## 2 Results
### Two Up-Regulated Genes in Rice Resistant Plants Encode Putative VAP Proteins  
_Mg707_ and _Mg11937_ share high homologies with the _Gr-VAPI_ gene from _Globodera rostochiensis_, an important effector able to modulate basal plant immunity [45; 46]. _Mg707_ and _Mg11937_ putatively encode proteins of 203 and 247 aa., respectively, with a signal peptide and a Sperm-Coating Protein (SCP) domain characteristic of VAP proteins (Figure 5a), designed also as the cysteine-rich secretory proteins/antigen5/pathogenesis-related1 (CAP) domain. _Mg707_ and _Mg11937_ only share \(\sim\)33% aa. identity. Deduced amino-acid sequences alignment of _Mg707_ and _Mg11937_ with _Mi-VAPI_[47], _Mi-VAP2_[48] and _Gr-VAPI_[46] indicated that _Mg707_ is close to _Mi-VAPI_ and _Gr-VAP1_, and _Mg11937_ is close to _Mi-VAP2_ (Figure 5b,c). Hence, we propose here to rename _Mg707_ as _Mg-VAP1_ and _Mg11937_ as _Mg-VAP2_.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline _M. graminicola_ & \multicolumn{3}{c}{**RNA Seq (R versus S)**} & \multicolumn{3}{c}{**qPCR (R versus S)**} \\ \cline{3-7} _Transcript_ & **Predicted Function** & \multicolumn{3}{c}{**2 dpi 4 dpi**} & **8 dpi** & **2 dpi** & **4 dpi** & **8 dpi** \\ \hline _Mg_449 & Endoglucanase & 2.17 * & 3.69 * & 2.99 * & 2.70 * & 5.14 * & 5.45 * \\ _Mg_693 & Glucuronoxylanase & 1.57 & 3.95 * & 4.38 * & 1.88 & 3.66 * & 5.82 * \\ _Mg_1702 & Neuropeptide _“nlp*_ & 0.07 & 1.40 & 2.19 * & 0.70 & 2.40 * & 4.98 * \\ _Mg_2129 & Neuropeptide _“flp”_ & 0.33 & 1.58 & 3.14 * & 1.03 & 2.06 & 4.69 * \\ _Mg_707 & VAP & 9.26 & 3.27 * & 2.57 * & 2.42 & 4.99 * & 5.62 * \\ _Mg_11937 & VAP & 1.38 & 5.03 * & 5.86 * & 2.08 & 5.63 * & 6.78 * \\ _Mg_757 & Unknown & 0.67 & 1.57 & 2.8 * & 1.11 & 2.83 * & 4.40 * \\ _Mg_4965 & CLE-like & \(-\)0.26 & 0.60 & 1.11 & 1.09 & 0.96 & 1.19 \\ _Mg_12322 & Unknown & \(-\)0.92 & \(-\)0.72 & 1.68 & \(-\)0.81 & \(-\)0.15 & 3.67 \\ _Mg_13532 & Unknown & \(-\)0.79 & \(-\)0.75 & \(-\)0.44 & \(-\)0.49 & \(-\)0.27 & 1.04 \\ _Mg_44061 & Unknown & 5.42 & 9.55 & 12.09 * & 0.001 & 1.89 & 5.02 \\ \hline \end{tabular}
\end{table}
Table 1: Validation of RNA-Seq data by quantitative PCR (qPCR). Values are expressed as Log2-Fold Change. For RNA-Seq, two independent biological assays were analysed (replicates A and B) and * indicate DEGs as defined by EdgeR (log2 FC \(>\) 2 or \(<\)\(-\)2, padj \(<\) 0.05). For Q-PCR, three independent biological assays were analysed (replicates A and B, both used for RNA-Seq, and replicate C) and * indicate significant differences (_t_-test, _p_\(<\) 0.05) between resistant (R) and susceptible plants (S).  
In order to study the role of _Mg-VAP1_ and _Mg-VAP2_ in _M. graminicola_ parasitism, we performed in vitro gene silencing by soaking J2s with specific siRNAs. Silencing of the _Mg-VAP1_ gene caused a significant reduction (\(p<0.05\)) of nematode colonization in Nipponbare rice roots as compared to the control treatments (Figure 6). A small difference in the number of nematodes in rice roots was also observed for _Mg-VAP2_ silencing but was not reported statistically significant from control treatments.  
Figure 5: _Mg707_ and _Mg11937_ encode venom allergen-like proteins (VAPs). (**a**) Schematic representation of the two predicted proteins. (**b**) Protein sequences alignment of Mg707 and Mg11937 with _M. incognita_ Mi-VAP1 (Genbank AAD01511, [47]), Mi-VAP2 (Genbank ABO38019, [48]) and _G. rostochiensis Gr-VAP1_ (Genbank AEL16453, [46]): the signal peptides are underlined, the conserved cysteines are highlighted in grey, the Venom Allergen conserved site is highlighted in black and the SCP domain (annotated as Smart00198 in NCBI) is delimited by black triangles. (**c**) Percent identity matrix of the SCP domain sequences.