MECANISMOS DE

PATOGENICIDAD Y VIRULENCIA
EN HONGOS Y OOMICETOS

Julio Rodríguez Romero

Departamento de Biotecnología y Biología Vegetal

julio.rodriguez.romero@upm.es
CBGP

1
 MECANISMOS DE VIRULENCIA EN HONGOS Y
OOMICETOS

Día 1 (Jueves 5 17-19): Introducción a los organismos

filamentosos y su interacción con las plantas. Diferentes
estrategias del hongo para la patogénesis de plantas.
Día 2 (Martes 10 17-19): Métodos moleculares y aproximaciones
“ómicas” usados para el estudio de organismos
filamentosos fitopatógenos.
Día 3 (Miércoles 11 15-17): Procesos biológicos reguladores de la
actividad patogénica: metabolismo - autofagia,
citoesqueleto - NADPH oxidasas, vías de señalización,
miRNAs. Bases moleculares de la patogénesis.
Día 4 (Martes 17 17-19): Determinantes de patogenicidad y
espectro de huésped: efectores y toxinas.
Día 5 (Miércoles 18 15-17): Seminarios.

2
 References
Books
- Vidhyasekaran, P. (2008). Fungal pathogenesis in plants and crops: Molecular biology and host defense mechanisms, 2nd edn. Boca Raton, FL
(USA): CRC Press.
- Lamour, K & Kamoun, S. (2009). OOMYCETE GENETICS AND GENOMICS. Diversity, Interactions, and Research Tools.
- Di Pietro, A. & Perez Martin, J. (2012). Morphogenesis and Pathogenicity in Fungi. Springer-Verlag Berlin and Heidelberg GmbH & Co. K,
Berlin.
Societies
British Mycological Society http://www.britmycolsoc.org.uk/
American Pathological Society (APS) http://www.apsnet.org/Pages/default.aspx
Journals
* in bold journals focused on reviews (not original research); Impact factor from Journal Citation Index 2014 (WoK)

Impact Factor > 14

Nature
Impact Factor = 5-8
Science New Phytologist
Cell PLoS Pathogen
Nature Reviews Microbiology PLoS Genetics
MICROBIOL MOL BIOL REVIEWS Fungal Diversity
Plant Journal
Impact Factor = 8-14 Seminars in Cell and Developmental Biology
Cell Host and Microbe
Nature communications Impact Factor = 3-5
EMBO J. Cellular Microbiology
Journal of Cell Biology Molecular Plant Pathology
PNAS Journal of Biological Chemistry
Current Biology Molecular Microbiology
PLOS Biology Genome Biology and Evolution
Plant Cell Molecular plant-microbe interactions
eLIFE Fungal Genetics and Biology
FEMS MICROBIOL REV BMC Microbiology
Trends in Plant Sciences Microbiology-SGM
ANNU REV MICROBIOL Plant Pathology
ANNU REV PHYTOPATH
Current Topics in Microb
Trends in Microbiology

3
 Clase 1
Introducción a los organismos filamentosos
y su interacción con las plantas.
Diferentes estrategias del hongo para la patogénesis de plantas.

4
Introducción a los organismos filamentosos y
su interacción con las plantas.

5
 Classification of organisms
Three domains
-Eukarya, eukaryotic, the Eukaryotes: nuclear envelop
-Bacteria, prokaryoric, the true Bacteria
-Archaea , prokaryoric, Archaebacteria

Ciccarelli FD, Doerks T, von Mering C, Creevey CJ, Snel B, et al. (2006) Toward automatic reconstruction of a highly resolved tree of
life. Science 311: 1283-1287.

