(Fig. 3C). Coimmunoprecipitation of LOV1 and
TRX-h5 was not successful, possibly because of
conditions required for solubilizing LOV1. Nonetheless, the cumulative data indicate that LOV1
and TRX-h5 interact in some manner at the plasma
membrane, consistent with the idea that TRX-h5
is guarded by LOV1.
The guard model accounts for plants having
immunity to a myriad of pathogens while possess-
ing a limited number of R genes (2, 3). R gene
limitation is possible because effector targets are
limited, and pathogens (however numerous) secrete
functionally redundant virulence effectors. This
implies that R genes across plant species evolve
to guard common targets (14). We have observed
victorin sensitivity in oats, Arabidopsis, barley,
rice, Brachypodium (15), and bean (fig. S6). Be-
cause victorin binds diverse thioredoxins (fig.
S1) and sensitivity is conditioned by a NB-LRR
gene (LOV1) in Arabidopsis, inseparable from
the Pc2 resistance gene in oats, and mapped to a
genomic region rich in NB-LRR genes in barley
(15), the data suggest that victorin sensitivity is
evoked by a common mechanism across these
species: by victorin binding to a thioredoxin that
is guarded by a NB-LRR protein. Given this and
the important defense functions of TRXs (6), it is
possible that multiple pathogens target thioredox-
ins to enhance virulence (i.e., redundant virulence
effectors). Notably, C. victoriae does not cause
disease in Arabidopsis in the absence of LOV1 or
in oats in the absence of Vb (5). This is important
because it implies that victorin production did not
evolve in C. victoriae to inhibit TRX-h5–conferred
defense. Rather, C. victoriae uses victorin solely in
its capacity as a defeated effector to exploit R gene–
mediated defense for disease susceptibility. This
suggests that other defeated effectors could confer
virulence if expressed by the appropriate pathogen.
References and Notes
1. J. M. Lorang, T. A. Sweat, T. J. Wolpert, Proc. Natl. Acad.
Sci. U.S.A. 104, 14861 (2007).
2. J. D. G. Jones, J. L. Dangl, Nature 444, 323 (2006).
3. B. J. De Young, R. W. Innes, Nat. Immunol. 7, 1243
4. T. A. Sweat, J. M. Lorang, E. G. Bakker, T. J. Wolpert,
Mol. Plant Microbe Interact. 21, 7 (2008).
5. T. J. Wolpert, L. D. Dunkle, L. M. Ciuffetti, Annu. Rev.
Phytopathol. 40, 251 (2002).
6. Y. Tada et al., Science 321, 952 (2008).
7. H. Cao, S. A. Bowling, A. S. Gordon, X. Dong, Plant Cell
6, 1583 (1994).
8. T. A. Sweat, T. J. Wolpert, Plant Cell 19, 673 (2007).
9. C. Laloi, D. Mestres-Ortega, Y. Marco, Y. Meyer,
J. P. Reichheld, Plant Physiol. 134, 1006 (2004).
10. See supplementary materials on Science Online.
11. T. J. Wolpert, V. Macko, W. Acklin, D. Arigoni, Plant
Physiol. 88, 37 (1988).
12. C. C. Mundt, Phytopathology 99, 1116 (2009).
13. A. Marmagne et al., Mol. Cell. Proteomics 6, 1980
14. T. Wroblewski et al., Plant Physiol. 150, 1733 (2009).
15. J. Lorang, A. Cuesta-Marcos, P. M. Hayes, T. J. Wolpert,
Mol. Breed. 26, 545 (2010).
16. E. D. Nagy, J. L. Bennetzen, Genome Res. 18, 1918
17. J. D. Faris et al., Proc. Natl. Acad. Sci. U.S.A. 107, 13544
Acknowledgments: We thank J. Chang and M. Behrenfeld
for valuable discussion. This work was supported in part by the
Agriculture and Food Research Initiative Competitive Grants
Program from the USDA National Institute of Food and
Agriculture (grants 2005-35319-15361 and 2008-35319-18651)
and by NSF grant IOS-0724954. OSU’s mass spectrometry
facility and core is in part supported by National Institute of
Environmental Health Sciences grant P30ES200210.
Materials and Methods
Figs. S1 to S6
29 June 2012; accepted 5 September 2012
Published online 18 October 2012;
Tug-of-War in Motor Protein Ensembles
Revealed with a Programmable
DNA Origami Scaffold
N. D. Derr,1,2,3* B. S. Goodman,1* R. Jungmann,4,5 A. E. Leschziner,6
W. M. Shih,2,3,5 S. L. Reck-Peterson1†
Cytoplasmic dynein and kinesin-1 are microtubule-based motors with opposite polarity that
transport a wide variety of cargo in eukaryotic cells. Many cellular cargos demonstrate bidirectional
movement due to the presence of ensembles of dynein and kinesin, but are ultimately sorted
with spatial and temporal precision. To investigate the mechanisms that coordinate motor
ensemble behavior, we built a programmable synthetic cargo using three-dimensional DNA origami
to which varying numbers of DNA oligonucleotide-linked motors could be attached, allowing for
control of motor type, number, spacing, and orientation in vitro. In ensembles of one to seven
identical-polarity motors, motor number had minimal affect on directional velocity, whereas
ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one
To dissect the biophysical mechanisms of
motor-driven cargo transport, we designed a programmable, synthetic cargo using three-dimensional
DNA origami (8, 9) (also see supplementary materials and methods). The cargo consisted of a 12-
helix bundle with 6 inner and 6 outer helices (Fig.
1A and fig. S1) (10). We refer to this structure as
a “chassis,” akin to an automobile chassis that
serves as a skeletal frame for the attachment of
additional components. The origami chassis was
made by rapidly heating and slowly cooling an
8064-nucleotide, single-strand DNA (ssDNA)
“scaffold” in the presence of 273 short, ssDNA
“staples” (fig. S1A and tables S1 to S3), which
hybridize with discontinuous regions of the
scaffold to fold it into a desired shape. Selective
inclusion of staples with extra “handle” sequences
that project out from the chassis provide site- and
sequence-specific attachment points for motors,
fluorophores, or other chemical moieties (Fig. 1B).
Cytoplasmic dynein and kinesin-1 (referred to as “dynein” and “kinesin” here) are opposite-polarity, microtubule-based motors
that are responsible for producing and maintaining subcellular organization via the transport of
many cargos in eukaryotic cells (1, 2). Defects in
these transport processes have been linked to neurological diseases (1, 3, 4). Microtubules contain
inherent structural polarity, polymerizing rapidly at
their “plus” ends and more slowly at their “minus”
ends (5), with dynein and kinesin driving most
minus- and plus-end–directed microtubule transport, respectively (2). Although some transport
tasks require a single motor type, many cargos use
both dynein and kinesin and move bidirectionally
on microtubules (1, 6, 7). The mechanisms that
allow ensembles of identical-polarity motors to coordinate their activity and ensembles of opposite-polarity motors to achieve both processive movement
and rapid switches in direction are unknown.
1Department of Cell Biology, Harvard Medical School, Boston,
MA 02115, USA. 2Dana-Farber Cancer Institute, Boston, MA
02115, USA. 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA
02115, USA. 4Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. 5Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA
02115, USA. 6Department of Molecular and Cellular Biology,
Harvard University, Cambridge, MA 02138, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail: