“ Plant immunity: towards an Plant immunity: towards an integrated view of plant pathogen integrated view of plant pathogen interaction and its implication in interaction and its implication in plant breeding plant breeding” 1 Pavan. R Department of Genetics and Plant Breeding University of Agricultural Sciences Bengaluru-65
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Plant immunity towards an integrated view of plant pathogen interaction and its implication in plant breeding
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“Plant immunity: towards an Plant immunity: towards an integrated view of plant pathogen integrated view of plant pathogen interaction and its implication in interaction and its implication in
plant breedingplant breeding”
1
Pavan. RDepartment of Genetics and Plant Breeding
University of Agricultural Sciences Bengaluru-65
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“For each resistance gene in the host there is a corresponding gene for avirulence in the
pathogen cnferring resistane and viceversa” Host plant genotype
Pat
hoge
nge
noty
peR1 r2 r1 R2
Avr1, avr2 I
I
C
Cavr1, Avr2
I - incompatible - no diseaseC - compatible - disease
Gene for gene hypothesis
H.H. Flor 6
It is a state of defense against infectious pathogens
Pathogens are like Bacteria, Fungi, Virus, Nematode, Oomycetes etc.
Mode of entry of pathogen depend on type of pathogen
Late blight, caused by the oomycete pathogen Phytophthora infestans,
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Genetic and physical maps of the genomic region
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BAC clones from the RB haplotype (filled boxes) and BAC clones from the rb haplotype (open boxes). Both 177O13 and CB3A14 contain one truncated and four complete RGAs. The direction of transcription of each gene(an arrow). The 3.6-kb deletion region between RGA2 and RGA-tris marked.
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Late blight screening of transgenic plants by using isolate US930287
Plants were scored as resistant (R) if the resistance score was >7.0 (< 25% infection) and plants were scored as susceptible was <6.9 (>25% infection). † Of the 14 resistant plants, nine plants had a score of 7 and five plants had ascore of 8.
Complementation analysis of putative RB genes
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(A–C) Transgenic Katahdin plants- RGA1-PCR,RGA2-PCR, and RGA4-PCR, respectively. (D) Control Katahdin plant. (E) Katahdin plant that was not inoculated. (F–I) Transgenic Katahdin plants containing constructs RGA1-BAC, RGA2-BAC,RGA3-BAC, andRGA4-BAC, respectively.
Structure of the RB gene and the deduced RB protein.
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Disadvantage of R genes …….?
Ectopic expression of R genes can
sometimes activate defence pathways in the
absence of pathogen
Reduced crop yields
Reduced Fitness
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3. Antifungal fusion proteins to induce immunity
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Fusarium head blight (FHB) or scab of wheat is a devastating disease in warm and humid regions at wheat-flowering periods worldwide.
Expression of pathogen-specific antibodies in plants has been proposed as a strategy for crop protection.
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An antibody fusion protein comprising a Fusarium-
specific recombinant antibody derived from chicken
and an antifungal peptide from Aspergillus giganteus
was expressed in wheat as a method for protecting
plants against FHB pathogens.
Plants expressing the antibody fusion displayed a
very significantly enhanced resistance in T2 and T3
generations upon single-floret inoculation with the
macroconidia of Fusarium asiaticum, the
predominant species causing FHB in China, indicating
a type II resistance.
Structure of AG-D2 fusion construct
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An antifungal peptide sequence from Aspergillus giganteus (AG) and a single-chain Fv (scFv) antibody coding region from chicken.
Connected by a sequence encoding a 10-amino-acid glycine-serine linker.
The AG-scFv fusion construct was inserted into the plant expression vector pAHC25 using EcoRI and SacI sites.
Ubi-Pro, maize ubiquitin promoter; UT: 5′ untranslated region of the petunia chalcone synthase gene; LP, leader peptide sequence; c-myc, c-myc epitope tag; His6, histidine 6 tag; Nos-T-Nos terminator.
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Integration and expression of AG-scFv fusion gene in transgenic wheat.
A, T3 transgenic wheat lines 2, and To detect the presence of the AG-scFv fusion gene with primers AGP1 and scFvP2.
B, RNA extracted from leaves of the plants in A was used in a RT- PCR assay to analyze expression of the AG-scFvfusion gene with the same set of primers in A.
C, Proteins extracted from leaves in A were fractionated by electrophoresis on a 12% SDS-PAGE and then subjected to immunoblot analysis with an antibody against the Histidine 6 tag
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Southern blot analysis of transgenic wheat.
Fusarium head blight resistance in T2 and T3 transgenic wheat
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Comparison of yield parameters between nontransgenic plants and transgenic plants
expressing the antibody fusion.
