Lecture 13: Small RNAs in Plants

Describe the petunia experiment that was meant to deepen flower colour, and say what happened instead

• Original Aim: The experiment aimed to modify and deepen flower color by introducing a sense construct of chalcone synthase (CHS), the enzyme at the start of the anthocyanin biosynthetic pathway.

• What Happened Instead: Rather than producing darker purple flowers, both sense and antisense gene constructs unexpectedly inhibited pigment production, resulting in white or variegated/pale flowers where both endogenous and transgene CHS were silenced

Explain why a sense construct and an antisense construct both silenced pigmentation, and say what that result ruled out.

• Effect: Both sense and transgene-derived antisense transcripts triggered the silencing of the endogenous CHS gene.

• What It Ruled Out: This result ruled out simple, direct translation blocking through the formation of sense-antisense RNA duplexes as the sole explanation, pointing instead to a catalytic, homology-dependent post-transcriptional gene silencing mechanism

Define co-suppression, and say which genes are silenced in a co-suppressed plant

• Definition: Co-suppression is the phenomenon where the introduction of a transgene causes the silencing of both the introduced transgene and the homologous endogenous gene.

• Silenced Genes: Both the transgene and the endogenous gene (such as CHS) are silenced

State the evidence that co-suppression is caused by small RNAs, and say where those small RNAs come from

• Evidence: Detection of distinct, extremely abundant small RNAs (siRNAs) associated with co-suppression states in plants.

• Origin: These small RNAs originate from the transcripts of the transgene and homologous endogenous genes through the action of RNA-dependent RNA polymerases and Dicer-like enzymes.

Describe the cross-protection experiment in Nicotiana: the three inoculation treatments, and what the low viral RNA level in the virus-then-virus plant shows

• Three Inoculation Treatments (Day 1 -> Day 22):

  1. Water -> Water (mock control)

  2. Water -> Virus

  3. Virus -> Virus (virus-then-virus)

• Significance of Low Viral RNA: The low viral RNA level in the virus-then-virus treatment shows that the first viral infection induced an RNA silencing defense mechanism, successfully preventing subsequent viral replication.

Explain what virus-induced gene silencing (VIGS) is, and say why co-suppression, VIGS and antiviral defence are all the same pathway

• Definition: Virus-induced gene silencing (VIGS) is a process where viral vectors or viruses trigger the homology-dependent silencing of target host or viral genes.

• Common Pathway: Co-suppression, VIGS, and antiviral defense are manifestations of the same small RNA-mediated silencing pathway because they all rely on the production of siRNAs, Dicer-like processing, and Argonaute (AGO) effector complexes to target homologous RNA sequences.

State what Hamilton and Baulcombe detected in silenced plants, and explain why a small antisense RNA was the result that mattered

• What Was Detected: A specific population of small antisense RNAs (approximately 25 nucleotides long, such as PVX asRNA) associated with post-transcriptional gene silencing.

• Why Small Antisense RNA Mattered: It provided foundational evidence that sequence-specific small RNAs act as the guiding molecules responsible for targeting and degrading homologous RNA in silencing pathways

Define paramutation, and describe what happens at the maize b1 locus when B′ and B-I meet in a heterozygote

• Definition: Paramutation is the interaction between two alleles at the same locus that results in a heritable change in the expression state of one allele induced by the other.

• What Happens at the b1 Locus: When the highly active allele (B-I) and the paramutagenic allele (B') meet in a heterozygote (B'/B-I), rather than showing dominant normal purple pigmentation, the B' allele paramutates B-I into a heritably silenced state, resulting in a pale plant.

State where the sequences required for b1 paramutation lie relative to the gene, and say why that location is notable

• Location: The sequences required for b1 paramutation lie approximately 100 kb upstream of the b1 coding gene.

• Why Notable: This region contains repeat sequences located far upstream in a non-coding regulatory region rather than inside the structural protein-coding sequence of the gene itself.

Explain how the genetics of Mop1 connects paramutation to RNA-directed DNA methylation, and give the steps of the working model

• Connection: Mop1 encodes an RNA-dependent RNA polymerase (homologous to RDR2), and mutations in Mop1 disrupt paramutation, connecting it directly to RNA-directed DNA methylation (RdDM).

• Working Model Steps:

  1. A transcript produced by RNA Pol IV is copied by RNA-dependent RNA polymerase (RDR2/Mop1) to generate double-stranded RNA.

  2. Dicer-like proteins (such as DCL3) cleave the double-stranded RNA into small interfering RNAs.

  3. Small RNAs associate with AGO proteins (such as AGO4) and guide them to target loci interacting with Pol V nascent transcripts.

  4. This complex recruits a methyltransferase (such as DRM2), resulting in targeted DNA methylation and transcriptional gene silencing