Transcript NSF project meeting presentation 2009

Mutant-assisted exploration of natural variation
underlying R gene-mediated immunity in maize
1. Survey the entire panel of maize diversity lines (association mapping
panel) for their ability to impact HR induced by Rp1-D21.
2. Clone and characterize Hrml1 by using a combination of linkage and
association mapping tools, especially the nested association
mapping (NAM) RIL populations.
3. Evaluate NAM RIL populations for additional Hrml genes/QTL and
clone at least three of them.
And the outreach effort
The idea is to find the missing pieces of the HR pathway
puzzle by taking advantage of natural variation in maize
MAGIC: Mutant-assisted gene identification and characterization
HR stands for:
Hypersensitive Reaction
(hypersensitivity, or HR cell death)
Hypersensitive Response
The term was first coined by Stakman (1915)
He defined ‘hypersensitiveness’ to indicate the abnormally rapid
host plant death when attacked by rust fungi’ (Stakman, 1915)
Compatible vs. incompatible plantpathogen interaction
Biochemical mechanisms underlying the HR response
Biochemical mechanisms underlying the HR response
The HR response - updated definition
Genetics of the HR response
HR is induced Specifically by the interaction of R genes and
their corresponding Avr genes following a gene-for-gene
interaction (Flor 1941)
The HR is controlled by R gene-encoded receptor
proteins, a major proportion of which are ‘nibblers’
CC/
Molecular basis of the HR response
• In the absence of the pathogen AVR proteins (now called effector
proteins), nibblers are quiescent.
• This seems to be because of their ability to undergo intermolecular
interactions, as well as association with a number of other proteins,
including chaperones such as Hsp90
Molecular basis of the HR response
R gene activation
Pathways linking R gene activation with the HR response
In Arabidopsis, two major signaling pathways have emerged
that link R genes with their downstream responses
NDR1 - non race-specific disease resistance
CC type NBS-LRR genes
EDS - enhanced disease susceptibility
TIR type NBS-LRR genes
R genes have two functions
• The first is to recognize the pathogen
• The second is to trigger the HR response following recognition
Means mutations can in either function that can cause the R gene
to fire HR constitutively, in the absence of the pathogen
In fact many R genes have been identified that are autoactive
The first cases of which came from the maize Rp1 locus
Rp1 - a complex locus
The Rp1-D allele – is made up of 9 NBS-LRR genes
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Undergoes unequal crossing over frequently
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Generation of the Rp1-D21 recombinant
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Rp1-D21
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A dominant lesion mimic
mutant
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The HR phenotype of Rp1-D21 in H95
Rp1-D21 plants exhibit all the hallmarks of the HR
response
Rp1-D21
WT
PR1
PR5
PRms
WIP1
18S rRNA
mut
The HR phenotype of Rp1-D21 is cell autonomous
Rp1-D21 expression in different genetic backgrounds
Expression of Rp1-D21 in B73 vs. Mo17
IBM RIL population
• 302 lines strong recombinant inbred lines (RILs) population
• Derived from a cross between B73 with Mo17
• F2 plants were allowed to intermate for 4 generations before
inbreds was derived by the single seed descent
method
MAGIC screen of the IBM population for modifiers of Rp1D21 (HR response)
- Rp1-D21::H95/+ was crossed with the entire IBM RIL population
- The F1 progenies were evaluated for the severity of the Rp1-D21
population
Other RILs significantly suppressed Rp1-D21
MAGIC screen of the IBM population for modifiers of Rp1D21 (HR response)
- Rp1-D21::H95/+ was crossed with the entire IBM RIL population
- The F1 progenies were evaluated for the severity of the Rp1-D21
population
- The phenotypic data thus collected was correlated with the
genotypic data (which was already available for all these
IBM RILs) using a QTL software (cartographer)
QTL scan
Identification of Hrml1 (HR modulating locus-1)
Chromosome 10
Rp1
To clone Hrml1 and to identify additional modifiers of the
HR response
We have started a MAGIC screen of the NAM resource
Nested association mapping (NAM) resource
Rp1-D21 phenotype in hybrids of H95 with the
NAM founders
In some cases HR becomes dependent on
temperature
Mutant-assisted exploration of natural variation
underlying R gene-mediated immunity in maize
1. Survey the entire panel of maize diversity lines (association mapping
panel) for their ability to impact HR induced by Rp1-D21.
