An introduction to the amazing world of the oomycetes, some of the most destructive plant pathogens.

2. Crop losses due to
fungi and oomycetes
(filamentous plant
pathogens)
Fisher et al. 2012

3. Crop losses due to
fungi and oomycetes
(filamentous plant
pathogens)
Fisher et al. 2012

4. Veredeling voor resistentie niet geluktThe Irish potato famine pathogen Phytophthora infestans
causes potato blight

5. Phytophthora
Irish potato famine pathogen
Greek for plant destroyer
fungus-like oomycete

6. Ü Infection cycle and diversity
Ü Evolutionary history
Ü Genome architecture and evolution
Ü Virulence mechanisms: effector biology
Ü Host resistance and evasion of resistance
Ü Breeding host resistance: prospects and challenges
What you will learn – Phytophthora and the oomycetes

8. Fifi the oomycete is a scary parasite,
With flagellated spores and hyphal threads
She kills crops and triggers blight.
Infection cycle and diversity

9. Infection cycle of Phytophthora infestans

10. Infection cycle of Phytophthora infestans
Zoospore
cyst
appressorium
Infection
vesicle
haustorium
sporangium

11. Phytophthora infestans
necrotrophy
biotrophy
Phytophthora infestans is a hemibiotroph –
undergoes a biotrophic phase and then becomes necrotrophic

12. Filamentous growth of Phytophthora

13. Appressoria – Infection structures that enable
penetration of plant tissue
appressorium

14. Haustoria – Infection structures that enable
nutrient uptake and secretion of effector proteins
Coffey and Wilson, 1983
Tolga Bozkurt, Imperial College
Extrahaustorial
membrane (EHM)
haustorium

15. Haustoria – pathogen infection structures that are
surrounded by a host-derived membrane
Bozkurt et al. Curr Opin Plant Biol 2012

16. Haustoria enable secretion of effector proteins
and nutrient uptake
suppress immunity,
alter host physiology
nutrients?

17. Asexual spores – finding the host and dispersal
Sporangia and zoospores

18. a d
c f
g h
↓↓
↓
↓
↓
↓↓
↓
↓
↓
b
↓ ↓ ↓ ↓
↓ ↓ ↓ ↓
↓ ↓
↓ ↓
↓↓
↓
↓
↓ ↓↓
↓
↓
↓
↓↓
e
↓↓
↓ ↓↓
↓↓
↓
↓
↓ ↓
*
Phytophthora palmivora:
Root tip (electrotaxis)
Pythium aphanidermatum:
Wound exudates (chemotaxis)
Zoospore-root
interactions

20. Fifi the oomycete is a scary parasite,
With flagellated spores and hyphal threads
She kills crops and triggers blight.
Infection cycle and diversity

21. Oomycete phylogeny and diversity
Colonized many ecological niches
yet more than 60% of species are
parasitic on plants

22. ■ Obligate biotrophic parasites: require living plants
to grow, usually do not kill host plants
■ Largest number of species
■ Typically host specific/specialized parasites: one
species infects one plant species
■ Best studied is Hyaloperonospora arabidopsidis
(Hpa); infects host plant Arabidopsis thaliana
Downy mildews

23. Variety of symptoms caused by downy mildews on plants

24. ■ Phytophthora are the most important pathogens of
dicot plants, causing tens of billions of dollars of
losses annually
■ Phytophthora infestans, the agent of late blight of
potato and tomato, causes more crop losses than
any other member of the genus
■ But there are many other species that are
damaging to natural and agricultural ecosystems
Phytophthora: “plant killers”

25. Phytophthora capsici causes blight on several vegetables

26. Phytophthora capsici causes blight on several vegetables

27. Phytophthora ramorum and Phytophthora kernoviae infect many native
British woodland species and are a threat to the environment

29. Fifi the oomycete is a heterokont, they say,
She’s fungus-like but the scientists
Know how she had plastids one day.
Evolutionary history

30. Phytophthora is an oomycete not a fungus

31. Oomycetes are fungus-like filamentous microbes:
a unique group of eukaryotic plant pathogens
Plant pathogens in greenAnimal parasites in redTree adapted from Embley and Martin (2006) Nature 440:623
(Heterokonts)

32. Christine Strullu-Derrien and Paul Kenrick
@ Natural History Museum
Oomycetes form an ancient
eukaryotic lineage
§ may have been parasitic ~300
million year ago
§ present in the 407 million year-old
Rhynie Chert, an ecosystem of
plants, fungi and oomycetes

33. ■ Unrelated to fungi, more closely related to brown
algae and diatoms in the Stramenopiles
(Heterokonts)
■ Supported by molecular phylogenies based on
ribosomal RNA sequences, compiled amino acid data
for mitochondrial proteins, and protein encoding
chromosomal genes
■ Exhibit fungal-like filamentous (hyphal) growth and
several fungal-like morphological structures
Oomycetes - Phylogeny

35. Fifi the oomycete has a big genome, they say,
Full of repeats but don’t call it junk
‘cause can be handy one day.
Genome architecture and evolution

36. Veredeling voor resistentie niet geluktWhy the misery? Why are oomycetes the scourge of
farmers worldwide?
§ Phytophthora are astonishing plant destroyers that can
wipe out crops in days but the secret of their success is
their ability to rapidly adapt to resistant plant varieties
§ How did Phytophthora and other oomycetes manage to
keep on changing and adapting to ensure their
uninterrupted survival over evolutionary time?

37. 2006
maland
e of the
with the
10, 38).
to ap-
ntrol. In
e vibra-
dth are
ence in
owever,
dths in
to ma-
ch long-
ization.
nterfer-
splaced
bility of
obser-
proper-
ocesses,
ted.
9 (2003).
990).
Phys. 92,
Mathies,
Acad. Sci.
.
U.S.A. 97,
1 (2001).
(2002).
7 (2002).
J. 85,
5 (2004).
ujimura,
r,
(2005).
ler,
90).
01, 6629
New York, 1981).
33. D. Gelman, R. Kosloff, J. Chem. Phys. 123, 234506 (2005).
34. J. Hauer, H. Skenderovic, K.-L. Kompa, M. Motzkus, Chem.
Phys. Lett. 421, 523 (2006).
35. D. Oesterhelt, W. Stoeckenius, in Methods in Enzymology,
vol. 31 of Biomembranes (Academic Press, New York,
1974), pp. 667–678.
36. The saturation energy is related to the absorption cross
section s as Es 0 1/strans (for a negligibly small
contribution of the excited-state emission), and s is
thank J.T.M. Kennis, Vrije Universiteit Amsterdam, for
helpful discussions of preliminary results.
Supporting Online Material
www.sciencemag.org/cgi/content/full/313/5791/1257/DC1
Materials and Methods
Figs. S1 to S5
References
1 June 2006; accepted 10 August 2006
10.1126/science.1130747
Phytophthora Genome Sequences
Uncover Evolutionary Origins and
Mechanisms of Pathogenesis
Brett M. Tyler,1
* Sucheta Tripathy,1
Xuemin Zhang,1
Paramvir Dehal,2,3
Rays H. Y. Jiang,1,4
Andrea Aerts,2,3
Felipe D. Arredondo,1
Laura Baxter,5
Douda Bensasson,2,3,6
Jim L. Beynon,5
Jarrod Chapman,2,3,7
Cynthia M. B. Damasceno,8
Anne E. Dorrance,9
Daolong Dou,1
Allan W. Dickerman,1
Inna L. Dubchak,2,3
Matteo Garbelotto,10
Mark Gijzen,11
Stuart G. Gordon,9
Francine Govers,4
Niklaus J. Grunwald,12
Wayne Huang,2,14
Kelly L. Ivors,10,15
Richard W. Jones,16
Sophien Kamoun,9
Konstantinos Krampis,1
Kurt H. Lamour,17
Mi-Kyung Lee,18
W. Hayes McDonald,19
Mo´nica Medina,20
Harold J. G. Meijer,4
Eric K. Nordberg,1
Donald J. Maclean,21
Manuel D. Ospina-Giraldo,22
Paul F. Morris,23
Vipaporn Phuntumart,23
Nicholas H. Putnam,2,3
Sam Rash,2,13
Jocelyn K. C. Rose,24
Yasuko Sakihama,25
Asaf A. Salamov,2,3
Alon Savidor,17
Chantel F. Scheuring,18
Brian M. Smith,1
Bruno W. S. Sobral,1
Astrid Terry,2,13
Trudy A. Torto-Alalibo,1
Joe Win,9
Zhanyou Xu,18
Hongbin Zhang,18
Igor V. Grigoriev,2,3
Daniel S. Rokhsar,2,7
Jeffrey L. Boore2,3,26,27
Draft genome sequences have been determined for the soybean pathogen Phytophthora sojae and
the sudden oak death pathogen Phytophthora ramorum. Oo¨mycetes such as these Phytophthora
species share the kingdom Stramenopila with photosynthetic algae such as diatoms, and the
presence of many Phytophthora genes of probable phototroph origin supports a photosynthetic
ancestry for the stramenopiles. Comparison of the two species’ genomes reveals a rapid expansion
and diversification of many protein families associated with plant infection such as hydrolases, ABC
transporters, protein toxins, proteinase inhibitors, and, in particular, a superfamily of 700 proteins
with similarity to known oo¨mycete avirulence genes.
P
hytophthora plant pathogens attack a
wide range of agriculturally and orna-
mentally important plants (1). Late blight
of potato caused by Phytophthora infestans re-
sulted in the Irish potato famine in the 19th cen-
tury, and P. sojae costs the soybean industry
millions of dollars each year. In California and
Oregon, a newly emerged Phytophthora species,
P. ramorum, is responsible for a disease called
sudden oak death (2) that affects not only the live
w.sciencemag.org SCIENCE VOL 313 1 SEPTEMBER 2006 1261
LETTERS
Genome sequence and analysis of the Irish potato
famine pathogen Phytophthora infestans
Brian J. Haas1
*, Sophien Kamoun2,3
*, Michael C. Zody1,4
, Rays H. Y. Jiang1,5
, Robert E. Handsaker1
, Liliana M. Cano2
,
Manfred Grabherr1
, Chinnappa D. Kodira1
{, Sylvain Raffaele2
, Trudy Torto-Alalibo3
{, Tolga O. Bozkurt2
,
Audrey M. V. Ah-Fong6
, Lucia Alvarado1
, Vicky L. Anderson7
, Miles R. Armstrong8
, Anna Avrova8
, Laura Baxter9
,
JimBeynon9
,PetraC.Boevink8
,StephanieR.Bollmann10
,JorunnI.B.Bos3
,VincentBulone11
,GuohongCai12
,CahidCakir3
,
James C. Carrington13
, Megan Chawner14
, Lucio Conti15
, Stefano Costanzo16
, Richard Ewan15
, Noah Fahlgren13
,
Michael A. Fischbach17
, Johanna Fugelstad11
, Eleanor M. Gilroy8
, Sante Gnerre1
, Pamela J. Green18
,
Laura J. Grenville-Briggs7
, John Griffith14
, Niklaus J. Gru¨nwald10
, Karolyn Horn14
, Neil R. Horner7
, Chia-Hui Hu19
,
3 18 2 2 20
Vol 461|17 September 2009|doi:10.1038/nature08358
2009

