CRISPR- mediated Mutation in Cinnamoyl- CoA Reductase 4 in Allohexaploid Oilseed Crop Camelina sativa, Revealed its Pivotal Role in Resistance Against Sclerotinia sclerotiorum.

Abstract

Background: Sclerotinia sclerotiorum (Ss) is a broad host range necrotrophic ascomycete fungus affecting over 400 plant species. Ss causes stem rot disease on Camelina sativa (Cs) an allohexaploid crucifer species that is promoted as a low input crop and industrial oil attributes suitable as biofuel and lubricant. Histochemical and molecular studies has linked resistance to Ss in C. sativa with the cell wall lignification (Eynck et al., 2012) and reported constitutive expression of Cinnamoyl-CoA Reductase 4 (CsCCR4) gene, in the Cs resistant line CN114263. Modern breeding efforts, such as gene editing, are needed to improve commercial lines and to limit the risk of crop loss which would be substantial to producers.Objectives: To investigate the importance of monolignol biosynthesis and the role of CsCCR4 in Camelina resistance to Ss we generated CsCCR4 knockout mutants of CN114263 Camelina line using CRISPR/Cas9-mediated gene editing.Materials and Methods: Thirty T1 plants were produced via floral dip transformation followed by glyphosate spraying that was used in the first step of screening procedures and were confirmed by PCR method. Transgene’s T-DNA copy number variation, T-DNA CNV, in T1 and T2 progenitors were determined using digital droplet PCR (ddPCR) and the occurrence of mutation in the three copies of CsCCR4 homeologues in T1 and T2 generations were scrutinized by drop-off assay technique. To make sure that if the created mutants in T2 plants are real, TOPO TA sequencing flanking the Cas9/gRNA specific hot point of cleavage for three of them was conducted.Results: In the T1 generation, 25 plants were confirmed which had between one to nine T-DNA copies in the corresponding Camelina genome. In T2 generation the population were screened for potential mutation in CsCCR4 gene. Various types of mutations, including insertions and deletions, were demonstrated in three copies of CsCCR4. In fact, CRISPR system could have cut one, two or three copies of the gene in events numbered T2-plant 10, T2-plant 15 and T2-plant 19, respectively. The T3-plant 19 which showed mutation in all versions of CsCCR4 in previous generation had susceptibility to S. sclerotiorum invasion and was kept as real csccr4 mutant material for further investigations of Camelina-Sclerotinia interaction. Mutation in CsCCR4 had occurred through error-prone none- homologous end joining (NHEJ) nucleus DNA repair pathway. Ss challenge on the early flowering T3 generation. The T3 plants with mutation causing premature stop codon at position 217 of CsCCR4 were compromised in their resistance to Ss compared to the wildtype resistant control parent CN114263.Conclusion: Using ddPCR it easily was possible to identify both the T-DNA CNV and occurrence of mutation in CsCCR4 homeologues in T1 and T2 progenitors. We illustrated that CRISPR/Cas9-mediated mutation is a decent technique that can be utilized to expedite the mutant line development which could assist to figure out the activity of a CsCCR4 gene in defense responses to the pathogens in C. sativa as prospective oilseed crop for biodiesel production.