L1: Synthetic Genomes Flashcards
What is epigenetics?
Study of heritable changes in gene expression, by methylation or histone modifications
What is vernalisation?
Where prolonged exposure to cold temperatures induces or enhances the flowering process in plants.
- Achieved by repressing a repressor FLC
- Found to be a quantitative response, with longer periods leading to more rapid flowering
What mechanisms are controlled by epigenetics in plants?
Vernalisation = Flowering Locus C (FLC) represses the FT gene that promotes flowering. H3K27 trimethylation leads to suppression of FLC, resulting in flowering. This is mediated by VRN1/2
Stress response = Dehydration-responsive element-binding (DREB) transcription factors to respond to drought, epigenetically controlled. Also memory of drought
Transposon silencing = important for maintaining genome stability. met1 and ddc mutants show loss of methylation maintenance and activation of TEs
What is euchromatin and heterochromatin?
Euchromatin - open and more active chromosomes
Heterochromatin - closed and inactive regions of the chromosomes
What is required for nucleosome remodelling?
ATP - leading to changes in the position and accessibility of regions of the chromosome
What are the differences between homotypic and heterotypic histones?
Combinations of either the same histone variant type or different.
Homo - commonly present and important in maintaining structural integrity of chromatin, regulating access for transcription, replication and repair
Hetero - commonly present for specialised chromatin function such as activation or silencing
What are some transgenerational effects of epigenetics?
Changes in gene expression that can be inherited without altering the DNA sequence
1. Changes to ripening - Colourless Non-ripening (CNR) gene in tomato. In cnr mutants, hypermethylation leads to suppression of other ripening-related genes. Crosses with WT leads to transient expression changes
2. Flowering times - FWA allele, different flowering times but not genetic difference between the two A.thaliana plants
3. Enhanced stress response to salinity (rice) and drought (Arabidopsis)
- greater response if parents had been exposed to stress, but if mutated in the RdDM pathway then priming and advantage disappears
What is paramutation? and an example
No change in DNA sequence, just a change in silent state from one allele to another.
The active homologue becomes paramutated (silenced)
= Inheritance is non-Mendelian
TAB2 in tomato is responsible for chlorophyll synthesis, however paramutation can lead to hypermethylation and silencing
Describe the example of epialleles with DEF1 in oil pam as an example
Bad karma - hypomethylation of the retrotransposon leads to ineffective silencing and mantled phenotype
Good karma - methylated transposon, promotes proper splicing and proper fruit development
(changes occur stochastically and propagation is via tissue culture)
Describe how CHIP-Seq works
Chromatin Immunoprecipitation high-throughput Sequencing
1. Cross link TF to the DNA
2. Shear the DNA
3. Use antibody to bind the protein
4. Wash off any unbound DNA
5. De-crosslink
6. Purify DNA sequence
What can be found by bisulfite sequencing?
Shows the proportion of methylated cytosines in the sample
Non-methylated cytosines (C) are converted to Ts, following bisulfite treatment
PCR and analysis of read to find which bases have undergone changes due to lack of methylation
Where does DNA methylation occur?
Addition of a methyl group onto the 5C of cytosine
What is semi-conservative replication? How is methylation retained?
Replication complex generates a complementary strand that is unmethylated
VIM recruits MET1, a methyl transferase to the replication complex
= Methylation of the new strand and inheritance of epigenetic marks
How might DNA methylation landscapes differ?
- Mosaic methylation
e.g. in fungi, methylation occurs at transposable elements - Global methylation
e.g. whole genomes except islands within the promoter may be methylated
What are the different DNA sequence contexts at which DNA methylation may occur?
Symmetric - CG or CHG methylation
Asymmetric - CHH methylation
Where H is any base that isn’t a G
What causes CG methylation? What is the evidence?
MET1 is responsible for CG methylation
met1 mutants had significantly lower levels of CG methylation
What is responsible for CHG methylation?
CMT3
Responsible for all CCG, CAG and CTG methylation with no clear preference
MET1 also found to methylate CCG
What is responsible for CHH methylation?
CMT2 or DRM2- depends on the length of TEs region and chromatin state
CMT2= maintains methylation of long TEs regions in a heterchromatic areas
DRM2 = maintains methylation of short TEs region in a euchromatic areas
What gene is responsible for de novo methylation? Evidence
DRM2 - can deposit methylation at any sequence context
Insertion of unmethylated FWA transgene causing late flowering into A.thaliana plant found that in drm1/2 mutants, late flowering was present. While in wild-type there is normal flowering, as the gene would become methylated
Describe the canonical RNA-directed DNA-methylation
Required for CHH maintenance, characterised by 24nt siRNAs
1. RNA PolIV is recruited to generate a copy
2. This is converted to a dsRNA by RDR pol 2 (RDR2)
3. dsRNA is detected and targeted by Dicer and Argonaute
4. AGO with the siRNA targets complementary transcript
5. Recruits DRM2 to promote methylation of the site
Describe the non-canonical method of RNA-directed DNA methylation
Important for initial establishment of DNA methylation, characterised by 21nt siRNAs
1. Uses RNA pol II instead of RNA Pol IV
2. Same method of dsRNA formation, dicer and argonaute targeting
Describe an example of transition from non-canonical RdD to canonical RdDM
Birth and death of a transposon
1. Release the suppression of the retrotransposon e.g. EVADE
2. Back-crossing to generate EpiRIL (Epigenetic Recombinant Inbred lines). Found an increase in copy number
3. Greater increase in 21nt then decrease, followed by increase in 24nt (canonical RdDM)
What are the two methods of DNA demethylation?
Passive - prevent maintenance of methylation
Active - glycosylates, enzymes such as ROS1 (important for transposon suppression and genomic stability) or DEMETER (DME) important for fertilisation and embryonic development
Describe the active way that ROS1 removes methylation
ROS1 carries out Base Excision Repair, removing the entire cytosine.
ROS1 is a glycosylase that functions as a demethylase by removing the entire methylated base
- Knockout of ros1 leads to enhanced methylation at the edges of the transposon and increased gene silencing
Regulation
Very rare, but ROS1 is activated by DNA methylation, generating a feedback loop
What is the effect of removing all DNA methylation?
met1, drm1/2 and cmt2/3 mutants lack methylation and full mutants show clear developmental defects
- more genes are upregulated
- genomic instability due to movement of Transposable Elements (TEs)
What are the different domains present in different epigenetic reader proteins?
- MBD and SRA domain recognises DNA methylation
- BROMO domain recognises acyl groups on HISTONES
- BAH and CHROMO domain recognises methylated histones
What is the role of the SRA domain in VIM? And the role of VIM itself
SET and RING associated domain (SRA) - recognises methylation of cytosine residues
When binding leads to flipping out of the methylated cytosine allowing interaction with proteins such as KRYPTONITE for methylation of nearby histones
@ the replication fork, hemi-methylated strands form and VIM is important in recognising this and recruits MET1
= met1 and vim mutant have the same methylation pattern, suggesting they work together
Describe the DNA methylation/ H3K9me2 loop
Feedback loop that couples DNA methylation with H3K9me2 methylation.
