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Lecture 3
Agrobacterium tumefaciens
The transfer f DNA from Agrobacterium tumefaciens into plants: a feast of
fundamental insights
Zupan et al (2000)
The Plant Journal 23(1) 11-28
Gene silencing
Identification of a novel RNA silencing suppressor, NSs protein of
Tomato spotted wilt virus(2005) Febs letters 53275-79
Atsushi Takedaa, Kazuhiko Sugiyamaa, Hideaki Naganob;1,
Masashi Moric, Masanori Kaido; Kazuyuki Misea;, Shinya Tsudab,
Tetsuro Okunoa
Glycosylation
From planta to pharma with glycosylation in the toolbox
Claude Saint-Jore-Dupas, Loı¨c Faye and Ve´ ronique Gomord
Trends in Biotech (2007) 25 (7)
pTi- tumor inducing plasmid
T-DNA is transferred from bac to
plant
25bp repeats on right and left
Virulence region on pTi
Vir induced by wounds in plant –
acetosyringone
T-complex = ssTDNA +VirD2 at 5’
and VirE2 on DNA
T-complex transporter –
transports from bac to plant
Plant cell
T-complex is imported into nucleus
Vir E2 targets DNA to nucleus with NLS
VirD2also NLS 5’ end to enter first
Vir E2 protects ssDNA against
degradation
T-DNA is made ds
Can get transient expression of genes
T-strand is integrated into the plant
chromosome
Vir D2 helps with integration into
chromosome at 5’ end
DNA transfer into plants with
Agrobacterium tumefaciens
Gene silencing
PTGS -Post-transcriptional gene silencing
 Defence of plant to viral infection
 Recognises dsRNA

 21-25nt long siRNA
Then degrades homologous DNA
 PTGS is induced in one site and then
spread systemically throughout plant

S-PTGS- sense gene induced
 Get dsRNA made by RNA dependent RNA
pol
 IP-PTGS (Inverse repeat)
 Induced by inverse repeats of transgene

Silencing suppressors
Made in response to silencing by virus
 NSs

 Tomato spotted wilt virus -TSWV

P19
 Tomato bushy stunt virus -TBSV
Silencing suppression
PTGS -Post-transcriptional gene silencing
Dicer is an RNaseIII-like enzyme.
siRNAs guide an RNA-induced silencing
complex (RISC) to mRNA degradation.
NSs could interfere with a step(s) for
generating the dsRNA in the S-PTGS
pathway. In addition, NSs might weakly
interfere with a later step(s) after the
generation of dsRNA.
TBSV p19 would bind siRNAs and
suppress S-PTGS and IR-PTGS
GFP
NSs
p19
Glycosylation of PMP
Glycosylation is covalent linkage of
oligosaccharide to proteins
 Most common post-translational modification
 More than half of human proteins are
glycosylated
 Glycosylation has effect on

 Half-life
 Targeting
 Biological activity
Glycosylation
Most common:
 N-glycosylation

 Oligosacchride is attached to Asn-asparagine
 To amide nitrogen
O-glycosylation
 Attached to Hydroxyl of

 Thr-Threonine,
 Ser – Serine
 Hyp - hydroxyproline
Mammlian cell lines
Glycosylation also varies from cell to cell
type
 CHO different to human glycosylation
 Plants perform N-glucosylations
 N-glycosylation starts in ER
 Precursor oligosacc transferred to
Asn-X-Ser/Thr (X any aa except Pro)

N-glycan liked to Asn undergoes
maturation as protein is transported along
secretory pathway
 Maturation:

 Removal of Glucose Glc and Mannose Man
 Addition of new sugars in ER and Golgi
Plant
Animal
3
1
2
1. β 1,2 Xylose
β mannose
2. α 1,3 fucose
α1,6 fucose
3. β1,3 galactose and fucose
β1,4 galactose
Plant glycosylation
Plants have Lewis glycosylation,
 β 1,2 Xylose
 α1,3 fucose
 These sugars are immunogenic

 Could be good or bad
Try and eliminate PTM in PMPs
 Glycoengineering of PMPs

 Target to ER
 Inactivate endogenous glycotranferases
 Express heterologous glycotranferases
ER targeting and Knockout plants

Add KDEL or HDEL to C-terminal
 Have high Manose type N-glycans

Knockout glycosyltransferase plants
 β 1,2 Xylosyltransferase
 α1,3 fucosyltransferase

Knock-in human like glycosylation
– β1,4 galactosyl transferase and sialyltransferase
– addition of sialic acid
 Second last galactose is capped by addition of sialic acid
 Sialylation increases ½ life of PMPs
Glycosylation