Molecular genetics of Pseudomonas syringae pathovar pisi: plasmid involvement in cultivar-specific...

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Journal of General Microbiology (l99l), 137, 223 1-2239. Printed in Great Britain 223 1

Molecular genetics of Pseudomonas syringae pathovar pisi: plasmid involvement in cultivar-specific incompatibility

ADRIAN D. BAVAGE, '~ ALAN VIVIAN,'* GRAHAM T. ATHERTON,'$ JOHN D. TAYLOR~ and AFSHAN N. MALIK' tj

Science Department, Bristol Polytechnic, Coldharbour Lane, Frenchay, Bristol BS16 1 Q Y, UK 2 Horticultural Research International, Wellesbourne, Warwick CV35 9EF, UK

(Received 13 March 1991 ; revised 9 May 1991 ; accepted 3 June 1991)

A mutant (PF24) of the race 1 strain, 2WA, of Pseudomonas syringrre pv.pisi has been characterized in terms of its interactions with pea (Pisum satiuum) cultivars. The mutant showed a changed reaction (avirulence to virulence) with a gruup of pea cultivars, including cvs. Belinda and Puget, previously thought to contain resistance genes R1 and R3. Avirulence towards cv. Puget was restored by transfer of any one of five cosmid clones from a race 3 (strain 870A) gene library to a rifampicin-resistant derivative of PF24. These observations were in agreement with a revised race-specific resistance genotype for Belinda and similar cultivars comprising a single resistance gene, R3. An incompatible interaction was observed between strain PF24 and cvs. Vinco (postulated to harbour race-specific resistance genes R1, R2, R 3 and R5) and Hurst's Greenshaft (R4 and possibly Rl), indicating that the mutant retains at least one avirulence gene (A1 or A1 and A4). Mutant PF24 showed loss of a cryptic plasmid (pAV212) compared with its progenitor, strain 299A. A subclone (pAV233) of one of the race 3 restoration clones showed strong hybridization with similar-sized digestion fragments in race 3 plasmid DNA, confirming the A3 gene to be plasmid-borne. Strong cross-hybridization was also observed with a single 3.27 kb EcoRI fragment of plasmid DNA present in strain 2WA but absent from strain PF24. This is consistent with the corresponding A3 determinant being located on pAV212 in the race 1 strain 2WA. The novel avirulence gene corresponding to A3 in strain 870A is provisionally designated aurPpi3. A spontaneous race-change variant (strain 1759, which expressed no avirulence phenotype toward the pea differential cultivars) was derived from the race 3 strain 870A. This race 6 strain and a wild isolate of a race 6 strain both lacked plasmid DNA sequences corresponding to the insert in pAV233.

Introduction

Pseudomonas syringae pathovar pisi is the cause of bacterial blight of pea (Pisum sativum). On the basis of cultivar specificity six races of the pathogen have been identified (Taylor et al., 1989). This specificity is postulated to reside in recognition between host and pathogen involving single resistance genes in the host matching with single avirulence genes in the pathogen (Flor, 1956). This gene-for-gene theory envisages the outcome of host-pathogen interactions being either compatible or incompatible. In a compatible interaction

t Present address: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.

$ Present address: Gene Regulation Group, Paterson Institute for Cancer Research, Christie Hospital & Holt Radium Institute, Wilmslow Road, Manchester M20 9BX, UK. 0 Present address: Department of Biochemistry, University of Wales

College of Cardiff, PO Box 903, Cardiff CFI IST, UK.

disease symptoms develop. In an incompatible interaction host defence mechanisms are induced, observed as a hypersensitive response (Klement, 1963) and absence of disease symptoms.

The race 1 strain 299A of P . s. pv. pisi harbours two cryptic plasmids, designated pAV212 and pAV213 (Malik, 1985). Transposon mutagenesis of the race 1 strain 299A using the Tn.5 vector pSUP2011 (Simon et al., 1983) generated three classes of mutants, showing altered virulence (Malik et al., 1987). The precise genetic basis of these mutants was unclear. They were thought, in most cases, to be probably due to insertion of part or all of the RP4 derivative present in the Escherichia coli host harbouring pSUP2011 (strain SM10). Class I11 mutants arose with a relatively high frequency, and about 40% of them showed apparent loss or rearrangement of the resident cryptic plasmids. One mutant, designated PF24, appeared to hate lost the larger of the two plasmids (pAV212) present in strain 299A (Malik et al., 1987).

