- Email: [email protected]
Characterization, growth, and scanning electron microscopy of mutants of Pseudomonas syringae pv. phaseolicola which fail to elicit a hypersensitive response in host and non-host plants
Physiological and Molecular Plant Pathology (1988) 33,443-457
Characterization, growth, and scanning electron microscopy of mutants of Pseudomonas syringae pv . phaseolicola which fail to elicit a hypersensitive response in host and non-host plants M . C . DEASEY
and A .
G . MATTHYSSE
Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280 U .S.A . Accepted, for publication May 1988)
Transposon mutants of Pseudomonas syringae pv . phaseolicola which fail to elicit a hypersensitive response in the non-host plant tobacco were isolated and compared with the parent strain . The mutants also failed to elicit a race-specific hypersensitive response in resistant bean . In susceptible bean the mutants were reduced in virulence on both leaves and pods . Both mutants were reduced in growth in resistant and susceptible bean leaves . However, one of the mutants grew at a rate similar to the parent strain in tobacco ; the growth of the other mutant was reduced . No differences were detected between the parent strain and the mutants grown in bacteriological medium in extracellular polysaccharide, lipopolysaccharide, motility, or surface proteins . When the parent and the mutant bacteria were injected into tobacco or resistant bean leaves and examined in the scanning electron microscope, fibrils were seen surrounding the parent bacteria after 8 h . No such fibrils were observed surrounding one of the mutant bacteria . The number of fibrils surrounding the other mutant was reduced compared to the parent bacteria . No fibrils were produced when the parent or mutant bacteria were injected into susceptible bean leaves . These results suggest that the fibrils may play a role in the hypersensitive response, although it is unclear if the fibrils are required for the development, or are the result, of the hypersensitive response .
INTRODUCTION
the causative agent of halo blight in bean . Like many other phytopathogenic pseudomonads and xanthomonads, P. syringae pv . phaseolicola induces a hypersensitive response (HR) when it is inoculated into a non-host plant such as tobacco . Saprophytic bacteria, symbiotic bacteria and bacteria not normally associated with plants fail to elicit a HR when inoculated in comparable numbers. In order to determine what aspects of the potentially pathogenic bacteria are recognized or required by the non-host plant for the elicitation of the HR, transposon mutants of the bacteria which fail to elicit a HR in the non-host plant Nicotiana tabacum were isolated and characterized . Pseudomonas syringae pv . phaseolicola is
MATERIALS AND METHODS Growth of bacteria and plants Virulent Pseudomonas syringae pv . phaseolicola
strain 19304 was obtained from the American Type Culture Collection . It was grown in Luria [6] or nutrient broth . Abbreviations used in text : HR, hypersensitive response . 0885-5765/88/060443+ 15 $03 .00/0
© 1988 Academic Press Limited
444 M . C . Deasey and A . G . Matthysse Escherichia coli SM10 pSUP1011 was obtained from Dr Paul Bishop, North Carolina State University, and grown in Luria broth with 100 pg ml - ' of chloramphenicol . Nicotiana tabacum variety Coker 319, and Phaseolus vulgaris varieties Top Crop and Red Mexican U134 were grown in the greenhouse . Plants which had been inoculated with bacteria were grown in a growth chamber at 27 °C with a 14 h day . Inoculated bean plants were covered to maintain a relative humidity in excess of 80°0 for the first day after inoculation . Leaves of tobacco and bean plants were inoculated by injecting bacteria into the leaf using a 0 . 5 ml syringe . For experiments in which the growth of the inoculated bacteria in the leaves was measured, the syringe was equipped with a device which allowed the delivery ofa predetermined volume (Tirdak stepper), 5 PI in the case of bean leaves and 50 gl in the case of tobacco leaves . Numbers of viable bacteria were determined by cutting the entire inoculated area out of the leaf, grinding the excised tissue in a Waring blender in phosphate buffered saline (Na 2 HPO, 7 g 1 -1 , KH 2PO,, 3 g 1 - 1 , NaCl 4 g I - ', and MgSO 4 :7 H 2O 0 . 2 g 1 - ' ), and determining the viable cell count on Luria agar plates . Bean pods were purchased at a local supermarket and inoculated by applying a drop of about 50µl of bacterial suspension containing about 10 9 bacterial ml - ' to the pod and making a needle wound through the drop . For experiments testing the virulence (rather than growth) of bacteria on bean leaves, the leaves were wounded with carborundum and 0 . 5 ml of a suspension of 10 9 bacterial ml - ' was pipetted on to the leaf. Scanning electron microscopy Tissue fixation and preparation for scanning electron microscopy were carried out as previously described [2] . Leaf discs coated with gold-pallidium were examined with an ETEC Autoscan scanning electron microscope . Discs were examined from at least three different injection sites on different leaves . Preparation and isolation of mutant bacteria Transposon mutagenesis of P . syringae pv . phaseolicola was carried out by introducing Tn5 from E . coli SM10 pSUP1011 which is a multiple auxotroph and contains Tn5 on the plasmid [18] . This conjugative plasmid is not capable of replicating in Pseudomonas . For bacterial conjugation E. coli and P . syringae pv . phaseolicola were grown to mid-log phase in flasks containing Luria broth on shakers at 37 and 25 ° C respectively . The bacteria were mixed on the surface of a Luria agar plate and incubated overnight at 25 °C . Transconjugants were selected for growth on minimal medium containing 60 µg ml - ' neomycin . Neither parent grew under these conditions . P . syringae pv . phaseolicola transconjugants containing Tn5 were screened for the ability to elicit a hypersensitive response in the non-host plant tobacco by injecting bacteria grown in Nutrient Broth into tobacco leaves . Two of the first 200 transconjugants failed to elicit a HR (Hyr-25 and Hyr-37) . These two mutants were kept for further study . They were resistant to neomycin, but sensitive to chloramphenicol to which pSUP1011 provides resistance . When their plasmid composition was examined by the lysis in the well technique [T], they contained only the indigenous plasmid contained in the parent strain 19304 . Characterization ofparent and mutant bacteria Growth of the parent and mutant bacteria in Luria broth was measured in a Klett photometer with a green filter . Bacterial motility was examined using a light microscope .