A: Single floret inoculation and B: Spray inoculation
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FHB-susceptible cv. Bobwhite,
FHB-resistant cv. Sumai3 at 21 days postinoculation with the conidia of Fusarium asiaticum.
A, Spikes of a single floret (indicated by an arrow) inoculated with the conidia of F. asiaticum.
B, Spikes by spray inoculation with the conidia of F. asiaticum.
C, Grains from a spike of a single-floret inoculation in A.
Phenotype of representative spikes and grains from T3 transgenic wheat line 2,
Phytoalexins are antimicrobial and often antioxidative
substances synthesized de novo by plants that accumulate
rapidly at areas of pathogen infection
They are broad spectrum inhibitors and are chemically
diverse with plant species.
Phytoalexins tend to fall into several classes including
terpenoids, glycosteroids and alkaloids
4. Use of phytoalexins to induce immunity
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1997
Stilbene synthase occurs in several plant species and
synthesizes the stilbene phytoalexin transresveratrol
Transfer of two genes from grapevine (Vitis Šinifera)
coding for stilbene synthase genes (vst1 and vst2 ) to
tomato by means of Agrobacterium tumefaciens
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The accumulation of the phytoalexin trans-
resveratrol, the product of stilbene synthase, for
resistance tomato to Phytophthora infestans (Late
blight of tomato).
Accumulation of resveratrol occurred after
inoculation with Botrytis cinerea (Gray mould in
tomato) and Alternaria solani (Early blight in
tomato)
Southern blot analysis of transgenic tomato plants of the T 3 progeny
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Southern blot analysis of transgenic tomato plants of the T3
progeny from regenerant To25 (lane 1±4), To42 (lane 5±8), and
transgenic oilseed rape as a positive control (lane c). Genomic DNA
was isolated from leaves and digested with EcoRI that generates
two fragments of 3.4 kb and 4.9 kb representing the two
transferred stilbene synthase genes.
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Northern blot analysis showing the transient accumulation of stilbene synthase mRNA in leaves
Northern blot analysis showing the transient accumulation
of stilbene synthase mRNA in leaves of a transgenic tomato
plant of the T3 progeny from To25 after inoculation with
P.infestans. No specific mRNA was detectable immediately
after inoculation.*Leaves were treated with tap water only.
Resveratrol (stilbenoid, a type of natural phenol, and a
phytoalexin) accumulation in leaves of a transgenic
tomato plant from the T2 progeny of regenerant To25
after inoculation with P. infestans and B. cinerea.61
Resveratrol contents of leaves of transgenic tomato plants
from T3 progeny of To25 4 days after inoculation with B.
cinerea, A. solani, and P. infestans
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Disease symptoms on leaves of a transgenic tomato plant from the T3 progeny of To25(right) and non-transformed tomato plant (left) 4 days (upper) and 6 days (lower) afterinoculation with P. infestans.
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Biological testing of transgenic tomato plants from progenies T2,
T3, and T4 of regenerant To25 and To42 for an increased
resistance to A. solani, B. cinerea, and P. infestans 4 days after
inoculation64
Development of P. infestans on transgenic tomato plant To25 (T 3 progeny) and non-transformed plant 6 days after
inoculation
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Incidence of P. infestans on transgenic tomato
plants and non-transformed plants in
dependence on the leaf insertion
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Probenazole (PBZ) is the active ingredient
of Oryzemate
Protection of rice plants from Magnaporthe
grisea (blast fungus)
PBZ pre-treatment increased accumulation
of SA and PR proteins in the eighth leaves
of adult plantsTakayoshi Iwai., et al 2008
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5. Use of chemicals to induce immunity
Phenotypes of blast fungus-inoculated leaves of young and adult rice plants. 68
Free SA and SAG levels in rice leaves after fungus inoculation and PBZ treatment. 69
Accumulation of rice PR proteins in M. grisea-infected leaves.
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Induced expression of the OsPR1a gene in M. grisea-infected leaves.
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Induced resistance to blast fungus by SA treatment. 72
6. RNAi-mediated silencing of pathogen’s genes
Parasitism genes expressed in esophageal gland
cells mediate infection and parasitism of plants by
root-knot nematodes (RKN).
Parasitism gene 16D10 encodes a conserved RKN
secretory peptide
Used in vitro and in vivo RNA interference to induce
immunity
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In Vitro RNAi of 16D10.
RNAi silencing of 16D10 in preparasitic M. incognita J2.
Fluorescence microscopy showing ingestion of FITC in the treated J2.