0001-6819 O 1991 SGM


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Table 1. Gene-for-gene relationship between pea cultivars and races of Pseudornonas syringae pu. pisi, conjirmed by genetic analysis

Based on Taylor et al. (1989) and unpublished data of J. R. Bevan, J. D. Taylor and P. J. Hunter. + , Susceptible; -, resistant; ?, no evidence for presence or absence of gene. A full stop indicates that a resistance or avirulence gene is absent.

Race . . . 1 2 3 4 5 6 7 PF24

1 ? 1 2 3

4 4 ? cultivar Resistance (R) genes 5 ?

Avirulence (A) genes Differential

_________________ ~

Kelvedon Wonder Early Onward Belinda Hurst's Greenshaft Partridge Sleaford Triumph Vinco Fortune

+ + . 2 . . . +

+ . . 3 . . - + ? . . 4 . - + ? . 3 4 . -

? 2 - 4 ? - - 1 2 3 . 5 - - ? 2 3 4 * - -

. . . . . -

The differential interactions observed between pea cultivars and races of P. s. pv. pisi permit prediction of the genotypes of both host and pathogen, assuming a series of five matched gene pairs for host resistance and pathogen avirulence (Taylor et al., 1989). A recent revision of the predicted genotypes for pathogen races and host cultivars is shown in Table 1. In this scheme race 1 strains of P. s. pv. pisi are thought to contain one or more expressed avirulence genes, designated A 1, A3 and A4. Here we describe the loss of an avirulence function associated with strain PF24, that can be restored by cloned D N A from a race 3 strain.

Methods

Bacterial strains and plasmids. These are listed in Table 2.

Confirmation oj'the identity of P . s. pv. pisi isolates. Tests to confirm the identity of mutant strain PF24 involved the detection of heat-stable antigen specific for P. s. pv. pisi and were performed as described previously (Taylor, 1972a; Taylor & Dye 1972).

Growth conditions. Strains of P . s. pv. phi were grown at 25 "C in aerated nutrient broth (NB; Oxoid no. 1) or on King's medium B (King et al., 1954). Where discrete colonies in close proximity to one another were required, cells were plated on nutrient agar, containing, g I-' : NB, 13; Difco Bacto Agar, 15. Where elimination of auxotrophic E. coli strains from mixtures was required, cells were plated on Minimal Salts medium (Clowes & Hayes, 1968), containing, g 1 - I : NH,CI, 20; NH,N03, 4; Na2S04 (anhydrous), 8; K2HP04, 12; KH2P04, 4; MgSO,. 7H20, 0.4; Difco Bacto Agar, 15.

Strains of E. coli were grown at 37 "C in aerated Luria-Bertani broth (LB), containing, g I - I : Difco Bacto Tryptone, 15; Oxoid Yeast Extract, 5; NaCI, 5; or on Luria-Bertani agar (LB with NaCl increased to 10 g I - ' and added Difco Bacto Agar, 15 g I-').

Antibiotics, freshly prepared as solutions in sterile water (except where indicated), were added to media at the following final concentrations for all bacteria, except where indicated (pg ml-I) : ampicillin, 125 (E. coli); chloramphenicol (dissolved in 0.1 volume ethanol and diluted in water, 80 mg ml-I), 40; kanamycin sulphate, 50; rifampicin (dissolved in methanol), 100; tetracycline hydrochloride, 10.

Gene library construction and use. This was as described previously by Vivian et al. (1 989).

Bacterial matings. Filter matings were done as described by Malik et al. (1987). Triparental matings were done by replica-plating as described by Vivian et al. (1989).

Plasmid D N A isolation. Plasmid DNA from E. coli was isolated by the method of Clewell & Helinski (1969). This method was modified to isolate plasmid DNA from P . s. pv. pisi, as follows. Cells (cultured in aerated NB, approx. 16 h) were harvested in sterile 250 ml pots at 5000 r.p.m., 4"C, 10 min. The supernatant was decanted and the cells resuspended, on ice, in 3.75 ml Tris-sucrose (50 mM-Tris/HCl pH 8.0, 25%, w/v, sucrose). The suspension was transferred to a sterile 50 ml polypropylene tube and 0.5 ml lysozyme (1 5 mg ml-1 in 0.25 M-EDTA pH 8.0) added. After 5 min on ice 3.75 mlO.25 M-EDTA was added and the tubes returned to ice for 5 min. The cells were lysed by the addition of 5 ml Brij-doc solution ( 1 %, w/v, Brij-58,0.4% sodium deoxycholate, 10 mM Tris/HCl pH 8.0). The tube contents were mixed by a single inversion on each addition. After 15 rnin on ice the lysate was cleared by centrifugation at 15000 r.p.m. for 45 rnin at 4 "C. Plasmid DNA was isolated from the cleared lysate by CsCl density-gradient centrifugation.