38. Features of sequenced oomycete genomes
Figure 2. Features of sequenced oomycete pathogen genomes. The representative phylogeny depicts oomycete pathogens with sequenced
genomes and was generated using Interactive Tree Of Life (iTOL) with National Center for Biotechnology Information (NCBI) taxonomy identifiers
(branch lengths are arbitrary). Pathogen lifestyles and major variations in effector gene families are indicated along the tree branches. Potential
loss of particular effector classes in a lineage is indicated by a red cross. The principal host, genome size, repetitive DNA content (as a percentage
of genome size), gene space (the percentage of the genome encoding genes), number of protein-coding genes and number and percentage of
proteins encoding predicted secreted proteins (secretome) are indicated from left to right for each pathogen. CRN, Crinkler; ND, not determined.
Pythium ultimum
Phytophthora ramorum
Phytophthora infestans
Phytophthora sojae
Phytophthora parasitica
Hyaloperonospora arabidopsidis
Saprolegnia parasitica
Albugo candida
Albugo laibachii
CRN
effectors
CHXC effectors
Host
Varied
Arabidopsis
Arabidopsis
Vegetables
Potato,tomato
Varied
Soybean
Varied
Fish
Woody plants
53
80
65
100
45
53
Genome
size (Mb)
% Repetitive
DNA content
ND
39
17
ND
43
22
Protein
coding genes
20,822
16,988
14,451
14,543
15,824
20,088
Secretome (%)
1,561
1,867
1,523
727
1,255
RefsOomycetes
% gene
space
32
28
31
17
43
49
(7.5)
(11.0)
(10.5)
(5.0)
(3.3)515
(6.2)
NPP1
-like
NPP1-like
64 19 1,17619,80529 (5.9)
240 74 18,155 1,588 (8.7)10
43 7 15,291 843 (5.5)44
37 28 13,032 413 (3.2)48
Plant Biotroph
Plant Necrotroph Fish Saprotroph/Necrotroph
Non-repetitive Repetitive Non-coding Coding
Phytophthora capsici
RXLR effectors
YxSL[RK]
& RXLR
effectors
YxSL[RK] effectors
[6]
[11]
[12]
[13]
[13]
[14]
[15]
[16]
[17]
[18]
Apoplastic effectors Cytoplasmic effecttors
Plant Hemibiotroph
Key (i): Key (ii):
Pais et al. Genome Biology 2013, 14:211
http://genomebiology.com/2013/14/6/211
Page 3 of 10
Pais et al. Genome Biology, 2013

