Beneficial interaction betweenplants and microbes

• Symbiosis:

• Beneficial interactions between plants and other organisms (fungi or bacteria)

• plant contribution: sucrose• contribution of plant partners:

– air compounds (N2)– soil components (water, minerals)

- Nitrogen-fixing bacteria- Mycorrhizal fungi

both are intracellular symbiosis, but not in plant cytoplasm

• mycorrhiza: 450 mio years old• appeared in first land plants• N2-fixing bacteria: much younger

• both use the same/similar recognition and signaling pathways

• common mechanism: recognition

Mycorrhiza

• VAM (vesicular-arbuscular) mycorrhiza• 80% of all plant families• VAM = endomycorrhiza

• many trees

• photoassimilates from plants• vs. • better assess to soil nutrients from fungi

Effect on growth

Better nutrient (P) uptake

Other effects:

resistance against abiotic stress

- drought, salt stress, heavy metals, toxins, cold, heat, nutrientstress, etc.

-resistance against biotic stress

- pathogens (virus, bacterial, fungi)

- nematodes

- insects

- parasites

- promotion of biomass

- promotion of seed yield

- promotion of fitness

Mycorrhiza confers droughttolerance

Prunus + fungus

Drought tolerance

Arabidopsis抗旱及抗寒

Heavy metal tolerance -phytoremediation

- fungus

Micorrhizal fungi can be up to 50% of root biomass

cellexternal

networkwithhyphae

and spores

Clover root naturally infectedby an arbuscular mycorrhizal fungus.

external network of fungal hyphae, bearing several large (up to 1 mm) spores of the fungus.

Fungal hyphae between the root cortical cells

Hyphae produce swollen vesicles in the root tissues, and tree-like branching structures (arbuscules, blue fuzzy areas)

within the root cells.

External hyphae are much biggerthan arbuscular structures

Arbuscules within root cortical cells.

Arbuscules within root cortical cells.

Small hyphae can penetrate thesoil much better than bigger lateral

roots

only 6 fungal species form VAM

- they all belong to Glomales (Zygomycetes)

- Initiation of interaction through germinating spores on plant plasma membrane

- Hyphae form appressorium (attachment sites)

- Formation of an extracellular hyphal system in the apoplast

- Formation of haustorium: penetration into the plant cell(intracellular arbuscules)

- Enlargement of interaction surface

- life time of arbuscle: a few days

appressorium

haustorium

Difficult molecular field

• Fungus: activates hexose import system• Plants: activate phosphate transporters

• Extracellular hyphae: collect nutrientsand transfer them to the fungus

• Crop plants: up to 4-fold higher yieldwith mycorrhizal fungi

Difficult molecular field• fungus grows only with host

• Fungal signal (?)– flavonoids– phenolic compounds– oligosaccharides of cell wall – peptides (modified)

• Signaling: receptor kinases, calcium

• Recognition and signaling in plants share componentswith rhizobacteria

Model plants: Lotus & Medicago

Lotus

Many mutants are impaired in mycorrhiza formation and nodulation

At least seven proteins (receptor kinases, signaling components and plastidproteins) are required for bothmycorrhiza formation and nodulation in Legumes.

Fungal and bacterial entry into plant celloccurs via the same mechanism

Ca2+ oscillations differ in response to fungi and rhizobacteria

Ca2+ oscillations in Medicago –visualized with a dye

Transgenic cameleon system issimilar to FRET

Transgenic aequorin system measuresluminescence

Pastor and Pollux is located in plastids

CCaMK is a nuclear Ca sensor required for bothmycorrhiza formation and nodulation

Ca could be released from internal stores – e.g. nuclear envelope

Many orchids require mycorrhizalfungi for seed germination

Extreme form of endomycorrhiza: primitive, non-photosynthetic orchids:

fungus delivers C to the plant

• fungus: Tulasnella (also parasites or saprophystes)• utilizes complex C-sources: cellulose• transfer of C to non-photosynthetic Orchids• fungi forms intracellular hyphae, „Knäuls“• Plant cell digests fungal „Knäuls“ through plant-specific

exoenzymes (leftover: chitin)

• „ancient“ orchids: C requirement• „modern photosynthetic orchids“: C requirement replaced by P

requirement.

