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Plant Physiol. (1999) 119: 935-950
Isolation of Ethylene-Insensitive Soybean Mutants That Are
Altered in Pathogen Susceptibility and Gene-for-Gene Disease
Resistance1
Thomas Hoffman,
J. Scott Schmidt,
Xiangyang Zheng, and
Andrew F. Bent*
Department of Crop Sciences, University of Illinois at
Urbana-Champaign, Urbana, Illinois 61801
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ABSTRACT |
Plants commonly respond to pathogen
infection by increasing ethylene production, but it is not clear if
this ethylene does more to promote disease susceptibility or disease
resistance. Ethylene production and/or responsiveness can be altered by
genetic manipulation. The present study used mutagenesis to identify
soybean (Glycine max L. Merr.) lines with reduced
sensitivity to ethylene. Two new genetic loci were identified,
Etr1 and Etr2. Mutants were compared with
isogenic wild-type parents for their response to different soybean
pathogens. Plant lines with reduced ethylene sensitivity developed
similar or less-severe disease symptoms in response to virulent
Pseudomonas syringae pv glycinea and
Phytophthora sojae, but some of the mutants developed
similar or more-severe symptoms in response to Septoria
glycines and Rhizoctonia solani. Gene-for-gene
resistance against P. syringae expressing
avrRpt2 remained effective, but
Rps1-k-mediated resistance against P. sojae races 4 and 7 was disrupted in the strong
ethylene-insensitive etr1-1 mutant.
Rps1-k-mediated resistance against P. sojae race 1 remained effective, suggesting that the
Rps1-k locus may encode more than one gene for disease
resistance. Overall, our results suggest that reduced ethylene
sensitivity can be beneficial against some pathogens but deleterious to
resistance against other pathogens.
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INTRODUCTION |
Ethylene is a growth hormone of relatively simple structure that
influences many aspects of plant growth, development, and productivity
(Matoo and Suttle, 1991 ; Abeles et al., 1992). Directed genetic
manipulation of plant ethylene production or responsiveness has been
demonstrated for plant species that include tomato, Arabidopsis, petunia, and tobacco (Bleecker et al., 1988; Hamilton et al., 1990;
Klee et al., 1991; Oeller et al., 1991; Ecker, 1995; Wilkinson et al.,
1997; Knoester et al., 1998). In many cases, these manipulations are
carried out to alter economically important traits such as fruit
ripening, floral senescence, or disease tolerance.
Ethylene signaling requires not only ethylene production, but also
ethylene perception and response. Ethylene signal transduction pathways
are currently the subject of extensive research. A number of
ethylene-response mutants have been identified in Arabidopsis, and
study of these mutants has been particularly revealing (Ecker, 1995;
Kieber, 1997). Arabidopsis ETR1 and its homologs encode proteins with ethylene-binding activity that apparently function as the
primary cellular receptors of ethylene. Mutations of ETR1 that cause insensitivity to ethylene are typically dominant to wild-type ETR1. Downstream signaling components of the
ethylene-perception pathway have also been identified by mutational
analysis, and the Arabidopsis CTR1, EIN2, and
EIN3 genes have been isolated and characterized. Mutant
alleles of these downstream genes are typically recessive. Ethylene
apparently acts by disrupting negative regulation of ethylene-response
pathways, and early stages of this pathway involve protein kinases with
similarity to the Raf kinase family of signal transduction proteins.
Ethylene-responsive promoter elements and transcription factors have
also been characterized (Ecker, 1995; Deikman, 1997). Although many
steps of the ethylene signal transduction pathway have been identified,
it is likely that others remain to be discovered.
Ethylene is produced by plants in response to a wide variety of biotic
or abiotic stresses (Matoo and Suttle, 1991; Abeles et al., 1992). The
production of ethylene is one of the earliest responses of plants to
pathogen attack, and ethylene production is observed in response to
diverse viral, bacterial, and fungal plant pathogens (Pegg, 1976;
Boller, 1991). Because ethylene formation occurs in many plant-pathogen
interactions, the question of its role in disease resistance and
disease susceptibility arises. Ethylene may be a stimulus for defense
responses that lead to resistance, or conversely, it may play a role in
disease symptom development and in the breakdown of endogenous
resistance (Boller, 1991; Abeles et al., 1992; Lund et al.,
1998).
Roles for ethylene in the activation of plant defense are suggested by
the known ability of ethylene to induce the accumulation of
pathogenesis-related proteins. Numerous workers have shown that
ethylene will induce synthesis of PR-1,  -1,3-glucanase, chitinase,
Phe ammonia-lyase, Hyp-rich glycoproteins, osmotin, and other
defense-associated proteins (Boller, 1991; Deikman, 1997).
Significantly, synthesis of many of these proteins can also be induced
by ethylene-independent pathways (Dixon and Lamb, 1990). However,
biological elicitation of some defense-associated proteins may require
a functional ethylene response (e.g. Penninckx et al., 1996), and a
rhizobacterium-stimulated "induced systemic resistance" response
was disrupted in Arabidopsis etr1 mutants (Pieterse et al.,
1998). Further evidence for a role of ethylene in resistance was
provided using tobacco lines made ethylene insensitive by expression of
a dominant mutant etr1 transgene from Arabidopsis (Knoester
et al., 1998). These ethylene-insensitive tobacco plants displayed
susceptibility to Pythium sylvaticum fungi that are usually
not pathogenic on tobacco. However, ethylene-insensitive Arabidopsis
and tomato lines have not shown excessive susceptibility to
Pseudomonas, Xanthomonas, Peronospora,
or Fusarium pathogens (Bent et al., 1992; Lawton et al.,
1995; Lund et al., 1998).
Although ethylene is involved in the expression of a number of
pathogenesis-related plant genes, it can also increase disease symptom
severity. Ethylene increased disease severity of verticillium wilt of
tomato and of gray mold on rose and carnation flowers and on detached
leaves of tomato, pepper, bean, and cucumber (Boller, 1991).
Similar correlations between ethylene production and
infection-related chlorotic or necrotic symptom development have been
demonstrated in other plant species (Gentile and Matta, 1975;
Goto et al., 1980; Stall and Hall, 1984; Ben-David et al., 1986). More
generally, ethylene is a well-known inducer of necrosis, ripening,
chlorosis, and senescence in plants (Matoo and Suttle, 1991;
Abeles et al., 1992). These findings imply that ethylene can promote
disease symptom development and general disease
susceptibility.
Because ethylene appears to increase disease severity, researchers have
asked whether insensitivity to ethylene may increase plant tolerance to
plant pathogens. Early evidence for this came from studies of a pepper
line with enhanced sensitivity to ethylene that exhibited more rapid
development of chlorosis in response to Xanthomonas
campestris pv vesicatoria, the causal agent of bacterial spot disease (Stall and Hall, 1984). Studies with
ethylene-insensitive mutants of Arabidopsis revealed enhanced disease
tolerance to Pseudomonas syringae pv tomato or pv
maculicola and Xanthomonas campestris pv
campestris pathogens (Bent et al., 1992). Disease tolerance
can be defined as quantitatively reduced symptom severity despite
pathogen growth that is similar to that observed in disease-susceptible control plants. A simple role for ethylene insensitivity in disease tolerance remained uncertain in the studies cited above with
Arabidopsis, because tolerance was observed only with ein2
mutants and not with etr1 (ein1) and
ein3 mutants (Bent et al., 1992). The ein2 mutations may disrupt a specific branch of the ethylene-response pathway or provide an especially complete block in ethylene signaling. Alternatively, mutation of EIN2 may exert direct or indirect
pleiotropic effects on other response pathways (e.g. Cary et al.,
1995). Arabidopsis ethylene-insensitive mutants were also used to show
that ethylene insensitivity does not disrupt gene-for-gene resistance
(resistance gene/avirulence gene-dependent disease resistance) (Bent et
al., 1992). In a separate study, systemic acquired resistance was not disrupted in these Arabidopsis ethylene-insensitive mutants (Lawton et
al., 1995). These studies suggested that it may be possible to use a
screen for ethylene insensitivity as a method to identify disease-tolerant lines in other plant species.
