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CELL BIOLOGY & MOLECULAR GENETICS

Expression of Soybean Cyst Nematode Resistance in Transgenic Hairy Roots of Soybean

^a Dep. of Agronomy and Plant Genetics, Univ. of Minnesota, St. Paul, MN 55108 USA
^b Dep. of Plant Pathology, Univ. of Minnesota, St. Paul, MN 55108 USA

somers{at}biosci.cbs.umn.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
Transgenic hairy roots of soybean [Glycine max (L.) Merrill] induced by Agrobacterium rhizogenes support the complete life cycle of soybean cyst nematode (SCN, Heterodera glycines Ichinohe) in vitro. However, expression of SCN resistance in hairy soybean roots has not been investigated. A transgenic hairy root system would be useful in developing an assay for candidate SCN resistance genes. The objectives of this study were to characterize transgene expression in SCN-infected hairy soybean roots and to evaluate a transgenic hairy root system for investigations of resistance to SCN. Seedling cotyledons of the SCN-susceptible cultivars, Agassiz and Parker, and SCN-resistant Bell and Faribault were infected with A. rhizogenes strain K599 transformed with T-DNA binary vectors containing the gusA gene fused to promoters from either the cauliflower mosaic virus (CaMV 35S), Arabidopsis thaliana phenylalanine ammonia lyase (PAL), or bean (Phaseolus vulgaris L.) chalcone synthase-8 (CHS) genes. Nine days after inoculating transgenic hairy roots with sterile J2 nematodes, CHS-regulated ß-glucuronidase (GUS) staining at infection sites increased in hairy roots of resistant Faribault and decreased in susceptible Agassiz. PAL-regulated GUS staining was absent at infection sites in hairy roots of resistant cultivars, but was increased in infection sites in susceptible cultivars. Thirty-five days after inoculation with SCN, the mean number of cysts formed on hairy roots of the resistant cultivars was about 14% of the mean number of cysts formed on hairy roots of the susceptible cultivars, indicating that the SCN resistance phenotypes were preserved in transgenic hairy roots. These results indicated that the transgenic hairy soybean root system will be useful for investigating differential transgene expression during nematode infection and evaluation of candidate SCN resistance genes.

Abbreviations: SCN, soybean cyst nematode • CaMV, cauliflower mosaic virus • CHS, chalcone synthase • PAL, phenylalanine ammonia lyase • GUS, ß-glucuronidase

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
SOYBEAN CYST NEMATODE

is an obligate, sedentary endoparasite of soybean that is found in soils throughout the world (Ichinohe, 1952). SCN is responsible for significant reduction in soybean yields (Sasser and Freckman, 1987; Noel, 1992). Among the various methods used for controlling SCN are crop rotations (Koenning et al., 1995), nematicide application, and planting resistant cultivars (Hartwig, 1985; Riggs et al., 1995). Resistant cultivars are the most widely used approach to SCN management (Riggs et al., 1995) and result in increased soybean yields concomitant with decreased nematode populations.

Resistance to SCN was identified soon after the discovery of the disease (Ross and Brim, 1957) and was quickly incorporated into breeding programs. Resistance to SCN is an oligogenic quantitative trait (Anand and Rao-Arelli, 1989; Young, 1996). Restriction fragment length polymorphism (RFLP) mapping and linkage analyses have shown that at least three quantitative trait loci (QTLs) exist among the sources of resistance studied (Concibido et al., 1997). Among these, a resistance locus on linkage group G was found to act in a race independent manner and to account for more than 50% of total expressed phenotypic resistance to SCN (Concibido et al., 1997). Further mapping and analysis has indicated the presence of a gene coding for resistance to SCN (rhg1) in this genomic region. Breeding for SCN resistance has been difficult because of its complex inheritance, linkage drag (Anand and Koenning, 1986), and associated yield depression (Mudge et al., 1997). Resistant cultivars are known to have generally lower yields and poor agronomic traits compared with uninfected susceptible cultivars (Hartwig, 1985). Hence, there is interest in improving SCN resistance through genetic engineering based on isolation and manipulation of the SCN resistance genes. The crucial step in this process is a transgenic assay system that is easy to manipulate genetically and quickly establishes the SCN resistance phenotype conferred by candidate genes.