• Between Peter’s and our lab, we have crossed most diversity inbreds
with Rp1-D21(H95). This includes almost all NAM founders.
• Peter also crossed some (~50) with Rp1-D21(B73)
• The resulting F1 progenies from all of these crosses were planted this
past summer (2009) at ACRE as well as at NCSU
• These were screened at multiple times using various approaches and
parameters
Mutant-assisted exploration of natural variation underlying
R gene-mediated immunity in maize
1. Survey the entire panel of maize diversity lines (association mapping
panel) for their ability to impact HR induced by Rp1-D21.
• Between Peter’s and our lab, we have crossed most diversity inbreds
with Rp1-D21(H95). This includes almost all NAM founders.
• Peter also crossed some (~50) with Rp1-D21(B73)
• The resulting F1 progenies from all of these crosses were planted this
past summer (2009) at ACRE as well as at NCSU
• These were screened at multiple times using various approaches and
parameters
• This strategy identifies dominant modifiers only
• To unveil and map recessive modifiers, we have also made F2
populations for a number of diversity inbreds (especially the NAM
founders) by making F1 x F1 crosses between WT plants with their
mutant F1 siblings
Mutant-assisted exploration of natural variation underlying
R gene-mediated immunity in maize
1. Survey the entire panel of maize diversity lines (association mapping
panel) for their ability to impact HR induced by Rp1-D21.
•
Between Peter’s and our lab, we have crossed most diversity inbreds with Rp1-D21(H95).
This includes almost all NAM founders.
• Peter also crossed some (~50) with Rp1-D21(B73)
• The resulting F1 progenies from all of these crosses were planted this past summer (2009)
at ACRE as well as at NCSU
• These were screened at multiple times using various approaches and parameters
• This strategy identifies dominant modifiers only
• To unveil and map recessive modifiers, we have also made F2
populations for a number of diversity inbreds (especially the NAM
founders) by making F1 x F1 crosses between WT plants with their
mutant F1 siblings
• In fact, nine such F2 populations were planted at ACRE, which involved
Mo17, A632 and 7 NAM founders.
• These were screened multiple times for various Rp1-D21 severity
parameters and sampled for DNA (mutant plants) for genotyping
purposes.
Mutant-assisted exploration of natural variation underlying
R gene-mediated immunity in maize
1. Survey the entire panel of maize diversity lines (association mapping
panel) for their ability to impact HR induced by Rp1-D21.
2. Clone and characterize Hrml1 by using a combination of linkage and
association mapping tools, especially the nested association
mapping (NAM) RIL populations.
3. Evaluate NAM RIL populations for additional Hrml genes/QTL and
clone at least three of them.
Mutant-assisted exploration of natural variation underlying R genemediated immunity in maize
1. Survey the entire panel of maize diversity lines (association mapping
panel) for their ability to impact HR induced by Rp1-D21.
2. Clone and characterize Hrml1 by using a combination of linkage and
association mapping tools, especially the nested association
mapping (NAM) RIL populations.
3. Evaluate NAM RIL populations for additional
Hrml loci and clone at least three of them.
Strategy:
Cross Rp1-D21(H95) mutants with 3,400 of the
5,000 NAM RILs, screen their F1
progenies at both Purdue and NCSU, and
then do QTL analysis just as it was done
in the experiment involving IBM RILs.
Mutant-assisted exploration of natural variation underlying R genemediated immunity in maize
1. Survey the entire panel of maize diversity lines (association mapping
panel) for their ability to impact HR induced by Rp1-D21.