More recently, studies with the Never ripe mutant of tomato
provided further evidence of enhanced disease tolerance in
ethylene-insensitive plant lines (Lund et al., 1998). Enhanced
tolerance was observed in response to both X. campestris pv
vesicatoria and Fusarium oxysporum, an important
fungal pathogen. It is interesting that the Never ripe
mutation disrupts a tomato homolog of Arabidopsis ETR1
(Wilkinson et al., 1995), and in earlier studies the Arabidopsis etr1 mutants did not show enhanced tolerance (Bent et al.,
1992). In tobacco lines made ethylene insensitive by expression of a dominant mutant etr1 transgene from Arabidopsis,
gene-for-gene resistance against tobacco mosaic virus remained
effective (Knoester et al., 1998). However, as mentioned above, these
ethylene-insensitive tobacco plants displayed susceptibility to
P. sylvaticum fungi that are not usually pathogenic on
tobacco (Knoester et al., 1998). A limited number of pathogens were
tested in the studies cited above, and different pathogens have very
different modes of pathogenicity. In light of the known role of
ethylene as an inducer of chlorosis and senescence, but also of
pathogenesis-related gene expression, it has remained unclear if the
ethylene-insensitivity trait could be used to confer enhanced disease
tolerance to plants or if it might, on balance, be more disruptive of
resistance.
To examine the potential contribution of ethylene insensitivity to
disease tolerance and/or disease susceptibility in an important field
crop, we identified ethylene-insensitive mutants of soybean (Glycine max). Soybean mutants displaying reduced ethylene
sensitivity were then compared with their parental lines in tests with
virulent Septoria glycines, Rhizoctonia solani,
Pseudomonas syringae pv glycinea, and
Phytophthora sojae pathogens chosen to represent different
taxonomic groups and different primary sites of infection. In addition,
gene-for-gene resistance responses were monitored using compatible and
incompatible races of P. sojae and P. syringae pv
glycinea. Mutations that reduce ethylene sensitivity altered plant resistance to some but not all of the soybean pathogens examined
in this study, and altered gene-for-gene resistance against some but
not all avirulent races of a single pathogen species.
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MATERIALS AND METHODS |
Plant Material
Mutagenized populations of soybean (Glycine max)
derived from 13 experimental breeding lines, including A90-312022, were
supplied as M3 seed by Dr. Walter Fehr (Iowa
State University, Ames). These M3 seeds were in
approximately 24,000 packets each derived from single
M2 individuals, with packets bundled to group
seed from 10 M2 individuals from each of
approximately 2,400 M1 plants. M1 plants were from seed that had been
mutagenized with either ethyl methanesulfonate, NMU, or
nitrosoguanidine; A90-312022 lines were mutagenized with NMU. To sample
the Fehr seed populations in the ethylene triple-response test, pools
of 72 seeds were obtained by removing 12 M3 seeds
from each of six packets from separate M2 plants
from a given M1 plant. Additional
M3 seed was furnished by Dr. James Specht
(University of Nebraska, Lincoln). This seed was grouped in
approximately 4,000 packets from individual M2 plants that came from 1,434 M1 individuals that
had been derived from the soybean var Hobbit 87 (Cooper et al., 1991)
by NMU mutagenesis. Twelve seeds from each Hobbit 87 M3 packet were used in the ethylene triple-response screen.
Screen for Ethylene-Insensitive Soybean Lines
The method of Bleecker et al. (1988) was modified for use with
soybean. Light-tight, gas-tight boxes (40 cm wide × 90 cm
long × 25 cm tall) made from one-fourth-inch opaque gray sheets
of polyvinyl chloride were constructed with welded seams and fitted with two brass gas ports. The removable top was fitted with a Neoprene
gasket and latches. For each ethylene test the box was filled with
autoclaved sand to a depth of approximately 2.5 cm, soybeans (typically
approximately 1700 seeds) were planted at a depth of 0.5 to 1.0 cm, the
sand was watered once, and the lid was secured. Compressed air with 10 µL L 1 (10 ppm) ethylene was bubbled through
water and then into one of the two gas ports; the port at the other end
released air through a second water trap to prevent inflow of room air.
In later tests the flow-through system was replaced with a sealed
system in which a small volume of pure ethylene was injected to achieve
the desired ethylene concentration, typically 18 µL
L1. After 6 d the box was opened and
seedlings were visually scored for the ethylene triple response (short
hypocotyl length, exaggerated curl of the hypocotyl hook, and radial
swelling of the hypocotyl). Any seedlings not exhibiting the wild-type
ethylene triple response were measured, rescued, acclimated in a
controlled environment, and then transplanted to 20-cm (diameter) pots
and grown to maturity in a greenhouse. The remainder of the seedlings
were transplanted in bulk to sand flats and moved to the greenhouse for
other mutant screens.
Genetic Analysis
Candidate mutant lines were grown in the field and genetic crosses
were performed by manual pollination. F1 and
F2 seedlings from self-fertilized
F1 plants were then tested for ethylene
sensitivity using the ethylene triple-response assay described above.
The female parent is listed first in the description of all crosses unless otherwise noted. Each ethylene sensitivity test of an
F1 or F2 population
typically included 10 or more individuals of both parents as the
internal controls. Where overlap in the range of hypocotyl elongation
phenotypes for wild-type and weak mutant parents was present, the
cutoff values for phenotypic categories were set to equalize as much as
possible the proportion of individuals from the parent data sets that
fell into the category of the opposite parent.
Septoria glycines Tests
The S. glycines strain used was a purified, virulent
strain isolated in Urbana, IL, in 1997 from a field-grown soybean
plant. For inoculum preparation, S. glycines spores
suspended in sterile distilled water were spread onto potato
dextrose agar plates and grown for 1 week at room temperature
(Dhingra and Sinclair, 1995). Plates were then flooded with distilled
water and gently rubbed with a glass rod to dislodge spores; the liquid
was then harvested for use. To compare ethylene-insensitive mutants
with their respective parents, experiments were set up as completely
randomized factorial designs with two treatments (inoculation with
S. glycines or mock inoculation with water), four to nine
soybean lines, and three or four replications, depending on the
experiment. Five seeds were planted in an 8-cm pot for each
replication. Plants were grown in a growth chamber set to a daylength
of 16 h with a day temperature of 23.3°C stepped at nightfall to
a night temperature of 18.8°C. Daybreak humidity was set to 68%,
increasing to 80% 2 h later and for the remainder of the day, to
100% at nightfall, and then decreasing to 82% over a 4-h period and
remaining at 82% until daybreak.