• Symbiosis is mutualistic, but metastabile: • => shift to parasitism (dominance of the fungus)• => digestion of fungal hyphae (dominance of the plant)

symbiosis of chlorophyll-free orchids

1st example: The chlorophyll-free orchid Neottianidus-avis (Nestwurz) digests hyphae which

penetrates into the vascular system.

The orchid lives exclusively from fungal compounds.=> from symbiosis to parasitism

outer layers inner layersundigested >>>> digested hyphae

2nd example: Extreme endomycorrhiza: fungal alcaloids

protect the plantLolium/Festuca vs. Epichloe/Neotyphodium:

- fungus produces alcaloids- plant is protected against herbivores

> Interaction not primarily due to nutrient exchanges

Extreme: broad band protection of plants againstherbivorous insects or animals

Close dependency of both partnersVegetative propragation of fungus via plant seeds

Ectomycorrhiza

Ecotomycorrhizal fungi form fruitbodies

Ectomycorrhiza• Almost all tress form ectomycorrhizas• Fungus does not enter plant cell• Fungus forms a net around the root

(hairs) to extent their access to soilnutrients

• Fungus colonizes the outer cell layersand forms a Hartig Net.

• (formation of a fungal mantle on top of the root)

• Optimization of nutrient exchanges• Hartig Net protects against pathogenic

fungi and soil bacteria.• Soil network that connects several

organisms.

• Fungus builds fruit bodies in the fall.

Ectomycorrhiza promotes growth of tree seedlings and germination of

seeds

Ectomycorrhiza promotes nutrientuptake of trees

Hartig Net

Ectomyccorhiza

Ectomycorrhiza

• Ectomycorrhizal nets in forests

older tries help younger tries

no species-specificity

crosstalk between different fungiand different trees

Beneficial fungi

• Activate defense genes like in pathogenic interactions

• - pathogen-related proteins, defensins• - H202 production

• Low activation• During initial phase• Decline during later phases

Mycorrhizal fungi activate thedefense-inducing MAPK4 pathway

Fungus may produce ROS throughthe NADH oxidase

Mutualism - Parasitism

• Unstable symbiosis• Change during the interaction• Depends on colonization• Depends on defense gene activation

Rhizobia interacting withLegumes – a second type of

beneficial interaction

Nitrogen assimilation: uptake of nitrate or ammonium from the soil

Rhizobia interacting withroots of Legumes

N2 fixation

• Haber-Bosch technic: N-fertilizer• nitrogen fixation by rhizobacteria and

cyanobacteria

N2 + 8H+ + 8e- + 16 ATP = 2NH3 + H2 + 16ADP + 16 Pi

Rhizobia

-nodules of Legumes fix nitrogen

- Industry: transfer of bacterial genes into plantsto uncouple nitrogen fixation

from the bacterium-no success because of the complex interaction

between the two partners

Nodules

Nodules

Nodules

Rhizobia strongly promotes growth under N limitations (alfalfa)

Lotus plants without/with rhizobia

Soybean with and without N fixation

Recognition of the two partners: formation of nodules

- Nod-factors induce nodule formation- best characteized factor: chitooligosaccharide- initiates meristematic activity (10-9 M)- Plant genes:- determine type of rhizobia and form on nodules

- Symbiosis as co-evolution

- Exclusion of oxygen

- Glutamine synthase removes toxic ammonium

- Nif genes from bacteria: nitrogenase

- nitrogenase is oxygen-sensitive: no nitrogen fixationof the free living bacteria.

Description of the interaction

- N-fixing bacteria are of polyphyletic origin

- Interaction is highly specific- Penetration occurs through root hairs

- Infection tube grows into the cells- bacteria cause a reduction in cell wall synthesis

- comparable to phagocytosis

- Bacteria from bacteroids- Cell division of bacteroids

Rhizobia

- Bacteria contain plasmids with nod genes

plants synthesizeflavones, flavonoids, flavanones, isoflavones,

chalcones

Induction of nod genes

nodD gene product: transcription factoractivates other nod genes

nod-box: 47 base pairs2 classes of nod genes (general/host-specific)