Southern transfer and hybridization. Separation of DNA fragments by agarose gel electrophoresis was as described by Maniatis et al. (1982). DNA was blotted to Hybond-N membranes (Amersham), which were used according to the manufacturer's instructions. Filters were washed twice in 2 x SSC (1 5 min) followed by 0.1 % (w/v) SDS in 2 x SSC (30 min) at 65 "C (1 x SSC is 0.15 M-NaCI, 0.015 M-triSodiUm citrate, pH 7.0). High stringency was obtained by a further wash in 0-1 % SSC for 10 min at 65 "C prior to autoradiography using Kodak X-OMAT X-ray film at - 70 "C with intensifying screens (Cronex Lightning Plus).


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Table 2. Bacterial strains and plasmids ~~~ ~~~ ~

Strain or plasmid Relevant properties Re ference/source

P. syringae pv. pisi 299A PF24 PF304 202 203 1441 870A 9748 1704B 1759

E. coli 5-53 SMlO 803

Plasmids pAV2 12 pAV213 pLAFR3 pRK2013 RP4 RP4-2-TC : MU pSUP2011 pAV233

pAV270 pAV271 pAV272 pAV275 pAV277 pAV278 pAV279 pAV280

Race 1, wild-type; derivatives prefixed PF Path- class I11 (Kmr) As PF24, but RIP Race 2 wild-type Race 2 wild-type Race 2 wild-type Race 3 wild-type Race 5 wild-type Race 6 wild-type Race 6 spontaneous from 870A

Pro- Met- Thr- Leu- Thi-; RP4-2-Tc : : Mu in chromosome recA Met-

Cryptic, approx. 54 kb plasmid in 299A Cryptic, approx. 50 kb plasmid in 299A

Taylor ( 1972 b) 299A; Malik et al. (1987) PF24

J

Clowes & Hayes (1968) Simon et al. (1983) Murray et al. (1977)

This paper This paper

Tra- Mob+ Tcr/RK2 replicon Tra+ Km'/ColE 1 replicon Apr Kmr Tc' Km' Tra+ Ap5 due to ISRZ in Tn l Apr Kmr Cmr Tn5/pBR325 replicon Tcr; 6.5 kb Hind111 junction fragmenl

Tcr; P. s. pv. pisi race 2 library clone Tcr; P. s. pv. pisi race 3 library clone Tcr; P. s. pv. pisi race 3 library clone Tc'; P. s. pv. pisi race 3 library clone Tcr; P. s. pv. pisi race 3 library clone Tcr; P. s. pv. pisi race 3 library clone Tcr; P. s. pv. pisi race 3 library clone Tc'; P. s. pv. pisi race 3 library clone

pLAFR3 replicon t

Staskawicz et al. (1987) Figurski & Helinski (1979) Datta et al. (1971) Simon et al. (1983) Simon et al. (1983) This paper of pAV277 insert in

Atherton (1987)

This paper 1 J

Pathogenicitj. testing. The stem inoculation procedure was as described by Malik rt al. (1987). Symptoms were recorded 7-14 d after inoculation : 0, null reaction, no water soaking or necrosis; -, no water soaking, obvious necrosis, typical resistant reaction associated with a hypersensitive response (Klement, 1963) in an incompatible interaction; +, compatible interaction, water soaking, no necrosis; inter- mediate reactions involved some water soaking and some necrosis.

Plusmid clrtection. Whole plasmids in P. s. pv. pisi were detected by the method of Eckhardt (1978).

Loss Of'acirulence phenotype in strain PF24

Strain PF24 was isolated from a mating of the race 1 strain 299A with E. coli strain SM10, which harboured the transposon vector pSUP2011. It was one of a number of mutants which showed reduced virulence in their interaction with pea cv. Early Onward and was designated as a class 111 Path- mutant (Malik et al., 1987). Testing of strain PF24 on a range of pea cultivars revealed a change of reaction from that of strain 299A. On cultivars postulated by Taylor et al. (1989) to contain

race-specific resistance genes R1 and R3, strain PF24 showed a compatible interaction whereas strain 299A was incompatible (Table 3). Assuming the loss of a single avirulence gene in PF24, these cultivars, previously thought to harbour R1 and R3, must harbour a single resistance gene, probably R3. Further analysis in the host has shown that these cultivars are unlikely to harbour more than one resistance gene. In crosses between cv. Kelvedon Wonder and cv. Belinda, segregation of resistance to races 1 and 3 was never observed (J . R. Bevan, personal communication). Consequently, incompatibility between strain 299A and cultivars of the group typified by Belinda, which includes Puget, resides in the specific matching of resistance gene R3 with an avirulence determinant A3. This avirulence gene has apparently been lost or inactivated in the creation of strain PF24. The behaviour of strain PF24 and wild-type P . s. pv. pisi strains with a range of cultivars suggests that PF24 retains at least one functional avirulence gene. Both strain 299A and strain PF24 exhibit incompatible interactions with cultivars Fortune, Hurst's Greenshaft and Vinco. This incompatibility suggested that strain PF24 retains A1 or A1 and A4 (Table 3). We therefore