6
See Tree of life project: http://tolweb.org 7
8
 THE FUNGAL KINGDOM

saprophytic symbiotic pathogenic

- About 6.0 million fungal species exist on Earth

saprophytic/symbiotic/pathogenic lifestyles

Only 100000 species are presently recognized ~20,000 species are

plant pathogenic

multicellular
- yeast-like or filamentous growth unicellular pseudohyphae hyphae

- Filamentous fungi: EUKARYOTIC MODEL ORGANISMS

- infection-related processes in animals and plants

- polar hyphal growth and multicellularity
absent in budding or
fission yeast
- production of secondary metabolites
- more diverse signalling pathways
- ageing – circadian cycle

9
 Glomalean fungi may have played a crucial role in facilitating the
colonization of land by plants
- Fossilised hyphae and spores support molecular estimates of Glomalean fungal phylogeny
(Ascomycota, Basiodiomycota and Glomeromycota) around ~600 mya

- Place the origin of Glomalean fungi before the appearance of vascular plants (~ 400-500 mya)

Redecker et al, Science (2000). Glomalean Fungi from the Ordovician

10
 Morphological
structures of genera of
phytopathogenic fungi

Crous PW, Hawksworth DL, Wingfield

MJ (2015) Identifying and Naming
Plant-Pathogenic Fungi: Past, Present,
and Future. Annu Rev Phytopathol 53:
247-267.

11
 Classification of the fungi
- Domain Eukarya

- Kingdom Fungi

- Phylum -mycota

- Class -mycetes

- Order -ales

- Family -aceae

- Genus Magnaporthe, Verticillium , Fusarium

- Species Magnaporthe oryzae, Verticillium dahliae, Fusarium oxysporum

12
 The rice blast fungus Magnaporthe oryzae
- Domain Eukarya

- Kingdom Fungi

- Phylum Ascomycota

- Class Pyrenomycetes

- Order Diaporthales

- Family Magnaporthaceae

- Genus Magnaporthe

- Species Magnaporthe oryzae

20 mm

13
 Blumeria, Botrytis (Mary Berbee, 2001)
Ascomycota Leotiomycetes
(Discomycetes)

Plant pathogens Dothideomycetes

Mammal pathogens
Lichens (Loculoascomycetes)
Mycorrhizal fungi Leptosphaeria, Septoria

Plectomycetes
Aspergillus

18S ribosomal
subunit
Neurospora
Pyrenomycetes
Fusarium Magnaporthe, Gaeumannomyces

Colletotrichum

Pezizomycetes
Mycorrhizal fungi etc

Candida Hemiascomycetes
Archaeascomycetes
Taphrinomycetes 14
Ustilago Basidiomycota
Classification of the fungi

(zoosporic fungi)

(Zygomicetes)

(Arbuscular mycorrhiza )

Current Biology 2009 Vol 19 No 18

15
MycoCosm
1000 FUNGAL GENOME

https://genome.jgi.doe.gov/programs/fungi/index.jsf

16
 Characteristics of fungi -1
• A highly versatile group of eukaryotic organisms
• non-vascular organisms
• reproduce by means of spores:
– Asexual Reproduction (conidiophores - conidia)

– Sexual Reproduction (fruiting bodies - sexual spores)

the asexual state is referred to as the anamorph

the sexual state of a fungus is termed teleomorph
• Both sexual (meiosis) and asexual (mitotic) spores may be produced,
depending species and conditions
– Spread over a wide area
– Spores can remain dormant until favorable conditions
17
 Characteristics of fungi - 2
• Vegetative body may be unicellular (yeasts)

or constructed of microscopic cylindrical filaments (multicellular) called

hyphae

• Network of hyphae = mycelium

• Cell walls: similar in structure to plants’ but different chemical composition.

Fungi cell walls contain mostly chitin, plant cell walls contain mostly
cellullose (plus lignin and secondary walls)

• Typically not motile, although a few (e.g. Chytrids) have a motile phase

18
 Characteristics of fungi - 3

• Fungal cell walls contain a unique sterol, ergosterol, which

replaces cholesterol found in mammalian cell membranes

• Lysine biosynthesis pathway in fungi is different

• Different type of tubule protein (present in microtubules formed

during nuclear division)

• Chitin biosnthesis occurs in fungi

19
 Characteristics of fungi - 4
• Heterotrophic, must feed on preformed organic material (in
comparison to autotrophic green plants)

• But unlike animals, fungi need to produce digestive exoenzymes

(secreted to the environment) to “convert” the food into
small molecules that can be absorbed by the fungus.

