It provided the first system-wide analysis of natural variation that modulates the HR response in plants, and identified the location of 44 genes that play a role in controlling the HR response. The HR cell death is an integral part of the plant immune response, yet our understanding of how it is triggered, executed and strictly contained remains rudimentary. This project has allowed us to unveil specific pathways and genes that are involved in the elicitation and containment of HR. Many of these genes and mechanisms seem to be involved in the control of redox homeostasis, programmed cell death, autophagy, ubiqutin-mediated protein degradation, and lignin biosynthesis, suggesting that HR is rather complicated and requires the function of multiple host processes to use cell suicide as an effective immune strategy. The identification of all these genes and mechanisms represents a significant step forward for the field. The next steps are to validate specific genes and or their alleles that we have identified here and characterize their role in HR in more detail.
The work done on the structure-function aspects of the RP1-D21 protein, which is a typical NBS-LRR protein, shows for the first time how a disease resistance gene of the NBS-LRR class can become autoactive as a result of simple genetic recombination. Rp1-D21 is a chimeric gene that came into existence as a result of recombination between two R gene paralogs (duplicates) at the Rp1 locus of maize. The 5’ region of Rp1-D21 is from gene 2 (Rp1-dp2) at the locus and the 3’ end is from gene 9 (Rp1-dp9) at this complex locus. Thus, the N terminal region of the RP1-
D21 protein, which contains largely the coiled coil (CC) domain, is from one gene and the C terminal region, which constitutes mostly the NBS and LRR domains of an R protein, are from a separate gene. These two ends are not compatible with each other and they fail to lock the NBSLRR immune receptor in an auto-inhibited configuration. This in turn leads to auto activation of the RP1-D21 protein, resulting in the triggering of the HR cell death response. Furthermore, we found the N-terminal coiled-coil domain from either Rp1-D21 (CC) or Rp1-dp9 was sufficient to induce HR, and that the NB domain was important for suppressing this phenotype. Interestingly, the LRR domain relieved this NBinduced suppression in Rp1-D21 but not in Rp1-dp9, again showing the incompatibility between different domains of RP1-D21. Experimental domain swaps between Rp1-dp2 and Rp1-dp9 (progenitors of Rp1-D21) identified precise regions associated with the autoactive nature of Rp1-D21-triggered HR. This work provides several novel insights into structural requirements for NBS-LRR function in plant immunity and informs efforts towards utilizing these proteins for engineering disease resistance.
Impact on other disciplines
Our study demonstrated that MAGIC is a very effective approach in exploring the genetic basis of a complex trait using natural variation. This simple genetic approach will be useful for identifying useful genetic variation that had heretofore been inaccessible. Although we focused largely on structured populations for this project - such as the maize IBM and NAM populations, MAGIC also showed great promise in revealing and harnessing HR variation from the germplasm that has never been characterized.
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 gene/QTL in the trait of interest. Thus, it allows even subtle changes in the mutant phenotype to be detected and therefore readily mapped.
MAGIC is especially useful in finding QTL with relatively large sizes no matter how rare they might be in the germplasm or populations, something that association mapping fails to achieve. MAGIC also simplifies the phenotypic evaluation of the trait of interest. In fact, it can convert a highly complex and subjective phenotype into one that become completely quantitative, allowing it to be measured precisely by anyone, even a novice.
MAGIC can also facilitate the build-up of ideal gene combinations that can enhance the performance of a trait of interest. Using this idea, we have been developing maize germplasm with both improved yield and disease resistance to multiple pathogens. We have also successfully used MAGIC to enhance the attractiveness of a western corn rootworm beetle-susceptible maize mutant (crw1) for use as a ‘trap’ crop to manage this pest better without having to rely on environmentally unsafe chemicals.