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Pivoting the Plant Immune System from Dissection to Deployment

Authors:
  • Two Blades Foundation

Abstract and Figures

Diverse and rapidly evolving pathogens cause plant diseases and epidemics that threaten crop yield and food security around the world. Research over the last 25 years has led to an increasingly clear conceptual understanding of the molecular components of the plant immune system. Combined with ever-cheaper DNA-sequencing technology and the rich diversity of germ plasm manipulated for over a century by plant breeders, we now have the means to begin development of durable (long-lasting) disease resistance beyond the limits imposed by conventional breeding and in a manner that will replace costly and unsustainable chemical controls.
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Switzerland, 22 to 24 October 2012; www.abim.ch/
fileadmin/documents-abim/Presentations_2012/
ABIM_2012_6_McDougall_John.pdf.
18. L. Zirngibl, Antifungal Azoles (Wiley-VCH, Weinheim,
Germany, 1998).
19. K.-J. Schleifer, in Pesticide Chemistry, H. Ohkawa,
H. Miyagawa, P. W. Lee, Eds. (Wiley-VCH, Wei nheim,
Germany, 2007), pp. 7788.
20. C. M. Tice, Pest Manag. Sci. 57,316 (2001 ).
21. C. M. Tice, Pest Manag. Sci. 58, 219233 (2002).
22. E. D. Clarke, J. S. Delaney, Chimia (Aarau) 57, 731734 (2003).
23. C. Lamberth, J. Dinges, in Bioactive Heterocyclic Compound
Classes - Agrochemicals, C. Lamberth, J. Dinges, Eds.
(Wiley-VCH, Weinheim, Germany, 2012), pp. 320.
24. P. Jeschke, in Modern Methods in Crop Protection Research,
P. Jeschke, W. Krämer, U. Schirmer, M. Witschel, Eds.
(Wiley-VCH, Weinheim, Germany, 2012), pp. 73128.
25. G. Theodoridis, in Fluorine and the Environment
Agrochemicals, Archaeology, Green Chemistry and Water,
A. Tressaud, Ed. (Elsevier, Amsterdam, 2006), pp. 121175.
26. N. Kurihara, J. Miyamoto, Eds., Chirality in Agrochemicals
(Wiley, Chichester, UK, 1998).
27. G. M. Ramos Tambo, D. Belluš, Angew. Chem. Int. Ed. Engl.
30, 11931215 (1991).
28. H.-U. Blaser, Adv. Synth. Catal. 344, 17 (2002).
29. J. Rheinheimer, in Modern Crop Protection Compounds,
W. Krämer, U. Schirmer, P. Jeschke, M. Witschel, Eds.
(Wiley-VCH, Weinheim, Germany, 2012), pp. 627639.
30. A.B.Charette,A.Beauchemin,Org. React. 58,1415 (2001).
31. C. Lamberth, in Bioactive Heterocyclic Compound
Classes - Agrochemicals,C.Lamberth,J.Dinges,Eds.
(Wiley-VCH,Weinheim,Germany,2012),pp.155162.
32. D. M. T. Chan, P. Y. S. Lam, in Boronic Acids,D.G.Hall,Ed.
(Wiley-VCH, Weinheim, Germany, 2005), pp. 205240.
33. J. Wenger, T. Niderman, C. Mathews, in Modern Crop
Protection Compounds, W. Krämer, U. Schirmer,
P. Jeschke, M. Witschel, Eds. (Wiley-VCH, Weinheim,
Germany, 2012), pp. 447477.
34. A. Schnyder, A. F. Indolese, T. Maetzke, J. Wenger,
H.-U. Blaser, Synlett 2006, 31673169 (2006).
35. H. Walter, in Bioactive Heterocyclic Compound
Classes - Agrochemicals, C. Lamberth, J. Dinges, Eds.
(Wiley-VCH, Weinheim, Germany, 2012), pp. 175193.
36. C. Lamberth, Bioorg. Med. Chem. 17, 40474063 (2009).
37. S. F. McCann, D. Cordova, J. T. Andaloro, G. P. Lahm,
in Modern Crop Protection Compounds, W. Krämer,
U. Schirmer, P. Jeschke, M. Witschel, Eds. (Wiley-VCH,
Weinheim, Germany, 2012), pp. 12571273.
38. S. F. McCann et al., Pest Manag. Sci. 57, 153164 (2001).
39. I. D. Kuntz, Science 257, 10781082 (1992).
40. G. Klebe, J. Mol. Med. 78, 269281 (2000).
41. A. C. Anderson, Chem. Biol. 10, 787797 (2003).
42. S. W. Kaldor et al., J. Med. Chem. 40, 39793985
(1997).
43. J. N. Varghese, Drug Dev. Res. 46, 176196 (1999).
44. A. nardeau et al., Bioorg. Med. Chem. Lett. 19,
24682473 (2009).
45. M. W. Walter, Nat. Prod. Rep. 19, 278291 (2002).
46. R. J. Howard, B. Valent, Annu. Rev. Microbiol. 50,
491512 (1996).
47. C. Bechinger et al., Science 285, 18961899 (1999).
48. T. Lundqvist et al., Structure 2, 937944 (1994).
49. D. B. Jordan et al., Bioorg. Med. Chem. Lett. 9,
16071612 (1999).
50. G. S. Basarab, D. B. Jordan, T. C. Gehret, R. S. Schwartz,
Z. Wawrzak, Bioorg. Med. Chem. Lett. 9, 16131618 (1999).
51. G. S. Basarab, D. B. Jordan, T. C. Gehret, R. S. Schwartz,
Bioorg. Med. Chem. 10, 41434154 (2002).
52. H. M. Berman et al., Nucleic Acids Res. 28,235242 (2000).
53. H. M. Berman, Acta Crystallogr. A 64,8895 (2008).
54. L. P. Yu, Y. S. Kim, L. Tong, Proc. Natl. Acad. Sci. U.S.A.
107, 2207222077 (2010).
55. A. rnberg, A. K. Tunemalm, F. Ekström, Biochemistry
46, 48154825 (2007).
56. R. E. Hibbs, E. Gouaux, Nature 474,5460 (2011).
57. N. Bocquet et al., Nature 457, 111114 (2009).
58. T. Nakao, S. Banba, M. Nomura, K. Hirase, Insect
Biochem. Mol. Biol. 43, 366375 (2013).