• Nutritional modes of life: Saprophytic

mycorrhizae
Parasitic
Mutualistic (symbionts - mycorrhizae)

20
 Characteristics of fungi - 5

Saprophytic fungi
- Use of non-living organic material

- Along with bacteria, fungi are important in recycling Carbon, Nitrogen, and
essential mineral nutrients

- Example: Neurospora crassa

Parasitic fungi

- Use of organic material from living organisms (harming them)

- Wide range of hosts: can also parasitize other fungi, algae, animals, insects…
- Different types of parasitism: Biotrophs-necrotrophs-hemibiotrophs

21
OOMYCETES
◦ Rudimentary mycelium (aseptate) Phytophtora, Pythium,
◦ Sexual reproduction by fusion of gametes Peronospora, Bremia

ASCOMYCETES
◦ Mycelium septate
◦ Sexual reproduction  ascospores in asci

BASIDIOMYCETES
◦ Mycelium septate
◦ Sexual reproduction  basidiospores carried by basidia

ZYGOMYCETES
◦ No Mycelium septate
◦ Sexual reproduction  Zygospores
◦ Zygomycetes synthesize chitosan (not chitin)

22
The Oomycetes are not fungi – they are
taxonomically grouped within algae

Fungi and
oomycetes both
grow as filamentous
hyphae but their
lineages diverged
before the split of
fungi from plants
and animals.

23
The Oomycetes are not true fungi
Animals

Fungi

Alveolates (dinoflagellates, ciliates, apicomplexa)

Stramenopiles (Oomycetes, diatoms, brown algae)
Rhodophyta (red algae)
Green plants (green algae, higher plants)
Other protists (cryptomonads, euglenids, glaucophytes)

24
 Distinguishing features of Oomycetes

Dispersed via zoospores (flagellate asexual spores)

Produce oospores (sexual spores)

Cellulose cell walls (versus chitin in true fungi)

Diploid (2N)

Unable to synthesise sterols from squalene

Use of secondary metabolites to regulate sexual compatibility and differentiation

25
Most relevant plant pathogenic oomycetes (Fawke et al, 2015)

Great Hunger was a period of mass starvation, disease,

and emigration in Ireland between 1845 and 1852

26
 The life cycle of the potato blight pathogen
Phytophthora infestans (oomycete)

https://www.youtube.com/watch?v=l9ikfDWZaT8

27
The life cycle of the potato blight pathogen
Phytophthora infestans (oomycete)

28
OOMYCETES
◦ Rudimentary mycelium (aseptate)
◦ Sexual reproduction by fusion of gametes

ASCOMYCETES Magnaporthe, Blumeria

◦ Mycelium septate (mildew), Septoria,
◦ Sexual reproduction  ascospores in asci Botrytis, Rhynchosporium,
Cercospora, Colletotrichum
Fusarium, Verticillium
BASIDIOMYCETES
◦ Mycelium septate
◦ Sexual reproduction  basidiospores carried by basidia

ZYGOMYCETES
◦ No Mycelium septate
◦ Sexual reproduction  Zygospores
◦ Zygomycetes synthesize chitosan (not chitin)

29
Around 75% of all described fungi are
Ascomycetes

30
 Around 75% of all described fungi are Ascomycetes

Nuclear Cell fusion

fusion
N+N Gametes
2N
Meiosis N

Hypha or yeast Conidia

Ascospore

Germination Germination

Perithecia Ascospores N
in ascus sacs

SEXUAL ASEXUAL

In yeast a single cell

becomes an ascus,
often with just four KEY DIAGNOSTIC FEATURE = THE ASCUS SAC
spores

31
32
33
 Blumeria, Botrytis (Mary Berbee, 2001)
Ascomycota Leotiomycetes
(Discomycetes)

