequences of
were edited using
of nematode
cellulase were searched in public databases
and checked for significance.
sequences from BLAST search together
with the three isolates sequences w
subjected to clustalW multiple
using BioEdit 7.13 followed by
editing. The phylogenetic tree was
constructed using Mega 5.50
evolutionary distances were computed
using the Kimura 2-parameter method. A
bootstrap analysis based on 1000 replicates
of NJ data was performed.
this, the cellulase genes from
were subjected to BCM
(http://searchlauncher.bcm.tmc.edu/seq
util/Options/sixframe.html
related celullase gene from the
frames. Further, the genes at the vicinity of
cellulase gene were
STRING (Search Tool for the Retrieval of
Interacting Genes/Protein)
Results DNA QUALITATIVE ANALYSIS
As shown in Figure 1, the DNA
greater than 12000bp and
concentration, this is depicted by the faint
bands.
Figure 1. Agarose gel electrophoresis of
genomic DNA extracted from
Bursaphelenchus xylophilus
(DNA ladder), Lane 1(AG), 2(HF), 3(Bm7), 4(20)
and 5(479).
PCR PRODUCT A
QUANTIFICATION
Amplification of the cellulase gene with
ENG00s and ENG00a
single fragment band of 600bp in HF
cellulase were searched in public databases
and checked for significance. Homologous
sequences from BLAST search together
three isolates sequences were then
multiple alignments
followed by manually
. The phylogenetic tree was
g Mega 5.50. The
evolutionary distances were computed
parameter method. A
bootstrap analysis based on 1000 replicates
of NJ data was performed. In addition to
cellulase genes from the isolates
BCM Search Launcher
http://searchlauncher.bcm.tmc.edu/seq-
util/Options/sixframe.html), to note a
related celullase gene from the six reading
genes at the vicinity of
cellulase gene were checked using
Search Tool for the Retrieval of
Interacting Genes/Protein).
NALYSIS
igure 1, the DNA size was
greater than 12000bp and was in low
depicted by the faint
Agarose gel electrophoresis of the
extracted from five
Bursaphelenchus xylophilus isolates. Lane M
1(AG), 2(HF), 3(Bm7), 4(20)
ANALYSIS AND
Amplification of the cellulase gene with
ENG00a primers yielded
single fragment band of 600bp in HF
Nematology, 2011
sample and the other samples were
negative. The results are shown in figure
Figure 2. Agarose gel electrophoresis of
amplification of five cellulase gene from 5 isolates
of B. xylophilus. Lane M (DNA ladder)
1(AG), 2(HF), 3(BM7), 4(20) and 5(479).
The quantification of the PCR product
using Quant-iT ™ dsDNA High
Sensitivity Assay Kit from Invitrogen
as follows: HF (1.56ng), 7C (1.25ng), HB
(0,521ng) and AS (0,396ng).
RECOMBINANT PLASMID ANALYSIS AFTER
DIGESTION WITH Not1
The approximate sizes of
restriction fragments from the rec
plasmids containing the gene of interest
are presented in Figure 2. The vector band
is 3015 bp and the gene of interest in AS,
7C1 and 7C2 is 600bp except that of HF
(500bp).
Figure 3. Agarose gel electrophoresis showing
restriction fragments of the recombinant plasmids
after digestion with Not1, Lane M
Lane 1(AS), 2(HF), 3(7C1), 4(7C2)
PHYLOGENY ANALYSIS
The sequence alignment included
24 sequences, 21 of which were obtained
sample and the other samples were
The results are shown in figure 2.