39. The 240 Mbp genome of Phytophthora infestans
– a repeat and transposon driven expansion

40. P. infestans
P. sojae
P. ramorum
P. sojae
P. infestans
P. ramorum
B. Haas, S. Kamoun et al. Nature, 2009
Phytophthora infestans genome architecture - repeat-
rich and gene-poor loci interrupt colinear regions

41. RXLR
Crinklers
Protease inhibitors
Phytophthora infestans genome: so many effectors!
Apoplastic
Host-translocated
~38
~550
~200
~250ψ

42. P. infestans
P. sojae
P. ramorum
P. sojae RXLR effector gene
P. infestans
P. ramorum
B. Haas, S. Kamoun et al. Nature, 2009
RSLR
43 47 100221
AVRblb2
Host translocation Effector activity
Signal Peptide
Phytophthora infestans effectors typically occur
in the expanded, repeat-rich and gene-poor loci

43. Length of intergenic regions (kb)
Nb of genes :
80-85
75-80
70-75
65-70
60-65
55-60
50-55
45-50
40-45
35-40
30-35
25-30
20-25
15-20
10-15
5-10
0-5
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
00-5
11-15
16-20
6-10
21-25
31-35
36-40
26-30
41-45
51-55
56-60
46-50
61-65
71-75
76-80
66-70
81-85
P. infestans (16442 genes)
Core orthologs (7580) RXLR effectors (520)
Phytophthora infestans effectors typically occur
in the expanded, repeat-rich and gene-poor loci
B. Haas, S. Kamoun et al. Nature, 2009

44. The “two-speed genome” of P. infestans
underpins high evolutionary potential
P. sojae
P. infestans
P. ramorum
§ Gene-sparse regions of genome show highest rates of structural
and sequence variation, signatures of adaptive selection
§ Gene-sparse regions underpin rapid evolution of virulence (effector)
genes and host adaptation; cradle for adaptive evolution

45. Several filamentous plant pathogens (fungi and
oomycetes) have a “two-speed” genome architecture
A C
D
B

46. O, Fifi the oomycete
Was as virulent as she’s been;
and scientists say she secretes her way
Inside potatoes and bean.
Virulence mechanisms: effector biology

47. bacterium
fungus
oomycete
haustorium
apoplas4c
effectors
plant
cell
Microbes alter plant cell processes
using secreted “effector” molecules
Win, J., Chaparro-Garcia, A., Belhaj, K., Saunders, D.G.O., Yoshida, K., Dong, S., Schornack, S., Zipfel, C., Robatzek, S., Hogenhout, S.A., and
Kamoun, S. Cold Spring Harbor Symposium on Quantitative Biology, 2013; Dodds, P.N., and Rathjen, J.P. Nature Reviews Genetics, 2010
Cytoplasmic
effectors
targets
targets
Alter
plant
cell
processes
Help
microbe
colonize
plant

48. Ü Effectors have been described in bacteria, oomycetes,
fungi, nematodes, insects etc.
Ü Current paradigm - effector activities are key to
understanding parasitism (and symbiosis)
Ü Operationally effectors are plant proteins - Encoded by
genes in pathogen genomes but function in (inside)
plant cells
Key concepts about plant pathogen effectors

49. Effectors - sensus Dawkins in “The extended
phenotype: the long reach of the gene” p. 210, 1981
“...parasite genes
having phenotypic
expression in host
bodies and behavior”
Phytoplasmas
see Saskia Hogenhout’s lectures

50. RXLR
Crinklers
Protease inhibitors
The diverse effectors of Phytophthora infestans
Apoplastic
Host-translocated
~38
~550
~200
~250ψ

51. Apoplastic effectors are typically
small cysteine-rich secreted proteins
- often get processed upon secretion
- disulfide bridges provide stability in apoplast
Targeting Function

52. (tomato cell)
(apoplast)
RCR3 PIP1 C14 CYP3 CatB1ALP CatB2
Immune response
EPIC1
EPIC2B
Phytophthora infestans
Oomycete
Phytophthora and fungal effectors inhibit the same
plant immune proteases
with Renier van der Hoorn
Cladosporium fulvum
Fungus
AVR2

53. RLLR SEER
21 44 59 147
Signal Peptide
1
RSLR DEER
21 51 72 1521
Signal Peptide
RFLR EER
23 54 69 1681
Signal Peptide
PEX-RD3
IPIO1
NUK10
AVR3a
RQLR GEERSignal Peptide
19 50 77 2131
Cytoplasmic effectors of oomycetes have
a conserved domain defined by the RXLR motif
Targeting Function