KRYPTONITE(SUVH4) works with SRA-domain containing proteins to detect DNA methylation, and methylate histones
CMT3 is capable of interacting with H3K9 methylation marks via chromo and BAH domains. CMT3 function is to methylate DNA
= Mutation in DNA methylation (drm1/2, cmt2/3) eliminates CHG and CHH methylation which results in reduced H3K9 methylation also
Describe the superman and clark kent epialleles
Superman and clark kent are epialleles capable of switching between phenotypes in different generations, suggesting epigenetic differences instead and genetic mutations.
SUPERMAN - prevents excessive number of male organs (stamens)
In mutant superman or Clark Kent epiallele, SUP gene is hypermethylated, silenced and so increase in stamen number
- Mutagenesis found that a mutation in KRYPTONITE (SUVH4) could cause would lead to reversion to WT
- Similarly mutation in cmt3 leads to WT phenotype as methylation to generate SUPERMAN phenotype is not possible
What is the role of the domains in KRYPTONITE?
SET domain = Histone methylatransferases
SRA domain = reader of DNA methylation
What are the roles of the domains present in CMT3?
BAH - recognising methylated histones
Chromo - protein-binding domain that typically recognises methylated histones
(BAH and Chromo domain found to have a high Kd to H3K9 methylation from Isothermal Titration Calorimetry (ITC) analysis
Isothermal Titration Calorimetry (ITC)
DNA Methyltransferase - addition of methyl onto DNA
What is the difference in the structure of SUVH4 and SUVH2/9?
SUVH2/9 lack a post-SET domain, this leads to non-functional histone methyltransferases
What are the roles of SUVH2/9?
Maintenance of non-CG RdDM by interaction with Defective in RNA-directed DNA Methylation 1 (DRD1)
- evidenced by Co-IP assays
What is the role of SUVH1/3?
SRA domain to bind methylated DNA
Lacks functional histone methyltransferases = STRICT reader function
- Interacts with transcriptional activators such as DNAJ1/2
- Required for expression of genes nearby to TEs
Summarise the roles of the different SUVH
SUVH4(KYP) - read and write, forming DNA methylation and H3K9 methylation feedback loop
SUVH2/9 - read and recruit DRD1, maintain non-CG methylation in RdDM = feedback loop
SUVH1/3 - read and recruit transcriptional activators, preventing TE related silencing = homeostasis
What are the MBD DNA methylation reader types?
Methyl CpG Binding Domain (MBD)
MBD5/6
- binds methylated CG and recruits SILENZIO to silence genes = regulates downstream signalling
MBD7
- Recruits demethylators such as ROS1 to prevent hypermethylation, binds to methylated CG = homeostatic regulation
What are some example of histone readers? (activators and repressors)
Repression = AGDP1 - binds H3K9me2
Activation = BRAT1 binds H4ac, recruiting ROS1 resulting in demethylation and promotion of transcription
Dual reader - EBS, has BAH (repression binds H3K27me3) and PHD (activator binds H3K4me3)
What epigenetic changes occur upon biotic stress?
De-methylation of transposable elements (TEs), especially of CHH methylation
- Evidenced by GUS reporter analysis that fused GUS to LTR transposon, and upon addition of flg22 there was an increase in GUS expression
What is the importance of demethylation on pathogen resistance?
Reduction in the suppression of genes leading to pathogen resistance
met1 and ddc (triple) mutants (unable to methylate) had lower leaf bacteria concentration
= Increase in pathogen related 1 (PR1) even in the absence of the pathogen
ACTIVE demethylation by enzymes such as ROS1 is required for activation of R genes
Describe the importance of ROS1 in stress-mediated chromatin changes
ROS1 is important for demethylation and activation of R genes
- ros1 mutants were hypermethylated in the W box of the promoter of an R gene RLP43
+ It was found that 72% of transcription factors are inhibited by DNA methylation
What changes occur in chromatin as a result of heat stress?
Pericentromeric decompaction
- Pericentromeres, which are rich in TEs that flank the centromeres are found to interact more with the rest of the genome
Evidenced by Hi-C results
What epigenetic changes occur as a result of salt stress?
DNA hypermethylation in CG, CHG and CHH regions
Priming - faster, greater response
Transgenerational stress memory assay found that plants that had ancestors exposed to salt stress were better able to respond to such stress
How does BABA act as a ‘priming agent’?
Treatment leads to enhanced disease resistance by the plant
- Doesn’t induce the transcription of PR1 but primes it for activation, so that levels increase more rapidly
What are some mechanisms for priming? and some examples (5)
- Increased chromatin accessibility
- hypomethylation in WRKY areas - Removal of DNA methylation
- removal of H3K27me3 increased drought/ salt tolerance - Removal of repressive histones
- Addition of activation associated histones
- activation to promote WRK29 expressio - Poised Pol II
- having Pol II already bound before TSS
NOTE: priming doesn’t have to involve methylation changes, and methylation changes doesn’t have to prime the plant
What gene is important for retaining memory of stress?
FORGETTER1 (FGT1)
Loss of function mutants exhibit reduced stress memory capacity
What are the downstream effects of stress induced epigenetic changes?
- Changes in expression patterns of genes
- Movement of transposons (potentially acting as a mutagen)
- Changes to regulatory pathways
How are transcriptional and methylation changes correlated?
Transcriptional changes can precede changes to DNA methylation
e.g. transcription can change 7 days post induction, while methylation changes can occur 21days later
Rate of reversal is also faster for transcriptional changes
Why are plants not always primed?
- Cost of priming is high (e.g. increase in protein amount)
- Priming might suppress other functions, for example general responses to light might be hindered by always being primed to high light
How else can memory to stress be generated?
- Epigenetic changes
- Visualised using whole genome bisulfite sequencing (WGBS) = increase in CMT2 led to greater DNA methylation
- Expression of memory transcripts were found to be regulated by DNA methylation changes in rice (Li et al., 2019) - Production of miRNAs - can degrade mRNAs e.g. in drought response in switchgrass
- Changes to phytohormone production - increase in JA and ABA was found to be associated with drought in rice
Describe first generation Sanger Sequencing
Chain termination PCR and addition of tagged bases that are subsequently read out
- 500-1500bp read length
- 99.99% accuracy
What are some second generation sequencing methods?
Illumina (sequencing by synthesis)
1. Fragments formed and adapters bind to the fragments
2. Adapters then bind to the flow cell
3. Strands are amplified to generate clusters
4. Sequence Sanger style
What are some negatives of second gen sequencing?
- Amplification bias
- Requires sufficient numbers for amplification and detection
- Read length limits (hard to map repetitive regions)
What are some third gen sequencing methods? Describe them
Pacbio
- incorporation of a fluorescent base which emits light that is then detected.
Oxford Nanopore
- strand is passed through a nanopore membrane which undergoes changes in voltage.
- specific changes in voltage are associated to different bases and with different modifications
What are some negatives of third gen sequencing techniques?
- PacBio had a lower accuracy pre Hi-Fi. HiFi allows for circularisation and multiple reads of the same strand
- Oxford Nanopore struggles with accuracy in regions that are homopolymers
How has third gen sequencing been beneficial?