Plant pathogens Dothideomycetes

Mammal pathogens
Lichens (Loculoascomycetes)
Mycorrhizal fungi Leptosphaeria, Septoria

Plectomycetes
Aspergillus

18S ribosomal
subunit
Neurospora
50 to 150 Mya
Pyrenomycetes
Fusarium Magnaporthe, Gaeumannomyces

Colletotrichum

Pezizomycetes
Mycorrhizal fungi etc

Candida Hemiascomycetes
Archaeascomycetes
Taphrinomycetes 34
Ustilago Basidiomycota
OOMYCETES
◦ Rudimentary mycelium (aseptate)
◦ Sexual reproduction by fusion of gametes

ASCOMYCETES
◦ Mycelium septate
◦ Sexual reproduction  ascospores in asci

BASIDIOMYCETES
◦ Mycelium septate
Ustilago, Puccinia (rust),
◦ Sexual reproduction  basidiospores carried by basidia
Uromyces

ZYGOMYCETES
◦ No Mycelium septate
◦ Sexual reproduction  Zygospores
◦ Zygomycetes synthesize chitosan (not chitin)

35
Basidiomycetes

The spore-bearing structure is the basidium

◦ Bears haploid basidiospores (usually four)

Mycelium is primarily dikaryotic

◦ The two nuclei brought together in mating do not fuse in
the vegetative hyphae (only in the basidium)

Hyphae usually have clamp connections

◦ This allows the two types of haploid nuclei to be
maintained in pairs in each cell

36
Generalised life cycle of Basidiomycetes
DIPLOID

2N
Nuclear Meiosis
fusion
Mitosis N+N N Mitosis

Cell fusion

ASEXUAL SEXUAL ASEXUAL

DIKARYOTIC HAPLOID

N+N 2N N
37
Generalised life cycle of Basidiomycetes

38
Formation of clamp connections for maintaining
a dikaryotic state

•One nucleus divides in the main hypha while the other divides into the clamp

•Septa are formed across the mitotic spindles

•The apex of the clamp cell fuses with the subapical cell, reestablishing the
dikaryotic state 39
OOMYCETES
◦ Rudimentary mycelium (aseptate)
◦ Sexual reproduction by fusion of gametes

ASCOMYCETES
◦ Mycelium septate
◦ Sexual reproduction  ascospores in asci

BASIDIOMYCETES
◦ Mycelium septate
◦ Sexual reproduction  basidiospores carried by basidia

ZYGOMYCETES
◦ No Mycelium septate-multinucleade Mucor, Rhyzopus,
◦ Sexual reproduction  Zygospores Blakeslea, etc
◦ Zygomycetes synthesize chitosan (not chitin)

40
41
Mucor circenelloides
Rhyzopus oryzae
Phycomyces blakesleeanus

42
 Phycomyces blakesleeanus
(Zigomicetos)
Max Delbrück (1906-1981)

”I am still convinced that Phycomyces is the most

intelligent primitive eukaryote and as such capable of
giving access to the problems that will be central in the
biology of the next decades.”

43
Fotorrespuestas del esporangióforo.
Fototropismo

M. Delbrück, HK Galle, HH Heunert. IWF 1975

44
Pilobolus crystallinus

20000 g

45
Different kinds of
plant pathogens

M. Möller, E. H. Stukenbrock, Nat. Rev. Microbiol. (2017),

doi:10.1038/nrmicro.2017.76.