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Table 3 . Reactions of race I strain 299A and the mutant strain PF24 on a range of pea cultivars

Pea seedlings were stem-inoculated as described in Methods and scored after further growth (7-1Od) in a controlled-environment glasshouse. For PF24 some necrosis typically accompanied all compatible reactions. +, Compatible; -, incompatible.

299A PF24 Proposed

Cultivar/No. genotype* Reaction nlrt Reaction nlrt

Kelvedon Wonder Ps88000 1

Belinda Ps870959

Jade Ps86000 1

Shasta Ps870094

English Maple Ps87098 1

Puget Ps870964

Early Onward Ps880002

Alderman P s8 6002 3

Martus Ps860004

Mars P s8 60 204

Sprite Ps860002

Meteor Ps8600 1 4

Sleaford Triumph Ps87094 1

Vinco Ps870927

Hurst's Greenshaft Ps870953

Partridge JDT 860

Fortune Ps870964

N o R genes

R3

R3

R3

R3

R3

R2

R2

R2

R2

R2

R2

?R1, R2, R4, ?R5

R1, R2, R3, R5

?R1, R4

?R1, R3, R4

?R1, R2, R3, R4

+ + + + + + + + + + + + + -

~~ ~ ~~ ~~~~ ~~~~

* See Table 1 ; ? before a resistance gene indicates a lack of evidence for presence or absence of that gene. No. of plants showing typical reaction as a fraction of the total tested.

concluded that the loss of avirulence in PF24 was probably due to the loss (or inactivation) of A3.

pAV271, 275, 277, 278 and 280) consistently resulted in the restoration of avirulence (Table 4).

Restoration of'phenotype in strain PF24 Role y f ' plasmids

An avirulence gene (A3) matched with pea resistance gene R3 is postulated to be the only functional avirulence gene in the race 3 strain 870A (Taylor et al., 1989). A strain 870A gene library (Atherton, 1987) was therefore introduced into strain PF24 in an attempt to restore avirulence towards cv. Puget. A total of 1100 cosmids were independently transferred into a rifampicin-resistant derivative of PF24 (PF304) and inoculated into pea cv. Puget (R3). Five of these cosmids (designated

Examination of the above cosmids digested with EcoRI revealed co-migrating bands of approx. 5.9 (except in pAV278), 5.6 (except in pAV277), 2.69 and 0.84 kb. These bands corresponded in size to fragments of similarly digested plasmid DNA from the race 3 strain 870A (Fig. 1 a) , suggesting that these cosmids contained 870A plasmid DNA. To investigate this further, digested plasmid DNAs from race 1 and race 3 strains together with the restoring cosmids described above were probed


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Fig. 1 . Relationships between DNA preparations of P. s. pv. pisi strains and race 3 cosmid clones. (a) Plasmid DNA was isolated and digested with EcoRI as described in Methods, and separated on a 0.5% (w/v) agarose gel run in Tris/acetate buffer at 40 V, 16 h. (b) Southern blot of (a) probed with 32P-labelled pAV271 at high stringency as described in Methods. Lanes: 1, PF24; 2,299A; 3, 1759; 4, 870A: 5 , pAV271; 6, pAV275; 7, pAV277; 8, pAV278; 9, pAV280; 10, pLAFR3; 11, pAV270; 12, 1441; 13, Iz standard (HindIII and HindIII + EcoRI mixture).

Table 4. Restoration of avirulence phenotype towards pea cu. Puget after introduction of race 3 library cosmids into

P . s. pu. pisi strain PF304

Seedlings (7 d) of pea cv. Puget (R3) were stem-inoculated with strains 299A, 870A, PF304 and PF304 harbouring cosmids from a race 3 (870A) library as described in Methods. Plant reactions were recorded after growth (14 d) in a contained glasshouse environment. Some degree of necrosis was associated with all interactions observed in this experiment. A typical hypersensitive response associated with an incompatible interaction was recorded as - . An indication of the variability observed is shown by the number of plants exhibiting a compatible reaction.

Strain No.