59. K. Tietjen, P. H. Schreier, in Modern Methods in Crops
Protection Research, P. Jeschke, W. Krämer, U. Schirmer,
M. Witschel, Eds. (Wiley-VCH, Weinheim, Germany,
2012), pp. 197216.
60. D. A. Erlanson, R. S. McDowell, T. OBrien, J. Med. Chem.
47, 3463 3482 (2004).
61. W. K. Brewster, et al., paper presented at the 244th ACS
National Meeting, Philadelphia, PA, 19 to 23 August 2012,
abstr. no. AGRO-241.
62. T. Bretschneider, R. Fischer, R. Nauen, in Modern Crop
Protection Compounds, W. Krämer, U. Schirmer,
P. Jeschke, M. Witschel, Eds. (Wiley-VCH, Weinheim,
Germany, 2012), pp. 11081126.
63. G.-F. Hao et al., J. Am. Chem. Soc. 134, 1116811176
(2012).
64. H. Sauter, W. Steglich, T. Anke, Angew. Chem. Int. Ed.
38, 1328 1349 (1999).
65. S. Kar, K. Roy, Expert Opin. Drug Discov. 8, 245261 (2013).
66. M. pez-Ramos, F. Perruccio, J. Chem. Inf. Model. 50,
801814 (2010).
67. R. Beffa, Pflanzenschutz Nachr. Bayer 57,4661 (2004)
(English edition).
68. S. Kamoun et al., Can. J. Plant Pathol. 24,69 (2002).
69. K. Okada et al., Planta 215, 339344 (2002).
70. M. Witschel, F. hl, R. Niggeweg, T. Newton, Pest
Manag. Sci. 69, 559563 (2013).
71. M. C. Witschel et al., Angew. Chem. Int. Ed. 50,
79317935 (2011).
72. M. Witschel, paper presented at the 244th ACS National
Meeting, Philadelphia, PA, 19 to 23 August 2012, abstr.
no. AGRO-242.
Acknowledgments: The authors are grateful to their colleagues
R. Viner and D. P. Kloer for helpful comments.
10.1126/science.1237227
REVIEW
Pivoting the Plant Immune System
from Dissection to Deployment
Jeffery L. Dangl,
1,2,3,4,5
* Diana M. Horvath,
6
* Brian J. Staskawicz
7
*
Diverse and rapidly evolving pathogens cause plant diseases and epidemics that threaten crop
yield and food security around the world. Research over the last 25 years has led to an increasingly
clear conceptual understanding of the molecular components of the plant immune system.
Combined with ever-cheaper DNA-sequencing technology and the rich diversity of germ plasm
manipulated for over a century by plant breeders, we now have the means to begin development
of durable (long-lasting) disease resistance beyond the limits imposed by conventional breeding
and in a manner that will replace costly and unsustainable chemical controls.
P
lants turn sunlight into sugar . Thus, plants
are rich sources of nutrients and water that
are, to no ones surprise, host to diverse mi-
crobial communities both above and below the
ground. Microbes are likely to have accompanied
the first plants that emigrated from water to land
400 to 500 hundred million yea r s ago. Many of
their descendant contemporar y microbes are
adapted to take advantage of the nutr i e n t n i c hes
afforded to them by the huge diversity of plants all
over the earth. Plants are protected from infection
by a skin, a waxy cuticular layer atop the cell
wall. Would-be pathogens breaching th is barr i er
encounter an active plant immune system that
specifically recognizes pathogen and altered-self
molecules generated during infection. Consequent
regulation of a network of inducible defenses can
halt pathogen proliferation and signal distal plant
organs to become nonspecifically primed against
further infection.
Nevertheless, fungal, oomycete, bacterial, and
viral pathogens cause devastating epidemics that
have affected human civilizations since the dawn
of agriculture (1). The late blight Irish potato fam-
ine of the 1840s was caused by the oomycete
Phytophthora infestans (2); the loss of the worlds
first mass-cultivated banana cultivar Gr os Michel
in the 1920s to Panama disease was caused by the
fungus Fusarium oxysporum (3); and the current
wheat stem, leaf, and yellow stripe rust epidemics
spreading from East Africa into the Indian sub-
continent caused by rust fungi Puccinia graminis
and P. s t r i i f o r m i s (4) are all testament to the recur-
ring impact of plant diseases. Plant pathogens can
spread rapidly over great distances, vectored by
water, wind, insects, and humans (http://rusttracker .
cimmyt.org/). Despite various cultural practices, crop
protection chemicals, and available disease-resistant
crop varieties, an estimated 15% of global crop
production is lost to preharvest plant disease (5).
Plant Breeding and Disease Resistance
Humans have selected for disease-resistant crops
throughout the history of agriculture, at times
unwittingly (6). As a practiced science, plant breed-
ing for disease resistance originated with Sir
Rowland Biffen in Cambridge, England, who
1
Department of Biology, University of North Carolina, Chapel
Hill, NC 27599, USA.
2
Howard Hughes Medical Institute, Uni-
versity of North Carolina, Chapel Hill, NC 27599, USA.
3
Curric-
ulum in Genetics and Molecular Biology, Un iversity of North
Carolina, Chapel Hill, NC 27599, USA.
4
Department of Micro-
biology and Immunology, University of North Carolina, Chapel
Hill, NC 27599, USA.
5
Carolina Center for Genome Sciences,
University of North Carolina, Chapel Hill, NC 27599, USA.
6
Two
Blades Foundation, 1630 Chicago Avenue, Evanston, IL 60201,
USA.
7
Department of Plant and Microbial Biology, 111 Koshland
Hall, University of California, Berkeley, CA 947203120, USA.
*These authors contributed equally and are listed alphabetically.
Corresponding author. E-mail: dangl@email.unc.edu
16 AUGUST 2013 VOL 341 SCIENCE www.sciencemag.org746
identified a single recessive gene for resistance
to wheat yellow rust caused by P. striiformis (7).
The ensuing century of breeding in nearly every
crop species resulted in deployment of disease
resistance (R) genes, many of which were in-
troduced by introgression from sexually com-
patible wild relatives. Dominant or semidominant
R genes were easier to breed into existing crop
cultivars, as they could be selected functionally in
each generation. We now know that R genes are
present in multigene clusters and can occur as
true alleles across naturally variant genetic back-
grounds. The function of each R protein is ac-
tivated by the product of a specific pathogen
virulence gene (8), now generically termed ef-
fector genes. Each pathogen isolate can express
an array of effectors, and the diversity of effec-
tors across the population of any pathogen species
can be stunning (9, 10).