P. Phaseolicola HR mutants in leaves
445
Bacterial lipopolysaccharide was extracted as previously described [9] and examined by SDS polyacrylamide gel electrophoresis using the method of Tsai and Frasch [21] . Bacterial surface proteins were extracted by the method of Sonoki & Kado [19] and examined by SDS-PAGE using the method ofLaemli [5] and the silver staining procedure of Merril et al . [10] . Bacterial exopolysaccharides were prepared by the method of El-Banoby & Rudolph [3] . Their size was examined by SDS-PAGE . Bacterial production of phaseolotoxin was examined by the method of Staskowicz & Panopoulos [20] . In order to determine whether the site of Tn5 insertion was the same in the two mutants, we examined the hybridization of Tn5 to Southern blots of EcoRl digest fragments of DNA extracted from the mutants as previously described [7] . Restriction endonuclease EcoR l was purchased from New England Biolabs and used under the conditions specified . In order to determine whether the Tn5 insertion was responsible for the mutant phenotype, the EroRI fragment containing the Tn5 insertion (Tn5 lacks an EcoRI site) was cloned into the EcoRI site in pBR325 using the techniques described by Maniatis et al. [6] . The cloned DNA fragment was reintroduced into the parent strain by marker exchange as previously described [7, 15] . Transconjugants which were resistant to neomycin and sensitive to tetracycline and thus contained Tn5, but lacked the other genes of pBR325, were tested for their ability to elicit the HR in tobacco leaves . Such transconjugants showed the mutant phenotype and failed to elicit a HR . RESULTS Interaction of the bacteria with tobacco When the parent strain was injected into tobacco leaves a visible hypersensitive response developed within 18 to 24 h . When the mutant bacteria Hyr-25 and Hyr-37 were injected into tobacco leaves no visible response was obtained even after 48 h (Fig . 1) . The growth of the parent and mutant bacteria in injected leaves was measured by injecting 50 pl of a stationary phase culture grown in nutrient broth into tobacco leaves at several locations . The injection site was marked and the entire site was cut out and the total number of viable bacteria was determined after various time intervals . The parent strain grew for the first 12 h after injection . However, by 30 h, when the hypersensitive response was well developed, the number of viable bacteria had declined to about 70% of those present at 12 h . Mutant Hyr-37 bacteria showed a similar pattern of growth and decline although no visible HR was elicited by these bacteria . Mutant Hyr-25 bacteria failed to grow in tobacco leaves and showed a decline in number by 7 h (Fig . 2) . The doughnut shaped area surrounding the injection site was also removed . The total number of viable bacteria in this location was less than 10 for both the mutants and the parent bacteria suggesting that the bacteria did not move out of the original injection site in tobacco leaves . The appearance of the bacteria injected into tobacco leaves was examined in the scanning electron microscope . At 3 h after injection the parent and mutant bacteria could be seen on the surface of the tobacco mesophyll cells . There was no visible response of the bacteria to the presence of the plant cells, nor of the tobacco cells to the presence of bacteria for either the parent or mutant bacteria [Figs 3(a) and 4(a)] . After 7 . 5 h the mutant bacteria appeared unchanged [Fig . 4(b)] ; however, small fibrillar projections on the surface of the parent bacteria were visible at this time [Fig . 3(b)] . After 25 h the
446
M . C . Deasey and A. G . Matthysse
FIG . 1 . Hypersensitive response in tobacco . Suspensions ofP . syringae pv . phaseolicola 19304, Hyr25 and Hyr-37 were injected into the underside of leaves of a young tobacco plant . Leaves were photographed 35 h after injection . (a) Parent strain 19304 ; (b) mutant Hyr-25 ; (c) mutant Hyr-37 . Neither mutant gave a hypersensitive response in tobacco .
10'
0
5
10
15
20
25
30
Time (h )
FIG . 2 . Time course of growth of bacteria injected into tobacco leaves . The total number of
viable bacteria in the injection site was determined . ( •) Parent strain 19304 ; (A) mutant Hyr-25 ; (∎) mutant Hyr-37 . The HR was first visible at about 20 h with the parent strain 19304 . No visible HR was produced by the mutants .
P. Phaseolicola HR mutants in leaves
447
FIG . 3 . Scanning electron micrographs of the interior of tobacco leaves injected with the parent strain 19304 . (a) 3 h after injection (b) 7 . 5 h after injection (,c) 25 h after injection ( the leaf tissue had collapsed by this time) . Note the fibrils surrounding the bacteria in (b) and (c) .
FIG . 4 . Scanning electron micrographs of the interior of tobacco leaves injected with mutant strain Hyr-37 . No HR was observed . (a) 3 h after injection ; (b) 7 . 5 h after injection ; (e) 25 h after injection . The appearance of the bacteria did not change with time after injection . Similar results were obtained with mutant strain Hyr-25 .
mutant bacteria still appeared unchanged [Fig . 4)c)] . The fibrils on the surface of the parent bacteria were more prominant at 25 h [Fig . 3(c)] . The HR elicited by the parent strain was apparent by this time . A similar lack of fibrils surrounding the injected bacteria was seen using the transconjugant bacteria in which the wild type gene was replaced by the Tn5 mutant gene by marker exchange . These transconjugant bacteria failed to elicit a HR . In these experiments the parent bacteria again elicited a HR and became surrounded by fibrils . More that 50% of the bacterial clusters examined in the interior of the leaf showed these fibrils for the parent strain . In order to compare these fibrils with those seen after the injection ofdead bacteria (and thus presumably produced by the plant cell) the appearance of injected heat-killed bacteria of the parent strain was
4 48
M . C . Deasey and A . G . Matthysse
FIG . 5 . Interior of tobacco leaves injected with heat-killed parent strain 19304 . No HR was observed . (a) Immediately after injection showing the rough appearance ofthe heat-killed bacteria ; (b) 3 h after injection (the bacteria appear to have a rougher sur(ace) ; (cj I 1 h after injection the bacteria arc covered by an amorphous material presumably made by the plant cells; .
examined . Immediately after injection the heat-killed bacteria had a rough bumpy appearance [Fig . 5(a)] . The bacterial surface appeared to be little changed at 3 h [Fig . 5(b)] . By 11 h the heat-killed bacteria were covered by an amorphous material [Fig . 5(c)] which was quite different in appearance from the fibrils seen with the living bacteria . The heat-killed bacteria did not elicit a visible HR . Interaction of bacteria with resistant bean The parent strain of P. syringae pv . phaseolicola, 19304, used in these studies was a race 1
strain and elicited a HR when the bacteria were injected into the leaves of the resistant bean cultivar Red Mexican U134 . When the mutants Hyr-25 and Hyr-37 were injected into the leaves of resistant beans they failed to elicit a HR . No visible response to their injection was seen . The growth of the parent and mutant bacteria in the leaves of Red Mexican beans was measured . The parent bacteria doubled in number during the first 20 h after injection. The number of viable bacteria then remained constant . The HR was visible at about 25 h after the injection of the parent stain . Mutant Hyr-37 bacteria showed only very slight growth, while Hyr-25 showed a slow decline in number of viable bacteria (Fig . 6) . The appareance of bacteria injected into leaves of resistant bean plants was examined in the scanning electron microscope . At 7 h after injection the parent and mutant could be seen on the surface of the mesophyll cells . Some fibrils were seen around the parent bacteria [Fig . 7(a)] . No fibrils were observed around either bacterial mutant [Figs 8(a) and 9(a)] . After 24 h, when the HR was just beginning to be apparent to the unaided eye, the parent bacteria were found as groups inside the bean leaves . Some groups of bacteria consisted mostly of smooth bacteria with an occasional rough bacterium with fibrils around it [Fig . 7(b)] ; other bacterial groups consisted almost entirely of rough bacteria surrounded by fibrils [Fig. 7(c)] . The bacterial mutant Hyr-25 was largely found in groups of smooth bacteria [Fig . 8(c)] . However, rare areas of debris
P. Phaseolicola HR mutants in leaves
449
10'
I
i
0
5
10
15
20
25 ~~
42
Time (h) FIG . 6 . Time course of growth of bacteria injected into leaves of the resistant bean cultivar Red
Mexican U134 . The total number of viable bacteria per injection site was determined . Sites injected with the parent strain showed a visible HR after about 24 h . Injection of the mutants produced no visible response . (0) Parent strain 19304 ; (A) mutant Hyr-25 ; (∎) mutant Hyr-37 .
Fte . 7 . Scanning electron micrographs of the interior of resistant bean leaves cultivar U134 injected with the parent bacteria . A HR was observed after 24 to 30 h . (a) 7 h after injection ; (b) and (c) 24 h after injection . Note the bacterium with fibrils in (b) and the large number of bacteria with fibrils in (c) .
containing some bacteria with fibrils were seen [Fig 8(b)] . These were much rarer than with the parent bacteria . After 24 h no fibrils were seen surrounding the mutant Hyr-37 [Fig . 9(a) and (b)] . Even in areas which contained debris no fibrils were seen associated with this mutant . Interaction of bacteria with susceptible bean
When susceptible beans were inoculated with the parent strain, yellow lesions with a water soaked centre were observed after 5 days . These lesions spread slowly and
450
M . C . Deasey and A . G . Matthysse
. 8 . Scanning electron micrographs of the interior of resistant beau leaves cultivar l'13 1 ; Ft( injected with mutant bacteria Hyr-25 . No HR was observed . (a 7 h after injection : (h' and (c 24 h after injection . Note the general absence of fibrils even in areas ofdebtis .
