Here I am explaining about the different Genetic Engineering approaches for development of disease resistance

1. DEVELOPMENT OF DISEASE RESISTANT PLANTS USING
GENETIC ENGINEERING APPROACHES
Presented by
Mr. Sabhavat Srinivasnaik
ID.No.RAD/21-25
Course In-charge
Dr. G. Uma Devi, Sr. Prof. & Univ. Head
Department of Plant Pathology
DEPARTMENT OF PATHOLOGY
COLLEGE OF AGRICULTURE, RAJENDRANAGAR
PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY

2. Biotechnology Applications
Protoplast Genetic Engineering
Biolistic method of Genetic Engineering
Genome editing approach for Genetic engineering
CONTENTS
RNA based approach for Genetic engineering
Genetic engineering approaches
Agrobacterium mediated Genetic engineering
RNA based approach for Genetic engineering
R genes based approach
Case Study and Conclusion

3. BIOTECHNOLOGY APPLICATIONS
Biotechnology
Medical Biotechnology
Vaccines
Therapeutics
Diagnostics
BioServices and
BioInformatics
Clinical
research
Contract
manufacturing
Contract
research
BioAgriculture
Transgenic crops
Biopesticides
Biofertilizers
Tissue culture
BioIndustrial
Enzymes
Diagnostics
Textiles
Food
Pharmaceuticals
Leather
Paper
Biofuels

4. GENETIC MODIFICATION IS AGE OLD PRACTICE
Ten thousand years of genetic modification
• Selection
• Crossing
• Mutagenesis
• Genome fusion
• Genetic engineering
Wild tomato Domestic tomato Domestication of corn
from weedy grass

5. GENETIC ENGINEERING NATURAL PHENOMENA
Agrobacterium tumefaciens mediated crown gall disease in plants
Transposable elements in maize

6. TRANSGENIC TECHNOLOGY
DNA as genetic
material governs
traits
Vector for plant
transformation
Agrobacterium-
mediated genetic
transformation
Regeneration and
differentiation

7. COMPARISON BETWEEN CONVENTIONAL
AND GENETIC ENGINEERING

8. PLANT GENETIC ENGINEERING APPROACHES
Approach Technique
Indirect Agrobacterium-mediated
gene transfer
A. tumefaciens, A.rhizogenes
Direct Gene transfer to protoplast Electroporation
PEG-mediated
Liposome fusion
Microinjection
Ligand polycation based
receptor mediated
Biolistic gene transfer Microprojectile
Agrolistic
Microtargeting
RNA based RNA intereference RNA based approach
DNA editing Genome editing approach CRISPR-Cas9, TALENs,

9. GENE TRANSFER
The transfer of desirable gene from one organism to another and the
subsequent integration & expression of a foreign gene in the host genome.
Why gene transfer?
•Crop improvement [quality, yield (QTL)]
•Resistance to biotic agents (insects, pathogens .etc)
•Resistance to abiotic agents (cold, stress tolerance)
•Improved performance
•Value-added traits
•Production of novel biochemicals & vaccines
Co transformation: Plants are produced with more than one gene/trait
simultaneously in the plant genome via transformation with two vectors.
These transgenes are integrated in a single locus
Multiple or re-transformation: Plant produced by integrative transformation
with vectors containing different transgenes/ traits.
These transgenes are integrated in multiple loci.

10. AGROBACTERIUM SPECIES AND HOST RANGE
The genus Agrobacterium has been divided into a number of species on the
basis of symptoms of disease and host range.
A. radiobacter is an “avirulent” species,
A. tumefaciens causes crown gall disease,
A. rubi causes cane gall disease,
A. rhizogenes causes hairy root disease and
A. vitis causes galls on grape and other plant species.
A. tumefaciens can transform a remarkably broad group of organisms including
dicots, monocots and gymnosperms. In addition, it can also transform fungi,
including ascomycetes, basidiomycetes and yeasts.
Agrobacterium tumefaciens is a soil-borne, gram negative, motile, rod shaped
bacterium.
Two mostly used species of Agrobacterium plasmids as vectors:
Ti (tumor inducing) plasmid - A. tumefaciens
Ri (root inducing) plasmid – A. rhizogenes

11. AGROBACTERIUM MEDIATED GENETIC TRANSFORMATION

12. TUMOR INDUCED BY AGROBACTERIUM
Agrobacterium contains a transfer DNA (T-DNA) located in its tumor-inducing
(Ti) plasmid that is transferred into the nucleus of an infected plant cell. The T-
DNA gets incorporated into the plant genome and is subsequently transcribed.
The T-DNA integrated into the plant genome carries not only oncogenic genes but
also opine synthesizing genes

13. AGROBACTERIUM CAUSE CROWN TUMORS BY TRANSFORMUNG PLANTS

14. MOLECULAR BASIS OF AGROBACTERIUM MEDIATED TRANSFORMATION
 The virulent strains of A. tumefaciens harbor large plasmids (140-235 kb)
known as tumor inducing (Ti) plasmid involving elements like 1.T-DNA 2.vir
region 3. origin of replication 4. region enabling conjugative transfer 5.
O-cat region (required for catabolism of opines).

15. FUNCTIONAL REGIONS OF Ti PLASMID
1) T-DNA: A 25 kb segment containing genes for the synthesis of opines and
oncogenes.
2) vir-region: genes induce the transfer of T-DNA but are themselves not
transferred.
3) Opine catabolism region: produce enzymes needed for the utilization of
opines by the bacterium produced by the infected plant.
4) Conjugative transfer (tra) region: helps in conjugative transfer of the
plasmid between bacteria.
5) Origin of replication: functions in the propagation in Agrobacterium.

16. T-DNA
• It is a small, specific segment of the plasmid, about 25 kb in size and found
integrated in the plant nuclear DNA at random site. This DNA segment is
flanked by right and left borders.
Genes on T-DNA
• The T-DNA contains two groups of genes, for the synthesis of opines and
oncogenes.
• Oncogenes for synthesis of auxins and cytokinins (phytohormones). The
over-production of phytohormones leads to proliferation of callus or tumour
formation.
• Opine synthesizing genes for the synthesis of opines (a product from amino
acids and sugar phosphates secreted by the crown gall infected cells and
utilized by A. tumefaciens as carbon and nitrogen sources).
• Thus opines act as source of nutrient for bacterial growth, e.g. Octopine,
Nopaline.
16

17. ORGANIZATION OF T-DNA
• T-DNA element is defined by its borders but not the sequences within.
So researchers can substitute T-DNA coding region with any DNA
sequence without any effect on its transfer from Agrobacterium to the
plant.

18. ORGANIZATION OF T-DNA
• vir region, about 40kb size, is located adjacent to the left border repeat of the
T-region.
• Virulence genes aid in the transfer of T-DNA into the host plant cell. Ti
plasmid contains 35 vir genes arranged in 8 operons, vir A to vir H.
• Mutations in the vir A, vir B, vir D & vir G operons eliminate tumor
formation while mutations in other loci (vir C, vir E, vir F & vir H) leads to
restriction in plant host range.
• Unlike the octopine Ti plasmid, the nopaline Ti plasmid lacks the vir F and
vir H operons

19. T DNA TRANSFER, INTEGRATION AND EXPRESSION

20. T DNA TRANSFER, INTEGRATION AND EXPRESSION
Step 1: Production of signal molecules from wounded plant cell;
Step 2: Recognition of signal molecules by bacterial receptors;
Step 3: Attachment of Agrobacterium to plant cell;
Step 4: Activation of Vir proteins which process ss-TDNA;
Step 5: Formation of immature T-complex;
Step 6: T-DNA transfer;
Step7: Assembly of mature T-complex and Nuclear transport;
Step 8: Random T-DNA integration in the plant genome;
Step9: Expression of bacterial genes and synthesis of bacterial proteins.

21. RECEPTORS INVOLVED IN INITIAL BINDING
• Plant vitronectin-like protein (PVN, 55kDa) was found on the surface of
plant cell. This protein is probably involved in initial bacteria/plant cell
binding.
• Aside from PVN, rhicadhesin-binding protein was found in pea roots.
• Also, rat1 (arabinogalactan protein; AGP) and rat2 (potential cell-wall
protein) are also involved.
Signal recognition by Agrobacterium: The wounded plant cells release certain
phenolic signal molecules acetosyringone and α-hydroxy acetosyringone
which are recognized by Agrobacterium.
• Acetosyringone which are strongly attractive at even very low
concentrations (10-7 Molar).