Spray-inoculation treatments were applied with a misting bottle when
plants were 10 d old, a stage at which the first set of trifoliate
leaves was expanded to near-full size. The S. glycines treatment contained S. glycines spores at a concentration of
approximately 2 × 106 spores
mL1 in distilled water plus the surfactant
Silwet L-77 (OSi Specialties, Danbury, CT) at 100 µL
L1. The control consisted of distilled water
with L-77 but no spores. Immediately after spraying, plants were
covered for 2 d with a tall, plastic dome and a shade cloth and
placed back in the growth chamber. After 2 d the domes were
removed and the plants were maintained under growth-chamber conditions
for the duration of the experiment. S. glycines symptoms
were evaluated 9 d after inoculation on a scale of 0 to 5: 0, no
symptoms; 1, slight chlorotic flecking; 2, a few tiny necrotic lesions;
3, many smaller necrotic lesions; 4, a few large lesions (>1 mm in
diameter); and 5, many large lesions. For the syringe-inoculation
method, a plastic syringe (no needle) was used to lightly rub
resuspended S. glycines spores or water alone (no surfactant
in either case) over a small area of the underside of one fully
expanded, unifoliate leaf on each plant. The rating scale for this
technique also ranged from 0 to 5: 0, no symptoms; 1, very small
lesions with no chlorosis; 2, small lesions with chlorosis; 3, lesions
of intermediate size (1-2 mm); 4, large lesions with little chlorosis;
and 5, large lesions with chlorosis.
Pseudomonas syringae pv glycinea
Tests
P. syringae pv glycinea race 4 (Kobayashi et
al., 1989) carrying pVSP61 (empty plasmid vector) or pV288 (pVSP61 + avrRpt2; Kunkel et al., 1993) were grown from frozen stocks
at 28°C on NYG agar (Daniels et al., 1984) and then subcultured in
NYG liquid, both containing rifampicin 50 µg/mL and kanamycin 25 µg/mL. Bacteria were harvested by centrifugation and resuspended in
10 mM MgCl2 to an optical
density at 600 nm of 0.04 (approximately 4 × 107 colony-forming units/mL) in experiments 1 to
4, and an optical density at 600 nm of 0.01 for experiments 5 and 6. The surfactant Silwet L-77 was then added at a rate of 50 µL
L1. Experiments 1 to 5 consisted of three
treatments (water/MgCl2/L-77 containing race 4 [pVSP61], race 4 [pV288], or no added bacteria), four to nine
cultivars, and three or four replications, depending on the experiment.
Experimental units (five seeds per 9-cm pot) were completely randomized
within a particular experiment. Leaves were inoculated by vacuum
infiltration when plants were approximately 2 weeks old and the first
trifoliate leaf was partially expanded. Plants were then returned to
the growth chamber and rated for symptom expression 1 week after
infiltration. In experiments 1 to 4, a scale of 0 to 5 was used for
rating unifoliate leaf symptoms: 0, healthy, no bacterial damage; 1, slight necrotic flecking; 2, more obvious necrotic lesions; 3, necrosis
converging in small, dead sectors; 4, large, dead sectors; and 5, unifoliate leaves completely dead. Experiments 5 and 6 used a new 0 to
5 rating scale focused on both necrosis and chlorosis: 0, healthy,
green leaves; 1, slight symptoms, leaves still very green; 2, more
visible necrosis and/or slight chlorosis; 3, dispersed chlorosis and/or necrosis covering 20% to 50% of the leaf; 4, large, necrotic areas but leaves still partly green; and 5, leaves senesced. For chlorophyll assays leaf discs were removed from a standardized location on one
unifoliate leaf from each of five plants per treatment, discs from each
treatment were pooled, and chlorophyll measurements were performed
according to the method of Lichtenthaler and Wellburn (1983).
Phytophthora sojae Tests
P. sojae strains of known race designation were
obtained from Dr. Cecil Nickell (Urbana, IL) and maintained on lima
bean agar plates at 4°C with minimal passaging. To foster rating of
tolerance/partial-resistance phenotypes, an inoculum layer test method
was used in which soybean seedlings were grown in vermiculite in
containers carrying a P. soja-covered disc of V-8 agar at a
depth 2 cm below the planted seeds (Schmitthenner and Bhat, 1994).
Experimental units were completely randomized within a given experiment
and each experiment consisted of three to five treatments (agar-only
control plus various races of Phytophthora), the cultivars
to be tested, and three to four replications each. Two weeks after
planting, plants were rated for leaf/shoot symptoms and then removed
from the vermiculite and rated for root rot symptoms. Shoots were rated
on a scale of 0 to 6: 0, leaves appear healthy; 1, slight leaf damage;
2, plants of normal height with stunted leaves; 3, short, stunted plants; 4, tiny, unexpanded unifoliate leaves visible; 5, seed germinated but only cotyledons visible; and 6, seedling rotted, no
leaves visible. Roots were rated on a scale of 0 to 7: 0, healthy roots; 1, slight root rot (browning at tips); 2, moderate root rot; 3, some healthy roots remain but severe rot; 4, many rotted roots present;
5, a few sick roots remaining; 6, all roots completely rotted; and 7, seedling rotted at emergence.
Rhizoctonia solani Tests
R. solani strain 2B-12, an isolate highly virulent on
soybean, was obtained from the laboratory of Dr. James Sinclair
(Urbana, IL) and maintained at 4°C with minimal passaging on potato
dextrose agar plates. To compare the reaction to R. solani
between soybean mutants and the parental lines from which they were
derived, experiments were set up as completely randomized designs with
two treatments (uninfested and R. solani-infested soil), six
to nine cultivars, and three to four replications per experiment.
R. solani inoculum was prepared from liquid cultures by
placing plugs of mycelium from potato dextrose agar plates in a flask
of potato dextrose broth on a shaker rotating at 80 rpm at a
temperature of 28°C. After approximately 10 to 14 d a large
mycelial mass developed that was harvested by centrifugation, blotted
for less than 1 min until relatively dry, weighed, placed in distilled
water, and ground into small pieces in a blender. This mycelial
suspension was then added gradually and mixed thoroughly into a soil
mixture of 1 part soil, 1 part sand, and 1 part vermiculite, to an
inoculation density of 100 to 200 mg fresh weight
kg1 soil. Five seeds per pot were planted
directly in the fungus-infested soil at a depth of approximately 1.5 cm
and grown in the greenhouse. After 14 d the soil was gently washed
from the roots, which were rated on a scale of 0 to 6: 0, roots
completely healthy; 1, slight root rot; 2, moderate root rot; 3, severe
root rot with some healthy roots remaining; 4, only rotted roots
present; 5, root system completely rotted away; and 6, seedling
completely rotted. Similarly, shoots were rated on a scale of 0 to 5:
0, healthy leaves; 1, normal plant height with stunted leaves; 2, mildly stunted plants; 3, small deformed plants; 4, seedling just
emerged; and 5, seedling completely rotted at emergence.
For disease tests with all four pathogen species, disease severity was
rated for randomized, numerically coded pots, and plant genotypes were
then matched to the numerical code after disease scores had been
recorded. Disease test data were analyzed using the GLM
procedure in the SAS package (SAS, 1989). Within GLM, contrasts were
written for each specific comparison between the parent and the mutant.
In analysis of variance, treatments and cultivars were considered fixed
and replications were considered random.
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RESULTS |
Characterization of the Ethylene Triple Response in
Soybean
We adapted the ethylene triple-response assay of Bleecker et al.
