Deployment of broad spectrum resistance against rice blast which includes gene pyramiding, deployment, transgenic approaches, marker assisted back cross breeding, pedigree by using major R genes and QTLs and phytoalexin genes.

1. Deployment of broad-spectrum Resistance against Rice
blast caused by Magnaporthe oryzae
Indian Agricultural Research Institute
Division of Plant Breeding and Genetics
Speaker - M. Ashajyothi

2. Plant innate immunity
Rice blast facts
Co-evolution of plant resistance and pathogen virulence
R genes – Sources, Mapped, Cloned
Breeding Methods for deployment of resistance genes
1. Conventional approaches
2. Molecular Breeding – MABB, Transgenic, Tissue culture
Case study 1
Case study 2
Challenges
Future prospects
Conclusion

3. Plant Innate Immunity
• Despite the lack of a cellular immune system, plants share with animals an
innate immune system.
• Plants contain two major innate immune responses: Pathogen-associated
molecular pattern (PAMP)-triggered immunity (PTI) (Boller and He, 2009) and
effector-triggered immunity (ETI) (Jones and Dangl, 2006).
• The PTI response includes activation of MAP kinases, induction of (ROS),
deposition of callose, and induction of (PR) genes (Nurnberger et al., 2004).
• ROS burst constitutes an early response to pathogen attack by strengthening
cell walls and by activating defense signaling components (Daudi et al., 2012).

4. • Pathogens deliver effectors into plant cells to inhibit the host PTI response
to create a favoring host cell environment.
• Plants have developed intracellular sensors encoded by resistance (R) genes
that perceive pathogen effectors directly or indirectly leading to ETI (Jones
and Dangl, 2006).
• ETI confers strong resistance which is limited to a few races of the pathogen
and not durable because pathogen effectors evolve quickly (Nurnberger et
al., 2004).

5. Leaf blast Node blast Panicle blast
• Blast is caused by the hemibiotrophic fungus Magnaporthe oryzae.
• The annual loss of rice production caused by blast could fulfil the annual rice
consumption of 60 million people (Parker et al., 2008).
• Magnaporthe oryzae can infect all parts of the rice plant, including the roots
(Duan et al., 2014).
• The fungus is able to develop resistance to both chemical treatments and
genetic resistance i.e. a threat to the effectiveness of blast-resistant rice
varieties.
Rice blast facts
Image- courtesy : Donald Groth, Louisiana State University AgCente

6. M. Variar et al.(2009)

7. • Host resistance is the most economical and environmentally friendly way of
disease control.
• The differences in the pathogenicity of the blast fungus strains were first
noticed by Sasaki (1922,1923).
• Unusually high degree of pathogenic instability within a single race, i.e. single
spore isolates from a single lesion (Ou, 1971; Ou et al.,1980) .
• Variation in pathogenicity of the blast fungus have been attributed : mutations,
sexual hybridisation, parasexuality and heterocaryosis (Valent and Chumley,
1991).
• Quamaruzzaman and Ou (1970) : Detailed studies for 21 months, by
monitoring the monthly changes in pathogenic races of the blast fungus at the
blast nursery at IRRI, Philippines.
Co-evolution of plant resistance and pathogen virulence

8. Co evolutionary Arms race
Limited success has been realized in durable resistance breeding programmes due to variability
of pathogen across locations.
Jonathan D. G. Jones & Jeffery L. Dangl
Nature (2006)

9. • Qualitative resistance is modulated by interaction between the products of a
major disease resistance (R) gene and an avr gene.
• Till date about 105 major resistance(R) genes have been identified, and 27of
them cloned and characterized (Ellur et al., 2016).
• Some of these genes Pi-1, Pi2, Pi9, Pi20, Pi27, Pi39, Pi40 and Pikh are reported
to have confers broad-spectrum resistance (BSR) (Liu et al., 2002).
• Some of them including Pia, Pib, Pii, Pi-km, Pi-t, Pi12 and Pi19 confers race
specific resistance (RSR) (Yang et al., 2008).
• Twenty five of the 27 characterized MR genes against M. oryzae encode NB-LRR-
type proteins (Ashkani et al., 2016).
Qualitative resistance

10. S.No Gene Chr Domain
Expression
pattern
1 Pib 2 NBS-LRR
Circadian stress
inducible
2 Pita 12 NBS-LRR
Circadian stress
inducible
3 Pi54 11 NBS-LRR Pathogen inducible
4 Pid2 6 Lectin receptor
Constitutive
membrane bound
5 Pi9 6 NBS-LRR Constitutive
6 Pi2 6 NBS-LRR Constitutive
7 Pizt 6 NBS-LRR Constitutive
8 Pi36 8 CC-NBS-LRR Constitutive
9 Pi37 1 NBS-LRR
Constitutive
cytoplasmic
10 Pikm 11 NBS-LRR Constitutive
11 Pi5 9 CC-NBS-LRR Pathogen inducible
12 Pit 1 CC-NBS-LRR Constitutive
13 Pid3 6 CC-NBS-LRR Constitutive
14 pi21 4 Proline-rich protein Slowly inducible
15 Pish 1 CC-NBS-LRR Constitutive
16 Pbl 11 CC-NBS-RR Panicle Blast
17 Pik 11 CC-NBS-LRR -
18 Pikp 11 CC-NBS-LRR -
19 Pikh 11 CC-NBS-LRR -
20 Pia 11 CC-NBS-LRR -
21 NLS1 11 CC-NBS-LRR
Constitutive age
dependent
22 Pi25 6 CC-NBS-LRR -
23
Pi54r
h
11 CC-NBS-LRR Pathogen inducible
24 Pi54of 11 CC-NBS-LRR Pathogen inducible
25
Pid3A
4
6 NBS-LRR -
26 Pi35 1 NBS-LRR
Quantitative but
consistent
27 Pigm* NBS-LRR
Epigenetic
regulation
- Not Known; MB - map based (Modified from Sharma et
al. 2012);*- Deng et al. (2017)
List of cloned and characterized blast
resistance genes in rice.

11. • Quantitative resistance is conferred by QTLs and is presumably race non-specific and
durable (Roumen, 1994).
• Major genes prevent life cycle completion , whereas QTLs reduce the sporulation of the
pathogen within a compatible interaction.
• Approximately 350 QTL have been mapped from 15 different populations, most of
which are derived from indica and japonica crosses (Chen et al., 2003).
• QTLs are difficult to identify and characterize in the presence of major genes due to
epistatic interactions.
• Four partial-resistance genes have been identified and described as specific, Pif , Pi21 ,
Pb1 and Pi34.
• This suggest that partial resistance is sometimes specific and does not necessarily have
a broader resistance spectrum than complete resistance.
• Gene and individual QTL pyramiding should be considered for durable resistance to blast
fungus.
Quantitative resistance

12. QTL Mapping
Ashkani et al. (2015)

13.  Allele mining is the commonly used approach to identify novel alleles or allelic variants
of a gene/or candidate genes of interest.
 Eco Tilling and sequence based allele mining are the two widely used approaches in
allele mining.
Allele mining

14. Ashkani et al. 2015

15. Methods for deploymet of rice blast resistance
• Kushibuchi et ai., (1971) suggested the use of mass selection for blast
resistance.
• Ikehashi and Khush (1979) proposed an approach for accumulating diverse
genes for resistance through multi location tests with different P. grisea
isolates.
• Bonman and Mackill (1988) proposed a procedure for breeding for blast
resistance. This includes: (1) selecting parents, (2) selecting agricultural
checks, (3) screening segregating populations and (4) evaluating advanced
lines.
• A number of cultivars possessing durable resistance to rice blast have been
identified and selected using these approaches
• There is a need to develop strategies providing long lasting disease
resistance against abroad spectrum of pathogens.

16. Conventional breeding
 Conventional approaches are important for producing novel genetic variants:
1. Backcrossing
2. Recurrent selection
3. Pedigree method
4. Mutation breeding
 Backcross breeding : It has been adopted in the South and Southeast Asia as breeding
strategy to improve elite varieties such as KDML105, Basmati and Manawthukha for
their resistances to blast (Sreewongchai et al., 2009).
 Recurrent selection : Upland cultivar CG-91 was developed with resistance to rice blast
(Guemaraes et al., 2000).
 In efficacy evaluation study observed 6.65 % genetic gain considering two cycles of
recurrent selection in the irrigated rice population CNA-IRAT 4 (Rangel et al., 2005).

17.  The pedigree method is highly suitable to develop rice with resistance to insects and
diseases if the resistance is governed by major genes.
 It is possible to combine genes for resistance to six or seven major diseases and insects
in a short period (Rangel et al., 2005)
Pedigree method

18.  Mutation Breeding
 An attempt was made to induce blast resistance in the high yielding variety
Ratna (IR8/TKm 6) through chemo mutagenesis with EMS 0.1 and 0.2 %
concentrations (Kaur et al., 1975).
 The Mtu 17 rice variety dES induced mutants had blast resistance, whereas
the parent Mtu 17 was susceptible (Gangadharan et al., 1976).
 Blast resistance mutant R917 was derived from the F1 progeny radiated by
10 krad 60Co c-ray (Zhang et al., 2003).

19. Molecular approaches for Blast resistance
 Molecular breeding approaches involving DNA markers:
1. Marker-assisted selection
2. Gene pyramiding
3. Tissue culture
4. Genetic transformation
 These techniques are low-cost, high-throughput alternative to conventional
methods allowing rapid introgression of disease resistance genes into
susceptible varieties.

20. Marker assisted Improvement of Basmati rice varieties
PB 1121, PB 6 and PB 1
 Basmati is highly susceptible to M. Oryzae and there is no resistance source found in
Basmati rice germplasm.
 Blast resistant donors developed through MAS are Pusa1602 (PRR78 + Pi2) and
Pusa1603 (PRR78 + Pi 54) to transfer these genes in to PB1121 and PB 6 (AK Singh et
al., 2012).
 It lead to the development of Pusa 1716 (PB 1121+Piz 5), Pusa 1717 (PB 1121+Pi 54),
Pusa 1726 (PB 6+ Piz 5), Pusa 1727 (PB 6 + Pi 54) , Pusa 1883 (PB 1121+Pi2 + Pi
54), Pusa 1884 (PB 6 + Piz 5+ Pi 54) (Ellur et al., 2013, 2016).
 Pusa 1850-27 (BPT 5204 +Pi54+Pi1+Pita) (Yet to be released).
 Both resistant genes transferred (Pyramiding ) to PRR background i.e., Pusa Basmati
1609 (PRR78 + Piz5+Pi54) (AK Singh et al., 2014).
Pusa 1612 (Pusa Sugandh 5 +Pi2+Pi54)
Pusa Basmati 1637 - (PB1+Pi9)

22.  For molecular screening, the NRVs were
genotyped for the presence of 12 major
blast resistance genes viz. Pib, Piz, Piz-
t, Pik, Pik-p, Pikm, Pik-h, Pita/Pita-
2, Pi2, Pi9, Pi1, a-2 and Pi5.
 A total of 17 markers available for above
twelve genes were collected and used for
molecular screening (Yadav MK et al.,2017)
Yadav MK et al. (2017)

23. Yadav MK et al. (2017)

24. Gene pyramiding Strategies
Marker Assisted Plant Breeding: Principles and Practices – BD Singh & AK Singh

25. Gene pyramiding
Ashkani et al. 2015 (Frontiers in Plant Science)

26.  Tissue culture: Information on soma clonal variation for blast resistance is
scanty.
• In Brazil, a high degree of partial resistance has been reported in progenies of
regenerated plants derived from immature panicles of a susceptible upland
rice cultivar IAC47 (Araujo et al., 1997).
 Gene transformation: Transgenic technologies allow multiple genes insertion
simultaneously into genome to obtain broad-spectrum resistant lines.
• There have been reports on increasing rice blast resistance through
transformation of 1. Chitinase gene (Nishizawa et al., 1991)
2. Plant antitoxin gene (Stark lorenzen et al., 1997) ,
3. Chitinase–glucanase gene (Feng et al., 1999),
4. Trichosanthin gene (Ming et al., 2000),
5. Wasabi phytoalexin gene (Kanzaki et al., 2002) and
6. Rice blast resistance genes Pi-ta, Pi-9, Pi-2 etc.

27.  Backcrossing for concentration of slow-blasting components
 Combination of major genes with slow-blasting components: Centre
International de Agricultural Tropical (CIAT) rice breeding programme attempted
adopting this strategy
 Mixtures of variety: Varietal mixtures are the way of reducing the development
of blast races consisting of 80–90 % resistant plants and 10–20 % susceptible
plants of similar varietal background.
 Multiple lines
• The durability resistance depends upon the rate of blast races develop, the
number of lines component in a mixture and the extent of planted area.
• Multiple line variety of ‘‘Sasanishiki’’ has been commercially cultivated on a
market scale since 1995.
 Deployment of gene
Distinct gene deployment in different maturity groups may help to improve the
durability of blast resistance in newly developing rice varieties.
Some components of breeding strategies suggest prolong durability of resistance which
generally can be adopted for stabilization and control of blast disease in rice are :

29. Hypothesis: A natural allele of a C2H2-domain transcription factor gene, bsr-d1,
confers broad-spectrum resistance to rice blast
Li et al., 2017, Cell 170, 114–126

30. • Digu is a rice variety carrying, high-level resistance to a broad-spectrum of M.
oryzae races (Li et al., 2016).
• It an excellent resource for discovery of novel genes conferring broad-
spectrum resistance.
• Identification of a novel allele, bsr-d1, involved in the broad-spectrum,
durable resistance to M. oryzae in Digu.
• Bsr-d1 encodes a C2H2-type transcription factor, which is directly regulated
by a MYB family transcription factor.
• These two transcription factors regulate expression of H2O2-degradation
enzymes to accomplish resistance to M. oryzae, constituting a novel
mechanism employed by rice blast resistance.
Background.....

31. Identification of the responsible SNP:
Digu X LTH
3,685 recombinant inbred lines
74 RILs that are morphologically indistinguishable from the LTH parent and that lack
both Pid2 and Pid3
42 are susceptible and 32 resistant to M. oryzae inoculation
Analyzed the SNPs
Identified an association between SNP33 and blast resistance
Pathogen inoculation gave 12 Digu type and 12 LTH type SNP33
Genome-wide Association Study Identifies the bsr-d1 Allele as Associated with Digu
Blast Resistance

32. Blast Resistance of Digu and LTH under Field Condition and Sequence Comparison of Bsr-d1
between Digu and LTH

33. Bsr-d1 RNA level LTH Before inoculation 2-fold higher than Digu
After inoculation 8-fold higher than Digu
• These results indicate that the Bsr-d1 gene is associated with susceptibility to
blast and is induced by blast inoculation in LTH, but not in Digu.
• M. oryzae induces Bsr-d1 expression, possibly as a strategy to suppress host
immunity.
M. oryzae Induces Bsr-d1 Expression in Susceptible Rice Cultivars, but Not in
Resistant Rice
Silencing, Overexpression, and CRISPR-Mediated Knockout of Bsr-d1 Validate the
Role of Bsr-d1 in Digu Resistance

34.  Cellular Responses to M. Oyrzae Infection
DAB staining at infection sites
Bsr-d1 Knockout Lines, Like Digu, Carry Resistance to a Broad Spectrum of Blast
Isolates and Elicit a Hypersensitive Response

35. BSR-D1 targets two Peroxidase Genes whose Induction by Blast Infection Is
suppressed in Digu
 Bsr-d1 directly regulates expression of the two peroxidase genes, the expression
levels of these peroxidase genes in the Bsr-d1KO lines reduced 3- to 8-fold compared to
those in TP309.
 BSR-D1 regulates expression of these two peroxidase genes differentially in Digu and LTH,
due to the different Bsr-d1 levels in these two rice varieties, leading to enhanced
resistance in Digu, but not in LTH.

36. The MYBS1 Transcription Factor Binds the Bsr-d1 Promoter Differentially between
Digu and LTH
 MYBS1 binds to the Bsr-d1 promoter and inhibits its expression.
 The EMSA results show that GST-MYBS1 binds MYS1, but not MYS2, and that
it has a high affinity for the Digu MYS1 than for the LTH MYS1.

37. Knockout of Mybs1 Leads to Elevated Bsr-d1 Expression and Enhanced Susceptibility
to M. oryzae

38. A Model for bsr-d1-Mediated Disease Resistance

39. Application of the bsr-d1-Mediated Resistance to the Blast Disease
in Breeding
• bsr-d1 also confers non-race-specific resistance to a broad-spectrum of M.
oryzae, constituting a superior disease-resistant characteristic for crop breeding.
• bsr-d1 identified as a variant allele from natural rice varieties.
• bsr-d1 located on chromosome 3, is not linked to any genes known to confer
undesirable grain quality or flavour.
• Therefore, bsr-d1 plants represent a better alternative genetic resource for rice
resistant breeding.

40. Summary
A single base change (SNP33-G) in the bsr-d1 promoter enhances binding to
MYBS1
Binding of MYBS1 to the bsr-d1 promoter suppresses bsr-d1 expression
BSR-D1 promotes peroxidase expression, suppressing immunity to M.
Oryzae
The SNP33-G allele is present in 10% of 3,000 surveyed rice varieties

41. Aim: To develop improved blast resistant line by pyramiding Pi46 & Pita

42. Materials
• Recurrent parent : HH179
• Donor parent of blast resistance : H4 (Pi46 and Pita)
Marker Assisted Selection and
Pathogenic assay
Work flow of marker-assisted backcross breeding
HH179×H4
F1×HH179
BC1F1×HH179
BC2F1×HH179
BC3F1×HH179
BC3F2
BC3F3

43. Screening of BC1F1, BC2F1 and BC3F1 generations
(17 individuals) BC1F1
8 resistant (carried Pi46 heterozygously)
GD0193 Isolate
Four found to harbor Pita
Dominant marker
YL155/YL87
HH179X
BC2F1(3 found heterozygous for both pita & pi46)
GD0193 Isolate
GD0193 Isolate
BC3F1(4 found heterozygous for both pita & pi46)
4 Self pollinated
BC3F2

44. Foreground selection for the BC3F2 population
BC3F2 (200 Individuals)
GD0193 Isolate
36 carry pita
102 (Aa) 49(AA)
155(R) 45(S)
Dominant marker (pi46)
RM224
Dominant marker (pita)
YL155/YL87
Dominant marker (pita)
YL183/YL87
23 (Bb) 13(BB)
Dominant marker (pita)
YL155/YL87
Dominant marker (pita)
YL183/YL87
12 (BB):21(Bb):12(bb)
 13 plants with Pi46 only
(designated as set “A”)
 12 plants with Pita only
(designated as set “B”)
 13 plants carrying
both Pi46 and Pita (designated
as set “C”).

45. • The recovery percentages of the recurrent genome of the three selected
individuals : set A - 92.31, 96.15 and 100.00% - R1791(Pi46)
set B - 92.31, 96.15 and 96.15% - R1792 (Pita)
set C - 92.31, 92.31 and 96.15% - R1793 (Pi46+Pita)
Background selection for the target BC3F2 individuals
BC3F3
NILs selfed
Resistance spectra of R1791, R1792 and R1793 were 91.1, 64.7 and 97.1%, respectively,
and were significantly above the recurrent parent, 23.5%
34 Isolates
• Heading date (days to 50% flowering)
• Plant height
• Tillers per plant
• Panicle length
• Number of grains per panicle
• Spikelet fertility
• 1 000-grain weight
• Yield per plant Agronomic traits considered

46. • MAS has been advocated as a highly efficient breeding method because it
allows to select the target gene rapidly and accurately.
• Successfully integrated Pi46 and Pita from the donor parent into HH179,
using MABB in three generations of backcrossing followed by two
generations of selfing.
• Three NILs, R1791 (Pi46), R1792 (Pita) and R1793 (Pi46+Pita) were
developed.
• The pyramided line, R1793, exhibited a broader resistance spectrum
during the seedling stage and also improved resistance to panicle blast
during the adult stage.
Summary

47. Challenges
• The major difficulty in controlling rice blast is the durability of genetic resistance.
• Pyramiding of multiple Pi genes to stack up the resistance spectrum to M. oryzae
is a very time-consuming task and may not be an effective way to combat the
fast evolving rice blast pathogen.
• We are not entirely sure what genetic constitution results in durable resistance.
• Not possible to select specific gene combinations, because of the epistatic
interactions among genes.
• Molecular marker technology offers the opportunity to overcome some of the
problems by improving the efficiency and resolution of genetic analysis.

48. Understanding of genetic identity of contemporary M. oryzae is important
for accurate deployment of rice cultivars with different R genes.
For combinations of different blast resistance genes in host plant in the
rice blast breeding programs, superior alleles of the targeted genes
should be considered.
Rice research should more focus on identifying more durably resistant
genes, tagging of these genes with molecular markers.
Candidate gene identification through rice functional genomics has great
potential for developing more durably resistant varieties.

49. The highly destructive and variable nature of rice blast has made it a disease of
immense importance for the whole of the world.
Deployment of genes conferring broad-spectrum, durable resistance is highly
favoured by breeders.
Need for the continuous research on development of durably resistant
cultivars, will always be there.