The involvement of the orbitofrontal cortex in the experience of regret.
ABSTRACT Facing the consequence of a decision we made can trigger emotions like satisfaction, relief, or regret, which reflect our assessment of what was gained as compared to what would have been gained by making a different decision. These emotions are mediated by a cognitive process known as counterfactual thinking. By manipulating a simple gambling task, we characterized a subject's choices in terms of their anticipated and actual emotional impact. Normal subjects reported emotional responses consistent with counterfactual thinking; they chose to minimize future regret and learned from their emotional experience. Patients with orbitofrontal cortical lesions, however, did not report regret or anticipate negative consequences of their choices. The orbitofrontal cortex has a fundamental role in mediating the experience of regret.
- SourceAvailable from: Claire M Gillan[show abstract] [hide abstract]
ABSTRACT: BACKGROUND: Obsessive-compulsive disorder (OCD) is a disorder of automatic, uncontrollable behaviors and obsessive rumination. There is evidence that OCD patients have difficulties performing goal-directed actions, instead exhibiting repetitive stimulus-response habit behaviors. This might result from the excessive formation of stimulus-response habit associations or from an impairment in the ability to use outcome value to guide behavior. We investigated the latter by examining counterfactual decision making, which is the ability to use comparisons of prospective action-outcome scenarios to guide economic choice. METHODS: We tested decision making (forward counterfactual) and affective responses (backward counterfactual) in 20 OCD patients and 20 matched healthy control subjects using an economic choice paradigm that previously revealed attenuation of both the experience and avoidance of counterfactual emotion in schizophrenia patients and patients with orbitofrontal cortex lesions. RESULTS: The use of counterfactual comparison to guide decision making was diminished in OCD patients, who relied primarily on expected value. Unlike the apathetic affective responses previously shown to accompany this decision style, OCD patients reported increased emotional responsivity to the outcomes of their choices and to the counterfactual comparisons that typify regret and relief. CONCLUSIONS: Obsessive-compulsive disorder patients exhibit a pattern of decision making consistent with a disruption in goal-directed forward modeling, basing decisions instead on the temporally present (and more rational) calculation of expected value. In contrast to this style of decision making, emotional responses in OCD were more extreme and reactive than control subjects. These results are in line with an account of disrupted goal-directed cognitive control in OCD.Biological psychiatry 02/2013; · 8.93 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Considering the neuroscientific findings on reward, learning, value, decision-making, and cognitive control, motivation can be parsed into three sub processes, a process of generating motivation, a process of maintaining motivation, and a process of regulating motivation. I propose a tentative neuroscientific model of motivational processes which consists of three distinct but continuous sub processes, namely reward-driven approach, value-based decision-making, and goal-directed control. Reward-driven approach is the process in which motivation is generated by reward anticipation and selective approach behaviors toward reward. This process recruits the ventral striatum (reward area) in which basic stimulus-action association is formed, and is classified as an automatic motivation to which relatively less attention is assigned. By contrast, value-based decision-making is the process of evaluating various outcomes of actions, learning through positive prediction error, and calculating the value continuously. The striatum and the orbitofrontal cortex (valuation area) play crucial roles in sustaining motivation. Lastly, the goal-directed control is the process of regulating motivation through cognitive control to achieve goals. This consciously controlled motivation is associated with higher-level cognitive functions such as planning, retaining the goal, monitoring the performance, and regulating action. The anterior cingulate cortex (attention area) and the dorsolateral prefrontal cortex (cognitive control area) are the main neural circuits related to regulation of motivation. These three sub processes interact with each other by sending reward prediction error signals through dopaminergic pathway from the striatum and to the prefrontal cortex. The neuroscientific model of motivational process suggests several educational implications with regard to the generation, maintenance, and regulation of motivation to learn in the learning environment.Frontiers in Psychology 01/2013; 4:98.
