BACKGROUND
the banana  is one of the world's most popular fruits and is regarded as the fourth most important crop in developing countries  <cit> .
it suffers from several diseases, the most famous being fusarium wilt disease , which is regarded as one of the most significant threats to banana production worldwide  <cit> .
this disease is caused by the fungus fusarium oxysporum f. sp.
cubense  and has been reported in all banana-growing regions of the world, including australia, asia, africa and central and south america  <cit> .
foc has been classified into four physiological races based on pathogenicity to host cultivars in the field.
foc <dig> infects the cultivar gros michel; foc <dig>  'bluggoe'; foc <dig>  heliconia spp.
; and foc <dig>  cavendish cultivars and all cultivars susceptible to foc <dig> and foc <dig>  <cit> .
earlier in the last century, foc <dig> infection nearly destroyed the world's banana industry, which was based on the gros michel cultivar.
consequently, gros michel was replaced by cavendish cultivars, which were resistant to foc <dig>  however, foc <dig>  which is capable of attacking cavendish cultivars, was reported in taiwan and africa in  <dig>  to date, foc <dig> has caused serious crop losses in asia, australia and africa  <cit> .
grimm  predicted that if foc <dig> hits the banana heartland in latin america, it could be game over for banana production in the region  <cit> .
the plant cell wall is a barrier to the penetration and spread of phytopathogenic bacteria and fungi, so many plant pathogens produce extracellular enzymes that can degrade cell wall polymers.
cell wall-degrading enzymes and their genes have been studied for their possible role in many aspects of pathogenicity, including penetration, maceration, nutrient acquisition, plant defense induction, and symptom expression  <cit> .
polygalacturonases  are pectic enzymes that hydrolyze polygalacturonan, and they are the key components of pectinases.
pgs are further classified into endopgs and exopgs, although some enzymes exhibit both endo- and exopg activities  <cit> .
endopgs  cleave the backbone of polygalacturonan internally, whereas exopgs  hydrolyze monomers progressively from the nonreducing end of the substrate.
exopgs have been reported in plant-pathogenic fungi  <cit>  and their role in disease has been studied in the fungal plant pathogens cochliobolus carbonum  <cit>  and fusarium oxysporum f.sp.
lycopersici  <cit> .
exopgs may have an important function in pathogen-plant interactions because they degrade oligogalacturonides released by endopgs to elicitor-inactive monomers  <cit> .
moreover, exopgs are not subject to inhibition by plant polygalacturonase-inhibiting proteins   <cit> .
in this study, we report for the first time the isolation and purification of an exopg  from the supernatant of the plant pathogen foc <dig>  we cloned the pgc <dig> genes of foc <dig> and foc <dig> and then expressed them in p. pastoris.
both recombinant pgc <dig> proteins retained their exopg activity.
further studies of these genes will provide valuable insights into the role of pgc <dig> in foc pathogenicity in banana cultivars.
RESULTS
purification of pgc2
pg activity in the foc <dig> culture supernatant could be detected when the fungi were grown in the presence of citrus pectin.
pgc <dig> was purified from foc <dig> through successive steps of ultra filtration, gel filtration chromatography and cation exchange chromatography.
during the purification process, the specific pg activity increased from  <dig>  to  <dig>  units mg protein- <dig> min- <dig> .
one faint single peak of pg activity was seen after culture was applied to a sephacryl s- <dig> 16/ <dig> gel filtration column.
subjecting the pooled pg fraction to cation exchange chromatography  resulted in a significant single pg peak.
sds-page showed one single protein band and indicated that pgc <dig> was purified to homogeneity .
according to the markers, the molecular weight of pgc <dig> was about  <dig> kda.
a one unit of pg activity was defined as the amount of enzyme that liberated one μmole of ga in one minute at 45°c.
characterization of pgc2
the end products of enzymatic hydrolysis of pga by purified enzyme were analyzed.
galacturonic acid was the only degradation product detected during enzyme activity assays, demonstrating that the purified enzyme is an exopg.
treatment of pgc <dig> with n-glycosidase f reduced the apparent mr of the enzyme from  <dig> kda to  <dig> kda, as determined by sds-page, indicating that pgc <dig> is a glycoprotein with n-linked carbohydrate .
a number of studies have shown that many functional proteins are glycoproteins.
glycosylation has been described in exopg <dig>  <cit>  and pg <dig>  <cit>  from f. oxysporum f.sp.
lycopersici.
the purified pgc <dig> was diluted with milli-q water and filtered through  <dig>  μm pore membranes, then subjected to esi-ms. mass spectrometry of the purified pgc <dig> demonstrated many signals  with a molecular mass of  <dig>  kda.
the result of esi-ms also showed that the purified pgc <dig> may be a glycoprotein, as above.
isolation of genes encoding exopolygalacturonase pgc <dig> from foc <dig> and foc4
the full-length cdna of pgc <dig> from foc <dig> and foc <dig> was cloned by 3'-race and 5'-race.
a full-length  <dig> nucleotide dna sequence was isolated and sequenced.
sequencing revealed the presence of an open reading frame  of  <dig> nucleotides, interrupted by four introns of  <dig>   <dig>   <dig> and  <dig> nucleotides, and encoding a predicted protein of  <dig> amino acids with a predicted mass of  <dig>  kda and a predicted pi of  <dig> .
analysis with signalp v <dig>  detected a putative n-terminal signal peptide sequence of  <dig> amino acids that would produce a mature protein when cleaved.
the complete nucleotide sequences of pgc <dig> genes from foc <dig> and foc <dig> were deposited in the genbank database under accession numbers gu <dig> and gu <dig>
the nucleotide sequences of the pgc <dig> genes from foc <dig> and foc <dig> and the pgx <dig> gene of fol   <cit>  were aligned .
among the three nucleotide sequences, nucleotide variation was detected at  <dig> positions.
the sequence of pgc <dig> from foc <dig> shared as high as  <dig> % and  <dig> % nucleotide sequence identity with pgc <dig> from foc <dig> and the exopg gene of fol, respectively, while the sequence of pgc <dig> from foc <dig> shared  <dig> % nucleotide sequence identity with fol.
all of the nucleotide variations were located in exons.
we noted the following differences between pgc2-foc <dig> and pgc2-foc4: 420-c to g, 690-g to c and 886-g to c.
standard protein-protein blast analysis revealed that both isolated pgc <dig> genes shared a high amino acid sequence identity with exopgs from other fungi.
figure  <dig> shows the multiple alignments for the pgc <dig> amino acid sequences of foc <dig>  foc <dig> and fol .
foc1-pgc <dig> shared the same amino acid sequence with fol and as high as  <dig> % amino acid sequence identity with foc4-pgc <dig>  we noted the following differences between foc1-pgc <dig> and foc4-pgc2: 230-k to n, 296-d to h. among all prosite motifs, we found a single difference between foc1-pgc <dig> and foc4-pgc <dig>  namely, an n-glycosylation site  present in amino acids 230- <dig> of foc4-pgc <dig> but not in foc1-pgc <dig>  in other words, there is no n-glycosylation site of foc1-pgc <dig> at this locus.
whether this variation has an impact on the function of pgc <dig> is still uncertain.
expression and purification of recombinant pgc2
recombinant pgc <dig> proteins from foc <dig> and foc <dig> were expressed in p. pastoris as secreted proteins r-foc1-pgc <dig> and r-foc4-pgc <dig>  culture samples taken at  <dig>   <dig> and  <dig> d post-induction were analyzed by sds-page .
proteins of about  <dig> kda were detected from the r-foc1-pgc <dig> and r-foc4-pgc <dig> transformant cultures, but were not observed in control p. pastoris transformed with ppiczαa vector.
specific pg activity was detected from the r-foc1-pgc <dig> and r-foc4-pgc <dig> transformant cultures but not from control p. pastoris transformed with ppiczαa vector.
both recombinant pgc2s were purified through successive steps of 2so <dig> precipitation and gel filtration chromatography.
during the purification process, the specific pg activity of r-foc1-pgc <dig> and r-foc4-pgc <dig> increased from  <dig>  to  <dig>  units and from  <dig> to  <dig>  units, respectively.
sds-page showed one single protein band for each, indicating that r-foc1-pgc <dig> and r-foc4-pgc <dig> were purified to homogeneity .
according to the markers, the molecular weight of both recombinant proteins was about  <dig> kda.
biochemical characterization of recombinant pgc2
the optimal ph and temperature for pg activity of the recombinant pgc <dig> proteins were investigated.
both enzymes exhibited the highest activity at ph  <dig>   and at 50°c .
the ph value and temperature optima for the pg activity from pg <dig> of fol were reported to be  <dig>  and 55°c  <cit>  and the temperature optimum for the pg activity from pg <dig> of fol was 55°c  <cit> .
to estimate the ph stability of recombinant pgc <dig>  samples were incubated in buffers of different ph values at 4°c for  <dig> h and the remaining pg activity was assayed.
both recombinant pgc <dig> retained > 70% activity at ph 3- <dig> .