Whole plant transformation of soybean has been carried out by Agrobacterium tumefaciens-mediated gene transfer (Hinchee et al., 1988; Chee et al., 1989), particle bombardment (McCabe et al., 1988), and electroporation (Dhir et al., 1992). However, production of transgenic soybean plants remains difficult and slow. In contrast, Agrobacterium rhizogenes, the causative organism of hairy root disease (for review, see Nilsson and Olsson, 1997), produces large numbers of transgenic hairy roots in soybean (White et al., 1985) and is cultivar independent (Owens and Cress, 1985). Agrobacterium rhizogenes induces the formation of transgenic hairy roots by transforming host plant cells with the T-DNA of the Ri plasmid (Nilsson and Olsson, 1997). In addition, A. rhizogenes also introduces novel genes into hairy roots from the T-DNA of binary vector plasmids (Bevan, 1984; Simpson et al., 1986; Hamill et al., 1987).

Hairy roots produced in sugar beet (Beta vulgaris L.) exhibit the resistance phenotypes of the whole plant (Paul et al., 1987; Paul et al., 1990; Verdejo et al., 1988). Furthermore, when susceptible sugar beet genotypes are cotransformed with A. rhizogenes to introduce the Hs1^pro-1 resistance gene, the hairy roots formed exhibit resistance to sugar beet cyst nematode (Cai et al., 1997). In soybean, Savka et al. (1990) reported the ability of H. glycines race 3 to complete its life cycle and form cysts in hairy roots of the susceptible cultivar Williams 82. However, no comparison of SCN phenotype variation in hairy soybean roots has yet been reported. The objectives of the present study were to characterize transgene expression in SCN-infected hairy soybean roots and to evaluate an in vitro transgenic hairy root system for investigations of resistance to SCN.

	Materials and methods

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Agrobacterium rhizogenes
Agrobacterium rhizogenes strain K599, a generous gift of P. Gresshoff, University of Tennessee, Knoxville, is a cucumopine strain isolated by Allan Kerr, Waite Agricultural Research Institute, Glen Osmond, Australia. Cultures were maintained as frozen stocks in 50% (v/v) glycerol and growth was initiated by streaking on Yeast Extract-Mannitol (YEM) agar plates (Bohlool and Schmidt, 1970) supplemented with antibiotics for 2 d at 28°C. Individual colonies were inoculated into shake flasks and grown overnight to a density of 2 x 10⁸ cells mL^-1.

Binary Plasmids
Three binary vectors—pAM194, pCHS, and pSO1—were used. pAM194 is a derivative of pBin19 (Bevan, 1984) with the gusA gene under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter kindly provided by C. Jung, Christian-Albrechts University of Kiel, Germany. The gusA gene was fused with the second intron of the ST-LS1 gene for exclusive expression in the plant cell (Vancanneyt et al., 1990). The chalcone synthase-8 (CHS) promoter from bean and the phenylalanine ammonia lyase (PAL) promoter from Arabidopsis thaliana were a generous gift of C. Lamb, Salk Institute of Biological Sciences, La Jolla, CA. The CHS promoter (Schmid et al., 1990) is a 1.4-kb fragment of the 5' sequence cloned upstream of gusA (Jefferson et al., 1987) in the binary vector pBI101.1, hereafter referred to as pCHS. The plasmid pSO1 has a 1816-bp EcoRI/BglII 5' fragment from a genomic PAL1 clone ligated between the SalI and BamHI sites of pBI101.1 (Ohl et al., 1990). The gusA gene of pBI101.1 lacks the ST-LS1 intron (Jefferson, 1987).

Agrobacterium Transformation
Binary vectors were introduced into wild-type A. rhizogenes K599 by electroporation (Nagel et al., 1990) with some modifications. Agrobacterium rhizogenes was grown in 40 mL of Terrific Broth (Sambrook et al., 1989) at 28°C at 250 rpm on a rotary shaker until the cell density exceeded an OD₆₀₀ of 1.5. Cells were isolated by centrifuging at 10 000 x g for 10 min, washed four times by centrifuging at 10 000 x g with 40 mL of chilled sterile distilled water and then with 40 mL of cold sterile 10% (v/v) glycerol. Washed cells were resuspended in 100 µL of 10% (v/v) glycerol to a final density of approximately 0.5 to 1.0 x 10¹⁰ cells mL^-1. Forty microliters of the suspension was transferred with 250 ng of plasmid DNA to a 1-mm gap electroporation cuvette and electroporated with a BTX Electro Cell Manipulator 600 (BTX Inc., San Diego). Four hundred microliters of YEM was added to the cuvette after electroporation and the bacterial suspension was transferred to sterile centrifuge tubes and shaken at 250 rpm for 30 min at 28°C. The suspension was further diluted with 860 µL of YEM broth and 325-µL aliquots were plated on YEM agar plates supplemented with 100 mg L^-1 of kanamycin. For freezer stocks, individual colonies were grown overnight in 5 mL of YEM broth supplemented with kanamycin. Aliquots of 750 µL of cell suspension were transferred to 2-mL screw cap centrifuge tubes and 750 µL of glycerol was added and the tubes were flash frozen in dry ice or liquid nitrogen and stored at -70°C.