- [show abstract] [hide abstract]
ABSTRACT: One of the most intriguing frontiers of current neuroscientific research is represented by the investigation of the possible neural substrates of morality. The assumption is that in humans an innate moral sense would exist. If this is true, with no doubt it should be regulated by specific brain mechanisms selected over the course of evolution, as they would promote our species' survival. In the last decade, an increasing number of studies have been carried out to explore the neural bases of human morality.The aim of this paper is to present a comprehensive review of the data regarding the neurobiological origin of the moral sense, through a Medline search of English-language articles from 1980 to February 2012.The available findings would suggest that there might be a main integrative centre for the innate morality, in particular the ventromedial prefrontal cortex, with its multiple connections with the limbic lobe, thalamus and brainstem. The subjective moral sense would be the result of an integration of multiple automatic responses, mainly associated with social emotions and interpretation of others' behaviours and intentions.Since converging observations outline how lesions of the proposed neural networks may underlie some personality changes and criminal behaviours, the implications of the studies in this field encompass many areas of the scientific domain.Annals of General Psychiatry 03/2013; 12(1):6. · 1.57 Impact Factor
The Involvement of the
Orbitofrontal Cortex in the
Experience of Regret
Nathalie Camille,1* Giorgio Coricelli,1,2* Jerome Sallet,1
Pascale Pradat-Diehl,3Jean-Rene ´ Duhamel,1Angela Sirigu1†
Facing the consequence of a decision we made can trigger emotions like
satisfaction, relief, or regret, which reflect our assessment of what was gained
as compared to what would have been gained by making a different decision.
These emotions are mediated by a cognitive process known as counterfactual
thinking. By manipulating a simple gambling task, we characterized a subject’s
choices in terms of their anticipated and actual emotional impact. Normal
subjects reported emotional responses consistent with counterfactual thinking;
they chose to minimize future regret and learned from their emotional expe-
rience. Patients with orbitofrontal cortical lesions, however, did not report
regret or anticipate negative consequences of their choices. The orbitofrontal
cortex has a fundamental role in mediating the experience of regret.
When faced with mutually exclusive options,
the choice we make is conditioned by what
we hope to gain, the economist’s “expected
value,” but it is also influenced by how we
hope we will feel afterward. The emotional
component of a decision may be the reason
why, once we are committed to a course of
action, we often prefer to ignore what would
have happened if we had chosen differently
(1), especially if the outcome turns out to be
unfavorable. Missed opportunities as a result
of wrong choices may indeed result in the
emotion of regret (2). Regret is a cognitively
mediated emotion triggered by our capacity
to reason counterfactually. Counterfactual
thinking is the mechanism by which we
compare “what is” with “what might have
been” (3, 4). Contrary to mere disappoint-
ment, which is experienced when a nega-
tive outcome happens independently of our
own decision, regret is an emotion strongly
associated with a feeling of responsibility
(5). Regret has a profound impact in deci-
sion making (6) and is a powerful predictor
of behavior because people’s choices are
often made to avoid this highly unpleasant
emotion (7, 8).
What are the cerebral structures mediating
such fundamental human emotions as regret?
One potentially critical player is the orbito-
frontal cortex, a structure that is connected
with the dorsolateral prefrontal regions active
in reasoning and planning, with limbic areas
such as the amygdala important for emotion,
and with other areas providing direct or indi-
rect access to multiple sensory modalities (9).
The orbitofrontal cortex is also active in re-
ward evaluation and comparison (10–13). Pa-
tients with lesions in this region show poor
social and individual decision-making skills
and abnormal anticipatory emotional re-
The orbitofrontal cortex thus appears to be
at the interface of emotion and cognition and
is ideally suited to control emotional experi-
ence through mechanisms such as counterfac-
tual reasoning. We adopted a decision-theory
framework to test the prediction that advan-
tageous choice behavior depends on the abil-
ity to anticipate and hence minimize regret.
We adapted an experimental paradigm in-
spired from the work of Mellers et al. (5) to
analyze the emotional impact of decisions in
terms of disappointment and regret and to test
whether the ability to experience these emo-
tions is mediated by the orbitofrontal cortex.
Normal subjects and patients with orbito-
frontal cortex lesions were presented with a
choice between two risky gambles with a
monetary reward (17) (Fig. 1). We tested
several predictions: (i) the same obtained out-
come will lead to different experienced emo-
tions depending on whether feedback about
the outcome of the unchosen option is avail-
able; (ii) as compared with the emotions of
normal subjects, the emotions of patients
with orbitofrontal lesions will not show an
effect of feedback about the outcome of the
unchosen option; and (iii) choice strategy
will develop as a result of the ability to take
1Institut des Sciences Cognitives, CNRS, 67, Boulevard
Pinel 69675 Bron, France.2Department of Economics,
University of Siena, Piazza San Francesco 7, 53100
Siena, Italy.3Service de Me ´decine Physique et Re ´ad-
aptation, Ho ˆpital de la Salpetriere, 47 Boulevard de
l’Ho ˆpital, 75013 Paris, France.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-
Fig. 1. Time course of a gambling
trial. Two wheels appeared on a
computer screen (gamble 1 and
gamble 2). Each wheel had two
sectors (black and light gray) asso-
ciated with different value pairs.
