Communicating Current Research and Educational Topics and Trends in Applied Microbiology

A. Mndez-Vilas (Ed.)
_____________________________________________________________________

Glycoproteins of sugarcane plants facilitate the infectivity of

Ustilago scitaminea and Xanthomonas albilineans, two sugarcane pathogens
M. Blanch, M.E. Legaz, A.M. Millanes and C. Vicente
Department of Plant Physiology, Faculty of Biology, Complutense University, 28040 Madrid, Spain.

Sugarcane plants produce heterofructans composed of homofructan domains consisting of -1,2-fructofuranoside chains which intercalate variable-length segments of polygalactitol. Sometimes, these heterofructans appear as the glycosidic moiety of glycoproteins. They are produced as a response to mechanical injuries and pathogen infections. These glycoproteins act as a defense mechanism against smut
(Ustilago scitaminea) by inducing homotypic adhesion and by inhibiting teliospore germination. When
smut teliospores are cultured on glycoproteins produced by resistant cultivars of sugarcane plants, the actin capping, which occurs before teliospore germination, is efficiently inhibited. Then, cell polarization is
not achieved and the growth of germinative tube is completely inhibited. However, inoculation of sensitive cultivars with smut teliospores induce glycoprotein fractions that promote teliospore polarity after
binding to their cell wall ligand, and are different from those obtained from resistant plants.
On the other hand, leaf scald, a bacterial-vascular disease of sugar cane, has Xanthomonas albilineans as
casual organism. The pathogen is confined mainly to the leaf and stalk vascular bundles, which are often
partly or completely occluded with a gum-like substance, identified as a xanthan-like polysaccharide. This
xanthan-like polysaccharide produced consists of a basal tetramer that is repeated to form the
macromolecule. This basal tetrasaccharide is composed by two molecules of glucose, one mannose rest
and a final glucuronic acid. Since xanthans contain glucuronic acid, the ability of these bacteria to
produce an active UDP glucose dehydrogenase (the enzyme that produces UDP glucuronic acid from
UDPG) is often seen as a virulence factor. Xanthomonas albilineans produces a UDP-glucose dehydrogenase growing on sucrose but the enzyme activity rapidly decays after hydrolysis of the enzyme by bacterial proteases. Thus, X. albilineans axenically cultured did not secrete xanthans to liquid media but the
use of inoculated sugarcane tissues for producing and characterizing xanthans is absolutely required.
Glycoproteins from sugarcane, the natural host of the bacterium, also assures the production of the active
enzyme by inhibiting bacterial proteases.
Keywords Ustilago scitaminea, Xathomonas albilineans, cell polarity, glycoproteins, infectivity, sugarcane, xanthans

1. Introduction
Resistance of plants to disease seems to be a multifactorial process. The response phase includes accumulation of different compounds such as: phytoalexins (i.e. low molecular mass antimicrobial compounds that accumulate at sites of infection); systemic enzymes that degrade pathogens (e.g. chitinases,
-1,3-glucanases and proteases); systemic enzymes that generate antimicrobial compounds and protective biopolymers (e.g. peroxidases and phenoloxidases); biopolymers that restrict the spread of pathogens (e.g. hydroxyproline-rich glycoproteins, lignin, callose); and regulators of the induction and/or
activity of defensive compounds (e.g. elicitors of plant and microbial origin, immune signals from
primed plants and compounds, which release immune signals) [1].Other glycoproteins are involved in
resistance responses. Three varieties of sugarcane, defined by their relative resistance to smut, have been
previously used to study the production of glycoproteins after smut infection.

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A. Mndez-Vilas (Ed.)
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2. Production of glycoproteins as a plant response to diseases caused by

microorganisms.
Sugarcane produces, after infection by several pathogens, two different pools of glycoproteins containing
a heterofructan as glycidic moiety and tentatively described as high molecular mass (HMMG) and midmolecular mass (MMMG) glycoproteins [2]. The hydrolysis of the polysaccharide moiety of both
HMMG and MMMG, HMMC and MMMC (high- and mid-molecular mass carbohydrates) respectively,
results in free fructose
Signal
molecules from
the host

Pathogen
N

Lytic enzymes
secreted from the
pathogen

Growth inhibition

lisis

chitinase
-1,3-glucanase
others

lysis

receptors

Plant
cell

Signal
transduction

fitoalexins
PAL

Nucleus

RP proteins

Figure 1. Chemical communication between the plant cell and its pathogen. The
pathogen secretes lytic enzymes to degrade the plant cell wall and some of these
lysis products can act as signal molecules
to bind to cell wall receptors of the pathogen. Metabolites from the microorganism
elicit plant responses of induced resistance
consisting of the production of phytoalexins or resistance proteins (RP) which impede the growth of the pathogen.

