## 3 Discussion  
In this study, we found that MgPDI2 encodes a typical PDI in the endoparasitic nematode _M. graminicola_. MgPDI2 is involved in _M. graminicola_ pathogenicity of host plants and it can also induce cell death in _N. benthamiana_. These results indicated that _MgPDI2_ might play a role in _M. graminicola_ virulence. In the 3-D tertiary structure prediction, MgPDI2 contained four conserved thioredoxin domains (a,b,b',a') which were found to be arranged in the shape of a twisted \"U\" in which the a and a' domains were located on the ends while the b and b' domains formed the rounded base of the U (Figure 1A). This thioredoxin domain arrangement is typical of members of the PDI family [20]. In addition, the a  
Figure 8: Determination of MgPDI2 domains required for cell death induction. (**A**) Schematic views of MgPDI2 and deletion mutants are shown on the left. The deletion mutants of MgPDI2 were transiently expressed in _N. benthamiana_ leaves. (**B**) Western blot analysis of the MgPDI2 deletion series. The anti-GFP primary antibody was used to detect the accumulation of fused proteins in the leaves of _N. benthamiana_ plants. 1: signal peptide deletion mutant; 2: “a” deletion mutant; 3: “b” deletion mutant; 4: “b” deletion mutant; 5: “a” deletion mutant; 6: CGHC in domain “a” deletion mutant; 7: CGHC in domain “a” replaced by AAAA mutant. The ladder used as size standards for the gel consists of the following molecular weight bands: 250, 150, 100, 80, 60, 50, 40, 30, 25, 20, 15, and 10 kDa. The presence of numerous bands might be due to the poor antibody specificity. and a' domains each contain one active site which is characterized by a Cys-Gly-His-Cys (CGHC) catalytic motif (Figure 1A). This CGHC motif is the most common conserved motif in PDIs [20], and the catalytic motifs appear to be highly conserved in the various eukaryotes. Therefore, MgPDI2 is annotated as a typical PDI. Phylogenetic analysis revealed that MgPDI and MgPDI2 are in two different clades (Figure 1B). MgPDI2 grouped with PDIs from _P. parasitica_ and _Brugia malayi_, while MgPDI grouped with PDIs from _Heterodera schuchti_ and _Caenorhabditis elegans_. The distant relationship between MgPDI and MgPDI2 indicates that these two proteins are distinct in amino acid sequences. During this study, it was also noted that the functions of both proteins are also completely different, MgPDI can suppress reactive oxygen species (ROS) production in _N. benthamiana_ induced by flg22 (unpublished data), while MgPDI2 can induce strong necrotic responses in _N. benthamiana_ which further supports the unique characteristics of MgPDI2 compared to MgPDI. _In situ_ hybridization results indicated that _MgPDI2_ was expressed in esophageal gland cells of pre-J2, which is thought to be one of the origins of nematode effector secretion (Figure 2A). These results suggested that MgPDI2 can be secreted into host tissues by _M. graminicola_ to facilitate infection [21]. In addition, our qRT-PCR data showed that the _MgPDI2_ transcription levels in sedentary stages of nematode were relatively high, particularly in the early parasitic stages, reaching its maximum at 3 dpi (Figure 2B). This expression pattern suggested that _MgPDI2_ might play a role in _M. graminicola_ infection. The spatial and temporal expression pattern of _MgPDI2_ is very similar to the expression pattern of _MgPDI_ and _HsPDI_ in _Heterodera schuchti_[12; 19]. Like the recombinant MgPDI protein from _M. graminicola_, recombinant MgPDI2 was highly expressed at 20 \({}^{\circ}\)C (Figure 3) [19]. Previous studies reported that PDIs have enzymatic activities relevant to oxidization, isomerization, and reduction [2; 22]. Here, we tested the reduction and antioxidant activities of MgPDI2. For the reductive assays of MgPDI2, a classical insulin reduction assay was performed, and the results indicated that MgPDI2 can reduce insulin disulfide bonds (Figure 4A). This reductive activity is consistent with that of other PDIs as observed in previous studies [2; 19]. However, compared to MgPDI of _M.