Examples for NOD factors

Red: bacteria in theroot cells

Nod factor recognition

NFR1/5 are receptor-like kinases

Downstream event: increase in intracellular Ca2+ elevation

- many Ca2+ channels in Arabidopsis

- Uptake from extracellular space- Release from internal stores

K+ uptake is downstream of Ca2+

uptake – requires AKT1

Signaling from Ca2+ to AKT1 isshort

Ca2+ – CBL1/9 – CIPK23 – AKT1

Phosphorylation cascade

Ca2+ may link nodule formation to K+ stress

Biochemistry in the nodule

- Expression of rhizobia-specific genes- bacterial genes: nod-genes- plant genes: nodulin genes

- leghemoglobin (protection against oxygen)- nitrogenase (N-fixation)

- glutamine synthase (N-detoxification)- uricase (N-detoxification)

Leghemoglobin accumulates in symbiosome membrane

The interiors of legume nodules are normally pink due to the presence of leghemoglobin (similar to the oxygen-carrying hemoglobin that causes theblood to be red).

Leghemoglobin has a high affinity for oxygen, and it locks up oxygen, thusfostering the oxygen-free conditions needed for nitrogen fixation.

Leghemoglobin is related to haemoglobin (chain A and B)

nitrogenase

The two components can beseparated on sucrose gradients

The nitrogenase contains the MoFe protein (in blue and purpleat the center) and two copies of the Fe protein dimer bound on

either end (shown in green). The iron-sulfur cluster, the P-cluster, and the FeMo-cluster are arranged in a row. The ATP

binding site is revealed by an ADP molecule.

GS is located around the centralveins and in the nodules –detoxification of ammonium

Detoxification of ammonium

Nodule formation is controlled byshoot-derived factors

Pathogenesis• Plants vs. Bacteria, fungi, parasites

• Wounding

• Compatible/incompatible interactions:– Plant susceptable, pathogen aggressive– leads to (cell) death

– incompatible interaction:– Plant is resistant against pathogen

– Gene-for-gene concept

Constitutive defense

• Protection through cuticula• Fungus produces penetration hyphae

• Hyphae secretes hydrolases• (cutinases, cellulases, pektinases)

•• Penetration hyphae grows into stomata• Haustorium penetrates into the cell and gains excess to the cytoplasm• Destruction of the plant plasma membrane occurs

at the end of the penetration process

Infection of other cells, propagation through spores

Plant response during constitutivedefense

• Available compounds block hyphal growth• (alcaloids, terpenes, cyanogene

glycosides, fungi-toxic cell wall components)

Induced defense• Synthesis of new cell wall material around the penetration hyphae• Callose synthesis: resistant against fungal hydrolases

• Hypersensitive reaction: necrosis at infection place, i.e. induced celldeath

• Synthesis of anti-hyphal compounds in surrounding cells• Induced through fungal elicitors which activate a plant defense

signaling pathway• (two-component system, receptor kinases, MAP kinase pathway)• Evolutionary relationship between defense and symbiosis• Plant defense: production of polyphenol compounds• Primary signal in plants: salicylic acid

• Arabidopsis mutants with lesions in salicylic acid metabolism: already sensitive to changes in environment.

Oxidative burst, defense and alarmsignals in plants and animals

• Pathogen response in blood:• Leucocyte activate a NAD-oxidase

complex in outer membrane, whichtransfer electrons from NADH (inner) via a flavoprotein and cytochrome b to 0xygen (outer)

• O2. and H2O2

. are toxic for bacteria and induce fever

NADH oxidase reaction in leucocytes

Oxidative burst in plants

• Tissue culture:• Oxidative burst after 3 min, generation of H2O2• (specific for compatible and incompatible reactions) • Function of H2O2: block of pathogen, cross-linkage of phenolic compounds

at invation place

• After 3 h, continuous production of H2O2• (specific for incompatible reactions)• H2O2 functions as signaling compound (i.e. synthesis of phytochelatine,

induction of hypersensitive response)

• H2O2 production in plant cells requires G proteins, protein kinases and Ca

Phytotoxin production of pathogen

• More than 120 phytotoxins• Highly effective in killing plant cells

– Weakening the defense response– Activate efflux carrer (pathogen gets efflux

compounds)

– Fusicoccin:– Receptor activation, H and K export, cell wall

loosening

Plant antibiotics: phytochelatine

Plant antibiotics: phytochelatine

• More than 200 compounds• Secondary metabolites• Block microbial growth unspecifically• Important: hypersensitive response• Induced by fungal elicitors (or stress)

• PR proteins, partially secreted into the cell wall, contain chitinases, glucanases, hydrolases, ethylen as second messanger.