Cultivar-strain specificity between Chrysanthemum morifolium and Agrobacterium tumefaciens

Physiological and Molecular Plant Patholo

309

(1991) 39, 309-323

Cultivar-strain specificity between Chrysanthemum morifolium and Agrobacterium tumefaciens ARLA L . BUSHt and STEVEN G . PUEPPKE Department of Plant Pathology, University of Missouri, Columbia, Missouri 65211, U .S.A . (Accepted for publication August 1991)

We have examined cultivar specificity between Chrvsanthemum morifolium and Agrobacterium tumefaciens strains Chry5 and B6 . Although 42 °;o of the 85 cultivars formed tumours after inoculation with both strains, some cultivars formed no tumours in response to Chry5, to B6, or to both strains . Four cultivars were utilized to examine these interactions in a leaf disc transformation system with a virE : :lacz fusion . We specifically tested the hypothesis that cultivar-strain specificity is due to differential vir gene induction by conditioned media from the cultivars, but could find no evidence to support this possibility . We also tested the hypothesis that specificity is due to differential T-DNA transfer, integration or expression . This was accomplished by transferring into each strain a scorable marker (/3-glucuronidase) contained between T-DNA borders . Leaf discs then were inoculated, and expression of enzyme activity was used to monitor transformation . Transfer of the T-DNA marker gene followed a pattern that reflected tumorigenicity in all cultivar-strain pairs, indicating that cultivar-strain specificity was due to differences in the transfer of T-DNA, or in subsequent integration processes . Analysis of the auxin and cytokinin levels of cultivars representative of different cultivar-strain interactions suggested that, although high hormone levels may be associated with high tumour mass, they do not appear to account for the innate susceptibility of a cultivar .

INTRODUCTION

Agrobacterium tumefaciens is a well-known phytopathogen that causes the disease crown gall . Although the species is able to infect a wide variety of plants [12, 13], individual strains are much more limited in their host range [2] . The host range of a strain is most commonly characterized as wide or narrow, depending on the number of plant species that are susceptible [27, 42] . Host range also can be more finely dissected, according to the different cultivars of a host species that can form tumours . Such cultivar specificity has been described for many different host species, including alfalfa [31 ], castor bean [16], cucurbits [38, 43], grape [25, 41 ], Kalanchoe [4], moth bean [15], oilseed rape [36], pea [19], potato [45], soybean [7, 26, 37], sunflower [5], and tomato [28], but never has been explained adequately . Cultivar specificity is particularly evident with Chrysanthemum morifolium [32] . A wide selection of Chrysanthemum cultivars was screened in the mid 1970s for susceptibility to Missouri Agricultural Experiment Station Journal Series No . 11453 . t'ho whom correspondence should be addressed . Present address : Department of Plant Pathology, Iowa State University, Ames, Iowa 50011, U .S .A . Abbreviation used in text : GUS, Escherichia coli : f-glucuronidase gene ; PBS, phosphate buffered saline ; IAA . indole acetic acid ; X-gluc, 5-bromo-4-chloro-3-indolyl-f-glucuronic acid . 0885 -5765/91/100309+15 $03 .00/0

1991 Academic Press Limited siPP 3o



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a chrysanthemum strain of A . tumefaciens and reference strain B6, and ,orne of these cultivars exhibited striking strain-specific responses to either or both strains . Although knowledge of the genetics of tumorigenicity has advanced substantially during the past 15 years, these host-specific interactions with chrysanthemum have not been confirmed systematically nor further examined . Cultivar-strain specificity between a naturally occurring isolate on its original host, and between this host and a thoroughly characterized strain of A . tumefaciens, makes Chrysanthemum an attractive system to examine this phenomenon . Consequently, we have obtained the original chrysanthemum strain (designated Chry5) which demonstrated cultivar specificity [32] and begun to examine it. Recently presented data show Chry5 is biologically distinct from previously described strains [6] . Here, we extend the previous observations of specificity in the chrysanthemum system [32] . In addition, we describe a leaf disc transformation system for chrysanthemum and use it to test two hypotheses that might explain the observed cultivar-strain specificity . The first hypothesis states that specificity is the result of differential vii gene induction by the different cultivars . This was tested by measuring activity of a

virE : : lacZ

fusion [39] in a medium conditioned by leaf discs of different cultivars . The second hypothesis states that cultivar-strain specificity is the result of differential T-DNA transfer, integration or expression . This was tested with an expression vector containing the Escherichia coli f-glucuronidase gene (GUS) with a eukaryotic promoter between T-DNA borders [8] . We show that cultivar-strain specificity in Chrysanthemum is due to differential transfer, integration or expression, and is not due to differential vir gene induction .

