## 3 Discussion
graminicola_, the reduction activity of MgPDI2 is likely stronger than that of MgPDI as the reduction of disulfide bonds using 0.625 \(\upmu\)M recombinant MgPDI2 is better than that of 2.5 \(\upmu\)M recombinant MgPDI [19]. To assess the antioxidant activity of MgPDI2, we used an MFO system to generate thiol radicals that damage DNA to assess how MgPDI2 was able to protect DNA integrity. Our results suggested that MgPDI2 can protect plasmid DNA from nicking in a dosage-dependent manner (Figure 4B). [PERSON] et al. [19] demonstrated that MgPDI also possessed this kind of antioxidant activity. It was reported that as PDI is a member of the thioredoxin superfamily, PDI plays a key role in several pivotal antioxidant and redox regulation processes [22]. Thioredoxin-mediated protection of DNA from oxidative damage was also demonstrated in _Haemontchus contortus_[23]. The two above enzymatic activities are consistent with the functions of typical PDIs which contain four conserved thioredoxin-like domains. Therefore, we confirmed that MgPDI2 is a functional PDI. To analyze whether _MgPDI2_ plays a role in the parasitism process of _M. graminicola_, in vitro RNAi targeting _MgPDI2_ was performed. We found that pre-parasitic J2s treated with ds_MgPDI2_ reduced to the MF 6.69 compared to an MF of 11.37 when treated with ds_GFP_ (Figure 6B). RNAi can be used to investigate the function of pathogenicity factors in both sedentary and migratory nematodes [24]. Our results are consistent with the conclusions drawn in previous studies, such as, [PERSON] et al. [12] reported that silencing _HsPDI_ by RNAi reduced the number of _H. schachtii_ eggs [12]; [PERSON] et al. [19] demonstrated that _MgPDI_ silencing caused a reduction in the MF of _M. graminicola_. Together, these findings suggest that PDI is essential in the reproduction of parasitic nematodes. Similar to _MgPDI_, 40 mM H2O2 can induce the expression of _MgPDI2_ (Figure 6A) [19]. Moreover, it is noted that both 20 and 40 mM H2O2 can significantly increase the mortality rate of _MgPDI2_ dsRNA-treated J2s (Figure 6B). These results suggest that MgPDI2 plays a role in protecting J2s from the impact of the exogenous H2O2. [PERSON] et al. [12] and [PERSON] et al. [24] also reported that _HsPDI_from _H. schuchtii_ and peroxiredoxins from _Meloidogyne incognita_ increased tolerance to exogenous H\({}_{2}\)O\({}_{2}\), respectively. Some pathogen effectors can elicit cell death in _N. benthamiana._ For example, it is demonstrated that the effector protein RBP-1 from _Globdera pallida_ can be recognized by the NB-LRR protein Gpa2 to elicit cell death in _N. benthamiana_[25]. PDI1 of _P. parasitica_ (PpPDI1) and _G. pallida_ (GpPDI1) also triggered cell death in _N. benthamiana_[11; 26]. In this study, we found that the secreted protein MgPDI2 from _M. graminicola_ induced noticeable cell death in _N. benthamiana_ 48 h after Agro-infiltration (Figure 7), indicating that MgPDI2 is sufficient for inducing cell death in _N. benthamiana_. However, deletion mutants lacking a, b, and b\({}^{\prime}\) domains lost the ability to induce cell death. These data indicate that an intact C-terminus containing domains a, b, and b\({}^{\prime}\) is required for activity. Furthermore, deleting or replacing the CGHC catalytic motif in domain a with alanine residues prevented cell death induction in _N. benthamiana_, indicating that the first active CGHC motif is also essential for cell death induction by MgPDI2 (Figure 8A). These results indicated that the first catalytic domain (a), particularly the CGHC catalytic motif in domain a, and the two non-active domains (b and b\({}^{\prime}\)) are required for cell death induction in _N. benthamiana_, while the signal peptide and the domain a\({}^{\prime}\) have no role in inducing cell death in _N. benthamiana_. However, it is unusual that PDI, a ROS scavenger, caused cell death in _N. benthamiana_.