No. of Cosmid plants

designation tested

No. of compatible reactions Compatibility

299A 810A PF304 PF304 PF304 PF304 PF304 PF304 PF304 PF304

None None None pAV211 pAV272 pAV275 pAV277 pAV278 pAV279 pAV280

7 7 7

26 25 13 15 16 14 16

0 0 6 1

21 1 0 3

10 1

with radiolabelled pAV271 , a cosmid which restored avirulence to PF24. Strong hybridization to several common-sized bands in the clones (Fig. 16, lanes 5-9) and plasmid DNA of strain 870A (lane 4) was observed. There was also strong hybridization with a number of fragments in digested 299A (lane 2) and PF24 (lane I) plasmid DNAs. A number of hybridizing fragments in strain 299A were absent in strain PF24. Similarly, several cross-hybridizing fragments in strain 870A (lane 4) were absent from strain 1759 (lane 3), a spontaneous variant, subsequently identified as a race 6 strain (no expressed avirulence genes) which arose from strain 870A in culture (Taylor et al., 1989).

In order to identify the specific region which restored avirulence to PF24, each cosmid was subcloned in pLAFR3. One subclone, pAV233, obtained by digestion of pAV277 with HindIII followed by religation, harboured approximately 6.5 kb of strain 870A DNA and retained the ability to restore avirulence to PF304 on cv. Puget. Strong hybridization was observed between pAV233 and three EcoRI fragments (5-6, 2.69 and 0.84 kb) present in pAV275 (Fig. 2, lane lo), each of which was also common to the cosmids pAV271,


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Fig. 2. Determination of common DNA fragments associated with avirulence phenotype conferred by race 3 cosmid clones. (a) Plasmid DNA was isolated and digested with EcoRI as described in Methods, and separated on a 0.5% (w/v) agarose gel run in Tris/acetate buffer at 40 V, 16 h. (b) Southern blot of (a) probed with 32P-labelled pAV233 at high stringency. Lanes: 1,1 standard (HindIII and Hind111 + EcoRI mixture); 2,299A; 3, PF24; 4,203; 5,1441 ; 6, genomic 870A; 7,974B; 8, 1704B; 9, 1759; 10, pAV275; 1 1 , pAV277; 12, pAV233; 13, pAV270; 14, 1 standard.

pAV278 and pAV280 (Fig. 1, lanes 5,8,9). In pAV277, a unique EcoRI band of 1.90 kb (also present in pAV233) replaced the 5.6 kb band (a faint band of hybridization at about 5.6 kb in pAV277 is probably a partial digestion product); we conclude that the 1.90 kb band is a junction fragment abutting the vector in both pAV277 and pAV233 (Figs 1 and.2). The three EcoRI bands were present and hybridized with plasmid DNA in race 3 strain 870A, although only the larger two bands were detectable in genomic DNA of 870A (Fig. 2). Plasmid DNA from the race 6 strain 1759 did not include these fragments (Fig. 2, lane 9). Strong cross-hybridization with pAV233 was limited to a 3-27 kb EcoRI fragment of strain 299A plasmid DNA. This fragment was absent from the plasmid DNA of strain PF24 (Fig. 2, lanes 2 and 3). Plasmid DNA from races 1,2,5 and 6 was probed at high stringency with pAV233 ; strong cross-hybridization was only observed in a single strain of race 1 and race 2 (Fig. 2, lanes 2 and 4).

Both pAV27 1 and pAV233 probes cross-hybridized with the cosmid clone pAV270 (Fig. 1, lane 11 ; Fig. 2, lane 13), which harbours an avirulence determinant, A2, isolated from the race 2 strain 203 (Vivian et al., 1989). The basis of this cross-hybridization is not apparent and is being further investigated.

The genetic basis of’ mutant PF24

Malik (1985) showed that strain PF24 had apparently lost one of two cryptic plasmids present in strain 299A.

Further confirmation of this observation was aided by analysis of the restriction fragments generated by digestion of plasmid DNA from strain 299A and strain PF24 (Table 5) . Two restriction enzymes were employed, EcoRI and HindIII. For both enzymes, no DNA fragments were detected in PF24 that were not also present in 299A (Table 5) . The absence of sequences corresponding to pAV212 in PF24 was confirmed by probing EcoRI-digested, genomic DNA from strains PF24 and 299A with plasmid DNA from strain 299A. The strain PF24 DNA failed to show hybridization of bands corresponding to pAV212 which were present in strain 299A DNA (Fig. 3). It has not been possible to attempt the restoration of PF24 with pAV212 due to the lack of any selective markers, and attempts to introduce Tn5 into pAV212 have been unsuccessful.