22. PLANT SIGNALS
 Wounded plants secrete sap with acidic pH (5.0 to 5.8) and a high content
of various phenolic compounds (lignin, flavonoid precursors) serving as
chemical attractants to agrobacteria and stimulants for vir gene expression.
 Among these phenolic compounds, acetosyringone (AS) is the most
effective.
 Sugars like glucose and galactose also stimulate vir gene expression
when AS is limited or absent. These sugars are probably acting through
chvE gene to activate vir genes.
 Low opine levels further enhance vir gene expression in the presence of AS.

23. ATTACHMENT OF AGROBACTERIUM
Attachment of Agrobacterium to plant cells: is two step process
i) Loosely bound step:- initial attachment through acetylated
polysaccharides-
ii) Strong binding step:- Mesh of cellulose fibres produced by the
bacterium, to stabilize the initial binding, resulting in a tight association
between Agrobacterium and the host cell.
virulence genes (chv genes) are also involved in the attachment of
bacterial cells to the plant cells

24. INDUCTION OF vir GENES
 Acetosyringone (about 10-5 to 10-4 Molar) activates the virulence genes VirA
on the Ti plasmid.
 Vir A is subsequently auto phosphorylated and thereby activating Vir G.
 VirG protein is a transcriptional activator of other vir genes and is inactive in
non-phosphorylated form. The activation of Vir G thus induces the
expression of the other vir genes.
 VirG interacts with the vir box to activate transcription.
 • VirH2 : detoxify phenolics secreted by plants; also mineralize some
phenolics, making them as agrobacteria’s carbon source.
 • KatA: catalase that can detoxify hydrogen peroxide so HR (hypersensitive
response) will be inhibited.

25. FORMATION OF DNA COMPLEX
•Vir D1/D2 border-specific endo nucleases recognize the left and right borders
of T-DNA.
•Vir D2 induces single stranded nicks in Ti plasmid causing the release of the
ss-T DNA.
•Vir D2 then attaches to the 5'-end of the displaced ss-T DNA forming an
immature T-complex.

26. INTERCELLULAR TRANSPORT
• The transfer of T-DNA complex to the plant cell is mediated by Type-IV
secretion system composed of proteins encoded by Vir D4 and 11 genes from
virB operon, only virB2 is essential for T-pilus biogenesis.
• VirB2 is involved in T-pilin export and T-pilus formation.
• Intercellular transport of T-DNA is probably energy dependent, requiring
ATPase activities from VirB4 and VirB11.
• VirD4 serves as a “linker” that helps in the interaction of the processed T-
DNA/VirD2 complex with the VirB2- encoded pilus.
• Other vir genes (Vir E2, Vir E3, VirF, Vir D5) also pass through this T-pilus to
aid in the assembly of T-DNA complex/vir protein complex in the plant
cytoplasm forming a mature T-complex.

27. INTERCELLULAR TRANSPORT
 Vir D2 and Vir E2 protect the ss-T DNA complex from nucleases inside the
plant cytoplasm by attaching to the 5'end.
 Both VirD2 and VirE2 proteins have nuclear localization signals (NLS)
which serves as pilot proteins to guide the mature T-complex to the plant
nucleus.
 The T-DNA-vir D2 complex covered with vir E2, crosses the plasma
membrane and the ssT-DNA becomes double stranded soon after entering
the plant cell. The efficiency of transfer is enhanced by VirC2 proteins,
which recognize and bind to the overdrive enhancer element.
 Some additional proteins like importins, VIP1 and VirF may interact with
the T-strand, either directly or indirectly, to form larger T-complexes in the
plant cell. Vir F directs the proteins coating T-complex (VIP1 and Vir E2)
for destruction in proteasome.
 NUCLEAR TRANSPORT
 Vir E2 protect T-DNA from nucleases, facilitate nuclear localization and
confer the correct conformation to the T-DNA/Vir D2 complex for passage
through the nuclear pore complex (NPC).
 The T-DNA/VirD2/VirF2/Plant protein complex enter the nucleus through

