Plant disease resistance through R genes.

1. PPlantlant DDiseaseisease RResistanceesistance GGenes andenes and
CCroprop BBreedingreeding
Janarthanan .P
qRT-PCR Lab
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2. Of 77 billionbillion
peoplespeoples
11 billions arebillions are
Hungry..Hungry..
World Food Demand Scenario
We need to increase food
production by 50% by 2030
and by 70–100% by 2050.
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Global harvest initiative -2011Global harvest initiative -2011
FAO -2010FAO -2010

3. Constrains in Food Production
 Plant diseases
 Major Threat to global food security
 Also reduces the Crop quality
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Jia et al., 2000

4. Plant Immune System
R-genes – Essential for Disease Resistance Breeding
Jones & Dangl (2006)

5.  Genes in plant genomes that convey
plant disease resistance against
pathogens by producing R proteins
 Encode putative receptors that
respond to the products of ‘Effector
genes’ expressed by the pathogen
during infection.
 Majority contains conserved domains
(essential for perception/signal
transduction activity)
• Nucleotide binding site (NBS),
• leucine rich repeats (LRR), and a
• Serine /threonine protein kinase.
What are Resistance Genes (R-Genes)
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6. (NBS: nucleotide-binding site; LRR: leucine-rich repeat; TIR: Toll-interleukin-1 receptor; CC: coiled-
coil; TM: transmembrane domain; PK: Protein Kinase; WRKY: WRKY domain; B-lectin: bulb-type
mannose specific binding lectin domain).
Classes of R-Genes
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Martin et al., 2003

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Plant proteins belonging to
the nucleotide-binding site–
leucine-rich repeat (NBS-
LRR) family are used for
pathogen detection.
(R-PROTEIN)
Harmful organism
Recognition by
resistance protein
Signal to cell
nucleus
Genetic
material
Defense
Response Defense protein
(R-PROTEIN)
Outsideplantcell
Insideplantcell
Diagram of a plant disease resistance protein in action. A portion of the protein
(MAROON) lies outside the cell and specifically recognises the harmful organism.
The remaining portion of the protein (RED) resides inside the cell and
communicates a signal to the plant’s genetic material, which in turn stimulates a
defense response against the invading organism.
R-Gene in Action
NBS-LRR
PROTEIN
(R-GENE)

8.  It has concerted responses can efficiently Halt the pathogen
growth with minimal collateral damage to the plant
 Dozens of R genes, against many different pathogens, have now
been cloned from a variety of plants
 The vast majority of genes cloned so far belong to the NBS-LRR,
LRR, or LRR-Kinase super families
 These super families were initially identified in tomato, tobacco
and Arabidopsis by map-based cloning or transposon tagging.
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9. GENE PLANT PATHOGEN YEAR ISOLATED
Pto Tomato Pseudomonas syringae
pv. tomato (avrPto)
1993
PBS1 Arabidopsis Pseudomonas syringae
pv. phaseolicola(avrPphB)
2001
RPS2 Arabidopsis Pseudomonas syringae
pv. maculicola (avrRpt2)
1994
N Tobacco Tobacco Mosaic virus 1994
Bs2 Pepper Xanthomonas campestris pv.
vesicatoria (avrBs2)
1999
RRS-1 Arabidopsis Ralstonia solanacearum 2002
Pi-ta Rice Magnaporthe grisea(avrPita) 2000
Cf-9 Tomato Cladosporium fulvum(Avr9) 1994
Ve2 Tomato Verticillium albo-atrum 2001
Xa-21 Rice Xanthomonas oryzae pv.oryzae 1995
Gururani et.al.,2012
Cloned R genes
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10. Features of R genes
 Follows gene for gene hypothesis
 Co-evolve
 predominantly inherited
 Highly polymorphic
 Majority of them code for specific receptors
(Proteins, enzymes, antimicrobial compounds)
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11. R gene – discovery approachesR gene – discovery approaches
Map based cloning
Molecular markers map + BAC libraries screening
Transposon tagging
Mutational R gene Enrichment Sequencing
(MutRenSeq)
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12. Map based cloning
 It otherwise called as
Positional Cloning.
 First plant resistance
gene (Pto) isolated using
Map Based cloning
 Using the genetic
relationship between a
gene and a marker as the
basis of Map based cloning.
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13.  Tomato Pto gene
 Provides resistance against
bacterial speck disease
 Pseudomonas syringa pv
 Rice Xa21 gene
 Resistance to Bacterial blight
disease
 Xanthomonas oryzae pv.
Rice pi9,pi2 gene
 Resistance to Rice blast disease
 Mangnaporthe grisea
Examples
Laborious,
Time Consuming
Expensive
Skilled man power
required
Infra structure
Limitations
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14. Transposon Tagging
 It was first recognized by McClintock
 It is an insertion based technique.
 Transposon mutagenesis in plants has become an increasingly
useful tool for gene discovery.
 A transposable element is a DNA sequence that has the ability to
change its location in the genome, i.e., it can transpose from one
location to another in the genome.
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15. Transposon tagging describes isolation of genes using transposable
elements as gene tags.
 When a transposon integrates within a gene, the gene function is
lost.
 The inserted fragment is usually a well characterized transposable
element, most of which has been sequenced
Gene can be easily recognize by sequence of the inserted fragment.
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Steps involved in Transposon tagging