M. Möller, E. H. Stukenbrock, Nat. Rev. Microbiol. (2017), doi:10.1038/nrmicro.2017.76. 46

 Different kinds of plant pathogens

Biotroph
◦ Entirely dependent on the plant as a source of nutrients
(i.e. does not grow in culture - OBLIGATE)
Necrotroph
◦ Kills the plant tissue as it grows within the host
(i.e. colonises dead tissue - FACULTATIVE)
Hemibiotroph
◦ Has both biotrophic and necrotrophic growth phases

47
Different kinds of plant pathogens
Biotroph
◦ e.g. Rusts and mildews; very little damage to the host
apart from dense sporulation (“Royas y Mildius”)
Necrotroph
◦ e.g. Botrytis cinerea and Pythium spp– Rots plant tissue

48
Examples of biotrophs
Stripe rust disease of wheat
(Puccinia striiformis)
(basidiomycete)

Roya amarilla de cereales

49
 Examples of biotrophs

Asparagus rust (Puccinia asparagi)

Roya del espárrago (basidiomycete)

50
Examples of biotrophs

Barley powdery mildew

(Blumeria graminis, previously
known as Erysiphe graminis)
(ascomycete)

Mildiu de la cebada

51
Examples of biotrophs
Rye Ergot fungus
(Claviceps purpurea)

52
Examples of biotrophs

Ergot fungus
(Claviceps purpurea)

(ascomycete)

Cornezuelo del centeno

53
 Examples of biotrophs

Corn smut disease on maize

(Ustilago maydis)
(basidiomycete)
54
Haustoria are specialised feeding
structures formed by biotrophic fungi

55
Examples of biotrophs
(Mutualism)

Arbuscular mycorrhizal fungus

(Glomus intraradices)
(Glomeromycetes)

56
Different kinds of plant pathogens

Biotroph
◦ e.g. Rusts and mildews; very little damage to the host
apart from dense sporulation
Necrotroph
◦ e.g. Botrytis cinerea and Pythium spp– Rots plant tissue
Hemibiotroph
◦ e.g. The tomato leaf spot fungus Septoria lycopersici;
the potato blight fungus, Phytophthora infestans; leaf
spot of sugarbeet, Cercospora beticola; Downy mildew
of lettuce, Bremia lactucae

57
The grey mould fungus, Botrytis cinerea
Necrotroph

•Macerates plant tissue

•Broad host range
•Ascomycete

Moho gris

58
Examples of necrotrophs – Pythium spp.
(oomycete)

blight in
turgrasses
Quemazón del césped

Potato pink rot

Stem rot in Raiz Rosada
Nicotiana
tabacum

59
Examples of necrotrophs – Rhizopus spp.
(Zygomycete)

Rhizopus microsporus
Quemazón de la semilla de arroz

60
Different kinds of plant pathogens

Biotroph
◦ e.g. Rusts and mildews; very little damage to the host
apart from dense sporulation
Necrotroph
◦ e.g. Botrytis cinerea and Pythium spp– Rots plant tissue
Hemibiotroph
◦ e.g. The tomato leaf spot fungus Septoria lycopersici;
the potato blight fungus, Phytophthora infestans; leaf
spot of sugarbeet, Cercospora beticola; Downy mildew
of lettuce, Bremia lactucae

61
 Examples of hemibiotrophs

The tomato pathogen Septoria lycopersici

(ascomycete)
Septoriosis o viruela del tomate

62
Examples of hemibiotrophs

Leaf spot of sugarbeet

(Cercospora beticola)
(ascomycete)

Cercospora de remolacha

63
Examples of hemibiotrophs

The potato blight pathogen,

Phytophthora infestans (oomycete)
Mildiu de la patata
64
 Examples of hemibiotrophs

Downy mildew of lettuce, Bremia lactucae

(oomycete)
Mildiu de la lechuga

65
 Fungal pathogens employ different
infection strategies
Necrotroph Biotroph Hemibiotroph

Means of attack Secreted cell wall-degrading Intimate intracellular Initial biotrophic phase
enzymes and/or toxins. contact with plant cells. then necrotrophic.

Specific features Plant tissue killed and then Plant cells remain alive Plant cells alive only in
colonised by the pathogen. throughout infection. the initial stages.
Extensive tissue damage. Minimal plant damage. Extensive damage at late
stages.

Host range Broad Narrow; often only a Intermediate

host species

Examples Rotting fungi (e.g. Mildews and rusts Tomato leaf spot (Septoria
Botrytis cinerea). lycopersici); potato late
blight (Phytophthora
infestans).