to investigate the thermostability of recombinant pgc <dig>  both enzymes were incubated at different temperatures in  <dig> mm potassium phosphate buffer, ph  <dig>  for  <dig> h., and then residual activity was determined.
both recombinant pgc <dig> retained > 50% activity at 10-50°c .
for hydrolysis of polygalacturonic acid  at ph  <dig>  and 45°c, the km and vmax of purified r-foc1-pgc <dig> were  <dig>  mg.ml- <dig> and  <dig>  units mg protein- <dig> min- <dig>  respectively .
the km and vmax of purified r-foc4-pgc <dig> were  <dig>  mg.ml- <dig> and  <dig>  units mg protein- <dig> min- <dig>  respectively .
active recombinant pgc <dig> causes tissue maceration and necrosis
purified r-foc1-pgc <dig> and r-foc4-pgc <dig> proteins were inoculated onto banana tissues to examine their ability to macerate tissue.
sterilized banana tissues were inoculated with  <dig> unit of r-foc1-pgc <dig> or r-foc4-pgc <dig> mixed with  <dig> ml of  <dig> mm sodium acetate buffer ph  <dig> , and maceration was evaluated after  <dig> h. the maceration activity of r-foc1-pgc <dig> to guangfen- <dig>  was higher than that of r-foc4-pgc <dig>  while the maceration activity of r-foc1-pgc <dig> to baxi  was lower than that of r-foc4-pgc <dig>  both exopgs showed higher maceration activity on guangfen- <dig> than baxi .
these same exopgs showed differences in their ability to macerate two banana cultivars.
these results suggest that pectins of guangfen- <dig> and baxi, as an important component of cell walls, might differ in polymer structure because baxi pectin is a poor substrate for pgc <dig> compared to that of guangfen- <dig>
five days after the stems of cavendish cultivar baxi plants were injected with  <dig> unit of r-foc1-pgc <dig> or r-foc4-pgc <dig>  the stem vascular tissues showed partial necrosis .
the r-foc1-pgc <dig> induced less necrosis compared to r-foc4-pgc <dig>  it seems that r-foc1-pgc <dig> had a lower activity than r-foc4-pgc <dig>  sterile double distilled water and  <dig> mm sodium acetate buffer ph  <dig> , used as controls, did not cause any symptoms when injected into stems of the bananas.
CONCLUSIONS
we demonstrated both that the pgc <dig> genes of two races of foc could be expressed in p. pastoris and that the recombinant proteins displayed pg activity.
the two exopgs have similar molecular structures and almost the same enzyme characteristics.
further studies of these gene products will lead to valuable insights into the role of pgc <dig> in foc pathogenicity in banana cultivars.

 the banana is one of the world's most popular fruits and is regarded as the fourth most important crop in developing countries. it suffers from several diseases, the most famous being fusarium wilt disease, which is regarded as one of the most significant threats to banana production worldwide. this disease is caused by the fungus fusarium oxysporum f. sp. cubense and has been reported in all banana-growing regions of the world, including australia, asia, africa and central and south america. foc has been classified into four physiological races based on pathogenicity to host cultivars in the field. foc infects the cultivar gros michel; foc'bluggoe'; foc heliconia spp.; and foc cavendish cultivars and all cultivars susceptible to foc and foc. earlier in the last century, foc infection nearly destroyed the world's banana industry, which was based on the gros michel cultivar. consequently, gros michel was replaced by cavendish cultivars, which were resistant to foc however, foc which is capable of attacking cavendish cultivars, was reported in taiwan and africa in to date, foc has caused serious crop losses in asia, australia and africa. grimm predicted that if foc hits the banana heartland in latin america, it could be game over for banana production in the region. the plant cell wall is a barrier to the penetration and spread of phytopathogenic bacteria and fungi, so many plant pathogens produce extracellular enzymes that can degrade cell wall polymers. cell wall-degrading enzymes and their genes have been studied for their possible role in many aspects of pathogenicity, including penetration, maceration, nutrient acquisition, plant defense induction, and symptom expression. polygalacturonases are pectic enzymes that hydrolyze polygalacturonan, and they are the key components of pectinases. pgs are further classified into endopgs and exopgs, although some enzymes exhibit both endo- and exopg activities. endopgs cleave the backbone of polygalacturonan internally, whereas exopgs hydrolyze monomers progressively from the nonreducing end of the substrate. exopgs have been reported in plant-pathogenic fungi and their role in disease has been studied in the fungal plant pathogens cochliobolus carbonum and fusarium oxysporum f.sp. lycopersici. exopgs may have an important function in pathogen-plant interactions because they degrade oligogalacturonides released by endopgs to elicitor-inactive monomers. moreover, exopgs are not subject to inhibition by plant polygalacturonase-inhibiting proteins. in this study, we report for the first time the isolation and purification of an exopg from the supernatant of the plant pathogen foc we cloned the pgc genes of foc and foc and then expressed them in p. pastoris. both recombinant pgc proteins retained their exopg activity. further studies of these genes will provide valuable insights into the role of pgc in foc pathogenicity in banana cultivars.  