54. P. infestans RXLR effectors target a variety host
proteins and processes
Ü AVR3a targets an E3 Ubiquitin Ligase CMPG1 to suppress
immunity (Bos et al. PNAS, 2010)
Ü AVR2 targets a Kelch repeat phosphatase BSL1 to suppress
immunity (Saunders et al. Plant Cell, 2012)
Ü PexRD54 binds ATG8 and antagonizes selective autophagy to
counteract plant defense (Dagdas, Belhaj et al. eLife 2016)
Ü AVRblb2 interferes with secretion of cysteine protease C14 to
counteract plant defense (Bozkurt et al. PNAS, 2011)

55. Kamoun & van der Hoorn Labs
EPIC
C14
P. infestans evolved distinct effectors and
mechanisms to interfere with apoplastic proteases

56. Kamoun & van der Hoorn Labs
EPIC
C14
C14
P. infestans evolved distinct effectors and
mechanisms to interfere with apoplastic proteases

57. Kamoun & van der Hoorn Labs
EPIC
C14
C14
P. infestans evolved distinct effectors and
mechanisms to interfere with apoplastic proteases

58. Kamoun & van der Hoorn Labs
EPIC
C14
AVRblb2
P. infestans evolved distinct effectors and
mechanisms to interfere with apoplastic proteases

59. Fifi the oomycete found
A resistant plant that day,
So she said, "Let's run and
There’ll be no fun
Until I mutate away."
Host resistance and evasion of resistance

60. Resistance to Phytophthora infestans
§ The Hypersensitive Response
(HR): a programmed cell
death response
§ Activation of a plant immune
receptor (R) by an effector

61. effectors
bacterium
fungus
oomycete
haustorium
plant
cell
Some effectors “trip the wire” and activate
immunity in particular plant genotypes
Alter
plant
cell
processes
targets
NB-­‐LRR/NLR
immune
receptors
NLR-­‐
triggered
immunity
Win, J., Chaparro-Garcia, A., Belhaj, K., Saunders, D.G.O., Yoshida, K., Dong, S., Schornack, S., Zipfel, C., Robatzek, S., Hogenhout, S.A., and
Kamoun, S. Cold Spring Harbor Symposium on Quantitative Biology, 2013; Dodds, P.N., and Rathjen, J.P. Nature Reviews Genetics, 2010

62. effectors
bacterium
fungus
oomycete
haustorium
plant
cell
Concept: Host Resistance and Susceptibility genes
Alter
plant
cell
processes
targets
NB-­‐LRR/NLR
immune
receptors
NLR-­‐
triggered
immunity
Win, J., Chaparro-Garcia, A., Belhaj, K., Saunders, D.G.O., Yoshida, K., Dong, S., Schornack, S., Zipfel, C., Robatzek, S., Hogenhout, S.A., and
Kamoun, S. Cold Spring Harbor Symposium on Quantitative Biology, 2013; Dodds, P.N., and Rathjen, J.P. Nature Reviews Genetics, 2010
R
S

63. Wheat
Potato
Tomato
Corn
Rice
Barley
Bacteria
Nematodes
Oomycetes
Insects
Fungi
Viruses
CCNB-ARCLRR(xxLxLxx)
Plants utilize a ubiquitous disease resistance toolkit
to defend against unrelated pathogens

64. NLR
/
NB-­‐LRR
immune
receptors
How do pathogen effectors evade immunity?
AVR
effector
§ Effectors evade activating
immune receptors by
acquiring stealthy mutations
target
§ But, they have to maintain
their virulence activity!
§ Still many effectors become
nonfunctional (pseudogenes)
or get deleted in the pathogen
genome

65. Functional redundancy among pathogen
effectors enables “bet-hedging”
Ü Many effectors are functionally
redundant; affect different steps
or converge on same target
Ü Functional redundancy enables
robustness in the face of the
evolving plant immune system
Ü “Bet-hedging”
Win, J., et al. Cold Spring Harbor Symposium on Quantitative Biology, 2013

66. For Fifi the oomycete
Keeps evolving in her way,
But don’t wave her goodbye,
Don't you even try,
She’ll be back again some day.
Breeding host resistance: prospects and challenges

68. An arms race between the plant breeder/
biotechnologist and the pathogen?
• Ability of the pathogen to adapt is astounding
• “Don’t bet against the pathogen” – silver bullet
solutions unlikely to be durable
• Framework to rapidly generate new resistance
specificities and introduce these traits into crop
genomes
• Can we generate and deploy new resistance
traits faster than the pathogen can evolve?

