## 7 Building a Molecular Toolbox for Engineering of Targeted Disease Resistance
### Results and Discussion of Candidate Wheat Orthologues of DORN1  
Because DORN1 is highly expressed in roots [47], DORN1-mediated resistance is dependent upon the jasmonate signal pathway [45; 46; 119], and wheat roots appear to possess an active jasmonate signal pathway [120], we hypothesize that expression of the wheat orthologue of the _DORN1_ gene will confer resistance to the necrotroph _R. solani_ AG-8, hemibiotroph _F. culmorum_, and possibly to nematodes that cause cellular damage while migrating through host root tissue.  
Searches of the wheat and barley databases using HMM and TMHMM resulted in 23 candidate barley DORN1 orthologues containing both lectin-binding and transmembrane domains, but not the kinase domain. In contrast, 205 candidate wheat orthologues were retrieved that contained lectin-binding and kinase domains, regardless of whether transmembrane domains were present. The numbers of candidates and the absence of at least one key domain discouraged us from pursuing cereal _DORN1_ genes at the time. More recently, three accessions (Traces_2 BS_80806 CA5F.1, Traces_2 AS_DE7 DCB294.1, and Traces_1 BS_A999A190E.3) from the IWGSC RefSeq v1.0 database were identified based on highest amino acid sequence identity to DORN1 from _A. thaliana_, _A. lyrata_, and _Camelina sativa_, respectively (Supplementary Table S2). Identification of the wheat orthologue was not clear-cut, as certain features of the DORN1 protein vary among species [42]. The Brassica amino acid sequences shared 90.4%-96.4% identity with each other, whereas the candidate wheat orthologues were 58.4%-59.3% identical to each other at the amino acid level. For the sake of clarity, the candidate wheat orthologues were compared only to the _A. thaliana_-deduced amino acid sequence (Figure 1). Wheat sequences 80806 CA5F.1, A999A190E.3, and DE7 DCB294.1 showed 45.9%, 45.5%, and 44.0% identity, respectively, to the Arabidopsis DORN1, but there was no single wheat orthologue that stood out for future gene transfer to wheat and subsequent testing in soilborne fungal pathogenicity assays. However, the pool of wheat DORN1 candidate sequences was narrowed from 205 to 3 genes.  
Identifying gene orthologues can be complicated by evolutionary differences among different species. For instance, recognition of extracellular ATP by a transmembrane receptor might depend on conservation of structural features rather than on conserved amino acid sequences. The wheat _DORN1_ gene, once identified, might function in barley and vice versa, due to the molecular relatedness of these species, whereas the Arabidopsis gene might or might not be operational in the small-grain cereals. The parasitism genes orthologues from _H. avenue_ and _M. gramitcola_ often did not share high sequence identity with known parasitism genes. [PERSON] and coauthors [92] noted significant diversity among G16B09 parasitism genes from _H. avenue_, _H. glycine_, _H. schachtii_, and two _Globodera_ spp. Likewise, the annexin orthologue from _H. avenue_ shared 76% amino acid sequence identity to the _G. pallida_-deduced protein, and differed substantially from proteins of other _Heterodera_ spp. and _Globodera_ spp. It is likely that the _H. avenue_ genes were the correct orthologues, based on their ability to suppress host defenses. In a comparison of fungal CYP51 proteins, [PERSON] and [PERSON] [121] found 80.9% and 64.8% identity between _F. graminearum_ and _F. solani_ or _Magnaporthe oryzae_, respectively. The application of _CYP51B_ HIGS for suppression of _R. solani_ AG-8 is being explored. In this case, there is substantial gene sequence diversity relative to _Fusarium CYP51B_ to merit design of a new RNAi molecule for _R. solani_.  
The relatively low efficiency of wheat transformation limits the number genes that can be reasonably tested in planta. However, rice is much more amenable to _Agrobacterium_ transformation [122; 123], and a rapid and efficient system for barley root transformation has been developed [124].