28. BASIC PROTOCOL USED AGROBACTERIUM GENE TRANSFER
1.Identify a suitable explant: Suitable plant tissue is removed and disinfection
of an explant.
2. Isolation and growth of the Agrobacterium vector.
3. Co-culture of the explant with the Agrobacterium: Leaf tissues is cut into
small pieces and then placed into a culture of Agrobacterium for about 30 min.
The explants are subsequently removed from the bacterial culture and placed
on to the MS medium that contain no selective agent. The incubation of
explants with Agrobacterium is allowed for days during which the
transformation occurs.
4. Kill the Agrobacterium with a suitable antibiotics: The explants are
removed from the medium add washed in antibiotic (cefotaxime) solution to
kill the Agrobacterium cell.

29. BASIC PROTOCOL USED AGROBACTERIUM GENE TRANSFER
5. Select for transformed plant cell: The explant are transferred to fresh solid
medium supplemented with a selective agent (Kanamycin).It also contain
cefotaxime.
The cefotaxime kills the agrobacterium cells and the kanamycin kills the non-
transformed cells.
The only left over are the transformed plant discs having the gene of interest
6.Regeneration of whole plant:- Auxin, cytokinin are used to encourage the
regeneration of by organogenesis. High cytokinin to auxin ratio promotes shoot
formation from the explants.
The shoot can be rooted by placing them on solid medium containing a high
auxin to cytokinin ratio.

30. + carbenicillin + kanamycin

31. GENE TRANSFER TO PROTOPLAST
ELECTROPORATION
 This method was first demonstrated by Wong and Neumann in 1982 to
study gene transfer in mouse cells.
 It is now a widely used method for the introduction of transgene either
stably or transiently into bacterial, fungal, plant and animal cells.
 It involves use of a large electric pulse that temporarily disturbs the
phospholipid bilayer, allowing the passage of polar molecules such as DNA.
 The basis of electroporation is the relatively weak hydrophobic/hydrophilic
interaction of the phospholipids bilayer and ability to spontaneously
reassemble after disturbance.
 A quick high voltage shock may cause the temporary disruption of areas of
the membrane and allow the passage of polar molecules. The membrane
reseals leaving the cell intact soon afterwards.

32. ELECTROPORATION
• The host cells are suspended suitable ionic solution and linearized
recombinant plasmid DNA is added to the solution.
32

33. ELECTROPORATION
33
Cells are subjected to:
1.Low voltage - long pulse: 300-400 V cm-1 for 10-50 microseconds
or
2.High voltage - short pulse: 1000-1500 V cm-1 for 10 microseconds ; high
frequency of stable transformation.
Electroporation has been used to produce stable transformed cell lines
and/or plants in several plant spp. e.g., tobacco, petunia, maize, rice,
wheat, etc.,

34. ELECTROPORATION

35. MICROINJECTION
• DNA microinjection was first proposed by Dr. Marshall A. Barber.
• It involves delivery of foreign DNA into a living cell (e.g. a cell, egg, oocyte,
embryos of animals) through a fine glass micropipette.

36. MICROINJECTION-PROCEDURE
 The delivery of foreign DNA is done under a powerful microscope using a
glass micropipette tip of (0.5-10.0µm) diameter.
 Cells to be microinjected are placed in a container. A holding pipette is
placed in the field of view of the microscope that sucks and holds a target
cell at the tip.
 The tip of micropipette is injected through the membrane of the cell to
deliver the contents of the needle into the cytoplasm and then the empty
needle is taken out.
 Microinjection techniques for plant protoplasts utilises a holding pipette for
immobilisng the protoplast.
 While an injection pipette is utilised to inject the macromolecule
 In order to manipulate the protoplasts without damage, the protoplasts are
cultured for from about 1 to 5 days before the injection is performed to
allow for partial regeneration of the cell wall.

37. MICROINJECTION-PROCEDURE
The methods are particularly useful for transformation of plant protoplasts
with exogenous genes.
As the process of microinjection is complete, the transformed cell is cultured
and grown to develop into a transgenic plant.
Transgenic tobacco, Brassica napus, alfalfa have been developed by this
approach.

38. MICROINJECTION-PROCEDURE
It is the method tried for artificial DNA transfer to cereal plants that show
ability to regenerate and develop into whole plants from cultured cells.
Needles used are with the diameter greater than cell diameter.
Around 0.3 ml of DNA solution is injected at a point above tiller node.
Timing of injection is important and should be 14 days before meiosis.
Successfully used in Rye.

