ArticlePDF AvailableLiterature Review

Pivoting the Plant Immune System from Dissection to Deployment

  • 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.
Content may be subject to copyright.
Switzerland, 22 to 24 October 2012;
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.
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
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
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.
Pivoting the Plant Immune System
from Dissection to Deployment
Jeffery L. Dangl,
* Diana M. Horvath,
* Brian J. Staskawicz
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.
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 . 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
Department of Biology, University of North Carolina, Chapel
Hill, NC 27599, USA.
Howard Hughes Medical Institute, Uni-
versity of North Carolina, Chapel Hill, NC 27599, USA.
ulum in Genetics and Molecular Biology, Un iversity of North
Carolina, Chapel Hill, NC 27599, USA.
Department of Micro-
biology and Immunology, University of North Carolina, Chapel
Hill, NC 27599, USA.
Carolina Center for Genome Sciences,
University of North Carolina, Chapel Hill, NC 27599, USA.
Blades Foundation, 1630 Chicago Avenue, Evanston, IL 60201,
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:
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).
Immunity gene
Bacterium Nematode Aphid
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] SCIENCE VOL 341 16 AUGUST 2013
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]
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
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. SCIENCE VOL 341 16 AUGUST 2013
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.
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
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
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
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 SCIENCE VOL 341 16 AUGUST 2013 751
... Microorganisms are among the significant and dominant components of the rhizosphere that release biochemical and antibiotic compounds and significantly serve as plant growth regulators, inducing plant immunity and suppressing soil-borne pathogens in the rhizosphere (Dangl et al., 2013). The plant microbiome holistically improves plant health and fitness, increases host tolerance to stresses, and increases disease resistance in a harsh environment. ...
Full-text available
The rhizosphere is a narrow and dynamic region of plant root-soil interfaces, and it’s considered one of the most intricate and functionally active ecosystems on the Earth, which boosts plant health and alleviates the impact of biotic and abiotic stresses. Improving the key functions of the microbiome via engineering the rhizosphere microbiome is an emerging tool for improving plant growth, resilience, and soil-borne diseases. Recently, the advent of omics tools, gene-editing techniques, and sequencing technology has allowed us to unravel the entangled webs of plant-microbes interactions, enhancing plant fitness and tolerance to biotic and abiotic challenges. Plants secrete signaling compounds with low molecular weight into the rhizosphere, that engage various species to generate a massive deep complex array. The underlying principle governing the multitrophic interactions of the rhizosphere microbiome is yet unknown, however, some efforts have been made for disease management and agricultural sustainability. This review discussed the intra- and inter- microbe-microbe and microbe-animal interactions and their multifunctional roles in rhizosphere microbiome engineering for plant health and soil-borne disease management. Simultaneously, it investigates the significant impact of immunity utilizing PGPR and cover crop strategy in increasing rhizosphere microbiome functions for plant development and protection using omics techniques. The ecological engineering of rhizosphere plant interactions could be used as a potential alternative technology for plant growth improvement, sustainable disease control management, and increased production of economically significant crops
... In addition to carbon, microorganisms discharge many more signaling chemical substances to the rhizosphere. Through them, the most prominent are phytohormones, extracellular enzymes, organic acids, antibiotics, volatile contents and surface factors, e.g., immunomodulatory precursors such as flagellins and lipopolysaccharides in Pseudomonas (Ping, 2004;Dangl et al., 2013). As a signaling molecule, quorum sensing, e.g., N-acyl-homoserine lactones (AHLs), when secreted, is used to regulate gene expression by plant-associated bacteria (Berendsen et al., 2012). ...
