Symbiosis: ⢠Beneficial interactions between plants and other organisms (fungi or bacteria). ⢠plant contribution: sucrose. ⢠contribution of plant partners:.
Beneficial interaction between plants 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, nutrient stress, etc. -resistance against biotic stress - pathogens (virus, bacterial, fungi) - nematodes - insects - parasites - promotion of biomass - promotion of seed yield - promotion of fitness
Mycorrhiza confers drought tolerance
Prunus + fungus
Drought tolerance
抗旱及抗寒
Arabidopsis
Heavy metal tolerance phytoremediation
- fungus
Micorrhizal fungi can be up to 50% of root biomass
cell external network with hyphae and spores Clover root naturally infected by 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 bigger than arbuscular structures
Arbuscules within root cortical cells.
Arbuscules within root cortical cells.
Small hyphae can penetrate the soil 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 nutrients and transfer them to the fungus • Crop plants: up to 4-fold higher yield with 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 components with rhizobacteria
Model plants: Lotus &
Medicago
Lotus
Many mutants are impaired in mycorrhiza formation and nodulation
At least seven proteins (receptor kinases, signaling components and plastid proteins) are required for both mycorrhiza formation and nodulation in Legumes. Fungal and bacterial entry into plant cell occurs via the same mechanism
Ca2+ oscillations differ in response to fungi and rhizobacteria
Ca2+ oscillations in Medicago – visualized with a dye
Transgenic cameleon system is similar to FRET
Transgenic aequorin system measures luminescence
Pastor and Pollux is located in plastids
CCaMK is a nuclear Ca sensor required for both mycorrhiza formation and nodulation Ca could be released from internal stores – e.g. nuclear envelope
Many orchids require mycorrhizal fungi 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 Neottia nidus-avis (Nestwurz) digests hyphae which penetrates into the vascular system. The orchid lives exclusively from fungal compounds. => from symbiosis to parasitism
undigested >>>> digested hyphae outer layers inner layers
2nd example: Extreme endomycorrhiza: fungal alcaloids protect the plant Lolium/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 against herbivorous insects or animals Close dependency of both partners Vegetative propragation of fungus via plant seeds
Ectomycorrhiza
Ecotomycorrhizal fungi form fruit bodies
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 soil nutrients • Fungus colonizes the outer cell layers and 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 nutrient uptake of trees
Hartig Net
Ectomyccorhiza
Ectomycorrhiza
• Ectomycorrhizal nets in forests older tries help younger tries no species-specificity crosstalk between different fungi and 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 the defense-inducing MAPK4 pathway
Fungus may produce ROS through the NADH oxidase
Mutualism - Parasitism • • • •
Unstable symbiosis Change during the interaction Depends on colonization Depends on defense gene activation
Rhizobia interacting with Legumes – a second type of beneficial interaction
Nitrogen assimilation: uptake of nitrate or ammonium from the soil
Rhizobia interacting with roots 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 plants to 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
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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 fixation of 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 synthesize flavones, flavonoids, flavanones, isoflavones, chalcones Induction of nod genes
nodD gene product: transcription factor activates other nod genes nod-box: 47 base pairs 2 classes of nod genes (general/host-specific)
Examples for NOD factors
Red: bacteria in the root 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 is short
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 the blood to be red). Leghemoglobin has a high affinity for oxygen, and it locks up oxygen, thus fostering the oxygen-free conditions needed for nitrogen fixation.
Leghemoglobin is related to haemoglobin (chain A and B)
nitrogenase
The two components can be separated on sucrose gradients
The nitrogenase contains the MoFe protein (in blue and purple at the center) and two copies of the Fe protein dimer bound on either end (shown in green). The iron-sulfur cluster, the Pcluster, and the FeMo-cluster are arranged in a row. The ATP binding site is revealed by an ADP molecule.
GS is located around the central veins and in the nodules – detoxification of ammonium
Detoxification of ammonium
Nodule formation is controlled by shoot-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 constitutive defense • 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 cell death 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 alarm signals in plants and animals • Pathogen response in blood: • Leucocyte activate a NAD-oxidase complex in outer membrane, which transfer 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.
