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Ubiquitination is a vital cellular biochemical process that has been shown to be involved in growth and development of eukaryotic organisms, including plants. The work presented in the dissertation investigates role of ubiquitination in regulation of programmed cell death (PCD) and in innate immune responses elicited by a devastating fungal pathogen Magnaporthe oryzae (Mo) which causes the rice blast disease. Lesion mimic mutant spl11 confers broad-spectrum resistance to variety of rice pathogens including Mo. SPL11,an E3 ubiquitin ligase enzyme, has been shown to be a negative regulator of PCD. To identify the genetic components in the SPL11-mediated PCD pathway a suppressor screen approach led to the identification of three spl11 suppressor (sds) mutants. Genetic analysis revealed that two of the mutants (sds2 and sds3) have gain-of-function mutations whereas the remaining suppressor (sds1) has one gain-of-function and one loss-of-function mutations that are responsible for the suppression of lesion mimic phenotype. Test of allelism excludes the mutations to be allelic to each other in the sds lines. Defense markers’ gene expression, protein levels and metabolite analysis along with Mo and Xanthomonas oryzae pv. oryzae (Xoo) infection indicate that the causal mutations might be involved in regulation of basal defenses. Molecular mapping of the sds2 has narrowed down the mutation in 131 kbp interval. Further fine mapping and cloning of the SDS2 will identify a genetic component in SPL11-mediated PCD pathway in rice.Mo effector protein AvrPiz-t interacts with a number of ubiquitination-related enzymes in rice. Of special importance to the work are two E3 ubiquitin ligase enzymes APIP6 and APIP10. Comprehensive immune response analysis revealed that the APIP6 and APIP10 are involved in the positive regulation of pathogen-associated molecular pattern (PAMP)- triggered immunity (PTI). As the PTI is a major component of the basal defense in rice against Mo infection, the fungus secretes AvrPiz-t to interfere with the enzymatic activity of the APIP6 and APIP10 in the rice cytoplasm. It promotes mutual turnover of the proteins via ubiquitin-proteasome system to suppress PTI responses that leads to enhanced fungal growth. Mutation analysis of the AvrPiz-t emphasizes importance of the lysine residues in the mutual turnover and overall virulence function of the AvrPiz-t. Interestingly, the work also shows that a rice NBS-LRR receptor (NLR) Piz-t acts as a guard protein for APIP10 and Piz-t protein levels are negatively regulated by the APIP10. In rice cultivars with Piz-t, activity of Mo secreted AvrPiz-t is sensed by Piz-t due to loss of the negative regulation by AvrPiz-t-mediated turnover of APIP10. The recognition leads to stronger immune responses that result in the resistance against the fungus. Thus, the work presented gives further evidence that the ubiquitination plays important role in innate immunity of the plants. In future, this knowledge can be translated to develop cultivars with durable and long lasting resistance because ever-evolving pathogens will challenge intensively cultivated global crop plants like rice. |