- De novo assembly
- reconstruction of whole genomes, including centromeres and other areas with high number of repetitive elements - Structural variation analysis
- allows for analysis of changes such as deletions and insertions which may have been hard to piece together when chromosome is fragmented - Long read sequencing of RNA
- allows for analysis of splice variants, doesn’t require conversion to cDNA which could generate biases
What is the role of the centromere?
Forms the link between DNA and spindle fibre which is important for proper segregation of chromosomes
- Failure to link properly can lead to aneuploidy and cell death
How have new sequencing techniques allowed for ground breaking study into crop breeding?
- Analysis of resistance genes
- useful for following R genes during introgression (movement of genetic material during hybridisation or crossing) e.g. TM-2 that provides resistance against TMV - Tracking structural variation
- Short reads provides bias towards detecting SNPs compared to other changes e.g. SIKLUH gene thought to increase fruit size due to a SNP in the promoter, but turns there was an additional copy of the gene - Haplotype phasing
-identifying which alleles are inherited together to look at inheritance patterns. whole sequencing of many individuals fast and cheap can be used to compare and phase haplotypes
How can base modifications be detected?
- Bisulfite sequencing
- conversion of non-methylated Cs to U and sequence to find differences in - Nanopore
- different voltage change when a methylated base is passed through compared to a non-methylated base - PacBio
- different time lag associated with the incorporation of the tagged base - CHIP-seq
- bind antibody that is complementary to specific histones around a DNA fragment, then fragment and isolate before sequencing
What are the 4 sustainable development goals surrounding agriculture?
- Change priorities towards healthy foods
- Sustainably intensify farming practices
- Global governance of land and oceans
- Reduce food waste
What is Mendel’s law of segregation?
During the formation of gametes (sperm and egg) two alleles for the trait will separate
Independent genes will independently assort these alleles (Independent assortment)
What is linkage?
The likelihood of two closely located genes to be inherited together
Degree of linkage is calculated by recombination frequency, which is the percentage of offspring in which recombination occurs
What are genetic maps? What units are present?
Location of genes and marks along a chromosome based on recombination frequencies
Measured in centiMorgans
- Important when looking at breeding at it shows chance of recombination
Why is the physical map not so important when looking at breeding?
May show two genes being closely located, however, if near the centromere were crossover is minimal then highly unlikely that these genes will cross
Describe the difficulties surrounding polygenic inheritance
Traits determined by multiple genes
- Makes it difficult to classify roles of certain genes as they may cause a gradient of changes
- In polyploid genomes (such as wheat) there may also be redundancy, so knock out of a gene may not show anything
(CRISPR has been useful to overcome this problem, allowing multiple copies of one gene to be knocked out in one go)
Why are quantitative traits in plants hard to measure?
- Influenced by the environment
- measurements may vary depending on temperature, soil pH e.g. Hydrangea flower colour - Regulation by multiple genes
- polygenic inheritance - Trait may be continuous e.g. height, unlike flower colour
What are the steps to studying genetics of quantitative traits?
- Acquire individuals with genetic variation
- Locate genetic markers that are specific to the gene
- Determine heritability of the phenotype and accuracy of measurement
- Statistically test
- See whether there is significant association between the phenotype and the genetic marker for the gene
Describe a QTL analysis
Quantitative Trait Locus analysis
1. have genetic markers that help distinguish between two parents
2. Use these markers to characterise the genome of the F2 population as either heterozygous/ homoA or homoB = genetic map . Uses the marker as the assay
3. Phenotype e.g. for length
4. Generate a table with the genotype at the different marker points
5. Generate a table with the average value of the phenotype from a certain genotype e.g. homoA (average between no of plants that are homoA at marker 1)
- used to find genes that are important for the phenotype by calculating the difference between homoB and homoA
(NOTE: if heterozygous is bigger this could suggest hybrid vigour)
How can GWAS be used for quantitative trait analysis? What are some positives and negatives?
Genome Wide Association Studies (GWAS)
Analysis genome for SNPs that are statistically associated to certain traits
= Manhattan plot higher dots mean greater association
Positives - Doesn’t require specific lines being crossed (useful for human analysis)
Negatives - requires many marker genes to annotate the whole genome
- for rarer alleles, recombination frequency below 5% doesn’t provide enough statistical power to generate conclusions as to whether the gene could be relating to trait
1. Measure the trait
2. Genotype the population
3. Statistically analyse the correlation between the trait and the phenotype
Describe MAGIC
Multi-parent Advanced Generation Intercross (MAGIC)
Hybrid between GWAS and QTL
- Less diversity than GWAS but uses known markers from the known set number of parents max around 12
Intercrosses are carried out to increase genetic diversity within the population
What are the key take-aways on which methods of QTL to use?
Depends on the population size and type
Also depends on the availability of markers
- More recombination means greater resolution as it allows for distinction between different markers
- Also can help show any linkage traits within the genome
What is Vavilov’s centre of origin theory? What is some evidence against this?
Proposed that areas with the greatest diversity in the wild are where humans first cultivated them
Against
- Hard to know where the crops originated
- Crops have moved around a lot and may have undergone major evolutionary changes in a certain area, rendering secondary places also very important. For example the movement of maize from low-lands to high lands in Central America
Describe in detail an example of a crop domestication event
Teosinte domesticated to what is now known as maize
- Increase in ear and protective leaf size
- Sweetening of kernels also
- shorter size due to dwarfing gene
- aim for determinate development so that plants mature at similar times for easier harvesting
Describe the hybridisation and origin of wheat
Polyploid formed from the cross over event between a tetraploid and diploid to form a hexaploid
- there has been greater genetic exchange with the tetraploid wild relative, while the D (diploid) genome is largely ‘locked’ showing little diversity
(suggested that D genome captures less than 10% of the diversity found in wild types)
++ Opposingly for Durum wheat, this was formed solely from the tetraploid
What is genetic bottlenecking and an example?
Bottlenecking - events that limit genetic variation in a population
e.g. only 45% of the variation found in wild type wheat is found in the domesticated AB genome, and only 10% in the D genome
How can diversity in wheat be increased?
- de novo domestication of wheat, would allow for introduction of variety from the D genome e.g from A.tauschii
- Genetic engineering and CRISPR techniques, e.g to knock out negative regulators
What are traditional breeding strategies?
Crossing of two plants with desirable phenotypes to get these in one plant
Describe the Green revolution
Norman Borlaug
Following the development of the Harber-Bosch process that produced nitrate for fertilisers
- Application of fertilisers led to extreme growth and an increase in height which caused lodging of plants
Dwarfing of the plant (Semidwarf1) meant that upon application of fertilisers less plants lodged
What is marker assisted selection? What are some uses?
The use of genetic markers within the chromosome to select for certain traits
+ faster way to quantify, easier also for R genes that require infection and pathogen to find real effect
+ Found the gene Flood Resistance 13A which was resistant to submergence and flooding, this was easily selected with IR64 that produces high yield
What are the different methods that can be used for crop breeding?
- Traditional breeding
- Marker assisted selection
- Mutation breeding
- Genetic modification
- Gene editing
What is mutation breeding and an example?