Unfortunately, the utility of most R alleles can
be short-lived in the field, because their deployment
in monoculture selects for pathogen variants, where-
in the corresponding effector allele has suffered
mutation or been lost. Effectors are virulence fac-
tors, but each typically contributes only partially to
virulence. Unrelated effectors can act redundantly
by altering the same host signaling pathway . There-
fore, effector genes can often be lost without
significant impact on pathogen virulence. Likely
exceptions to this principle are core effectors,
defined operationally by their wide distribution
across the population of a particular pathogen and
their substantial contribution to pathogen virulence.
Genomics-based identification of core effectors and
their utilization to functionally define new R alleles
that they activate in diverse plant germ plasm is a
particularly promising strategy for research and
deployment that we discuss below.
The Plant Immune System
Research using both tractable experimental sys-
tems (Arabidopsis) and the irreplaceable germ
plasm toolkits provided by plant breeders and
plant pathologists (notably in flax, tomato, and
barley) led to the isolation of the first pathogen
effector genes (11)andplantR genes (12). Ad-
ditional fundamental discoveries demonstrated
that plants could perceive diverse structures gen-
erally encoded by microbes via high-affinity cell
surface pattern-recognition receptors (PRR) (13).
These lines of research converged to describe a
plant immune system that consists of two inter-
connected tiers of receptors, one outside and one
inside the cell, that govern recognition of microbes
and response to infection (1418).
The first tier of the plant immune system is
governed by extracellular surface PRRs that are
activated by recognition of evolutionarily con-
served pathogen (or microbial)associated molec-
ular patterns (P AMPs or MAMPs). These receptors
are typically leucine-rich repeat kinases and ly-
sine motif (L ysM) kinases (although some lack
the kinase domain and thus require a co-receptor
to provide signaling function) and are broadly
analogous to T oll-like receptors in animals. Ac-
tivation of PRRs leads to intracellular signal i n g ,
transcriptional reprogramming, and biosynthesis of
a complex output response that limits microbial
colonization (13) (Fig. 1, step 1).
Successful pathogens use their effector re-
pertoire to subvert PRR-dependent responses, to
facilitate nutrient acquisition, and to contribute
to pathogen dispersal. Effector repertoires have
been described from pathogens with diverse life-
styles. These include effectors from extracellular
plant bacterial pathogens that are delivered into
host cells by the type III secretion system (TTSS)
(9, 19); effectors from oomycetes and fungi (10, 20)
that invaginate specialized feeding organelles,
called haustoria, into host cells; and salivary
proteins delivered to plant cells during aphid and
nematode feeding (21) (Fig. 1, step 2). Effector
suites from at least two evolutionarily dive r s e
pathogens interact with a limited set of plant
targets, a high proportion of which have im-
mune system functions (Fig. 1, step 3) (22).
Most R genes encode members of an ex-
tremely polymorphic superfamily of intracellular
nucleotide-binding leucine-rich repeat (NLR) re-
ceptors, which function intracellularly and an-
chor the second tier of the plant immune system
(1418). Specific NLR proteins are activated by
specific pathogen effectors. This can be via direct
in teraction, as receptor and ligand, respectively
(23) (Fig. 1, step 4a). Alternatively, an effector
can modify its host cellular target (or a molecular
de c o y of that target), and a specific NLR asso-
ciated with the target or decoy can be activated
by the modificatio n (14, 24) (Fig. 1, steps 4b and
4c). NLR activation coordinates effector-triggered
im munity, a rapid and high-amplitude reboot of
effector-suppressed, PRR-dependent outputs that
limits pathogen proliferation (Fig. 1, step 5). Ani-
mal NLR proteins are likely to follow similar ac-
tivation models (25).
PAMPs/MAMPs
PTI
Nucleus
Immunity gene
expression
PRR
PRR
Co-
PRR
NLR
activation
ETI
Effectors
Effectors
Effectors
Effectors
Bacterium Nematode Aphid
Fungus/
Oomycete
Haustorium
Stylet
TTSS
Intracellular
NLRs
Decoy
4a
5
1
2
4c
4b
3
E
x
t
r
a
c
e
l
ul
ar
s
p
a
c
e
/
c
e
l
l
w
a
l
l
Fig. 1. Schematic of the plant immune system. Pathogens of all lifestyle classes (color coded and
labeled) express PAMPs and MAMPs as they colonize plants (shapes are color coded to the pathogens). Plants
perceive these via extracellular PRRs and initiate PRR-mediated immunity (PTI; step 1). Pathogens deliver
virulence effectors to both the plant cell apoplast to block PAMP/MAMP perception (not shown) and to the plant
cell interior (step 2). These effectors are addressed to specific subcellular locations where they can suppress PTI and
facilitate virulence (step 3). Intracellular NLR receptors can sense effectors in three principal ways: first, by direct
receptor ligand interaction (step 4a); second, by sensing effector-mediated alteration in a decoy protein that
structurally mimics an effector target, but has no other function in the plant cell (step 4b); and third, by sensing
effector-mediated alteration of a host virulence target, like the cytosolic domain of a PRR (step 4c). It is not yet
clear whether each of these activation modes proceeds by the same molecular mechanism, nor is it clear how, or
where, each results in NLR-dependent effector-triggered immunity (ETI). [Modified from (17) by Sarah R. Grant]
www.sciencemag.org SCIENCE VOL 341 16 AUGUST 2013
747
SPECIALSECTION
The molecular architectures of NLR proteins in
their resting, transition, and active signaling states
are poorly de f i n e d (26, 27). There are limited and
conflicting data on the role of self-association or
oligomeriz ation fo r se n sor N LR protein function,
at both pre- and postactivation steps (28). Resting
state oligomerization (in some cases), activation-
dependent intramolecular rearrangements (in es-
sentially all cases), and activation-dependent
N-terminal signaling domain dimerization (in many
but not all cases) have been documented. Some
effector-triggered responses require a pair of NLR
proteins (28): One is activated by the effector and is
a sensor NLR; the other is required for its function
and is a helper NLR (27, 29). Heteromeric pairing
could expand NLR repertoires (30, 31). Similar
NLR pairs can function in animal NLR systems
(32, 33). Exceptions abound, and generalizable
models for NLR activation may not exist; evolu-
tion may have favored a mix of mechanisms that
were refined by coevolutionary conflict between
effectors, targets or decoys, and sensor NLRs.
The cellular site(s) of NLR activation and
action are likely to be diverse. Some NLRs may
require nucleocytoplasmic shuttling for function,
whereas others appear to be activated at the
plasma membrane (18, 27). These different sites
of activation suggest a more idiosyncratic model
for NLR function, dictated in part by the localiza-
tion of, and functional constraints on, the effector
tar gets whose integrity each NLR monitors.
The presence of NLRs with diverse N-terminal
signaling domains in both plants and animals
suggests that this architecture confers a funda-
mental advantage in host defense. This advantage
may include recruitment of diverse cofactors after
activation, as suggested by the functionally relevant
interaction of NLR N-terminal domains with tran-
scription factors in some cases (34). NLRs may
facilitate tightly regulated cooperative threshold
responses to ligands within an evolutionarily flex-
ible scaff old that permits innate immune systems
with limited germ lineencoded repertoires to keep
pace with functionally diverse pathogen effectors
acting at a variety of intracellular sites.
Engineering Disease Resistance in Crops:
Early Successes
Successful transgenic disease resistance was dem-
onstrated in 1986. Constitutive in planta expres-
sion of viral coat protein gene sequences conferred
virus resistance via small RNAs, now understood
to be a widely applicable mechanism for inhibiting
viral replication (35). By combining coat protein
genes from three different viruses, scientists devel-
oped squash hybrids with field-validated, multivir al
resistance (Table 1). The Asgrow Seed Company
obtained regulatory approvals for transgenic com-
mercial squash in 1994, and these continue to be
sold by Seminis today . Similar levels of resistance
to this variety of viruses had not been achieved by
conventional breeding.
Table 1. Published examples of transgenic disease resistance in crops and development status.
Pub. year Crop Disease resistance Mechanism Development status Ref.
2012 Tomato Bacterial spot R gene from pepper 8 years of field trials (46)
2012 Rice Bacterial blight and
bacterial streak
Engineered E gene Laboratory (56)
2012 Wheat Powdery mildew R gene from wheat, overexpressed 2 years of field trials at time of publication (82)
2011 Apple Apple scab fungus Thionin gene from barley 4 years of field trials at time of publication (83)
2011 Potato Potato virus Y Pathogen-derived resistance 1 year of field trial at time of publication (84)
2010 Apple Fire blight Antibacterial protein from moth 12 years of field trials at time of publication (85)
2010 Tomato Multibacterial resistance PRR from Arabidopsis Laboratory scale (43)
2010 Banana Xanthomonas wilt Novel gene from pepper Now in field trial (86)
2009 Potato Late blight R genes from wild relatives 3 years of field trials (87)
2009 Potato Late blight R gene from wild relative 2 years of field trials at time of publication (88)
2008 Potato Late blight R gene from wild relative 2 years of field trials at time of publication (89)
2008 Plum Plum pox virus Pathogen-derived resistance Regulatory approvals, no commercial sales (90, 91)
2005 Rice Bacterial streak R gene from maize Laboratory (92)
2002 Barley Stem rust RLK gene from resistant barley cultivar Laboratory (93)
1997 Papaya Ring spot virus Pathogen-derived resistance Approved and commercially sold
since 1998, sold into Japan since 2012
(94, 95)
1995 Squash Three mosaic viruses Pathogen-derived resistance Approved and commercially sold since 1994 (96)
1993 Potato Potato virus X Mammalian interferon-induced enzyme 3 years of field trials at time of publication (97)
Fig. 2. Hawaiian papaya plot in 2011. Hawaiian papaya plot showing diseased, devastated, non-
transformed trees in the foreground and healthy transgenic trees behind. [Photo courtesy of Dennis
Gonsalves, Agricultural Research Service, U.S. Department of Agriculture, Hawaii]
16 AUGUST 2013 VOL 341 SCIENCE www.sciencemag.org
748
A similar strategy was deployed to combat
papaya ringspot virus, which, by 1994, threat-
ened to destroy Hawaiis papaya industry. Field
trials demonstrated excellent efficacy and high
fruitquality(Table1),andby1998,thefirsttrans-
ge nic virus-resistant papaya was approved for
sale in Hawaii. Disease resistance has been du-
rable for over 15 years of commercial use, and
transgenic papaya currently accounts for ~85%
of Hawaiian production (Fig. 2). The fruit is
now approved for sale in Canada and Japan.
Since the approval and commercialization of
these two crops in the late 1990s, not a single new
crop with engineered disease resistance has reached
the market. Research successes exist (Table 1),
and there is still potential to reduce yield losses
and chemical inputs associated with crop disease.
Effector-Targeted Strategies for Durable
Disease Resistance An Emerging Paradigm
R gene isolation using genetics and genomics is
now a reality in even the most complex plant
genomes (36, 37). Rapid and inexpensive DNA-
sequencing technologies can provide the ge-
nomes of natu ral field iso lates of plant pathogens
with impact on breeding strategies for durable con-
trol of plant diseases (38). It is now possible to
define the genomes, and thus the effector comple-
ment, of plant pathogens isolated from infected
plants in a rapid and efficient manner . Defining
core effectors facilitates identification of suites of
corresponding R genes from wild germ plasm by
using transient coexpression assays, followed by
either marker-assisted breeding or transgenic deploy-
ment (Box 1). V alidation of these new R genes could
be enhanced by new genome-editing methods that
use transcription activatorlike effector nuclease
(TALEN) (39) and clustered regulatory interspac ed
short palindromic repeat (CRISPR) technologies
(40, 41).
The function of any particular R gene is likely
to be durable only if the effector that activates it is
present and important for virulence in the path-
ogen strains that one is trying to control. Knowl-
edge of the effector content in local pathogen
isolates can inform R gene deployment or chemi-
cal treatment in the control of potato late blight
(38). Another exmaple is Xanthomonas axonopodis
pv . manihotis (Xam), the causal agent of cassava
bacterial blight (42). This disease devastates a
staple crop in East Africa. The sequence of ~65
Xam strain s collected over a 70-year time frame,
from 12 countries and three continents, revealed
a core effector set that can now serve as targets to
define R genes activated by them in wild species
of Manihot and potentially other related plants in
the Euphorbiaceae.
