CRISPR-Cas9 Functional Characterisation of NLR Immune Receptors in Cassava for Durable Resistance to Cassava Mosaic Begomoviruses in Nigeria

Cassava mosaic disease caused by begomoviral complexes is the primary yield-limiting disease constraint in Nigerian cassava production, and while host resistance is the most sustainable management strategy, the molecular and genetic architecture of cassava NLR immune receptor-mediated resistance to begomoviruses remains poorly characterised. This doctoral research employs CRISPR-Cas9 functional genomics to characterise a selected panel of NLR immune receptors in cassava with predicted recognition specificity for the AC2 and AC4 silencing suppressor proteins of African Cassava Mosaic Virus and East African Cassava Mosaic Virus, the predominant species in Nigeria. The research involves bioinformatic prediction of candidate NLR-effector interaction pairs using structure-based modelling, CRISPR-Cas9-mediated knockout of five prioritised NLR candidates in susceptible cassava variety TMS 30572, and Agrobacterium-mediated virus-induced gene silencing assays to validate target gene function in resistance responses. Inoculation of edited lines with Nigerian begomovirus isolates confirms the functional contribution of each targeted NLR to resistance specificity and durability. The work also employs co-immunoprecipitation and yeast-two-hybrid assays to characterise direct NLR-effector protein interactions. Original theoretical contributions include a mechanistic model of cassava begomovirus recognition incorporating the guard hypothesis adapted for cassava-begomoviral systems, and a prioritised list of NLR targets for integration into conventional breeding and transgenic resistance programmes. This research represents the first application of CRISPR-Cas9 functional NLR analysis in cassava in West Africa. Keywords: CRISPR-Cas9, NLR, cassava, begomovirus, resistance.