L14 - Signalling in root symbioses Flashcards
Describe how forwards and reverse genetics approaches work (these are used to identify signalling genes)
Forward genetics:
- Mutated plants grown
- Lines with desired altered phenotype cloned
- Genes causing mutation identified
Reverse genetics
- Candidate genes for mutation first identified e.g. via transcriptomics
- Target genes mutated and mutants grown to assess function
What are some of the requirements for forward and reverse genetics approaches and the difficulties?
Forward genetics:
- Efficient mutagenesis programme needed
- Unambiguous throughput screens needed
Reverse genetics:
- Knowledge of candidate genes needed
Requirements for both:
- Genetically tractable organism, possible for ectomycorrhizal fungi + bacteria, hard for AM
- Short lifecycles - hard for ectomycorrhizal fungi (trees + shrubs), easier for AM (grasses, legumes)
Give an example of a protein used in mycorrhizal fungi to establish symbiosis that was identified using transcriptomics and reverse genetics
Give the wider significance of this discovery
- Mycorrhiza Induced Small Secreted Protein 7 (MiSSP7)
- From ectomycorrhizal fungus Laccaria bicolor
- Interferes w/ Jasmonic Acid (JA) signalling, preventing induction of plant defence genes
- Missp7 mutants unable to infect host
- Beneficial fungi use identical strategies as pathogens
How has phylogenomics combined with reverse genetics been used to identify AM-symbiosis associated genes?
- NGS = more plant genomes available for analysis
- Common genes between 39 host and 11 non-host plant species identified 138 genes associated w/ AM trait
- Reverse genetics confirmed these genes were needed for establishment of symbiosis
Is there a link between the signalling pathways used for nodulation and AM symbiosis?
What observations lead to this
- Yes, common genetic programmes underpin establishment of both symbioses
- Nodulation mutants were re-screened for AM phenotypes
- Subset of mutants also impaired in AM interaction
- Fungi developed hyphopodia + enter epidermal cell but further cortex invasion compromised
Evolutionarily, what is thought to be the reason for the common genetic programmes between RNS and AMS?
- AMS evolved 450 Mya, well before RNS (60- 100Mya)
- RNS may have “hijacked” genetic programmes from AMS
What is the largest difference between RNS and AMS?
What are some similarities seen between RNS and AMS in signalling?
- Development of new organ, the nodule, in RNS, not seen in AMS
- Both release lipo-chitooligosaccharides (LCOs) into rhizosphere that triggers Ca spiking in root cells
- Signals perceived by LysM domain containing RLKs e.g Nod-Factor Receptor NFR1 and NFR5
Draw a sketch of the common signalling pathway in RNS and AMS in legumes and describe the function of each of the 7 sections (if known)
(See diagram on pg 3)
1) NFR1/5 previously described
2) Leucin Rich Repeat (LRR) SYMRK:
- Receptor-like kinase localised to PM
- Extracellular signals perceived not known
3) CASTOR and POLLUX:
- High sequence similarity to each other
- Encode K permeable cation channels in nuclear envelope
- Possibly counter-ion channels, compensating for rapid charge imbalance from Ca2+ spiking
4) Calcium spiking
5) CCamK:
- Calcium-calmodulin dependent protein kinase
- Thought to decipher calcium signatures
6) CYCLOPS:
- TF that’s phosphorylated by CCaMK
7) NSP2:
- GRAS TF strongly required for RN in legumes + in monocotyledons for AM
Beyond the shared signalling pathway for RNS and AMS a fork occurs where each type of symbiosis then relies on specific signalling components.
Add this fork to the previous diagram and describe the function of the specific signalling components on each branch
RNS:
1) TFs NIN and RPG
- Required for IT formation
AMS:
1) Complex of GRAS TFs
- Needed for arbuscle formation
- E.g. RAM1 needed for induction of biosynthesis of fatty acids to feed fungus
- Unanswered how specificity downstream of common signalling path causes either RNS or AMS
How is knowledge of this shared signalling pathway between RNS and AMS being used in crops?
- Enabling N2 fixing symbiosis into cereals could reduce costly N fertiliser use
- RNS could be engineered using knowledge of the pre-existing signalling pathway in cereals for AMS
- E.g. ENSA attempting this for small holder farms in Sub-Saharan Africa
Give an example illustrating the importance of AMS in crops
How does nutrient availability impact ability for AMS and why is this important?
- Inability to engage w/ AM fungi in medium-input field conditions reduced maize yield by 30%
- Nutrient availability inversely correlated w/ ability of plants to engage w/ AM fungi
- E.g. higher phosphate fertilisation = lower root colonisation
- Enabling continuous colonisation therefore necessary to optimise mycorrhizal benefit
Describe a recent discovery of a pathway that controls the ability of a plant to engage in AMS
- Rice dwarf 14-like (d14l) mutant showed complete loss of susceptibility to AM fungi:
- no fungal hyphopodia
- no transcriptional response to fungal spore exudates
- D14L encodes alpha/beta hydrolase previously known for perception of karrikin
- Perception of karrikin recruits F-box protein involved in ubiquitination of negative regulator SMAX1
- Leads to degradation of SMAX1 + de-repression of programmes for symbiosis + strigolactone biosynthesis
- Links attraction of fungus w/ enabling AMS
- Hypothesis: Under nutrient limiting conditions, endogenous ligand activates D14L signalling pathway
- Enables AMS
- Enables continuous colonisation of AMS, very useful!