The size of each sector indicated
the outcome probability. The two
possible outcomes are formed by
any pair of the following values:
?50, –50, ?200, –200 (units cor-
respond to former French francs),
associated with different outcome
probabilities (0.8, 0.2, 0.5). The
subject selected one of the two
wheels by clicking a mouse. A
rectangular box appeared around
the selected wheel. In partial feed-
back blocks, a spinning arrow ap-
peared only in the selected wheel,
rotated for a variable duration,
and stopped in one of the two
sectors. Only the outcome of the
selected wheel could be seen. In
complete feedback blocks, a spin-
ning arrow appeared in both the
wheels. The arrows rotated and
stopped, allowing the subject to
each trial, subjects rated their af-
fective state using a rating scale
from –50 (extremely sad) to ?50
R E P O R T S
www.sciencemag.orgSCIENCE VOL 304 21 MAY 2004
into account the outcome of the unchosen
option in normal subjects but not in orbito-
The subjective emotions experienced in
this gambling task depend on the values of
the obtained outcome and the unobtained
outcome. Other things being equal, subjects
express more pleasant emotions when the
obtained value is positive than when it is
negative. The effect of the unobtained out-
come strongly modulates that of the obtained
outcome. In the partial feedback condition,
disappointment is expressed in the perception
of losses as more unpleasant and gains as less
pleasant if the unobtained outcome from the
same gamble wins 200 (units correspond to
former French francs) instead of losing 200
(Fig. 2A, Wilcoxon signed-rank test, Z ?
–3.703, P ? 0.001, for –50 obtained; Z ?
–3.637, P ? 0.001, for ?50 obtained). The
emotional reaction is modulated more strong-
ly in the complete feedback condition, show-
ing the effect of regret. Losing 50 when the
unchosen alternative wins 200 induces a
strong negative feeling, whereas the same
outcome is perceived as indifferent when the
other gamble loses more (Fig. 2C, Wilcoxon,
Z ? –3.237, P ? 0.0012). Even a gain of 50
can produce unhappiness if the other option
wins more (Wilcoxon, Z ? –3.680, P ?
0.001), whereas it produces a pleasant sensa-
tion when the other gamble loses (18).
Direct comparisons between the two
conditions show different levels of emo-
tional involvement under complete and par-
tial feedback. Affective ratings for a given
outcome obtained in the face of a more
favorable outcome of 200 for the unchosen
gamble are more negative than in the face
of an unobtained outcome of 200 for the
chosen gamble (Wilcoxon, P ? 0.001, for
both –50 and ?50 obtained outcomes).
This is the signature of regret: an unpleas-
ant emotion triggered by knowledge of the
rejected alternative’s outcome.
Skin conductance response (SCR) in-
creases when learning the outcome of the
gamble, revealing the emotional nature of
this information. The distinction between dis-
appointment and regret expressed by subjec-
tive affective ratings is confirmed by the
physiological index of emotional reactivity,
because viewing the outcome of the rejected
alternative enhances SCR as compared with
viewing only the outcome of the chosen gam-
ble (Fig. 2E, paired t test, t ? –2.124, P ?
0.0406, two-tailed). This effect is particularly
pronounced when losing as compared with
winning 50, suggesting that regret potentiates
more strongly an already negative emotion
(t ? –2.007, P ? 0.031).
A very different pattern of results was
observed in patients with orbitofrontal le-
sions. Like normal subjects, they are gener-
ally happier when winning than when losing
(Wilcoxon, Z ? –3.296, P ? 0.001), and their
SCR demonstrates clear emotional arousal
when learning the outcome of the gamble.
The disappointment effect, i.e., the effect of
the unobtained outcome of the chosen option,
is present but without as much contrast as that
seen in normal subjects. When losing, pa-
tients were somewhat sadder if the unob-
tained outcome was a large gain than if it was
a greater loss (Fig. 2B, Wilcoxon, Z ?
