Molecular cloning of a polygalacturonate lyase gene from Xanthomonas campestris pv. campestris and role of the gene product in pathogenicity

Physiological and Molecular Plant Pathology (1989) 35, 113-120

Molecular cloning of a polygalacturonate lyase gene from Xanthomonas campestris pv . campestris and role of the gene product in pathogenicity J . MAXWELL Dowt, DAWN E . MILLIGAN, LEE JAMIESON, CHRISTINE E . BARBER and MICHAEL J . DANIELS Sainsbury Laboratory, John Innes Institute, Colney Lane, „Norwich YR4 7UH, U .K. (Accepted for publication January 1989)

A plasmid, designated pIJ3051, encoding a pectolytic activity from Xanthomonas campestris pv . campestris was identified by screening a genomic library in the non-pectolytic X . campestris pathovars translucens and vesicatoria on nutrient agar plates containing polygalacturonate . The pectolytic activity encoded by the clone was a transeliminase with the same ion-exchange pv . properties and molecular weight as polygalacturonate lyase isozyme I of X ..4campestris campestris . Sub-cloning and Tn5 mutagenesis located the structural gene to a 1 kb region of pIJ3051 . Mutants of X. campestris pv. campestris specifically lacking isozyme I were constructed by marker exchange of negative Tn5 insertions into the chromosome of the wild-type . These mutants were as virulent as the wild type in pathogenicity tests on mature leaves or seedlings of turnip (Brassica campestris) . These results suggest that isozyme I of polygalacturonate lyase is not absolutely necessary for black rot pathogenesis indicating a redundancy amongst the three major isozymes . INTRODUCTION Xanthomonas campestris pv . campestris Pammel (Dowson) is the causal agent of black rot disease of crucifers [22] . This organism produces a number of extracellular enzyme activities including polygalacturonate lyase (PGL), carboxymethylcellulase, amylase and protease [4, 7, 14] all of which may play an essential role in plant tissue maceration . Mutants which are pleiotropically defective in the synthesis or export of these enzymes are non-pathogenic and we have described genes which mediate these processes [4, 7, 21] . We have now begun to establish the role of individual extracellular enzymes in the disease process . This has been achieved for protease and carboxymethylcellulase through the construction of mutants, specifically defective in these activities by marker exchange mutagenesis using the cloned structural genes [9, 19] . Comparison of the virulence of these mutants with that of the wild type in a number of different plant tests has then allowed the contribution of the particular enzyme to pathogenesis to be j''ho whom correspondence should be addressed . Abbreviations used in text : PGL, polygalacturonate lyase ; PGA, polygalacturonic acid ; FPLC, fast protein liquid chromatography ; CTAB, cetyltrimethylammonium bromide ; MMX, Xanthomonas minimal medium ; NYGA, peptone, yeast extract, glycerol agar . 0885-5765/89/080113+08 $03 .00/0

© 1989 Academic Press Limited



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assessed . In this paper we report the cloning of the structural gene for one of the three major isozymes of PGL which we have previously described in X.c. campestris [7] and an analysis of the role of this enzyme in black rot pathogenesis .

MATERIALS AND METHODS

Organisms and growth conditions The Xanthomonas campestris pathovars used in this work are listed in Table 1 . Growth in liquid culture was in minimal medium (MMX medium) [7], and on plates on peptone, yeast extract, glycerol agar (NYGA medium) [5] . For induction of PGL, neutralized polygalacturonic acid (PGA, Sigma) was added to MMX medium to a final concentration of 0 . 25 % (w/v) . For screening for PGL clones on plates, NYGA medium was supplemented with 0 . 25 % (w/v) PGA .