Deployment of Immune System Receptors
Research aimed at deployment of the two classes
of immune receptors currently follows two main
strategies. One is to transfer PRRs that detect
common microbial products into species that lack
them. For example, the Arabidopsis PRR EF-Tu
receptor (EFR) recognize s the bac te r ial trans l at io n
elongation factor EF-Tu. Deployment of EFR
into either Nicotiana benthamiana or Solanum
lycopersicum (tomato), which cannot recognize
EF-T u, conferred resistance to a wide range of
bacterial pathogens (43). The expression of EFR
in tomato was especially effective against the
widespread and devastating soil bacterium
Ralstonia solanacearum. Also, the tomato PRR
Verticillium 1 (Ve1) gene can be transferred from
tomato to Arabidopsis, where it confers resist-
ance to race 1 isolates of Verticil lium (44). Iden-
tification of functional PRRs and their transfer to
a recipient species that lacks an orthologous re-
ceptor could provide a general pathway to addi-
tional examples of broadened PRR repertoires (13).
The second strategy exploits immune responses
in contexts where multiple NLR genes are de-
ployed simultaneously, a breeding strategy known
as stacking. Such cultivars, generated by either
DNA-assisted molecular breeding or gene trans-
fer , should provide more durable disease resistance
because pathogen evasion would require mutations
in multiple effector genes. Recent breakthroughs
in DN A seq u e n ci n g al l ow acce s s to the huge ge-
netic diversity of our major crops and their rela-
tives to functionally mine NLR genes directe d
against different core effectors. This approach will
ultimately overcome inherent barriers to traditional
crop breeding (Box 1). Illustrative examples follow .
The first effector -rationalized search for a
potentially durable R gene was predicated on the
finding that the avrBs2 effector gene from Xan-
thomonas perforans, the causal agent of bacte-
rial spot disease of pepper and tomato, is found
in most species of Xanthomonas that cause dis-
ease and is required for patho gen fitness (45). The
Bs2 NLR gene from the wild pepper, Capsicuum
chacoense, was transformed into tomato, where
it inhibited growth of pathogen strains that con-
tained avrBs2. Successful field trials of trans-
genic tomato plants that express Bs2 demonstrated
ro bust resistance to X. perforans without bacte-
ricidal chemicals (46). However, rare strains of
Xanthomonas have overcome Bs2-mediated re-
sistance in pepper by acquisition of avrBs2 muta-
tions that avoid recognition but retain virulence
(47). Stacking of multiple R genes that each rec-
ognize a different core effector could delay or
prevent this problem.
The oomycete Phytophthora infestans causes
late blight disease of potato (2). Cultivated po-
tato, Solanum tuberosum, is tetraploid and clo-
nally propagated via cuttings, which significantly
h ampers introgression of disease resistance from
diploid wild species in the genus Solanum. Fur-
thermore, the pathogen is aggressive and has re-
peatedly adapted to evade host resistance mediated
by single R genes and chemical treatments. Most
potato cultivars are thus susceptible to P. infestans
in fection, which necessitates continual updating
of chemical treatments.
Genome-wide definition of effector suites
across pathogen isolates collected worldwide and
of R gene distribution across Solanum sp. will
have a major impact on management of resist-
ance to P. infestans (38). Sequ en cin g of several
P. infestans genomes has identified a core set of
effectors that can now be used to identify new
sources of disease resistance acro s s th e gen u s
Solanum. This approach has been validated in the
potato cultivar Sarpo mira, which contains four
naturally stacked R genes activated by already
known P. infestans effectors (48). Rational stack-
ing of R genes is a general approach (49, 50)and
the method of choice for producing sustainable,
durable disease resistance that will require fewer
chemical inputs.
In modern wheat and its many relatives, more
than 50 different loci have been described that
confer disease resistance against wheat stem,
leaf, and yellow stripe rust pathogens. A few
were known to confer resistance to the pandemic
wheat rust isolate Ug99 and its derivatives, but
these were not readily incorporated into hexa-
ploid wheat or provide only partial resistance.
The Stem rust 35 (Sr35) NLR gene was very re-
cen tly cloned from a diploid relative of cultivated
wheat, Triticum monococcum, and transferred
into cultivated hexaploid wheat to derive resist-
ance to Ug99 (36). Similarly, the Stem rust 33
(Sr33) NLR gene from the wheat relative Aegi-
lops tauschii was also very recently cloned and
shown to encode a wheat ortholog to the barley
Mla powdery mildewresistance genes (37). Both
Sr35 and Sr33 are fairly rare in wheat and its
relatives, which accentuates the importance of d i-
verse germ plasm screening to identify usefu l new
R genes. It is hoped that Sr35 and Sr33, combined
with the Sr2 gene that is known to act additively
with at least Sr33 (51), could provide durable
disease resistance to Ug99 and its derivatives.
Deployment of Executor-Mediated
Disease Resistance
In contrast to PRRs and NLRs, another class
of plant disease resistance genes has evolved to
coopt pathogen virulence functions and open a
trap door that stops pathogen proliferation. Xan-
thomonas and Ralstonia transcription activator
like (TAL) effectors are DNA-binding proteins
delivered into plant cells, where they activate host
gene expression to enhance pathogen virulence
(39). In a neat evolutionary trick, however , both
the rice and pepper lineages independently evolved
TAL-effector binding sites in the promoters of
genes whose products induce hypersensitive host
cell death when up-regulated and thus inhibit
pathogen proliferation. The known executor
genes, Xa27 from rice (52)andBs3 and Bs4c
from pepper (53, 54), encode plant proteins of un-
known function that share no homology. Exec-
utor genes are not expressed in the absence of
infection, but expression of each is strongly in-
duced by a specific TAL effector.
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Engineered executor genes provide unique
opportunities to deliver enhanced and poten-
tially durable disease resistance. This was demon-
strated by successfully redesigning the pepper
Bs3 promoter to contain two additional binding
sites for TAL effectors from disparate patho-
gen strains (55). Subsequently, an engineered ex-
ecutor gene was deployed in rice by adding five
di ffer e nt TAL e ffector binding sites to the Xa27
promoter. The synthetic Xa27 construct was ac-
tivated by TAL effectors from, and conferred
resistance against, both bacterial blight and bac-
terial leaf streak species of Xanthomonas (56)
(Table 1).
Defining and Deploying Altered Host
Susceptibility Alleles to Control
Plant Diseases
Most plant pathogens reprogram host plant gene
expression patterns to directly benefit pathogen
fitness, as exemplified above for TAL effectors.
Host genes reprogrammed by pathogens that are
required for pathogen survival and proliferation
can be th o ugh t of a s disease-susceptibility genes.