Irradiate seeds to mutagenise and analyse phenotypic effects
Commonly done to generate fruits such as grapes and oranges with no seeds
Adv
- Generates mutations that may not exist
- Rapid development of changes, compared to traditional strategies
Disadv
- Unpredictable
- Regulatory hurdles, whether they are classed as genetically modified
Describe what genetic modifications can be made in crops
Insertion of new genes that leads to acquisition of a new trait
Transgenics - transfer of genetic material from one species to another
e.g. sweet potato has bacterial DNA in it
e.g. transfer of pathway that stores oils from algae as omega3 in fish to plants. reducing fishmeal for fish farming
Cisgenics - transfer of material between different varieties but same species
e.g. transfer of R genes from a wild potato from Peru into Maris species lead to greater resistance in the absence of pesticides
What is a KASP assay?
Kompetitive Allele Specific PCR
- for detection of SNPs
Uses 3 types of primers
Common primer - binds to region of DNA that is identical in both alleles
Allele-specific primer - primers that are specific to different alleles (pairing to the different SNPs)
PCR product generated is distinct to which allele-specific primer is used, with fluorescence being specific to either A, B or both alleles
Describe capture sequencing
Uses oligonucleotide baits that selectively enrich certain areas of the genome
- Allows for focused analysis and reduces cost, especially for large genomes such as wheat
e.g. Exon capture to look at coding regions
Describe MutRenSeq
Mutation Resistant Gene Enrichment Sequencing
1. Mutate seeds that are resistant to the pathogen
2. Grow them and look for susceptibility
3. Use NLR baits to capture R genes and then sequence
4. Use multiple independent lines to confirm that mutations in that specific R gene is the cause for susceptibility
(as there are many R genes and many mutations can occur, accuracy of pinpointing which gene is responsible is hard without multiple lines)
Why is cassette development for resistance breeding important?
Generates the ability to introduce multiple R genes at once to prevent the pathogen from evolving resistance
- introduction of one or two, the pathogen may be able to overcome resistance
What changes were made in the domestication of physalis/ Groundcherry?
Previously small fruit, indeterminate structure
- Target CLAVATA to have more locules in the fruit
Mutated repressors of traits, in order to enhance certain traits
Describe the de novo domestication of tetraploid ric
Tetraploid rice (O.alta) had many favourable traits such as drought and waterlogging resistance
- Mutations were made in the tetraploid genome to enhance the yield and growth characteristics of the tetraploid rice to have the beneficial traits that Oryza sativa has
(this was done by introducing a casette targeting many genes in one go, much easier than trying to transfer multiple genes that give rise to a quantitative trait from O.alta in O.sativa)
Challenges
- successful transformation of the polyploid
- ensuring heritability (of these genes in the rice example is not known)
What are the positive and negatives of a polyploid plant?
- Genetic robustness
- increase in genetic diversity with additional chromosomes carrying various alleles - Hidden variation
- redundancy against genetic loss, preventing loss of crucial functions
- only clear phenotypes may be seen in higher order mutants which are hard to generate if gene is not fully known. This is commonly overcome by looking at sequence similarity of genes in diploids
e.g. mutation to increase yield may be 5% in wheat while 20% in rice, however, knockout of the genes in all chromsomes can lead to 20% increase in yield in wheat - Large chromosomes
- makes it hard and expensive to sequence, in the case of wheat the genome is also highly repetitive
- takes longer to backcross and select, however, new technologies have helped overcome this - Gene dosage effect
- increase in fruit size and yield due to greater gene dosage
Describe the characteristics of Teff and its agronomic issues
C4 cereal that is commonly grown in Africa, gluten-free alternative
- Produces very small seeds that are sewn by dispersal = harder for mechanised farming
- Prone to lodging which leads to yield loss = SD1 gene targeted to reduce height, however, the straw from the plants are also useful for building and liveihoods
How can transformation of plants be enhanced?
Addition of growth regulators GRF4-GIF1
- promote the proliferation of the callus which can be transformed in AgBac media
BabyBoom, Wuschel and GIF1 - are important for retaining undifferentiated state to promote proliferation
Describe the concept behind regulatory gene editing
- Improvements in quantitative traits are commonly associated with changes in the expression of genes in terms of strength and location of expression
e.g. fas in tomato, 294kb inversion causing change in the expression profile that increases locule number. This leads to quantitative variation with many different locule numbers
NOTE: hard to see if it is due to expression as these analyses commonly involve grinding tissue and which includes many cells that can mask the changes in expression
What evidence is there of regulatory gene editing having an effect on spatial expression?
Mutation in cis-regulatory element of VRT2 found changes to spatial patterning, visualised by in situ hybridisation with labelled probes
- Found ectopic expression (expression where not commonly found)
VRT2- plays a role in floral development and vernalisation
What are the three types of transposons?
- Retrotransposon (Class I)
- encodes a reverse transcription that generates a dsDNA strand that can be integrated
- move via an RNA intermediate - Class II
- encodes a transposase that allows for cut and paste of the element
- move as DNA - Helitron
- doesn’t have border repeats, replicates via rolling circle
What are the effects of transposon movement?
- Inhibition of gene function (transfer within gene)
- Change expression of gene (transfer into promoter)
- Promote re-arrangement of chromosomes
- Attract heterochromatin resulting in silencing e.g. by H3K9me2
What is the C-value paradox?
Genome size doesn’t reflect complexity
-Many plant genomes are large due to highly repetitive regions
What are ways in which heterochromatin can be analysed?
- Cytological analysis
- DAPI binds to AT rich regions of DNA
- Heterochromatic areas stain more brightly due to high intensity packing of DNA - HiC
- analysis of DNA-DNA interactions across the chromosomes
- achieved by crosslinking closely located DNA using formaldehyde - CHIP-seq
- look for the areas in which certain proteins such as histones bind to - ATAC-seq (assay for transposase-accessible Chromatin)
- transposase that fragments DNA that is easily accessible to add primer and tag
-
Is heterochromatin silent?
-HP1 (methylation reader attached to GFP), photobleaching of the area saw rapid recovery suggesting the area is active
- Gene switching between different epigenetic states can occur
- transcription of non-coding RNAs that are responsible for maintaining heterochromatic state suggests the area is not silenced
- regulation of the epigenetic status of TEs is variable, with more TEs opening up in stress conditions
What is position effect variegation?
Changes in the expression of a gene due to changes in its position along a chromosome
- For example transfer nearby a heterochromatic region can lead to its silencing
e.g. movement of the white gene causing changes in Drosophila eye colour
Name an example in which mutations in a gene resulted in spreading of DNA methylation
Mutation in IBM1 a gene encoding a H3K9me2 demethylase
- This resulted in an increase in methylation which silenced the gene Bonsai
- This caused the A.thaliana phenotype to be small
What are the roles and features of the siRNAs produced at heterochromatic regions?
21-24nt in length
- Responsible for maintaining methylation and modifications within a specific region of TEs
What are the pieces of evidence for RdDM?