As noted, the precise event(s) responsible for the observed phenotype of strain PF24 is (are) unclear. It was possible that some of the class I11 mutants derived from strain 299A retained part or all of the RP4-2- Tc : : Mu, as discussed by Malik et al. (1987). Plasmid and genomic DNAs from P. s. pv. pisi strains PF24 and 299A, and E. coli strain SMlO, harbouring pSUP2011 (Simon et al., 1983) were digested with EcoRI. The resultant digests were blotted and independently probed with 32P-labelled pSUP2011 and RP4. Positive hybridization was observed between RP4 and pSUP2011, in both total and plasmid DNA from strain SM lO(pSUP2011). Probing with 32P-labelled pSUP2011 showed strong hybridization with pSUP2011 DNA and


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Table 5 . Fragments produced from digestion of plasmid DNA in strains of P . s. pv. pisi

These results were the product of several independent digestions of purified plasmid DNA (see Methods). Fragments of 1 kb or less were not visible in all digestions. Fragments: +, present; -, absent; ND, not detected.

Strain no. . . . Race . . .

29919 PF24 870A 1759 1 l(m) 3 6

299A PF24 1 1 (m)

EcoRI (kb)

* 24 19.3* 12.9 12.3 9.1 7.6 7.0 6-5 5.9 5.6 5.3 5.2 5.0 4*36* 3-79 3.70 3.27 3.16 2.69 2.45 2.36 2.24 2.19 2.02 1*74* 1.55 1.45 1 *36 1.16 1-06* 0-99 0.84 0.75 0.56

< 0.3

- + + + + + + + + + + + + + + + + + + +

-

-

-

-

-

-

-

-

- - + + + + -

15.8 13.5 11.0 9.4* 8.1 * 6.6 5.9 5.3 5.1 2.95 2-63 2.04 1.33 0.85

+ + + + + + + + + + + + + +

+ + + -

+ -

+ + + + + + + + + + +

-

-

+ +

- + + + -

- +

+ + + -

ND ND ND ND ND

- + + ND

- + + ND

* These bands represent two co-migrating fragments in strain 299A.

with similar-sized fragments in digested total DNA from strain SM lO(pSUP2011). Neither probe cross-hybridized to either total or plasmid DNA from strains PF24 or 299A (data not shown). These results indicated that retention of an insertion of part either of the RP4 derivative or of pSUP2011 was not responsible for the phenotype of strain PF24.

was incompatible with cv. Belinda, strain PF24 was compatible. Avirulence towards cultivars of the Belinda group was restored by functional complementation after transfer of any one of five cosmid clones carrying plasmid-derived sequences from the race 3 strain 870A. A subclone, pAV233, obtained by digestion and religation of the library cosmid pAV277, retained the ability to functionally complement PF24. This permitted the identification of three EcoRI bands (5.6, 2-69 and 0.84 kb) which probably contain the avirulence gene.

Taylor et al. (1989) postulated that cultivars such as Belinda harbour two resistance genes, R1 and R3. The interactions of races 1, 3, 4 and mutant PF24 indicate that the R-gene responsible for the observed reactions in

Discussion In this paper we describe the characteristics of a mutant, PF24, whose loss of a cryptic plasmid, pAV212, appeared to correlate with a change in host cultivar specificity. Whereas the progenitor race 1 strain 299A


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Fig. 3. Total and plasmid DNA preparations of P. s. pv. pisi strains blotted and probed with plasmid DNA of race 1 strain 299A. (a) Total and plasmid DNA from strains 299A and PF24 were digested with EcoRI and separated on a 0.5% (w/v) agarose gel in Tris/acetate buffer run at 40 V for 16 h. (b) Southern blot of (a) probed with 32P-labelled plasmid (pAV212 and pAV213) DNA from strain 299A at high stringency as described in Methods. Lanes: 1 , 299A total; 2, PF24 total; 3, 299A plasmid; 4, A standard (HindIII and HindIII + EcoRI mixture); 5, PF24 plasmid.

these cultivars must be R3 (Taylor et al., 1989; Table 3). This has been further supported by genetical studies with the host ( J . R. Bevan, personal communication).

The novel avirulence gene, designated avrPpi3, responsible for the race specificity of strain 870A, must have a functionally equivalent, possibly allelic, gene present in the race 1 strain 299A but which has been lost in strain PF24. The clone pAV233 cross-hybridized strongly to a single fragment of 3.27 kb in EcoRI- digested plasmid DNA from strain 299A. This fragment was derived from pAV212 and was absent in plasmid DNA from strain PF24, and therefore is the probable location of the counterpart of avrPpi3 in race 1.

