A plant pathogen virulence factor inhibits the
eukaryotic proteasome by a novel mechanism
Michael Groll1*, Barbara Schellenberg2*, Andre ´ S. Bachmann3,4, Crystal R. Archer3,4, Robert Huber5,6,7,
Tracy K. Powell8, Steven Lindow8, Markus Kaiser9& Robert Dudler2
Pathogenic bacteria often use effector molecules to increase viru-
lence. In most cases, the mode of action of effectors remains
unknown. Strains of Pseudomonas syringae pv. syringae (Pss)
secrete syringolin A (SylA), a product of a mixed non-ribosomal
a virulence factor because a SylA-negative mutant in Pss strain
B728a obtained by gene disruption was markedly less virulent
on its host, Phaseolus vulgaris (bean). We show that SylA irrevers-
ibly inhibits all three catalytic activities of eukaryotic protea-
somes, thus adding proteasome inhibition to the repertoire of
modes of action of virulence factors. The crystal structure of the
yeast proteasome in complex with SylA revealed a novel mech-
anism of covalent binding to the catalytic subunits. Thus, SylA
defines a new class of proteasome inhibitors that includes glido-
bactin A (GlbA), a structurally related compound from an
unknown species of the order Burkholderiales2, for which we
demonstrate a similar proteasome inhibition mechanism. As pro-
teasome inhibitors are a promising class of anti-tumour agents,
the discovery of a novel family of inhibitory natural products,
which we refer to as syrbactins, may also have implications for
the development of anti-cancer drugs3. Homologues of SylA and
GlbA synthetase genes are found in some other pathogenic bac-
teria, including the human pathogen Burkholderia pseudomallei,
the causative agent of melioidosis4. It is thus possible that these
bacteria are capable of producing proteasome inhibitors of the
Pss is a pathogen of many plant species, causing, for example,
brown spot disease on bean (P. vulgaris)5. Some Pss strains secrete
SylA, a peptide derivative synthesized by a mixed non-ribosomal
peptide synthetase (NRPS)/polyketide synthetase (PKS) encoded
by a gene cluster which was previously cloned from the strain Pss
B301D-R2. To examine the functional role of SylA, we constructed a
SylA-negative mutant by targeted gene disruption of the sylC gene in
Pss B728a, a strain that causes brown spot disease on bean (P. vul-
garis) and whose SylA synthetase gene cluster is almost identical to
that of strain B301D-R2. The sylC gene encodes the first NRPS
module of the SylA synthetase, and disruption of sylC in the B301D-
bean plants revealed a significantly reduced virulence of the mutant
strain. The number of brown spots developing on bean leaves after
spray inoculation was reduced to 29621% compared with the wild
type in four independent experiments (Fig. 1). Thus, SylA was iden-
tified as a virulence factor in this plant–pathogen interaction.
Previously performed transcript profiling revealed that exogenous
application of SylA on wheat and Arabidopsis thaliana leads to pro-
nounced changes in global gene activity6. Intriguingly, transcripts
encoding all proteasome subunits and many heat-shock proteins
for yeast and mammalian cells after treatment with proteasome
inhibitors7,8. Therefore, we tested whether SylA was a proteasome
inhibitor. The eukaryotic 20S proteasome contains three catalytic
subunits (b1, b2 and b5) conferring caspase-like, trypsin-like and
chymotrypsin-like proteolytic activities, respectively3. In vitro experi-
measurement of proteasome activity in the presence of SylA revealed
of the irreversible proteasome inhibitor epoxomicin and in contrast
to the effect of the reversible peptide aldehyde inhibitor MG-132
(Fig. 2a), suggesting that SylA actsas an irreversible proteasome inhi-
bitor. This finding was confirmed by removing inhibitors and sub-
of substrate to dialysed proteasomes previously inhibited by MG-132
led to renewed activity, whereas proteasomes that had been inhibited
chymotrypsin-like activity was found to be most sensitive to SylA
inhibition, whereas higher concentrations of SylA were necessary to
*These authors contributed equally to this work.
1Center for Integrated Protein Science at the Department Chemie, Lehrstuhl fu ¨r Biochemie, Technische Universita ¨t Mu ¨nchen, Lichtenbergstrasse 4, Garching D-85747, Germany.