–1.671, P ? 0.094, for –50 obtained). When
patients won, the effect of the unobtained
outcome was not significant (Wilcoxon, Z ?
–1.483, P ? 0.138, for 50 obtained). This
result suggests that they were somewhat able
to think counterfactually on the chosen gam-
ble. However, the emotions expressed by
these patients are not modulated at all by the
feedback on the outcome of the unchosen
gamble, and they seem to experience no re-
gret whatsoever (Fig. 2D). Sadness expressed
at losing 50 is not more intense if the rejected
alternative wins 200, nor is the joy felt at
winning 50 tarnished by seeing that the gain
would have been larger had the alternative
gamble been selected [see fig. S1 for patients’
individual performance and (17) for detailed
statistical analyses]. It should be stressed that
the absence of a regret effect cannot be ex-
plained by a less differentiated emotional ex-
pression or by a reluctance to use the ex-
tremes of the rating scales, because patients,
like normal subjects, were shown to use the
full range of the affective rating scale with
the larger values of obtained outcomes (i.e.,
–200 and 200, fig. S2). SCR data confirm a
lack of emotional reaction of the orbitofrontal
patients to the outcome of the rejected gam-
ble (Fig. 2F).
Three control patients with frontal lesions
sparing the orbital area participated in the
experiment. Emotional ratings show the ef-
fects of the unobtained outcome in both the
partial and complete feedback condition, in-
dicating that they responded with disappoint-
ment and regret in a manner comparable to
Fig. 2. Effect of the
of the gamble in par-
feedback. (A and C)
Mean emotional rat-
ings made by 18 nor-
mal control subjects
for two obtained out-
comes (–50 or 50) as a
function of the unob-
tained outcome (blue
–200; red line and
symbols, 200) in the
partial and complete
partial condition, the
unobtained value of
the chosen gamble. In
the complete condi-
tion, it corresponds to
gamble. (B and D)
Mean emotional rat-
ings made by five or-
bitofrontal patients in
the partial and com-
plete feedback condi-
Conventions as in (A)
and (C). (E) Mean skin
(? standard error) of
normal subjects, mea-
sured at the end of
arrow rotation, for the
conditions in which
the unobtained out-
come is more advantageous than the obtained outcome (corresponding to the red curves in the
graphs above). Gray bars (partial feedback) and black bars (complete feedback) are physiological
markers of disappointment and regret, respectively. Regret is correlated with stronger emotional
arousal. (F) Same data for orbitofrontal patients, showing no regret effect.
R E P O R T S
21 MAY 2004VOL 304 SCIENCEwww.sciencemag.org
normal subjects (fig. S3), clearly showing the
selectivity of the effects to the orbital region
within the frontal lobe (see Fig. 3 for location
of maximum lesion overlap in orbitofrontal
patients). It should be stressed that the lack of
regret observed in orbitofrontal patients is not
due to a general lack of interest in potential
monetary gains. They responded emotionally
to winning and losing, as shown by the basic
affective ratings. They saw the actual piles of
coins building up (or down) from one trial to
the next and kept track of their earnings.
Neither was this indifference due to an in-
ability to orient attention to more than one
gamble at a time. Throughout the task the
experimenter verified that the patients had
correctly registered the outcome of each gam-
ble before recording the affective rating.
To determine the influence of anticipated
emotions of disappointment and regret on the
decision process, we tested a model of choice
incorporating these emotional variables as
well as the expected values of the two gam-
bles. The outcome structure of the experi-
ment was defined so that the two gambles
always differed in their expected values, but
the gamble with the highest expected value
won less often on average that the one with
the lowest expected value. This was done to
ensure that the subject would experience neg-
ative emotions on a sufficient number of
trials. Under this condition, subjects could
learn to choose advantageously by anticipat-
ing future emotional reactions and trying to
avoid negative emotions.
We tested the model exclusively with
data from the complete feedback condition,
considering that in the partial condition the
feedback provided did not elicit regret (Ta-
ble 1, regression analysis) (17). Patients
chose only according to the expected values
of the gambles (the coefficient of e is pos-
itive and significant), whereas the normal
subjects anticipated regret (the coefficient
of r is positive and highly significant). The
results of our model show that the variable
d (anticipated disappointment) is not sig-
nificant for either group.
