## 7 Building a Molecular Toolbox for Engineering of Targeted Disease Resistance
### Results and Discussion of the Pratylenchus-Wheat Root Transcriptome  
Properties of the RNA and RNA libraries generated for the wheat root-_Pratylenchus_ transcriptomes are shown in Supplementary Table S1. Total RNA concentrations were sufficient for library preparation. RNA quality (RIN), ranging from 3.9 to 5.4, indicated partial degradation, likely occurring during the root washing step prior to freezing in liquid nitrogen. However, the RNA produced libraries of sufficient quantity and size for Illumina HiSeq sequencing, and allowed us to analyze RPKM data for candidate pathogen-inducible promoters. A total of 262,908 wheat transcript groups were annotated using Blast2 GO (BioBam Bioinformatics S.L.) and the IWGSC coding sequence database for hexaploid wheat; 4,17 groups harbored at least one treatment having an RPKM value \(\geq\)50.0. The 24 non-paired read files are available as accession numbers SAMN10686471-10686492 under NCBI BioProject ID PRJNA512537. We selected 44 transcript groups in which induction of \(\geq\)2.0 was observed for one or more of the _Pratylenchus_ treatments relative to the non-challenged control (Table 1). One important caveat of our RNA-seq data was that initial RNA sample quality was lower than ideal. Therefore, induction of an annotated gene was deemed more reliable if they were represented by multiple transcript groups, all of which were up-regulated by nematode treatment. Nine annotated transcripts, such as those encoding three late embryogenesis abundant proteins, were comprised at least two members and accounted for 36 of the 44 transcript groups. Induction was often specific to one nematode species, indicating that the two species of _Pratylenchus_ were interacting differently with the roots of cv. Scarlet. For instance, ascorbate peroxidase and catalase peroxidase genes showed induction by _P. neglectus_, and the glutathione S-transferases and late embryogenesis abundant proteins were induced by _P. thornei_ at the time of root harvest. A similar degree of induction was observed in the corresponding PnPt treatments, indicating that the presence of the second species did not have a strongly positive or negative effect on transcription. The CAP superfamily, actin-11, and three transcript groups of the major pollen allergen appeared to be induced by both Pn and Pt. Induction of the CAP superfamily also seemed to be additive. Authentication of induction will require additional experiments, such as quantitative reverse-transcription PCR. Gene expression will also be quantified using the resistant Scarlet mutant [112]. A similar approach could be used to find regulatory DNA segments that respond to other pathogens. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline  & & & **RPKM 1** & & & **Fold-induction 2** \\ \hline \hline
**Annotation/locus ID 3** & **Cont** & **Pn** & **Pt** & **Pn+Pt** & **Pn** & **Pt** & **Pn+Pt** \\ \hline
**Actin-11 LENGTH=377** & & & & & & & \\ Treses\_SDS\_54253C6084.1 & 7.51 & 66.43 & 15.96 & 67.9 & 8.84 & 2.12 & 9.04 \\
**Treses\_SDS\_54253C6084.2** & 5.12 & 66.32 & 20.31 & 55.05 & 12.95 & 3.97 & 10.75 \\
**Alodes reductase** & & & & & & & \\ Treses\_2 DL\_78780003.1 & 16.81 & 16.47 & 77 & 75.05 & ns & 4.58 & 4.46 \\
**Ascorbate peroxidase** & & & & & & & \\ Tres\_4 BL\_19 FA6 DCAD.1 & 71.83 & 411.76 & 87.15 & 374.33 & 5.73 & ns & 5.21 \\
**Tres\_4 BL\_19 FA5 DCAD.2** & 54.76 & 392.67 & 61.9 & 331.86 & 7.17 & ns & 6.06 \\
**CAP superfamily** & & & & & & & \\ Treses\_3 AL\_AS024B15.1 & 9.84 & 50.01 & 21.86 & 68.9 & 5.08 & 2.22 & 7 \\ Treses\_3 DL\_4345A6 DD4.1 & 10.04 & 53.19 & 27.22 & 79.85 & 5.3 & 2.71 & 7.95 \\
**Catalase peroxidase** & & & & & & & \\ Tres\_4 AS\_SPERADEC1.1 & 150.71 & 758.44 & 185.84 & 706.01 & 5.03 & ns & 4.68 \\ Tres\_4 DL\_8 cED055F15.1 & 109.26 & 436.8 & 126.79 & 421.09 & 4 & ns & 3.85 \\ Tres\_4 DL\_8 CC055F15.3 & 87.02 & 314.44 & 101.59 & 300.4 & 3.61 & ns & 4.18 \\ \hline \hline \end{tabular}
\end{table}