- Viroid mediated RdDM
- Tobacco transformed with DNA copy of Potato Spindle Tuber viroid
- Infection with virus led to increase in DNA methylation evidenced by a southern blot where the size of DNA fragment was larger, because the enzyme was unable to cut (due to more methylation) - Promoter hairpin-driven silencing
- Trigger and target transgene, with transgene conferring resistance against kanamycin
- solely have target leads to kanamycin resistance, while trigger has a inverted promoter sequence of target leading to complementarity and dsRNA forming
- Southern blot found changes in the DNA fragment size, suppression of methylators returned fragment sizes to unmethylated control
Describe virus induced gene silencing (VIGS) and an example of it
Virus detected as foreign and the plant generates small RNAs to reduce the transcript load of the virus
- In plant that is expressing GFP under a 35S promoter, addition of TRV with part of the GFP sequence will lead to the plant suppressing the expression of GFP due to VIGS
- Similarly when the virus contains part of the 35S promoter that is driving the GFP
- Transcriptional run on analysis found that in the TRV 35S plant transcription is suppressed, while in the TRV:GFP modification is post-transcriptional
Heritability
- promoter suppression that is transcriptional is heritable due to methylation changes, while TRV:GFP suppression is not inherited
Give an example where de novo methylation occurred/ was discovered
Analysis of the FWA gene in A.thaliana that regulates flowering
- Commonly the FWA promoter region is methylated and leads to normal flowering
= introduction of unmethylated FWA in a drm2 mutant leads to lack of de novo methylation and late flowering
- however, introduction of FWA in wildtype, the gene is methylated and flowering is normal
Describe the players involved in RdDM
- SHH1 helps recruit PolIV to heterochromatic mark
- Pol IV along with RDR2 generates dsRNA
- C terminal domain of PolIV is important for SHH1 recruitment which is a histone reader - The siRNAs then recruit DRM2 to the site for DNA methylation
- Which is sensed by SHH1, generating a loop
How do archaebacterial histones vary from eukaryotic histones?
Archaebacterial lack long tails
*long tails in eukaryotes are important for post-translation modifications
- However, function the same by binding and wrapping DNA to form a nucleosome like structure
What is constitutive and facultative heterochromatin?
Constitutive - always present e.g. at transposons
Facultative - depends on cell type and conditions of the cell as to whether present or not e.g. vernalisation
What factors regulate flowering time?
- Circadian clock CONSTANS
- Temperature - vernalisation
- Light
= APETALA genes need to be switched on
What is the role of FLC?
MADS transcription factor that represses floral integrator genes
FLC is promoted by FRIGIDA (FRI)
What evidence is there of memory of vernalisation?
Northern blot showing that FLC levels remained low after 20days no cold which followed 3 or more week of vernalisation
VRN2 + LHP1 - responsible for vernalisation memory, as mutants had levels of FLC increasing again after only 10-15days
= CHIP-Seq found an increase in heterochromatic marks on the promoter of the FLC gene
How were genes involved in vernalisation discovered?
Mutagenesis of Arabidopsis plants that didn’t respond to vernalisation (so after cold didn’t flower)
Found VRN1/2/5 and VIN3
What is the role and function of VRN2?
Retain memory of vernalisation
Homologue of Polycomb Group that is responsible for gene silencing
- An orthologue was found to be responsible for H3K27methylation
: Although the mRNA levels of VRN2 was not found to differ in cold conditions
What is the role of VIN3 and how was it discovered?
Plays a role in vernalisation and its transcript levels increase in cold conditions, as FLC decreases
- has a PHD (chromatin reading) and FIN III domain (protein interaction)
Suggested to play a role in establishment of vernalisation prior to VRN1/2 complex that regulates FLC
Experiment
- Mutation looks like a vernalisation mutant, where in FLC:LUC assays, FLC is still expressed and flowering is suppressed
What is the proposed model for vernalisation?
- Before cold VRN2 is bound but not active
- After vernalisation VRN2 is activated by VIN3 and VRN5
- Early in vernalisation there is an increase in COOLAIR transcript (antisense of FLC to support suppression) - This leads to stable repression of FLC by H3K27methylation of the promoter region
- With upregulation of antisense FLC transcript to antagonise sense also
How is vernalisation reset between generations?
mutant analysis
ELF6 - histone demethylase required for the removal of H3K27 methylation
- Measurement of FLC transcript n mutant found no change in the in vernalisation FLC suppression response, but FLC levels were lower during resetting
Overexpression leads to global demethylation
What is unique about plant germline specialisation?
Occurs post-embryonically
Meaning changes can occur during lifetime and seeds from different branches on a tree may be genetically different
e.g. Shorea tree found a linear increase in the number of mutations with branch distance
What are the different types of DNA damage?
- Single stranded break
- Mismatch
- Damaged base
- Intra-strand cross-link
- Inter-strand cross-link
What are the main types of DNA damage repair? (5)
- Base excision repair
- Nucleotide Excision repair
- Homologous recombination
- Non-homologous end joining
- Mismatch repair
- Show some redundancy as seen in experiments that have knocked out pathways and radiated plants and they still look healthy
What are common base change mutations that occur?
C:G to A:T transition due to the deamination of cytosine to uracil which can be fixed to cytosine
HOWEVER - 5-methyl cytosine is deaminated to thymine, so harder to judge which is correct
Is mutation completely random?
Essential genes - mutations are less likely
Mutations are more likely to occur in areas depleted of nucleosomes e.g. upstream of the TSS
- It is possible that epigenetic marks help recruit machinery involved in DNA repair, so there are less mutations that remain
What are epimutation rates?
Mutation and changes in the epigenome e.g. methylation to non-methylation
- These rates are faster than DNA mutation rates
- Could be important for adaptability of organisms to new environments by changing gene expression
Describe NHEJ
Fixes double stranded breaks
- Homology independent, so RAD51 independent
Error prone due to the lack of template strand
Experiment: Require ligase IV, which is responsible for rejoining broken strands
Alternative NHEJ - uses microhomology in the overhanging strands that can result in sections of the DNA being lost
How does CRISPR cas rely on NHEJ?
CRISPR-Cas9 introduces a break in the DNA strand
- Relies on NHEJ to introduce a mutation due to error prone repair
Describe HR
Homology dependent, uses a template strand
1. Strand invasion, transferring genetic material from one strand to another
(if they are two chromatids then unlikely that a difference will be seen, however, if this is during meiosis then differences in SNPs may be seen)
EVIDENCE
- GUS analysis with breaks and complementary strands in different positions along the chromosome were able to repair to generate a functional GUS protein
What is the role of the meganuclease I-SceI
Recognises a specific 18bp sequence and cleaves to generate an overhang
- Base pair sequence that is unlikely to occur in most genomes, so can introduce a transgene with this sequence and introduce a DSB cut at a specific location by adding the enzyme
What is the role of RAD51?