Soybean Cultivars
Four soybean cultivars adapted for cultivation in Minnesota were used. Agassiz (Orf and Kennedy, 1994a) and Parker (Orf and Kennedy, 1994b) were used as susceptible controls. Bell and Faribault were resistant cultivars that derived their resistance from PI 88788 (Nickell et al., 1990) and PI 209332 (Orf and MacDonald, 1995), respectively. The cultivar Lee was used as the universal suscept. Seeds were surface sterilized by incubating overnight in an atmosphere of chlorine gas produced by adding 3.5 mL of 12 M HCl to 100 mL of 5.25% (v/v) sodium hypochlorite (Fox-Chlor, Fox Packaging Co., St. Paul, MN) in a sealed desiccator in an exhaust hood.

Soybean Transformation
Surface sterilized soybean seeds were planted in magenta boxes (Magenta Corp., Chicago, IL) in 50 g L^-1 sucrose solidified with 0.8% (w/v) phytagar (GibcoBRL, Grand Island, NY). Magenta boxes were placed under 20 to 40 µmol m^-2 s^-1 light supplied by cool white fluorescent lights on an 18-h photoperiod. Seed coats were removed from 7-d-old seedlings. With a sterile forceps and scalpel, several slices were made across the abaxial surface of the cotyledons and inoculated with 12 to 16 µL of the A. rhizogenes suspension. Fresh A. rhizogenes cultures were initiated from frozen stocks on YEM plates containing kanamycin. Individual colonies were inoculated into 30 mL YEM liquid broth under selection and grown overnight until a density of OD₆₀₀ = 1.0 was attained. Cells were pelleted at 5800 x g for 15 min and resuspended in YEM without antibiotics and used for cotyledon inoculations. The inoculated plants in magenta boxes were returned to the culture room for 21 d.

Hairy Root Cultures
Cotyledons exhibiting hairy root formation were removed from the plants and transferred to six-well culture plates (Costar Corp., Cambridge, MA) 21 d after inoculation with A. rhizogenes. At this stage, roots extended several centimeters from the cotyledon surface. The rooty cotyledons were incubated in 4 mL of Monmor broth (Savka et al., 1990) without sucrose and supplemented with 300 mg L^-1 of cefotaxime to suppress bacterial growth. Rooty cotyledons were transferred to fresh Monmor medium daily for 3 d while decreasing the strength of cefotaxime to 100 mg L^-1. Cotyledons were incubated in the dark at 28°C for 10 d in 10-cm petri dishes containing Monmor medium supplemented with 20 g L^-1 sucrose and 100 mg L^-1 of cefotaxime solidified with 0.75% (w/v) phytagar.

Histochemical GUS Staining
GUS activity of hairy roots was determined by removing a segment of the root and incubating 16 to 24 h in 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide Cyclohexylammonium salt (X-Gluc) staining solution (15 mg X-Gluc dissolved in 20% [v/v] methanol, with 0.08% [v/v] Triton X-100 and 0.16% [w/v] potassium ferrocyanide, 8 mM EDTA, and 80 mM Na₂HPO₄, pH = 7.0). Alternatively, the entire surface of the petri plate was bathed overnight in 5 mL of X-Gluc solution.

For characterizing the progression of SCN in hairy roots, 1200 freshly sterilized nematodes were added per plate for inoculating hairy roots growing in the low salt Lauritis medium (Lauritis et al., 1983). Infected roots were stained for GUS activity by adding X-Gluc at different times after inoculation. After development of histochemical staining (48 h), plates were immersed in a boiling water bath to free roots of agar and stained for nematodes with acid fuchsin. Nematode staining procedure was a modification of Byrd et al. (1983) and involved boiling the roots for 30 s in a solution containing 30 mL of water with 1 mL of stain (0.35% [w/v] acid fuchsin in 25% [v/v] acetic acid). Excess stain was removed by washing in 1.0% (w/v) NaOCl (20 mL 5.25% bleach in 85 mL of water) and rinsing in tap water to remove excess NaOCl. Stained roots were observed using a Nikon SMZ-10 microscope at 4x magnification (Nikon Inc., Melville, NY). Nematodes were counted visually and data collected by taking photographs with a Nikon M35FA 35-mm camera mounted on the microscope.