2Zurich–Basel Plant Science Center, Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland.3Cancer Research Center of Hawaii, University of
651 Ilalo Street, Honolulu, Hawaii 96813, USA.5Max-Planck-Institut fu ¨r Biochemie, D-82152 Martinsried, Germany.6School of Biosciences, Cardiff University, Cardiff CF10 3US, UK.
7Zentrum fu ¨r medizinische Biotechnologie, Universita ¨t Duisburg-Essen, D-45117 Essen, Germany.8Department of Plant and Microbial Biology, University of California, 111 Koshland
Hall, Berkeley, California 94720-3102, USA.9Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, D-44227 Dortmund, Germany.
Lesions per trifoliate leaf
Exp. 1 Exp. 2Exp. 3
**P < 0.01
*P < 0.05
Figure 1 | Syringolin-negativemutantexhibitsreducedvirulence. Fivepots
per experiment (Exp), each with eight 18-day-old bean plants, were spray-
inoculated with 105cells per millilitre of wild-type or SylA-negative (sylC
the five replica pots are given. p, error probability (two-sided t-test).
Vol 452|10 April 2008|doi:10.1038/nature06782
inhibit trypsin-like and caspase-like activities (Fig. 2b–d). In contrast
to MG-132, which also inhibits cysteine proteases, SylA inhibition of
the proteasome was specific because both papain (Fig. 2e) and the
serineprotease trypsin were notaffected by SylA, even at significantly
higher concentrations (up to 500mM). SylA also inhibited the
chymotrypsin-like activity of the proteasome in crude extracts of
etiolated bean seedlings (Supplementary Fig. 2).
We next determined whether SylA inhibits in vivo proteasome
function in Arabidopsis seedlings and human cultured cells. To this
end, an Arabidopsis line homozygous for a CyclinB1;1::uidA (GUS)
reporter fusion gene9was used. The mitotic CyclinB1;1 gene is
expressed only around the G2/M transition of the cell cycle10. The
fusion gene contains a cyclin destruction box leading to the pro-
teasomal degradation of the protein at the end of the M phase11.
Proteasomal dysfunction due to amutation has been shown to result
in strong GUS staining of dividing cells12. Indeed, much stronger
staining of dividing cells in the root meristem of 3-day-old seedlings
treated with 10mM SylA solution compared with controls was
observed (Supplementary Fig. 3). Incubation of human SK-N-SH
neuroblastoma cells with SylA for 2h resulted in a dose-dependent
decrease of proteasome activity (Supplementary Fig. 4a). As
expected13, incubation of SK-N-SH cells with 25mM SylA resulted
in a time-dependent accumulation of ubiquitinated proteins (Sup-
plementary Fig. 4d). The SylA treatment of SK-N-SH cells was
further accompanied by a time-dependent increase in the protein
levels of the tumour suppressor protein p53, a known target of the
proteasome and an important regulator of apoptosis and cell-cycle
progression (Supplementary Fig. 4d).
To elucidate the binding mode of SylA to the proteasome we co-
crystallized SylA with the yeast 20S proteasome and collected data to
2.9A˚resolution. Crystallographic refinement started from the coor-
dinates of the yeast 20S proteasome14followed by anisotropic overall
temperature factor correction and positional refinement using the
Crystallography & NMR system (CNS) software suite (Rcrys/Rfree5
21.8/24.8; Supplementary Table 1)15. Electron-density maps calcu-
lated with phases after twofold averaging allowed a detailed inter-
pretation of SylA (Fig. 3c), revealing that SylA covalently binds to
the hydroxy group of the active site amino (N)-terminal threonine
by a novel mechanism: Thr1Ocof the proteasome performs a
Michael-type 1,4-addition to the double bond located at C4 in the
12-membered ring system of the inhibitor. The resulting covalent
ether bond withthe 12-membered ring system causes theirreversible
inhibition.Thisreactionisfacilitated byGly47N,which stabilizesthe
carbonyl anion of SylA in its activated transition intermediate state
by hydrogen bonds (oxyanion hole) (Fig. 3b). Furthermore, the 12-
membered ring structure of SylA containing the functional reactive
group has a constrained conformation favouring high-affinity bind-
functional reactive double bond at the 12-membered ring is com-
bined with a dipeptide located in its close proximity (Fig. 3a). This
dipeptide is essential for stabilization of SylA by formation of an
the mean residence time of the inhibitors at the active centre for
completing the covalent ether bond formation with Thr1Oc.