Coats single stranded DNA that invades the strand
- Promotes finding of homology in another chromosome
knockout of rad51 results in impairment of repair mechanisms that require another chromosome
Describe SSA and SDSA
Both part of HR
Single-strand annealing (SSA) - no donor
- can lead to large deletions of the genome
1. 5’ resection of the strand
2. Binding of the strand in another place due to homology e.g. due to repetitive sequences, generating flaps are cleaved
3. Gaps again are filled with polymerase
Synthesis-Dependent strand annealing (SDSA)
1. Resection of DNA, followed by strand invasion into ‘donor’
2. Generates a Displacement (D) loop in the donor strand
3. Starts synthesising the new strand using a donor as a template until homology is found with the broken strand
4. Broken strands find homology and bind, gaps are filled by polymerase
- repair without excising large regions of DNA, preserving more genetic information
How is DNA damage signalled?
ATM/ATR kinase signalling
- Break sites leads to activation and phosphorylation of targets
= DNA repair mechanism recruitment or apoptosis
- In animals cells a target is p53
- In plants a target is H2A.X that is phosphorylated and recruits repair mechanisms
How can DNA recombination in mitosis and meiosis differ?
Reason
- Mitosis DNA recombination uses the strand for repair
- Meiosis DNA recombination uses the strand to generate diversity by crossing over
DSB formation
- Mitosis DSB formation is random
- Meiosis SPO11 can promote DSB to promote cross-over
Factors and complex involved
- Mitosis RAD51
- Meiosis RAD51, DMC1 and synaptonemal complex
Types of DNA recombination
- Mitosis uses HR and NHEJ (no RAD51, no homology required)
- Meiosis uses HR and not NHEJ (no crossing over)
How have eukaryotic innovations facilitated the increase in genetic diversity ?
- Chromosome expansions
- Telomeres to prevent degradation
- Histone code and RNAi to suppress deleterious genes from expressing
- Meiosis (crossing over)
What are homologous chromosomes?
Pair of chromosomes one from maternal and one from paternal origin
Contain the same genes in the same order, however have allelic variation
- Form and align during prophase I in meiosis
Describe the process of meiosis
Prophase I - synapsis occurs when homologous chromosome pairs align and the synaptonemal complex forms. Recombination occurs which can lead to crossing over
First division occurs where the homologues separate
Second division occurs where the sister chromatids separate
Describe the process of mitosis
Cohesin rings hold chromatids together until anaphase
Following anaphase when the cell divides the chromatids are split
Formation of progeny identical to parent
Compare and contrast meiosis and mitosis
Similarities
- In both cases the genome is duplicated at the start
- Cohesin rings are present
Differences
- Meiosis, crossover occurs in DNA recombination
- Orientation of the centromere differs
- Meiosis has two rounds of division
Describe the evolution of recombination and maintenance/ importance of sexual recombination
Sexual recombination allows for integration and sharing of genes and mutations
In asexual populations, for an individual to have all the changes present would require sequential adaptation and mutation in the same lineage
= Sexual mutation better for evolution of new traits which is essential for survival
Describe the changes that occur between the first and second round of division in meiosis
SHUGOSHIN1 (SGO1) is a phosphatase that removes Pi added by CDPK
- This protects the cohesin from being degraded and keeps the sister chromatids together
At the second round of division SGO1 is no longer present so the two chromatids can be separated
Describe the structure of the synaptonemal complex
Complex forms between two homologous chromosome pairs
Promotes breakage, crossing over and repair of the chromosome
ZYP1- axial protein that ensures recombination between homologous and not non-homologous pairs
Recombination nodule - with MSH4/ MLH1
SPO11 to introduce DSB
Describe the tethered loop axis for recombination
Chromosomes are arranged in loops extending from the middle axis
Following DSB the broken strand is moved into the axis for recombination
- The broken strand is coated by DMC1 that likely interacts with proteins that drive this movement
What are Class I and Class II recombination pathways?
Class I - interfering 85% of most recombination
Class II - non-interfering
Describe the cross over frequency across the chromosome
Few crossovers occur at the centromeres
- likely due to heterochromatic marks
More crossovers occur at the ends, evidenced by comparison between physical and genetic map and fluorescent staining of recombination nodules that are found to be located near the ends
Describe the pattern of crossovers
Many DSB are made however few actually cross over
Requirement of at least 1 crossover to ensure proper segregation
Interference - reduces the chances of multiple crossovers in an area
e.g. fusion of chromosomes in C.elegans which elongated the physical length of the chromosome still only had 1 crossover
e.g. non-linear relationship between size of the genome and number of cross-overs
What is the role of SPO11? What experiments have been carried out?
Topoisomerase that regulates DNA unwinding
- Generates a break in the phosphate backbone to reduce torsion
1. Becomes covalently bound to DNA via tyrosine residue
2. Cleaved off and retains a DNA tag
3. This can be purified and sequenced to find where SPO11 binds
= Found that SPO11 binds inter-genic regions e.g. promoters where there is a lack of nucleosome
What evidence is there that the chromatin state can effect DNA recombination?
- Knock out of RdDM genes leads to increased recombination due to a lack of methylation
(except for knockout of CG methylators ddm1 and met1) - Location of SPO11 binding is in nucleosome free areas with minimal epigenetic marks
- Recombination rate is much lower in heterochromatic regions such as the centromere
How is interference generated?
Distribution of the E3 ligase HEI10
Coarsening model - suggests that accumulation of HEI10 leads to aggregate HEI10 and further accumulation targeting recombination to that area and away from others
Experiment: transformation of HEI10 gene to increase copy number was found to increase the amount of recombination
Describe the difference in meiosis in female and male parts of a plant
Female - single cell undergoes meiosis in the ovule’s megaspore mother cell
Male - occurs in anthers, in the 4 locules so much easier to see
What is the importance of crossovers?
- Enable breeding
- conventional breeding to select for an agronomic trait - Limit breeding
- unevenly distributed along a chromosome, can prevent crossover of 25% of genes in cross-over cold spots
What is linkage drag?
When genes bring along other genes commonly closely located
What is the need for overcoming cross-over limitations?
- Increase in genetic diversity
- 25% of protein coding genes are located in cross-over cold spots
How can cross-over recombination be enhanced?
- Reducing cross-over suppressors
- while many breaks occur, only few cross-overs form
- e.g. fancm, mutagenised zip4 plants and found fancm mutants to be fertile. FANCM is responsible for unwinding D loops and preventing holiday junction formation - Promoting cross-over promoters
- e.g. ZIP4 promotes cross-over, mutants show no cross-overs and plants are infertile
What are the two different types of cross-over and their relation to ZIP4 and FANCM?
Class I - promote by ZIP4 (85%)
Class II - repressed by FANCM (15%)
(fancm mutants had higher rates of class II non-interfering cross-over)
What is the evidence that suggests that epigenetic leads to changes in cross-over patterns?
- cmt3 mutants that had reduced CG methylation can open up the centromere and lead to an increasing in crossover (cM)
- suvh5/6 mutants that had reduced H3K9e2 also had increase in cM
- Differences between melon and cucumber arise from TE expansion which can suppress crossing over
Why is removing epigenetic modifications in crops not desirable?
Increase in the number and movement of transposable elements
- This can lead to upregulation of deleterious genes
- Additionally can cause genomic instability
What are the types of recombination that can occur as a result of targeted recombination?
Targeted recombination using Cas9 or Cas12
- Homologous recombination occurs when there is cross-overto finsih
What is the difference between cross-over and non-crossover?