MATERIALS AND METHODS

Bacterial media and strains The A . tumefaciens strains

and their sources are as follows : B6 (A . Matthysse, University of North Carolina, Chapel Hill), Chry5 (R . E . Stall, University of Florida, Gainesville), and the pTi cured strain A136 (E . Nester, University of Washington, Seattle) . E. coli strains D5a(pSM358) and D5a(pMSG) were from R . Morris and L . Castle, University of Missouri, Columbia . Working cultures were maintained on LB [30 ], or gluconatemannitol medium [3] plus appropriate antibiotics at 5 ° C ; long-term storage was at - 70 ° C in 15 °,o glycerol . Prior to use, bacterial cultures were grown overnight in LB, AB minimal medium [10], or gluconate-mannitol medium at 30 ° C on a gyratory shaker at 150 r mint , harvested by centrifugation for 1 min in a tabletop microcentrifuge, and resuspended to the appropriate concentration, in either liquid growth medium or phosphate-buffered saline (PBS, g 1 -1 : 0 . 43 g KH 2 PO 4 , 1 . 48 g Na 2 HPO 4 , 7 . 2 g NaCl, final pH 7 . 2), as indicated for each experiment . Triparental bacterial matings were performed essentially as described by Ditta et al . [14], with E. coli strain DH5a containing pSM358 (virE2 : : Tn3HoHol to form a virE : : lacZ fusion gene, Kin' and Cb') [39] or pMSG (T-DNA binary vector with linked /3-glucuronidase under the control of a mannopine synthase promoter and npt genes between T-DNA borders, Tc') [8] as the donor . Helper functions were provided by pRK2013, and A . tumefaciens strains B6 and Chry5 served as the recipients . Trimethoprim (50 mg 1 - ') was used to counter-select against E. coli . B6(pSM358),



Cultivar-strain specificity

311

Chry5(pSM358), B6(pMSG) and Chry5(pMSG) were selected and maintained on appropriate antibiotics . Plant culture and greenhouse cultivar screen Cultivars of Chrysanthemum morifolium were donated by Yoder Brothers (Barberton,

Ohio) as rooted cuttings . Plants were grown in 15-cm pots in autoclaved soil, two or three plants per pot under greenhouse conditions (usually 21-27 ° C, occasionally up to 38 °C in the summer) without supplemental illumination . Plants were watered daily, and fertilized bi-weekly with a commercial fertilizer (Peters 15-16-17) . Plants were inoculated with strain A136 as a non-tumorigenic control), B6 or Chry5 when the cuttings had at least five or six nodes with fully expanded leaves . In a preliminary experiment, 84 cultivars were leaf-inoculated as described [32] . Four leaves on two plants were inoculated with each strain and scored for tumour formation at 28 days after inoculation . In the second experiment with 22 cultivars, 15 plants of each cultivar were stem-inoculated by piercing the stem once with a sterile hypodermic needle and depositing a droplet of inoculum (10 8 cells ml- ' in PBS) on the wounds created on opposing sides of the stem . Plants were scored for tumour formation weekly from 28 to 76 clays after inoculation . Leaf disc tumorigenesis assay

