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Induction of a 58,000 dalton protein during goldenrod gall formation
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ]348-]352
Vol. 152, No. 3,1988 May 16,1988
INDUCTION OF A 58,000 DALTON PROTEIN DURING GOLDENROD GALL FORMATION
P. Carango, K.D. McCrea, W.G. Abrahamson and M.I. Chernin,*
Bucknell University Biology Department, Lewisburg, PA 17837
Received March 7, 1988
Despite the widespread occurrence of plant-gallmaker interactions, little is known about the actual mechanisms of gall formation. To further characterize this type of parasite-host interaction, the mechanism of gall formation in Solidago altissima, tall goldenrod, by the larva of the tephritid fly Eurosta solidaginis was studied. Proteins produced by galled and ungalled tissues were examined, and the hyperinduction of a 58 kilodalton protein was observed in galled tissues for the second and third week of gall growth. The presence of this protein suggests that a substance secreted by the larva may function as a trans-acting gene regulator. ©1988AcademicPress, Inc.
ABSTRACT:
The debate over the biological nature of the plant gall is nearly as old as the study of natural history.
Theophrastus (ca. 300 B.C.) described
several types of oak galls, but believed the association of insect with gall to be incidental.
Later thought to be a defensive encapsulation initiated by
the host plant in response to an invading organism, gall formation and growth is now known to be caused by the parasite itself.
To further characterize
this type of parasite/host interaction, we studied the mechanism of gall formation in S o i i d a g o altissima (SolidaEo canadensis var. scabra), the tall goldenrod (i) by the larva of the tephritid fly Eurosta solidaginis (Fitch). Proteins produced by galled and ungalled tissues were examined, and the hyperinduction of a native plant protein was observed in galled tissues for the second and third week of gall growth. Gallmaking parasites, or cecidozoans, compose a wide array of organisms, including bacteria, fungi, nematodes, and insects.
It is the insects which
are by far the most numerous, comprising approximately 15,000 species across six different orders.
Interestingly, most gallmakers are host-specific,
and
confine their activities to one single plant species or a few closely related species within one genus.
Accordingly, few plant genera escape gallmaker
* To whom reprint request should be addressed.
0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Vol. 152, No. 3, 1988
attack (2).
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The gall itself is a region of highly differentiated and
organized tissue which functions both as protection and food source for the developing gallmaker.
The form and location of the gall on the host plant is
also species specific, each type of gallmaker producing a structure unique unto itself (3). Despite the widespread occurrence of plant/gallmaker interactions, little is known about the actual mechanisms of gall formation.
Induction by
purely mechanical means (oviposition, feeding of larva, etc.) has already been rejected (3,4), and studies now point to a chemical basis for gall formation. Hovanitz (5) recognized the presence of gall-inducing agents, or cecidotoxins, in the ovipositional fluids of the sawfly (Pontania pacifica), but was unable to identify any of the compounds involved.
A later study by McCalla et al.
succeeded in identifying some of the constituents of the sawfly ovipositional fluids, revealing the presence of glutamic acid, uridine, uric acid, and several as yet unidentified compounds (6).
Both Hovanitz and McCalla were
able to induce gall formation on willow leaves with sawfly extract, and McCalla achieved limited success with each of the individual compounds isolated.
Other studies indicate the importance of several cedidozoan larvalJ
secreted chemicals in gall formation, among them cytokinins or substituted adenines (7,8), auxins or auxin precursors (9,10), gibberellins (Ii), and free amino acids (12). The above studies dealt with the problem of gall formation in terms of larval secretions altering normal plant growth (all used insect gallmaker systems), but working within the existing genetic framework of the plant. Cornell (13), however, suggests that an actual transfer of DNA in the form of a virus or viroid may take place between gallmaker and host, and the information needed to produce the gall is contained within this piece of DNA. Furthermore, the virus may be dependent on the insect's secretions altering the host plant cells in some way, such alterations rendering the plant tissues more susceptible to viral attack.
This idea could explain the fact that many
galls require a constant input of cecidotoxin to reach full development.
The
concept of such a mutualistic relationship between a virus and a eukaryotic organism is not unknown (14), but it has not yet been shown for a gallmaker/plant system. With the incredible number of gallmaking organisms, it is obvious that no one mechanism is likely to be used in gall formation.
However, gall
induction may be accomplished by a certain type of mechanism across a broad range of organisms.
What type of mechanism is used by E. solidaginis in
inducing its characteristic ball gall on S. altissima?
To answer this
question, the mRNA of both galled and ungalled tissue were examined to determine whether the proteins produced by each kind of tissue differed.
1349
Vol. 152, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS
Galled and ungalled ramets of a single genet (clonal plant genotype) were obtained from the field at one week intervals for a period of three weeks from the first appearance of the galls (collection dates: 6-9-87, 6-16-87, 625-87). This three week period covered the major growth of the galls; gall development drops off shortly thereafter (15). Total RNA was extracted by the method of Chirgwin et al. (16). Poly A + (messenger) RNA was isolated by oligo(dT) cellulose column chromatography (17). These RNA's were translated with a rabbit reticulocyte lysate translation system (Promega) using [35S]methionine as a label. Trichloroacetic acid precipitable counts were used to determine the efficiency of translation, and translated proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Samples from all three weeks were run simultaneously to show differences between galled and ungalled tissues from one week to another.
RESULTS AND DISCUSSION
The gel showed an induction of a protein (M r 58,000 daltons) for week 2, and a reduced but continued induction in week 3. week I (Fig. i).
No induction was visible in
These data concur with the time course of gall growth
obtained by McCrea and Abrahamson,
in which gall growth is shown to
approximately fit a bell shaped curve and is seen to peak at -2 weeks development (18).
a
b
c
d
e
f
66I
0 ~" ×
45-
3629-
Fig. I.
SDS-polyacrylamide gel eleetrophoresis of proteins translated from mRNA extracted from week i galled (a) and ungalled (b) tissue, week 2 galled (e) and ungalled (d) tissue and week 3 galled (e) and ungalled (f) tissue. Arrows mark the location of the induced 58 kd protein. All protein samples werestandardized to 106 counts and prepared according to Laemmli (22). Molecular weight markers are indicated to the left of lane a. 1350
Vol. 152, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
There also appears to be a slight induction of a protein of M r 46,000 daltons.
However, these data are difficult to interpret as other experiments
indicate that this protein may be developmentally regulated independent of gall formation. Apparently, a native plant protein is being hyperinduced by some action of the larva.
This 58,000 dalton protein may be regulatory in nature and act
to stimulate later events in the gall forming process. effect is not without precedence.
This presumed cascade
Ashburner (19) has shown that in fruit
flies ecdysone and an early polytene chromosome puff product (presumably a regulatory protein) are necessary for late puff stimulation. The presence of a hyperinduced plant protein in developing gall tissues suggests that a substance secreted by the larva may function as a trans-acting gene regulator, overriding the host plant's normal growth systems to produce the gall.
Several facts about gall formation support this idea.
First, a gall must be formed from meristematic tissue if it is to be fully developed (4,20).
Furthermore, this formation does not seem to be a
stimulation of growth into new tissue types, but is rather a change in the pattern by which normally developed tissues are laid down (2,4,21).
Were gall
growth caused by the introduction of new genetic material, one would expect to find structures not endemic to the host (4). Secondly, gall growth depends on the presence of a living Eurosta larva, indicating that a constant supply of cecidotoxin is required (4,20).
It is
unlikely that this continuous cecidotoxin input is necessary to provide a suitable environment for viral infection in light of what is known about viral particles as infectious agents.
A single infection should be sufficient to
transfer the needed information to the host plant's cells.
Subsequent g[owth
and proliferation of infected cells would then be directed by the information coded in the viral DNA.
However, if gall growth and development is governed
by a trans-acting inducer, the insect would need to provide this inducer in constant supply in order to maintain control over normal plant growth. An alternative hypothesis could be that the protein induction is a hyperinduced wound or defense response to gall initiation, formation, and/or larval feeding and development; not a protein necessary in gall formation.
It
should be noted however, that not all ovipositional attempts are successful. The plant has been "wounded" in the ovipositional attempt but gall formation does not occur.
This is consistent with earlier studies (3,4).
In conclusion, the evidence seems to indicate that goldenrod ball gall formation is caused by a larval secretion altering existing plant growth mechanisms, rather than through the impartation of genetic information to the host.
However, more work is needed to determine the identities of the
hyperinduced plant protein and cecidotoxin(s) of the Eurosta-Solidago system,
1351
VOI. 152, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
as well as their exact relationship within this intriguing parasite/host interaction.
REFERENCES
i. 2.
3.
4, 5. 6. 7 8 9 i0 ii 12 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Abrahamson, W.G. and McCrea, K.D. (1985) Bull. Tort. Bot. Club 112, 414420. Rohrfritsch, O. and Shorthouse, J.D. (1982) In Molecular biology of plant tumors (G. Kahl and J. Schell, Eds.), Academic Press, New York, p. 131152. Abrahamson, W.G. and Weis, A.E. (1987) In Nutritional ecology of insects, mites, spiders, and related invertebrates (F. Slansky and J.G. Rodriguez, Eds.), John Wiley and Sons, New York, p. 235-258. Beck, E.G. (1947) PhD disser., Univ. of Mich., Ann Arbor. Hovanitz, W. (1959) Sci. Am. 201, 151-162. McCalla, D.R., Genthe, M.K. and Hovanitz, W. (1962) Plant Physiol. 37, 98-103. Elzen, G.W (1983) Comp. Biochem. Physiol. 76, 17-19. VanStaden, J. and Davey, J.E. (1978) Bot. Gaz. 139, 36-41. Mapes, C.C and Davies, P.J. (1984) Plant Physiol. (Suppl.) 75, 27. Nuorteva, P. (1956) Suom. Hyonteistiet. Aikak. 22, 108-117. Byers, J.A., Brewer, J.W. and Derma, D.W. (1976) Marcellia 39, 125-134. Heady, S.E., Lambert, R.G. and Covell, C.V. (1982) Gomp. Biochem. Physiol. 73, 641-644. Cornell, H.V. (1983) Am. Mid1. Nat. ii0, 225-232. Edson, K.M., Stoltz, D.B. and Summers, M.D. (1981) Science 211, 582-583. Weis, A.E. and Abrahamson, W.G. (1985) Ecology 66, 1261-1269. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Biochemistry 18, 5294-5299. Aviv, H. and Leder, P. (1972) Proc. Nat. Acad. Sci. U. S. A., 69, 14081412. McCrea, K.D., Abrahamson, W.G. and Weis, A.E. (1984) Ecology 66, 19021907. Ashburner, M. (1974) Dev. Biol. 39, 141-157. Dieleman, F.L. (1969) Ent. Exp. App1. 12, 745-749. Miles, P.W. (1968) Ann. Rev. Phytopath. 6, 137-164. Laemmli, U.K. (1970) Nature 227, 680-684.
1352
Vol. 152, No. 3,1988 May 16,1988
INDUCTION OF A 58,000 DALTON PROTEIN DURING GOLDENROD GALL FORMATION
P. Carango, K.D. McCrea, W.G. Abrahamson and M.I. Chernin,*
Bucknell University Biology Department, Lewisburg, PA 17837
Received March 7, 1988
Despite the widespread occurrence of plant-gallmaker interactions, little is known about the actual mechanisms of gall formation. To further characterize this type of parasite-host interaction, the mechanism of gall formation in Solidago altissima, tall goldenrod, by the larva of the tephritid fly Eurosta solidaginis was studied. Proteins produced by galled and ungalled tissues were examined, and the hyperinduction of a 58 kilodalton protein was observed in galled tissues for the second and third week of gall growth. The presence of this protein suggests that a substance secreted by the larva may function as a trans-acting gene regulator. ©1988AcademicPress, Inc.
ABSTRACT:
The debate over the biological nature of the plant gall is nearly as old as the study of natural history.
Theophrastus (ca. 300 B.C.) described
several types of oak galls, but believed the association of insect with gall to be incidental.
Later thought to be a defensive encapsulation initiated by
the host plant in response to an invading organism, gall formation and growth is now known to be caused by the parasite itself.
To further characterize
this type of parasite/host interaction, we studied the mechanism of gall formation in S o i i d a g o altissima (SolidaEo canadensis var. scabra), the tall goldenrod (i) by the larva of the tephritid fly Eurosta solidaginis (Fitch). Proteins produced by galled and ungalled tissues were examined, and the hyperinduction of a native plant protein was observed in galled tissues for the second and third week of gall growth. Gallmaking parasites, or cecidozoans, compose a wide array of organisms, including bacteria, fungi, nematodes, and insects.
It is the insects which
are by far the most numerous, comprising approximately 15,000 species across six different orders.
Interestingly, most gallmakers are host-specific,
and
confine their activities to one single plant species or a few closely related species within one genus.
Accordingly, few plant genera escape gallmaker
* To whom reprint request should be addressed.
0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
1348
Vol. 152, No. 3, 1988
attack (2).
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The gall itself is a region of highly differentiated and
organized tissue which functions both as protection and food source for the developing gallmaker.
The form and location of the gall on the host plant is
also species specific, each type of gallmaker producing a structure unique unto itself (3). Despite the widespread occurrence of plant/gallmaker interactions, little is known about the actual mechanisms of gall formation.
Induction by
purely mechanical means (oviposition, feeding of larva, etc.) has already been rejected (3,4), and studies now point to a chemical basis for gall formation. Hovanitz (5) recognized the presence of gall-inducing agents, or cecidotoxins, in the ovipositional fluids of the sawfly (Pontania pacifica), but was unable to identify any of the compounds involved.
A later study by McCalla et al.
succeeded in identifying some of the constituents of the sawfly ovipositional fluids, revealing the presence of glutamic acid, uridine, uric acid, and several as yet unidentified compounds (6).
Both Hovanitz and McCalla were
able to induce gall formation on willow leaves with sawfly extract, and McCalla achieved limited success with each of the individual compounds isolated.
Other studies indicate the importance of several cedidozoan larvalJ
secreted chemicals in gall formation, among them cytokinins or substituted adenines (7,8), auxins or auxin precursors (9,10), gibberellins (Ii), and free amino acids (12). The above studies dealt with the problem of gall formation in terms of larval secretions altering normal plant growth (all used insect gallmaker systems), but working within the existing genetic framework of the plant. Cornell (13), however, suggests that an actual transfer of DNA in the form of a virus or viroid may take place between gallmaker and host, and the information needed to produce the gall is contained within this piece of DNA. Furthermore, the virus may be dependent on the insect's secretions altering the host plant cells in some way, such alterations rendering the plant tissues more susceptible to viral attack.
This idea could explain the fact that many
galls require a constant input of cecidotoxin to reach full development.
The
concept of such a mutualistic relationship between a virus and a eukaryotic organism is not unknown (14), but it has not yet been shown for a gallmaker/plant system. With the incredible number of gallmaking organisms, it is obvious that no one mechanism is likely to be used in gall formation.
However, gall
induction may be accomplished by a certain type of mechanism across a broad range of organisms.
What type of mechanism is used by E. solidaginis in
inducing its characteristic ball gall on S. altissima?
To answer this
question, the mRNA of both galled and ungalled tissue were examined to determine whether the proteins produced by each kind of tissue differed.
1349
Vol. 152, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS
Galled and ungalled ramets of a single genet (clonal plant genotype) were obtained from the field at one week intervals for a period of three weeks from the first appearance of the galls (collection dates: 6-9-87, 6-16-87, 625-87). This three week period covered the major growth of the galls; gall development drops off shortly thereafter (15). Total RNA was extracted by the method of Chirgwin et al. (16). Poly A + (messenger) RNA was isolated by oligo(dT) cellulose column chromatography (17). These RNA's were translated with a rabbit reticulocyte lysate translation system (Promega) using [35S]methionine as a label. Trichloroacetic acid precipitable counts were used to determine the efficiency of translation, and translated proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Samples from all three weeks were run simultaneously to show differences between galled and ungalled tissues from one week to another.
RESULTS AND DISCUSSION
The gel showed an induction of a protein (M r 58,000 daltons) for week 2, and a reduced but continued induction in week 3. week I (Fig. i).
No induction was visible in
These data concur with the time course of gall growth
obtained by McCrea and Abrahamson,
in which gall growth is shown to
approximately fit a bell shaped curve and is seen to peak at -2 weeks development (18).
a
b
c
d
e
f
66I
0 ~" ×
45-
3629-
Fig. I.
SDS-polyacrylamide gel eleetrophoresis of proteins translated from mRNA extracted from week i galled (a) and ungalled (b) tissue, week 2 galled (e) and ungalled (d) tissue and week 3 galled (e) and ungalled (f) tissue. Arrows mark the location of the induced 58 kd protein. All protein samples werestandardized to 106 counts and prepared according to Laemmli (22). Molecular weight markers are indicated to the left of lane a. 1350
Vol. 152, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
There also appears to be a slight induction of a protein of M r 46,000 daltons.
However, these data are difficult to interpret as other experiments
indicate that this protein may be developmentally regulated independent of gall formation. Apparently, a native plant protein is being hyperinduced by some action of the larva.
This 58,000 dalton protein may be regulatory in nature and act
to stimulate later events in the gall forming process. effect is not without precedence.