Cross-over -> cut and swap event, reciprocal exchange of genetic information. Genes are physically swapped
– Detected by changes in flanking markers –
Non-crossover -> copy paste, where genes around the DSB will be replaced using a copy of the homologous chromosome
Overall what are the conclusions from attempts at unlocking cross-over limitation?
- Targetting of cross-over suppressors or activators still doesn’t unlock heterochromatic regions
- Targeted recombination could used to overcome this and to separate linkage drag
- Although there can be more breaks, the actual amount of cross-over that occurs isn’t as high
- Unlocking by removing epigenetic marks in crops is not feasible and can lead to genomic instability
What are the challenges of targeted somatic recombination?
- Doesn’t for all loci
- Low transmission to the next generation
- Can lead to whole chromosome loss
What are some examples of experiments of targeted somatic recombination?
Tomato
- had two mutant alleles, one for yellow (mutation in the gene), and one that generated bicolor phenotype (mutation in the promoter)
- Generated an F1 hybrid that was bicolor then induced DSB in the region between the two mutation sites by cas9
- in NHEJ more mutation would lead to yellow fruit, HR would lead to spotted or red, depending on whether repair was early in fruit development, or if the gene was fully repaired e.g. by crossing over and generating un-mutated fully functioning PSY1 gene
Maize
- Generated a targeted cut site for recombination and cross over to occur then backcrossed to one of the homozygous parent. This easily showed if the crossed parent had undergone reciprocal exchange (crossover)
- Saw a 10 fold increase in occurrence of recombination around the DSB cut site, and these changes were heritable
What are the roles of COOLAIR in vernalisation?
PRE- VIN3 and other histone remodelling
1. Antisense transcript helps reduce amount of FLC
- mutant had a slower decrease in FLC transcript
2. Destabilises H3K36me3 that acts as an activator for FLC
- mutant had no reduction in this activator histone mark
3. Promotes chromatin remodelling
How do gene inversions influence cross-overs? What is the relevance to agriculture?
- Inversions doesn’t allow for cross-overs due to the differences in orientation
- Drive high recombination rates to the sub-telomeric regions to compensate. Also supporting the idea that cross-overs are required for proper segregation
- Used to lock important stretch of genes to prevent loss
- Can also result in genetic bottlenecking as recombination can’t occur
- Presence of inversions identified due to next gen sequencing techniques
What evidence is there for gene inversions being overcome?
Targeted CRISPR can invert a whole section of the genome
- Evidenced by increase in recombination frequency
How can homozygosity be synthetically reached faster?
- Colchicine
- binds to tubulin and prevents spindle fibre formation - In vivo - chromosome elimination
Describe the process of chromosome elimination.
Histone variant (CENH3) in the centromere is responsible for binding microtubules
In chromosome elimination
- asynchronous cell division
- CENH3 fails to load onto one of the centromeres and so chromosome is lost and this generates a micronuclei
How can chromosome elimination be used in crop breeding? What is an example of this (GFP)?
- Haploid induction easier
- Intraspecies induction
CENH3 tail modification for GFP - When crossed with WT, the chromosome segregation was weaker so produced: Uniparental haploid (desired), Hybrid diploid (normal segregation) and aborted seeds
Describe the phenomenon of co-operative binding in chromosome elimination
- Haploid inducer will have less functional CENH3 than the WT
- When crossed, CENH3 from WT will bind to kinetochores
- This leads to segregation and haploids with WT chromosomes being formed
- HI chromsomes are lost as they couldn’t bind to kinetochore due to competition from WT
What is reverse breeding? How is it achieved
Generate parents, from desirable lines, that when crossed will regenerate these lines
1. Prevent further crossing over
- Achieved by silencing DMC1 or MSH5
2. Inducing haploid
3. Doubling the haploid
How can hybrid vigour be maintained in offspring?
Inducing clonal reproduction by MiMe and parthogenesis (fertilisation without meiosis)
1. Inhibiting crossovers by inhibiting SPO11 from generating DSB
2. REC8 - cohesin ring that hold sister chromatids together is removed, to allow for sisters to separate
3. OSD1 - omits second meiotic division to generate diploid and not haploids
How can doubling of genome with MiMe be prevented?
By haploid induction
What are the different ways for engineering parthogenesis?
- Baby Boom 1 (BBM1) expression
- not normally expressed in the egg cell, overexpession results in parthenogenesis (embryo without fertilisation) - PAR expression
- not commonly present, but results in embryogenesis without fertilisation
What is endoreduplication?
DNA doubling without the cell dividing
What is the use of apomixis in agriculture?
- Formation of clonal seeds e.g. from hybrids
- Fixing of specific traits
What is autoploidy progressive heterosis? How could this be used in crop breeding?
Combination of two plants already showing hybrid vigour to generate another plant with greater phenotype
Crop breeding
- Generation of tetraploid tomato that was larger in size, but cost of not being able to produce seed
- Greater resistance to stressors commonly seen in polyploid plants
-VE may lead to genome instability
What are the roles of effectors?
- Colonise the host
- Modulate host defence
- Promote nutrient uptake
- Alter plant morphology and secretion
What are the characteristics of PAMP?
Cell surface receptors: RLKs or RLPs
- Detect apoplastic effectors
- Commonly require a co-receptor
Give an example of PTI, against flg22
- Detected by FLS2
- Uses BAK1 as a co-receptor - Pi and activates BIK1
- Kinase that activates immunity
What are NLRs?
Nucleotide binding Leucine-rich Repeats
N-terminal domain = TIR/CC
NB-ARC domain = modular activation (ATP=on)
LRR = pathogen recognition site
- Intracellular receptor in ETI
How do NLRs recognise effectors?
Direct
Indirect - decoy or guardee binding that results in conformational change that is detected by the NLR
- integrated domain e.g. HMA domain in rice NLRs
=> Greater robustness one NLR can detect more effectors
Describe how singular CC-NLRs, such as ZAR1, are activated and elicit a response
ZAR1 binds and detects
Forms a pentameric structure known as a resistosome that is inserted into the plasma membrane and acts as a channel promoting ion flow
CC-domain is responsible for triggering cell death
How do TIR-type NLRs trigger cell death?
Don’t move and form a channel, instead function as enzymes
- Binding (not ATP) leads to activation
Describe the evolution of the NRC network and its function
Sensor and Helper NLRs work together
- Sensors don’t have MADA motif that is responsible for triggering cell death
- Binds and associates with Helper NLRs
- Generates a degree of redundancy where some sensor NLRs can work through multiple helper NLRs which helps generate a robust immune system
What is the evidence for mutual potentiation?
- Intracellular receptors can increase the expression of BIK1 and MAPKs
- Pre-activating ETI an lead to greater ROS production when flg22 is detected (just activating ETI by AvrRPS4 didn’t trigger ROS increase)
- PTI potentiates ETI-induced cell death
What is an example of a decoy?
ZED1 a pseudokinase
- modification is sensed by ZAR1
Describe the structure of the NLR genome
Organised in clusters
Commonly in sub-telomeric regions
Generated by tandem duplications
High recombination rates
What is RenSeq?