Identification and isolation of these would pro-
vide useful sources for breeding disease resistance:
th eir loss or alteration of function would deprive
the pathogen of a host factor required for its pro-
liferation (57, 58). We highlight a few here.
Recessive disease-resistance genes, long known
to breeders, are candidates for disease-susceptibility
genes. For example, a loss-of-function mutation
in an Arabidopsis gene encoding pectate lyase,
an enzyme involved in cell wall degradation, con-
fe r r ed resistance to the powdery mildew patho-
gen Golovinomyces (syn. Erysiphe) cichoracearum
(59). Similarly , the Barley mlo gene has been de-
ployed against powdery mildew for more than
70 years, and it is required for pathogen invasion
(60). Spontaneous mutations in pea and tomato
MLO orthologs confer resistance to powdery mil-
dew pathogens of these plants (61, 62). And the
Pseudomonas syringae bacterial effector HopZ2
targets the Arabidopsis ortholog, MLO2, to con-
tribute to bacterial virulence (63).
Similarly illustrative is the cloning and de-
ployment of Lr34, a gene that provides partial
resistance to leaf and yellow rusts and powdery
mildew in wheat and that has been durable for
nearly a century . Lr34 encodes an adenosine tri-
phosphate (ATP)binding cassette (ABC) trans-
porter . The dominant allele that provides disease
resistance was recently derived in cultiva ted wheat
(it is not present in wild progenitors of wheat)
and, like mlo, is associated with ectopic plant cell
death that may establish a sensitized defense
state or accelerate senescence. Transfer of the wheat
Lr34 re sistance allele provides broad-spectrum
resistance in barley , although with the expected
cell deathlesion formation (6466). It is unclear
whether the wheat allele that provides durable
resistance is also functional for the inferred ABC
transporter activity of Lr34, and thus, the mech-
anism by which Lr34 confers disease resistance
remains obscure.
Naturally occurring alleles of the host trans-
lation elongation initiation factors eif4e and eif4g
double as recessive viral-resistance genes. Some
have been deployed to control important potyvi-
ruses in barley, rice, tomato, pepper, pea, lettuce,
and melon (67). The discovery of natural recessive
alleles prompted a successful mutant screen for
chemically induced eif4e alleles in tomato (68).
Natural variation in the promoters of key
plant-susceptibility genes can also lead to the
evolution of recessive disease-resistance alleles.
For example, the recessive resistance gene xa13
in rice is an allele of Os-8N3. Os-8N3 is tran -
scriptionally activated by Xanthomonas oryzae
pv. oryzae strains that express the TAL effector
PthXo1. The xa13 gene has a mutated effector-
binding element in its promoter that eliminates
PthXo1 binding and renders these lines resistant
to strains of the pathogen that rely on PthXo1 as
their essential virulence factor. This finding also
demonstrates that Os-8N3 is required for sus-
ceptibility (69).
The deployment of mutant alleles of host
disease-susceptibility genes can be problematic
if the disease-susceptibility phenotype comes at
the cost of altered function in other cellular and
developmental processes. This is the case for
Xa13/Os-8N3, which is also required for pollen
development (70). Nevertheless, it is possible to
separate disease susceptibility from normal de-
velopment. For example, mutations in the Os1 1N3
(OsSWEET14) T AL effectorbinding element were
made by using TAL effectors fused to nucleases
(TALE Ns). Genome-edited rice plants with altered
Os1 1N3 binding sites were resistant to Xanthomonas
oryzae pv . oryzae infection, but they were unaltered
for the normal Os1 1N3 (OsSWEE T14)develop-
mental function (71).
The identification of new susceptibility genes
in crops will come from forward genetic screens that
uncover new recessive disease-resistance genes
which may , indeed, turn out to be host-susceptibility
genesand from identification of host targets of
effectors. For example, mutant screens in Arabidopsis
identified additional recessive mutations that
confer recessive resistance to the obligate bio-
trophs, G. cichoracearum (72)andHyaloper onospora
arabidopsidis (73). These genes have orthologs in
other plants, thus making them obvious targets
for identification of mutant alleles in crop species
(Box 1).
Looking Forward: Future Challenges,
Technical and Societal
In the past century of disease-resistance breeding,
we were largely limited to germ plasm from sex-
ually compatible wild species that can recognize
and resist infection, without a priori knowledge
of the effector R gene mediating the outcome
(Box 1). This strategy is slow , and field efficacy
is often shortened by selection of effector gene
mutants that evade host recognition. Our current
challenge is to leverage evolutionary genomic
information stored in the worldwide germ plasm
diversity. The goal is to define and to stack mul-
tiple resistance specificities active against the
daunting array of economically important path-
ogens, including Phytophthora, Magnaporthe,
Box 1. Breeding for disease resistance.
Current practices involved in breeding for disease resistance
1. Discover single R genes in wild relative species and cross into agronomic cultivars by
interspecific hybridization, followed by successive generations of recurrent selection
for resistance. This process is slow.
2. Use pathogen inoculations to test plant germ plasm for resistance without a priori
knowledge of which effector is being detected by the new R gene.
3. R genemediated disease resistance can be short-lived, as pathogens can mutate to
evade activating R function.
4. Interspecific hybrid breeding is sometimes difficult because of sexual incompatibilities
and/or linkage drag of undesirable traits.
Improved practices for breeding durable resistance by genomic strategies
1. Use next-generation sequencing technologies to sequence and assemble pathogen genomes
causing disease in local fields.
2. Use computational biology to identify the most highly successful core effectors in these strains.
3. Identify R genes that are activated by those effectors.
4. Deploy multiple, stacked R genes that recognize defined core effectors to reduce the chance
that pathogens will overcome resistance.
5. Identify and edit within the genome disease-susceptibility genes to reduce pathogen growth
and symptom development.
6. Identify and deploy antipathogenic probiotic and/or antipathogenic microbial mixtures as
seed coats.
16 AUGUST 2013 VOL 341 SCIENCE www.sciencemag.org
750
Fusarium, Pseudomonas, Ralstonia, Xanthomonas,
and gemini and potyvirus es (74).Atthesametime,
we must maintain complex agronomic traitssuch
as yield, form, and flavorand avoid yield penal-
ties. The precision offered by transgenic and ge-
nome editing technologies offers considerable
advantages over conventional breeding (Box 1).
Prospects for the development of durable dis-
ease resistance have improved markedly because
of the ongoing molecular dissection of the plant
immune system and the advent of ever-faster,
ever-cheaper genome-sequencing technologies.