39. BIOLISTIC GENE TRANSFER
PARTICLE BOMBARDMENT
 Prof Sanford and colleagues at Cornell University (USA) developed the
bombardment concept in 1987 and coined the term “biolistics” (short for
“biological ballistics”) for both the process and the device.
 Also termed as particle bombardment, particle gun, micro projectile
bombardment and particle acceleration.
 It employs high-velocity micro projectiles to deliver substances into cells
and tissues.
USES
 This method is commonly employed for genetic transformation of plants
and many organisms.
 This method is applicable for the plants having less regeneration capacity
and those which fail to show sufficient response to Agrobacterium-
mediated gene transfer in rice, corn, wheat, chickpea, sorghum and pigeon-
pea.

40. WORKING SYSTEM OF PARTICLE BOMBARDMENT GUN

41. PARTICLE BOMBARDMENT APPARATUS
 It consists of a bombardment chamber which is connected to an outlet for
vacuum creation.
 The bombardment chamber consists of a plastic rupture disk below which
macro carrier (macro projectiles) is loaded with micro carriers
(microprojectiles).
 These micro carriers consist of tungsten or gold particles (1-2 µm) coated
with DNA, are carried by macro carriers (macro projectiles).
 The apparatus is placed in Laminar flow while working to maintain sterile
conditions.
 The target cells/tissue is placed in the apparatus and a stopping screen
is placed between the target cells and micro carrier assembly.
 The passage of high pressure helium (about 1000 p.s.i). ruptures the
plastic rupture disk propelling the macro carrier and micro carriers.
 The stopping screen prevents the passage of macro projectiles but
allows the DNA coated micro pellets to pass through it thereby,
delivering DNA into the target cells.

42. SILICON CARBIDE FIBRE MEDIATED TRANSFORMATION
 The silicon carbide fibres (SCF) are about 0.3-0.6 µm in diameter and 10-
100 µm in length.
 These fibres are capable of penetrating the cell wall and plasma membrane,
and thus can deliver DNA into the cells.
The DNA coated silicon carbide fibres are vortexed with plant
material (suspension culture, callus).
During this mixing, DNA adhering to the fibres enter the cells and get
stably integrated with the host genome.
The silicon carbide fibres with trade name Whiskers are available in
the market.

43. SONOPORATION
 Sonoporation involves the use of ultrasound for temporary
permeabilization of the cell membrane allowing the uptake of DNA from
the extracellular environment.
 Immersion of explant in sonication buffer containing plasmid DNA and is
then sonicated with ultra sonic pulse regenerator at 0.5w/cm2
acoustic cavitation intensity for 30 mins
 Then the sample are rinsed in buffer solution, and then cultured for growth
and differentiation

44. RNA Interference Technology
 RNA-dependent gene silencing process, which is initiated by a RNAse III enzyme
(Dicer) that cleaves a long double-stranded RNA (dsRNA) into double stranded
small (∼20-25 bp nucleotide) interfering RNAs (siRNAs) with a two-nucleotide
overhang at the 3′ end.
 Each siRNA is composed of a passenger (sense) strand and a guide (antisense)
strand.
 Guide strand is incorporated into an active RNA-induced silencing complex
(RISC).
 The guide strand of the siRNA-RISC complex then base-pairs with the
complementary mRNA target sequences and initiates endonucleolytic cleavage
through the action of induced Argonaute protein (AGO; catalytic component of the
RISC complex) preventing translation of the target transcript.

45. RNA Interference Technology

46. RNA Interference Technology
The protocol for RNAi mediated gene silencing involves designing siRNAs, delivering
them into cells, and analyzing the knockdown efficiency.