A leaf disc infection protocol for chrysanthemum was devised for mechanistic studies of transformation under uniform culture conditions and with maximum efficiency of space utilization . Based on the patterns of reaction with B6 and Chry5 in the greenhouse studies, cultivars Gem, Neptune, Puritan and Splash were chosen for these experiments . Plants of each were maintained in a growth chamber with a 16 h illumination daily at approximately 20 pE m -2 s -- ' of photosynthetically active radiation at 25 ° C . Leaf discs were prepared and inoculated according to a modified version of the protocol described by Horsch et al . [21 ] . Healthy leaves were washed in soapy water, surface sterilized by a 1 min submersion in 950" ethanol, rinsed in autoclaved distilled water, submerged in 1 °,io sodium hypochlorite plus one drop Tween 20 for 5 min and then rinsed three times in autoclaved distilled water . Leaves were cut with a hand-held paper punch, avoiding the main vein, but including branch veins at random, to create 6 . 5-mm discs . Prior to co-cultivation with bacteria, discs were preincubated for 1 day on MS agar medium [17], which was designated MS+ when supplemented with hormones (1 mg kinetin and 0 . 1 mg 2,4-D 1 - '), and MS when hormones were not added . Discs were incubated abaxial side up, at 25 ° C and 16 h illumination (40 .tE m- ' s - ') . After preincubation for 1 day, discs were immersed for 5 min in PBS containing Chry5 or B6 at 10 8 cells ml - ' . The negative control consisted of discs immersed in PBS lacking bacteria . Discs were blotted dry on autoclaved paper towels and placed in groups of approx . eight discs per section in Y-plate Petri dishes (Falcon No . 1004) containing 8 ml of 0.8 % water agar per section . Discs from each preincubation and cocultivation treatment were placed in separate sections . After co-cultivation for 3 days with bacteria, discs were transferred to Y-plate dishes, each section of which contained 8 ml of MS- medium supplemented with cefotaxime (250 mg 1 - ') and nystatin (10 mg l- ') . Separation of treatments was maintained . Discs were transferred to fresh 22_'



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A . L . Bush and S . G . Pueppke

plates of MS- medium containing cefotaxime and nystatin 17 days after the start of co-cultivation . Twenty-eight days after the start of co-cultivation, the discs were removed from the agar medium, dried for 5 days at 65 ° C, and weighed individually . The difference in weight between control and bacterially treated discs indicated the amount of tumour formation . Experimental sets consisted of batches of 22-48 discs from each preincubation treatment, which then were divided into three groups for cocultivation with B6, Chry5 or cultivation in the absence of bacteria . The experiment was repeated three times for Neptune and Splash and four times for Gem and Puritan . Data from all experiments were pooled .

Hormone analysis The hormone content of leaf tissue of selected cultivars was determined . Free and total forms (free, ester and amide) of indole acetic acid (IAA) were extracted and quantified from 0 . 5 g samples by gas chromatography and mass spectroscopy as described by Cohen et al . [11] and Chen ei al . [9] . Ten gram leaf samples were extracted for cytokinin analysis of free and glycoside-bound cytokinins according to the methods of Morris [35], and the concentrations in zeatin riboside equivalents were measured by enzyme-linked immunoabsorbent assay [29, 34] .

Assay of vir gene induction Strains B6(pSM358) and Chry5(pSM358) were employed to monitor the induction of the vir genes by conditioned media from cultivars Gem and Puritan . The liquid MS medium to be used for cultivating discs, adjusted to pH 5 . 6, contained 5 mm MES, (2-(N-morpholino)ethanesulphonic acid), and was supplemented with hormones as indicated . Leaf discs were prepared as described above . Batches of 10 freshly prepared discs were cultured in 10 ml of liquid MS + or MS - medium in 20 mm x 125 mm culture tubes, and incubated at 25 ° C for 3 days on a gyratory shaker at approximately 100 r min -1 . The conditioned medium thus produced was filtered through a 0 . 22 tm nitrocellulose filter, aseptically dispensed into Eppendorf-type tubes in I ml amounts for induction experiments, and inoculated immediately . Cells of B6(pSM358) and Chrys(pSM358) were concentrated to a turbidity of 2 .5 at 600 nm in MS- medium ; 1 ml of conditioned medium was inoculated with 10 µl of bacterial suspension and incubated at 25 ° C for 24 h with shaking as above . Fifty microlitres of the culture were mixed with 450 µl of Z buffer [33], and fl-galactosidase activity was determined as described [39] for two replicate samples per culture . The data from four experiments were pooled to give the average induction .