As a result of this anticipated emotional
process, the normal control subjects more
often chose the advantageous gamble, ending
up with net gains. The mean of earnings for
the normal subjects was 366.66. By contrast,
the orbitofrontal patients more often chose
the disadvantageous gamble, ending up with
net losses (mean earnings ? –110). The dif-
ference in earnings between the two groups
was statistically significant (Mann-Whitney
U test, Z ? 2.513, P ? 0.0120). The normal
subjects earned significantly more in the
complete condition than in the partial condi-
tion (297.22 versus 69.44, Wilcoxon, Z ?
–2.902, P ? 0.0037), whereas there was no
significant difference between patients’ earn-
ings in the partial and complete condition
(Wilcoxon, P ? 1).
In contrast to the standard theory in deci-
sion making (19), our results show that the
emotions related to experiencing gains or
losses are not independent from the alterna-
tive outcomes. Indeed, it is the counterfactual
thinking between the obtained and unob-
tained outcomes that determines the quality
and intensity of the emotional response (20).
Regret and disappointment are elicited by
two different counterfactual comparisons
characterized by two different levels of per-
sonal responsibility for the consequence of
one’s own choices (21, 22). The absence of
regret in orbitofrontal patients suggests that
these patients fail to grasp this concept of
liability for one’s own decision that colors the
emotion experienced by normal subjects.
We showed that regret generates higher
physiological responses and is consistently
reported by normal subjects as more intense
than disappointment. This was not the case in
orbitofrontal patients, demonstrating that dis-
tinct neural processes generate these two
emotions. The specificity of the orbitofrontal
region in mediating regret is strengthened by
the finding that three control patients with
lesions in other parts of the frontal lobes
showed normal regret levels and choice be-
havior in our gambling task.
Previous work implicating the orbitofrontal
cortex in emotion-based decision making prin-
cipally emphasized bottom-up influences of
emotions on cortical decision processes (14,
16). We propose a different role whereby the
orbitofrontal cortex exerts a top-down modula-
tion of emotions as a result of counterfactual
thinking, after a decision has been made and its
consequences can be evaluated. As shown by
the model of choice, the feeling of responsibil-
ity for the negative result, i.e., regret, reinforces
the decisional learning process. The orbitofron-
tal cortex integrates cognitive and emotional
components of the entire process of decision
inability to generate specific emotions such as
regret, which has a fundamental role in regulat-
ing individual and social behavior.
Fig. 3. Lesion overlap in the orbitofrontal cortex
for the five patients. Lesion locations were re-
constructed from individual magnetic reso-
nance imaging scans. The three slice levels (in
Talairach coordinates) show the region of com-
mon cortical damage, which is located in the
basal and ventromedial sector of the prefrontal
cortex and which includes Brodmann’s areas
10, 11, 32, 24, and 47.
Table 1. Dynamic model of choice: regression analysis. Given that Pr(g1) ?
1 – Pr(g2), where Pr(g1) and Pr(g2) are the probabilities of choosing gamble 1
and gamble 2, respectively, we define the probability of choosing g1in terms
of three factors affecting the choice: anticipated disappointment (d), antici-
pated regret (r), and expected value (e). Let us call x1, y1, and x2, y2the two
possible outcomes of the first (g1) and the second (g2) gambles, respectively,
with x1? y1, and x2? y2. The probability of x1is p and the probability of y1
is (1 – p). The probability of x2is q and the probability of y2is (1 – q). The
model is Pr (g1it) ? F [dit, rit, eit], where i is individual and t is time. The
function F [?] denotes the function exp(?) / [1 ? exp(?)]. The dependent
variable, “choice of g1,” is 1 when the subject chooses g1and 0 when the
subject chooses g2. Independent variables are d, r, e, where anticipated
disappointment choosing g1, d ? [?y2– x2? (1 – q)] – [?y1– x1? (1 – p)];
anticipated regret choosing g1, r ? [?y2– x1? – ?y1– x2?]; and maximizing
expected value choosing g1, e ? EV(g1) – EV(g2) ? [p x1? (1 – p) y1] – [q
x2? (1 – q) y2]. EV, expected value. Data is from 18 normal control subjects
and 5 orbitofrontal patients, in complete feedback condition. Panel logit
procedure with individual random effects yields the following results.