17.  Transposon tagging has been used to isolate several genes in
 Maize (e.g. A1, A2, BZ2, C1, C2, opaque2, R, P, etc.),
 Tomato (cf-9, Dem, etc.)
 Tobacco (cf-4A, N)
A major limitation of the method is the low frequency
of transposition.
Most species lack active transposons
Limitations:
Examples :
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18. Mutational R gene ENrichment Sequencing
(MutRenSeq)
 Scientists at the John Innes Centre (JIC) and The Sainsbury
Laboratory (TSL) have pioneered a new gene detecting technology
called MutRenSeq
 It is used to accurately pinpoints the location of disease resistance
genes in large plant genomes.
 We can very quickly locate resistance genes from crops, clone them
and stack multiple resistance genes into our elite variety.
 It has reduced the time it takes to clone the genes into crops from 5
to 10 years down to 2 years.
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19. MutRenSeqMutRenSeq -- 3 Step method
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20.  Using Ethyl Methane Sulfonate, a chemical known for causing
genetic variations.
 Mutagenized seeds called M1 generation, the plants are generated
self pollinated and make M2 Families
 EMS mutagenesis of resistant plant, creation of independent M2
families and screening for susceptible mutants
EMS Mutagenesis
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21. R gene ENrichment Sequencing
 RenSeq - involves capturing fragments from a genomic or cDNA
library using biotinylated RNA oligonucleotides designed to be
complementary to the NBS-LRR-encoding genes of a reference
genome
Target enrichment using a NBS-LRR–specific bait library and
sequencing of the wild-type and susceptible mutants
Sequences sources were derived from publicly available gene
annotations
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22. A de novo assembly of the enriched sequences of the resistant
wild type is used as a reference for mapping.
Subsequent SNV and presence/absence calls are integration and
scoring.
Data analysis and candidate calling
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23. Wild type Resistant Plant
Clear phenotype associated with single gene
EMS screen and identify loss-of-function mutants
Assumption of the structure of target gene (e.g. NB-LRR)
 Advantages:
No reference required
No fine mapping
No BAC library
No problems due to suppressed recombination
Time saving
 Requirement
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24. Case StudyCase Study
Wheat RustWheat Rust
(Puccinia triticina)
(Puccinia striiformis)
(Puccinia graminis)
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Burkhard et al., Apr 2016.

25. Wild type Resistant plant
EMS 6 Susceptible
mutants (1300
M2 Plants)
RenSeq
Wild-type & 6
mutants
Data
Analysis
 Total mutations ~ 44 to 84
 23 contigs that were mutated in 2 mutants
 3 contigs that were mutated in 3 mutants
 Single 3,408-bp contig, that contained
independent mutations in 5 of the 6 mutants
Wild-type assembly
&
Identified a contig
carried mutation in N-
terminal region of the
same gene
Joined the contigs to obtain the
full-length sequence of the
predicted open reading frame
CandidateCandidate
genegene (Sr22(Sr22))
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Work flow

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Susceptible Transgenic (Sr22)
Sr22 & Sr45 Validation
Introgression (Sr45)

27.  Pesticides can control these diseases but they are harmful to
environment and very expensive.
 Wild relatives of domesticated crops contain many useful disease
resistance (R) genes. Introducing this natural resistance is an smart way
of managing disease.
 Benefits of using the plant resistance genes in resistance breeding
programs include
 Efficient reduction of pathogen growth,
 Minimal damage to the host plant,
 Zero input of pesticides from the farmers (Economical)
Eco friendly nature of such crops
 We have to adapt new technologies that helps to enhance the
disease resistance and productivity.
Conclusion
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