(1988) for use with soybean. When seedlings of most species are
germinated in the dark in air, they become etiolated (tall, spindly,
and chlorotic). In the ethylene triple-response assay, seedlings are
germinated in the dark in an atmosphere containing ethylene. Plants
with wild-type ethylene sensitivity typically remain quite short and
exhibit thickening of the hypocotyl and excessive curling at the
hypocotyl hook under these conditions (Knight et al., 1910; Ecker,
1995). Our initial experiments tested the ethylene triple response of
wild-type soybean lines (Fig. 1). Soybean
plants germinated in the dark in air typically grew to a hypocotyl
length of 12 to 18 cm after 6 d in our experimental system. In
contrast, plants germinated in the dark in 10 µL
L1 ethylene gave a classic triple response,
with an average hypocotyl length of 2 cm, obvious radial thickening of
the hypocotyl (diameter increased approximately 2-fold at thickest
point), and exaggerated curvature of the hypocotyl hook to an angle
exceeding 180o. As in other plant species, the
response of soybean to ethylene in this assay was dose dependent. In
establishing the lower limits of the response, we observed a 2- to
3-fold reduction in hypocotyl length in 0.5 µL
L1 ethylene but only a slight response in 0.1 µL L1 ethylene (data not shown). The
hypocotyl thickening and hook curling of wild-type soybeans became
slightly more extreme as ethylene levels were increased up to 1000 µL
L1, but hypocotyl length was essentially
saturated at 10 µL L1 (Figs. 1 and
2).
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| Figure 1.
Ethylene triple response of etiolated soybean
seedlings. Plants were germinated for 6 d in complete darkness in
sealed vessels containing air or air supplemented with ethylene to the
concentrations noted. The typical triple response to ethylene includes
shortening of the hypocotyl, radial thickening of the central
hypocotyl, and excessive curling of the hypocotyl hook.
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| Figure 2.
Hypocotyl elongation of etiolated soybean
seedlings germinated in ethylene. Mutants are presented with their
respective nonmutagenized parent line Hobbit 87 (A) or A90-312022 (B).
Seedlings were germinated for 6 d in complete darkness in sealed
vessels containing air supplemented with ethylene to the concentrations
noted. Values are means ± SE. T10N5, T10N6, and T15
are sibling lines carrying the same mutation (see text). True-breeding
progeny of originally identified mutants were used in these and
subsequent studies.
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Isolation of Ethylene-Insensitive Mutants of Soybean
In the absence of immediately available M2
soybean seed, mutant screens for plants displaying an altered ethylene
triple response were initiated using M3
populations that were the kind gifts of Drs. Walter Fehr and James
Specht. Approximately 142,000 M3 seeds representing approximately 1,985 M1 lines
were tested from the Fehr seed populations, and approximately
48,000 M3 seeds representing 1,434 M1 lines were tested from the Specht seed
population. Plants from 84 different M2 families
were saved as possible ethylene-insensitive lines, although many were
only slightly taller than their parents in the initial ethylene
triple-response screen. Candidate mutants were grown to maturity and
allowed to self-fertilize, and the progeny were tested for the
ethylene triple response. Most of the lines resembled the wild type
upon retesting, but seven lines showed reproducible reduction in
ethylene sensitivity.
Figure 2 presents data from one of the many ethylene-sensitivity tests
of the mutant lines that were used in further studies. The mutants
presented in Figure 2 displayed full hypocotyl elongation when
germinated in the dark in air with no added ethylene (data not shown;
hypocotyl length typically 12-18 cm, depending on the experiment).
When the "strong" ethylene-insensitive mutant T119N54 was
germinated in 10 µL L1 ethylene, hypocotyls
extended to a length similar to that of etiolated seedlings grown in
air (Fig. 2A). The "intermediate" mutants such as T123N37 or
T124N38 displayed an obvious reduction in ethylene sensitivity relative
to the parent line, but still exhibited some inhibition of average
hypocotyl length when germinated under ethylene (Fig. 2A). The
"weak" ethylene-insensitive mutants displayed a modest but
reproducible reduction in their responsiveness to ethylene relative to
the wild-type parent (Fig. 2B). Because average hypocotyl length varied
somewhat from experiment to experiment, data comparisons focused on
plants within a given experiment, and parental genotypes were routinely
included as internal controls in these and subsequent ethylene
triple-response tests.
All of the mutants retained some degree of ethylene sensitivity. Even
in line T119N54, hypocotyls were more strongly shortened after
germination at extremely high ethylene levels than they were at the 10 µL L1 ethylene level that was saturating for
the wild type (Fig. 2). At low ethylene levels the transition between
ethylene-induced shortening and full hypocotyl elongation occurred at
similar concentrations in the wild type and in the partial or weak
ethylene-insensitive mutants. Partial loss of hypocotyl shortening was
observed when ethylene concentrations decreased from 2.5 to 0.5 µL
L1, and essentially full hypocotyl elongation
was observed in 0.1 µL L1 (data not shown).
Genetic Analysis of Ethylene-Insensitive Mutants
Genetic studies of the inheritance and allelism of the mutations
that cause ethylene insensitivity were performed, with a particular
focus on mutants that were isolated earlier in the project. Line
T119N54 was originally isolated from an M3 family in which three plants showed no triple response (hypocotyl
approximately 15 cm), three were short (>3 cm), and six were
intermediate (average approximately 10 cm). This 1:2:1 phenotypic ratio
suggested the possibility of a semidominant trait. Subsequent retests
of more than 200 progeny from the T119N54 family provided further
support for this hypothesis. In ethylene triple-response tests the
progeny from the tall phenotype plants were uniformly tall (12-19 cm), the progeny from the short plants were short (1.5-2.5 cm), and the
progeny from the intermediate plants segregated for the short, intermediate, and tall phenotype in ratios resembling 1:2:1 (data not
shown). Further genetic analyses focused on progeny from reciprocal crosses between a true-breeding ethylene-insensitive T119N54 line and
its parent, Hobbit 87. F1 plants from these
crosses exhibited an intermediate phenotype in the ethylene
triple-response test (Table I). The
F2 plants segregated in a manner consistent with a 1:2:1 ratio (Table II; Fig.
3). These data suggest that the ethylene-perception defect in line T119N54 is caused by mutation in a
single nuclear locus and that the mutant allele confers ethylene insensitivity in a semidominant fashion. We propose the name
Etr1 for this gene, and use etr1-1 to designate
the mutant allele in line T119N54. True-breeding ethylene-insensitive
T119N54 progeny are subsequently referred to as Hobbit 87 etr1-1, or simply etr1-1.
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Table I.
Dominance and complementation tests for the
ethylene-sensitivity trait among mutant and wild-type soybean lines
Seedlings were germinated in darkness for 6 d in 18 µL
L1 ethylene. For each designated cross, combined data for
progeny of multiple reciprocal crosses are presented. Results of
Student's t tests for similarity to parent lines (data at
top of table) are presented below cross data, accept similarity if P > 0.05. Values are means ± SE.
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Table II.
Segregation of ethylene-sensitivity trait in
F2 populations
Seedlings were germinated in darkness for 6 d in 18 µL
L1 ethylene. Column subheadings define hypocotyl length
ranges (cm) for the respective phenotypic classes (see ``Materials and Methods''); integer values for each plant line are the number of
individual plants within the phenotypic class.  2 and P
values are for 2 tests of similarity of the data to the
noted ratio; accept ratio if P > 0.05.
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| Figure 3.
Distribution of hypocotyl-length phenotypes in
parents and segregating F2 populations germinated under
etiolating conditions in 20 µL L1 ethylene. Data on a
given graph are from plants tested concurrently within the same
chamber. A, Hobbit 87 (broken line), Hobbit 87etr1-1
(heavy, solid line), and F2 of Hobbit 87 × Hobbit
87etr1-1 (solid line with fill). B, A90-312022 (broken
line), etr2-1 mutant T10N5 (heavy, solid line), and
F2 of A90-312022 × T10N5 (solid line with fill). C,
A90-312022 (broken line), T58N5 (heavy, solid line), and F2
of T58N5 × A90-312022 (solid line with fill).
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The partially ethylene-insensitive mutants included T10N5, T10N6, and
T15N23. All of these lines were derived from the experimental breeding
line A90-312022 after mutagenesis with NMU. The mutants T10N5 and T10N6
were derived from two separate M2 lines derived from the same M1 parent, and T15N23 was a second
line selected from the same M2 parent as T10N5.
Thus, it was very likely that T10N5, T10N6, and T15N23 all carry the
same mutant allele. Whereas traits of lines T10N5, T10N6, and T15N23
such as flower color, leaf shape, and pubescence color resembled those
of A90-312022, seed derived from the M1 parent of
T10N5, T10N6, and T15N23 also segregated for brown and black hilum
color independent of the ethylene-insensitivity trait. The line from
which these three ethylene-insensitive mutants was derived apparently
carried a separate mutation affecting hilum color.
When lines T10N5, T10N6, and T15N23 were crossed reciprocally to the
parental line A90-312022, F1 seedlings displayed
wild-type ethylene sensitivity in the triple-response test (Table I),
indicating that the mutant ethylene-insensitivity trait in these lines
is recessive to the wild type. Tests were then performed using 10 different F2 populations from reciprocal crosses
of the A90-312022 parental line and the mutants T10N5, T10N6, and
T15N23. These F2 populations displayed the range
of hypocotyl-elongation phenotypes that would be expected for a
recessive mutation at a single locus, and segregation ratios consistent
with a 3:1 hypothesis could be derived from the resulting
F2 data sets (see examples in Table II and Fig.
3). However, in some tests the overlapping bell-shaped distributions of
hypocotyl length for the controls (parental wild-type and weak
ethylene-insensitive lines) required that a maximally differentiating
but imperfect dividing value for phenotypic categories be chosen (see
``Materials and Methods''). Figure 3 is presented to give a more
precise example of the distribution of phenotypes obtained in these
experiments, which were consistent with segregation of a single
recessive gene.
T10N5, T10N6, and T15N23 were crossed reciprocally with each other for
complementation tests to confirm allelism. Most of the
F1 plants from these crosses exhibited the mutant
ethylene-insensitivity phenotype, although some T10N5 × T10N6
plants were of a size intermediate between the parental line A90-312022
and the two mutants (Table I). The complementation data again suggest
that these three closely related lines carry a mutation in the same
gene. Complementation tests between the etr1-1 mutant
T119N54 and T15N23 gave data that were inconsistent with allelism but
consistent with the hypothesis that these lines carry mutations in
separate genes. F1 plants from reciprocal crosses
of etr1-1 and T15N23 exhibited strong ethylene
insensitivity, as expected given the semidominant nature of the
etr1-1 mutation. F2 plants from the
etr1-1 × T15N23 cross segregated 12:1:3 for
ethylene-sensitivity phenotypes resembling etr1-1
homozygotes or heterozygotes, T15N23, and the wild type, respectively
(Table II and data not shown). No F2 plants would be expected in the small (wild type, <3 cm) category if
etr1-1 and T15N23 carried allelic mutations. We propose,
therefore, the name Etr2 for this second gene, and use
etr2-1 to designate the mutant allele in lines T10N5, T10N6,
and T15N23. In subsequent studies, data for T10N5, T10N6, and T15N23
were combined and presented as data for the etr2-1 lines.
A separate partially ethylene-insensitive mutant, T58N5, was also
isolated from NMU-mutagenized parental line A90-312022 seed but was
derived from a different M1 plant than the parent
of the etr2-1 line. The F1 for a cross
of T58N5 and the wild-type parent gave F1
individuals with ethylene-response phenotypes that were intermediate
between the parents (Table I). The F2 segregation ratios were not consistent with a 3:1 ratio, but were consistent with a
1:2:1 ratio (Table II; Fig. 3). The mutant phenotype in line T58N5 is
apparently conferred by a single locus that is semidominant with
respect to the wild type. Complementation tests with the T58N5 line
were complicated by the semidominant nature of the mutation in T58N5
and the overlap in the range of hypocotyl elongation phenotypes for
homozygous mutant, heterozygote, and homozygous wild-type plants. Tests
carried out with the F1 and
F2 of crosses of T58N5 to etr2-1
(T10N5, T10N6, and T15N23) and etr1-1 (T119N54) suggested
that this line carries a mutation in a third Etr gene (Table
I and data not shown), but the overlap in hypocotyl phenotypes mentioned above reduced our level of confidence in such a conclusion.
Response to S. glycines
Previous work with other plant species has shown that some
ethylene-insensitive lines exhibit enhanced disease tolerance against some pathogens, and we sought to examine the response of our
ethylene-insensitive soybean mutants to four diverse soybean pathogens.
S. glycines is a fungal pathogen that causes brown-spot
disease, a foliar disease characterized by necrotic leaf lesions that
turn brown with age and develop chlorosis around the margins (Sinclair
and Backman, 1989; McGee, 1991). Under moderate to heavy S. glycines disease pressure, chlorotic areas merge and entire leaves
senesce and drop from the plant. Despite extensive efforts, no strong genetic resistance to S. glycines has been identified in
soybean (Sinclair and Backman, 1989; McGee, 1991). In the present study S. glycines tests were performed using two different
methods. In the first method, plants were spray inoculated with
S. glycines spore suspensions, maintained in a humid
environment for 2 d, shifted to normal growth conditions, and then
scored for disease development 9 d after inoculation. In test 1 of
Table III, the highly
ethylene-insensitive etr1-1 mutant did not appear to exhibit altered sensitivity to S. glycines infection. In contrast,
the mutant T58N5 exhibited greater disease severity in this test than did the A90-312022 parent from which it was derived (Table III). Plants
were scored at a second time point 14 d after inoculation, and
similar results were obtained (data not shown). Mock-inoculated controls showed no significant differences between mutant and parental
lines.
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Table III.
Disease severity in ethylene-insensitive mutants
in response to S. glycines
Disease symptoms on unifoliate leaves, presented as mean disease
severity on a scale ranging from 0 (no lesions) to 5 (many large
lesions). -; Not tested. Test number is indicated at the top of the
column. Statistical comparisons are between the mutant and its
respective parent, Hobbit 87 or A90-312022.
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The S. glycines spray-inoculation method gave reasonably
reproducible results between replications within single experiments, but levels of plant infection varied substantially from experiment to
experiment, so a different inoculation method was pursued. This second
method was referred to as the syringe method for S. glycines
application. Here a small area (approximately 0.5 × 2 cm) on the
underside of one unifoliate leaf of each plant was rubbed gently with a
plastic syringe (no needle) filled with a concentrated spore
suspension. This treatment was designed to slightly wound the leaf
epidermis so that spores might more easily become established in the
leaf interior. Nine to fourteen days after inoculation, leaves of
control plants (syringe inoculated with water only) remained green and
healthy except for slight browning that did not extend past the abraded
area. Leaves that had been inoculated with S. glycines
typically developed severe necrosis that spread to double or triple the
inoculated area. The rating scale used for syringe inoculation was
based on lesion size and chlorosis. An experiment was conducted with
the ethylene-insensitive lines in which the spray- and
syringe-inoculation methods were used side by side. The two inoculation
techniques produced overlapping but different results (Table III, tests
2a and 2b). The spray-inoculation technique once again produced
symptoms that were more severe than those in the parent only for T58N5
and not for the other ethylene-insensitive lines. Syringe inoculation
gave pronounced S. glycines lesions on all of the lines
tested. The etr1-1 mutant and T124N38 exhibited significantly larger lesion size than the Hobbit 87 parent from which
they were derived. T58N5 also developed slightly larger lesions than
its parent when the syringe-inoculation method was used.