Full-text available
Plant microbiome (or phytomicrobiome) engineering (PME) is an anticipated untapped alternative strategy that could be exploited for plant growth, health, and productivity under different environmental conditions. It has been proven that the phytomicrobiome has crucial contributions to plant health, pathogen control, and tolerance under drastic environmental (a)biotic constraints. Consistent with plant health and safety, in this article we address the fundamental role of the plant microbiome and its insights into plant health and productivity. We also explore the potential of plant microbiome under environmental restrictions and the proposition of improving microbial functions that can be supportive for better plant growth and production. Understanding the crucial role of plant-associated microbial communities, we propose how the associated microbial actions could be enhanced to improve plant growth-promoting mechanisms, with a particular emphasis on plant-beneficial fungi. Additionally, we suggest possible plant strategies to adapt to a harsh environment by manipulating plant microbiomes. However, our current understanding of the microbiome is still in its infancy, and the major perturbations, such as anthropocentric actions, are not fully understood. Therefore, this work highlights the importance of manipulating the beneficial plant microbiome to create more sustainable agriculture, particularly under different environmental stressors.
... Plants detect microbes through recognition of specific microbial structures by pattern recognition receptors (PRRs), which always localize at the plasma membrane and work with coreceptors [3]. PRRs consist of receptor-like kinases (RLKs) and receptor-like proteins (RLPs): RLKs possess an intracellular kinase domain, a transmembrane domain, and an extracellular domain, while RLPs lack the intracellular domain but contain the other two domains [4][5][6][7]. ...
Full-text available
Plants must balance both beneficial (symbiotic) and pathogenic challenges from microorganisms, the former benefitting the plant and agriculture and the latter causing disease and economic harm. Plant innate immunity describes a highly conserved set of defense mechanisms that play pivotal roles in sensing immunogenic signals associated with both symbiotic and pathogenic microbes and subsequent downstream activation of signaling effector networks that protect the plant. An intriguing question is how the innate immune system distinguishes “friends” from “foes”. Here, we summarize recent advances in our understanding of the role and spectrum of innate immunity in recognizing and responding to different microbes. In addition, we also review some of the strategies used by microbes to manipulate plant signaling pathways and thus evade immunity, with emphasis on the use of effector proteins and micro-RNAs (miRNAs). Furthermore, we discuss potential questions that need addressing to advance the field of plant–microbe interactions.
... As the first plant immune barrier, PTI plays an important role in the plant's immune response. In PTI, cell surfacelocalized plant pattern recognition receptors (PRRs), including receptor-like kinases (RLKs), and receptor-like proteins (RLPs), recognize PAMPs [3,4], activating immune responses, including a reactive oxygen species (ROS) burst, defense-related gene expression, and antimicrobial compound production, including chitinase secretion [5][6][7][8][9][10]. To inhibit PTI, fungal pathogens secrete effectors to escape recognition by PRRs. ...
Full-text available
The shoot blight of Bambusa pervariabilis × Dendrocalamopsis grandis caused by Arthrinium phaeospermum made bamboo die in a large area, resulting in serious ecological and economic losses. Dual RNA-seq was used to sequence and analyze the transcriptome data of A. phaeospermum and B. pervariabilis × D. grandis in the four periods after the pathogen infected the host and to screen the candidate effectors of the pathogen related to the infection. After the identification of the effectors by the tobacco transient expression system, the functions of these effectors were verified by gene knockout. Fifty-three differentially expressed candidate effectors were obtained by differential gene expression analysis and effector prediction. Among them, the effectors ApCE12 and ApCE22 can cause programmed cell death in tobacco. The disease index of B. pervariabilis × D. grandis inoculated with mutant ΔApCE12 and mutant ΔApCE22 strains were 52.5% and 47.5%, respectively, which was significantly lower than that of the wild-type strains (80%), the ApCE12 complementary strain (77.5%), and the ApCE22 complementary strain (75%). The tolerance of the mutant ΔApCE12 and mutant ΔApCE22 strains to H2O2 and NaCl stress was significantly lower than that of the wild-type strain and the ApCE12 complementary and ApCE22 complementary strains, but there was no difference in their tolerance to Congo red. Therefore, this study shows that the effectors ApCE12 and ApCE22 play an important role in A. phaeospermum virulence and response to H2O2 and NaCl stress.