. Agarose gel electrophoresis of the PCR
five cellulase gene from 5 isolates
M (DNA ladder), Lane
4(20) and 5(479).
of the PCR product
iT ™ dsDNA High-
Sensitivity Assay Kit from Invitrogen were
as follows: HF (1.56ng), 7C (1.25ng), HB
NALYSIS AFTER
The approximate sizes of
restriction fragments from the recombinant
plasmids containing the gene of interest
The vector band
is 3015 bp and the gene of interest in AS,
7C1 and 7C2 is 600bp except that of HF
. Agarose gel electrophoresis showing
restriction fragments of the recombinant plasmids
M (DNA Ladder),
2(HF), 3(7C1), 4(7C2)
equence alignment included
24 sequences, 21 of which were obtained
from public databases by
(shown in Table 1). The phylogen
analysis was inferred from analysis of
cellulase gene and it showed that 7C and
HF are in a clade consisting of Asian
(BxCh-China, BxJT4
Japan, BxKBG-Korea)
isolates (BxPt68ps, BxPt73F2, BxMad3F,
BxMad4sv1, BxPt66F and Bx71TV)
there is 55% bootstrap support.
phylogeny tree is shown in figure
Nevertheless, the bootstrap consensus tree
(figure 5) revealed that the isolate 7C is
closely related to BxPt68
BxOt73F2Por and BxCh.
closely related to BxMad3F Portugal
tree show that our isolates, HF and
a distant away from an isolate from China
(BxJx) , AB179544Bx (most probably
from USA) and USA
isolates which are at the basal of the
xylophilus clade. In
consensus tree (figure 5
clade of BxPt245, BxMad18SCD, BxCh
BxUSA618USA and AB179544Bx is
observed and isolate AS
to Bursaphelenchus mucronatus
Portugal (BmPto) are form the
group. Moreover the BLAST search
reveals that the cellulase gene of
xylophilus is related to fungi
orthologs, Muccor circinelloides
AB175928, Staphylotrichum coccosporum
AB248917 and Gibberella zeae
AY342397 which forms a clade with
cellulase genes of AS and BxPto with 93%
bootstrap support, though it was not
strongly supported in the phylogeneitic
tree with other cellulases from
xylophilus. They are found at the basal
position of the clade.
public databases by BLAST search
. The phylogenetic
analysis was inferred from analysis of
and it showed that 7C and
HF are in a clade consisting of Asian
China, BxJT4-Japan, BxJS10-
Korea) and Portugal
(BxPt68ps, BxPt73F2, BxMad3F,
1, BxPt66F and Bx71TV) and
% bootstrap support. The
ee is shown in figure 4.
ootstrap consensus tree
revealed that the isolate 7C is
closely related to BxPt68PS and also near
BxOt73F2Por and BxCh. However HF is
closely related to BxMad3F Portugal. The
tree show that our isolates, HF and 7C are
an isolate from China
, AB179544Bx (most probably
USA (Bx618, Bx745)
isolates which are at the basal of the B.
In the bootstrap
5), a monophyletic
clade of BxPt245, BxMad18SCD, BxChjx,
BxUSA618USA and AB179544Bx is
AS is closely related
Bursaphelenchus mucronatus from
are form the outer
Moreover the BLAST search
reveals that the cellulase gene of B.
is related to fungi cellulase
Muccor circinelloides
Staphylotrichum coccosporum
Gibberella zeae
which forms a clade with
of AS and BxPto with 93%
though it was not
strongly supported in the phylogeneitic
with other cellulases from B.
are found at the basal
Nematology, 2011
Figure 4. Unrooted phylogenetic trees of different
isolates of Bursaphelenchus xylophilus
orthologs from fungi.
Figure 5. Bootstrap consensus tree
from Bursaphelenchus and fungi
probability (PP) support values are indicat
corresponding nodes, and those supported by
hylogenetic trees of different
Bursaphelenchus xylophilus and
. Bootstrap consensus tree of cellulase
from Bursaphelenchus and fungi Posterior
probability (PP) support values are indicated at
corresponding nodes, and those supported by
bootsrap values higher than 75 % maximum
likelihood showed high similarity.
Table 1. Homologous sequences from Basic Local
Alignment Search Tool using AS, HF and 7C
others from previous sequences provided for
phylogeny analysis.