70. HOME / SCIENCE : THE STATE OF THE UNIVERSE.
No, You Shouldn’t Fear GMO
Corn
How Elle botched a story about genetically modified food.
By Jon Entine Posted Wednesday, Aug. 7, 2013, at 2:45 PM
|
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71. ... for peace of mind
in potato cultivation
UNDER DEVELOPMENTFOR YOURCONVENIENCE
On top of that, you have to ensure that the
weather is favorable in terms of wind strength
and precipitation. If you want to put an end to
precisely that situation, Fortuna is the right
potato variety for you, as it provides lasting
protection against late blight. So there’s no
more need for you to regularly inspect your
fields and check for signs of this disease. With
Fortuna, you no longer have to worry about the
right time and the right weather. And best of all,
when you plant out your crops you can look for-
ward to an extremely high yield and outstanding
quality without any stress.
What is Fortuna?
Fortuna is a further development of the leading
European potato variety used for making fries.
Fortuna is identical to its parent variety, but has
one key additional benefit: It provides complete
protection against late blight. Fortuna therefore
offers impressively high tuber yield and thanks
to its tuber form, color and taste, it satisfies all
the requirements to be used in making fries or
as fresh produce.
How was Fortuna developed?
Dutch researchers have undertaken extensive
research into the special resistance of the wild
potato. They managed to identify the genes
which give the wild potato protection against
late blight. Agricultural and biotechnological
experts at BASF then transferred these genes
to the leading French fries potato variety using
the soil bacterium Agrobacterium. This resulted
in Fortuna which is equipped with the wild po-
tato‘s effective protection against late blight –
without modifying its outstanding agronomic
characteristics.
Why is Fortuna so useful to you?
If you’re a potato farmer, you’re sure to be fami-
liar with the following situation. July and August
are mainly wet. Late blight attacks all of your
potato fields, which you cared for intensively
throughout the whole year. All you can do is
wait until the rain subsides and machinery can
move on the soil again. Now you have the prob-
lem of, on the one hand, choosing the right
time to use fungicides to protect against late
blight. On the other hand, you don‘t want to risk
driving on the land and causing soil compaction.
20 30
P
40
P+A
50
P P P
Application against Alternaria (A) and Phytophthora (P; 5-12 treatments in “normal” years)
60
P+A
70
P P PP P P
80
Fungicide treatments in conventionally improved potatoes
20 30 40
A
50
Application against Alternaria (A) and Phytophthora (P)
60
A
70 80
Fungicide treatments in Fortuna (due to lasting Phytophthora resistance)
Fortuna, the potato variety for your peace of mind.
With natural resistance to Phytophthora
tato‘s effective protection against late blight –
without modifying its outstanding agronomic
characteristics.
20 30
P
40
P+A
50
P P P
Application against Alternaria (A) and Phytophthora (P; 5-12 treatments in “normal” years)
60
P+A
70
P P PP P P
80
Fungicide treatments in conventionally improved potatoes

72. ... for peace of mind
in potato cultivation
UNDER DEVELOPMENTFOR YOURCONVENIENCE
On top of that, you have to ensure that the
weather is favorable in terms of wind strength
and precipitation. If you want to put an end to
precisely that situation, Fortuna is the right
potato variety for you, as it provides lasting
protection against late blight. So there’s no
more need for you to regularly inspect your
fields and check for signs of this disease. With
Fortuna, you no longer have to worry about the
right time and the right weather. And best of all,
when you plant out your crops you can look for-
ward to an extremely high yield and outstanding
quality without any stress.
What is Fortuna?
Fortuna is a further development of the leading
European potato variety used for making fries.
Fortuna is identical to its parent variety, but has
one key additional benefit: It provides complete
protection against late blight. Fortuna therefore
offers impressively high tuber yield and thanks
to its tuber form, color and taste, it satisfies all
the requirements to be used in making fries or
as fresh produce.
How was Fortuna developed?
Dutch researchers have undertaken extensive
research into the special resistance of the wild
potato. They managed to identify the genes
which give the wild potato protection against
late blight. Agricultural and biotechnological
experts at BASF then transferred these genes
to the leading French fries potato variety using
the soil bacterium Agrobacterium. This resulted
in Fortuna which is equipped with the wild po-
tato‘s effective protection against late blight –
without modifying its outstanding agronomic
characteristics.
Why is Fortuna so useful to you?
If you’re a potato farmer, you’re sure to be fami-
liar with the following situation. July and August
are mainly wet. Late blight attacks all of your
potato fields, which you cared for intensively
throughout the whole year. All you can do is
wait until the rain subsides and machinery can
move on the soil again. Now you have the prob-
lem of, on the one hand, choosing the right
time to use fungicides to protect against late
blight. On the other hand, you don‘t want to risk
driving on the land and causing soil compaction.
20 30
P
40
P+A
50
P P P
Application against Alternaria (A) and Phytophthora (P; 5-12 treatments in “normal” years)
60
P+A
70
P P PP P P
80
Fungicide treatments in conventionally improved potatoes
20 30 40
A
50
Application against Alternaria (A) and Phytophthora (P)
60
A
70 80
Fungicide treatments in Fortuna (due to lasting Phytophthora resistance)
Fortuna, the potato variety for your peace of mind.
With natural resistance to Phytophthora
tato‘s effective protection against late blight –
without modifying its outstanding agronomic
characteristics.
20 30
P
40
P+A
50
P P P
Application against Alternaria (A) and Phytophthora (P; 5-12 treatments in “normal” years)
60
P+A
70
P P PP P P
80
Fungicide treatments in conventionally improved potatoes

