Understanding The Roles Of NtGSNOR1a/1b In Tobacco By Simultaneously Knocking Out Four Copies Of NtGSNOR1a/1b Using CRISPR/CAS9 Technology

GSNOR1?S-nitrosoglutathione reductase 1?is the key enzyme that regulates cellular homeostasis of S-nitrosylation across kingdoms.Although extensively studied in model plant Arabidopsis,the role of GSNOR1 in Nicotiana species has not been investigated previously.To study the role of GSNOR1 in cell death and disease resistance in Nicotiana species,transgenic CRISPR/CAS9 lines knocking out the two NtGSNOR1 genes simultaneously were generated in N.tabacum cv Samsun carrying a TMV resistant gene,N.To our surprise,a natural spontaneous cell death occurred on the leaves of the CRISPR/CAS9 lines but not on that of the wildtype?WT?,suggesting that NtGSNOR1negatively regulates cell death in Nicotiana species.Both nitric oxide?NO?and H₂O₂ were over-accumulated in the NtGSNOR1a/1b knock out leaves,implying that the cell death on the CRISPR/CAS9 lines could be a results of interactions between over-accumulated NO and H₂O₂.Paraquat is a herbicide that can kill plants by inducing over-production of ROS.Unexpectedly,we found that CRISPR/CAS9 lines displayed a significantly enhanced resistance to Parquat-induced cell death compared to WT,suggesting that NtGSNOR1functions as a positive regulator of the Paraquat-induced cell death.The increased resistance to the Paraquat-induced cell death of CRISPR/CAS9 lines seemed to be correlated with the reduced level of ROS accumulation because the Paraquat-induced ROS accumulation level on the leaves of the NtGSNOR1a/1b knockout lines was significantly lower than on that of WT plants.The fact that the reduced level of free SA level was observed in the leaves of the NtGSNOR1a/1b knockout lines relative to in WT leaves,implying that the naturally occurred spontaneous cell death on the leaves of the NtGSNOR1a/1b knockout lines is independent of SA pathway.Interestingly,whereas the N gene-mediated resistance to Tobacco mosaic virus?TMV?was significantly enhanced,the resistance to bacterial pathogen Pseudomonas syringae pv.Tomato DC3000 was significantly reduced in the NtGSNOR1a/1b knockout lines.In summary,our results indicate that GSNOR1 in Nicotiana species functions as both positive and negative regulator of cell death under different cellular conditions and displays distinct effects on resistance against viral and bacterial pathogens.