Assay for T-DNA transfer Strains B6(pMSG) and Chry5(pMSG) were used to monitor T-DNA transfer as described [8, 23] . Leaf discs were prepared, preincubated on MS- or MS+ medium and co-cultivated with B6(pMSG), Chry5(pMSG) or in the absence of bacteria as described for leaf disc tumorigenesis experiments . After 3 days, discs were transferred to MS+ medium containing cefotaxime (100-250 mg 1 -1 ), nystatin (10 mg 1 - ') and antibiotic G418 (50 mg 1 -1 ) . The latter was used to select cells carrying the npt gene for aminoglycoside resistance conferred by the T-DNA of pMSG . Ten days after the start of co-cultivation, discs were harvested and incubated overnight in 5-bromo-4-chloro-



313 Cultivar-strain specificity ; Research Organics, Cleveland, Ohio) at 3-indolyl-fl-glucuronic acid (X-gluc 0) containing 10 mm EDTA . After 0 . 1 mg ml -' in 0. 1 M phosphate buffer (pH 7 . incubation for 12-16 h on a gyratory shaker (approx . 100 r mini ) at 37 °C, discs were rinsed with water and their margins were examined with a dissecting microscope for cells containing the insoluble blue dye . A total of 83-129 control discs and 100-216 bacterially treated discs were examined for each cultivar, divided between five (Neptune and Splash) or six (Gem and Puritan) experiments . Data from all experiments were pooled . RESULTS Greenhouse cultivar screen

A preliminary screening of chrysanthemum cultivars confirmed the following cultivarstrain specificity : 35 cultivars formed tumours in response to both Chry5 and B6 42 °%,), 16 in response to neither strain (16 %), 24 in response to Chry5 only (28 0 ) , /

TABLE 1

Response of Chrysanthemum morifolium to Agrobacterium tumefaciens strains B6 and Chry5

Percentage of plants' with tumours after inoculation with : B6 Cultivar

28 days

Chry5 76 days

28 days

76 days

Cultivars that produced tumours in response to both strains Goldstrike 100 100 100 Neptune 100 100 100 Ping Pong 80 100 93 Roll Call 0 67 100 Stardom 40 54 100 Yellow Delaware 0 13 100 Yellow Dignity 0 7 100 Indian Summer 0 7 0

100 100 100 100 100 100 100 80

Cultivars that produced tumours in response to neither strain Cloud-9 0 0 0 Nob Hill 0 0 0 Polaris 0 0 0 0 Splash 0 0 0 0 0 White Sands Yellow Nob Hill 0 0 0

0 0 0 0 0 0

Cultivars that produced tumours in response to Chry5 only Gem 0 0 100 White Marble 0 0 100 Super Yellow 0 0 47 Yellow Mandalay 0 0 0 Mandalay 0 0 0 Mandarin 0 0 0

100 100 54 33 7 7

Cultivars that produced tumours in response to B6 only Puritan 0 27 Starburst 7 7

0 0

' A total of 15 plants were stem-inoculated with each strain .

0 0

0



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A . L . Bush and S . G . Pueppke

and nine in response to B6 only (I 1 "

. Details of these experiments are available from us upon request . Table I shows the results of the second experiment, which was more carefully controlled to decrease the probability of false positives arising from crosscontamination, which included 21 potentially interesting cultivars from the preliminary experiment plus Super Yellow . Again, all four categories of response were represented . Only eight of the nine cultivars that originally appeared to be specifically susceptible to B6 were available for retesting . Of these, two (Puritan and Starburst) survived the second screen . Four of the others 1 Cloud-9, Nob Hill, Polaris and Yell Nob Hill l did not respond to either strain, even after 76 days . Indian Summer gave a delayed response with both strains, and Stardom appeared to be uniformly susceptible i Table 1 In general, the responses of the cultivars from the other three categories were the same at 28 days in both experiments, although the response of Roll Call to B6 was delayed . After 76 days, however, several cultivars that originally had appeared resistant were forming a few tumours in response to either B6 (Yellow Delaware, Yellow Dignity) or Chry5 (Yellow Mandalay, Mandalay and Mandarin) . Thus, in some cultivars, tumour formation either was delayed . or tumours grew so slowly that considerable time was required before they were detectable . Leaf disc tumorigenesis Four chrysanthemum cultivars were selected to represent the four response categories found in the greenhouse assay, and conditions were determined for leaf disc tumorigenesis . On whole plants, tumours were produced by Gem in response to Chry5 only, by Puritan in response to B6 only, by Neptune in response to both strains, and by Splash in response to neither strain . In addition to determining the sensitivity of leaf discs of these cultivars to A . tumefaciens strains B6 and Chry5, the effect of disc preincubation on medium containing or lacking hormones was examined . Photographs of discs from a representative experiment are shown in Figs 1 and 2 ; Table 2 quantified tumorigenesis based on average disc dry weights . When Gem discs were co-cultivated with Chry5, 100 0 of the discs formed more or less confluent tumorous masses, regardless of whether the discs were preincubated in the presence or absence of hormones (Fig . 1) . The non-bacterial control discs produced a small amount of callus, a response that was enhanced by hormone pretreatment . The discs co-cultivated with B6 were very similar to the controls, both in appearance and in dry weight in the minus hormone pretreatment . Although discs in the hormone pretreatment appeared to be slightly heavier and have slightly larger masses than their control counterparts, the difference was not statistically significant (Table 2) . Neptune produced tumours in virtually 1000)) of the discs, regardless of hormone pretreatment or strain (Fig . 1 ; . The frequency of' the tumours increased when hormones were present in the preincubation medium (Fig . 1), as did disc dry weights (Table 2) . There was very little callus production by the control discs in either hormone pretreatment . Although tumours were formed by Puritan in response to B6 in both hormone pretreatments, the presence of hormones increased the size and frequency of the response (Fig . 2) . Although B-inoculated discs from the MS- preincubation medium were not significantly heavier than the Chry5-inoculated discs (Table 2), an average