Mock-inoculated controls showed no differences between mutants and
parental lines for either inoculation method (data not shown). Thus,
the weaker mutant allele in T58N5 and the stronger mutant alleles
in etr1-1 and T124N38 each made the plant more
susceptible to damage by S. glycines, but in an
assay-dependent fashion.
Response to P. syringae pv
glycinea
Soybean bacterial blight, caused by P. syringae pv
glycinea, superficially resembles S. glycines
brown spot in being a predominantly foliar disease characterized by
necrotic lesions with chlorotic margins (Sinclair and Backman, 1989;
McGee, 1991). However, the disease is caused by a bacterial pathogen
with different virulence mechanisms and different responses to host
defenses. Bacterial blight can be controlled using resistant cultivars,
including cultivars carrying race-specific resistance genes (Sinclair
and Backman, 1989; McGee, 1991). In the present study a number of tests
were performed to compare mutants to parental lines for their reaction
to virulent P. syringae pv glycinea. Additional studies examined reactions to an isogenic P. syringae pv
glycinea strain that expresses the cloned avirulence gene
avrRpt2 and is avirulent on the soybean lines used in this
study. Tests concentrated on the etr1-1 and
etr2-1 mutants. The mutants T58N5, T123N37, and T124N38 were
identified at a later date and were included in later tests. In tests 1 to 4 of Table IV, mock-inoculated
controls were included and no significant differences between mutants
and parental lines were observed in these controls (data not shown).
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Table IV.
Disease severity in ethylene-insensitive mutants in
response to virulent P. syringae pv glycinea and to an isogenic
avirulent strain that expresses avirulence gene avrRpt2
Data are from five separate tests; test number is indicated at top of
the column. Data presented are mean disease severity scores for
unifoliate leaves, rated on a scale ranging from 0 (no lesions) to 5 (leaves completely necrotic); -, not tested. Note that test 5 used a
different rating scale based on necrosis and chlorosis (see text).
Statistical comparisons are between the mutant and its respective
parent, Hobbit 87 or A90-312022.
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In studies with the virulent P. syringae pv
glycinea strain, the more strongly ethylene-insensitive
etr1-1 and T124N38 mutants were not significantly different
from the parent Hobbit 87 in their disease-severity scores (Table IV).
Infected leaves of etr1-1 often seemed to develop less
chlorosis, but the rating scale used in tests 1 to 4 focused instead on
necrosis. In test 3, leaf samples were taken from etr1-1 and
Hobbit 87 and chlorosis was quantified by chlorophyll assay (Fig.
4). There was significantly less
chlorosis in the ethylene-insensitive mutant. However, in test 5 a
new rating scale was used that focused on leaf necrosis and chlorosis
rather than on lesion size alone, and in that test no significant
difference was observed between the strongly ethylene-insensitive
etr1-1 line and Hobbit 87. In tests 1 to 5 the more weakly
ethylene-insensitive lines gave disease-severity scores that often were
not significantly different from those of the parent. However, in two
of the five tests the etr2-1 mutants had significantly fewer
disease lesions (Table IV). To summarize the results, most mutants
performed as well as their parental lines, and in the etr1-1
and etr2-1 lines there was a trend toward reduced disease
severity in response to virulent P. syringae pv
glycinea.
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| Figure 4.
Chlorophyll content in leaves from mock-inoculated
plants and plants infected by virulent P. syringae pv
glycinea. Leaf discs were sampled 9 d after
inoculation; values are means ± SE for four separate
groups of 5 plants (total of 20 plants per pathogen treatment). wt,
Wild type.
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P. syringae pv glycinea that express the
avirulence gene avrRpt2 elicit a HR in resistant soybean
hosts, and multiplication of such strains is restricted in these
resistant hosts (Whalen et al., 1991; Innes et al., 1993; data not
shown). The avrRpt2 gene also controls avirulence on
Arabidopsis, and for that interaction the corresponding resistance gene
RPS2 has been cloned and characterized (Bent et al., 1994;
Mindrinos et al., 1994). For the experiments in this study, avirulent
P. syringae pv glycinea bacteria were introduced
uniformly into the mesophyll of entire leaves at relatively high
initial population densities so that resistance-associated HR lesions
developed in patches that were visible to the naked eye. Chlorosis
developed around these lesions to a variable degree. Tests in Table IV
that have the same number were run and rated simultaneously with
virulent and avirulent P. syringae pv glycinea. The statistically significant resistance elicited by expression of
avrRpt2 was evident when mean disease scores for a given
plant genotype were compared for inoculations with the isogenic
virulent and avirulent strains (Table IV, statistical data not shown).
The primary focus of Table IV is on a statistical comparison between
the responses of parent and mutant plants to the same pathogen strain.
The etr1-1 mutant exhibited lower HR severity than Hobbit 87 in response to avirulent P. syringae pv glycinea (avrRpt2+) in two of the four tests (Table
IV). Overall, disease severity on the ethylene-insensitive Hobbit 87 mutants was similar to or less severe than disease severity on
wild-type Hobbit 87 after infection with avirulent P. syringae pv glycinea
(avrRpt2+). The mutations causing partial
ethylene insensitivity in the etr2-1 line and in T58N5 also
had a variable effect on the response to avirulent P. syringae pv glycinea
(avrRpt2+). As mentioned above, avirulence
gene-dependent resistance remained effective overall (Table IV; compare
the reactions of a given mutant to the isogenic virulent and avirulent
P. syringae strains). When reactions to avirulent P. syringae pv glycinea were compared between
etr2-1 and its nonmutagenized parent, lesion scores for tests 1 to 4 (in which scoring focused on HR lesion size) revealed more
extensive reactions in the etr2-1 mutant. However, the other two experiments gave no significant difference. In test 5 a
severity scale was used that focused on overall leaf browning and
chlorosis rather than on lesion size alone. In that case, as was
observed for etr1-1 in the experiment reported in Figure 4,
the etr2-1 line had significantly less chlorosis than the
nonmutagenized parent. T58N5, on the other hand, had a more severe
reaction than did the parent.
During the course of these studies five other mutant lines, T14N2B,
T14N3A, T14N10, T35N20A, and T38N5, were originally selected as
possible weak ethylene-insensitive mutants. In retests these lines
failed to exhibit the ethylene-insensitive phenotype, but in the
intervening period these lines were tested for their reaction to
virulent and avirulent P. syringae pv glycinea.
None of these mutants exhibited disease reactions that were
significantly different from those of the parental lines from which
they were derived (data not shown). Of the lines tested in these
studies, only the ethylene-insensitive lines exhibited altered
reactions to P. syringae pv glycinea infection.
Reactions to P. sojae
P. sojae is a soil-borne oomycete that is one of the
most economically destructive soybean pathogens in the United States (Sinclair and Backman, 1989; Doupnik, 1993). Damage is caused primarily
by rotting of the root and lower stem tissue, and disease control
relies heavily on genetic strategies that involve both single
race-specific resistance genes and multigenically controlled "field
tolerance" (Sinclair and Backman, 1989; McGee, 1991). To test
ethylene-insensitive mutants for their reaction to P. sojae, multiple races of Phytophthora were used. Hobbit 87 carries the Rps1-k resistance locus that conditions
race-specific resistance to many common Phytophthora races,
including races 1, 4, and 7, but not race 20 (Cooper et al., 1991;
Kasuga et al., 1997). We found that race-specific resistance mediated
by Rps1-k was partially compromised in the strongly
ethylene-insensitive etr1-1 and T124N38 mutants (Table
V). In particular, resistance against
race 4 was significantly compromised and resistance against race 7 was
also compromised in some of the tests. However, resistance against Phytophthora race 1 remained effective and was even
improved. Hobbit 87 does not carry any other known
Phytophthora resistance genes, and this differential effect
of ethylene insensitivity on the reaction to different avirulent races
suggests that races 1, 4, and 7 may stimulate different subsets of the
plant defense response (see ``Discussion''). Race 20 strains are
virulent on Rps1-k lines such as Hobbit 87, and the
reactions of the Hobbit 87 mutants etr1-1 and T124N38 to
race 20 were not significantly different from those of the parental
line (Table V).