Induced
resistance

Gene expression
related to the
defence response

and galactitol. Quantitation of products of the acidic hydrolysis reveals differences of the polymerization
degree of these polysaccharides. MMMC is thought to be a [Fructose2:Galactitol3]n polymer while
HMMC results to be a [Fructose4:Galactitol5]n polysaccharide [3]. HMMC and MMMC consist of a
minor chain of (12) fructofuranoside, hydrolyzed by invertase, on which a heteropolymer composed
of fructose-galactitol is attached. Hexitol-hexose bond showed to be sensitive to acidic hydrolysis but
unaccesible to invertase action. This ether linkage can be hydrolyzed by a sugarcane glycosidase that
produced three times more fructose than galactitol. Enzyme activity was clearly dependent on the presence of Mn2+ in the reaction mixtures. Purified native glycosidase has an apparent molecular mass of
13.2 kDa.
The occurrence of soluble polysaccharides in sugarcane juices causes a reduction in the amount of
sucrose recovered during the industrial process of crystallization [4] by forming molasses which affect
the purity of the syrup, changing its physico-chemical properties.
Analysis of both HMMG and MMMG by capillary electrophoresis reveals that MMMG fraction
contains two cationic and four anionic components whereas only one cationic and four anionic proteins
are separated from the HMMG fraction [5]. These glycoproteins can be considered as factors of
biological resistance to smut [6] since their amount increases in resistant cultivars but decrease in
susceptible varieties after infection with smut teliospores. In addition, Fontaniella et al. [7] found that
these glycoproteins selectively bind to smut teliospores, preferently those defined as anionic peptides.
After binding, cell aggregation occurs in parallel to a loss of the germination ability of recruited
teliospores and the emergence of the germinating tube is completely impeded.

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3. Sugarcane smut
Sugarcane smut is caused by the dimorphic basidiomycete Ustilago scitaminea. The characteristic
symptom of culmicolous smut is the formation of a long whip-like sorus in which diploid, airborne
teliospores are produced [8]. Spore germination originates a pro-mycelium which give rise to four
haploid sporidia after meiosis. When two compatible sporidia form germ tubes that fuse, a dikaryotic,
infecting mycelium is produced [9].
Hyphal growth occurs throughout the
infected plant but mostly in the
HMMG
parenchyma cells of the cane stalk
internode. The formation of the dikarion
CW
is readily recognized in vitro when culture
Glycase
changes from a yeast-like appearance to
HMMC
HMMGor
white, fuzzy mycelial growth [10] .
MMMC
or MMMC

PM
PLGV
HMMG

GA

TGN
Apo-HMMG

ER

Figure 2. Parenchymatous cells of sugarcane

stalks are able to produce proteins (ApoHMMG) which are glycosylated (HMMG) in
Golgi cisternae (TGN) to be moved through
the cytoplasm (PLGV) and secreted across the
plasma membrane (PM) into the cell wall
(CW). A glycosidase (Glycase) system catalyzes the total or partial hydrolysis of these
glycoproteins and the resulting polysaccharides
(HMMC and MMMC) are removed from the
cell wall to appear as soluble macromolecules
in the cytoplasm.

One of the major changes previous to, or during spore germination concerns cytoplasm polarization, a
process mediated by actin [11-13], that is essential for fungal tip growth. Ustilago maydis, the causal
pathogen of corn smut disease, divides by budding at its unicellular yeast-like stage. During budding, the
actin cytoskeleton polarizes to sites of active growth [14]. Because the bud tip is the site of the
incorporation of new cell wall material during bud growth, the concentration of actin at this site suggests
that actin plays an active role in the secretion of cell wall polymers.