Many exciting challenges are emerging to exploit
that knowledge. We can contemplate rational,
stacked deployment of multiple NLRs that each
recognize a different core effector (Box 1). We
will eventually be able to engineer novel NLR
recognition specificities, though this requires
detailed structural knowledge only now begin-
ning to be unraveled (75). Combinations of stacked
NLRs, new PRRs, and genome-edited disease-
susceptibility alleles that reduce or stop patho-
gen proliferation are realistic possibilities. We
can now monitor pathogen populations and their
effector complements in the field over space and
time to inform deployment of better-suited cul-
tivars requiring less chemical control (38). We
harbor ambitions to enhance plant immune sys-
tem function by man aging defined probiotic, anti-
pathogenic microbial consortia isolated from the
plants own microbiome (76, 77). A holistic,
mechanism-based approach will ultimately im-
prove plant immune system function to deliver
durable and sustainable disease resistance, with
minimum or no chemical input, where it is needed
most in the future.
Among the greatest challenges remaining
for deployment of next-generation disease-
resistant plants are those posed by regulatory and
consumer acceptance hurdles. V irus resistance
in modified papaya and squash has been du-
rable, and the crops have been safely consumed
for nearly 20 years, with no negative environ-
mental impacts (78). Nevertheless, significant an-
xiety remains. Sadly , commercial deployme nt by
BASF Corporation (Badische Anilin Soda Farbrik,
AG) of a potentially valuable potato cultivar ,
Fortuna, containing two stacked and potentially
durable NLR genes from a wild potato species,
was canceled because of pressure from lobbies
opposing genetic modification, despite the fact
that it would likely eliminate some or all of the
up to 25 fungicide treatments required in North-
ern Europe per year to control late blight (79). If
the examples of the introduction of coffee as a
beverage, and the use of hybrid crops, such as
corn, serve as guidelines, acceptance of trans-
genic crops should become mainstream in about
50 to 200 years (80, 81). That timeline is simply
too long to wait to confront the issues of food
security and environmental sustainability posed
by the plethora of microbes that value our crops
as food sources as much as we do.
References and Notes
1. G. N. Agrios, Plant Pathology (Academic Press, San
Diego, 1988).
2. K. Yoshida et al., eLife 2, e00731 (2013).
3. D. Koep pel, Banana: The Fate of the Fruit That Changed
the World (Penguin Books, New York, 2008).
4. R. P. Singh et al., Annu. Rev. Phytopathol. 49, 465481
(2011).
5. J. Popp, K. Hantos, Stud. Agric. Econ. 113, 47 (2011).
6. P. Piffanelli et al., Nature 430, 887891 (2004).
7. R. H. Biffen, J. Agric. Sci. 1, 4 (1905).
8. H. H. Flor, Annu. Rev. Phytopathol. 9, 275296 (1971).
9. D. A. Baltrus et al., PLoS Pathog. 7, e1002132 (2011).
10. S. Raffaele et al., Science 330, 15401543 (2010).
11. B. J. Staskawicz, D. Dahlbeck, N. T. Keen, Proc. Natl.
Acad. Sci. U.S.A. 81, 60246028 (1984).
12. B. J. Staskawicz, F. M. Ausubel, B. J. Baker, J. G. Ellis,
J. D. G. Jones, Science 268, 661667 (1995).
13. J. Monaghan, C. Zipfel, Curr. Opin. Plant Biol. 15,
349357 (2012).
14. J. L. Dangl, J. D. Jones, Nature 411, 826833 (2001).
15. J. D. Jones, J. L. Dangl, Nature 444, 323329 (2006).
16. S. T. Chisholm, G. Coaker, B. Day, B. J. Staskawicz,
Cell 124, 803814 (2006).
17. P. N. Dodds, J. P. Rathjen, Nat. Rev. Genet. 11,539548
(2010).
18. T. Maekawa, T. A. Kufer, P. Schulze-Lefert, Nat. Immunol.
12, 817826 (2011).
19. A. Block, J. R. Alfano, Curr. Opin. Microbiol. 14,3946 (2011).
20. M. Koeck, A. R. Hardham, P. N. Dodds, Cell. Microbiol.
13, 18491857 (2011).
21. J. I. Bos et al., PLoS Genet. 6, e1001216 ( 2010).
22. M. S. Mukhtar et al., Science 333, 596601 (2011).
23. P. N. Dodds et al., Proc. Natl. Acad. Sci. U.S.A. 103,
88888893 (2006).
24. R. A. van der Hoorn, S. Kamoun, Plant Cell 20, 20092017
(2008).
25. L. M. Stuart, N. Paquette, L. Boyer, Nat. Rev. Immunol.
13, 199206 (2013).
26. F. L. Takken, A. Goverse, Curr. Opin. Plant Biol. 15,
375384 (2012).
27. V. Bonardi, K. Cherkis, M. T. Nishimura, J. L. Dangl,
Curr. Opin. Immunol. 24,4150 (2012).
28. T. K. Eitas, J. L. Dangl, Curr. Opin. Plant Biol. 13,472477 (2010).
29. V. Bonardi et al., Proc. Natl. Acad. Sci. U.S.A. 108,
1646316468 (2011).
30. M. Narusaka et al., Plant J. 60, 218226 (2009).
31. D. Birker et al., Plant J. 60, 602613 (2009).
32. E. M. Kofoed, R. E. Vance, Nature 477, 592595 (2011).
33. B. Zhao, D. Dahlbeck, K. V. Krasileva, R. W. Fong,
B. J. Staskawicz, PLoS Pathog. 7, e1002408 (2011).
34. C. Chang et al., Plant Cell 25, 11581173 (2013).
35. J. A. Lindbo, W. G. Dougherty, Mol. Plant Microbe
Interact. 5, 144153 (1992).
36. C. Saintenac et al., Science 341, 783786 (2013).
37. S. Periyannan et al., Science 341,786788 (2013).
38. V. G. A. A. Vleeshouwers et al., Annu. Rev. Phytopathol.
49, 507531 (2011).
39. S. Schornack, M. J. Moscou, E. R. Ward, D. M. Horvath,
Annu. Rev. Phytopathol. 10.1146/annurev-phyto-
082712-102255 (2013).
40. M. Jinek et al., eLife 2, e00471 (2013).
41. T. Gaj, C. A. Gersbach, C. F. Barbas 3rd, Trends
Biotechnol. 31, 397405 (2013).
