Abstract
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 NtGSNOR1, 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 NtGSNOR1 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 (H2O2). 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 NtGSNOR1 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 H2O2 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 NtGSNOR1 functions as both positive and negative regulator of cell death under different conditions and displays distinct effects on resistance against viral and bacterial pathogens.
Highlights
Nitric oxide (NO) is a reactive free radical gas molecule with a plethora of functions in both animals and plants (Wendehenne et al, 2014)
Pst DC3000 multiply better on the leaves of the NtGSNOR1a/1b knockout plants than on the leaves of the WT plants (Figure 6C), indicating that NtGSNOR1a/1b positively regulates the basal resistance to a bacterial pathogen. These results indicate that knocking out NtGSNOR1a/1b has opposite effects on resistance to different types of pathogens with different infecting strategies. We found that both anti- and pro-cell death effects were observed in the NtGSNOR1a/1b knockout plants, reinforcing the notion that NO can play either anti- or prodeath roles depending on cellular conditions (Wang et al, 2013; Wendehenne et al, 2014) and the altered NO homeostasis plays key roles in switching its functions during the hypersensitive response (HR)
The cell death phenotype of the NtGSNOR1a/1b knockout plants under natural conditions was not reported for the gsnor1-3/hot5/par2-1 mutants (Feechan et al, 2005; Lee et al, 2008; Chen et al, 2009) or antisense lines of GSNOR1 in Arabidopsis (Rustérucci et al, 2007), spontaneous cell death was observed on the leaves of GSNOR1-silenced tomato plants by virus-induced gene silencing (VIGS) (Liu et al, 2017) and an accelerated cell death was observed on the leaves of gsnor1-3 mutant upon infections by avirulent bacterial strains or by an avirulent oomycete isolate (Rustérucci et al, 2007; Yun et al, 2011)
Summary
Nitric oxide (NO) is a reactive free radical gas molecule with a plethora of functions in both animals and plants (Wendehenne et al, 2014). Roles of NtGSNOR1 in Immunity (He et al, 2004), growth/development (Yun et al, 2016), and many other processes (Lamattina et al, 2003; Wendehenne et al, 2014) Among all these processes, the most significant function of NO is to potentiate the induction of hypersensitive cell death by reactive oxygen species (ROS) (Durner et al, 1998; Delledonne et al, 2001). S-nitrosylation, attachment of NO moiety to a target protein, is a newly emerged mechanism by which NO regulates the function of various target proteins and various biological processes (Hess and Stamler, 2012; Wendehenne et al, 2014). The S-nitrosylated cysteine residues of some target proteins have been identified, and the functions of this posttranslational modifications are revealed (Lindermayr et al, 2006, 2010; Romero-Puertas et al, 2007; Tada et al, 2008; Yun et al, 2011; Skelly et al, 2019; Gupta et al, 2020)
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