73. Ü Genomics-enabled gene mapping and cloning
Ü Impacting plant breeding: from Marker Assisted Selection
(MAS) to Genomic Selection (GS)
Ü Applicable to both Resistance and Susceptibility genes
Next-generation crop (disease resistance) breeding
A RT I C L E S
Genome sequencing reveals agronomically important
loci in rice using MutMap
Akira Abe1,2,7, Shunichi Kosugi3,7, Kentaro Yoshida3, Satoshi Natsume3, Hiroki Takagi2,3, Hiroyuki Kanzaki3,
3,4 3 3 3 5 6
ved.
A RT I C L E S
Genome sequencing reveals agronomically important
loci in rice using MutMap
Akira Abe1,2,7, Shunichi Kosugi3,7, Kentaro Yoshida3, Satoshi Natsume3, Hiroki Takagi2,3, Hiroyuki Kanzaki3,
Hideo Matsumura3,4, Kakoto Yoshida3, Chikako Mitsuoka3, Muluneh Tamiru3, Hideki Innan5, Liliana Cano6,
Sophien Kamoun6 & Ryohei Terauchi3
The majority of agronomic traits are controlled by multiple genes that cause minor phenotypic effects, making the identification
of these genes difficult. Here we introduce MutMap, a method based on whole-genome resequencing of pooled DNA from a

74. BACTERIA MAY NOT ELICIT MUCH SYMPA-
thy from us eukaryotes, but they, too, can get
sick. That’s potentially a big problem for the
dairy industry, which often depends on bac-
teria such as Streptococcus thermophilus to
make yogurts and cheeses. S. thermophilus
breaks down the milk sugar lactose into tangy
lactic acid. But certain viruses—bacterio-
phages, or simply phages—can debilitate the
pany now owned by DuPont, found a way to
boost the phage defenses of this workhouse
microbe. They exposed the bacterium to
a phage and showed that this essentially
vaccinated it against that virus (Science,
23 March 2007, p. 1650). The trick has
enabled DuPont to create heartier bacterial
strains for food production. It also revealed
something fundamental: Bacteria have a
become important for more than food scien-
tists and microbiologists, because of a valu-
able feature: It takes aim at specific DNA
sequences. In January, four research teams
reported harnessing the system, called
CRISPR for peculiar features in the DNA of
bacteria that deploy it, to target the destruc-
tion of specific genes in human cells. And in
the following 8 months, various groups have
The CRISPR CrazeA bacterial immune system yields a potentially
revolutionary genome-editing technique
IENCE/SCIENCESOURCE
Fighting invasion. When
viruses (green) attack
bacteria, the bacteria
respond with DNA-targeting
defenses that biologists
have learned to exploit
for genetic engineering.
onAugust23,2013www.sciencemag.orgDownloadedfrom
www.sciencemag.org SCIENCE VOL 341 23 AUGUST 2013
BACTERIA MAY NOT ELICIT MUCH SYMPA-
thy from us eukaryotes, but they, too, can get
sick. That’s potentially a big problem for the
dairy industry, which often depends on bac-
teria such as Streptococcus thermophilus to
make yogurts and cheeses. S. thermophilus
breaks down the milk sugar lactose into tangy
lactic acid. But certain viruses—bacterio-
phages, or simply phages—can debilitate the
bacterium, wreaking havoc on the quality or
quantity of the food it helps produce.
In 2007, scientists from Danisco, a
Copenhagen-based food ingredient com-
pany now owned by DuPont, found a way to
boost the phage defenses of this workhouse
microbe. They exposed the bacterium to
a phage and showed that this essentially
vaccinated it against that virus (Science,
23 March 2007, p. 1650). The trick has
enabled DuPont to create heartier bacterial
strains for food production. It also revealed
something fundamental: Bacteria have a
kind of adaptive immune system, which
enables them to fight off repeated attacks
by specific phages.
That immune system has suddenly
become important for more than food scien-
tists and microbiologists, because of a valu-
able feature: It takes aim at specific DNA
sequences. In January, four research teams
reported harnessing the system, called
CRISPR for peculiar features in the DNA of
bacteria that deploy it, to target the destruc-
tion of specific genes in human cells. And in
the following 8 months, various groups have
used it to delete, add, activate, or suppress tar-
geted genes in human cells, mice, rats, zebra-
fish, bacteria, fruit flies, yeast, nematodes,
and crops, demonstrating broad utility for the
The CRISPR CrazeA bacterial immune system yields a potentially
revolutionary genome-editing technique
CREDIT:EYEOFSCIENCE/SCIENCESOURCE
for genetic engineering.
Published by AAAS
infects plant scientists