Receptor-like kinases (RLKs) are among the diverse array of plant defense components that perceive specific ligands and transduce defense signaling by cascades of phosphorylations mediated by transmembrane and cytoplasmic kinases for ultimate defense response. Though RLKs are one of the most exploited subgroups of the abundant plant protein family, kinases, their defense role against fungal pathogens across plant species is fragmented and less comprehensively understood. The association of other proteins with RLK for coreception and RLK-mediated cascades of downstream defense-signaling are crucial for signal transduction and eventual defense response. This section is aimed at exhaustively discussing the diversities and roles of RLKs in pathogen ligand perception and downstream defense-signaling for triggering plant immunity against fungal pathogens. This chapter is, therefore, believed to provide important insights into the roles of RLKs in plant defense, which could be helpful for researchers working on genetic resistance breeding.
Fusarium verticillioides is a key maize pathogen and produces fumonisins, a group of mycotoxins detrimental to humans and animals. Unfortunately, our understanding on how this fungus interacts with maize to trigger mycotoxin biosynthesis is limited. We performed a systematic computational network-based analysis of large-scale F. verticillioides RNA-seq datasets to identify gene subnetwork modules associated with virulence and fumonisin regulation. F. verticillioides was inoculated on two different maize lines, moderately resistant line hybrid 33K44 and highly susceptible line maize inbred line B73, to generate time-course RNA-Seq data. Among the highly discriminative subnetwork modules, we identified a putative hub gene FvLCP1, which encodes a putative a type-D fungal LysM protein with a signal peptide, three LysM domains, and two chitin binding domains. FvLcp1 is a unique protein that harbors these domains amongst five representative Fusarium species. FvLcp1 is a secreted protein important for fumonisin production with the LysM domain playing a critical role. The chitin-binding domain was essential for in vitro chitin binding. Using Magnaporthe oryzae, we learned that FvLcp1 accumulates in appressoria, suggesting that FvLcp1 is involved in host recognition and infection. Full length FvLcp1 suppressed BAX-triggered plant cell death in Nicotiana benthamiana. This unique type-D LysM secreted protein with a chitin-binding domain in F. verticillioides was shown to be potentially involved in suppressing host cell death and promoting fumonisin biosynthesis while the pathogen colonizes maize kernels.
Full-text available
Polysubstituted phenylisoxazoles were designed and synthesized to discover new antibacterial agents via [3 + 2] cycloaddition. Thirty-five compounds with a phenylisoxazole scaffold were characterized by NMR, HRMS, and X-ray techniques. After being evaluated against Xanthomonas oryzae (Xoo), Pseudomonas syringae (Psa), and Xanthomonas axonopodis (Xac), 4-nitro-3-phenylisoxazole derivatives were found to better antibacterial activities. Further studies have shown that the EC 50 values of these compounds were much better than that of the positive control, bismerthiazol.
Grapevine downy mildew is one of the most devastating diseases in grape production worldwide, but its pathogenesis remains largely unknown. A thorough understanding of the interaction between grapevine and the causal agent, Plasmopara viticola, is helpful to develop alternative disease control measures. Effector proteins that could be secreted to the interaction interface by pathogens are responsible for the susceptibility of host plants. In this study, a Crinkler effector, named PvCRN17, which is from P. viticola and showed virulent effects towards Nicotiana benthamiana previously, was further investigated. Consistently, PvCRN17 showed a virulent effect on grapevine plants. Protein–protein interaction experiments identified grapevine VAE7L1 (Vitis protein ASYMMETRIC LEAVES 1/2 ENHANCER 7‐Like 1) as one target of PvCRN17. VAE7L1 was found to interact with VvCIA1 and VvAE7, thus it may function in the cytosolic iron–sulphur cluster assembly (CIA) pathway. Transient expression of VAE7L1 in Vitis riparia and N. benthamiana leaves enhanced the host resistance to oomycete pathogens. Downstream of the CIA pathway in grapevine, three iron–sulphur (Fe‐S) proteins showed an enhancing effect on the disease resistance of N. benthamiana. Competitive co‐immunoprecipitation assay showed PvCRN17 could compete with VvCIA1 to bind with VAE7L1 and VvAE7. Moreover, PvCRN17 and VAE7L1 were colocalized at the plasma membrane of the plant cell. To conclude, after intruding into the grapevine cell, PvCRN17 would compete with VCIA1 to bind with VAE7L1 and VAE7, demolishing the CIA Fe‐S cluster transfer complex, interrupting the maturation of Fe‐S proteins, to suppress Fe‐S proteins‐mediated defence responses. Two indispensable components of the cytosolic iron–sulphur cluster assembly pathway in grapevine are abducted to the plasma membrane by a virulent CRN effector from Plasmopara viticola to impede grapevine defence responses.