4. The role of sugarcane glycoproteins in the inhibition/promotion of the

germination of smut teliospores.
HMMG and MMMG fractions recovered from Barbados 42-231 cv. contain chitinase activity, the level
of which was minor in HMMG than in the second fraction, disappearing from the first one and getting
down in the second one when they were obtained from inoculated plants. Put both fractions, HMMG or
MMMG, in contact with smut teliospores, chitinase of healthy plants fastly and completely binds to
those, whereas the low amount of MMMG chitinase obtained from inoculated plants binds to
theliospores only in a 36 %.
The differences among these glycoproteins and their activities obtained from resistant or susceptible
cultivars to smut can be summarized in a following way:

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1 The proteomic endowment of Mayar 55-14 cv., resistant to the disease, shows a high degree of
diversity and microheterogeneity, estimated by means of capillary electrophoresis [15], whereas a low
level of complexity is revealed for glycoproteins obtained from. Barbados cv, sensitive to smut.
2 The cv Mayar shows high chitinase activity in both HMMG and MMMG and high capacity of
interaction with teliospores, whereas it is minor in Barbados and very low if the plant has been before
infected [16].

Germinative pole
Actin (inact.)

Cell wall
ligand

profilin

Actin (act.)

GTPase(act.)
cAMP
+

GTPase(inact.)

Figure 3. Signal molecules from the plant

cells (HMMG or MMMG) bind to specific receptors in the cell wall of smut
teliospores to activate a metabolic cascade
involving GTPase activated by cyclic
AMP and profiling that promotes actin
polarization. This rearrangement of the
cytoskeleton indicates the position of the
germinative pole through which the protrusion and growth of the fungal hypha is
achieved.

Signal molecule

3 Glycoproteins from the cv. Mayar show a high capacity of secretion of proteins from teliospores
of the pathogenic fungus, which makes them detectable for the plant, whereas the above mentioned
capacity is very low in the cv. Barbados, which significantly reduces its ability to recognize the
pathogen.
4 The ability of cane glycoproteins to join specific receptors has been studied by labelling with
fluorescein isothiocyanate and by the
Sugarcane
glycoproteins

U. scitaminea
N

Lytic enzymes and

glycoproteins from
the extracellular
matrix secreted
from the pathogen

receptors

Sugarcane
cell

Glycoprotein
neosynthesis

Signal
transduction

Figure 4. Smut teliospores secrete signal molecules (lytic enzymes and other glycoproteins)
which act as ligands for several plant cell wall
receptors. The binding promotes the biosynthesis
of both HMMG and MMMG which activates
teliospore germination when they are produced
by sensitive cultivars, or inhibition of the germination when they are produced by resistant cultivars.

Nucleus

observation of teliospores treated with labelled glycoproteins by fluorescence microscopy. The binding
of the glycoproteins of the resistant cultivar to the wall of smut teliospores disable cell polarization and
the reorganization of its cytoskeleton in order to mark the germinative pole. This effect was observed by
labelling the fungal actin by reaction with phaloidin to which the suitable fluorophore was previously

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A. Mndez-Vilas (Ed.)
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bound [15]. On the contrary, glycoproteins from Barbados are unable to disable this reorganization but
they promote cellular polarization and spore germination.
5 Consistent with this capability to bind to the walls of smut teliospores, HMMG and MMMG from
the resistant Mayari cv. show high affinity to bind to N-acetyl-D-glucosamine residues in glycoproteins
of the teliospore wall whereas the efficiency of Barbados HMMG and MMMG for the same receptor is
very low. This interaction has been studied after a selective extraction of glycoproteins from teliospore
walls and their retention in a bead of activated agarose to which HMMG or MMMG from both cultivars
had joined. The major recovery of fungal protein from the agarose bead was achieved by elution with a
50 mM N-acetyl-D-glucosamine solution.

5. Leaf scald disease

Xanthomonas albilineans is a member of Pseudomonaceae, a yellow-pigmented, Gram-negative plant
pathogenic bacterium [17]. This microorganism produces a xanthan-like polysaccharide which occludes
both xylem and phloem producing desiccation of leaves [18]. This xanthan-like polysaccharide consisted
of a basal tetramer composed by two molecules of glucose, one mannose rest and a final glucuronic acid
that is highly repeated to form the macromolecule [19]. The occurrence of glucuronate rest in the
polysaccharide requires the action of an UDP-glucose dehydrogenase which catalyses the NAD+dependent oxidation of UDP-glucose to UDP-glucuronic acid. It belongs to a small group of
dehydrogenases that are able to carry out the two-fold oxidation of an alcohol to an acid without the
release of an aldehyde as intermediate [20]. This enzyme has a wide range of functions. In plants, UDPglucose dehydrogenase is the main enzyme in the pathway of synthesis of hemicelluloses and pectins,
which are the components of newly formed cell walls [21].

