Reka Toth

Question 1

biolistics

Answer 1

important in a non-model species without approved transformation protocol

Question 2

mechanical stimuli

Answer 2

not reproducible

Question 3

how can you probe a pathway?

Answer 3

• receptors
• sensors
• kinases
• interacting proteins/co-factors

Question 4

how do you compare between species

Answer 4

• transcriptomics and DEGs
• heterologous complementation assays
• think necessary and sufficient, and then phylogeny
• draw attention to the different between homology and essentiality
• compare between 3 to find an intermediate stage

Question 5

double mutants show

Answer 5

genetic epistasis

Question 6

if they have tested expression of genes

Answer 6

have they tested accumulation of product?

Question 7

have they mentioned any signalling pathways which

Answer 7

they did not test?

Question 8

pleiotropic defects

Answer 8

• if you’re using a reporter, have they tested for this? important to note; can cite this experiment
• don’t know how much of the observed response is actually due to the transgene
• hard to avoid if you’re working with something that affects many pathways

Question 9

how many genes have they tested?

Answer 9

and are they in the same gene family?

Question 10

be careful of confusing

Answer 10

• the expression of a gene regulator and the
• expression of the genes that they regulate

Question 11

if they are investigating a particular stress response

Answer 11

does the responder affect many different stress pathways?

Question 12

if they mention genes

Answer 12

have they functionally annotated them?

Question 13

RNA methods

Answer 13

• RNA Seq
• qPCR

Question 14

Protein detection and protein-protein interaction

Answer 14

• western blotting

Question 15

localisation

Answer 15

• fluorescence
• immunohistochemistry
• GUS assay

Question 16

DNA binding and epigenetic

Answer 16

• chromatin immunoprecipitation (ChiP-Seq)

Question 17

gene mapping

Answer 17

• GWAS
• QTL

Question 18

What is protoplast generation?

Answer 18

• isolating cells without their cell walls and introducing new genetic material
• gene editing, plant transformation, and cell culture studies
• Somatic cell fusion

Question 19

transient expression

Answer 19

• Agroinfiltration and protoplast transformation

Question 20

GC-MS

Answer 20

for chemical detection

Question 21

flow cytometry

Answer 21

Cells or particles can be labeled with fluorescent dyes or antibodies that emit light of a specific wavelength when exposed to a laser

Question 22

How does co-IP show protein detection or protein/protein interactions?

Answer 22

• pull down a specific protein and identify interacting partners
• An antibody that specifically recognizes the target protein
• eluted proteins can be analyzed by methods like Western blotting, mass spectrometry
• Mapping Signaling Pathways
• Studying Post-translational Modifications (PTMs)

Question 23

What is GC-MS?

Answer 23

• used to identify and quantify compounds in a sample
• pesticides, herbicides, volatile organic compounds

Question 24

How does yeast 2 hybrid (Y2H) show protein detection or protein/protein interactions?

Answer 24

• reconstitution of a transcription factor in yeast that is split into two parts: the DNA-binding domain (BD) and the activation domain (AD)
• If these two proteins interact with each other, the transcription factor is reconstituted, leading to the expression of a reporter gene
• LacZ
• Negative controls
• Mapping Protein-Protein Interaction Networks
• Identifying Novel Protein Interactions

Question 25

How does immunohistochemistry show localisation?

Answer 25

ABS

Question 26

How does chromatin immunoprecipitation (ChiP-Seq) show DNA binding and epigenetic effects?

Answer 26

- Cells are treated with formaldehyde or other crosslinking agents, which covalently bond proteins to the DNA. This "locks" protein-DNA interactions in place, ensuring that transient or weak interactions are captured - fragmented chromatin is incubated with an antibody specific to the protein or epigenetic modification of interest

Question 27

How does GWAS work?

Answer 27

- identifying a phenotype you want to study - population: large and diverse to represent a variety of genetic backgrounds - collect DNA and genotype for SNPs - compare SNPs between cases and controls

Question 28

How does QTL work?

Answer 28

- two genetically distinct parental lines - measure the phenotypic variation for the quantitative trait in the offspring population - genotype using molecular markers; tracks inheritance - linkage mapping + ANOVA

Question 29

Somatic cell fusion

Answer 29

Protoplasts from two different plant species can be fused to create hybrid cells with genetic material from both parents (useful in plant breeding).

Question 30

Protoplast transformation

Answer 30

- electroporation uses an electrical pulse to create temporary pores in the protoplast membrane, allowing the foreign DNA to enter the cell - Gene function analysis - GE

Question 31

How does mass spectrometry work?

Answer 31

- Protein Extraction - Peptide Sequencing: tandem mass spectrometry (MS/MS) - protein database