Cultivar-strain specificity

MS-hormones

31 5

MS + hormones

Fin . 1 . Leaf disc tumorigenesis in response to A . tumefaciens B6, Chry5 or to controls lacking bacteria, and the effect of hormones on tumor formation . Discs in all plates are arranged as shown in the upper left plate : B6, B6 inoculated ; Chry5, Chry5 inoculated ; C, control .

of 67 0/o of the B6-inoculated discs were tumorous in the four experiments . This increased to 98 0o when hormones were included in the preincubation medium . Discs co-cultivated with Chry5 were non-responsive in almost all instances, but occasionally a tiny tumour or thickening of the disc edge occurred . There was essentially no callusing by the control discs . In contrast with the uniformly negative whole plant response, leaf discs of the cultivar Splash followed the same pattern as Gem, but to a lesser extent . The control discs produced some callus on the plus hormone pretreatment, and an occasional mass in the minus hormone pretreatment (Fig . 2) . Discs co-cultivated with B6 were the same as the controls, both in appearance and dry weight (Fig . 2, Table 2) . Chry5 caused tumour formation in 100% of the discs in both hormone pretreatments, with an

31 6

A . L . Bush and S . G . Pueppke

MS-hormones

MS + hormones

FIG . 2 . Leaf disc tumorigenesis in response to d . lumefaeieu B6, Chry5 or controls lacking bacteria, and the effect of hormones on tumor formation . Discs in all plates are arranged as shown in the upper left plate : B6. B6 inoculated : Chrv5, Chry5 inoculated ; C . control .

increase in tumour frequency and disc dry weight when hormones were included in the preincubation medium (Fig . 2, Table 2) . Endogenous hormone levels

Endogenous levels of IAA and cytokinin were measured in the four cultivars used to examine specific interactions . These measurements are highly labour-intensive, therefore the number of samples was small, and the data should be interpreted accordingly . Hormone concentrations were as follows : Levels of free and total IAA were 8 . 3 and 24. 6 ng g - ' for Gem, 6 . 7 and 12 . 2 ng g- ' for Neptune, 5 . 8 and 10. 2 ng g - ' for Puritan and 5 . 7 and 8 . 5 ng g- ' for Splash . Data for free IAA were from three Gem samples and two samples each for Neptune, Puritan and Splash . Total IAA values were



Cultivar-strain specificity

317 TABLE

2

Dry weight of leaf discs offour C . morifolium cultivars after co-cultivation with A . tumefaciens strains B6, Chry5, or in the absence of bacteria

Bacterial strain Cultivar Gem Neptune Puritan Splash

Preincubation medium'

None

MSMS+ MSMS+ MS_ MS+ NISMS+

6 .8+0. 9° 8-0+1-6 3 .8±0. 8 4. 3±1 . 2 3-2+0-7 3-6+1-0 1.0 6-2+1-0 8-1±1 . 3

B6

Chry5

6. 8+1-0 8-8+2-5 80±3 . 8 132±3 . 8 4-4+1-5 7-9+2-9 6-4+0-9 80±1 . 1

13 . 4+2 . 3 16-5+2-7 7 . 5±2 . 4 11 . 9±2 .8 4-0+0-8 4-8+1-2 10-3+2-4 2 .4 12 . 2±3. 1

' NIS-, no hormones added to preincubation medium . MS+, 1 . 0 mg kinetin and 0 . 1 mg 2,4-D-1-r added to preincubation medium . 'Dry weight (mg per disc) of leaf discs+ SD .