42. R. Bart et al., Proc. Natl. Acad. Sci. U.S.A. 109,
E1972E1979 (2012).
43. S. Lacombe et al., Nat. Biotechnol. 28, 365369 (2010).
44. E. F. Fradin et al., Plant Physiol. 156, 22552265 (2011).
45. B. Kearney, B. J. Staskawicz, Nature 346, 385386 (1990).
46. D. M. Horvath et al., PLoS ONE 7, e42036 (2012).
47. W. Gassmann et al., J. Bacteriol. 182, 70537059 (2000).
48. H. Rietman et al., Mol. Plant Microbe Interact. 25,
910919 (2012).
49. H. J. Kim et al., Theor. Appl. Genet. 124,923935 (2012).
50. S. Zhu, Y. Li, J. H. Vossen, R. G. Visser, E. Jacobsen,
Transgenic Res. 21,8999 (2012).
51. M. Ayliffe et al., Mol. Plant Microbe Interact. 26,658667 (2013).
52. K. Gu et al., Nature 435, 11221125 (2005).
53. P. mer et al., Science 318, 645648 (2007).
54. T. Strauss et al., Proc. Natl. Acad. Sci. U.S.A. 109,
1948019485 (2012).
55. P. mer, S. Recht, T. Lahaye, Proc. Natl. Acad. Sci. U.S.A.
106, 2052620531 (2009).
56. A. W. Hummel, E. L. Doyle, A. J. Bogdanove, New Phytol.
195, 883893 (2012).
57. F. Gawehns, B. J. C. Cornelissen, F. L. W. Takken, Microb.
Biotechnol. 6, 223229 (2013).
58. S. Pavan, E. Jacobsen, R. G. Visser, Y. Bai, Mol. Breed.
25,112 (2010).
59. J. P. Vogel, T. K. Raab, C. Schiff, S. C. Somerville, Plant
Cell 14, 20952106 (2002).
60. M. Humphry, C. Consonni, R. Panstruga, Mol. Plant
Pathol. 7, 605610 (2006).
61. S. Pavan et al., Theor. Appl. Genet. 123,14251431 (2011).
62. Y. Bai et al., Mol. Plant Microbe Interact. 21,3039 (2008).
63. J. D. Lewis et al., BMC Genomics 13, 8 (2012).
64. S. G. Krattinger et al., Theor. Appl. Genet. 126, 663672 (2013).
65. S. G. Krattinger et al., Plant J. 65, 392403 (2011).
66. J. M. Risk et al., Plant Biotechnol. J. 10,477487 (2012).
67. A. Wang, S. Krishnaswamy, Mol. Plant Pathol. 13,
795803 (2012).
68. F. Piron et al., PLoS ONE 5, e11313 (2010).
69. B. Yang, A. Sugio, F. F. White, Proc. Natl. Acad. Sci. U.S.A.
103, 1050310508 (2006).
70. Z. Chu et al., Genes Dev. 20, 12501255 (2006).
71. T. Li, B. Liu, M. H. Spalding, D. P. Weeks, B. Yang,
Nat. Biotechnol. 30, 390392 (2012).
72. J. Vogel, S. Somerville, Proc. Natl. Acad. Sci. U.S.A. 97,
18971902 (2000).
73. M. Van Damme et al., Mol. Plant Microbe Interact. 18,
583592 (2005).
74. R. N. Strange, P. R. Scott, Annu. Rev. Phytopathol. 43,
83116 (2005).
75. Z. Hu et al., Science 341, 172175 (2013).
76. D. Bulgarelli, K. Schlaeppi, S. Spaepen,
E. Ver Loren van Themaat, P. Schulze-Lefert, Annu. Rev.
Plant Biol. 64, 807838 (2013).
77. J. A. Vorholt, Nat. Rev. Microbiol. 10, 828840 (2012).
78. M. Fuchs, D. Gonsalves, Annu. Rev. Phytopathol. 45,
173202 (2007).
79. C. Dixelius, T. Fagerström, J. F. Sundstm, Nat. Biotechnol.
30, 492493 (2012).
80. R. C. Sutch, Henry Agard Wallace, the Iowa corn yield tests,
and the adoption of hybrid corn (National Bureau of Economic
Research working paper 14141, NBER, Cambridge, MA, 2008).
81. C. Juma, “‘Satans drink and a sorry history of global
food fights, Financial Times, 6 February 2006.
82. S. Brunner et al., Plant Biotechnol. J. 10,398409 (2012).
83. F. A. Krens et al., Transgenic Res. 20, 11131123 (2011).
84. F. Bravo-Almonacid et al., Transgenic Res. 21, 967982 (2012).
85. E. Borejsza-Wysocka, J. L. Norelli, H. S. Aldwinckle,
M. Malnoy, BMC Biotechnol. 10, 41 (2010).
86. L. Tripathi, H. Mwaka, J. N. Tripathi, W. K. Tushemereirwe,
Mol. Plant Pathol. 11, 721731 (2010).
87. S. J. Foster et al., Mol. Plant Microbe Interact. 22,
589600 (2009).
88. J. M. Bradeen et al., Mol. Plant Microbe Interact. 22,
437446 (2009).
89. D. Halterman, L. Kramer, S. Wielgus, J. Jiang, Plant Dis.
92, 339343 (2008).
90. T. Malinowski et al., Plant Dis. 90, 10121018 (2006).
91. J. Polák et al., J. Plant Pathol. 90 (suppl.) , S1.33S1.36 (2008).
92. B. Zhao et al., Proc. Natl. Acad. Sci. U.S.A. 102,
1538315388 (2005).
93. H. Horvath et al., Proc. Natl. Acad. Sci. U.S.A. 100,
364369 (2003).
94. S. Ferreira et al., Plant Dis. 86, 101105 (2002).
95. S. Lius et al., Mol. Breed. 3, 161168 (1997).
96. D. M. Tricoli et al., Biotechnology 13, 14581465 (1995).
97. E. Truve et al., Biotechnology 11, 10481052 (1993).
Acknowledgments: We thank our many colleagues who
provided insight and unpublished information that contributed
to the development of this review. J.L.D. is a cofounder of
AgBiome, LLC. J.L.D. is a Howard Hughes Medical Institute
(HHMI) Investigator, and work in his lab on this topic is funded
by HHMI, the Gordon and Betty Moore Foundation, and grants
from NSF. B.J.S. is a cofounder of Mendel Biotechnology, Inc.,
and is funded by grants from NSF and NIH.
10.1126/science.1236011
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