VL 7: RNAi II

Question 1

What is Viral induced gene silencing (VIGS)?

Answer 1

1. Most plant viruses are RNA viruses that replicate through a double-stranded RNA intermediate -> Virus-encoded RNA-dependent RNA polymerase (RdRP)
2. Double-stranded RNA is cleaved by DCL to produce siRNA which associates with AGO to silence virus replication and expression.

-> Plants can recover from viral infection and become resistant, because the first viral infection has induced RNA silencing, preventing subsequent viral replication. Virus infection causes systemic siRNA accumulation

Question 2

How does RNA silencing spread systemically?

Answer 2

-> Silencing can spread locally: Often the silencing spreads over up to 15 cells, probably by diffusion of the silencing signal through the plasmodesmata (connection between plant cells)

-> Silencing can spread systemically: through the phloem

1. To examine gene silencing, GFP-encoding inverted- repeat (IR) DNA is introduced into the GFP- expressing cells. (wildt-type leaf red, GFP plant green)
2. When GFP is silenced, the red chlorophyll fluorescence becomes visible.

experiment in Tobacco

Question 3

What is the role of RNA-dependent RNA polymerase (RdRP) in RNA silencing?

Answer 3

RdRP amplifies the RNA silencing signal by converting target mRNA into dsRNA, which is then processed into secondary siRNAs.
-> secondary siRNAs enhance the silencing effect and spread the silencing signal systemically. These duplexes can move between cells.

(small RNAs can move from shoot to root in ARabdopsis)

Question 4

How do viral suppressor proteins interfere with RNA silencing?

Answer 4

Viral suppressor proteins can inhibit RNA silencing by targeting various components of the RNA silencing machinery, such as Dicer-like enzymes or Argonaute proteins, thereby allowing the virus to escape plant defenses.

Question 5

Viral-induced gene silencing summary slide

Answer 5

• RNA-mediated gene silencing is an important tool in plant defense against pathogens
• siRNAs interfere with viral replication
• siRNAs act systemically to aid in host plant recovery and resistance
• Most viruses produce suppressor proteins that target components of the plant’s siRNA defense pathway; these proteins are important tools for dissecting RNA silencing pathways

Question 6

What are microRNAs (miRNAs), and what is their role in gene regulation?

Answer 6

miRNAs are small non-coding RNAs encoded by MIR genes that regulate gene expression
* by slicing target mRNAs or
* interfering with their translation.

They play crucial roles in developmental timing, stress responses, and other physiological processes.

Question 7

How are miRNAs processed and how do they function?

Answer 7

1. miRNAs are transcribed as primary miRNAs (pri-miRNAs)
2. pri-miRNAs fold into double-stranded structures and are processed by Dicer-like enzymes into mature miRNAs.
3. These miRNAs associate with Argonaute proteins to regulate target mRNAs.

They are highly conserved and are important gene regulators –> neraly half of there targets are transcription factors

Question 8

How do miRNAs regulate the vegetative phase change in plants?

Answer 8

• miRNAs like miR156 regulate the transition from juvenile to adult growth.
• miR156 targets SPL genes, which are promoters of phase change.
• As miR156 levels decrease with plant age, SPL gene expression increases, leading to vegetative phase change. (Pflanze wächst zu adulter flanse heran, bekommt manchmal andere blätternnn)

Question 9

What happens in miR156 overexpression or loss-of-function mutants in plants?

Answer 9

Overexpression of miR156 prolongs the juvenile phase, while loss-of-function mutants exhibit precocious (früreif) phase change, demonstrating miR156’s role in controlling the timing of developmental transitions.

Question 10

microRNAs Summary Slide

Answer 10

• miRNAs are thought to have evolved from siRNAs, and are produced and processed somewhat similarly
• Plants have a small number of highly conserved miRNAs, and a large number of non-conserved miRNAs
• miRNAs are encoded by specific MIR genes but act on other genes – they are trans-acting regulatory factors
• miRNAs in plants regulate developmental and physiological events

Question 11

Conclusion slide about Small RNAs

Answer 11

• Small RNAs contribute to the regulation and defense of the genome, and confer silencing specificity through base-pairing
• siRNA targets include repetitive-rich heterochromatin, transposons, viruses or other pathogens
• miRNAs and tasiRNAs targets include regulatory genes affecting developmental timing or patterning, nutrient homeostasis and stress responses