Nematode Maintenance, Sterilization, and Infection
Strain UMN10 inoculum, a race 3 field isolate of SCN, was maintained on the universal susceptible soybean cultivar Lee. Though not an inbred, this isolate has exhibited stable race characteristics over several years and has been used successfully in experiments to map SCN resistance loci (Concibido et al., 1997). Seeds were germinated in Ray Leach cone-tainers (Stuewe and Sons, Inc., Portland, OR) placed in sand filled 4-L buckets and inoculated 7 d after germination with 3000 eggs of H. glycines and grown in a water bath at a temperature of 28°C. Cysts were isolated 30 d after inoculation by uprooting the plants and washing roots with pressurized water and collecting cysts in a wire mesh sieve. Cysts were crushed with a tissue crusher and eggs counted with a hemocytometer.

Eggs were hatched by incubation in 4 mM ZnSO₄ in a sterile piepan. Paper towels were used to retain the eggs in the upper pan and hatched second stage juveniles settled to the bottom of the pan. Second stage juvenile (J2) nematodes were collected daily for sterilization and infection of hairy root plates. Nematodes were collected on a sterile 0.45-µm filter (Micron Separations Inc., Westboro, MA) and washed with 100 mL sterile 0.1% Triton X-100 followed by 100 mL of sterile water and resuspended in 1% (w/v) sodium dodecyl sulfate (SDS) and 2 mg mL^-1 streptomycin sulfate for 1 h. The nematodes were washed again with 100 mL each of Triton X-100 sterile solution and then water. This procedure was repeated a second time before suspending the nematodes in sterile water for root inoculation. The sterilization procedure that we developed resulted in almost 100% nematode viability, but about 60% of the root culture plates became contaminated with fungi when cultured for 35 d after SCN inoculation (data not shown). These contaminated plates exhibited poor nematode infection of roots and were not scored. To compensate for plates lost to contamination, large numbers of hairy root cultures were established and inoculated with SCN.

Approximately, 1200 freshly sterilized J2 nematodes were used per petri plate for inoculating hairy root cultures. Transgenic hairy roots expressing GUS, usually two 3- to 7-cm-long roots per petri plate, were excised from the cotyledons and cultured in the dark for 7 d in low salt Lauritis medium (Lauritis et al., 1983) supplemented with 20 g L^-1 sucrose adjusted to pH 5.8, and solidified with 0.75% (w/v) phytagar before infection with nematodes. Inoculated plates were incubated in the dark at 28°C. Final cyst counts were taken at 35d. At this stage, root growth and branching were substantial and cyst counts were expressed as the total cysts per petri plate.

Experimental Design
Twelve magenta boxes, divided equally among the four cultivars with four seedlings each, were used in each experiment, which was replicated twice. Each petri plate with two cotransformed hairy roots formed a unit for statistical analysis for cyst count experiments. Significant differences in mean frequency of GUS-positive roots were evaluated using Duncan's Multiple range test (Walpole, 1968). Significant differences in mean frequency of GUS-positive roots among the different cultivars of soybean were analyzed using a homogeneity test (Devore and Peck, 1997).

	Results

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Hairy Root Formation
Cotyledons on 7-d-old seedlings of Agassiz, Parker, Bell and Faribault were wounded and inoculated with A. rhizogenes strain K599 carrying both its native Ri plasmid and one of three binary vectors, pAM194, pCHS or pSO1, each of which carried the gusA gene fused to different promoters as a reporter gene cassette. Hairy roots were visible at wound sites on the cotyledons of all genotypes within 7 d post-infection and grew several cm within 20 d. No roots were observed in mock inoculations of cotyledons with water, indicating that all roots found on cotyledons were induced by A. rhizogenes-mediated transformation events. Hairy root formation frequency ranged from 4.9 to 10.8 roots formed per cotyledon and no differences were observed among the four genotypes tested (Table 1) .