Normal control subjects
Log likelihood ? –211.68417
Wald chi2(3) ? 128.23
Prob ? chi2? 0.0000
Log likelihood ? –116.60227
Wald chi2(3) ? 54.89
prob ? chi2? 0.0000
zP ? ?z?
R E P O R T S
www.sciencemag.orgSCIENCEVOL 30421 MAY 2004
References and Notes
1. D. Kahneman, D. Miller, Psychol. Rev. 93, 136 (1986).
2. Consider the following example: “Eight years ago a
man from Liverpool in UK, committed suicide. He
decided to end his life after he knew that he missed
out on a £2 million prize in the National Lottery.
While watching television he saw the numbers of his
winning combination, 14, 17, 22, 24, 42, and 47,
appearing one by one on the screen. He always
played these numbers, but on this occasion, he had
not renewed his ticket on time which actually expired
the previous Saturday” (23). Although extreme, this
example illustrates the profound regret this man felt
once he knew the opportunity he missed to become
3. N. J. Roese, J. M. Olson, What Might Have Been: The
Social Psychology of Counterfactual Thinking (Erl-
baum, Mahwah, NJ, 1995).
4. R. M. J. Byrne, Trends Cogn. Sci. 6, 426 (2002).
5. B. Mellers, I. Ritov, A. Schwartz, J. Exp. Psychol. Gen.
3, 332 (1999).
6. M. Zeelenberg, J. Beattie, J. van der Pligt, N. K. de
Vries, Organ. Behav. Hum. Decis. Process. 65, 148
7. D. E. Bell, Oper. Res. 30, 961 (1982).
8. G. Loomes, R. Sugden, Econ. J. 92, 805 (1982).
9. E. T. Rolls, Cereb. Cortex 10, 284 (2000).
10. H. C. Breiter, I. Ahron, D. Kahneman, A. Dale, P.
Shizgal, Neuron 30, 619 (2001).
11. R. Elliott, J. L. Newman, O. A. Longe, W. J. F. Deakin,
J. Neurosci. 23, 303 (2003).
12. L. Tremblay, W. Schultz, Nature 398, 704 (1999).
13. R. J. Dolan, Science 298, 1191 (2002).
14. A. Bechara, A. R. Damasio, H. Damasio, S. W. Ander-
son, Cognition 50, 7 (1994).
15. V. Goel, J. Grafman, J. Takik, S. Gana, D. Danto, Brain
120, 1805 (1997).
16. A. Bechara, H. Damasio, A. R. Damasio, Cereb. Cortex
10, 295 (2000).
17. Materials and methods are available as supporting
material on Science Online.
18. Avoiding a loss of 200 changes the negative impact
of losing 50 into a neutral affective response. Data
for ?50 obtained / –200 unobtained versus ?50
obtained / –50 unobtained show that the emotional
evaluation (elation) is higher in the first case com-
pared with the second one. Thus, the magnitude of
the avoided monetary loss does influence emotional
response, indicating that relief is present (table S3).
19. J. Von Neumann, O. Morgenstern, Theory of Games
and Economic Behavior (Princeton University Press,
Princeton, NJ, 1944).
20. One could argue that the regret effect is not inde-
pendent from the disappointment induced by the
unobtained outcome of the chosen gamble. However,
when reanalyzed as a function of the latter variable
(fig. S4), the affective ratings in the complete feed-
back condition are much less contrasted then when
analyzed as a function of the unchosen gamble’s
21. N. H. Frijda, P. Kuipers, E. ter Schure, J. Pers. Soc.
Psychol. 57, 212 (1989).
22. M. Zeelenberg, W. W. van Dijk, A. S. R. Manstead, J.
Van der Pligt, Cognition and Emotion 14, 521 (2000).
23. M. Zeelenberg, E. van Dijk, in D. Mandel, D. Hilton, P.
Catelani, Eds., The Psychology of Counterfactual
Thinking (Routledge, London, 2004).
24. We thank M. Thevenet, L. Granjon, and B. Messaoudi
for technical support. This work was supported by
Action Concerte ´e Incitative (Syste `mes complexes en
sciences humaines et sociales) from CNRS.