Fie . 9 . Scanning electron micrographs of the interior of resistant bean leaves cultivar U134 injected with mutant bacteria Hyr-37 . No HR was observed . (a) 7 h after injection ; (b) 24 h after injection . Note the absence of fibrils .
eventually fused to kill the leaf. No visible symptoms were produced by the inoculation of mutant Hyr-25 . Mutant Hyr-37 bacteria gave rise to fewer and smaller lesions than the parent strain and the lesions spread only very slowly (Fig . 10) . The growth of bacteria injected into a single site in leaves of susceptible beans was measured . The parent strain grew by a factor of 10 4 during the first 95 h after injection . Neither mutant grew as rapidly as the parent strain although both mutants showed some growth (Fig . 11) . P. syringae pv. phaseolicola also produces water soaked lesions when inoculated into bean pods . Virulence on bean pods has been reported to be controlled in part by host and
P. Phaseolicola HR mutants in leaves
451
FIG . 10 . Virulence on bean leaves . Bacteria were inoculated on to carborundum wounded leaves and the plants were photographed 11 days after inoculation . (a) Uninoculated plant ; (b) leaf inoculated with the parent strain 19304 ; (c) leaf inoculated with mutant Hyr-25 ; (d) leaf inoculated with mutant Hyr-37 . The two mutants appeared to be only weakly virulent when compared with the parent strain .
pathogen genes which differ from those involved in virulence on leaves [4] . Both Hyr-25 and Hyr-37 appeared to be weakly virulent when inoculated into bean pods . The lesions produced by the mutants were much more highly variable than those produced by the parent strain (Fig . 12) . The appearance of bacteria injected into leaves of susceptible bean plants was examined in the scanning electron microscope . Both parent and mutant bacteria were visible on the surface of mesophyll cells 7 h after injection . The bacteria were single or in small groups . Most of the parent bacteria and all of the mutant bacteria appeared to have a smooth surface . No fibrils were visible [Figs 13(a), 14(a) and 15(a)] . After 24 h larger groups of bacteria were observed . No fibrils were seen associated with either the parent or mutant bacteria [Figs 13(b), 14(b) and 15(b)] . However, in the case ofmutant Hyr-37 much debris was observed in some locations [Fig . 15(c)], and occasionally the
M . C . Deasey and A . G . Matthysse
452
f
20 40 60 80 100 Time (h) FIG . 11 . Time course of growth of bacteria injected into leaves of the susceptible bean cultivar
Top Crop. The total number of viable bacteria per injection site was determined . Symptoms were first visible about 100 h after the injection of the parent strain . The minor symptoms produced by the mutant strains were not visible until about 150-200 h . ( •) Parent strain 19304 ; (A) mutant
Hyr-25; (∎) mutant Hyr-37 .
FIG . 12 . Virulence on bean pods . Bacteria were inoculated into needle wounds on bean pods . Photographs were taken 6 days following inoculation. (a) Parent strain 19304 ; (b) mutant Hyr-25 ; (c) mutant Hyr-37 ; (d) water inoculated . The two mutants appear to be only weakly virulent when compared with the parent strain . The black dot near the inoculation site was made by a marking pen used to number the inoculation sites .
P . Phaseolicola HR mutants in leaves
453
FIG . 13 . Scanning electron micrographs of the interior of susceptible bean leaves injected with the parent bacteria . Disease symptoms were visible after 5 days . (a) 7 h after injection ; (b) 24 h after injection .
FIG . 14 . Scanning electron micrographs of the interior of susceptible bean leaves injected with mutant Hyr-25 bacteria. Variable disease symptoms were visible after five to 10 days . (a) 7 h after injection; (b) 24 h after injection . Most of the visible bacteria were single .
bacteria appeared to be covered by a blanket of amorphous material [Fig . 15(d)] . The nature and origin of this material is unknown . Characterization of the mutant bacteria
The mutant bacteria showed no detectable difference from the parent strain in any property examined using bacteria grown in bacteriological media . The parent and both mutants were prototrophic and had indistinguishable colony morphologies . They all grew at the same rate in Luria broth (Fig . 16) . They were all motile and all produced phaseolotoxin . When the surface components of the bacteria were extracted and examined, no differences were seen in amount or size of extracellular polysaccharide,
454
M . C . Deasey and A . G . Matthysse
FIG . 15 . Scanning electron micrographs of the interior of susceptible bean leaves injected with mutant Hyr-37 bacteria. No disease symptoms were apparent even after 14 days . (a) 7 h after injection ; (b), (c) and (d) 24 h after injection . After 24 h both single bacteria and bacterial groups were visible . In rare cases (d) the bacteria appeared to be covered by some amorphous material .
lipopolysaccharide, or surface proteins . Thus the differences between the mutant bacteria and the parent may either be small subtle changes in components present in bacteria grown in bacteriological media or in substances which are made by the bacteria only in the presence of plant cells . The location of the Tn5 insertion in the two mutants was examined . The parent strain contained one endogenous plasmid . When this plasmid was prepared from the two mutants and the pattern resulting from agarose gel electrophoresis of an EcoRI digest of the plasmid DNA was compared with that from the parent strain, no differences were seen, suggesting that the Tn5 insertion was into the bacterial chromosome rather than in the plasmid (data not shown) . Total bacterial DNA was extracted from the parent and mutant bacteria, digested with EcoRl (Tn5 has no EcoRl sites), separated by gel electrophoresis, the fragments transferred to nitrocellulose, the resulting filter hybridized to radioactive Tn5 DNA and the results examined using autoradiography . The parent strain showed no hybridization to Tn5 . Both mutants showed hybridization of a band approximately 18 kb in size . Mutant Hyr-25 also showed hybridization of a band about
455
P. Phaseolicola HR mutants in leaves
100 80 60
20
10 0
5
10
24 Time (h)
Fic . 16 . Time course ofgrowth of bacteria in Luria broth at 25 ° C . ( •) Parent strain 19304 ; (A) mutant Hyr-25 ; (∎) mutant Hyr-37 . The parent and mutant strains showed no difference in growth rate .
I
2
3
Fic . 17 . Location of Tn5 in the mutant bacteria . Autoradiogram of hybridization of radioactive Tn5 DNA to a Southern transfer of an EcoRl digest of DNA separated by electrophoresis in 0 .7°,,, agarose . Channel 1 : DNA from the parent strain 19304 showed no hybridization to Tn5 DNA ; 2 : DNA from mutant Hyr-25 showed two bands of hybridization ; 3 : DNA from mutant Hyr-37 showed one band of hybridization .
6 kb in size (Fig . 17) . When the 18 kb band from both mutants was cloned and reintroduced into the parent strain by marker exchange, the HR-minus phenotype of the mutants was reconstituted, suggesting that in both mutants the mutant phenotype resulted from the Tn5 (6 kb) insertion in an original 12 kb EcoR 1 DNA fragment . DISCUSSION Bacterial mutants which fail to elicit a hypersensitive response in a non-host plant were obtained by transposon mutagenesis at a frequency of about 1 0% of surviving
456 M . C . Deasey and A . G . Matthysse nonauxotrophic mutants . If the insertion is random then this suggests that about 1 of the genome is required for the ability of the bacteria to elicit a HR . The two mutants studied failed to elicit a HR on the non-host plant tobacco ; they also failed to elicit a racespecific HR on the resistant cultivar ofbean Red Mexican U134 . In addition the mutants were reduced in virulence on susceptible bean leaves and pods . These results suggest that the genes affected by the transposon insertion are also involved in these other interactions of the bacteria with plants . Bacterial mutants with similar ranges of effects have been obtained by Panapoulos et al . [13] and by Anderson et 1. [1,12] from P . syringae pv . phaseolicola . In fact these pleiotropic mutants, in which several aspects of the bacterial interaction with plants are altered, seem to be the most common type of mutant obtained, whether the original screening is for alterations in virulence, or in the ability to elicit a HR . When the ability of the mutants to grow in plants in which their inoculation did not produce any visible symptoms was examined no clear picture emerged . Both mutants were reduced in growth in resistant and in susceptible bean . However, in the non-host plant tobacco, mutant Hyr-37 grew at a rate similar to the parent strain while mutant Hyr-25 was much reduced in growth . Thus the lack of response of the plant to the mutant bacteria cannot always be attributed to lack of bacterial growth . No difference in the ability of the bacteria to grow in bacteriological media could be detected, nor could any difference in the mutant and parent bacteria grown in bacteriological culture be detected in exopolysaccharide, motility, lipopolysaccharide, toxin production, or surface proteins. However, it should be noted that only size and amount of exopolysaccharide and lipopolysaccharide were examined . Thus changes in composition which would not markedly alter size would not have been detected . The failure to detect any differences between the parent and mutant bacteria grown in bacteriological media suggests that they may differ in minor components or in substances which are produced only in response to the presence of the plant cells . When the interaction of the bacteria with tobacco was examined in the scanning electron microscope, the parent bacteria were seen to change with time after inoculation, with fibrils appearing around the bacteria after about 7 h . No detectable changes were seen with time after inoculation with the mutant bacteria, which appeared to sit on the surface of the plant cells unchanged for 25 h . Fibrils were also seen when the parent bacteria were injected into resistant bean leaves, but fibrils were apparent only at early times when the bacteria were injected into susceptible bean leaves . No fibrils were seen after the injection of the mutant Hyr-37 into resistant bean or tobacco . A much reduced number of fibrils was observed after the injection of Hyr-25 into resistant bean . The chemical nature of the fibrils produced around the parent bacteria is unknown . It is also not known whether the fibrils are produced by the plant or the bacteria . The fibrils appear to be distinct from the amorphous pellicle surrounding the bacteria produced by the plant in response to the injection of dead bacteria [16] . Similar fibrils have been seen by Sigee and Al-Issa in tobacco leaves injected with P . syringae pv . pisi [17] . Thus these fibrils may be produced during the interaction of P . syringae with resistant tobacco or bean cells . The absence or reduction in number offibrils in mutant bacteria which fail to elicit a HR suggests that the fibrils play a role in the development of the HR, but it is unclear whether they participate in causing the HR or are produced as a result of the development of the HR .
P. Phaseolicola HR mutants in leaves
457
This research was supported by NSF grant DBC-8416282 . REFERENCES I. ANDERSON, D .
M . & MILLS, D . (1985) . The use of transposon mutagenesis in the isolation of nutritional and virulence mutants in two pathovars of Pseudomonas syringae . Phytopathology75, 104-108 . 2 . DEASEY, M . C . & MATTHYSSE, A . G . (1984) . Interactions of wild-type and a cellulose-minus mutant of Agrobacterium tumefaciens with tobacco mesophyll and tobacco tissue culture cells . Ph_ytopathology 74, 991-994 . 3 . EL-BANOBY, F . E . & RUDOLPH, K . (1980) . Purification of extracellular polysaccharides from Pseudomonas phaseolicola which induce water-soaking in bean leaves . Physiological Plant Pathology 16,425-437 . 4 . HILL, K ., COYNE, D . P . & SCHUSTER, M . L . (1972) Leaf, pod and systemic chlorosis reactions in Phaseolus vulgaris to halo blight controlled by different genes . Journal ofthe American Horticultural Society 97,49"98 . 5 . LAEMLI, U K . (1970) . Cleavage ofstructural proteins during the assembly of the head of bacteriophage T4 . Nature 227, 680-685 . 6 . MANIATIS, T ., FRITSCH, E . F . & SAMBROOK, J . (1982) . Molecular cloning . A laboratory manual . Cold Spring Harbor Laboratory, New York . 7 . MATTHYSSE, A . G . (1983) . Role of bacterial cellulose fibrils in Agrobacterium tumejaciens infection . Journal of Bacteriology 154, 906-915 . 8 . MATTHYSSE, A . G . (1987) . Characterization of mutants of Agrobacterium tumefaciens which fail to bind to plant cells . Journal of Bacteriology 169, 313-323 . 9 . MATTHYSSE, A . G ., WYMAN, P . M . & HOLMES, K . V . (1978) . Plasmid-dependent attachment of Agrobacterium tumefaciens to plant tissue culture cells . Infection and Immunity 22, 516-522 . 10 . MERRIL, C . R ., GOLDMAN, D ., SEDMAN, S . A. & EBERT, M . H . (1981) . Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins . Science 211, 1437-1438 . 11 . MILLER, J . H . (1972) . Experiments in Molecular Genetics, pp . 320-322 . Cold Spring Harbor Laboratory, Cold Spring Harbor, New York . 12 . NIEPOLD, F ., ANDERSON, D . & MILLS, D . (1985) . Cloning determinants of pathogenesis from Pseudomonas syringae pathovar syringae. Proceedings of the National Academy ofSciences, United States ofAmerica 82, 406110 . 13 . PANOPOULOS, N . J ., LINDGREN, P . B ., WILLIS, D . K. & PEET, R . C . (1985) . Clustering and conservation of genes controlling the interactions of Pseudomonas syringae pathovars with plants . In Plant Gell/Cell Interactions . Ed . by I . Sussex, A. Ellingboe, M . Crouch and R . Malmberg, pp .69-71 . Cold Spring Harbor Laboratory, New York . 14 . PoLITIs, D . J . & GOODMAN, R . N . (1978) . Localized cell wall appositions : incompatibility response of tobacco leaf cells to Pseudomonas pisi . Phytopathology 68, 309-316 . 15 . RUVKUN, G . B . & AusuBEL, F . M . (1981) . A general method for site-directed mutagenesis in prokaryotes . Nature 289, 85-89 . 16 . SEQUEIRA, L ., GAARD, G . & DEZOETEN, G . A. (1977) . Interaction ofbacteria and host cell walls : its relation to mechanisms of induced resistance . Physiological Plant Pathology 10, 43-50 . 17 . SIGEE, D . C . & AL-IssA, A . N . (1983) . The hypersensitive reaction in tobacco leaf tissue infiltrated with Pseudomonas pisi 4 . Scanning electron microscope studies on fractured leaf tissue . Ph_ytopathologisehe zeitschrift 106, 1-15, 18 . SIMON, R ., PRIEFER, U . & PUHLER, A . (1983) . A broad host range mobilization system for in vivo genetic engineering : transposon mutagenesis in Gram negative bacteria . Biotechnology7, 784-791 . 19 . SONOKI, S . & KADO, C . I . (1978) . Proteins conferred by the virulence-specifying plasmid ofAgrobacterium tumefaciens C58 . Proceedings of the National Academy of Sciences, United States of America 75, 3796-3800 . 20 . STASKOWIcz, B . J . & PANOPOULOS, N . J . (1979) . A rapid and sensitive microbiological assay for phaseolotoxin . Phytopathology 69, 663-666 . 21 . TSAI, C .-M . & FRASCH, C . E . (1982) . Sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels . Analytical Biochemistry 119, 115-119 .