This presumed cascade
Ashburner (19) has shown that in fruit
flies ecdysone and an early polytene chromosome puff product (presumably a regulatory protein) are necessary for late puff stimulation. The presence of a hyperinduced plant protein in developing gall tissues suggests that a substance secreted by the larva may function as a trans-acting gene regulator, overriding the host plant's normal growth systems to produce the gall.
Several facts about gall formation support this idea.
First, a gall must be formed from meristematic tissue if it is to be fully developed (4,20).
Furthermore, this formation does not seem to be a
stimulation of growth into new tissue types, but is rather a change in the pattern by which normally developed tissues are laid down (2,4,21).
Were gall
growth caused by the introduction of new genetic material, one would expect to find structures not endemic to the host (4). Secondly, gall growth depends on the presence of a living Eurosta larva, indicating that a constant supply of cecidotoxin is required (4,20).
It is
unlikely that this continuous cecidotoxin input is necessary to provide a suitable environment for viral infection in light of what is known about viral particles as infectious agents.
A single infection should be sufficient to
transfer the needed information to the host plant's cells.
Subsequent g[owth
and proliferation of infected cells would then be directed by the information coded in the viral DNA.
However, if gall growth and development is governed
by a trans-acting inducer, the insect would need to provide this inducer in constant supply in order to maintain control over normal plant growth. An alternative hypothesis could be that the protein induction is a hyperinduced wound or defense response to gall initiation, formation, and/or larval feeding and development; not a protein necessary in gall formation.
It
should be noted however, that not all ovipositional attempts are successful. The plant has been "wounded" in the ovipositional attempt but gall formation does not occur.
This is consistent with earlier studies (3,4).
In conclusion, the evidence seems to indicate that goldenrod ball gall formation is caused by a larval secretion altering existing plant growth mechanisms, rather than through the impartation of genetic information to the host.
However, more work is needed to determine the identities of the
hyperinduced plant protein and cecidotoxin(s) of the Eurosta-Solidago system,
1351
VOI. 152, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
as well as their exact relationship within this intriguing parasite/host interaction.
REFERENCES
i. 2.
3.
4, 5. 6. 7 8 9 i0 ii 12 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Abrahamson, W.G. and McCrea, K.D. (1985) Bull. Tort. Bot. Club 112, 414420. Rohrfritsch, O. and Shorthouse, J.D. (1982) In Molecular biology of plant tumors (G. Kahl and J. Schell, Eds.), Academic Press, New York, p. 131152. Abrahamson, W.G. and Weis, A.E. (1987) In Nutritional ecology of insects, mites, spiders, and related invertebrates (F. Slansky and J.G. Rodriguez, Eds.), John Wiley and Sons, New York, p. 235-258. Beck, E.G. (1947) PhD disser., Univ. of Mich., Ann Arbor. Hovanitz, W. (1959) Sci. Am. 201, 151-162. McCalla, D.R., Genthe, M.K. and Hovanitz, W. (1962) Plant Physiol. 37, 98-103. Elzen, G.W (1983) Comp. Biochem. Physiol. 76, 17-19. VanStaden, J. and Davey, J.E. (1978) Bot. Gaz. 139, 36-41. Mapes, C.C and Davies, P.J. (1984) Plant Physiol. (Suppl.) 75, 27. Nuorteva, P. (1956) Suom. Hyonteistiet. Aikak. 22, 108-117. Byers, J.A., Brewer, J.W. and Derma, D.W. (1976) Marcellia 39, 125-134. Heady, S.E., Lambert, R.G. and Covell, C.V. (1982) Gomp. Biochem. Physiol. 73, 641-644. Cornell, H.V. (1983) Am. Mid1. Nat. ii0, 225-232. Edson, K.M., Stoltz, D.B. and Summers, M.D. (1981) Science 211, 582-583. Weis, A.E. and Abrahamson, W.G. (1985) Ecology 66, 1261-1269. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Biochemistry 18, 5294-5299. Aviv, H. and Leder, P. (1972) Proc. Nat. Acad. Sci. U. S. A., 69, 14081412. McCrea, K.D., Abrahamson, W.G. and Weis, A.E. (1984) Ecology 66, 19021907. Ashburner, M. (1974) Dev. Biol. 39, 141-157. Dieleman, F.L. (1969) Ent. Exp. App1. 12, 745-749. Miles, P.W. (1968) Ann. Rev. Phytopath. 6, 137-164. Laemmli, U.K. (1970) Nature 227, 680-684.
1352