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.

References

BELLAFIORE, S., SHEN, Z., ROSSO, M.N.,

ABAD, P., SHIH, P. & BRIGGS, S.

(2008). Direct identification of the

Meloidogyne incognita secretome

reveals proteins with host cell

reprogramming potential. PLoS

Pathogens 4, e1000192.

BRAASCH, H., BURGERMEISTER, W. &

PASTRIK, K.H. (1995).

Differentiation of three

Bursaphelenchus species by means

of RAPD-PCR. Nach-richtenbl.

Deut. Pflantzenschutzd 47, 310–

314.

CAO, A.X., LIU, X.Z., ZHU, S.F. & LU, B.S.

(2005). Detection of the pinewood

nematode, Bursaphelenchus

xylophilus, using a real-time

polymerase chain reaction assay.

Phytopathology 95, 566–571.

DANCHIN, E.G., ROSSO, M.N., VIEIRA, P.,

DE ALMEIDA-ENGLER, J.,

Nematology, 2011

COUTINHO, P.M., HENRISSAT, B.,

ABAD, P. (2010) Multiple lateral

gene transfers and duplications

have promoted plant parasitism

ability in nematodes. Proceedings

of the National Academy of

Sciences of the U S A 107, 17651–

17656.

DE BOER, J., YAN, Y., WANG, X., SMANT,

G., HUSSEY, R., DAVIS, E. & BAUM,

T. (1999). Mol. Plant-

Developmental expression of

secretory beta-1, 4- endoglucanes

in the sub ventral esophagous

glands of Heterodera glycines.

Microbe Interactions 12, 663-669.

DE BOER, J.M., YAN, Y., WANG, X.,

SMANT, G., HUSSEY, R.S., DAVIS,

E.L. & BAUM, T.J. (1999).

Developmental expression of

secretory beta-1, 4-endoglucanases

in the subventral oesophageal

glands of Heterodera glycines.

Molecular Plant-Microbe

Interactions 12, 663– 669.

DE MEUTTER, J., VANHOLME, B., BAUW,

G., TYTGAT, T., GHEYSEN, G. &

GHEYSEN, G. (2001). Preparation

and sequencing of secreted proteins

from the pharyngeal glands of the

plant parasitic nematode

Heterodera schachtii. Molecular

Plant Pathology 2, 297–301.

GAO, B., ALLEN, R., DAVIS, E.L., BAUM,

T.J. & HUSSEY, R.S. (2004b).

Molecular characterisation and

developmental expression of a

cellulose-binding protein gene in

the soybean cyst nematode

Heterodera glycines. International

Journal of Parasitology 34, 1377–

1383.

GAO, B., ALLEN, R., MAIER, T., DAVIS,

E.L., BAUM, T.J., HUSSEY, R.S.

(2003). The parasitome of the

phytonematode Heterodera

glycines. Molecular Plant-Microbe

Interaction 16, 720–726.

GOELLNER, M., SMANT, G., DE BOER, J.M.,

BAUM, T.J. & DAVIS, E.L. (2000).

Isolation of beta-1, 4-

endoglucanase genes from

Globodera tabacum and their

expression during parasitism.

Journal of Dermatology 32, 154–

165.

GOELLNER, M., WANG, X. H., & DAVIS,

E.L. (2001). Endo-β-1, 4-glucanase

expression in compatible plant–

nematode interactions. Plant Cell

13, 2241-2255.

HAEGEMAN, A., JACOB, J., VANHOLME, B.,

KYNDT, T. & GHEYSEN, G. (2008).

A family of GHF5 endo-1, 4-beta-

glucanases in the migratory plant-

parasitic nematode Radopholus

similis. Plant Pathology. 57, 581–

590.

HAEGEMAN, A., JOSEPH, S. & GHEYSEN, G.

(2011a). Analysis of the

transcriptome of the root lesion

nematode Pratylenchus coffeae

generated by 454 sequencing

technology. Molecular and

Biochemistry Parasitology 178, 7–

14.

HENRISSAT, B., & BAIROCH, B. (1993).

New families in the classification

of glycosyl hydrolases based on

amino acid sequence similarities.

Biochemistry Journal 293, 781–

788 (1993).

IRDANI, T. (2000). Genetic analysis of

three Bursaphelenchus species by

random amplified polymorphic

DNA. Nematologia Mediterranea

28, 11–120.

IWAHORI, H., KANZAKI, N. & FUTAI, K.