purification of pgc2 pg activity in the foc culture supernatant could be detected when the fungi were grown in the presence of citrus pectin. pgc was purified from foc through successive steps of ultra filtration, gel filtration chromatography and cation exchange chromatography. during the purification process, the specific pg activity increased from to units mg protein- min-. one faint single peak of pg activity was seen after culture was applied to a sephacryl s- 16/ gel filtration column. subjecting the pooled pg fraction to cation exchange chromatography resulted in a significant single pg peak. sds-page showed one single protein band and indicated that pgc was purified to homogeneity. according to the markers, the molecular weight of pgc was about kda. a one unit of pg activity was defined as the amount of enzyme that liberated one μmole of ga in one minute at 45°c. characterization of pgc2 the end products of enzymatic hydrolysis of pga by purified enzyme were analyzed. galacturonic acid was the only degradation product detected during enzyme activity assays, demonstrating that the purified enzyme is an exopg. treatment of pgc with n-glycosidase f reduced the apparent mr of the enzyme from kda to kda, as determined by sds-page, indicating that pgc is a glycoprotein with n-linked carbohydrate. a number of studies have shown that many functional proteins are glycoproteins. glycosylation has been described in exopg and pg from f. oxysporum f.sp. lycopersici. the purified pgc was diluted with milli-q water and filtered through μm pore membranes, then subjected to esi-ms. mass spectrometry of the purified pgc demonstrated many signals with a molecular mass of kda. the result of esi-ms also showed that the purified pgc may be a glycoprotein, as above. isolation of genes encoding exopolygalacturonase pgc from foc and foc4 the full-length cdna of pgc from foc and foc was cloned by 3'-race and 5'-race. a full-length nucleotide dna sequence was isolated and sequenced. sequencing revealed the presence of an open reading frame of nucleotides, interrupted by four introns of and nucleotides, and encoding a predicted protein of amino acids with a predicted mass of kda and a predicted pi of. analysis with signalp v detected a putative n-terminal signal peptide sequence of amino acids that would produce a mature protein when cleaved. the complete nucleotide sequences of pgc genes from foc and foc were deposited in the genbank database under accession numbers gu and gu the nucleotide sequences of the pgc genes from foc and foc and the pgx gene of fol were aligned. among the three nucleotide sequences, nucleotide variation was detected at positions. the sequence of pgc from foc shared as high as % and % nucleotide sequence identity with pgc from foc and the exopg gene of fol, respectively, while the sequence of pgc from foc shared % nucleotide sequence identity with fol. all of the nucleotide variations were located in exons. we noted the following differences between pgc2-foc and pgc2-foc4: 420-c to g, 690-g to c and 886-g to c. standard protein-protein blast analysis revealed that both isolated pgc genes shared a high amino acid sequence identity with exopgs from other fungi. figure shows the multiple alignments for the pgc amino acid sequences of foc foc and fol. foc1-pgc shared the same amino acid sequence with fol and as high as % amino acid sequence identity with foc4-pgc we noted the following differences between foc1-pgc and foc4-pgc2: 230-k to n, 296-d to h. among all prosite motifs, we found a single difference between foc1-pgc and foc4-pgc namely, an n-glycosylation site present in amino acids 230- of foc4-pgc but not in foc1-pgc in other words, there is no n-glycosylation site of foc1-pgc at this locus. whether this variation has an impact on the function of pgc is still uncertain. expression and purification of recombinant pgc2 recombinant pgc proteins from foc and foc were expressed in p. pastoris as secreted proteins r-foc1-pgc and r-foc4-pgc culture samples taken at and d post-induction were analyzed by sds-page. proteins of about kda were detected from the r-foc1-pgc and r-foc4-pgc transformant cultures, but were not observed in control p. pastoris transformed with ppiczαa vector. specific pg activity was detected from the r-foc1-pgc and r-foc4-pgc transformant cultures but not from control p. pastoris transformed with ppiczαa vector. both recombinant pgc2s were purified through successive steps of 2so precipitation and gel filtration chromatography. during the purification process, the specific pg activity of r-foc1-pgc and r-foc4-pgc increased from to units and from to units, respectively. sds-page showed one single protein band for each, indicating that r-foc1-pgc and r-foc4-pgc were purified to homogeneity. according