A second RNAi construct for targeting chestnut blight fungus genes

Our first gene construct is using RNAi to target the OAH gene of the blight fungus, we targeted that gene due to our previous success using an enzyme called Oxalate Oxidase (OxO) to detoxify the the acid produced by the blight fungus (Powell et al. 2019). We decided that it would be a good idea to also target genes that were not involved in the oxalic acid pathway in a second RNAi gene construct. Luckily, there have been decades of research where scientists have disrupted or deleted genes within the fungus itself and observed the effects of those gene disruptions. Some of the effects of those gene disruptions include reducing the virulence of the fungus during the infection process of chestnuts (Chun et al. 2022), and the disruption of some genes even results in the overall reduction of the health of the fungus (MyoungJu et al. 2004). One of the incredible capabilities of RNAi is the ability to target multiple genes in a single RNAi molecule (Czarnecki et al. 2016). With this knowledge in mind, we selected five separate C. parasitica genes for simultaneous silencing, designed and ordered synthetic sequences targeting those genes, and cloned the synthetic sequences into our pKannibal vectors used for RNAi production. We named this multi-gene disrupting construct Cp5x (C. parasitica-gene disruption x5). This new construct was transformed into Agrobacterium tumefaciens cells which were used to transform American chestnut embryos as described in McGuigan et al. 2020. We currently have multiple transgenic embryo lines carrying the Cp5x RNAi construct that are being moved through the American chestnut tissue culture process to be regenerated into whole plants for testing against chestnut blight fungal infection. (Mcguigan et al. 2020) (Powell et al. 2019) (Czarnecki et al. 2016) (Myoungju et al. 2004)  (Chun et al. 2022)