66
 Fungal pathogens employ different
infection strategies
Necrotroph Biotroph Hemibiotroph

Means of attack Secreted cell wall-degrading Intimate intracellular Initial biotrophic phase
enzymes and/or toxins. contact with plant cells. then necrotrophic.

Specific features Plant tissue killed and then Plant cells remain alive Plant cells alive only in
colonised by the pathogen. throughout infection. the initial stages.
Extensive tissue damage. Minimal plant damage. Extensive damage at late
stages.

Host range Broad Narrow; often only a Intermediate

host species

Examples Rotting fungi (e.g. Mildews and rusts Tomato leaf spot (Septoria
Botrytis cinerea). lycopersici); potato late
blight (Phytophthora
infestans).

67
 Fungal pathogens employ different
infection strategies
Necrotroph Biotroph Hemibiotroph

Means of attack Secreted cell wall-degrading Intimate intracellular Initial biotrophic phase
enzymes and/or toxins. contact with plant cells. then necrotrophic.

Specific features Plant tissue killed and then Plant cells remain alive Plant cells alive only in
colonised by the pathogen. throughout infection. the initial stages.
Extensive tissue damage. Minimal plant damage. Extensive damage at late
stages.

Host range Broad Narrow; often only a Intermediate

host species

Examples Rotting fungi (e.g. Mildews and rusts Tomato leaf spot (Septoria
Botrytis cinerea). lycopersici); potato late
blight (Phytophthora
infestans).

68
 Fungal pathogens employ different
infection strategies
Necrotroph Biotroph Hemibiotroph

Means of attack Secreted cell wall-degrading Intimate intracellular Initial biotrophic phase
enzymes and/or toxins. contact with plant cells. then necrotrophic.

Specific features Plant tissue killed and then Plant cells remain alive Plant cells alive only in
colonised by the pathogen. throughout infection. the initial stages.
Extensive tissue damage. Minimal plant damage. Extensive damage at late
stages.

Host range Broad Narrow; often only a Intermediate

host species

Examples Rotting fungi (e.g. Mildews and rusts Tomato leaf spot (Septoria
Botrytis cinerea). lycopersici); potato late
blight (Phytophthora
infestans).

69
Lo Presti L, Lanver D, Schweizer G, Tanaka S, Liang L, et al. (2015) Fungal effectors and plant susceptibility. Annu Rev Plant Biol 66: 513-545.

70
Disease symptoms caused by phytopathogenic fungi with different lifestyles:

(a) Botrytis cinerea – tomato

(b) Sclerotinia sclerotiorum –

rapeseed

(c) Uromyces viciae-fabae – bean

(d) Blumeria graminis – barley

(e) Ustilago maydis – maize

( f ) Cladosporium fulvum - tomato

( g) Colletotrichum higginsianum -
mustard spinach

(h) Magnaporthe oryzae - rice

Lo Presti L, Lanver D, Schweizer G, Tanaka S, Liang L, et al. (2015) Fungal

71
effectors and plant susceptibility. Annu Rev Plant Biol 66: 513-545.
 MECANISMOS DE VIRULENCIA EN HONGOS Y
OOMICETOS

Día 1 (Jueves 5 17-19): Introducción a los organismos

filamentosos y su interacción con las plantas. Diferentes
estrategias del hongo para la patogénesis de plantas.
Día 2 (Martes 10 17-19): Métodos moleculares y aproximaciones
“ómicas” usados para el estudio de organismos
filamentosos fitopatógenos.
Día 3 (Miércoles 11 15-17): Procesos biológicos reguladores de la
actividad patogénica: metabolismo - autofagia,
citoesqueleto - NADPH oxidasas, vías de señalización,
miRNAs. Bases moleculares de la patogénesis.
Día 4 (Martes 17 17-19): Determinantes de patogenicidad y
espectro de huésped: efectores y toxinas.
Día 5 (Miércoles 18 15-17): Seminarios.

72