The major focus of this project was to expand the knowledge of the plant hypersensitive
cell death response (HR) using maize as a model system. This was inspired by a novel
enhancer/suppressor genetic screen that we designed to specifically detect and
delineate natural variation. Because this approach is based on the use of the phenotype
of a mutant as a reporter of natural variation, it has been named it Mutant-Assisted
Gene Identification and Characterization (MAGIC).
The HR is an integral part of the plant immune response. Efforts over the past two
decades to characterize the genetic control of HR have relied mostly on the analysis of
artificially-induced variants. Despite these efforts, our knowledge of the components and
mechanisms underlying HR have remained limited. We reasoned that natural variation,
which represents a vast and largely untapped resource of genetic variation, may prove
complementary in revealing additional genes and mechanisms that take part in
executing the HR response. The problem with natural variation, however, is that it does
not yield readily to genetic analyses and most approaches devised to explore it are both
complicated and cumbersome and are very time and resource extensive. MAGIC was
designed as a direct forward-genetic approach that not only helps identify germplasm
with significant variation of interest but also allows to quickly map and locate the genes
responsible for such variation. An additional focus of the project was to test the power of
MAGIC in revealing variation underlying such a complex and complicated trait as the
plant immune system as a model for the analysis of other traits.
Three specific objectives of the project were:
1. MAGIC interrogation of the NAM RILs and subpopulations with Rp1-D21 to
detect and map QTL and genes capable of significantly modulating the HR
response.
2. MAGIC interrogation of the association mapping panel inbreds with Rp1-D21 to
identify genes capable of significantly modulating the HR response.
3. Attempt to clone and confirm a few (up to 3) genes capable of modulating the HR
response.
Another write-up for the project focus taken from the 2013 report
The overall goal of this project is to enhance our understanding of the genes and
mechanisms that underlie the hypersensitive response (HR). Although HR is the plant
world’s most important immune response, our understanding of the genetic network that
controls it remains poor. Since approaches based on artificially induced mutagenesis
have had limited success in turning up the genetic components of HR, we decided to
make use of the variation naturally present in the maize germplasm. To accomplish this,
we are making use of a novel, innovative genetic strategy that allows natural
suppressors and enhancers of the HR to be quickly discovered and characterized in a
forward genetics screen. Because this approach is based on the use of the phenotype
of a tester mutant (in a trait of interest) as a reporter of natural variation, we have
named it MAGIC, for Mutant-Assisted Gene Identification and Characterization.
The underlying idea of MAGIC is that the changes in the phenotype of the tester mutant
that happen in diverse genetic backgrounds serve both to uncover the nature of the
natural variation – enhancing or suppressing - and to genetically dissect the factors
responsible for this transgressive change in the phenotype of the tester mutant. All that
it takes is crossing the mutant tester with diverse lines and then evaluating the resulting
segregating populations for changes – both up and down - in the phenotype of the
reporter mutation. MAGIC can be conducted on both structured and unstructured
populations and also on individual accessions or lines that are either elite or exotic. A
key benefit of MAGIC is that it has the potential to reveal genetic variation that normally
remains hidden or cryptic and therefore inaccessible to genetic dissection. By having a
mutant in the background, MAGIC serves to hypersensitize the segregating populations
to potential changes in the reporter phenotype, thereby reducing the background noise
and amplifying the magnitude of the relevant variation (gene or QTL) in the trait of
interest. Thus, it allows even subtle changes in the mutant phenotype to be detected
and therefore readily mapped.
For this project, our reporter mutation is a partially dominant autoimmune R gene, Rp1D21, which triggers HR ectopically, leading to macroscopic necrotic spots all over the
plant. We have shown that the strength of the HR phenotype associated with Rp-D21 is
substantially dependent on genetic background - indicating that natural genetic variation
exists that controls the strength of HR.