The peptide structure of SylA is closely related to glidobactin A
(GlbA; Fig. 3a), an acylated peptide derivative reported to have anti-
tion. Indeed, GlbA blocked the chymotrypsin-like activity irrever-
sibly at low concentrations, whereas the trypsin-like activity was less
sensitive (Fig. 2d) and the caspase-like activity was not inhibited at
the concentrations tested (up to 20mM). The crystal structure of
GlbA in complex with the proteasome was elucidated at 2.7A˚
resolution, yielding Rcrys/Rfree523.4/26.2 (Supplementary Table 1)
(Fig. 3d). As expected, the complex structure revealed the same
mechanism of inactivation of the chymotrypsin-like (Fig. 3f) and
trypsin-like activities as SylA, whereas GlbA did not bind to the
caspase-like active site.
The results described above show that SylA is a virulence factor
that inhibits proteasome function, thus adding proteasome inhibi-
tion to the repertoire of modes of action of such factors. Although
secretion systems17–19, SylA is thought to be secreted by an exporter
belonging to the major facilitator superfamily2. This, together with
the fact that exogenous application of SylA leads to proteasome
of apoptosis20in mammalian cancer cells, as well as changes in gene
activity typical for proteasome inhibition in Arabidopsis and wheat,
0 20 4060 1030 50
8.0 × 104
1.0 × 105
1.2 × 105
1.4 × 105
1.6 × 105
1.8 × 105
25 µM MG132
50 µM MG132
100 µM MG132
25 µM SylA
50 µM SylA
100 µM SylA
Inhibitor Inhibited activity
843 ± 8.4 nM (n = 3) 863 ± 106 M–1 s–1 (n = 6; 100–200 nM)
6.7 ± 0.7 µM (n = 6) 94 ± 12 M–1 s–1 (n = 6; 150–600 nM)
6 ± 0.3 M–1 s–1 (n = 6; 20–40 µM)
49 ± 5.4 nM (n = 3) 3,377 ± 341 M–1 s–1 (n = 6; 40–60 nM)
2.0 ± 0.6 µM (n = 6) 141 ± 21 M–1 s–1 (n = 6; 250–500 nM)
0 2040 6080100120
1.6 µM MG132
0.2 µM Epo
1.6 µM Epo
0.2 µM SylA
1.6 µM SylA
0 204060 80100 120
1.25 µM SylA
5 µM SylA
40 µM SylA
0 20 40 6080
5 µM SylA
20 µM SylA
80 µM SylA
Figure 2 | SylA inhibits the eukaryotic proteasome. a–c, Inhibition of
chymotrypsin-like (a), trypsin-like (b) and caspase-like (c) activities of
mammalian proteasomes by SylA, epoxomicin (Epo) and MG-132 using
fluorogenic substrates. RFU, relative fluorescence units. d, Apparent Ki9
values and rates of covalent inhibition (kassociation) over inhibitor
concentrations given in parentheses were derived from plots as shown in
a–c (see Methods). Values represent means6s.d. e, Cleavage of fluorescent
casein by papain in the presence of MG-132 and SylA.
NATURE|Vol 452|10 April 2008
indicates that SylA is taken up by cells. As SylA is a hydrophilic
molecule, it probably cannot passively cross membranes, and
thus an uptake transporter must be postulated, which remains to
Previous studies have shown that the host proteasome has been
instrumentalized by plant pathogens to suppress host defence reac-
tions by type III effectors mediating proteasomal degradation of
specific host proteins2,21,22. On the other hand, there is increasing
evidence that the ubiquitin–proteasome degradation pathway is
essential for pathogen defence and disease immunity of plants23,24.
Pss appears to capitalize on this by inhibiting the host proteasome
using SylA, thereby suppressing host defence reactions directly or
indirectly. It also remains to be seen whether the proteasome is the
sole target of SylA.