7 ∎ E ∎ ®

B6(pSM358)-horm B6(pSM358)+horm Chry5(pSM358)-horm Chry5(pSM358)+horm

Puritan

Gem Cultivar

Fin . 3 . Induction of a virE : : lacZ gene fusion in conditioned medium containing (+) or lacking -) hormones. Levels of induction are expressed as the ratio of /3-galactosidase activity in conditioned medium to that in non-conditioned plant culture medium . Bars represent SD of pooled data from four experiments .

from one sample per cultivar . Measured as zeatin riboside equivalents, Gem contained g_°, 0. 29 and 0 . 15 ng g - ', Neptune 0 . 21 and 0 . 03 ng Puritan 0 . 19 and 0 . 09 ng g-1 and Splash 0 . 21 and 0 . 08 ng g- ' of free and glycoside bound cytokinin, respectively . Data were from two samples per cultivar in all cases except the free form in Neptune, which was based on one sample . Vir gene induction Fig . 3 shows the induction of a plasmid-borne copy of virE : :lacZ in strains Chry5(pSM358) and B6(pSM358) as a function of host cultivar and the presence of hormones in the induction medium . Although Gem formed tumours specifically with



318

A . L . Bush and S . G . Pueppke

Chry5, conditioned medium from Gem was equally effective in inducing virE in Chry5 and B6 . The presence of hormones in the medium enhances induction, but this effect is not strain-specific . Puritan, in contrast, formed tumours only with B6 . Induction of virE by conditioned medium from Puritan was uniformly high, and was affected neither by the strains containing the virE : : lacZ fusion nor by the presence or absence of hormones . The average level of virE induction in non-conditioned plant culture medium ranged from 91 .5 to 958 Miller units [331 . The presence of hormones in the non-conditioned medium had no significant effect on this value, and the basal level of

virE induction was not strain specific . T-DNA transfer GUS activity is a marker for the successful transfer, integration and expression of the engineered T-DNA contained in the pMSG plasmid . Transformation was quantified by counting the number of transformation sites, which were either single blue cells, or clumps of blue cells that appeared to arise from a single transformation event . Fig . 4 (a)

100 (a)

∎ B6(pMSG) ® Chry5(pMSG)

U

v'

80

0 v 40 o+ 0 c 20 a I Gem

Neptune

I Puritan

p Splash

Cultivar

Fia . 4 . GUS expression in leaf discs preincubated in the absence of hormones, and co-cultivated with strains B6(pMSG) and Chry5(pMSG) . (a) Percentage of discs with a positive response. (b) Average number of GUS-expressing spots per positive disc . Bars represent SD values from five (Neptune and Splash) or six (Gem and Puritan) pooled experiments .



Cultivar-strain specificity

319

presents the percentages of discs containing GUS expressing cells, and Fig . 4(b) gives the average number of transformation sites per positive disc . Preincubation in the presence of hormones slightly increased the efficiency of GUS transfer, similar to the effect on tumour formation, but did not significantly change cultivar x strain interactions . Thus, for the sake of simplicity, Fig . 4 contains only the data for the minus hormone treatment . GUS expression showed the same striking pattern of cultivar-specificity as tumorigenesis on leaf discs . Chry5, but not B6, formed tumours on Gem . Over the course of five experiments with Chry5(pMSG), 49 of 63 Gem discs (78 %) had at least one transformation site, with an average of seven sites per positive disc . The response to B6(pMSG) was very rare ; there were just two positive discs out of 70 (3 %), and each of those contained only a single site . In contrast, Puritan responded more strongly to B6(pMSG) than to Chry5(pMSG) . B6 formed tumours on this cultivar but Chry5 did not . After co-cultivation with B6(pMSG), 16 of 108 discs (15%) were positive, averaging over one site per disc . The corresponding rate of success for Chry5(pMSG) was very low (a single site on one of 69 discs) . Splash leaf discs produced tumours in response to Chry5 but not B6 . This cultivar gave a uniformly negative response to B6(pMSG), but 15 out of 50 discs treated with Chry5(pMSG) (30%) produced an average of over three sites per positive disc . In contrast, four of 54 (7 %) Neptune discs co-cultivated with B6(pMSG) were positive, and co-cultivation with Chry5(pMSG) resulted in 10 positive discs out of 37 (27 ;o . Both strains produced tumours on this cultivar . Discs co-cultivated with B6(pMSG) contained approximately one site per positive disc, while those discs co-cultivated with Chry5(pMSG) contained an average of two sites .

DISCUSSION

Chrysanthemum cultivars vary strikingly in their responses to inoculation with A . tumefaciens strains Chry5 and B6 . Our results and those of Miller et al . [32] are generally similar . Discrepancies are mainly quantitative and involve cultivars reported as developing small delayed galls, or no galls . For example, cv . White Sands earlier produced tiny, delayed galls in response to B6, but in our hands it did not respond to B6 at all . The total number of Chrysanthemum cultivars examined for susceptibility to Agrobacterium is now 259, the largest number of cultivars of a single species that has ever been tested . Our study demonstrates for the first time that Chrysanthemum cultivars exhibit quantitatively different responses to inoculation by the two strains, as measured by the percentage of plants that form tumours . B6 tends to form tumours on a lower percentage of plants than Chry5 on the cultivars susceptible to both strains . This is particularly evident on cultivars such as Yellow Delaware, Yellow Dignity and Indian Summer, and it suggests that strain B6 is more limited than Chry5 in ability to cause tumours on any particular cultivar and on any individual plant of a susceptible cultivar . It is not surprising that a majority of cultivars show a bias in favour of tumour formation in response to Chry5, since this strain occurs naturally on Chrysanthemum [32], as opposed to the apple strain, B6 . The two cultivars, Puritan and Starburst, that responded in a reverse fashion are the ones of special interest . Unlike cultivars that



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A . L . Bush and S . G . Pueppke

formed no tumours at all, Puritan and Starburst are amenable to transformation, but tumour formation in response to the chrysanthemum strain is somehow inhibited . Levels ofendogenous hormones are suspected to play a role in tumour formation, but their importance is not clear [40 1 . Although the limited numbers of samples and cultivars subjected to hormone analysis preclude firm conclusions based on the hormone levels detected, some inferences can be made . First, endogenous auxin and cytokinin levels do not appear to correlate with the susceptibility of a cultivar to infection, but there may be some relation to the size of the tumours that do form . The cultivar Gem contains slightly higher levels of auxin and cytokinin in their free forms, and distinctly higher levels of both in their bound forms, than the other three cultivars . Gem also is the cultivar on which (i tumours formed in confluent masses on leaf discs inoculated with a tumorigenic strain, (ii) hormone preincubation resulted in only a limited enhancement of tumour formation, and (iii) control discs formed significant amounts of callus . Thus, Gem may contain adequate levels of endogenous hormones to support tumour formation, whereas the other three cultivars, with lower endogenous levels, respond more to exogenously supplied hormones in compatible cultivar-strain pairs . Whole plants are not amenable to mechanistic studies of transformation . We therefore selected a cultivar representative of each response category and re-evaluated