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Table V.
Disease severity in ethylene-insensitive mutants in
response to races of P. sojae that are virulent or avirulent on the
respective soybean genotypes
Disease symptom ratings for above ground plant tissues, presented as
mean disease severity on a scale ranging from 0 (healthy plantlet) to 6 (seedling completely rotted); -, not tested. Data are presented from
five separate tests; test number is indicated at the top of the column.
Hobbit 87 and the three Hobbit 87 mutants carry Rsp1-k for
resistance to races 1, 4, and 7 (data in bold); all other host-pathogen
combinations involve compatible rather than gene-for-gene interactions.
Statistical comparisons are between the mutant and its respective
parent, Hobbit 87 or A90-312022.
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Races 1, 4, 7, and 20 are all virulent on plants of the A90-312022
genotype, which does not carry known genes for resistance to these
Phytophthora races. The partially ethylene-insensitive lines
derived from A90-312022 exhibited similar disease severity to the
parent in most of the tests (Table V). However, in more than one-fourth
of the tests disease severity was significantly reduced in the mutants,
and there were no cases in which the mutants appeared worse than the
parent. Mutation of the Etr2 locus in particular apparently
caused a partial reduction in the extent of disease caused by virulent
P. sojae.
Reactions to R. solani
R. solani is a genetically diverse species that causes
root-rotting diseases on a wide variety of plant hosts (Agrios, 1997). As for many other root-rotting diseases, the effect is most pronounced on germinating seeds and young seedlings. Taxonomic subgroups within
R. solani show a more limited host range, but single
resistance genes against R. solani are not known and genetic
resistance against this pathogen, where available, is multigenically
controlled and only partially effective (Sinclair and Backman, 1989;
McGee, 1991). Tests comparing the ethylene-insensitive mutants with
their respective parental lines for their reaction to R. solani are reported in Table VI.
Once again, a trend could be detected but statistical significance was
not consistently observed. In one test etr2-1 and T58N5
exhibited more severe root rot than their parental line, A90-312022
(Table VI). In a separate test the shoots of etr2-1 and
T58N5 were more severely affected by R. solani than their parental line, but differences in disease severity in roots were not
significant. As for the other disease tests reported above, we present
the full data set to illuminate the true variability observed in
replicated experiments. The response of ethylene-insensitive soybean
mutants to R. solani was variable, but more severe disease was observed in multiple instances.
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Table VI.
Disease severity in ethylene-insensitive mutants in
response to R. solani
Disease symptom ratings for aboveground tissues or for root and
hypocotyl tissues, presented as mean disease severity on a scale
ranging from 0 (very healthy) to 6 (seedling completely rotted); -,
not tested. Data are presented from four separate tests; test number is
indicated at the top of the column. Statistical comparisons are between
the mutant and its respective parent, Hobbit 87 or A90-312022.
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DISCUSSION |
The hormone ethylene modulates a number of traits that are
relevant to plant growth and productivity. Plant lines can be generated with genetic modifications that alter ethylene production or
responsiveness to ethylene. It is important to examine the effects of
altered ethylene signaling on a wide variety of traits in these plants, including responses to infection by pathogens. The present work initiated such studies with soybean. Mutant plant lines with reduced ethylene sensitivity were identified, and the response of these lines
to diverse virulent and avirulent pathogens was assessed. Reduction of
ethylene sensitivity had a neutral or beneficial effect on the plant
response to some pathogens, but a detrimental effect on others.
Furthermore, ethylene insensitivity had differential effects on
gene-for-gene resistance against different strains of the same pathogen
species.
Ethylene-Insensitive Mutants
Strategies for the production of plants with reduced ethylene
responses can target production of ethylene or responsiveness to
ethylene (Bleecker et al., 1988; Hamilton et al., 1990; Klee et al.,
1991; Oeller et al., 1991; Ecker, 1995; Wilkinson et al., 1997;
Knoester et al., 1998). Either trait can be altered by mutation or
through transgenic strategies. Variation within existing germplasm represents a third source of plants with reduced ethylene responses (Xie et al., 1996), but natural variants are less well suited for
controlled comparisons because of the heterogeneity of genetic backgrounds among such plants. In light of the significant difficulty of soybean transformation and the regulatory and intellectual property
issues that surround release of transgenic plant varieties to breeders
and growers, we chose to pursue a mutational strategy. Characterization
of the ethylene triple response of wild-type soybean seedlings revealed
phenotypic behavior and dose-response relationships that are quite
typical of previously studied dicotyledonous species. We were then able
to adapt the screening method of Bleecker et al. (1988) and identify a
number of mutant lines displaying reduced ethylene sensitivity. The
spectrum of strong and weak ethylene-insensitive mutants that we
identified resembles the outcome of similar screens performed with
Arabidopsis (Ecker, 1995; Kieber, 1997).
Genetic studies were carried out on a subset of the identified mutants.
Both semidominant and recessive mutations were observed and two new
genetic loci of soybean, Etr1 and Etr2, were
identified. Extensive work with Arabidopsis has shown that a very
common class of ethylene-insensitive mutants contains dominant
mutations in the ETR1 gene, which encodes an ethylene
receptor (Ecker, 1995; Kieber, 1997). Mutations in ETR1
homologs can also be dominant (Hua et al., 1995; Sakai et al., 1998),
whereas mutations in other ethylene-sensitivity loci are usually
recessive (Ecker, 1995; Kieber, 1997). In tomato the Never
ripe mutation confers semidominant ethylene insensitivity due to
alteration of the Le-ETR3 gene, a homolog of Arabidopsis
ethylene-receptor genes (Wilkinson et al., 1995). By analogy, the
semidominance of the soybean etr1-1 mutation suggests that
soybean Etr1 may encode a soybean ethylene receptor. The
recessive etr2-1 mutation identified in the present study of
soybean may be analogous to mutations in Arabidopsis EIN2,
EIN3, or other genes. Three other lines with mutations that reduce sensitivity to ethylene were also identified and used in this
study.
The incomplete ethylene insensitivity conferred by etr2-1
and by the unnamed mutations may be attributable to the retention of
partial function in the mutant gene products, or to functional redundancy with other genes of soybean. In this regard it is
interesting to note that modern soybeans, although diploid in overall
genetic behavior, are believed to be descended from a diploidized
tetraploid or from more complex polyploid ancestors (Shoemaker et al.,
1996). Numerous recessive mutations that confer obvious phenotypes have been identified in soybean, but genetic redundancy remains a
particularly strong possibility for any given gene in this species.
Because of this, mutations with dominant behavior, such as the
etr1-1 mutation, are particularly valuable. However, in
previous work on responses to pathogen infection it was the recessive
Arabidopsis ein2 mutants that displayed enhanced disease
tolerance (Bent et al., 1992). In light of this fact, the other soybean
mutants remained of interest. Genetic study of other more recently
identified ethylene-insensitive soybean lines is being initiated in the
present growing season. These newer studies may allow identification of
additional soybean loci involved in responsiveness to ethylene.