Fusarium wilt disease, caused by Fusarium oxysporum f. sp. cucumerinum (Foc), leads to widespread yield loss and quality decline in cucumber. However, the molecular mechanisms underlying Foc resistance remain poorly understood. We report the mapping and functional characterization of CsChi23, encoding a cucumber class I chitinase with antifungal properties. We assessed sequence variations at CsChi23 and the associated defense response against Foc. We functionally characterized CsChi23 using transgenic assay and expression analysis. The mechanism regulating CsChi23 expression was assessed by genetic and molecular approaches. CsChi23 was induced by Foc infection, which led to rapid up‐regulation in resistant cucumber lines. Overexpressing CsChi23 enhanced fusarium wilt resistance and reduced fungal biomass accumulation, whereas silencing CsChi23 causes loss of resistance. CsHB15, a homeodomain leucine zipper (HD‐Zip) III transcription factor, was found to bind to the CsChi23 promoter region and activate its expression. Furthermore, silencing of CsHB15 reduces CsChi23 expression. A single nucleotide polymorphism variation ‐400bp upstream of CsChi23 abolished the HD‐Zip III binding site in susceptible cucumber line. Collectively, our study indicates that CsChi23 is sufficient to enhance fusarium wilt resistance and reveals a novel function of an HD‐Zip III transcription factor in regulating chitinase expression in cucumber defense against fusarium wilt.
Full-text available
Wheat stem rust, caused by the fungus Puccinia graminis f. sp. tritici, afflicts bread wheat (Triticum aestivum). New virulent races collectively referred to as "Ug99" have emerged, which threaten global wheat production. The wheat gene Sr33, introgressed from the wild relative Aegilops tauschii into bread wheat confers resistance to diverse stem rust races, including the Ug99 race group. We cloned Sr33, which encodes a coiled-coil, nucleotide-binding, leucine-rich repeat protein. Sr33 is orthologous to the barley (Hordeum vulgare) Mla mildew resistance genes that confer resistance to Blumeria graminis f. sp. hordei. The wheat Sr33 gene functions independently of RAR1, SGT1, and HSP90 chaperones. Haplotype analysis from diverse collections of Ae. tauschii placed the origin of Sr33 resistance near the southern coast of the Caspian Sea.
Full-text available
NOD-like receptor (NLR) proteins oligomerize into multiprotein complexes termed "inflammasomes" when activated. Their autoinhibition mechanism remains poorly defined. Here, we report the crystal structure of the mouse NLRC4 in a closed form. The ADP-mediated interaction between the central nucleotide-binding domain (NBD) and the winged-helix domain (WHD) was critical for stabilizing the closed conformation of NLRC4. The helical domain (HD2) repressively contacted a conserved and functionally important α-helix of the NBD. The C-terminal leucine-rich repeat (LRR) domain is positioned to sterically occlude one side of the NBD domain and consequently sequester NLRC4 in a monomeric state. Disruption of ADP-mediated NBD-WHD or NBD-HD2/NBD-LRR interactions resulted in constitutive activation of NLRC4. Together, our data reveal the NBD-organized cooperative autoinhibition mechanism of NLRC4 and provide insight into its activation.
Full-text available
Transcription activator-like (TAL) effectors are encoded by plantpathogenic bacteria and induce expression of plant host genes. TAL effectors bind DNA on the basis of a unique code that specifies binding of amino acid pairs in repeat units to particular DNA bases in a one-to-one correspondence. This code can be used to predict binding sites of natural TAL effectors and to design novel synthetic DNA-binding domains for targeted genome manipulation. Natural mechanisms of resistance in plants against TAL effector-containing pathogens have given insights into new strategies for disease control. Expected final online publication date for the Annual Review of Phytopathology Volume 51 is August 04, 2013. Please see for revised estimates.
