## 4 Materials and Methods
### Graminicola Culture Conditions  
The _M. graminicola_ isolate ZJJH was maintained on rice (_Oryza sativa_ cv. \"Nipponbare\") in potting soil in a grow chamber under a 16 h/8 h light/dark photoperiod at 28 \({}^{\circ}\)C/26 \({}^{\circ}\)C [27].  
Cloning and Sequence Analyses  
The full-length cDNA sequence of _MgPDI2_ was obtained by rapid amplification of cDNA ends using the SMART RACE cDNA Amplification kit (Clontech, Carlsbad, CA, USA) according to the manufacturer's instructions based on _M. graminicola_ transcriptome data [28]. All primers used in this study are listed in Table 2. An online protein structure homology-modeling server, SWISS-MODEL, was used to predict MgPDI2 tertiary structure. The signal peptide of MgPDI2 was predicted using the SignalP-5.0 Server ([[http://www.cbs.dtu.dk/services/SignalP/](http://www.cbs.dtu.dk/services/SignalP/)]([http://www.cbs.dtu.dk/services/SignalP/](http://www.cbs.dtu.dk/services/SignalP/))). The conserved domain search for MgPDI2 was performed using the NCBI CD search program ([[https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi](https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)]([https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi](https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi))) [29]. The phylogenetic tree was constructed using maximum likelihood algorithms in MEGA 6, and PDI amino acid sequences from different organisms are described as the following: MgPDI (MH392200), _Phtyophthora parasitic_ (XP_008914616.1), _Ascaris suum_ (ERG84937.1), _Caenorhabditis elegans_ (NP_491995.1), _Homo sapiens_ (CAA89996.1), _Leishman major_ (AAN75008.1), _Heterodera schachtii_ (ANA05342.1), and _Brugia malayi_ (XP_001899304.1).  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Primer Name** & **Primer Sequences (5’-3’)** & **Use** \\ \hline \hline MgPDI2-F & GTCATGATCCCTTCTTCTTCTTC & open reading frame (ORF) \\  & & verification \\ MgPDI2-R & CAATGAAAAGAACAACAACAGGAGG & ORF verification \\ MgPDI2-Bamhl & CGCGATCCGAGAAGG & Vector construction \\ MgPDI2-XholI & CCGCTCGAAGCTCAGTTTTCCTC & Vector construction \\ MgPDI2-RT-F & TGATGAGGACTGCCTGACT & qRT-PCR \\ MgPDI2-RT-R & CTCCACCAAAAATGACGGC & qRT-PCR \\ Mg-ACT-Q-F & AAGATCCCTGAGCCTGGTTAC & qRT-PCR \\ Mg-ACT-Q-R & CTTGACCGTCAGCCAATTCATAGC & qRT-PCR \\ MgPDI2-P & TTATGATCCGCCTTGC & In situ \\ MgPDI2-AP & CAAAGAATGAGACGAACAGC & hybridization \\ MgPDI2-T7-P & TAATACGACTACTATAGGTTATGTTCTCCCGTTGC & In situ \\  & & hybridization \\ MgPDI2-T7-AP & TAATACGACTACTATAGGCTTATGTTCTGCCTTGC & In situ \\  & & hybridization \\ MgPDI2-dSRNA-P & CCAAGGATCCCTGATT & dsRNA \\ MgPDI2-dsRNA-AP-T7 & TAATACGACTACTATAGGTTCTCACACATGAGACACG & dsRNA \\ MgPDI2-dsRNA-P-T7 & TAATACGACTACTATAGGCCAAGGACACGG & dRNA \\ MgPDI2-dSRNA-AP & TCTCAGCATTAGGTTTT & dsRNA \\  & & Vector \\ MgPDI2-GFP-P & TCTACAATCTCTCTGGATCCATGACTTCTTCTTCTTCTTCTT & construction \\ MgPDI2-GFP-AP & TCGCCCTTCTCACCATGATGACCCTGACCCTG & Vector construction \\ MgPDI2-NSP-P & TCTACAATCTACTCTGATCCATGGAAAAGGTTTTCCTAC & MgPDI2 mutants \\ MgPDI2-NSP-AP & TGCCCCTTCTCACCATGATGACCACCCTGCTGCTCTCT & MgPDI2 mutants \\  & & MgPDI2 mutants \\ MgPDI2-a-AP & AAGAGAGAAGAATGAAAAGACTGGACCTCC & MgPDI2 mutants \\  & & MgPDI2 mutants \\ MgPDI2-b-AP & TGGAGGTCCAGCTTCTTCTT & MgPDI2 mutants \\  & & MgPDI2 mutants \\  & & MgPDI2-b-P & AGAAGACTGGACCCCAAGAATTCCTTTTTCAGA & MgPDI2 mutants \\  & & MgPDI2-b-AP & TGGGCTCAAATG