Transcript Document
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