Response of Ethylene-Insensitive Mutants to Virulent Pathogens
When the ethylene-insensitive soybean mutants were exposed to
plant pathogens, altered responses were observed in many cases. Significantly, although the ethylene-insensitive mutants displayed reduced disease severity in response to some pathogens, more severe disease was observed in other plant-pathogen pairings. To enhance the
reliability of the disease assays, pathogen tests were repeated on
separate dates and plant samples within a test were typically replicated, numerically coded, and randomized to allow blinded scoring.
Data analysis focused on the comparison of each ethylene-insensitive mutant line with its near-isogenic ethylene-sensitive parent. Although
some host-pathogen combinations gave consistent differences between
parent and mutant, in other cases significant differences were observed
only in a subset of the repeats of similar tests.
Our results with S. glycines suggested that ethylene
insensitivity, if anything, makes the plant more susceptible to this foliar fungal pathogen (Table III). The mutant allele in T58N5 may
influence the host response to infection in a different manner than
etr1-1 and the mutant allele in T124N38. T58N5 plants
displayed greater symptom severity after inoculation by either of two
methods, whereas the etr1-1 and T124N38 plants displayed
enhanced disease severity only when the pathogen was applied to a
gently wounded area.
In contrast to the results with S. glycines, the
ethylene-insensitive mutants performed similarly to or better than
their isogenic parents in response to virulent P. syringae
pv glycinea (Table IV; Fig. 4). S. glycines and
P. syringae, two very different pathogens, both infect
foliar tissues and induce lesions surrounded by chlorosis. In
previously published work, reduced symptom development was observed
in the response of Arabidopsis ethylene-insensitive mutants to virulent
P. syringae (Bent et al., 1992). However, in those studies
tolerance was observed only in Arabidopsis ein2 mutants and
not in ein3 or etr1 mutants. In the present study the data for experiments with virulent P. syringae pv
glycinea were variable. In general, however, the ethylene
mutants performed as well as their parental lines in response to
virulent P. syringae pv glycinea, and the
etr1-1 and etr2-1 mutant lines exhibited a
trend toward reduced disease severity.
The ethylene-insensitive mutants also performed as well as or better
than their parents in response to virulent P. sojae (Table V). No differences were observed with the strong ethylene-insensitive lines, but in 6 of 23 tests involving the weak ethylene-insensitive lines, the disease severity was significantly less severe on the ethylene-insensitive mutants. With R. solani a consistent
theme was less prominent, but in four of the eight tests involving the weak ethylene-insensitive lines, root or shoot disease severity in
response to R. solani was more severe on the mutant than on the parent (Table VI). Overall, for foliar pathogens (S. glycines and P. syringae) or for root/lower stem
pathogens (P. sojae and R. solani), the direction
in which ethylene insensitivity altered the reaction to virulent
pathogens was not constant within a given type of plant tissue, but was
instead dependent on the particular pathogen species. The plant
reaction to virulent P. syringae (bacteria) and P. sojae (oomycete) was unaffected or modestly improved by mutations
that reduce ethylene sensitivity. However, these same mutations led in
many cases to more severe disease symptoms in response to the fungi
S. glycines and R. solani.
Alternative interpretations of these results must also be considered.
For example, although numerically unlikely, it is possible that the
lines carrying mutations that cause ethylene insensitivity also carry
separate mutations that are the cause of altered interaction with
pathogens. This possibility could be tested using extensively backcrossed lines, or using a population of homozygous
etr1/etr1-1 and homozygous Etr1/Etr1 lines
derived from F2 individuals segregating for the
ethylene-insensitivity trait.
A separate issue concerns the possible pleiotropic effects of mutations
that cause ethylene insensitivity. It must be emphasized that
alterations in the response to pathogens in these lines do not
necessarily imply a direct role for ethylene signaling in a particular
pathogen-response pathway. However, even if the effect is indirect, it
remains relevant that genetic alterations that cause ethylene
insensitivity can alter the response of plants to pathogens.
In considering how ethylene and ethylene insensitivity might influence
disease resistance, it is interesting to note the involvement of
ethylene in jasmonic acid-mediated defense signaling. Multiple studies
have provided evidence that signaling through jasmonic acid and
salicylic acid pathways can show a degree of interaction and a tendency
toward mutual exclusivity (for review, see Dong, 1998; see also
Pieterse et al., 1998).
Response of Ethylene-Insensitive Mutants to Avirulent Pathogens
Soybean plants exhibiting reduced ethylene sensitivity were
altered in their response to avirulent pathogens. When the strong ethylene-insensitive mutants were tested for their
Rps1-k-mediated resistance against Phytophthora
races 1, 4, and 7, two very interesting results were obtained. First,
in contrast to previous findings with other species (Bent et al., 1992;
Knoester et al., 1998), some gene-for-gene resistance interactions were
significantly hindered by mutations causing strong ethylene
insensitivity in soybean. Second, resistance based on the
Rps1-k resistance locus was disrupted only for some and not
all of the avirulent races against which Rps1-k is
effective. The response of soybean to an avirulent strain of P. syringae pv glycinea expressing avrRpt2 was
less obviously affected by ethylene insensitivity. Alterations in the
HR to P. syringae pv glycinea were observed, but
overall, avrRpt2-specific resistance remained effective.
The finding that ethylene insensitivity can inhibit gene-for-gene
resistance suggests that ethylene signaling can influence some
gene-for-gene defense-signaling pathways. It seems very unlikely that
ethylene is globally required for gene-for-gene signaling given that
ethylene insensitivity did not perturb resistance in Arabidopsis-Pseudomonas interactions mediated by resistance
genes RPS2 and RPM1, or in the
N-gene-mediated reaction of tobacco to tobacco mosaic virus
(Bent et al., 1992; Knoester et al., 1998). Even an essential role for
ethylene in all Rps1-k-mediated resistance of soybean
against P. sojae is unlikely given the successful resistance to Phytophthora race 1 in the ethylene-insensitive mutants.
Instead, we more narrowly conclude that ethylene signaling modulates
plant processes that are also modulated in Rps1-k-mediated
resistance to Phytophthora races 4 and 7.
The bifurcation of resistance responses controlled by the single
Rps1-k locus might be explained by the production of
different Rps1-k-recognized avirulence factors in race 1, as
opposed to races 4 and 7. A clear example of this is provided by
RPM1-mediated resistance in Arabidopsis. RPM1 provides
resistance against P. syringae pathogens that express either
avrB or avrRpm1, two genes that encode very
different protein avirulence factors (Bisgrove et al., 1994). However,
in the case of RPM1 it is known that the same
RPM1 gene controls both responses (Grant et al., 1995). No substantial differences have been identified in the defense signaling that is activated downstream of RPM1 after stimulation by
these two different avirulent pathogens.
The differential effect of etr1-1 on
Rps1-k-mediated resistance to different P. sojae
races might arise if the Rps1-k locus carries multiple
tightly clustered but distinct resistance genes that confer separate
pathogen specificities. The tomato-P. syringae pv
tomato interaction involving the resistance genes
Pto, Fen, and Prf and elicitation by
AvrPto or fenthion provides an excellent model for this type of finding
(Bent, 1996; Hammond-Kosack and Jones, 1997). The
Pto/Fen/Prf gene cluster was treated previously as a single
resistance gene. Prf is a resistance gene encoding an
NBS-LRR protein that mediates responses to both AvrPto and fenthion
(Salmeron et al., 1996). Pto and Fen encode two
closely related but distinct protein kinases; Pto is apparently the
primary binding site for the AvrPto ligand, whereas Fen controls the
plant response to fenthion (Martin et al., 1994; Zhou et al., 1995; Scofield et al., 1996; Tang et al., 1996 |
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Abstract
Introduction