1.survey the entire panel of maize diversity lines (association mapping panel) for their ability to...

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 gene-mediated immunity in maize

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 rapidhost plant death when attacked by rust fungi’ (Stakman, 1915)

Compatible vs. incompatible plant-pathogen interaction

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

NDR1NDR1 - - nnon race-specific on race-specific ddisease isease rresistanceesistance EDSEDS - - eenhanced nhanced ddisease isease ssusceptibilityusceptibility

CC type NBS-LRR genesCC type NBS-LRR genes TIR type NBS-LRR genesTIR 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

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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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X

X

7 98

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2/91Rp1-D21

Generation of the Rp1-D21 recombinant

A dominant lesion mimic mutant

The HR phenotype of The HR phenotype of Rp1-D21Rp1-D21 in H95 in H95

Rp1-D21 plants exhibit all the hallmarks of the HR response

18S rRNA

PR1

Rp1-D21

WT mut

PR5

PRms

WIP1

The HR phenotype of The HR phenotype of Rp1-D21Rp1-D21 is cell autonomous 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 Rp1-D21 (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 Rp1-D21 (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

Rp1

Chromosome 10

Identification of Hrml1 (HR modulating locus-1)

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



• 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




• 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




• 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.



3. Evaluate NAM RIL populations for additional Hrml genes/QTL and clone at least three of them.




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.



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


• 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)

M1 - few thousand seed has been generated

(Treated with EMS)


• 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 Mu-

active 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



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)



• 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



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) X Rp1-D21(H95)

F1 (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 Rp1-Kr1N 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 Rp1- nc3 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

F1 hybrid

To perform MAGIC:

X

Diversity parent

mutant F2 progeny

X

EMS pollen

M1Dwarf mutant M2 population

X

EMS pollen

M1Dwarf mutant M2 population

Second-site suppressor screen following intentional mutagenesis

X

P1

P2

F1

X

P1

P2

F1

X

P1

P3

F1

X

P1

P3

F1

MAGIC is even more effective with a dominant mutant

1.survey the entire panel of maize diversity lines (association mapping panel) for their ability to...

Documents

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gene identification

link r genes