Invasion of the pathogens

Lecture 3

Not all plant associated micrboes are pathogenic!

• All parts of the plant can support microbial flora

• But mainly the leaves and roots that are best-studied niches

  • Microbes can occur inside plants without causing disease but this community is not well understood!

Why is studying plant-pathogen interaction important?

• at least 25% of crop production lost to pathogenes

• 1 in 3 people malnourised

• 1 in 9 bed hungry

• population unlikley to stabilise for at least 25 years

• Climate chnage will affect food sercruit too

Example of a virus on crops

Potato late blight

• Phytophthora infestans

• resulted in deaths of millions

What studying plant-pathogen interaction also helps with?

Providing key insights into

• physiology

• development

• molecular biology

of healthy plants

Where do plants harbour complex microbial communities

1. The Rhizosphere and Rhizoplane

2. Phyllosphere and Phylloplane

3. Endophytic: internal plant environment

1. Rhizosphere and Rhizoplane

The root habitat

• Rhizoplane= microbes on the surface of the root

• Rhizopshere= Rizoplane and micrones in immediate soil surrounding root

• Provides rich growth envrionemetn for microbs

• Plants exude >20% of their fixed carbon via the roots

How much photosynthate is given to the microbes IN RHIZOPHERE

20% of photosnythate leached out

(much more than at the roots)

2. Phyllosphere

Leaf niche envrionment

• Phylloplane= leaf surface itself

Phyllosphere compared to Rhizosphere

• much harsher conditions

• fewer resources

  • over which microbes must compete

• e.g only 1% of photosynthase it exuded for microbes (compared to 20% in the roots)

What do resident microbial population have to look out for?

Readily availible food supply and no deleterious effect on the plant so only look out for

1. microbes compete with each other for resources

2. cope with adverse envrionmental conditions

Amensalism

Presence of one microbe may diminish the ability of another to colonise

Amernsalism e.g1

• Ethanol produced by yeasts

• grow on grape skins

• inhibits bacterial growth

• Antibiosis

Amensalism e.g2

• When fungal spores germinate, they leach carbon

• But then reabsorb it

• BUT

  • bacteria and yeasts van compete for it

  • this decreases the fungal growth

Compeition (esp. for Fe) may be fierce

Why competition for Fe fierce?

It is at low concentration in the soils

• this is why e.g bacteria use the siderophores with high Fe affinity

Plant pathogen major trophic strategies (2)

1. Necrotrophs

2. Biotrophs

1. Necrotrophs

• Only use substrate from dead tissue

• Must kill host cells first

  • or use already dead material (saprotrophy)

  • can grow and reproduce away from a plant host

2. Biotrophs

• Get material from living host tissue

• must complete life cycle within living host

  • cannot be saprophytes

  • some cannot grow outside the host at all

  • (e.g viruses)

  • This makes them hard to cultivate on petri dish when find out about disease (see later)

Example of fungal biotrophs: powdery mildew fungus

• developed specialised feeding strucutures within the host

  • haustorium

  • similar to arbuscle:

  • large surface area and host plasma membrane is not breached

Hemi-biotrophy: P.infestans

Initial stage of infection involves extraction of nutrients from host cells through

• simple haustoria (biotrophic)

• then →

• more aggressive necrotrophic stage

What are viruses

• Obligate intracellular parasites

• incapable of replicating outside the host cell

  • biotrophs

Difference of virus compared to other obligate intracellular parasites

• no cells (non-cellular)

• intracellular parasited

• During replication:

  • not separated from the host cellcontents by lipid bilayer membrane

  • Not binary fission

  • instead:

    • assembly line type process

Virion

Virus particle containing:

1. viral nucleic acid

   • RNA or DNA

2. protein coat

   • protective

A fully assembled infectious virus is called a virion

• Virion= the extracellular form of a virus

  • a fully formed infectious particle

• Virus= encompasses its intracellular activties

Virions of most plant-infecting viruses

Shape:

• Icosahedral

  • e.g Cucumber mosaic virus CMV

• Rod shaped

  • e.g TMV

Nucleic acid:

• relatively small RNA or DNA

Compared to bacteriophage

• not as large cirons

Virus structures

• most plant-infecting viruses possess very simple particles

  • virions

    • comprise of coat protein molecules in simple geometric arrangement

    • e.g helical rods, icosahedra

    • surrounfing the viral nucleoid acid

  • Majority 70% of plant viruses have genome of RNA and the rest DNA

How do viruses spread through plants (short distance)

Symplastically

• between cells

• via plasmodesmata

Virus gating

• virus modulate the pore diameter of plasmodesmata

  • why?

  • fascilitate its own movement using a virus-coded factor

    ‘movement protein’

Long distance travel

Via the phloem

• same pattern as photosynthate transport

• from source to sink

• not passive

Mutations affecting virus travel

• virus unable to exploit the phloem

• remain trapped in the inoculation zone

Unlike necrotrophic fungi and bacteria…

• plant viruses rarely kill plants

  • but

• can seriously reduce crop yield and quality

  • how?

• due to the effects of specific viral gene products

• NOT a simple effect of ‘viral load’

Infection and disease are facilitaded by…

• pathogen-encoded Pathogenicity Determinants

Pathogen transmission mainly through

Other organisms

• Insects snd other invertebrates act as vectors

How are plant viruses tranmitted?

• 60% of plant viruses are transmitted by insects

  • e.g aphids and whiteflies

What normally spreads plant bacteria

Beetles

• e.g flea beetle transmises Pantoea stewartii to maize

What carries fungal spores

• e.g Fusarium carried on surface of herbivores

  • insects

really useful coz the aphids do not kill the plant

• good for biotrophic pathogensPlant -plant spread of bacteria

How do bacterial pathogens enter plants

Natural entry sites

• wounds

• stomata

• lenticels

  • raised pores in stem of a woody plant that allows gas exchange

• hydrathodes

  • ‘water stomata

  • release of water droplets

plant-plant spread of bacteria

Aided by rain splash

• bacteria lofted into clouds

• might be transported long distances this way

Flagellate oomycete spores

e.g Phytophthora

1. attracted to opening

   • chemotropically

2. encyst

   • (swell up and lose flagella)

3. then, send germ tubes into the host

Why crucial for pathogens to adapt to natural opening

Race to aquire host nutrients

• before their own run out

• evolved to recognise natural opening

  • e.g Bean rust fungus, Uromyces appendiculates

How bean rust fungus grows into bean plant

1. germ tube makes contact with stoma

2. hyphal apex differentiates into infection strucutre

   1. appressorium (bullbous strucutre)

3. This is positioned over the stomatal pore

4. Infection hypha grows from its lower surface into the sub-stomatal cavity

How does the germ tube recognise the stomata?

• bean guard cells have ridges

  • mean height 0.487 micrometers

• germintate spores on artificial surface

  • with ridges 0.5 micrometers high

  • form appressoria

• If ridges are higher or lower: no appressoria formed

Overall: thigmotropic

• can sense and react to changes in surface contour

How can they recognise precise ridge height?

• stretch-activated (mechnosensitive) cation channel

  • permeable to K+ and Ca2+

How discovered this

1. digested cell wall with enzymes

2. released apical portion of protoplast

3. exposed plasma membrane for electrophysiological analysis:

   • patch clamping revealed stretch mediated cation channel

How the channel works

1. blocked by extracellular Gd3+ at concentration that also inhibit appressorial differentiation

2. apex hits the ridge

3. range of membrane tensions of this hit opens the channel

4. nascent cell wall and membrane deform

5. apical region wall is flexible enough to allow sufficient deformation to affect stretch activated channels

Role of Ca2+ influx

Could be a signal to differentiate

Role of K+ entry

Contribute to the solute potential

• needed for appressorial swelling

Other ways to enter

• enzymes

• hydrostatic force

mainly together

What can do forced entry though plant cell wall

• only filamentous fungi

How do they do this?

Some

• require exoenzymes to break down cell wall

  • and its outer coatings of pectin and cutin

Others

• produced specialised infections structures

  • e.g rice blast fungus

  • Magnaporthe oryzae

Case study: Magnaporthe oryzae: how penetrate rice plant

Mechanical penetration by an appressorium

• punctures the plant surface

How does the fungus adhere to the surface

• germ tube secretes hydrophobins from apex

  • amphipathetic peptides

• reduce water loss

• permit adhesion to hydrophobic surface

Overall process of forcing its way into plant tissue

1. appressorium forms

2. adheres to leaf surface with hydrophobins

3. 3M glycerol accumlates to draw in water

4. generatures 8MPa pressure

   • but melanin keeps appressorium water-tight

5. pressure→ penetration hyphae formed at base of appressorium

What does penetration hyphae do

• force exerted over a period of several hours

  • concentrated to narrow diameter hyphae

    • more pressure for penetration

• Pressure is able to go through solid Mylar plastic

→Punches through

What does the melanin also do

protect against

• UV

• desiccation

Any enzyme involved too?

Cutinase esterase

• encoded by CUT2 gene

• helps to soften cuticle waxes

What are Woronin bodies role in thise?

Must have a crucial role because HEX1 (hex1delta) mutants cannot

• penetrate via appressoria

• survive nitrogen starvation

Pathogenicity determinants

• specific genes that confer pathogenicity

Why would some fungi be pathogenic but some closely related be saptrotroph?

• genes are not needed in vitro or native habitats for saprotroph

• but essential for pathogenesis

How to identify pathogenicity determinants

• knock out gene

  • insert antibiotic resistance gene

  • compare to original strain

e.g Moryzae (bean rust fugi)

• MPG1 gene

  • encodes a secreted 15 kDa hydrophobin

• knocked out:

  • germ tubes cannot attach to leaf

  • no pathogenicity

How do we identify pathogen, if the symptoms are similar?

1. isolate microbe from infection point

   • petri dish

2. inoculate into host of same species

   • but healthy

3. same symptoms appear

   • microbe must be the pathogen

4. re-isolate same microbe from diseased plant

Problems with this for biotrophic pathogens?

• will not be able to survive on the petri dish without living plant there

also??

• may be that the plant doesn’t have many symptoms?

• because plant needs to stay alive for the pathogen to stay alive??