Characterization, growth, and scanning electron microscopy of mutants of Pseudomonas syringae pv . phaseolicola which fail to elicit a hypersensitive response in host and non-host plants M . C . DEASEY
and A .
G . MATTHYSSE
Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280 U .S.A . Accepted, for publication May 1988)
Transposon mutants of Pseudomonas syringae pv . phaseolicola which fail to elicit a hypersensitive response in the non-host plant tobacco were isolated and compared with the parent strain . The mutants also failed to elicit a race-specific hypersensitive response in resistant bean . In susceptible bean the mutants were reduced in virulence on both leaves and pods . Both mutants were reduced in growth in resistant and susceptible bean leaves . However, one of the mutants grew at a rate similar to the parent strain in tobacco ; the growth of the other mutant was reduced . No differences were detected between the parent strain and the mutants grown in bacteriological medium in extracellular polysaccharide, lipopolysaccharide, motility, or surface proteins . When the parent and the mutant bacteria were injected into tobacco or resistant bean leaves and examined in the scanning electron microscope, fibrils were seen surrounding the parent bacteria after 8 h . No such fibrils were observed surrounding one of the mutant bacteria . The number of fibrils surrounding the other mutant was reduced compared to the parent bacteria . No fibrils were produced when the parent or mutant bacteria were injected into susceptible bean leaves . These results suggest that the fibrils may play a role in the hypersensitive response, although it is unclear if the fibrils are required for the development, or are the result, of the hypersensitive response .
INTRODUCTION
the causative agent of halo blight in bean . Like many other phytopathogenic pseudomonads and xanthomonads, P. syringae pv . phaseolicola induces a hypersensitive response (HR) when it is inoculated into a non-host plant such as tobacco . Saprophytic bacteria, symbiotic bacteria and bacteria not normally associated with plants fail to elicit a HR when inoculated in comparable numbers. In order to determine what aspects of the potentially pathogenic bacteria are recognized or required by the non-host plant for the elicitation of the HR, transposon mutants of the bacteria which fail to elicit a HR in the non-host plant Nicotiana tabacum were isolated and characterized . Pseudomonas syringae pv . phaseolicola is
MATERIALS AND METHODS Growth of bacteria and plants Virulent Pseudomonas syringae pv . phaseolicola
strain 19304 was obtained from the American Type Culture Collection . It was grown in Luria [6] or nutrient broth . Abbreviations used in text : HR, hypersensitive response . 0885-5765/88/060443+ 15 $03 .00/0
© 1988 Academic Press Limited
444 M . C . Deasey and A . G . Matthysse Escherichia coli SM10 pSUP1011 was obtained from Dr Paul Bishop, North Carolina State University, and grown in Luria broth with 100 pg ml - ' of chloramphenicol . Nicotiana tabacum variety Coker 319, and Phaseolus vulgaris varieties Top Crop and Red Mexican U134 were grown in the greenhouse . Plants which had been inoculated with bacteria were grown in a growth chamber at 27 °C with a 14 h day . Inoculated bean plants were covered to maintain a relative humidity in excess of 80°0 for the first day after inoculation . Leaves of tobacco and bean plants were inoculated by injecting bacteria into the leaf using a 0 . 5 ml syringe . For experiments in which the growth of the inoculated bacteria in the leaves was measured, the syringe was equipped with a device which allowed the delivery ofa predetermined volume (Tirdak stepper), 5 PI in the case of bean leaves and 50 gl in the case of tobacco leaves . Numbers of viable bacteria were determined by cutting the entire inoculated area out of the leaf, grinding the excised tissue in a Waring blender in phosphate buffered saline (Na 2 HPO, 7 g 1 -1 , KH 2PO,, 3 g 1 - 1 , NaCl 4 g I - ', and MgSO 4 :7 H 2O 0 . 2 g 1 - ' ), and determining the viable cell count on Luria agar plates . Bean pods were purchased at a local supermarket and inoculated by applying a drop of about 50µl of bacterial suspension containing about 10 9 bacterial ml - ' to the pod and making a needle wound through the drop . For experiments testing the virulence (rather than growth) of bacteria on bean leaves, the leaves were wounded with carborundum and 0 . 5 ml of a suspension of 10 9 bacterial ml - ' was pipetted on to the leaf. Scanning electron microscopy Tissue fixation and preparation for scanning electron microscopy were carried out as previously described [2] . Leaf discs coated with gold-pallidium were examined with an ETEC Autoscan scanning electron microscope . Discs were examined from at least three different injection sites on different leaves . Preparation and isolation of mutant bacteria Transposon mutagenesis of P . syringae pv . phaseolicola was carried out by introducing Tn5 from E . coli SM10 pSUP1011 which is a multiple auxotroph and contains Tn5 on the plasmid [18] . This conjugative plasmid is not capable of replicating in Pseudomonas . For bacterial conjugation E. coli and P . syringae pv . phaseolicola were grown to mid-log phase in flasks containing Luria broth on shakers at 37 and 25 ° C respectively . The bacteria were mixed on the surface of a Luria agar plate and incubated overnight at 25 °C . Transconjugants were selected for growth on minimal medium containing 60 µg ml - ' neomycin . Neither parent grew under these conditions . P . syringae pv . phaseolicola transconjugants containing Tn5 were screened for the ability to elicit a hypersensitive response in the non-host plant tobacco by injecting bacteria grown in Nutrient Broth into tobacco leaves . Two of the first 200 transconjugants failed to elicit a HR (Hyr-25 and Hyr-37) . These two mutants were kept for further study . They were resistant to neomycin, but sensitive to chloramphenicol to which pSUP1011 provides resistance . When their plasmid composition was examined by the lysis in the well technique [T], they contained only the indigenous plasmid contained in the parent strain 19304 . Characterization ofparent and mutant bacteria Growth of the parent and mutant bacteria in Luria broth was measured in a Klett photometer with a green filter . Bacterial motility was examined using a light microscope .