Phytophthora infestans, the cause of potato late blight, is infamous for having triggered the Irish Great Famine in the 1840s. Until the late 1970s, P. infestans diversity outside of its Mexican center of origin was low, and one scenario held that a single strain, US-1, had dominated the global population for 150 years; this was later challenged based on DNA analysis of historical herbarium specimens. We have compared the genomes of 11 herbarium and 15 modern strains. We conclude that the 19th century epidemic was caused by a unique genotype, HERB-1, that persisted for over 50 years. HERB-1 is distinct from all examined modern strains, but it is a close relative of US-1, which replaced it outside of Mexico in the 20th century. We propose that HERB-1 and US-1 emerged from a metapopulation that was established in the early 1800s outside of the species' center of diversity.
The Protein Data Bank (PDB; ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource.
Transgenic inbred squash lines containing various combinations of the cucumber mosaic virus (CMV), watermelon mosaic virus 2 (WMV 2) or zucchini yellow mosaic virus (ZYMV) coat protein (CP) genes were produced using Agrobacterium-mediated transformation. Progeny from lines transformed with single or multiple CP gene constructs were tested for virus resistance under field conditions, and exhibited varying levels of resistance to infection by CMV, WMV 2 or ZYMV. Most transgenic lines remained nonsymptomatic throughout the growing seasons and produced marketable fruits, while other lines showed a delay in the onset of symptoms and/or a reduction in symptom severity. A few lines failed to display any level of resistance. Depending on the CP gene used, 40 to 95% of the transgenic lines containing single CP constructs of either CMV, WMV 2 or ZYMV were resistant to the virus from which the CP gene was derived. Transgenic lines transformed with a double CP construct containing the CP genes from CMV and WMV 2, designated CW, displayed high level of resistance to CMV and WMV 2. A transgenic line, designated ZW-20, which contained the CP genes from ZYMV and WMV 2 displayed excellent resistance to ZYMV and WMV 2 in that most of the plants showed complete resistance. A few plants developed localized chlorotic dots or blotches, yet fruits remained asymptomatic. Southern blot analysis revealed that the CP inserts of some resistant plants of line ZW-20 were no longer linked to the neomycin phosphotransferase II (NPT II) gene. This loss of linkage allowed the marker gene to be separated from the virus resistance trait by Mendelian segregation. Further analysis of these plants showed that they contained multiple WMV 2 inserts which were designated B and H, the latter consisting of two hybridization signals. Analysis of inoculated plants showed that plants with the H inserts were symptomless or developed only chlorotic dots, while those without the H insert developed more prominent chlorotic blotches. In addition to lines with resistance to two viruses, a line with resistance to three viruses was also identified. Transgenic line CZW-3, transformed with the triple CP construct containing the CMV, WMV 2 and ZYMV CP genes, exhibited resistance to all three viruses. These two transgenic inbred lines, ZW-20 and CZW-3, have allowed for the development of commercial squash hybrids with multiple virus resistance.
Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a devastating disease that can cause severe yield losses. A previously uncharacterized Pgt race, designated Ug99, has overcome most of the widely used resistance genes and is threatening major wheat production areas. Here, we demonstrate that the Sr35 gene from Triticum monococcum is a coiled-coil, nucleotide-binding, leucine-rich repeat gene that confers near immunity to Ug99 and related races. This gene is absent in the A-genome diploid donor and in polyploid wheat but is effective when transferred from T. monococcum to polyploid wheat. The cloning of Sr35 opens the door to the use of biotechnological approaches to control this devastating disease and to analyses of the molecular interactions that define the wheat-rust pathosystem.
Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research. These chimeric nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific genomic locations. Here, we review achievements made possible by site-specific nuclease technologies and discuss applications of these reagents for genetic analysis and manipulation. In addition, we highlight the therapeutic potential of ZFNs and TALENs and discuss future prospects for the field, including the emergence of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases.