Transgene Silencing and RNA

Transgene Silencing

• Transgenes are genes transferred from one organism to another, with Agrobacterium facilitating this in plants.
• mRNA is a complementary copy of the antisense DNA strand, while a sense transcript mirrors the normal RNA product of a gene.
• An anti-sense transcript is the "reverse complement" of normal mRNA.

Pigmentation Modification

• Co-suppression is when endogenous genes & introduced RNA are silenced via siRNA

Virus Resistance

• Viral resistance studies in plants have unveiled the existence of small RNAs.
• Virus-induced resistance in plants resembles that of humans, as new leaves exhibit resistance.
• Virus induced resistance utilizes siRNA mediated silencing.
• Small RNA homologous to viral RNA are found in inoculated and distal systemic leaves, and not in the mock-infected leaves.
• All small RNAs start as double stranded RNAs, though viruses use single stranded RNAs.

Small RNA Production & Function

• Small RNAs impact endogenous protein production by changing chromatin state, cleaving mRNA transcripts, or inhibiting translation.
• Small RNAs biogenesis involves DCL cleaving dsRNA, HEN1 methylating small RNA duplexes, and AGO binding small RNAs to interact with mRNA.
• MIR genes are transcribed into microRNA genes (miRNAs).
• The primary miRNA (pri-miRNA) transcript folds back into a doublestranded structure and is processed by DCL1.
• The pri-miRNA* strand is degraded
• DICER-LIKE (DCL) proteins cleave long dsRNA or hairpin RNA into 21–24 nt fragments.
• DCLs uniform size chopping structure enables RNA quantification
• Plants possess at least four DCL proteins, suggesting pathogen response flexibility.
• Plant miRNAs and siRNAs are processed by related but different DCL proteins.
• ARGONAUTE (AGO) proteins bind small RNAs which target mRNA
• Small RNAs associate with AGO to form the core of the RISC

AGO Types & Functions

• AGO1 binds miRNAs or tasiRNAs to cleave target mRNAs, or it binds miRNAs but inhibits translation.
• AGO2 binds diRNAs and mediates DSB repair
• AGO4 binds 24 nt siRNAs and mediates DNA methylation
• Arabidopsis AGO10 and rice AGO18 act as decoys for miR165/166 and miR168, respectively.

MicroRNA Structure & Diversity

• Conserved structure is common among microRNA transcripts, while the sequence between orthologs differs significantly
• Highly conserved miRNA region is related to its conserved miRNA binding site
• Six microRNA families exist within land plants, many microRNAs are specific to a species or genus.

miRNA's Role in Leaf Morphology

• Flat leaf blades require coordinated cell growth in central and marginal regions.
• Marginal cells that grow more slowly create a cup shape, while those that grow more rapidly will be crinkly shaped
• CINCINNATA (CIN) mutations cause crinkly leaves because the distal to proximal wave of cell division stops
• A concave wave front results in more marginal cell proliferation

Gene Discovery via Activation Tagging

• Random insertion of a transgene containing enhancer elements overexpresses nearby genes enabling cloning sequence identification
• Screening based on plant phenotype helps find mutants with irregular leaves.

MicroRNA & CIN Expression

• MicroRNA miR-JAW controls CIN expression.
• Overexpression of miR-JAW causes TCP RNA degradation

Degradome Sequencing & Translational Repression

• MicroRNA and siRNA transcript cleavage can be monitored genome-wide using degradome sequencing.
• Detection of translational repression occurs via fusion of a microRNA target gene with a reporter.

Summary of Small RNAs

• MicroRNAs often belong to large multi-gene families, and have almost identical sequences between family members
• MicroRNA target genes frequently group into multi-gene families.
• Target gene divergence can be high, target site remains highly conserved.
• Expressed miRNA sequence should detectable changes mRNA levels within a miRNA mutant
• Translational repression requires direct measurement of protein levels
• Small RNAs contribute regulation and defense through base pairing
• Targets for siRNA and microRNA:
  • siRNA - heterochromatin, transposons, viruses, and pathogens.
  • microRNA - regulatory genes governing development, nutrient balance, and stress responses.

Plant Hormones

• Phytohormones function as small endogenous signaling molecules influencing plant growth and development.
• Examples include ABA, Auxin/IAA, JA, and SA.

Auxin's Effect on Plant Growth

• Growth promoting chemical at stem apex diffuses into stem.
• Increased auxin accumulation on shady portion of plant stems generates uneven growth through asymmetric transport

Auxin Resistant Mutants

• AUXIN mutated Arabidopsis lines produce long roots in auxin

Auxin in Protein Degradation

• AXR1 cloned by position to EMS mutations
• TIR 1 cloned via T-DNA tagging
• This type of complex is responsible for ubiquitin mediated protein degradation15 by the proteosome.
• Ubiquitin, comprised of 72 amino acids, covalently attaches to lysine residues on a target protein.

Auxin Protein Degradation

• Fusing domain II to LUC reporter confers auxininduced degradation
• “degron”: Short amino acid sequence that is necessary and sufficient enough to alter protein structure
• TIR1 can bind to the conserved degron in domain II of Aux/IAA proteins

Auxin Plant Hormones with Signaling Abilities Cont.

• A type of Aux/IAA protein, are short lived
• ARF proteins are short lived, nuclear-based proteins that promote activate auxin-induced gene expression
• ARE (Auxin Response Element) DNA sequence is what’s important in Auxin production (i.e cell elongation and phototropism)
• .Auxin triggers distinct impacts according to cell type.
• Each type of auxin features varying affinities for other binding partners based on individual genetic characteristics
• High, yet adjusted expression of both cellular attributes and corresponding DNA is key for proper auxin production (i.e precise cellular fine-tuning) for best downstream function

Agrobacterium Tumefaciens

• Bacteria known to trigger tumor like symptoms in certain plants by altering plant hormone production as well as cell function; may contain opines
• Transferrable genes can enter new species by creating self inserting sequences called transposons

Plant Hormone Transfer

• Can be caused specifically during cellular conjugation by different transferrable factors
• Agrobacteria is needed to initiate the transfer process from cell to cell
• Transferrin insertion can cause cell morphing (tumors) and is often hard to reverse because it is very stable

Gene Encoding Properties & Location

• Genes encoding proteins like nopaline are passed via tDNA in certain tumor cell lines
• The T-DNA itself can be altered to remove hormones and opines using separate antibiotic resistance genes,
• A more specific location is made available as scientists can take the newly inserted gene and conduct further, more precise operations
• T-DNA transfers to either the egg or sperm in cell reproduction and self pollination occurs This allows the discovery of certain genes in offspring

Methods in Cell Selection

• T-DNA tagged plants are isolated from wild type strands so that genetic expression can be viewed much more clearly from more “pristine genetic stock”

Pathogens

• Includes microbes and large herbivores that use animals for transport
• Pathogens enter plants by various methods including entry points such as fungus, wounds and seeds

Defense Mechanisms

• Biotrophs get their nutrients from living plant tissues, and often use infection points to penetrate the plant cell without causing extensive damage, necrtrophs do the opposite
• Hemibiotrophs switch between methods given environmental changes
• Necrotophs must kill, biotrophs often do not affect survival

Plant Immune Function

• Includes Pattern Triggered Immunity and Effector Triggered Immunity
• PTI happens before attack, by distinguishing structural anomalies
• ETI happens after cellular attack, using more reactive compounds