P. Phaseolicola HR mutants in leaves
445
Bacterial lipopolysaccharide was extracted as previously described [9] and examined by SDS polyacrylamide gel electrophoresis using the method of Tsai and Frasch [21] . Bacterial surface proteins were extracted by the method of Sonoki & Kado [19] and examined by SDS-PAGE using the method ofLaemli [5] and the silver staining procedure of Merril et al . [10] . Bacterial exopolysaccharides were prepared by the method of El-Banoby & Rudolph [3] . Their size was examined by SDS-PAGE . Bacterial production of phaseolotoxin was examined by the method of Staskowicz & Panopoulos [20] . In order to determine whether the site of Tn5 insertion was the same in the two mutants, we examined the hybridization of Tn5 to Southern blots of EcoRl digest fragments of DNA extracted from the mutants as previously described [7] . Restriction endonuclease EcoR l was purchased from New England Biolabs and used under the conditions specified . In order to determine whether the Tn5 insertion was responsible for the mutant phenotype, the EroRI fragment containing the Tn5 insertion (Tn5 lacks an EcoRI site) was cloned into the EcoRI site in pBR325 using the techniques described by Maniatis et al. [6] . The cloned DNA fragment was reintroduced into the parent strain by marker exchange as previously described [7, 15] . Transconjugants which were resistant to neomycin and sensitive to tetracycline and thus contained Tn5, but lacked the other genes of pBR325, were tested for their ability to elicit the HR in tobacco leaves . Such transconjugants showed the mutant phenotype and failed to elicit a HR . RESULTS Interaction of the bacteria with tobacco When the parent strain was injected into tobacco leaves a visible hypersensitive response developed within 18 to 24 h . When the mutant bacteria Hyr-25 and Hyr-37 were injected into tobacco leaves no visible response was obtained even after 48 h (Fig . 1) . The growth of the parent and mutant bacteria in injected leaves was measured by injecting 50 pl of a stationary phase culture grown in nutrient broth into tobacco leaves at several locations . The injection site was marked and the entire site was cut out and the total number of viable bacteria was determined after various time intervals . The parent strain grew for the first 12 h after injection . However, by 30 h, when the hypersensitive response was well developed, the number of viable bacteria had declined to about 70% of those present at 12 h . Mutant Hyr-37 bacteria showed a similar pattern of growth and decline although no visible HR was elicited by these bacteria . Mutant Hyr-25 bacteria failed to grow in tobacco leaves and showed a decline in number by 7 h (Fig . 2) . The doughnut shaped area surrounding the injection site was also removed . The total number of viable bacteria in this location was less than 10 for both the mutants and the parent bacteria suggesting that the bacteria did not move out of the original injection site in tobacco leaves . The appearance of the bacteria injected into tobacco leaves was examined in the scanning electron microscope . At 3 h after injection the parent and mutant bacteria could be seen on the surface of the tobacco mesophyll cells . There was no visible response of the bacteria to the presence of the plant cells, nor of the tobacco cells to the presence of bacteria for either the parent or mutant bacteria [Figs 3(a) and 4(a)] . After 7 . 5 h the mutant bacteria appeared unchanged [Fig . 4(b)] ; however, small fibrillar projections on the surface of the parent bacteria were visible at this time [Fig . 3(b)] . After 25 h the
446
M . C . Deasey and A. G . Matthysse
FIG . 1 . Hypersensitive response in tobacco . Suspensions ofP . syringae pv . phaseolicola 19304, Hyr25 and Hyr-37 were injected into the underside of leaves of a young tobacco plant . Leaves were photographed 35 h after injection . (a) Parent strain 19304 ; (b) mutant Hyr-25 ; (c) mutant Hyr-37 . Neither mutant gave a hypersensitive response in tobacco .
10'
0
5
10
15
20
25
30
Time (h )
FIG . 2 . Time course of growth of bacteria injected into tobacco leaves . The total number of
viable bacteria in the injection site was determined . ( •) Parent strain 19304 ; (A) mutant Hyr-25 ; (∎) mutant Hyr-37 . The HR was first visible at about 20 h with the parent strain 19304 . No visible HR was produced by the mutants .
P. Phaseolicola HR mutants in leaves
447
FIG . 3 . Scanning electron micrographs of the interior of tobacco leaves injected with the parent strain 19304 . (a) 3 h after injection (b) 7 . 5 h after injection (,c) 25 h after injection ( the leaf tissue had collapsed by this time) . Note the fibrils surrounding the bacteria in (b) and (c) .
FIG . 4 . Scanning electron micrographs of the interior of tobacco leaves injected with mutant strain Hyr-37 . No HR was observed . (a) 3 h after injection ; (b) 7 . 5 h after injection ; (e) 25 h after injection . The appearance of the bacteria did not change with time after injection . Similar results were obtained with mutant strain Hyr-25 .
mutant bacteria still appeared unchanged [Fig . 4)c)] . The fibrils on the surface of the parent bacteria were more prominant at 25 h [Fig . 3(c)] . The HR elicited by the parent strain was apparent by this time . A similar lack of fibrils surrounding the injected bacteria was seen using the transconjugant bacteria in which the wild type gene was replaced by the Tn5 mutant gene by marker exchange . These transconjugant bacteria failed to elicit a HR . In these experiments the parent bacteria again elicited a HR and became surrounded by fibrils . More that 50% of the bacterial clusters examined in the interior of the leaf showed these fibrils for the parent strain . In order to compare these fibrils with those seen after the injection ofdead bacteria (and thus presumably produced by the plant cell) the appearance of injected heat-killed bacteria of the parent strain was
4 48
M . C . Deasey and A . G . Matthysse
FIG . 5 . Interior of tobacco leaves injected with heat-killed parent strain 19304 . No HR was observed . (a) Immediately after injection showing the rough appearance ofthe heat-killed bacteria ; (b) 3 h after injection (the bacteria appear to have a rougher sur(ace) ; (cj I 1 h after injection the bacteria arc covered by an amorphous material presumably made by the plant cells; .
examined . Immediately after injection the heat-killed bacteria had a rough bumpy appearance [Fig . 5(a)] . The bacterial surface appeared to be little changed at 3 h [Fig . 5(b)] . By 11 h the heat-killed bacteria were covered by an amorphous material [Fig . 5(c)] which was quite different in appearance from the fibrils seen with the living bacteria . The heat-killed bacteria did not elicit a visible HR . Interaction of bacteria with resistant bean The parent strain of P. syringae pv . phaseolicola, 19304, used in these studies was a race 1
strain and elicited a HR when the bacteria were injected into the leaves of the resistant bean cultivar Red Mexican U134 . When the mutants Hyr-25 and Hyr-37 were injected into the leaves of resistant beans they failed to elicit a HR . No visible response to their injection was seen . The growth of the parent and mutant bacteria in the leaves of Red Mexican beans was measured . The parent bacteria doubled in number during the first 20 h after injection. The number of viable bacteria then remained constant . The HR was visible at about 25 h after the injection of the parent stain . Mutant Hyr-37 bacteria showed only very slight growth, while Hyr-25 showed a slow decline in number of viable bacteria (Fig . 6) . The appareance of bacteria injected into leaves of resistant bean plants was examined in the scanning electron microscope . At 7 h after injection the parent and mutant could be seen on the surface of the mesophyll cells . Some fibrils were seen around the parent bacteria [Fig . 7(a)] . No fibrils were observed around either bacterial mutant [Figs 8(a) and 9(a)] . After 24 h, when the HR was just beginning to be apparent to the unaided eye, the parent bacteria were found as groups inside the bean leaves . Some groups of bacteria consisted mostly of smooth bacteria with an occasional rough bacterium with fibrils around it [Fig . 7(b)] ; other bacterial groups consisted almost entirely of rough bacteria surrounded by fibrils [Fig. 7(c)] . The bacterial mutant Hyr-25 was largely found in groups of smooth bacteria [Fig . 8(c)] . However, rare areas of debris
P. Phaseolicola HR mutants in leaves
449
10'
I
i
0
5
10
15
20
25 ~~
42
Time (h) FIG . 6 . Time course of growth of bacteria injected into leaves of the resistant bean cultivar Red
Mexican U134 . The total number of viable bacteria per injection site was determined . Sites injected with the parent strain showed a visible HR after about 24 h . Injection of the mutants produced no visible response . (0) Parent strain 19304 ; (A) mutant Hyr-25 ; (∎) mutant Hyr-37 .
Fte . 7 . Scanning electron micrographs of the interior of resistant bean leaves cultivar U134 injected with the parent bacteria . A HR was observed after 24 to 30 h . (a) 7 h after injection ; (b) and (c) 24 h after injection . Note the bacterium with fibrils in (b) and the large number of bacteria with fibrils in (c) .
containing some bacteria with fibrils were seen [Fig 8(b)] . These were much rarer than with the parent bacteria . After 24 h no fibrils were seen surrounding the mutant Hyr-37 [Fig . 9(a) and (b)] . Even in areas which contained debris no fibrils were seen associated with this mutant . Interaction of bacteria with susceptible bean
When susceptible beans were inoculated with the parent strain, yellow lesions with a water soaked centre were observed after 5 days . These lesions spread slowly and
450
M . C . Deasey and A . G . Matthysse
. 8 . Scanning electron micrographs of the interior of resistant beau leaves cultivar l'13 1 ; Ft( injected with mutant bacteria Hyr-25 . No HR was observed . (a 7 h after injection : (h' and (c 24 h after injection . Note the general absence of fibrils even in areas ofdebtis .