47. GENOME EDITING APPROACH-CRISPR-Cas9
 The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -
CRISPR-associated (Cas) is a widely-used genome editing platform. CRISPR-Cas
originates from a prokaryotic acquired immune system against viruses. Cas9
recognizes and cuts at DNA targets complementary to the variable region on the
guide RNA with an adjacent protospacer adjacent motif (PAM).
 The variable region of guide RNA can thus be programmed to direct Cas9 to
generate double stranded breaks at specific targets in a given genome. Subsequent
cellular DNA repair processes often introduce modifications at the DNA breakpoints

48. GENOME EDITING APPROACH-CRISPR-Cas9

49. GENOME EDITING APPROACH-ZFNs
 Zinc-finger nucleases are synthetic restriction enzymes that can cleave any long
stretch of double-stranded DNA sequences .
 ZFN monomer is an artificial nuclease engineered by fusing two domains: a non-
specific DNA cleavage domain of the Flavobacterium okeanokoites I (FokI) and a
Cys2-His2 zinc finger domain.
 Digestion of target DNA can be achieved when two ZFN monomers bind to their
respective DNA target sequences. The two ZFN monomers will flank a 5- to 6-bp-
long sequence within the DNA target sequence, allowing the FokI dimer to digest
within that spacer sequence. Upon dimerization, FokI introduces a tailor-made DSB
in the spacer sequence surrounded by two zinc finger array binding sites.
 Multiple resistance against various begomoviruses, including Tomato yellow leaf
curl China virus (TYLCCNV) and Tobacco curly shoot virus (TbCSV) was
achieved by targeting a specific site in the viral DNA Resistance to bialaphos in
maize , resistance to herbicides in tobacco and ABA-insensitive phenotype
in Arabidopsis were achieved with ZFN technology.

50. GENOME EDITING APPROACH-TALENS
 TALENS are translocated by Xanthomonas bacteria through their type III secretion
system into the plant cells. TALENs can be engineered to bind any desirable DNA
sequence that when fused to a nuclease (TALEN) can introduce DNA breaks at any
specific location.
 Rice bacterial blight is controlled by the interaction between TALE of Xanthomonas
oryzae pv. oryzae (Xoo) and the host target S genes. TALENs bind to the effector
binding elements (EBEs) in the promoter region of the S genes, resulting in
disruption of the EBEs and impairment of the molecular interaction between TALEs
and the host S genes and subsequent improvement in disease resistance
against Xoo strains.

51. R GENES BASED APPROACH
 Plant defense responses are triggered by the recognition of Pathogen
Associated Molecular Patterns (PAMPs) by the plant extracellular receptor
like kinases (RLK) and rapidly trigger a signaling cascade through MAP
kinases resulting in basal immunity.
 Plants then develop and obtain immunity to the pathogens through expression
of distinct R-genes

52. CASE STUDY

53. CONCLUSION
Recently, genome editing tools are also used for development of disease
resistant plants
Biotechnology has a great role in development of disease resistant plants
using genetic engineering approach
Genetic engineering is not a new concept, it is being evolved from the
nature itself
The GE can done using Agrobacterium spp., Direct gene transfer
(Protoplast fusion), Biolisitc approach, RNA based approach (RNA
interference)
RNA interference is not only used in development of disease resistant
plants but also insect pest management field also.
The development of GM crops is useful to meet the demand of increased
population but safety is a major concern governed by GEAC at national
level

54. REFERENCES
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resistance in plants. F1000Research, 1-8
Chandrashekara, K.N and K. Boopal. 2012. Transgenics: A Genetic Engineering Approach to
Develop Disease Resistance in Plants, In: Eco-friendly Innovative Approaches in Plant
Disease Management: 303-324
Oliver Xiaoou Dong and Pamela C. Ronald. 2019. Genetic Engineering for Disease Resistance in
Plants: Recent Progress and Future, 1-58
Owen Wally and Zamir K. Punja. 2010. Genetic engineering for increasing fungal and bacterial
disease resistance in crop plants. GM Crops 1:4, 199-206
Peter van Esse, H, T. Lynne Reuber and Dieuwertje van der Does. 2020. Genetic modification to
improve disease resistance in crops. New Phytologist. 225: 70-86.
Piquerez, J. M., Sarah E. Harvey, Jim L. Beynon and Vardis Ntoukakis.2014. Improving crop
disease resistance: lessons from research on Arabidopsis and tomato Sophie. Frontiers in
Plant Science: 1-13
Swati Tyagi, Robin Kumar, Vivak Kumar, So Youn Won and Pratyoosh Shukla. 2021.
Engineering disease resistant plants through CRISPR-Cas9 technology, GM Crops & Food,
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