A unique model completely explaining the synthesis of the
peptide part of SylA, including the functionally important double
bond at C4 of the 12-membered ring system, has been proposed
based on the architecture of the sylC and sylD genes in the SylA
synthetase gene cluster2. The model allowed the recent cloning of
genes responsible for GlbA synthesis, including the sylC and sylD
homologues glbF and glbC, from K481-B101, the strain from which
GlbA was originally isolated16and which belongs to an unknown
species of the order Burkholderiales4. A National Center for
genomes revealed homologues of these genes in the insect pathogen
Photorhabdus luminescens and the human pathogen B. pseudomallei,
the causative agent of melioidosis4. These organisms are therefore
hypothesized to be capable of synthesizing proteasome inhibitors
of the syrbactin class. If this prediction is confirmed, it will be inter-
esting to determine whether the respective compounds are involved
Proteasome inhibitors form a promising new therapeutic class of
drugs against cancer and other diseases, causing intense interest in
such molecules3,25. Indeed, SylA was recently shown to inhibit pro-
cells20. Elucidation of the structural inhibition motif of syrbactins
opens new perspectives in the identification of natural products with
proteasome inhibition potential, as well as the design and creation of
bitors derived from such natural products can find their potential
application in drug development.
Mutant construction and plant inoculations. Construction of the SylA-
negative mutant by plasmid insertion into the sylC gene of Pss B728a was as
describedforthecorresponding mutantinstrainPssB301D-R2. VirulenceofPss
Figure 3 | Structural basis for proteasome inhibition by syrbactins.
a, Chemical structure of SylA and GlbA. Red, a,b-unsaturated carbonyl
group reacting with Thr1Oc; green, dipeptide bond stabilizing the inhibitor
upon proteasome binding; blue, molecule part determining active site
specificity; yellow, aliphatic tail of GlbA. b, Mechanism of binding of SylA/
GlbAto the active siteThr1. c, d, Stereorepresentation ofthechymotryptic-
like active site (rose, subunit b5; light blue, subunit b6) in complex with
(c) SylA (green; PDB accession code 2ZCY) and (d) GlbA (green, aliphatic
tail in yellow; PDB accession code 3BDM). Magenta, covalent linkage of
residues performing specific interactions with SylA and GlbA. Electron-
density maps (grey) are contoured from 1s in similar orientations around
Thr1. e, Electrostatic potential surface (contoured from 115kT/e (intense
blue) to 215kT/e (intense red)) of SylA bound to subunit b5. f, Structural
superposition of SylA (green) with GlbA (yellow) bound to subunit b5.
NATURE|Vol 452|10 April 2008
strains was assessed by counting the number of lesions on leaves of bean (P.
vulgaris) plants sprayed with suspensions of 105cells per millilitre (ref. 26).
Isolation of SylA and GlbA. SylA and GlbA have been isolated as described2,4,27.
SylA solutions with defined concentrations were prepared by dissolving
weighted amounts of SylA in water. Concentrations of GlbA solutions were
tion coefficient of 35,000M21cm21(ref. 16). For proteasome inhibition assays,
stock solutions of GlbA in DMSO were diluted with water.
Proteasome/protease inhibition assays. In vitro proteasome inhibition assays
were performed in 96-well microtiter plates with human erythrocyte 20S pro-
teasomes using the Assay Kit for Drug Discovery AK-740 (Biomol). In vivo
proteasome inhibition in cultured mammalian cells was measured using the
proteasome-Glo cell-based luminescent assay (Promega) in solid white 96-well
microtiter cell culture plates. Bovine pancreas trypsin (Pierce) and Papaya latex
papain (Sigma-Aldrich) inhibition assays were performed with the SensoLyte
Green Protease Fluorometric Assay Kit (AnaSpec).
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 10 October 2007; accepted 28 January 2008.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank U. Grossniklaus and J. Celenza (Boston University)
grateful to L. Eberl for use of equipment. C.R.A was supported by a graduate
student research assistantship from the Cell and Molecular Biology Graduate
Program, University of Hawaii. Support by grants from the Swiss National Science
Foundation to R.D. is acknowledged.