2. Clone and characterize Hrml1 by using a combination of linkage and
association mapping tools, especially the nested association
mapping (NAM) RIL populations.
To confirm that we have cloned the right gene for Hrml1, we are
following at least two strategies:
• Directed EMS mutagenesis of Hrml1
• Transposon tagging of Hrml1
Mutant-assisted exploration of natural variation underlying R genemediated immunity in maize
• Directed EMS mutagenesis of Hrml1
Using A632 for this purpose because:
• it has been shown to have an effective Hrml1 gene capable of
suppressing Rp1-D21
• A632 plants homozygous for Rp1-D21 produce pollen
H95
x
Rp1-D21/Rp1-D21(A632)
(Treated with EMS)
M1 - few thousand seed has been generated
Mutant-assisted exploration of natural variation underlying R genemediated immunity in maize
• Transposon tagging of Hrml1
• Using both B73 and A632
• Crosses were made this summer (2009) to transfer Mutator
(Mu) into B73 and A632.
- The progenies will be selfed/sib-mated to generate Muactive plants homozygous for Hrml1
- Such plants will be selected and propagated by either
selfing or sib-mating.
- Next summer (2010), these plants will be planted in the
field and fertilized with Rp1-D21(H95) pollen to
generate at least 50,000 F1 seed, which will be
screened in the greenhouse/field for plants with
enhanced severity of the Rp1-D21 phenotype
Mutant-assisted exploration of natural variation underlying R genemediated immunity in maize
• Transposon tagging of Hrml1
Next summer (2010), these plants will be planted in the
field and fertilized with Rp1-D21(H95) pollen to
generate at least 50,000 F1 seed, which will be
screened in the greenhouse/field for plants with
enhanced severity of the Rp1-D21 phenotype
B73 (Mu)
X
Rp1-D21(H95)
F1
(Screen for F1 plants that have a relatively
severe Rp1-D21 phenotype)
Mutant-assisted exploration of natural variation underlying R genemediated immunity in maize
• Transposon tagging of Hrml1
• The same procedure can be used to tag and clone
additional Hrml loci that we find in other lines (e.g.,
Tzi8, Co255, CML228)
• We will start crossing these lines with Mu stocks soon
Mutant-assisted exploration of natural variation underlying R genemediated immunity in maize
• Transposon tagging of Hrml1
In fact, the same procedure can be used to tag and clone additional
Hrml loci, and this includes enhancers.
For example,
TX303 and M37W have a very strong enhancing effect on Rp1-D21
in their hybrids with H95
TX303 (Mu)
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F1
Rp1-D21(H95)
(Screen for F1 plants that no longer have a
severe Rp1-D21 phenotype)
EMS mutagenesis of seed may work even better in knocking out the enhancer
Other intellectual considerations
• Why Rp1-D21 is constitutively active?
• Why the HR underlying Rp1-D21 does not express in very young
leaves?
• Why the HR underlying Rp1-D21 becomes cold sensitive in certain
genetic backgrounds?
• What about some other recombinant alleles of Rp1 that exhibit a
relatively weaker HR response? These alleles include Rp1Kr1N and Rp1-nc3. The latter normally exhibits a recessive
inheritance in most genetic backgrounds but we have found
one exception thus far. In the Mo20W background, the Rp1nc3 phenotype can be observed in a heterozygous condition.
Should we raise antibodies against the Rp1-D21 protein to address these
questions?
Mutant-assisted detection and capture of useful variation
By MAGIC
Its underlying principle is:
The phenotype of a mutant (in the trait of interest) can be
used to unveil relevant variation present in any line,
and also to consolidate this variation in individual plants which can then be used for breeding purposes
To perform MAGIC:
X
mutant
Diversity
parent
F1 hybrid
F2 progeny
Second-site suppressor screen following intentional mutagenesis
EMS
pollen
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Dwarf mutant
M1
M2 population
MAGIC is even more effective with a dominant mutant
F1
P2
P1
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F1
P3
P1
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