Selection of R genes for amplification, carried out by generating probes that bind to conserved domains such as the NB-ARC domain
- Sample then purified and don’t have to sequence the whole genome
How can plant ecology and lifestyle influence immune receptor evolution?
- Lifespan differences
- e.g. trees require more - Pathogenic load
- more load = more stress = more variation required - Plant size
- greater target for pathogens - Pathogen types
- airborne, aquatic etc. - Plant lifestyle
- parasitic plants with minimal roots have lost NB-ARC genes
- crops grown in high density such as rice and maize have greater repertoire of resistance genes
How can NLR death occur?
- Gene loss
- e.g. truncated FLS2 gene in Melon that wasn’t able to confer resistance against flg22 - Polymorphism
- mutations in R genes that results in susceptibility due to lack of resistance
How can NLRs be ‘born’?
- Tandem duplications resulting in
- sub-functionalisation = slightly different functions (2functions)
- neo-functionalisation = completely new function
- specialisation = more specific function - New gene fusions
- Integration of new domains
- helper and sensor commonly genetically linked
What evidence is there for gene fusion of NLRs occurring?
Unequal cross-over resulting in first half of NLR1 and second half of another NLR joining in Chinese spring variety
- Not tested whether the new NLR actually works
- Requires full genome sequencing to see that there is a large indel (missing stretch)
Describe the patterns of evolution of the NRC network
Sensor = balancing evolution, expansion to detect more effectors
Helper = purifying selection, reduced variation and greater redundancy
What is an example of NLR subfunctionalisation?
Slightly different functions
- NRC3 in benthi has subfunctionalised and diverged from potato and tomato
- In Solanum NRC3a works with Rpi-Avrblb2 to provide resistance against Avrblb2 from P.infestans
- Divergence likely due to higher pathogen load on potato and tomato than benthi
What is an example of neo-functionalisation?
Rx and Gpa2 are near identical NLRs with 88% sequence similarity
Rx- confers resistance to virus
Gpa2 confers resistance to nematode
How are NLRs regulated?
- Transcriptional level
- Epigenetic modification regulating accessibility
- WRKY TFs regulating transcription - Post-transcriptional level
- miRNA silencing
3 Post-translational level
- Chaperones regulating stability
- Ubiquitination - Inter NLR regulation
- e.g. RGA5 and RGA4
Give an example of interspecies transfer of PRRs
EFR cell surface receptor that detects elf18
- In Brassicaceae moved to tomato and generated greater resistance
What are the considerations for selecting immunity genes to integrate?
- Function and effectiveness in different host species
- Broad or specific resistance
- Number of R genes that need to added (need to know pathogen load/ diversity)
- Interaction between different immunity genes
Give an example of restricted taxonomic function
Transferance of certain genes from one species to another isn’t able to generate resistance
e.g. transfer of Bs2 gene from Solanaceae that confers resistance against Xanthomonas by detecting AvrBs2 to non-solanaceae
- due to it being part of the NRC network
Describe previous and more recent methods for receptor engineering
Previous
- Random mutagenesis found that single amino acid change in R3a increases target range of Avr3a molecules
Newer
- Targeted (structure-guided) mutagenesis
- Single amino acid mutation meant that Sr50 was able to detect new variation of AvrSr50 QCMJC
How can immune receptors be resurrected?
- single amino acid change in the NRC2/3 prevented it from being targeted by the SS15 effector
! Important to ensure that mutation doesn’t alter how the protein oligomerises! - Resistance was still present before as the sensor could act via NRC4
Describe the idea of plant vaccines
- Immunise llama with effectors and antigens from the plant pathogen
- The mammalian immune system will generate nanodies (like antibodies but smaller)
- Isolate these nanobodies
- Integrate these nanobodies in place of the ID domain in NLRs = formation of pikobody
- Confers plant immunity to the pathogen
Describe how decoys and integrated domains can be engineered
Decoys
- Modification of a decoy (e.g. PBS1) so that is can be targeted by more effectors
- Ensure its recognition site by NLRs (RPS5) isn’t modified and can still be detected
Integrated domains
- HMA from rice introduced to other NLRs to confer resistance against more effectors
Summarise the ways in which plant immunity can be engineered
- Promoter engineering - changing expression of genes
- Plant vaccines
- Engineering decoys
- Resurrecting NLRs
- Receptor engineering
- Transferring R cassettes between plants
What are the components and the importance of industrialisation of biology?
- Innovation - new ideas
- Manufacturing requiring standardisation
- e.g. certain enzymes, processes etc. are used
Describe the Golden Gate Assembly
- Type IIS restriction enzyme that cut outside recognition site
- Generates overhangs that can be matched with DNA ligase
- Cleaved and the recognition site disappears so can’t be cut again
What are the different forms of gene regulation?
- Simple on/off
- Feedback
Positive - generate oscillatory dynamics
Negative - suppresses induction, damping effect
Describe a repressilator design
Three repressors that repress each other in a loop
- generates a cycling of readouts
What was the production of artemisin industrialised?
Antimalarial drug
- Study of the processes and then pathway synthetically generated
Describe the Gibson Assembly
Cloning technique that allows you to assemble DNA fragments that have overlapping ends, without using restriction enzymes or ligation
1. Exonuclease creates overhangs
2. DNA pol fills gaps in annealed areas
3. DNA ligase seals the nick
What are some challenges with synthetic genomes and editing?
- Larger genomes of crops hard to edit
- Epigenetic marks and their regulatory role
- Compatibility in different species
- Repetitive sequences making design harder
How has different regulation of GM crops affected research into GM?
EU has less GM crop than USA due to stricter regulations
UK having left the EU are revising the precision breeding act and it may be feasible to sell GM crops
Describe CRISPR (Source and function)
Natural bacterial systems that target phage DNA
Function
- generates a DSB at a target
- promotes error prone homologous recombination to generate a mutation in the target gene
What are some target genes in tomato for gene editing?
WUSCHEL- maintains stem cell identity, so can changes locule number
FASCIATED - mutants have larger locules, greater fruit size
What is QTL used?
To identify genes associated with a quantitative trait (dynamic and governed by multiple genes)
- Also help with marker assisted selection in breeding programmes
What happens when mutants are made of all the DNA methyltransferases?
- Severe growth phenotypes
- met1, drm1/2, cmt2/3
= Suggests important for genome stability and transposon regulation
What happens when mutants are made of DNA demethylators?
Not much change in phenotype, despite the increase in methylation
- Early flowering in quadruple mutant
- dme, ros1, dml1/2
Describe the response of the epigenome to immune stress
- Changes in DNA methylation around TEs, increasing accessibility of machinery to neighbouring defence genes
- Overall demethylation to remove transcriptional silencing
- Changes in histone modification e.g. monoubiquitination by HBU1/2, mutant shows increased susceptibility
- Mutants of rddm pathway are more susceptible to P.syringae
- In crops important to be specific as 80% TEs
What is TEgenesis?
Chemical inhibition of Pol II to reduce transcript of siRNA 21nt as part of the non-canonical pathway
- This leads to less methylation and greater TE movement
