Jeff McDermott*, Richard Meilan#, and Robert Thornburg*+
*Department of Biochemistry and Biophysics
Iowa State University
Ames, IA 50011
#Department of Biochemistry
University of Missouri
Columbia, MO 65211
+Correspondence should be addressed to:
Phone Number: (515) 294-7885
FAX Number: (515) 294-0453
Submitted for publication: June 1996
Keywords: hackberry, insect galls, proteolytic processing, jumping plant lice, psyllids,
One entomological problem that affects hackberries in the midwestern United States is infestation by the
jumping plant lice. This is a group of insects belonging to the family Psyllidae. These insects look much
like adult cicadas, except that the psyllids are much smaller (4 to 5 mm long). The psyllids are often
severe insect pests of hackberry trees, causing a variety of galls on the
foliage of the trees. One species in particular, Pachypsylla celtidis-mamma Fletcher, causes
the infestation known as the hackberry nipple gall (1).
The hackberry nipple gall maker usually has one generation of insects per year, with the adults emerging
from crevices in the rough bark of the hackberry where they overwinter. Mating and egg laying occurs
over a 2 to 3 week period beginning when the leaves emerge in the spring. The eggs hatch after 7 to 10
days and the nymphs begin feeding on the foliage. The feeding causes morphological changes in the cells
of the leaf of the hackberry which results in the growth of a pouch or a gall that grows up around the
nymphs. The nymphs live within the gall throughout the summer and they emerge as adults in September
(2, 3, 4).
Gall forming insects, thus have the ability to alter the development of plant tissues to cause the formation
of tumor-like growths that surround the insect to protect it from the environment and supply it with a
source of food. However, the mechanisms of gall formation by plants in response to the insect attack
remain largely unknown. In addition, the stimuli which trigger gall formation are also unknown. This
work was undertaken to begin to study the initial events of gall formation by first identifying proteins in
the gall that differ from those in the leaf.
Reagents for PAGE were from Sigma, St. Louis, MO. The tritiated trialcohols of zeatin riboside
([³H]-ZRTA) and isopentenyl adenosine ([³H]-IPTA) were prepared according to the
protocol of Weiler and Spanier (5). The [³H]-NaBH4 was obtained
from New England Nuclear (12.6 Ci/µmol). All other materials were obtained locally and were of
the highest quality available.
Each HPLC fraction was divided into thirds and assayed separately. The cytokinins in each sample were
quantified using radioimmunoassay (10).
The hackberry nipple gall represents a unique plant insect interaction that occurs when the trees respond
to infestation by insect pests of the genus Pachypsylla. A typical gall is a small dome approximately 8 mm
tall that protrudes from the leaf surface (Figure 1, panel A). The fully formed
galls typically contain several tissue types that are organized in layers (11). The outermost layers are the plant's epidermis, and
inside the epidermis is a layer composed of thick-walled, highly lignified cells, which serves to protect the
young insect (see Figure 1, panel B). Inside of this protective layer, is a layer
nutritive cells lining the central chamber. These cells provide a rich source of proteins, sugars and other
nutrients needed for insect growth and development (12).
Because the morphology of the gall is quite different from the leaf, we expected there to be differences in
the proteins expressed in the gall and normal leaf tissues. Therefore, we initially examined the proteins of
the galls and of uninfested hackberry leaves.
Because the galls contain insect nymphs (Figure 1, panel C), the gall tissue was
harvested from the leaves of hackberry trees and the insect larvae were dissected out of the tissue prior to
extracting the proteins. We compared the proteins from the gall and leaf tissues under two different
polyacrylamide concentrations (15% and 6%) so that we could compare both small and large molecular
weight proteins. As shown in Figure 2, the protein profile of the gall tissue
indeed differ from the protein profile of the normal leaf. Many of the major leaf proteins are expressed at
reduced levels in the galls. Further, there are several proteins that are expressed in the gall tissue and are
not strongly expressed in the leaf blade (see arrows). Those
proteins that accumulate preferentially in the galls rather than in the leaves include a set of three large
molecular weight proteins (between 85 kDa and 110 kDa) as well as a protein of 37 kDa. There was also
a 31 kDa protein that was found to be at higher levels in the gall than in the leaf. However, this protein
was also expressed in the normal leaf, and was of less interest to us than the 37 kDa protein.