At least one further avirulence gene in strain 299A is retained in strain PF24 and it is therefore concluded that this gene is not located on pAV212. It is not possible to tell whether this avirulence is due to A1 or A1 and A4 from the resistance gene combinations available in the present cultivar lines (the interaction with cv. Vinco carrying R1, R2, R3 and R5 excludes the possibility that A4 alone is present in PF24).

Strain 299A has an estimated total plasmid DNA content of - 107 kb (EcoRI) and 108 kb (HindIII) based on the respective digests shown in Table 5. This is in reasonably good agreement with the approximate sizes for pAV212 (54 kb) and pAV213 (50 kb) estimated from whole plasmid visualization (Malik, 1985). Since no DNA fragments were detected in PF24 which were not also present in 299A, it is probable that pAV213 is retained without substantial deletion in PF24. The fragments which comprise each plasmid cannot be ascertained with absolute certainty because it is difficult to resolve the presence of putative double molarity fragments in PF24. Deletion of either pAV212 or pAV213, associated with the generation of PF24 and consistent with the absence of specific restriction fragments, could only occur if the putative deletions were coincident with both HindIII and EcoRI recognition sites. We therefore conclude that the balance of evidence favours the loss of an entire pAV212 plasmid and the retention of pAV213 in PF24.

The events leading to the creation of mutant PF24 are uncertain. We have demonstrated that it is unlikely any part of Tn5 or the E. coliltransposon vector system, SMlO(pSUP2011), remains in PF24. I t remains a possibility that all or part of the RP4 derivative in SMlO or of pSUP2011 may have been responsible for a deletion or rearrangement of chromosomal DNA in strain PF24 in addition to the plasmid loss. Incompatibility between a transferred RP4 derivative and pAV212 remains a likely possibility for the origin of PF24, with subsequent loss of the RP4-2-Tc : :Mu.

Tamaki et al. (1988) reported that an avirulence gene from P . s. pv. glycinea (avrC) was plasmid-borne in race 0. Subsequently aurD from P. s. pv. tomato, together with similar genes present in all races of P. s. pv. glycinea, were shown to be located on large indigenous plasmids (Keen et al., 1989). Further analysis showed that avrD was present on an approx. 75 kb plasmid in P . s. pv. tomato (Kobayashi et al., 1990).

In Xanthomonas campestris pv. vesicatoria, avrBsl was located on a plasmid pXvCu 1 , that also conferred copper resistance on race 2 strains (Stall et al., 1986; Swanson et al., 1988).

Taylor et al. (1989) reported the spontaneous deriva- tion of a race 6 strain from the race 3 strain 870A. Clearly such a strain could arise from the loss of the plasmid carrying avrPpi3 in 870A. Examination of plasmid DNA and hybridization with pAV233 probe suggest that a loss of plasmid DNA including regions homologous to pAV233 (which harbours avrPpi3 specificity) has oc- curred in this strain.

The demonstration here that avr genes in P. s. pv. pisi may be located on a plasmid, as is the case with avrPpi3, has important implications for the evolution of novel


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Cultivar-spec@ avirulence and plasmids 2239

races of phytopathogenic pseudomonads. It also indicates the potentially important role of plasmids in controlling the interactions of pathogen races and host cultivars.

In summary, we have described the discovery of a novel avirulence gene, corresponding to A3 in the model proposed by Taylor et al. (1989). This gene, designated avrPpi3, is postulated to interact with a single host resistance gene R3. It should be possible to confirm the precise gene-for-gene nature of the interaction postulated to occur here between avrPpi3 and R3, by sequential inoculations with 299A, PF24 and 870A in the F2 progeny from crosses showing segregation of R3.

This work was supported by grants to A.V. and J.D.T. from the AFRC. This work was carried out under MAFF licences numbers PHF678/65(38), PHF678/66(38) and PHF428A/42(96) issued under the Plant Pests (Great Britain) Order 1980.

References ATHERTON, G. T. ( 1987). The genetics ofpathogenicity and host speciJicity

of’ Pseudomonas qvringae pathouar pisi. PhD thesis, CNAA, Bristol Polytechnic.

CLEWELL, D. B. & HELINSKI, D. R. (1969). Supercoiled circular protein-DNA complex in Escherichia coli: purification and induced conversion to an open circular DNA form. Proceedings of’ the National Academj. of’ Sciences of’ the United States of’ America 62,

CLOWES, R. C. & HAYES, W. ( 1 968). E.uperiments in Microbial Genetics. Oxford & Edinburgh : Blackwell Scientific Publications.

