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S-nitrosoglutathione reductase 1 (GSNOR1) is the key enzyme that regulates cellular homeostasis of  S- nitrosylation. Although extensively studied in  Arabidopsis , the roles of GSNOR1 in tetraploid  Nicotiana  species have not been investigated previously. To study the function of  Nt GSNOR1, we knocked out two  NtGSNOR1  genes simultaneously in  Nicotiana tabacum  using clustered regularly interspaced short palindromic repeats (CRISPR)/caspase 9 (Cas9) technology. To our surprise, spontaneous cell death occurred on the leaves of the CRISPR/Cas9 lines but not on those of the wild-type (WT) plants, suggesting that  Nt GSNOR1 negatively regulates cell death. The natural cell death on the CRISPR/Cas9 lines could be a result from interactions between overaccumulated nitric oxide (NO) and hydrogen peroxide (H 2 O 2 ). This spontaneous cell death phenotype was not affected by knocking out two  Enhanced disease susceptibility 1  genes  (NtEDS11a/1b)  and thus was independent of the salicylic acid (SA) pathway. Unexpectedly, we found that the  NtGSNOR1a/1b  knockout plants displayed a significantly ( p  < 0.001) enhanced resistance to paraquat-induced cell death compared to WT plants, suggesting that  Nt GSNOR1 functions as a positive regulator of the paraquat-induced cell death. The increased resistance to the paraquat-induced cell death of the  NtGSNOR1a/1b  knockout plants was correlated with the reduced level of H 2 O 2  accumulation. Interestingly, whereas the  N  gene-mediated resistance to  Tobacco mosaic virus  (TMV) was significantly enhanced ( p  < 0.001), the resistance to  Pseudomonas syringae  pv.  tomato  DC3000 was significantly reduced ( p  < 0.01) in the  NtGSNOR1a/1b  knockout lines. In summary, our results indicate that  Nt GSNOR1 functions as both positive and negative regulator of cell death under different conditions and displays distinct effects on resistance against viral and bacterial pathogens.

Dual Roles of GSNOR1 in Cell Death and Immunity in Tetraploid Nicotiana tabacum