Library screening and enzyme assays Details of the construction of the genomic library in pLAFR I are given elsewhere [4] . Pectolytic colonies on NYGA-PGA plates were revealed by staining with ruthenium TABLE I

Bacteria and plasmids

Strain or plasmid

Genotype or relevant characteristics

Source or reference

Bacteria X.c . pv . campestris

8004 8415 8416

8417 8418 8419

rif-14

[4]

8004 : :Tn5 mutants derived by marker exchange of Tn5 insertions 73 and 718 respectively in pIJ3053, PGL I' 8004 : : Tn5 mutants derived by marker exchange of Tn5 insertions 14, 512 and 95 respectively in pIJ3053, PGL I-

This work

This work

X.c . pv . vesicaloria

2593 2593R

Rif'

[NCPPB] This work

Spc'

[1]

recA met

[12] [21]

X.c . pv . translucens

XT02 E. coli

ED8767 PCT800 Plasmids pLAFRI pLAFR3 pKT230 pIJ3051 pIJ3053

As ED8767 but : : Tn5 Tc' Derivative of pLAFRI, Tc'

[8] [18]

[2]

Library clone, PGL' Sub-clone of pIJ3051, PGL'

This work This work



Molecular cloning of âpolygalacturonate lyase gene

115

red (0 . 05 % w/v) after washing the colonies off the plate with 1 °% (w/v) CTAB . Positive colonies showed a yellow zone of clearing on a magenta background . Spectrophotometric measurements of PGL activity were made by following the change in absorbance at 235 nm using PGA as substrate as described previously [7] . One unit of activity causes the liberation of 0 . 5 µmole of unsaturated product per minute .

Genetic methods Antibiotic selection, plasmid transfer, Tn5 mutagenesis and other genetic techniques have been described previously [4, 20] . Restriction enzymes were used according to the manufacturer's instructions and DNA fragments generated by digestion were separated on a 0 . 8 %, agarose gel . Ligations were done as described by Maniatis et al . [13] .

Other analytical methods PGL enzymes from culture filtrates were prepared for FPLC and analysed by ionexchange chromatography on a mono S column as described previously [7] . SDSpolyacrylamide gel electrophoresis was performed on 11 % separation gels as described previously [7] . Culture filtrates were concentrated by addition of ammonium sulphate to 80 °i(, saturation .

Plant tests Pathogenicity tests on turnip (Brassica campestris) using needle-inoculated seedlings and infiltrated leaves were as described by Daniels et al . [5] . Inoculations by seed soaking and at mature leaf margins were as described by Gough et al . [9] .

RESULTS

Isolation of a pectolytic clone from a genomic library of X .c . pv . campestris The genomic library of X.c . pv . campestris constructed in pLAFR1 and maintained in E . coli ED8767 (Table 1) was originally screened for PGL clones by mating into the non-pectolytic X.c . pv . translucens . A positive colony, harbouring the recombinant plasmid designated pIJ3051, showed a zone of clearing on NYGA-PGA plates after ruthenium red staining . The expression of this putative cloned PGL gene was tested in a number of other X . campestris pathovars which were non-pectolytic on plate tests . Higher activity (as judged by larger zones of clearing) was seen in X.c . pv . vesicatoria strain 2593R, which is a rifampicin-resistant mutant of strain 2593 (Table 1) and subsequent work on the characterization of the gene was carried out using expression in this particular pathovar . ED8767/pIJ3051 showed no pectolytic activity even after lysis of the cells after exposure to chloroform vapour [19] .

Characterization of the cloned gene product Culture filtrates of 2593R and 2593R/pIJ3051 grown in MMX medium were assayed for PGL spectrophotometrically . Wherease 2593R/pIJ3051 showed an increase in absorbance at 235 nm consistent with the action of a PGL, 2593R showed no such activity . SDS-polyacrylamide gel electrophoresis showed a protein band of molecular weight 33 kD in the culture filtrate of 2593R/pIJ3051 which was not present in 2593



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J . M . Dow et al.

29-

1

2

3

4

5

6

FIG . 1 . SDS-Polyacrylamide gel electrophoresis of the PGL encoded by pIJ3051 and comparison with the PGL isozymes of X.c . pv . campestris . Lane I : Culture filtrate of X.c . pv . aesicatoria strain

2593R ; lane 2 : culture filtrate of 2593R/pIJ3051 ; lane 3 : PGL purified from the culture filtrate of 2593R/p1J3051 ; lanes 4, 5 and 6 : PGL isozyme fractions I, II and III respectively of X.c . pv. campestris . Molecular mass standards (in kD) are indicated on the left .

(Fig . 1) . The culture filtrate of 2593R/pIJ3051 was fractionated by ion-exchange FPLC as described previously for the PGL isozymes of the wild-type . The PGL activity eluted from the column as a single peak ; SDS-polyacrylamide gel electrophoresis of the most active fraction showed a single protein band of molecular mass 33 kD (Fig . 1) . The PGL encoded by pIJ3051 thus had the same molecular mass and ion-exchange properties as isozyme I of wild-type X.c. pv . campestris . The PGL activity of strain 2593R/pIJ3051 showed only slight induction by PGA . PGL levels were twofold higher after 24 h growth in MMX medium containing PGA compared with MMX alone . Sub-cloning and transposon mutagenesis of p1J3051

Digestion of pIJ3051 with BamH 1 gave fragments of approximate size 11 . 1, 7 . 0, 6. 1 and 3 . 7 kb in addition to a large fragment of 23 . 3 kb containing the vector moiety . These insert fragments were separately cloned into pLAFR3 (Table 1) cut with the same enzyme . The recombinant plasmid containing the 11 . 1 kb fragment was PGL positive on plate tests and was designated pIJ3053 . 2593R/pIJ3053 gave similar PGL activity to 2593R/pIJ3051 in MMX medium, but the activity was no longer inducible by PGA . The location of the PGL gene on pIJ3053 was established by transposon mutagenesis with Tn5 . The PGL activity of 28 transconjugants containing Tn5 at different mapped locations within pIJ3053 were tested both by the plate test and spectrophotometrically . All the insertions giving a PGL - phenotype mapped to a



Molecular cloning of a polygalacturonate lyase gene 73

95 14

7 8

512

b



117

,

1 kb FIG . 2 . Restriction map of p1J3053 showing positions of Tn5 insertions . V, Insertions which do not lead to a loss of PGL activity ; V, insertions which lead to a loss of PGL activity . The numbered insertions are those which were marker exchanged into the chromosome of X.c . pv . campestris to generate specific mutants for pathogenicity testing . Restriction sites for EcoRl(R), BamH l (B), Sall (S) and Hindl l l (H) are shown . Thick lines represent vector (pLAFR3) sequences .

region flanked by two positive insertions 1 .4 kb apart (Fig . 2) . All of the negative Tn5 insertions and one of the flanking positive insertions were in a 2 .04 kb EcoRl fragment . This fragment was cloned into the EcoRl site of pKT230 to give pIJ3054 . This subclone was PGL - although the 11 . 1 kb BamHI fragment cloned into the BamH 1 site of pKT230 was PGL+ . This suggests that the PGL gene has an EcoRI site within it located close to one end of the gene . Marker exchange of

Tn5

insertions

Three different PGL - Tn5 insertions (14 512 and 95, Fig . 2) were introduced into the wild type X.c . pv . campestris genome by marker exchange as described by Turner et al . [21] to give strains 8417, 8418 and 8419 . These marker exchange mutants were still PGL + ; fractionation of the PGL activity from the culture filtrates of these marker exchange mutants showed the presence of isozymes II and III but the absence of isozyme I (Fig . 3a, b) . Induction of PGL activity by PGA was unaffected in the mutants . As a control for pathogenicity testing the two positive insertions flanking the negative region (73 and 718, Fig . 2) were also introduced into the genome of X.c . pv . campestris to give strains 8415 and 8416 . Pathogenicity testing

The marker exchange mutants specifically lacking PGL isozyme I were compared with the wild type strain 8004 and positive marker exchange mutants in pathogenicity tests on turnip leaves and seedlings . In direct inoculation tests into seedlings, no differences in the rate or extent of disease symptom development were seen between the mutants and the wild type inoculated at 10 4 or 103 bacteria per seedling . Similar progression of disease symptoms with all the tested strains were also seen after inoculation of seedlings by a seed soaking method . No differences in the appearance of symptoms were seen when mutants and wildtype were inoculated directly into mature leaves at bacterial concentrations from 105 to 10 8 bacteria ml - ' . Inoculation of mature leaves through the leaf margins via notches in the vein endings also failed to reveal differences between the PGL isozyme I - mutants and the wild type or PGL isozyme I + mutants in the rate of spread of symptoms ; at all initial concentrations of bacteria from 10 5 to 10 8 ml - ', the chlorosis symptoms induced by 8417, 8418 and 8419 spread from the leaf margins as rapidly as those induced by 8415, 8416 or the wild type .