DATTA, N., HEDGES, R. W., SHAW, E. J., SYKES, R. B. &RICHMOND, M. H. (1971). Properties of an R factor from Pseudomonas aeruginosa. Journal 41’ Bacteriologj- 108, 1244- 1249.

ECKHARDT, T. (1978). A rapid method for the identification of plasmid DNA in bacteria. Plasmid 1, 584-588.

FIGURSKI, D. H. & HELINSKI, D. R. (1 979). Replication of an origin- containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proceedings of’ the National Academy of’ Sciences of’ the United States yf’ America 76, 1648- 1652.

FLOR, H. H. (1956). The complementary genetic systems in flax and flax rust. Adrances in Genetics 8, 29-54.

KEEN, N. T., TAMAKI, S., KOBAYASHI, D., STAYTON, M., GERHOLD, D., SHEN, H., GOLD, S., LORANG, J. & THORDAL-CHRISTENSEN, H. (1989). Characterization and function of avirulence genes from Pseudomonas syringae pv. tomato. In Signal Molecules in Plants and Plant-Microbe Interactions (NATO AS1 series, vol. H36), pp. 183- 188. Edited by B. J. J . Lugtenberg. Berlin & Heidelberg: Springer- Verlag.

1159-1 166.

KING, E. O., WARD, M. K. & RANEY, D. E. (1954). Two simple media for the demonstration of pyocyanin and fluorescin. Journal of’ Laboratory and Clinical Medicine 44, 301-337.

KLEMENT, Z. (1963). Rapid detection of pathogenicity of phytopathogenic Pseudomonads. Nature. London 199, 299-300.

KOBAYASHI, D. Y., TAMAKI, S. J. & KEEN, N. T. (1990). Molecular characterization of avirulence gene D from Pseudomonas syringae pv. tomato. Molecular Plant-Microbe Interactions 3, 94- 102.

MALIK, A. N. (1985). Genetic studies with Pseudomonas syringae pathovar pisi. PhD thesis, CNAA, Thames Polytechnic.

MALIK, A. N., VIVIAN, A. &TAYLOR, J. D. (1987). Isolation and partial characterization of three classes of mutant in Pseudomonas syringae pathovar pisi with altered behaviour towards their host, Pisum sativum. Journal of‘ General Microbiology 133, 2393-2399.

MANIATIS, T., FRITSCH, E. F. & SAMBROOK, J. (1982). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

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.

SIMON, R., PRIEFER, U. & PUHLER, A. (1983). A broad host range mobilization system for in vitro genetic engineering : transposon mutagenesis in Gram negative bacteria. BiolTechnology 1, 784-789.

STALL, R. E., LOSCHKE, D. C. & JONES, J . B. (1986). Linkage of copper resistance and avirulence loci on a self-transmissible plasmid in Xanthomonas campestris pv. vesicatoria. Phytopathology 76, 240-243.

STASKAWICZ, B., DAHLBECK, 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.

SWANSON, J., KEARNEY, B., DAHLBECK, D. & STASKAWICZ, B. (1988). Cloned avirulence gene of Xanthomonas campestris pv. cesicatoria complements spontaneous race-change mutants. Molecular Plant- Microbe Interactions 1, 5-9.

TAMAKI, S., DAHLBECK, D., STASKAWICZ, B. & KEEN, N. T. (1988). Characterization and expression of two avirulence genes cloned from Pseudomonas syringae pv. glycinea. Journal of’Bacteriology 170,4846- 4854.

TAYLOR, J. D. (1972~). Specificity of bacteriophages and antiserum for Pseudomonas pisi. New Zealand Journal of’ Agricultural Research 15,

TAYLOR, J. D. (19726). Races of Pseudomonas pisi and sources of resistance in field and garden peas. New Zealand Journal of’ Agricultural Research 15, 441 -447.

TAYLOR, J. D. & DYE, D. W. (1972). A survey of the organisms associated with bacterial blight of peas. New Zealand Journal of’ Agricultural Research 15, 432-440.

TAYLOR, J. D., BEVAN, J. R., CRUTE, I. R. & READER, S. L. (1989). Gene tic relationship between races of Pseudomonas syringae pat ho- var pisi and cultivars of Pisum sativum. Plant Pathology 38, 364-375.

VIVIAN, A., ATHERTON, G . T., BEVAN, J. R., CRUTE, I. R., MUR, L. A. J. & TAYLOR, J. D. (1989). Isolation and characterization of cloned DNA conferring specific avirulence in Pseudomonas syringae pv. pisi to pea (Pisum sativum) cultivars, which possess the resistance allele, R2. Physiological and Molecular Plant Pathology 34, 335-344.

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