6. Regulation of UDPglucose dehydrogenase by sugarcane glycoproteins

X. albilineans in liquid culture is able to produce an enzyme which catalyses a redox reaction using
UDPG as substrate and NADPH as a cofactor. However, the absence of redox reaction by using NAD+
or NADP+ instead of NADPH implies that the enzyme does no catalyse the conventional reaction of
UDPG dehydrogenase (EC 1.1.1.22):
UDP-glucose + NAD+ + H2O UDP-glucuronic acid + NADH
as described for the protein identified from animals, plants and bacteria [22-25], but the alternative
reaction
UDP-glucose + NADPH + O2 UDP-glucuronic acid + NADP+ + H2O
as deduced from the oxygen dependence found for the enzymatic oxidation of NADPH.
The time-course of UDPG dehydrogenase activity shows that it strongly decreased in bacterial cells
growing in Wilbrink media for 15 h culture to remain unchanged to 24h at very low level. The initial loss
of UDPG dehydrogenase activity was repeated by bacterial cells growing in Wilbrink media supplied
with HMMG but strongly increased later, from 15h to 24h culture. When bacteria were grown on media
supplemented with MMMG, the initial decrease of UDPG dehydrogenase activity was abolished and the
enzyme activity strongly increased at the end of the time of culture.
By including 0.1 mM 8-azaguanine in culture media, the activity of the enzyme was maintained
unchanged along the time of culture, even when HMMG or MMMG were included in the culture media,
although values of activity obtained when sugarcane glycoproteins were added to the media were always

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Figure 5. Elicitors produce by bacteria invading plant tissues promote the synthesis of sugarcane glycoproteins
(HMMG) that are internalized into the bacterial cells. When bacteria synthesized UDPG dehydrogenase
(UDPGDH), the enzyme can be inactivated by bacterial proteases (BP), impeding the production of UDPglucuronic acid and, consequently, the synthesis and secretion of xanthans. However, cane glycoproteins synthesized
by sensitive cultivars act as powerful inhibitors of these bacterial proteases, facilitating then the synthesis of xanthans.

slightly higher than those obtained when the inhibitor of transcription was used alone. Then, it could be
concluded that sugarcane glycoproteins do not compete with 8-azaguanine to enhance transcription of
the corresponding mRNA. When 0.1 mM chloramphenicol was included in the Willbrink media, only
MMMG seemed to compete with the inhibitor by enhancing the enzyme production at 24h culture. Both
inhibitors, 8-azaguanine and chloramphenicol nullified the reported loss of UDPG dehydrogenase
activity in the absence of sugarcane glycoproteins. UDPG dehydrogenase activity was completely
nullified by increasing the concentration of inhibitors from 100 M to 300 M. These results could be
explained by considering that 100 M 8-azaguanine or chloramphenicol mainly inhibit protease
synthesis whereas the same inhibitors at a concentration of 300 M could inhibit the synthesis of both
proteases and UDPG dehydrogenase.
Dow et al. [26,27] reported that proteases could be used to degrade protein in the plant cell wall,
allowing bacterial spread or overcome host defences. The role of proteases in the pathogenicity depends
on the hostpathogen system [28], in which proteases act in the cleavage of peptide bonds and play a role
in many physiological functions [29]. However, in absence of the plant host, bacterial proteases could be
used to hydrolyze bacterial proteins, such as UDPG dehydrogenase. Since sugarcane glycoproteins
inhibit bacterial proteases in vitro and permit then the production of UDPG dehydrogenase, this can be

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A. Mndez-Vilas (Ed.)
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interpreted as an interdependence between host and pathogen, probably derived from a coevolution
process. This could explain why X. albilineans did not produce xanthans in culture whereas the gum was
secreted from bacteria invading sugarcane tissues [30].
Acknowledgements This work was supported by a grant, BFU2006-14263, from the Ministerio de
Educacin (Spain). We are grateful to Miss Raquel Alonso for the excellent technical support.

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