to the markers, the molecular weight of both recombinant proteins was about kda. biochemical characterization of recombinant pgc2 the optimal ph and temperature for pg activity of the recombinant pgc proteins were investigated. both enzymes exhibited the highest activity at ph and at 50°c. the ph value and temperature optima for the pg activity from pg of fol were reported to be and 55°c and the temperature optimum for the pg activity from pg of fol was 55°c. to estimate the ph stability of recombinant pgc samples were incubated in buffers of different ph values at 4°c for h and the remaining pg activity was assayed. both recombinant pgc retained > 70% activity at ph 3-. to investigate the thermostability of recombinant pgc both enzymes were incubated at different temperatures in mm potassium phosphate buffer, ph for h., and then residual activity was determined. both recombinant pgc retained > 50% activity at 10-50°c. for hydrolysis of polygalacturonic acid at ph and 45°c, the km and vmax of purified r-foc1-pgc were mg.ml- and units mg protein- min- respectively. the km and vmax of purified r-foc4-pgc were mg.ml- and units mg protein- min- respectively. active recombinant pgc causes tissue maceration and necrosis purified r-foc1-pgc and r-foc4-pgc proteins were inoculated onto banana tissues to examine their ability to macerate tissue. sterilized banana tissues were inoculated with unit of r-foc1-pgc or r-foc4-pgc mixed with ml of mm sodium acetate buffer ph, and maceration was evaluated after h. the maceration activity of r-foc1-pgc to guangfen- was higher than that of r-foc4-pgc while the maceration activity of r-foc1-pgc to baxi was lower than that of r-foc4-pgc both exopgs showed higher maceration activity on guangfen- than baxi. these same exopgs showed differences in their ability to macerate two banana cultivars. these results suggest that pectins of guangfen- and baxi, as an important component of cell walls, might differ in polymer structure because baxi pectin is a poor substrate for pgc compared to that of guangfen- five days after the stems of cavendish cultivar baxi plants were injected with unit of r-foc1-pgc or r-foc4-pgc the stem vascular tissues showed partial necrosis. the r-foc1-pgc induced less necrosis compared to r-foc4-pgc it seems that r-foc1-pgc had a lower activity than r-foc4-pgc sterile double distilled water and mm sodium acetate buffer ph, used as controls, did not cause any symptoms when injected into stems of the bananas.  we demonstrated both that the pgc genes of two races of foc could be expressed in p. pastoris and that the recombinant proteins displayed pg activity. the two exopgs have similar molecular structures and almost the same enzyme characteristics. further studies of these gene products will lead to valuable insights into the role of pgc in foc pathogenicity in banana cultivars. 
cubense race and maitain banana production after gros michel was destructed by race a new race race was found to infect cavendish. pgc hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. pgc had an apparent mr of kda by sds-page and kda by mass spectrometry. collectively, these results suggest that pgc is the first exopg reported from the pathogen foc, and we have shown that fully functional pgc can be produced in the p. pastoris expression system. although cavendish banana cultivars are resistant to fusarium oxysporum f.sp. an exopolygalacturonase was isolated and purified from the supernatant of the plant pathogen fusarium oxysporum f.sp.
 fusarium wilt is an economically devastating disease that affects banana production. although cavendish banana cultivars are resistant to fusarium oxysporum f.sp. cubense race and maitain banana production after gros michel was destructed by race a new race race was found to infect cavendish.  an exopolygalacturonase was isolated and purified from the supernatant of the plant pathogen fusarium oxysporum f.sp. cubense race. pgc had an apparent mr of kda by sds-page and kda by mass spectrometry. the enzyme was n-glycosylated. pgc hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. to obtain adequate amounts of protein for functional studies between the pgc proteins of two races of the pathogen, pgc genes encoding pgc from race and race, both bp in length and encoding amino acids with a predicted amino-terminal signal sequence of residues, were cloned into the expression vector ppiczaa and then expressed in pichia pastoris strains of smd the recombinant pgc products, r-foc1-pgc and r-foc4-pgc were expressed and purified as active extracellular proteins. optimal pgc activity was observed at 50°c and ph the km and vmax values of purified r-foc1-pgc were mg.ml- and units mg protein- min- respectively. the km and vmax values of purified r-foc4-pgc were mg.ml- and units mg protein- min- respectively. both recombinant pgc proteins could induce tissue maceration and necrosis in banana plants.  collectively, these results suggest that pgc is the first exopg reported from the pathogen foc, and we have shown that fully functional pgc can be produced in the p. pastoris expression system. 