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A

(a)

60

E

0 .2

C

0 •I 20

10

20

30

40

30

0 •I 10

10

20

30

40

Fraction no . FIG . 3 . Ion-exchange FPLC separation on mono S of PGL isozymes from culture filtrates . Q-Q, PGL activity ; -, absorbance at 280 nm . Fractionations shown are of: (a) 8004 (wild type) ; (b) 8417, representative PGL isozyme I-minus mutant .



Molecular cloning of a polygalacturonate lyase gene

119

DISCUSSION

It is probable that the recombinant plasmid pIJ3051 from the X.C . pv . campestris genomic library carries the structural gene encoding PGL isozyme I ; the clone directs the synthesis of a single PGL activity with the same molecular weight and ion-exchange properties as this isoform and marker exchange mutants of X.c . pv . campestris specifically lack this isozyme . As far as we are aware this is the first report of the cloning of a gene for a pectolytic enzyme from Xanthomonas . The role in pathogenesis of pectic enzymes produced by phytopathogenic bacteria has been most widely studied in the genus Erwinia [6, 10] ; relatively little is known of their role in other bacterial genera . Marker exchange mutants of different strains of Erwinia chrysanthemi lacking specific PGL isozymes have been described [3, 15, 16] . Pathogenicity tests with these mutants have shown that simultaneous production of three of the five PGL isozymes of strain 3937 (isozymes a, d and e) seems to be necessary for soft-rot pathogenesis . The other two isozymes (b and c) are not apparently necessary . Similar results were seen with E . chrysanthemi strain CUCPB 1237 [16], where mutants specifically lacking isozymes b or c show no loss in ability to macerate potato tuber tissue compared to the wild type . A marker exchange mutant of E . carotovora subsp . carotovora specifically lacking an endopolygalacturonase activity has been described [23] ; the mutant was as virulent as the parent strain . The major polygalacturonase of Pseudomonas solanacearum has been characterized as an endo-polygalacturonase and strains specifically lacking this enzyme have been constructed by marker exchange mutagenesis [17] . In this case the mutants had reduced virulence on tomato plants. Similarly an extracellular pectate lyase has been implicated as a major pathogenicity factor of the soft rotting bacterium Pseudomonas viridiflava through analysis of the pathogenicity of transposon insertion mutants [11] . However the mutants of Xanthomonas described here (which specifically lack PGL isozyme I) appear to be equally virulent as the wild type in a number of different pathogenicity tests . These results suggest that PGL isozyme I is not absolutely required for pathogenesis . These are similar conclusions to those reached for pectate lyases b and c of Erwinia chrysanthemi [3] . The Sainsbury Laboratory is supported by a grant from the Gatsby Foundation . This work was also supported by the Agricultural and Food Research Council via a Grantin Aid to the John Innes Institute, and was carried out according to the provisions of MAFF Licence No . PHF1185/8(48) issued under the Plant Health (Great Britain) Order, 1987 (statutory instrument 1758) .

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pathovar

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with plasmid

DANIELS, M . J . DNA . Journal

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of General

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3.

(1981) . Specific-purpose plasmid cloning vectors II . Broad host range, high copy number, RSF1010derived vectors, and a host-vector system for gene cloning in Pseudomonas . Gene 16, 237-247 . BOCCARA, M ., DIOLEZ, A ., ROUVE, M . & KOTOUJANSKY, A. (1988) . The role of individual pectate lyases of Erwinia chrysanthemi strain 3937 in pathogenicity on saintpaulia plants . Physiological and Molecular Plant Pathology 33, 95-104 .



