MECHANISTIC ROLES OF DNA POLYMERASE DELTA SUBUNIT 1 IN RESISTANCE TO DNA GEMINIVIRUSES

Cassava is an important crop for small, medium, and large-scale farmers. It is a hardy plant that can be grown without irrigation, fertilizer and pesticides and is highly productive even when grown on marginal land. As such, it is likely to become an even more important crop in the face of global climate change. Cassava Mosaic Disease (CMD), caused by geminivirus species, is endemic across the African continent and has recently spread to Asia. CMD is primarily controlled by a single dominant locus,CMD2.We previously demonstrated that passage of CMD2-type cassava cultivars through tissue culture deactivates CMD2-mediated resistance and CMD2-type resistance is caused by specific amino acid changes within the DNA Polymerase delta subunit 1 (POLD1) protein. The current model to explain these results is that CMD2-type cultivars of cassava are periclinal chimeras with the resistance allele present in only specific cell layers. De novo morphogenesis that occurs during tissue culture yields plants derived from the susceptible cell layer. Because cassava is clonally propagated, this chimerism may have arisen several decades ago. Exploration of publicly available genomic data suggests that similar alleles exist in germplasm collections from tomato, cotton, and several other important crops.Having discovered that specific alleles of MePOLD1 confer dominant resistance to CMGs is a major advance, but also raises many questions. How does this resistance work on a molecular level? Does it block replication of the virus, movement, or both? With the resistant allele present in only some cell types, does this indicate the involvement of a mobile signal? Can this resistance mechanism function in other geminivirus pathosystems? And, given the importance of this source of resistance to the lives of millions of people, why have CMGs not (yet) mutated around this type of resistance? This research will elucidate the molecular mechanism behind CMD2-resistance, identify additional genes important during the resistance response, and test whether this mechanism can function in other plants.The goals of this research are as follows:Goal 1: Test whetherMePOLD1resistant alleles affect viral replication, interaction with other proteins, and/or viral movement.Objective 1.1: Elucidate whetherMePOLD1-based geminivirus resistance functions through altered rate and/or fidelity of DNA replication.Objective 1.2: Evaluate physical interaction of POLD1-R proteins with viral proteins and viral DNA.Objective 1.3: Investigate MePOLD1 protein structures and potential interaction with viral proteins.Goal 2: Characterize different POLD1 resistance alleles for interaction with the plant antiviral responseObjective 2.1. Generate a series of POLD1 alleles.Objective 2.2: Generate baseline data for strength of resistance for each MePOLD1 resistance allele.Objective 2.3: Develop new methods of in planta detection of geminivirusesGoal 3: Test whether POLD1-mediated resistance control geminivirus diseases in distinct plants?Objective 3.1. Test for POLD1-based resistance in Arabidopsis.Objective 3.2 Genomic investigation of POLD1 as a source of geminivirus resistance in cotton and tomato.