Isolates
Bursaphelenchus xylophilusBxPt68ps BxPt73F2Portugal
BxCh
BxMad3F
BxMad4sv1
BxJT4
BxJS10
AB179544Bx
BxUSA618
BxPt245Pt
BxMad18SCD
BxPt73FZ
BxPt66F
BxKBG
BxPt24S
BxChJX
BxUSA745
EU6602070
Bx71TV
Fungi Muccor circinelloides AB175928
Staphylotrichum coccosporum
AB248917
Gibberella zeae AY342397
...-unknown origin* Discussion
The comparison
sequences with various database
homologous sequence revealed similarities
of our sequences to known cellulase genes
of Bursaphelenchus xylophilus
fungi species. The results suggest that 7C
and HF together with other Portugal
isolates within the clade of Asian
probably originated from Asian
as they are far less related to the isolates
from USA which occupied the basal
position in the
monophyletic group. This gives a blue
print that Asian isolates have originated
bootsrap values higher than 75 % maximum
likelihood showed high similarity.
. Homologous sequences from Basic Local
Alignment Search Tool using AS, HF and 7C and
previous sequences provided for
Origin
Bursaphelenchus xylophilus
Portugal Portugal
China
Portugal
Portugal
Japan
Japan
..
USA
Portugal
Portugal
Portugal
Portugal
Korea
Portugal
China
USA
Europe
Portugal
AB175928 Japan
Staphylotrichum coccosporum Japan
AY342397 ..
omparison of the isolates
with various database
revealed similarities
to known cellulase genes
Bursaphelenchus xylophilus and other
The results suggest that 7C
together with other Portugal
within the clade of Asian isolates
probably originated from Asian countries
as they are far less related to the isolates
from USA which occupied the basal
sition in the Bursaphenchus
monophyletic group. This gives a blue
print that Asian isolates have originated
Nematology, 2011
from USA. However the best way to track
the origin or evaluation of genetic diversity
of B. xylophilus is the molecular analysis
of the rDNA region including the 18S and
5.8S coding regions and the noncoding
ITS-1 and ITS-2 regions as they proved to
be helpful (Irdani, 2000; Kanzaki & Futai,
2002) and the region is used for diagnostic
purposes (Braasch et al., 1995; Iwahori et
al., 2000; Liao et al., 2001; Kang et al.,
2004; Matsunaga & Togashi, 2004; Cao et
al., 2005; Takeuchi et al., 2005).
To investigate the evolutionary
relationships between the Bursaphelenchus
cellulase and the representative fungi
cellulase orthologs from the database, a
neighbour-joining tree of the cellulase
gene family was constructed. Remarkably,
the GHF45 endoglucanases from
Bursaphelenchus xylophilus show the
highest homology to fungal sequences.
This suggests that, B. xylophilus cellulase
gene was transferred from fungi through
lateral gene transfer. To support this, B.
xylophilus is a facultative fungal feeder;
share the common niche and this appears
to have evolutionary relevance in support
of say the gene was acquired from fungi
(Kikuchi et al., 2004). Furthermore, in the
vicinity of the cellulase gene there were no
co-transcribed genes after analysis of the
cellulase gene with STRING. The absence
of co-transcribed genes with cellulase gene
strongly reinforces the hypothesis of the
acquisition of nematode GH45 cellulases
via lateral gene transfer (LGT) from fungi.
Remarkably and astonishingly, the
cellulase present with B. xylophilus bears
little resemblance to the cellulases of other
plant parasitic nematodes, the tylenchids
but is most analogous to GHF45 cellulases
from fungi. Combining all this and other
body of evidences (Kikuchi et al., 2004),
strongly suggests that the cellulase gene
was acquired by an ancestor of
Bursaphelenchus by LGT from fungi.
However, the LGT hypothesis should be
handled cautiously since the dilemma is
that there are no strict objective set of laws
to examine whether a given gene was
acquired from another non-related
organism via LGT. Nevertheless, the most
commonly used method to claim this is
that no homologous genes can be found in
other eukaryotes, but exclusively found in
fungi (Mitreva et al., 2009). Acknowledgements I would like to express my gratitude to EC,
Professor Solange Oliviera for the
provision of all necessary materials for the
project and also for carrying some tasks of
the project in our absentia. Sincerest
thanks also are extended to Anna
Alexandra and Marta Laranjo for their
input particularly on bioinformatics.
Finally, I would like to humbly share the
success of this work with my classmates
for the inspiration and unending support.
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