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E ., TURNER, P . C ., SAWCZYC, M . K ., BYRDE, R . J . W . & FIELDING, A . H . (1984) . Cloning of genes involved in pathogenicity of Xanthomonas campestris pv campestris using the broad host range cosmid pLAFR L EMBO Journal 3, 3323-3328 . 5 . DANIELS, M . J ., BARBER, C. E ., TURNER, P. C ., CLEARY, W . G . & SAWCZYC, M . K . (1984) . Isolation of mutants of Xanthomonas campestris pv campestris showing altered pathogenicity . Journal of General Microbiology 130, 2447-2455 . 6 . DANIELS, M . J ., Dow, J. M . & OSBOURN, A. E . (1988) . Pathogenicity genes in phytopathogenic bacteria . Annual Reviews of Phytopathology 26, 285-312 . 7 . Dow, J . M ., SCOFIELD, G ., TRAFFORD, K ., TURNER, P. C. & DANIELS, M . J . (1987) . A gene cluster in Xanthomonas campestris pv campestris required for pathogenicity controls the excretion of polygalacturonate lyase and other enzymes . Physiological and Molecular Plant Pathology 31, 261-271 . 8 . FRIEDMAN, A . M ., LONG, S. R ., BROWN, S. E ., BUIKEMA, W . J . & AUSUBEL, F. (1982) . Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium . Gene 18, 289-296 . 9 . GOUGH, C . L ., Dow, J . M ., BARBER, C . E . & DANIELS, M . J . (1988) . Cloning of two endoglucanase genes from Xanthomonas campestris pv campestris : role of the major enzyme in pathogenesis . Molecular Plant Microbe Interactions 1, 275-281 10. KOTOUJANSKY, A . (1985) . Molecular genetics of pathogenesis by soft-rot erwinias . Annual Review of Phytopathology 25, 405-430 . 11 . LIAO, C .-H ., HUNG, H .-Y. & CHATTERJEE, A. K . (1988) . An extracellular pectate lyase is the pathogenicity factor of the soft-rotting bacterium Pseudomonas viridfava . Molecular Plant Microbe Interactions 1, 199-206 . 12 . MURRAY, N . E ., BRAMMAR, W . J . & MURRAY, K . (1977) . Lambdoid phages that simplify the recovery of in vitro recombinants. Molecular and General Genetics 150, 53-61 . 13 . MANIATIS, T ., FRITSCH, E. F . & SAMBROOK, J . (1982) . Molecular Cloning : A Laboratory Manual . Cold Spring Haber Press, New York . 14 . NASUNO, S . & STARR, M . P. (1967) . Polygalacturonic acid trans-eliminase of Xanthomonas campestris . Biochemical journal 104, 178-185 . 15 . RIED, J . L . & COLLMER, A . (1988) . Construction and characterization of an Erwinia chrysanthemi mutant with directed deletions in all of the pectate lyase structural genes . Molecular Plant Microbe Interactions 1, 32-38 . 16 . ROEDER, D . L . & COLLMER, A . (1985) . Marker-exchange mutagenesis of a pectate lyase isozyme gene in Erwinia chrysanthemi . Journal of Bacteriology 164, 51-56 . 17 SCHELL, M . A ., ROBERTS, D . P. & DENNY, T . P. (1988) . Analysis of the Pseudomonas solanacearum polygalacturonase encoded by pg1A and its involvement in phytopathogenicity . Journal of Bacteriology 170, 4501-4508 . 18 . STASKAWICZ, B ., DAHBECK, D ., KEEN, N . & NAPOLI, C . (1987) . Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv . glycinea. Journal of Bacteriology 169, 5789-5794 . 19 . TANG, J . L ., GOUGH, C . L ., BARBER, C . E ., Dow, J . M . & DANIELS, M . J . (1987) . Molecular cloning of protease gene(s) from Xanthomonas campestris pv campestris : Expression in Escherichia coli and role in pathogenicity. Molecular and General Genetics 210, 443-448 . 20. TURNER, P ., BARBER, C . E . & DANIELS, M . J. (1984) . Behaviour of the transposons Tn5 and Tn7 in Xanthomonas campestris pv campestris. Molecular and General Genetics 195, 101-107 . 21 . TURNER, P ., BARBER, C . E . & DANIELS, M . J . (1985) . Evidence for clustered pathogenicity genes in Xanthomonas campestris pv campestris . Molecular and General Genetics 199, 338-343 . 22 . WILLIAMS, P. H. (1980) . Black rot : a continuing threat to world crucifers . Plant Disease 64, 736-742 . 23 . WILLIS, J . W ., ENGWALL, J . K. & CHATTERJEE, A . K. (1987) . Cloning of genes for Erwinia carotovora subsp . carotovora pectolytic enzymes and further characterization of the polygalacturonases . Phytopathology 77, 1199-1205 . 4 . DANIELS, M . J ., BARBER, C .