Fie . 9 . Scanning electron micrographs of the interior of resistant bean leaves cultivar U134 injected with mutant bacteria Hyr-37 . No HR was observed . (a) 7 h after injection ; (b) 24 h after injection . Note the absence of fibrils .
eventually fused to kill the leaf. No visible symptoms were produced by the inoculation of mutant Hyr-25 . Mutant Hyr-37 bacteria gave rise to fewer and smaller lesions than the parent strain and the lesions spread only very slowly (Fig . 10) . The growth of bacteria injected into a single site in leaves of susceptible beans was measured . The parent strain grew by a factor of 10 4 during the first 95 h after injection . Neither mutant grew as rapidly as the parent strain although both mutants showed some growth (Fig . 11) . P. syringae pv. phaseolicola also produces water soaked lesions when inoculated into bean pods . Virulence on bean pods has been reported to be controlled in part by host and
P. Phaseolicola HR mutants in leaves
451
FIG . 10 . Virulence on bean leaves . Bacteria were inoculated on to carborundum wounded leaves and the plants were photographed 11 days after inoculation . (a) Uninoculated plant ; (b) leaf inoculated with the parent strain 19304 ; (c) leaf inoculated with mutant Hyr-25 ; (d) leaf inoculated with mutant Hyr-37 . The two mutants appeared to be only weakly virulent when compared with the parent strain .
pathogen genes which differ from those involved in virulence on leaves [4] . Both Hyr-25 and Hyr-37 appeared to be weakly virulent when inoculated into bean pods . The lesions produced by the mutants were much more highly variable than those produced by the parent strain (Fig . 12) . The appearance of bacteria injected into leaves of susceptible bean plants was examined in the scanning electron microscope . Both parent and mutant bacteria were visible on the surface of mesophyll cells 7 h after injection . The bacteria were single or in small groups . Most of the parent bacteria and all of the mutant bacteria appeared to have a smooth surface . No fibrils were visible [Figs 13(a), 14(a) and 15(a)] . After 24 h larger groups of bacteria were observed . No fibrils were seen associated with either the parent or mutant bacteria [Figs 13(b), 14(b) and 15(b)] . However, in the case ofmutant Hyr-37 much debris was observed in some locations [Fig . 15(c)], and occasionally the
M . C . Deasey and A . G . Matthysse
452
f
20 40 60 80 100 Time (h) FIG . 11 . Time course of growth of bacteria injected into leaves of the susceptible bean cultivar
Top Crop. The total number of viable bacteria per injection site was determined . Symptoms were first visible about 100 h after the injection of the parent strain . The minor symptoms produced by the mutant strains were not visible until about 150-200 h . ( •) Parent strain 19304 ; (A) mutant
Hyr-25; (∎) mutant Hyr-37 .
FIG . 12 . Virulence on bean pods . Bacteria were inoculated into needle wounds on bean pods . Photographs were taken 6 days following inoculation. (a) Parent strain 19304 ; (b) mutant Hyr-25 ; (c) mutant Hyr-37 ; (d) water inoculated . The two mutants appear to be only weakly virulent when compared with the parent strain . The black dot near the inoculation site was made by a marking pen used to number the inoculation sites .
P . Phaseolicola HR mutants in leaves
453
FIG . 13 . Scanning electron micrographs of the interior of susceptible bean leaves injected with the parent bacteria . Disease symptoms were visible after 5 days . (a) 7 h after injection ; (b) 24 h after injection .
FIG . 14 . Scanning electron micrographs of the interior of susceptible bean leaves injected with mutant Hyr-25 bacteria. Variable disease symptoms were visible after five to 10 days . (a) 7 h after injection; (b) 24 h after injection . Most of the visible bacteria were single .
bacteria appeared to be covered by a blanket of amorphous material [Fig . 15(d)] . The nature and origin of this material is unknown . Characterization of the mutant bacteria
The mutant bacteria showed no detectable difference from the parent strain in any property examined using bacteria grown in bacteriological media . The parent and both mutants were prototrophic and had indistinguishable colony morphologies . They all grew at the same rate in Luria broth (Fig . 16) . They were all motile and all produced phaseolotoxin . When the surface components of the bacteria were extracted and examined, no differences were seen in amount or size of extracellular polysaccharide,
454
M . C . Deasey and A . G . Matthysse
FIG . 15 . Scanning electron micrographs of the interior of susceptible bean leaves injected with mutant Hyr-37 bacteria. No disease symptoms were apparent even after 14 days . (a) 7 h after injection ; (b), (c) and (d) 24 h after injection . After 24 h both single bacteria and bacterial groups were visible . In rare cases (d) the bacteria appeared to be covered by some amorphous material .
lipopolysaccharide, or surface proteins . Thus the differences between the mutant bacteria and the parent may either be small subtle changes in components present in bacteria grown in bacteriological media or in substances which are made by the bacteria only in the presence of plant cells . The location of the Tn5 insertion in the two mutants was examined . The parent strain contained one endogenous plasmid . When this plasmid was prepared from the two mutants and the pattern resulting from agarose gel electrophoresis of an EcoRI digest of the plasmid DNA was compared with that from the parent strain, no differences were seen, suggesting that the Tn5 insertion was into the bacterial chromosome rather than in the plasmid (data not shown) . Total bacterial DNA was extracted from the parent and mutant bacteria, digested with EcoRl (Tn5 has no EcoRl sites), separated by gel electrophoresis, the fragments transferred to nitrocellulose, the resulting filter hybridized to radioactive Tn5 DNA and the results examined using autoradiography . The parent strain showed no hybridization to Tn5 . Both mutants showed hybridization of a band approximately 18 kb in size . Mutant Hyr-25 also showed hybridization of a band about
455
P. Phaseolicola HR mutants in leaves
100 80 60
20
10 0
5
10
24 Time (h)
Fic . 16 . Time course ofgrowth of bacteria in Luria broth at 25 ° C . ( •) Parent strain 19304 ; (A) mutant Hyr-25 ; (∎) mutant Hyr-37 . The parent and mutant strains showed no difference in growth rate .
I
2
3
Fic . 17 . Location of Tn5 in the mutant bacteria . Autoradiogram of hybridization of radioactive Tn5 DNA to a Southern transfer of an EcoRl digest of DNA separated by electrophoresis in 0 .7°,,, agarose . Channel 1 : DNA from the parent strain 19304 showed no hybridization to Tn5 DNA ; 2 : DNA from mutant Hyr-25 showed two bands of hybridization ; 3 : DNA from mutant Hyr-37 showed one band of hybridization .