Supporting Online Material
Materials and Methods
Figs. S1 to S4
Tables S1 to S4
10 December 2003; accepted 17 March 2004
Definition of a Bacterial
Type IV Secretion Pathway
for a DNA Substrate
Eric Cascales and Peter J. Christie*
Bacteria use conjugation systems, a subfamily of the type IV secretion systems,
and mechanism of action of the channel mediating DNA transfer across the
a DNA substrate (T-DNA) and 6 of 12 components of the VirB/D4 conjugation
system of the phytopathogen Agrobacterium tumefaciens. Our results define
the translocation pathway for a DNA substrate through a bacterial conjugation
machine, specifying the contributions of each subunit of the secretory appa-
ratus to substrate passage.
The translocation of nucleic acids across
membrane barriers is central to many cel-
lular processes. Bacterial conjugation sys-
tems are a subfamily of the type IV secre-
tion systems (T4SS), which collectively
mobilize the transfer of macromolecules
such as monomeric proteins, multimeric
toxins, and DNA-protein complexes across
the cell envelope (1, 2). Conjugation sys-
tems mediate horizontal gene transfer, thus
contributing to genome plasticity, evolu-
tion of infectious pathogens, and dissemi-
nation of antibiotic resistance and other
virulence traits (3). Since the early discov-
ery of the Escherichia coli F plasmid trans-
fer system, many regulatory and mechanis-
tic features of this and other T4SS have
been described (1, 2, 4). Surprisingly, how-
ever, we still lack a fundamental under-
standing of the channel through which
DNA substrates are delivered across the
donor cell envelope.
In nature, Agrobacterium tumefaciens uses
the VirB/D4 conjugation system (fig. S1) to
deliver oncogenic transfer DNA (T-DNA) and
effector proteins to susceptible plant cells, often
agriculturally important crop species. In the
laboratory, the capacity of this bacterium to
transfer DNA between kingdom boundaries has
been exploited to genetically engineer a large
number of plant, fungal, and other eukaryotic
species (5). Here, we sought to define the trans-
location route for the T-DNA through this ar-
chetypal T4SS (1).
We developed a sensitive assay termed
transfer DNA immunoprecipitation (TrIP) to
identify close contacts between the T-DNA
substrate as it exits the cell and subunits of
the VirB/D4 T4SS (6) (fig. S2). This assay
was adapted from the chromatin immunopre-
cipitation (ChIP) assay commonly used to
study chromatin and transcription complexes
in eukaryotic cells (7). In this three-stage
assay, we treat vir gene–induced A. tumefa-
ciens cells with formaldehyde to cross-link
proteins to DNA in vivo, and then we precip-
itate a Vir protein of interest from detergent-
solubilized cell extracts. Finally, we assay for
coprecipitation of DNA by the polymerase
chain reaction (PCR). We amplify DNA with
two sets of primers, one specific for the left
end of the transmissible TL-DNA carried on
the tumor-inducing (Ti) plasmid pTiA6NC of
strain A348. The second set is specific for the
nontransferred octopine catabolism region
(ophDC) positioned ?25 kb from the T-DNA
on the Ti plasmid. We further developed a
quantitative version of TrIP to compare levels
of DNA substrate recovered in the immuno-
precipitates (fig. S3).
Initially, we defined the genetic require-
ments for two early reactions associated with
type IV translocation: substrate processing
and recruitment to the secretory apparatus.
Reminiscent of the processing of conjugative
plasmids (8), the A. tumefaciens VirD2 relax-
ase binds origin of transfer–like T-DNA bor-
der sequences and cleaves the strand destined
for transfer (T-strand). The relaxase is
thought to remain covalently bound to the 5?
end of the T-strand, resulting in a VirD2–T-
strand nucleoprotein particle. We isolated
this presumptive transfer intermediate by im-
munoprecipitation with antibodies to VirD2
(Fig. 1A). The antibodies precipitated VirD2
as well as the T-strand—but not the ophDC
Ti plasmid control fragment—from extracts
of wild-type cells as well as mutants (table
S1) defective for synthesis of the secretory
Department of Microbiology and Molecular Genetics,
University of Texas Medical School at Houston, 6431
Fannin, JFB1.765, Houston, TX 77030, USA.
*To whom correspondence should be addressed. E-
R E P O R T S
21 MAY 2004VOL 304 SCIENCE www.sciencemag.org