Inheritance of Resistance to Crown Gall in Pisum sativumI - NCBI

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Steven L. Robbs, Martha C. Hawes*, Hao-Jan Lin, Steven G. Pueppke, and Laura Y. Smith ... found to bethe cause of crown gall disease by Smith and.
Received for publication July 12, 1990 Accepted September 20, 1990

Plant Physiol. (1991) 95, 52-57 0032-0889/91 /95/0052/06/$01 .00/0

Inheritance of Resistance to Crown Gall in Pisum sativumI Steven L. Robbs, Martha C. Hawes*, Hao-Jan Lin, Steven G. Pueppke, and Laura Y. Smith Sugarcane Field Station, Canal Point, Florida 33438 (S.L.R.); Departments of Plant Pathology and Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85721 (M.C.H., H.J.L.); Department of Plant Pathology, University of Missouri, Columbia, Missouri 65211 (S.G.P.); and Department of Plant Sciences, Montana State University, Bozeman, Montana (L.Y.S.) are hampered by our lack of information about host genes that function in tumorigenesis. Although genotypic variation in susceptibility has been observed with several species (for example, refs. 4, 8, 15, 18), studies on inheritance have been limited to grapevine (12, 20). We recently developed an assay that results in a 95 to 100% infection frequency on susceptible pea cultivars and used it to demonstrate the occurrence of quantitative variation in susceptibility of 34 cultivars (4). The objectives of the current study were to identify variation in responses of more than 1300 pea genotypes. We characterized resistance observed in one cultivar and identified three 'supersusceptible' genotypes that developed unusually large tumors.

ABSTRACT We screened a total of 1365 pea (Pisum sativum) lines for response to inoculation with Agrobacterium tumefaciens, strain B6, and characterized resistance in one cultivar, Sweet Snap. Sweet Snap seedlings were highly resistant to tumorigenesis under most conditions. Resistance was overcome at inoculum concentrations of greater than 109 bacteria per milliliter. At such high concentrations, very small tumors developed on Sweet Snap in response to four wide-host-range Agrobacterium strains, but tumors on other cultivars were two- to sevenfold larger than those that formed on Sweet Snap. The hypervirulent strain A281 induced larger tumors on Sweet Snap than did other Agrobacterium strains, but tumors on other genotypes were more than 100% larger than those on Sweet Snap. Physiological experiments suggested that tumorigenesis in Sweet Snap is not blocked in early stages of infection, and genetic analysis indicated that inheritance of resistance to crown gall is a quantitative trait. In addition to the observed resistance in Sweet Snap, three 'supersusceptible' genotypes, which developed very large tumors, also were identified.

MATERIALS AND METHODS Plant Material A total of 1331 plant introduction lines and breeding lines of pea (Pisum sativum L.) were a generous gift from the late Dr. G. A. Marx, Department of Horticulture, New York State Agricultural Experiment Station, Geneva. Seed of genotypes of interest was increased by planting in steam-pasteurized potting soil in 20-cm pots in a greenhouse. Plants were watered daily and fertilized weekly. Plants were allowed to self fertilize, and seed was harvested approximately 4 months after planting. Sources of commercial varieties used in this study are listed in a previous report (4). Breeding lines are available to interested readers upon request.

The soilborne bacterium Agrobacterium tumefaciens was found to be the cause of crown gall disease by Smith and Townsend in 1907 (19). Rapid progress has been made in understanding the molecular biology of the infection process since it was discovered that symptoms occur as a result of transfer of a bacterial DNA region (T-DNA) into the host genome (reviewed in refs. 2, 14, 24). A number of steps in the process can now be generally outlined: Bacterial cells bind to the plant cell surface and respond to plant phenolic signals by activation of virulence (vir) genes that function in the excision and transfer of T-DNA into the host cells. Upon stable incorporation of the T-DNA into the nuclear genome, T-DNA genes for hormone production are expressed, resulting in the overproduction of auxins and cytokinins, which induce cell proliferation that, in turn, leads to the development of galls. A large assortment of Agrobacterium strains and mutants have been used to dissect the pathogen's contribution to this disease. Efforts to understand the infection process in detail

Bacterial Strains

Agrobacterium tumefaciens strain B6 was used in screening for crown gall response and in other experiments unless otherwise indicated. Other biovar I strains used in these tests were A. tumefaciens B6 (16), T37 (16), A281 (9), A723 (3), C58 (16), and A. rhizogenes strain RIOOO (22). The Ti plasmid-cured strain A136 (3) was included as a control in all experiments. Single colony isolates were grown overnight on solidified yeast extract-mannitol medium and were suspended in sterile water for assays. Bacterial concentration was estimated turbidimetrically and confirmed by dilution plating. For all strains used in these experiments, an A620 of 1.0 equals 2.1 to 2.7 X 109 bacterial cells/mL.

'This study was supported by grants No. 86-CRCR- 1-2224 and No. 88-37234-4009 from the U.S. Department of Agriculture. This is journal series No. 7177 of the Arizona Agricultural Experiment

Plant Screening For all experiments, seeds were surface-sterilized by consecutive 5-min rinses in 95% ethanol and 50% commercial

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INHERITANCE OF RESISTANCE TO CROWN GALL IN PEA

bleach (the final concentration of sodium hypochlorite was 2.5%), but seeds were not chemically pretreated to inhibit microbial infection. For initial screening, individual seedlings were planted in 70-mL plastic urine collection vials (Fisher Scientific) filled with water-saturated vermiculite. Shoots were inoculated when they were about 2 mm long. A sterile dissecting needle was used to make two, about 1 mm deep, wounds on opposite sites of the stem. A l0-,uL droplet of bacterial suspension (5 x 109 cells/mL) was immediately placed onto each wound site. Water loss was minimized by affixing a plastic Whirl-Pak bag (Fisher Scientific) to the mouth of each vial. Each bag was slit to permit exchange of gases, and the vials were incubated under fluorescent lights (400 MuE- m-2 *-') for 14 to 16 h d. Tumorigenesis was rated 3 weeks after inoculation. Three individuals from each genotype were tested, and one control inoculated with strain A 136 was included. The response was considered positive if a macroscopically visible tumor developed on at least one of the three plants in response to inoculation to B6 but not to inoculation with A136. With the exception of genotypes that developed unusually large tumors, a given genotype was not evaluated further if any of the test plants developed visible tumors. When all three plants were negative, or all developed unusually large tumors in the initial screen, second and third tests of three individuals each were repeated in a growth pouch assay (4). Seeds were germinated on water agar. When roots were about 1 cm long, an incision 1 mm deep and 1 mm long was made with a scalpel. Seedlings were immersed for 5 min in strain B6 inoculum (5 x I09 bacteria/mL), planted in pouches, and maintained at 23°C day/20°C night, with 12 h of fluorescent light/d. The appearance of visible tumors was scored after 2 to 3 weeks. When the results were consistent-either none developed tumors, or all developed large tumors-in all three tests, plants were grown to maturity, allowed to self-fertilize, and seed was harvested. At least 20 individual progeny were assayed to confirm that the observed phenotype was not confined to the original seed lot. Physiology of Resistance

The growth pouch assay was used to compare responses of Sweet Snap with responses of other pea genotypes to inoculation with different strains of A. tumefaciens and A. rhizogenes. In two independent experiments of 20 to 30 plants each, seedlings were inoculated as described above and were grown in pouches for 2 weeks before tumors were harvested. A 5-mm segment containing the site of inoculation was excised from each root and weighed. To derive tumor weight, mean weight of at least 30 sections from control plants inoculated with A 136 was subtracted from the value for plants inoculated with virulent strains. A root cap cell binding assay (5, 6) was used to compare binding of A. tumefaciens to cells of Sweet Snap and other cultivars. Root cap cells were isolated from aseptically germinated seedlings grown on water agar (0.8%) overlaid with filter paper. Root tips were immersed for 20 to 60 s in 50 ML of sterile distilled water in wells of a microtiter plate, and the water was agitated to release the cells. For binding assays, cells were adjusted by direct counts to a concentration of approximately 100 cells/100 ,L of water. The concentration of

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bacteria was adjusted to 2 x 107/mL, and 50 ML were added directly to root cap cell samples. After incubation for 2 h at 25°C, a 20-ML sample of cells was washed over a 10-,um mesh screen to remove unbound bacteria. Binding was evaluated by direct microscopic observation in one focal plane of the number of bacteria bound to the perimeter of each cell (5, 6). A swarm plate chemotaxis assay (7) was used to assess attraction of A. tumefaciens to wounded root tissue from Sweet Snap and Wando. Sections of root tissue were placed at the edge of a Petri plate (5 cm in diameter) containing 5 mL of water solidified with 0.2% agar. A l0-ML droplet containing I07 bacteria was placed into the center ofthe plate. After 16 h, the ratio of the distance that the bacterial swarm moved toward roots and away froum roots, the chemotaxis ratio, was measured (7). To test the possibility that resistance to crown gall exists because Sweet Snap lacks chemicals needed to induce vir genes in A. tumefaciens, acetosyringone (70 uM), a known inducer of vir genes (24), was incubated with B6 inoculum for 24 h before the seedlings were inoculated as described above. Experiments were carried out to test the possibility that a reduced ability of A. tumefaciens to survive and grow in wounds of Sweet Snap accounts for the observed resistance. A l0-,uL droplet containing about 107 bacteria was placed onto a 1 mm x 1 mm wound in the root crown of Sweet Snap or Wando seedlings. After 24 h, a 1-cm segment of tissue containing the wound site was excised and macerated in sterile distilled water in a tissue grinder, and the extracts were diluted serially and plated onto nutrient medium to enumerate bacteria. Inheritance of Resistance

Sweet Snap self-crosses or crosses with Wando and Target were carried out in a greenhouse. Plants were grown in a greenhouse and were fertilized weekly and watered daily. When plants flowered after approxiumately 5 weeks, individuals were either allowed to self-cross or reciprocal crosses were made between Sweet Snap and Wando or Target. Seeds were harvested approximately 4 months after planting. Progeny were inoculated as described above, and after 2 weeks the total weight of the root section containing either a tumor or a healed wound site was determined for each plant. Alternatively, Fl plants were allowed to mature and to self-cross, and F2 progeny were inoculated and root sections containing inoculation sites were weighed. RESULTS Resistance None of the 1365 tested pea genotypes exhibited immunity to tumorigenesis in response to the concentrated inoculum utilized in screening. In initial tests, four genotypesP1164568, P1236493, PI164568, and P1244245-failed to develop tumors in triplicate tests and appeared to be immune to infection. However, up to 50% of the progeny from all four lines developed tumors. In a previous study, we found that individuals from bulked seed of the commercial cultivar Sweet Snap were much less

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Figure 1. Excised root sections (including those that did not develop visible tumors) from plants 2 weeks after inoculating with strain B6 (top row) or with Ti plasmid-cured strain Al 36 (bottom row) at a concentration of 5 x 109 bacteria/mL. Left to right: Sweet Snap, Wando, Target. Mean tumor weights were 4 ± 2, 18 ± 4, and 44 ± 5, respectively. All photos were taken at the same magnification.

susceptible to Agrobacterium tumefaciens than other tested cultivars (4). The current study revealed that Sweet Snap plants were iummune to tumorigenesis at concentrations of inoculum lower than 109 bacteria/mL; none of more than 100 plants inoculated with 5 x 108 bacteria/mL developed any visible tumors. Even at 109 bacteria/mL, Sweet Snap seedlings were highly resistant to disease development. Tumors that formed on Sweet Snap were much smaller (Fig. la) than those on susceptible cultivars like Wando (Fig. lb) and Target (Fig. lc). In addition, tumors on Sweet Snap plants remained sumall even after 8 weeks (Fig. 2A), whereas tumors on Wando were much larger at maturity than after 2 weeks (Fig. 2B). Physiology of Sweet Snap Resistance to Tumongenesis

Sweet Snap responses to inoculation with wide-host-range strains other than strain B6 were tested to determine if the observed resistance was strain-specific. When inoculated at 5 x I09 cells/mL with A. tumefaciens T37, A28 1, and A723, or with A. rhizogenes RIOOO, tumors that formed on five other genotypes were consistently two- to sevenfold larger than those on Sweet Snap (Table I). ln these studies, pea developed tumors in response to A. rhizogenes RIOOO rather than the hairy root disease typically associated with this Agrobacterium species (22). Weights of tumors that developed on all genotypes in response to inoculation with A28 1 were 2.5- to 5-fold greater than those induced by other strains.

Physiological assays revealed no differences in binding, chemotaxis, or in survival in wound sites between Sweet Snap and other genotypes. Each root cap cell of Sweet Snap as well as those ofother varieties bound an average of 13 ± 3 bacterial cells per perimeter, and there were no significant differences among the varieties. Strain B6 was strongly attracted to excised roots of Sweet Snap in the chemotaxis assay, and there was no difference in attractiveness of Sweet Snap roots compared with other varieties (data not shown). Addition of acetosyringone to inoculum prior to inoculation of Sweet Snap seedlings had no measurable effect on tumorigenesis (data not shown). In tests with commercial bulk seed, Sweet Snap seedlings frequently developed a rapid and localized necrosis in response to inoculation with A1 36 or with virulent strains. The necrosis occurred in response to wounding alone, however, and did not occur in progeny of the original seed lot. There were no differences in the numbers of viable bacteria recovered from wound sites of Sweet Snap compared with those of Wando (data not shown). Inheritance of Resistance

We carried out tests to confirm that the resistance observed in Sweet Snap is a heritable trait. In initial tests, Sweet Snap was self-crossed or crossed with the moderately susceptible genotype Wando (4), and progeny seedlings were inoculated with 5 x I09 bacteria/mL. The percentages of individuals with mean root section weights in the indicated size classes Table I. Mean Weight (mg) of Excised Tumors from Pea Cultivars Inoculated with High Concentrations (5 x 109/mL) of Different Strains of A. tumefaciens and A. rhizogenes Plants were grown in pouches for 2 weeks before tumors were harvested: a 5-mm segment containing the site of inoculation was excised from the root, and mean weight of at least 30 such sections from control plants was subtracted from the total weight to derive the tumor weight. Values are based on the averages of at least 50 replicates in two independent experiments. Agrobacterium Strain

Cultivar

Figure 2. Tumors (arrows) on Sweet Snap (A) or Wando plants (B) 8 weeks after inoculation with A. tumefaciens strain B6.

B6

A723

T37

4±1 5±1 8±1 SweetSnap DwarfGreySugar 12±1 16±2 16±2 13± 1 21±4 25±3 Alaska 19±3 39±3 27±2 Freezonian 21 ± 3 25 ± 7 25 ± 3 Thomas Laxton 28±3 16±3 15±3 SugarSnap

R1000 6±1

37±4 32±5 26±3 31 ± 6 27±3

A281

20±3 43±5 41±6 50±5 53 ± 6 52±7

INHERITANCE OF RESISTANCE TO CROWN GALL IN PEA

(including those from seedlings that did not develop tumors) from Wando and Sweet Snap parents, and from F2 progeny, are plotted in Figure 3. The mean weights of sections from inoculated Sweet Snap, Wando, and F2 progeny, 22 ± 4, 37 + 11, and 26 ± 8, respectively, were statistically distinct from each other, but ranges from the three categories overlapped. Mean weights of sections from control Sweet Snap, Wando, and F2 progeny (inoculated with Ti-plasmid minus strain A136) were 16 ± 5, 17 ± 4, and 16 ± 4 mg, respectively, and did not differ from each other. In subsequent experiments, Sweet Snap was self-crossed or was crossed with Target, which was found in a previous study to form larger tumors in response to inoculation with strain B6 than Wando (3). In these experiments, inoculum was reduced to 5 x 108 bacteria/mL, and responses were classified as 'no tumors', a healed wound site with no swelling (root section weight 10-19 mg); small to medium swellings or tumors (21-30 mg); or large tumors (31-40 mg). When seedlings were inoculated as described above and inoculated root sections were weighed, the ratio of Sweet Snap individuals with no tumors, small to medium tumors, or large tumors, was 40:0:0. For Target individuals, the ratio of individuals in each category was 0:0:35, and for FH progeny the ratio was 2:48:0. 35

0

0

0

20

125 10

0

(Li 20

-

Weight of Inoculated Root Sections (mg) Figure 3. Complex inheritance of resistance to crown gall in pea. Roots were inoculated by making a 1 mm x 1 mm incision with a scalpel and then dipping into inoculum (5 x 108 bacteria/mL) for 5 min. After 2 weeks, 5-mm sections containing the site of inoculation (including those that did not develop visible tumors) were excised and weighed. The total numbers of tested individuals of parent genotypes Sweet Snap (0) and Wando (+), and F2 progeny from Sweet Snap x Wando crosses (*), respectively, were 102, 91, and 236.

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Supersusceptibility Relative susceptibility of the 1331 pea lines was assessed based on tumor size as well as number. Tumor size was evaluated nondestructively by measuring tumor diameter. Most varieties developed tumors with mean diameters of up to 8 mm. Three of the lines-P1272185, B586-105(l), and B786-287( 1)-developed unusually large tumors (more than 9 mm in diameter), and this 'supersusceptible' phenotype was transmitted to Fl and F2 progeny from selfed parents. Tumor response phenotypes were also compared with those of 34 commercial cultivars whose responses were documented in a previous study (4). Tumors on the three supersusceptible lines (for example, Fig. 4a,b) were substantially larger than those that developed on highly susceptible commercial cultivars such as Target (Fig. 4c). Most tumors on supersusceptible lines were rounded (Fig. 4a), but tumors that developed on 12 of 20 B586-105(1) seedlings exhibited a 'rooty' phenotype (Fig. 4b). On most plants with rooty tumors, normal root proliferation was greatly reduced (Fig. 4b). DISCUSSION All of more than a thousand pea lines tested appear to have the basic machinery required to take up and express T-DNA genes to form tumors when inoculum levels are sufficiently high. However, one genotype, Sweet Snap, was immune to strain B6 except when inoculated at a concentration more than six orders of magnitude higher than that required to cause tumors on other cultivars (4). The resistance is not specific to strain B6: with three diverse wide host range strains, tumors on Sweet Snap were consistently much smaller than on other genotypes. Strain A281 has been reported to be hypervirulent on several species, including pea (8-10), as a result of enhanced activity of vir genes due to increased expression of the transcriptional activator virG (10). Sweet Snap as well as other genotypes responded to increased vir gene activity in A28 1, but tumors induced on other genotypes were more than 100% larger than those that developed on Sweet Snap. We carried out physiological assays designed to test whether Sweet Snap is deficient in several traits that have been proposed to represent early steps in infection, including chemotaxis, binding, and survival of bacteria in wound sites (1, 14). Responses of Sweet Snap in these in vitro assays were indistinguishable from those of susceptible cultivars. Previous workers (17) have found that addition of the vir gene-inducing compound acetosyringone to bacteria prior to inoculation can enhance tumorigenesis in certain species, suggesting that, in these species, the presence of vir gene inducers in wound exudates may be a limiting factor in susceptibility. The fact that preincubation of inoculum with acetosyringone had no effect on tumorigenesis in Sweet Snap is consistent with the assumption that vir gene inducers are not limiting in this cultivar. We do not know the basis for the observed resistance, but our data do not support the hypothesis that it involves the induction of a hypersensitive response, as has been observed in grapevine (12, 20). In tests with coummercial bulked seed of Sweet Snap, we observed a localized necrosis reminiscent of hypersensitive resistance, but the phenotype was not

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Figure 4. Tumor phenotypes on supersusceptible pea lines. a, Rounded tumor (arrow) on P.1. No.1272185 pea seedling; b, rooty tumor induced on pea genotype No. B586-105(1), showing proliferation of roots at the site of inoculation (arrow), with inhibition of normal root development; c, typical 'large' tumor on the coummercial cultivar Target (3). (For clarity, cotyledons were removed from plants before photographing tumors.)

transmitted to progeny and thus was apparently a physiological response confined to the original seed lot. The resistance phenotype was transmitted to progeny in self-crosses or in crosses with susceptible cultivars, confirming that the resistance to strain B6 observed in Sweet Snap is a heritable trait. The results from these experiuments do not support the possibility that the phenotype is simply inherited, as has been reported for resistance in grapevine (20). In contrast, the absence of distinct genetic classes among Sweet Snap, Wando, and F2 progeny suggests that multiple genes control resistance to tumorigenesis. In these experiments, two factors made it difficult to identify sharply defined phenotypic classes: First, plants were inoculated with 109 bacteria/mL, which results in the development of some tumors on Sweet Snap. Second, Wando is only moderately susceptible to strain B6, and some plants developed tumors that were no larger than those that formed on Sweet Snap. In subsequent experiments, we crossed Sweet Snap with Target, a cultivar that develops large tumors on all plants when inoculated with 1o8 bacteria/mL. These conditions allowed much better discriumination of parent and progeny responses into discrete, nonoverlapping phenotypic classes, and should in future experiments make it possible to determine how many genes control response of Sweet Snap to A. tumefaciens and how they are inherited. The two Target x Sweet Snap Fl individuals with no tumors are likely to represent escapes rather than a segregating population, since a small percentage of inoculated individuals from moderately susceptible genotypes frequently fail to develop tumors in the growth pouch assay (4).

The predominant occurrence of an intermediate tumor size phenotype in Fl progeny from Target x Sweet Snap crosses is consistent with the hypothesis that resistance to crown gall in pea is a complex trait. Under the conditions we used to qualitatively screen the majority of pea lines in the current study, in which we discarded plants that developed any visible tumors in response to inoculation with 5 x 109 bacteria/mL, we would not have detected lines such as Sweet Snap that are immune to lower levels of inoculum. The discovery of heritable resistance in one genotype in a quantitative assay of only 34 cultivars, in our initial survey (4), suggests that such resistance is not uncommon. Many of the 1365 genotypes we screened, perhaps including the four lines that appeared to be immune in initial tests, are likely to have been capable of exhibiting resistance such as that observed in Sweet Snap. We do not know the physiological basis for the development of large tumors in the supersusceptible genotypes. However, variation in hormone balance in roots of the pea genotype B586-105(1) may contribute to its supersusceptible phenotype. Most B586-105(1) plants developed symptoms in response to strain B6 that are typically associated with 'rooty' T-DNA mutants of A. tumefaciens or with hairy root disease caused by A. rhizogenes. In contrast to other pea genotypes in which shoot growth is inversely proportional to the size of tumors on roots or root crowns (4, 11), in B586-105(1) normal root development was stunted in inverse proportion to the amount of 'hairy root' growth at the site of inoculation. Whereas hairy root disease induced by A. rhizogenes occurs

INHERITANCE OF RESISTANCE TO CROWN GALL IN PEA as a result of a change in cellular sensitivity to auxin, the phenomenon in this genotype may involve a higher level of auxin or lower level of cytokinins in the infected host cells.

ACKNOWLEDGMENTS

This paper is dedicated to the memory of Dr. G. A. Marx, with much gratitude for his generosity with his time. We thank Kim Graves and Leslie Gaffney for their assistance in screening pea varieties.

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