75. REVIEW Open Access
Plant genome editing made easy: targeted
mutagenesis in model and crop plants using the
CRISPR/Cas system
Khaoula Belhaj†
, Angela Chaparro-Garcia†
, Sophien Kamoun*
and Vladimir Nekrasov*
Abstract
Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant
breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing
site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases
(TALENs). However, these technologies have not been widely adopted by the plant research community due to
complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an
easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic
repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA
guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end
joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent
applications of the CRISPR/Cas technology in plants.
Keywords: CRISPR, Cas9, Plant, Genome editing, Genome engineering, Targeted mutagenesis
Introduction
Targeted genome engineering has emerged as an alter-
native to classical plant breeding and transgenic (GMO)
methods to improve crop plants and ensure sustainable
food production. However, until recently the available
methods have proven cumbersome. Both zinc finger
nucleases (ZFNs) and TAL effector nucleases (TALENs)
based on the Cas9 nuclease and an engineered single
guide RNA (sgRNA) that specifies a targeted nucleic acid
sequence. Given that only a single RNA is required to gen-
erate target specificity, the CRISPR/Cas system promises
to be more easily applicable to genome engineering than
ZFNs and TALENs.
Recently, eight reports describing the first applications
PLANT METHODS
Belhaj et al. Plant Methods 2013, 9:39
http://www.plantmethods.com/content/9/1/39

76. Humans have been modifying the genomes
of animals and plants…
…using mutagens (chemicals, irradiation) and selective breeding
§ We are now moving into an era of precise genome editing
(synthetic biology, biohacking etc.); ultimate in editing
§ Accessing and generating genetic diversity will not be a
limiting factor anymore; key is to know which genes
influence agronomically important traits

77. 500 -
400 -
300 -
bp
1 2 8 10WT
500 -
400 -
300 -
bp
200 -
1 2 83 4 5 6 7 9 10
ACATAGTAAAAGGTGTACCTGTGGTGGAGACTGGTGACCATCTTTTCTGGTTTAATCGCCCTGCCCTTGTCCTATTCTTGATTAACTTTGTACTCTTTCAGG!
ACATAGTAAAAGGTGTACCTGTGGTGGAGACTGGTGACCATCTTTTCTGGTTTAATCGCCCTGCCCTTGTCCTATTCTTGATTAACTTTGTACTCTTTCAGG!
ACATAGTAAAAGGTGTACCTGTGGTGGA------------------------------------------------CTTGATTAACTTTGTACTCTTTCAGG -48!
ACATAGTAAAAGGTGTACCTGTGGTGGA------------------------------------------------CTTGATTAACTTTGTACTCTTTCAGG -48!
ACATAGTAAAAGGTGTACCTGTGGTGGA------------------------------------------------CTTGATTAACTTTGTACTCTTTCAGG -48!
ACATAGTAAAAGGTGTACCTGTGGTGGA-------------------------------------------------TTGATTAACTTTGTACTCTTTCAGG -49!
WT
Plant 1
Plant 2
Plant 8
Plant 10
PAM PAMTarget 1 Target 2
500 -
400 -
300 -
8-1
500 -
400 -
300 -
bp 8-2 8-3 8-4 8-5 WT 8-6
SlMlo1
T-DNA
8-4
T-DNA: - - +-
WT slmlo1&
8-6
b c
d
e
f
Figure 1
Transform with
Cas9/sgRNAs
Callus
tissue
T0
plantlets
T1 seeds slmlo1
T-DNA
segregating
Regenerate T0
plants and screen
for slmlo1
homozygotes
Screen T1 generation
for T-DNA-free plants
3.5 months
T2 seeds
slmlo1
T-DNA-free
0 3 6 9.5
Time, months3 months 3 months 3.5 months
Tomelo – A DNA deletion results in fungus resistance
Tomelo – A DNA deletion in the S gene mlo
results in resistance to powdery mildew fungus
Vladimir Nekrasov

78. Deletion of CMPG1 in tomato reduces infection by P. infestans
Lesionsize(mm2)
Wild-Type
cmpg1 mutants
Mutant 1 Mutant 2 Mutant 3
P. infestans 88069 (5 dpi)
Wild-Type
cmpg1 mutant
Angela Chaparro-Garcia