In Vitro SCN Infection
Hairy roots expressing GUS activity were detached from cotyledons, cultured on low salt Lauritis medium (Lauritis et al., 1983) and infected with sterile J2 stage nematodes from a race 3 field isolate. Attempts to characterize the early reactions of hairy roots to nematode infection 3 to 6 d after inoculation were unsuccessful because staining conditions dislodged nematodes from roots (data not shown). At 9 d after inoculation, nematodes were attached to the hairy roots (Fig. 1) . Females were most easily identified by their enlarged bodies, although males could also be identified occasionally. Nematodes at the J3 and J4 stages were most frequently observed at 9 d after inoculation (data not shown). At this stage of infection, the number of juvenile nematodes attached to hairy roots was independent of the soybean genotype. A few adult female nematodes were identified in roots of susceptible cultivars, Agassiz and Parker, and occasionally in the resistant cultivar Faribault, but rarely in Bell.

View larger version (61K): [in this window] [in a new window]   Fig. 1 Histochemical GUS staining of transgenic hairy roots infected with SCN (stained red with acid fuchsin). Hairy roots of (A) SCN-susceptible Agassiz, and (B) SCN-resistant Faribault cotransformed with bean chalcone synthase-gusA. Hairy roots of (C) Agassiz, and (D) Faribault cotransformed with Arabidopsis phenylalanine ammonia lyase-gusA

	Discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

The main advantage of the hairy root system for evaluating plant-parasite interactions with SCN is related to the difficulty in producing transgenic whole soybean plants. Transgene expression in plants is extremely variable; thus evaluation of transgenic SCN resistance genes or investigating soybean gene regulation in response to SCN infection requires large numbers of independent transgenic events. Production of large numbers of transgenic soybean plants is very difficult and labor intensive. Transgenic hairy roots can be readily produced in soybean and each root represents an independent transformation event. Further, our results show that these hairy roots retain the resistance phenotype of soybean cultivars from which they are derived. The main difficulty of the hairy soybean root system we encountered was in maintaining axenic culture conditions. The hairy root system used in this study involved three organisms, G. max, A. rhizogenes, and SCN, indicating a high degree of complexity in terms of optimizing conditions for studies of their interactions. Root initiation and development up to the point of nematode inoculation required several treatments and culture conditions that greatly increased the chances of contamination. Further improvements in sterilization procedures will be required to maximize the usefulness of the system.

Halbrendt et al. (1992) showed that only 49 to 77% of J2 nematodes that successfully penetrate and infect soybean roots are able to complete their life cycle in susceptible cultivars of soybean. Initial plant responses to nematode infection include cell wall thickening and deposition of lignin (Endo, 1991), and the production of pathogenesis-related proteins, proteinase inhibitors and peroxidases (Bowles et al., 1991). The phytoalexin, glyceollin-I, a product of the phenylpropanoid pathway, has been shown to specifically accumulate locally to high levels at infection sites of SCN in roots of resistant cultivars of soybean (Huang and Barker, 1991). Edens et al. (1995) found changes in transcript levels of CHS and PAL in whole roots of soybean infected by SCN and Meloidogyne incognita. These results indicated an important role for CHS and PAL during early response to SCN infection in soybean. We used heterologous CHS and PAL promoters derived from P. vulgaris and A. thaliana, respectively, to determine the temporal and spatial expression patterns of CHS and PAL during early stages of infection of SCN. Expression patterns of these promoters in uninfected hairy soybean roots were similar to published results in P. vulgaris (Schmid et al., 1990) and A. thaliana (Ohl et al., 1990). When transgenic hairy roots carrying the CHS-GUS construct were infected with SCN, localized increases in CHS expression were observed in infection sites in resistant cultivars 9 d after inoculation. At the same time, a decrease in CHS-gusA staining was visible in hairy roots derived from susceptible soybean cultivars. PAL-gusA expression increased in hairy roots derived from susceptible cultivars of soybean and decreased in resistant cultivars. It is possible that the local effects of PAL and CHS expression could be induced by the nematode or result from host defense mechanisms induced in response to pathogen infection. Our results demonstrate the utility of the transgenic hairy root system for studying soybean-SCN interactions at the molecular genetic level and for evaluation of candidate SCN resistance genes.

	NOTES

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Contribution no. 991130114 from the Minnesota Agric. Exp. Stn. This work was supported in part by USDA/95-37300-1593.

Received for publication March 8, 1999.

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