(2000). A simple, polymerase

chain reaction-restriction fragment

length polymorphism-aided

diagnosis method for pine wilts

disease. Forest Pathology 30, 157–

164.

JAUBERT, S., LEDGER, T.N., LAFFAIRE,

J.B., PIOTTE, C., ABAD, P. &

ROSSO, M.N. (2002). Direct

identification of stylet secreted

proteins from root-knot nematodes

by a proteomic approach.

Nematology, 2011

Molecular and Biochemistry

Parasitology 121, 205–211.

JONES, J.T., FURLANETTO, C. & KIKUCHI,

T. (2005). Horizontal gene transfer

from bacteria and fungi as a driving

force in the evolution of plant

parasitism in nematodes.

Nematology 7, 641–646.

KARIM, N., JONES, J.T., OKADA, H. &

KIKUCHI, T. (2009). Analysis of

expressed sequence tags and

identification of genes encoding

cell-wall-degrading enzymes from

the fungivorous nematode

Aphelenchus avenae. BioMed

Central Genomics 10, 525.

KANG, J.S., CHOI, K.S., CHIN, S.C., MOON,

I.S., LEE, S.G. & LEE, S.H. (2004).

Development of an efficient PCR-

based diagnosis protocol for the

identification of the pinewood

nematode, Bursaphelenchus

xylophilus (Nematoda:

Aphelenchoididae) Nematology. 6,

279–285.

KANZAKI, N. & FUTAI, K. (2000). A PCR

primer set for determination of the

phylogenetic relationship of

Bursaphelenchus species within the

xylophilus group. Nematology. 4,

35–41.

KIKUCHI, T., JONES, J.T., AIKAWA, T.,

KOSAKA, H. & OGURA, N. (2004).

A family of GHF45 cellulases from

the pine wood nematode

Bursaphelenchus xylophilus. FEBS

Letters 572, 201-205.

KIKUCHI, T., LI, H., KARIM, N., KENNEDY,

M.W., MOENS, M. & JONES, J.T.

(2009). Identification of putative

expansin-like genes from the pine

wood nematode, Bursaphelenchus

xylophilus, and evolution of the

expansin gene family within the

Nematoda. Nematology 11, 355–

364.

KIKUCHI, T., SHIBUYA, H. & JONES, J.T.

(2005). Molecular and biochemical

characterization of an endo beta-1,

3-glucanase from the pinewood

nematode Bursaphelenchus

xylophilus acquired by horizontal

gene transfer from bacteria.

Biochemistry Journal 389, 117–

125.

KURODA, K. (1989). Terpenoids causing

tracheid-cavitation in Pinus

thunbergii infested by the pine

wood nematode (Bursaphelenchus

xylophilus). Annals of the

Phytopathological Society of Japan

55, 170–178.

LEDGER, T.N., JAUBERT, S., BOSSELUT, N.,

ABAD, P. & ROSSO, M.N. (2006).

Characterization of a new beta-1,

4-endoglucanase gene from the

root-knot nematode Meloidogyne

incognita and evolutionary scheme

for phytonematode family 5

glycosyl hydrolases. Gene 382,

121–128.

LIAO, J.L., ZHANG, L.H., ENG, Z.X. (2001).

Reliable identification of

Bursaphelenchus xylophilus by

rDNA amplification. Nematologia

Mediterranea 29, 131–135.

MAMIYA, Y. & ENDA, N. (1972).

Transmission of Bursaphelenchus

lignicolus (Nematoda:

Aphelenchoididae) by

Monochamus alternatus

(Coleoptera: Cerambycidae).

Nematologica 18, 159–162.

MAMIYA, Y. (1983). Pathology of the pine

with disease caused by

Bursaphelenchus xylophilus.

Annual Reviews of Phytopathology

21, 201–220.

MATSUNAGA, K., & TOGASHI, K. (2004).

Among-tree difference in the

inhibition of systemic dispersal of

Bursaphelenchus xylophilus

(Nema-toda: Aphelenchoididae) by

Pinus densiflora. Applied

Entomology and Zoolology 39,

271–277.

MITREVA, M., SMANT, G. & HELDER, J.

(2009). Role of horizontal gene

transfer in the evolution of plant

parasitism among nematodes. In:

Nematology, 2011

Gogarten MB, Gogarten JP,

Olendzenski LC (eds) Horizontal

gene transfer—Genomes in flux.

Humana Press, New York, pp517–

535.

MOTA, M.M TAKEMOTO, S., TAKEUCHI, Y.,

HARA, N., & FUTAI, K. (2006).

Comparative Studies between

Portuguese and Japanese Isolates of

the Pinewood Nematode,

Bursaphelenchus xylophilus.

Journal of Nematology 38(4), 429–

433.

ODANI, K., SASAKI, S., YAMAMOTO, N.,

NISHIYAM, Y. & TAMURA, H.

(1985). Differences in dispersal

and multiplication of two

associated nematodes,

Bursaphelenchus xylophilus and

Bursaphelenchus mucronatus in

pine seedlings in relation to the

pine wilt disease development.

Journal of Japanese Forestry

Society 67, 398–403.

OPPERMAN, C.H., BIRD, D.M,

WILLIAMSON, V.M., ROKHSAR,

D.S., BURKE, M., COHN, J.,

CROMER, J., DIENER, S., GAJAN, J.,

GRAHAM, S., HOUFEK, T.D., LIU,

Q., MITROS, T., SCHAFF, J.,

SCHAFFER, R., SCHOLL, E.,

SOSINSKI, B.R., THOMAS, V.P. &

WINDHAM, E. (2008). Sequence

and genetic map of Meloidogyne

hapla: a compact nematode

genome for plant parasitism.

Proceedings of the National

Academy of Sciences 105, 14802–

14807.

REHMAN, S., BUTTERBACH, P., POPEIJUS,

H., OVERMARS, H., DAVIS, E.L.,

JONES, J.T., GOVERSE, A.,

BAKKER, J. & SMANT, G. (2009a).

Identification and characterization

of the most abundant cellulases in

stylet secretions from Globodera

rostochiensis. Phytopathology 99,

194–202.

ROSSO, M.N., FAVERY, B., PIOTTE, C.,

ARTHAUD, L., DE BOER, J.M,

HUSSEY, R.S., BAKKER, J., BAUM,

T.J. & ABAD, P. (1999). Isolation

of a cDNA encoding a beta-1, 4-

endoglucanase in the root-knot

nematode Meloidogyne incognita

and expression analysis during

plant parasitism. Molecular Plant-

Microbe Interactions 12, 585–591.

ROZE, E., HANSE, B., MITREVA, M.,

VANHOLME, B., BAKKER, J.,

SMANT, G. (2008). Mining the

secretome of the root-knot

nematode Meloidogyne chitwoodi

for candidate parasitism genes.

Molecular Plant Patholology 9, 1–

10.

SMANT, G., STOKKERMANS, J.P., YAN, Y.,

DE BOER, J.M., BAUM, T.J., WANG,

X., HUSSEY, R.S., GOMMERS, F.J.,

HENRISSAT, B., DAVIS, E.L.,

HELDER, J., SCHOTS, WANG, X.,

MEYERS, D., YAN, Y., BAUM, T.,

SMANT, G., HUSSEY, R. & DAVIS,

E. (1999). In planta localization of

a beta-1, 4-endoglucanase secreted

by Heterodera glycines. Molecular

Plant-Microbe Interactions 12, 64–

67.

SMANT, G., STOKKERMANS, J.P., YAN, Y.,

DE BOER, J.M., BAUM, T.J., WANG,

X., HUSSEY, R.S., GOMMERS, F.J.,

HENRISSAT, B., DAVIS, E.L.,

HELDER, J., SCHOTS, A. & BAKKER,

J. (1998). Endogenous cellulases in

animals: isolation of beta-1, 4-

endoglucanase genes from two

species of plant-parasitic cyst

nematodes. Proceedings of the

National Academy of Sciences of

the U S A 95, 4906–4911.

TAKEUCHI, Y., KANZAKI, N. & FUTAI, K.

(2005). A nested PCR-based

method for detecting the pine wood

nematode, Bursaphelenchys

xylophilus, from pine wood.

Nematology 7, 775–782.

YAN, Y., SMANT, G., STOKKERMANS, J.,

QIN, L., HELDER, J., BAUM, T.,

SCHOTS, A. & DAVIS, E. (1998).

Genomic organization of four beta-

Nematology, 2011

1, 4-endoglucanase genes in plant-

parasitic cyst nematodes and its

evolutionary implications. Gene

220:61–70.

YAN, Y., SMANT, G., DAVIS, E. (2001).

Functional screening yields a new

beta-1, 4-endoglucanase gene from

Heterodera glycines that may be

the product of recent gene

duplication. Molecular Plant-

Microbe 14(1), 63-71. Interactions

14, 63–71.