This document discusses various biotechnological approaches for plant disease management, including tissue culture, recombinant DNA technology, and transgenic approaches. Tissue culture techniques like meristem culture can produce disease-free planting materials. Recombinant DNA technology allows generation of resistant plants by expressing genes conferring resistance to bacterial, fungal or viral diseases. Transgenic approaches discussed include pathogen-derived resistance utilizing viral coat protein or movement genes, as well as expressing plant disease resistance genes, ribosome-inactivating proteins, and genes involved in systemic acquired resistance.

2. Topic : Biotechnological
Approaches in Plant
Disease Management
Rahul M
M Sc Genetics and
Plant Breeding

3. Biotechnological
approaches
Tissue culture
r DNA
technology

4. Tissue culture
• Use of vegetative part for clonal propagation
• Confers disease free planting materials(apical
meristem culture)
• Development of resistant plants

6. • Somaclonal variation for disease
resistance .
• Screening
• Cell selection (resistant to toxin)
Mutants
from plant
cell culture
• Pollen or megaspore culture
• Screening for resistant haploid
• Treatment with colchicine
Dihaploids
production
• Hybrid cell – fusion of protoplast of
two cell
• Cybrid cellls – fused cell contains
cytoplasm of one cell and nuclei of
other cell
• Useful in combining two resistant
haploid cells
Protoplast
fusion

7. Recombinant DNA Technology
Transformati
on of plants
Cloning
of
vector

9. Transformation of plants

10. Resistance to fungal and bacterial
diseases
• Occurrence of disease is explained by gene for gene
hypothesis.
• It may be classified in to incompatible and
compatible reaction.
Resistance
gene
Virulence gene
Incompatibility compatibility
A1 a1 A1 a1
R1 Resistance susesptible Resistance Resistance
r1 susesptible susesptible Resistance susesptible

11. Incompatible reaction
• Seen in biotropic
pathogens.
• Interaction of specific
compounds produced
by host R gene and
pathogen A gene results
in resistance.
• Rust, Smuts .
Compatible reaction
• Seen in heterotropic
pathogens.
• Lack of interaction
between products of
host r gene and
pathogen a gene results
in resistance.
• Helminthosporium leaf
blight.

12. Recombinant DNA Technology
• Disease resistance can be achieved by expressing
(bacterial and fungal diseases)
Gene encoding insensitive target enzyme
Gene specifying toxic inactivation
Antibacterial peptides
Bacterial lysozyme
Genes specifying artificially programed cell death

13. Heterologous phytoalexins
Gene encoding ribosome inactivation protein
Heterologous thionins
Ectoip expression of pathogenesis related proteins
Ectoip expression of chitinases

14. Artificially programed cell death
• PCD is brought out by endogenous genes in
response to specific stimulus (elicitors).
• Hypersensitive response.
• artificial PDC requires exact interaction of avr
and R genes.
• Two schemes are
1.two component system
2.one component system

15. Two way component system
• Two precisely matched
transgenes are expressed in cell
• One is avr ,other is R
• avr must be expressed exactly
after pathogen attack in only
infected cells.
• Elicitor produced by avr is
recognised by R grne prodect
leading to PCD
One way component system
• It is based on expression of
toxic polypeptide.
• Trance gene should produce
toxin ribonucleases which kill
cell.
• Pathogen attack leads to
expression of this genes and
causes PCD.
• barenase gene from
B. amyloliquefaciens

16. Two way component system
path
ogen
avr R
elicitors
receptor
attack
PCD
cell

17. One way component system
cell
bar
RNase
PDC
pathogen

18. Important viral diseases

19. Based on source of gene
Pathogenic
(PDR)
Other source

20. Pathogen-Derived Resistance
• Pathogen-Derived Resistance (PDR) : A part, or a
complete viral gene is introduced into the plant,
which, subsequently, interferes with one or more
essential steps in the life cycle of the virus
• This was first illustrated in tobacco by Roger Beachy
and coworkers.
• Introduced the coat protein (CP) of tobacco mosaic
virus (TMV) into tobacco and observed TMV
resistance in the transgenic plants.

21. Viral diseases
Viral coat protein gene
DNA copy of viral
satellite RNA
Defective viral
genome
Antisense constructs
of gene and ribozymes

22. Coat protein
• The use of viral CP as a transgene for producing virus
resistant plants.
• This was first demonstrated for Tobacco mosaic
virus (TMV) in tobacco by Powel-Abel in 1986.
• The resistance was manifested as delayed appearance
of symptoms as well as a reduced titre of virus in the
infected transgenic plants,