To demonstrate that the proteins expressed in the galls are different from those present in the insect, we
examined galls after removal of the insect nymphs and the insect nymphs after removal from the galls.
The results of this are shown in Figure 3. There is little similarity between the
protein profiles of the insects and the galls. Therefore, those proteins which are present in the gall and not
in the leaf are newly induced plant proteins and not insect proteins.
In addition to those proteins that are expressed at higher levels in the gall, several leaf proteins show
reduced expression in the gall. Two of these are the two major leaf proteins which have molecular
weights of 57 kDa and about 12 kDa. Because ribulose bisphosphate carboxylase (RUBISCO) shows large
and small subunits of these same sizes, we decided to probe the presence of this enzyme in the leaf and
gall tissues. Figure 4 shows a western blot analysis of leaf and gall tissues
probed with antiserum raised against the large subunit of Amaranth RUBISCO (this antiserum was kindly
provided by Dr. Basil Nikolau, Iowa State University). The anti-Amaranth RUBISCO does indeed show
good cross-reactivity to the hackberry large subunit in leaf tissues (lane 1). However, RUBISCO was
expressed in gall tissues at significantly reduced levels (lane 2). When RUBISCO was quantitated by
cutting and counting the bands from the blot, we found the level of RUBISCO present in the gall was
about 5% of the level in the leaf. Therefore, RUBISCO expression is significantly reduced in the gall
tissues relative to the leaf tissues.
In order to better characterize the proteins that accumulate specifically in the gall, we prepared antiserum
against the 37 kDa protein and examine its expression in leaf and gall tissues by western blot analysis.
Antiserum against the 37 kDa protein was prepared as described in Materials and Methods. When the
antiserum was tested in western blot assays against the gall extracts, it recognized the 37 kDa protein (Figure 5, lane 2), but it also recognized a 52 kDa protein and a 59 kDa protein.
When the proteins were extracted from leaves without galls, we also found cross-reacting material in
uninfested leaves (Figure 5, lane 1). Thus, gall formation appears to result in a
unique proteolysis of a protein that is normally present in the leaf. This proteolytic processing gives rise
to the 37 kDa protein. Because the 37 kDa protein does not accumulate in uninfested leaves, gall
formation may induce a unique proteolytic pathway. Because the 52 kDa intermediate protein also
accumulates, it appears that proteolytic degradation proceeds in a two step process, first removing 7 kDa,
and subsequently removing an additional 15 kDa. Alternatively, it is possible that the 57 kDa protein in
leaves is normally turned over very rapidly and that gall tissues show altered proteolytic degradation of
this protein which allows the 52 and 37 kDa proteins to accumulate. It is not yet clear which of these
alternatives is occurring. Future studies will address this problem.
The structure of the nipple gall indicates that cell proliferation is likely to be involved in its development.
Cytokinins are known to be involved in the control of plant cell division. In addition, they have also been
shown to stimulate the production of certain mRNAs and proteins (13, 14). We therefore sought to determine the levels of
cytokinins present in the leaf and the gall tissues. We utilized the immunological purification and
quantification system of MacDonald and Morris (10) for these studies.
Both uninfested leaf tissue (2.0 g) and nipple gall tissue (0.5 g) were extracted and the cytokinins were
purified by immunoaffinity chromatography and fractionated by HPLC. Those HPLC fractions which
eluted at the same time as authentic cytokinin standards were assayed in triplicate by radioimmunoassay.
In both the leaf and the gall tissues, cytokinins were detected only in those HPLC fractions corresponding
to the retention time of isopentenyladenosine (iPA). The average iPA level in uninfested hackberry leaf
tissue was low. However, in marked contrast to this, the average iPA level in the hackberry nipple gall
tissue was nearly 50 fold higher than the uninfested leaf tissues (see Table
Whether these elevated levels of cytokinins are responsible for the altered protein patterns observed
between the leaf and gall has not yet been determined. Nor is it known whether the source of the
cytokinin is the plant or the insect. It would also be of interest to examine whether exogenous application
of cytokinin could result in the either the production of 37 kDa protein or in growth of gall-like structures
on uninfested hackberry leaf tissues.
The authors would like to thank the State of Iowa and the Iowa State Biotechnology Council for support;
Dr. Sanggyu Park and Dr. Djoko Santoso for their assistance in the laboratory in performing these
experiments; and Dr. R.O. Morris for providing the facilities and the antibodies for conducting the