Author Contributions M.K., M.G., S.L., R.D. and A.S.B. designed experiments,
analysed results and wrote the manuscript. R.D. constructed the sylA-negative
mutant, T.K.P. performed the bean inoculation experiments, B.S. isolated SylA and
mammalian cell-based proteasome inhibition assays and immunoblot studies.
Author Information Atomic coordinates have been deposited in the RCSB Protein
proteasome–syringolin A complex) and 3BDM (yeast 20S
proteasome–glidobactin A complex). Reprints and permissions information is
available at www.nature.com/reprints. Correspondence and requests for
materials should be addressed to R.D. (firstname.lastname@example.org), A.S.B.
(email@example.com) or M.K. (firstname.lastname@example.org).
NATURE|Vol 452|10 April 2008
METHODS Download full-text
In vitro proteasome/protease assays and determination of constants.
Proteasome inhibition assays with human erythrocyte 20S proteasomes (AK-
740, Biomol) were performed at 37uC in 100ml reaction volumes containing
2mgml2120S proteasome and 100mM Suc-LLVY-AMC, Boc-LRR-AMC or
Z-LLE-AMC for assaying the chymotrypsin-like, trypsin-like and caspase-like
activity, respectively. Fluorescence was monitored with an MWGt Sirius HT
plate reader (BIO-TEK Instruments) equipped with 360nm excitation and
460nm emission filters.
Fluorescence values (RFU) measured in proteasome inhibition assays were
plotted versus time (t). The fluorescence data (see Fig. 2) were fitted by the least
squares method using SigmaPlot version 10.0 software (Systat Software) to the
equation f5f01vst1[(vi2vs)/kobs][12exp(2kobst)], where viand vsare
initial and final velocities, respectively, and kobsis the pseudo-first-order asso-
ciation rate constant13,28. Fittings with R2values.0.99 were usually obtained.
The rate of covalent inhibition (kassociation) which equals kobs/[I], was calculated
over the range of inhibitor concentrations given in parentheses in Fig. 2d. The
initial velocities (vi) were determined from the time derivative of these curves
and plotted against the corresponding inhibitor concentration ([I]). Where
concentration ([I]). Using SigmaPlot, the data were fitted to the hyperbolic
equation vi5c1[v0/(Ki91[I])], where v0 is the velocity of the control.
Usually, R2values.0.98 were obtained.
Bovine pancreas trypsin (Pierce) and Papaya latex papain (Sigma-Aldrich)
inhibition assays were performed with the SensoLyteTM Green Protease
Fluorometric Assay Kit (AnaSpec) in 100ml volumes containing 200ngml21
enzyme at 33uC. For detection, 485/550nmexcitation/emission filters were
Inhibition assays of plant proteasomes were performed with crude lysates of
7-day-old etiolated bean seedlings. Hypocotyls and emerging first leaves of
seedlings were ground in liquid nitrogen and extracted with twice the amount
(v/w) of extraction buffer (50mM Tris-HCl, pH 7.5, 2mM EDTA, 100mM
NaCl, 10mM sodium orthovanadates). After centrifugation at 500g for
15min, the supernatant was used for inhibition experiments of the proteasome
chymotrypsin-like activity in microtitre plates. Each reaction contained 20ml
lysate (about 100mg protein), 100mM Suc-LLVY-AMC substrate, desired con-
centrations of inhibitors and assay buffer (AK-740, Biomol) to a volume of
100ml. In some experiments, the cysteine protease inhibitor E64 (Sigma) was
included in each reaction at a final concentration of 50mM. Reactions were
monitored at room temperature every 4min as described above. Reaction rates
were approximately linear between the 8 and 32min time points.
In vivo proteasome inhibition and immunoblot analysis. Source and main-
For in vivo proteasome inhibition assays, wells of solid white 96-well microtiter
cell culture plates were seeded with 1.13105cells per millilitre (90ml per well).
After 24h, 10ml SylA was added at different concentrations (0–100mM) and
incubated for 2h. The proteasome inhibitor epoxomicin (0–1mM) and the
apoptosis-inducing agent cerulenin (0–50mgml21)30,31were used as positive
and negative controls, respectively (Supplementary Fig. 4). Microtiter plates
were then equilibrated to room temperature for 15min before 100ml of
proteasome Glo reagent (containing the bioluminescent substrate Suc-LLVY-
manufacturer’s instructions (Promega).