23. • Several important crops have
been engineered for virus
resistance using CPMR
approach and released for
commercial cultivation.
• Tomato resistant to TMV,
• Tomato mosaic virus (ToMV)
and cucumber mosaic virus
(CMV);
• The transgenitically expressed CP sub-units are believed to re-coat
the nascent disassembled viral RNA which leads to a decreased
pool of the available viral RNA for translation, resulting in
resistance
• Cucumber resistant to CMV,
squash resistant to zucchini yellow
mosaic virus (ZYMV) cantaloupe
resistant to ZYMV, WMV2 and
CMV;
• Potato resistant to PVX, potato
virus Y (PVY) and potato leafroll
virus (PLRV);
• Transgenic papaya (var. sunset)
with CP gene was grown from
1991 to 1993, and remained virus-
free for 25 months(in Hawaii)

24. Movement protein
• Movement proteins (MP) are essential for cell-
to-cell movement of plant viruses.
• These proteins have been shown to modify the
gating function of plasmodesmata, thereby
allowing the virus particles or their
nucleoprotein derivatives to spread to adjacent
cells
• The conferred resistance is believed to be
based on the competition between wild-type
virus encoded MP and the preformed
dysfunctional MP to bind to the
plasmodesmatal sites.
• example resistance against TMV could be
achieved in tobacco using the MP derived from
brome mosaic virus.

25. Satellite RNA
• Some strains of CMV encapsidate satellite RNA
(sat RNA) in addition to the tripartite messenger
sense, single-stranded RNA genome
• The presence of sat-RNA modulates the
symptoms induced by the Helper Virus and often
depresses HV accumulation in different host
species.
• By combining sat-RNA and CMV CP high-level
of tolerance to CMV conferred in tomato.

26. Defective-interfering viral nucleic
acids
• In several viruses, genomic components are
often detectable in infected tissues, which
interfere with the replication of the genomic
components. These species of DNA are also
called defective interfering (DI) DNA

27. OTHER SOURCES
• Non-pathogen-derived resistance: It is based on
utilizing host resistance genes and other genes
responsible for adaptive host processes, elicited in
response to pathogen
• utilization of plant
disease resistance
genes,
• the ribosome-
inactivating proteins,
• plant proteinase
inhibitors,
• human interferon-like
systems,
• antiviral antibodies in
plants,
• systemic acquired resistance
• secondary metabolite
engineering

28. Post-transcriptional gene silencing
• Post-transcriptional gene silencing (PTGS) is a
specific RNA degradation mechanism of any
organism that takes care of aberrant, unwanted
excess or foreign RNA intracellularly in a
homology-dependent manner.
• This activity could be present constitutively to
help normal development or induced in response
to cellular defence against pathogens

30. Plant and viral genes inducing or
repressing PTGS

31. Plant disease resistance genes
• R genes in plants are defined by the classical gene-for gene
hypothesis, which states that for every incompatible host
pathogen interaction, there exist matching R genes in the
host and avr genes in the pathogen
• All known R genes encode products having two basic
functions: to act as sensors for the corresponding avr
factors/elicitors and to initiate signalling cascades for the
expression of defence-related genes.
• These include leucine-rich repeat (LRR), nucleotide-
binding site (NBS), serine-threonine kinase, leucine zipper,
toll-interleukin region (TIR), etc

32. R genes against viruses and
corresponding avr gene products

33. Ribosomal inactivating proteins
• Several plants have been found to contain antiviral
proteins, commonly termed as ribosome-inactivating
proteins (RIPs).
• RIPs inhibit the translocation step of translation by
catalytically removing a specific adenine base from 28S
ribosomal RNA.
• The genes for RIPs have been isolated from a number
of plant sources. The cDNAs for PAP (Pokeweed),
MAP (Mirabilis jalapa), Trichoxanthin (Trichoxanthes
kirilowi), Dianthin (Dianthus caryophyllus), Ricin
(Ricinus communis).

34. Protease inhibitors from plants
• Many viruses, namely poty-, tymo-, nepo-, como-,
and closteroviruses need cysteine protease activity
to process their own polyproteins for their
replication and propagation.
• Hence plants expressing cysteine protease
inhibitors might resist the growth of viruses

35. Systemic acquired resistance
• viral infections, develops an active resistance which is at first
localized only at the site of infection, but spreads
systemically in due course. This resistance, called systemic
acquired resistance (SAR),
• is characterized by the coordinate activation of several genes
in uninfected, distal parts of the inoculated plants.
• associated with accumulation of salicylic acid (SA),
enhanced expression of pathogenesis related (PR) proteins,
activation of phenylpropanoid pathway, synthesis of higher
phenolic compounds, reinforcement of cell wall by the
deposition of lignin and suberin.

36. • Involvement of SA in TMV resistance has been
shown by expressing the bacterial salicylate
hydroxylase (NahG) gene in tobacco plant
• Transgenic tobacco plants were developed that
expressed catalase 1 (Cat1) or catalase 2 (Cat2) gene
in an antisense orientation.
• Antisense catalase transgenic plants exhibiting
severe reduction in catalase activity (approximately
90% or more), developed chlorosis or necrosis on
lower leaves. These plants also showed high level of
SA and PR accumulation as well as enhanced
resistance to TMV.