Agrobacterium

tumorigenesis with leaf discs, using dry weights and photographic records to indicate the degree of tumour formation . The leaf disc responses of Neptune and Puritan were the same as in whole plants . Gem leaf discs gave the same response as the whole plants in that Chry5 is strongly tumorigenic, but there was some ambiguity in the B6 x Gem combination . The callus tissue formed on Gem control discs showed that the hormonal status of this cultivar allows at least limited growth on medium lacking hormones . Thus, hormone-independent growth is not a reliable marker for transformation in this cultivar over the time course of this experiment . Splash was the only cultivar in which whole plant and leaf disc responses were distinct . Stem inoculations of Splash did not produce tumours in response to either Chry5 or B6, but the leaf discs did form tumours in response to Chry5 . Leaf discs appeared to be more sensitive than the whole plant, as indicated by the limited amount of tumorous tissue that was formed in cultivar x strain combinations that did not produce tumours in the whole plant . It was unclear if this tissue was transformed or was a wound response, since the control discs of all cultivars formed varying amounts of callus . Increased sensitivity in in vitro transformations of leaf discs or leaf-petiole explants compared to whole plant inoculations have been observed in petunia and soybean [22, 26], so this differential is not unusual . While leaf disc tumorigenesis was less clear-cut than the whole plant inoculations, the cultivar-strain specificity still was evident, making leaf discs an appropriate system for further investigations of the phenomenon . Earlier work suggested that the extent of vir gene induction correlates with tumorigenicity [1, 20, 24, 44] . Thus, our first hypothesis was that vir gene induction is greater in the compatible combinations than in incompatible combinations . One explanation for this might be that the cultivars produce different inducers which may have a strain-specific effect on the vir genes . In particular, this hypothesis predicts that conditioned medium from Gem induces vir genes in Chry5 to a greater extent than



Cultivar-strain specificity 321 those in B6, while the opposite is true for Puritan . These predictions were not supported by induction experiments with a virE : : lacZ fusion plasmid in otherwise wild-type strains . Each cultivar induced the vir genes in both strains to approximately the same degree, but Puritan-conditioned medium had inherently greater activity than that of Gem . We conclude that vir induction is not the limiting factor in transformation . Since the presence of hormones in the preincubation medium generally enhanced tumour formation, their effect on vir induction also was examined . Hormones in the preincubation medium increased the response of Puritan to B6 more so than that of Gem to Chry5, as indicated by tumour formation on leaf discs, In the vir-induction assays, however, the presence of hormones enhanced induction in Gem-conditioned medium, but had no effect in Puritan-conditioned medium . Therefore, the enhancement of tumorigenicity by preincubation in the presence of hormones does not appear to be the result of increased vir induction . We also tested the hypothesis that cultivar-strain specificity is due to differential transfer and integration, or expression of T-DNA . A GUS transformation vector was used to distinguish between these possibilities . If the strains differ in their ability to transfer or integrate T-DNA into different cultivars, a process controlled by bacterial genes lying outside the T-DNA borders [18] and the plant genome, then there should be similar differences in the transfer of the GUS-containing T-DNA . Alternatively, if specificity is due solely to differential expression of T-DNA genes of Chry5 or B6, or the response of a cultivar to these genes, then the GUS marker gene should be transferred and expressed with equal efficiency in each cultivar-strain pair . Our results in fact confirmed a pattern of GUS expression that faithfully reflected the tumorigenicity in all cultivar-strain pairs . This implicates non-transferred bacterial genes, the host genome, or the interaction between the two as major determinants of specificity in this system . Compared to tumour formation, GUS expression occurs with low efficiency . This was illustrated by comparison of the percentage of discs that formed tumours to those that had any GUS expression . For example, whereas 100 0 of the Neptune discs inoculated by either Chry5 or B6 produced tumours, only 27 °0 and 7 % of the discs inoculated by those strains carrying pMSG expressed detectable GUS . McCormick et al . [28] also noted that transfer of T-DNA in a binary vector system may be less efficient than that of a wild-type strain with integrated T-DNA . In conclusion, we compared the response of whole plants and leaf discs of Chrysanthemum morifolium to Agrobacterium tumefaciens strains Chry5 and B6, and we examined cultivar-strain specificity as demonstrated by cultivars Gem, Neptune, Puritan and Splash . We found that leaf discs were more sensitive to tumour formation than whole plants, and that preincubation of the leaf discs on medium containing hormones could increase tumour formation, but not change the qualitative response of a cultivar to a strain . Cultivar-strain specificity in Chrysanthemum does not appear to result from differential vir gene induction, but rather from differences in effective transfer or integration of T-DNA . 1 0

We thank R . O . Morris and J . Cohen and the members of their respective laboratories for their assistance in the hormone analyses . We thank Linda Castle for the gift of pMSG . We also thank A . Novacky and D . Blevins for reviewing the manuscript .



322

A . L . Bush and S . G . Pueppke A . L . B . was supported by a pre-doctoral fellowship for the Food of the 21st Century Program, University of Missouri . This is journal Series No . 11453 of the Missouri Agricultural Experiment Station .

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