For immunoblot analysis, cells were seeded in six-well cell culture plates at a
concentration of 3.83105cells per millilitre (1.98ml per well). After 24h, 20ml
of SylA (final concentration 25mM) or cell culture medium (control, C) was
samples at the same time after 24h. Cell lysates were prepared as previously
reported29. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS–PAGE), electro-transfer to PVDF Immobilon-P membranes (Millipore)
and antibody incubations were performed according to standard procedures.
Sigma), mouse monoclonal tumour suppressor protein p53 (1:250) (sc-126;
Santa Cruz Biotechnology) and rabbit monoclonal a-tubulin (1:1,500) (11H10;
Cell Signalling Technology). Horseradish peroxidase (HRP)-conjugated anti-
mouse or anti-rabbit antibodies (1:5,000) were used as secondary antibodies.
Membranes were developed using the ECL Plus kit following the manufacturer’s
protocol (Amersham Biosciences) and exposed to Blue Lite Autorad Film (ISC
HCl, pH 6.7, 2% SDS, 100mM b-mercaptoethanol) for 20min at 55uC and
times with similar results, as shown in Supplementary Fig. 4d.
Treatment and GUS-staining of transgenic Arabidopsis carrying a CyclinB1;
1::uidA fusion gene. A homozygous line containing a transgene derived from
pCDG in which the CycB1;1 promoter plus the sequence encoding the first 150
amino acids of the CYCB1;1 protein including a cyclin destruction box were
fused in frame to the uidA reporter gene encoding b-glucuronidase (GUS)9.
Seeds of this line, which were provided by U. Grossniklaus, University of
Zurich, were germinated on vertically oriented MS plates (4.4gl21Murashige
and Skoog basal medium with Gamborg’s vitamins (Sigma), 3% sucrose and
to 3-day-old seedlings. After an 18h incubation period, seedlings were trans-
ferred to a 96-well microtiter plate, stained for GUS activity for 60min as
described32and observed under a Leica DMR microscope.
Co-crystallization. Crystals of the 20S proteasome from S. cerevisiae were
grown in hanging drops at 24uC as described14,33and incubated for 60min with
in Tris-HCl (10mM, pH7.5) and EDTA (1mM). The drops contained 3ml of
protein and 2ml of the reservoir solution (30mM magnesium acetate, 100mM
morpholino-ethane-sulphonic acid (pH 7.2) and 10% MPD).
The space group belongs to P21with cell dimensions of about a5134A˚,
b5302A˚, c5144A˚and b5112u (see Supplementary Table 1). Data to 2.9A˚
for the yeast 20S proteasome–SylA- and to 2.7A˚for the 20S proteasome–GlbA
complexes were collected using synchrotron radiation with l51.05A˚on
the BW6-beamline at DESY, Hamburg, Germany, and with l51.0A˚on the
X06SA-beamline at SLS, Villingen, Switzerland, respectively. Crystals were
soaked in a cryoprotecting buffer (30% MPD, 20mM magnesium acetate,
100mM morpholino-ethane-sulphonic acid, pH 6.9) and frozen in a stream
of liquid nitrogen gas at 90K (Oxford Cryo Systems). X-ray intensities were
evaluated by using the DENZO program package34and data reduction was
performed with CCP435. The anisotropy of diffraction was corrected by an
overall anisotropic temperature factor by comparing observed and calculated
structure amplitudes using the program CNS15. Electron density was improved
by averaging and back transforming the reflections ten times over the twofold
non-crystallographic symmetry axis using the program package MAIN36.
Conventional crystallographic rigid body, positional and temperature factor
refinements were performed with CNS15using the yeast 20S proteasome struc-
ture as starting model14. Model building was performed using the program
MAIN. Apart from the bound inhibitor molecules, structural changes were only
noted in the specificity pockets. Temperature factor refinement indicates full
occupancies of all inhibitor-binding sites. The inhibitors have been omitted for
28. Fenteany, G. et al. Inhibition of proteasome activities and subunit-specific amino-
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