6 kb in size (Fig . 17) . When the 18 kb band from both mutants was cloned and reintroduced into the parent strain by marker exchange, the HR-minus phenotype of the mutants was reconstituted, suggesting that in both mutants the mutant phenotype resulted from the Tn5 (6 kb) insertion in an original 12 kb EcoR 1 DNA fragment . DISCUSSION Bacterial mutants which fail to elicit a hypersensitive response in a non-host plant were obtained by transposon mutagenesis at a frequency of about 1 0% of surviving
456 M . C . Deasey and A . G . Matthysse nonauxotrophic mutants . If the insertion is random then this suggests that about 1 of the genome is required for the ability of the bacteria to elicit a HR . The two mutants studied failed to elicit a HR on the non-host plant tobacco ; they also failed to elicit a racespecific HR on the resistant cultivar ofbean Red Mexican U134 . In addition the mutants were reduced in virulence on susceptible bean leaves and pods . These results suggest that the genes affected by the transposon insertion are also involved in these other interactions of the bacteria with plants . Bacterial mutants with similar ranges of effects have been obtained by Panapoulos et al . [13] and by Anderson et 1. [1,12] from P . syringae pv . phaseolicola . In fact these pleiotropic mutants, in which several aspects of the bacterial interaction with plants are altered, seem to be the most common type of mutant obtained, whether the original screening is for alterations in virulence, or in the ability to elicit a HR . When the ability of the mutants to grow in plants in which their inoculation did not produce any visible symptoms was examined no clear picture emerged . Both mutants were reduced in growth in resistant and in susceptible bean . However, in the non-host plant tobacco, mutant Hyr-37 grew at a rate similar to the parent strain while mutant Hyr-25 was much reduced in growth . Thus the lack of response of the plant to the mutant bacteria cannot always be attributed to lack of bacterial growth . No difference in the ability of the bacteria to grow in bacteriological media could be detected, nor could any difference in the mutant and parent bacteria grown in bacteriological culture be detected in exopolysaccharide, motility, lipopolysaccharide, toxin production, or surface proteins. However, it should be noted that only size and amount of exopolysaccharide and lipopolysaccharide were examined . Thus changes in composition which would not markedly alter size would not have been detected . The failure to detect any differences between the parent and mutant bacteria grown in bacteriological media suggests that they may differ in minor components or in substances which are produced only in response to the presence of the plant cells . When the interaction of the bacteria with tobacco was examined in the scanning electron microscope, the parent bacteria were seen to change with time after inoculation, with fibrils appearing around the bacteria after about 7 h . No detectable changes were seen with time after inoculation with the mutant bacteria, which appeared to sit on the surface of the plant cells unchanged for 25 h . Fibrils were also seen when the parent bacteria were injected into resistant bean leaves, but fibrils were apparent only at early times when the bacteria were injected into susceptible bean leaves . No fibrils were seen after the injection of the mutant Hyr-37 into resistant bean or tobacco . A much reduced number of fibrils was observed after the injection of Hyr-25 into resistant bean . The chemical nature of the fibrils produced around the parent bacteria is unknown . It is also not known whether the fibrils are produced by the plant or the bacteria . The fibrils appear to be distinct from the amorphous pellicle surrounding the bacteria produced by the plant in response to the injection of dead bacteria [16] . Similar fibrils have been seen by Sigee and Al-Issa in tobacco leaves injected with P . syringae pv . pisi [17] . Thus these fibrils may be produced during the interaction of P . syringae with resistant tobacco or bean cells . The absence or reduction in number offibrils in mutant bacteria which fail to elicit a HR suggests that the fibrils play a role in the development of the HR, but it is unclear whether they participate in causing the HR or are produced as a result of the development of the HR .
P. Phaseolicola HR mutants in leaves
457
This research was supported by NSF grant DBC-8416282 . REFERENCES I. ANDERSON, D .
M . & MILLS, D . (1985) . The use of transposon mutagenesis in the isolation of nutritional and virulence mutants in two pathovars of Pseudomonas syringae . Phytopathology75, 104-108 . 2 . DEASEY, M . C . & MATTHYSSE, A . G . (1984) . Interactions of wild-type and a cellulose-minus mutant of Agrobacterium tumefaciens with tobacco mesophyll and tobacco tissue culture cells . Ph_ytopathology 74, 991-994 . 3 . EL-BANOBY, F . E . & RUDOLPH, K . (1980) . Purification of extracellular polysaccharides from Pseudomonas phaseolicola which induce water-soaking in bean leaves . Physiological Plant Pathology 16,425-437 . 4 . HILL, K ., COYNE, D . P . & SCHUSTER, M . L . (1972) Leaf, pod and systemic chlorosis reactions in Phaseolus vulgaris to halo blight controlled by different genes . Journal ofthe American Horticultural Society 97,49"98 . 5 . LAEMLI, U K . (1970) . Cleavage ofstructural proteins during the assembly of the head of bacteriophage T4 . Nature 227, 680-685 . 6 . MANIATIS, T ., FRITSCH, E . F . & SAMBROOK, J . (1982) . Molecular cloning . A laboratory manual . Cold Spring Harbor Laboratory, New York . 7 . MATTHYSSE, A . G . (1983) . Role of bacterial cellulose fibrils in Agrobacterium tumejaciens infection . Journal of Bacteriology 154, 906-915 . 8 . MATTHYSSE, A . G . (1987) . Characterization of mutants of Agrobacterium tumefaciens which fail to bind to plant cells . Journal of Bacteriology 169, 313-323 . 9 . MATTHYSSE, A . G ., WYMAN, P . M . & HOLMES, K . V . (1978) . Plasmid-dependent attachment of Agrobacterium tumefaciens to plant tissue culture cells . Infection and Immunity 22, 516-522 . 10 . MERRIL, C . R ., GOLDMAN, D ., SEDMAN, S . A. & EBERT, M . H . (1981) . Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins . Science 211, 1437-1438 . 11 . MILLER, J . H . (1972) . Experiments in Molecular Genetics, pp . 320-322 . Cold Spring Harbor Laboratory, Cold Spring Harbor, New York . 12 . NIEPOLD, F ., ANDERSON, D . & MILLS, D . (1985) . Cloning determinants of pathogenesis from Pseudomonas syringae pathovar syringae. Proceedings of the National Academy ofSciences, United States ofAmerica 82, 406110 . 13 . PANOPOULOS, N . J ., LINDGREN, P . B ., WILLIS, D . K. & PEET, R . C . (1985) . Clustering and conservation of genes controlling the interactions of Pseudomonas syringae pathovars with plants . In Plant Gell/Cell Interactions . Ed . by I . Sussex, A. Ellingboe, M . Crouch and R . Malmberg, pp .69-71 . Cold Spring Harbor Laboratory, New York . 14 . PoLITIs, D . J . & GOODMAN, R . N . (1978) . Localized cell wall appositions : incompatibility response of tobacco leaf cells to Pseudomonas pisi . Phytopathology 68, 309-316 . 15 . RUVKUN, G . B . & AusuBEL, F . M . (1981) . A general method for site-directed mutagenesis in prokaryotes . Nature 289, 85-89 . 16 . SEQUEIRA, L ., GAARD, G . & DEZOETEN, G . A. (1977) . Interaction ofbacteria and host cell walls : its relation to mechanisms of induced resistance . Physiological Plant Pathology 10, 43-50 . 17 . SIGEE, D . C . & AL-IssA, A . N . (1983) . The hypersensitive reaction in tobacco leaf tissue infiltrated with Pseudomonas pisi 4 . Scanning electron microscope studies on fractured leaf tissue . Ph_ytopathologisehe zeitschrift 106, 1-15, 18 . SIMON, R ., PRIEFER, U . & PUHLER, A . (1983) . A broad host range mobilization system for in vivo genetic engineering : transposon mutagenesis in Gram negative bacteria . Biotechnology7, 784-791 . 19 . SONOKI, S . & KADO, C . I . (1978) . Proteins conferred by the virulence-specifying plasmid ofAgrobacterium tumefaciens C58 . Proceedings of the National Academy of Sciences, United States of America 75, 3796-3800 . 20 . STASKOWIcz, B . J . & PANOPOULOS, N . J . (1979) . A rapid and sensitive microbiological assay for phaseolotoxin . Phytopathology 69, 663-666 . 21 . TSAI, C .-M . & FRASCH, C . E . (1982) . Sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels . Analytical Biochemistry 119, 115-119 .