**6. Biotechnological Applications and Perspectives**

The use of GM crops resistant to pests such as fungi, nematodes and insects is an appealing strategy considering the current need of efficiently increasing the yield of the agricultural production with less impact in the environment and health [121]. Since their discovery in the 80′ s, the use of transgenic crops has been dominated by the Bt technology. Nevertheless, some insect species are not susceptible to them and its intensive application has led to the development of resistance [19,122]. Considering the fact that IDPs do not need to fold in a proper way to be biologically active, this feature can potentially be an advantage regarding their expression in transgenic plants. In the case of Jaburetox, its disordered structure gives the peptide the capacity to withstand a vast range of temperatures and pH without losing its biological activity [69], a desirable feature for a biotechnological tool. Moreover, the conformational flexibility of Jaburetox and Soyuretox allows them to interact with several binding partners with different subcellular distributions, leading ultimately to diverse targets. This feature gives them the ability of avoiding or at least delaying the generation of resistance.

In this context, three types of transgenic crops expressing urease-derived peptides have been developed with promising results [87,88,123]. Soybean plants overexpressing Soyuretox were challenged with the root-knot nematode *Meloidogyne javanica*, a major agricultural pest in several countries [123]. As a result, the average reproductive factor of the nematode was significantly reduced. On the other hand, Didoné [87] reported that maize (*Zea mays*) expressing Jaburetox fed to the important polyphagous pest *S. frugiperda* led not only to a 39% lethality of larvae, but also to other sub-lethal statistically significant effects, such as body weight reduction, decreased ingestion and remarkably, fertility decline. In addition, Ceccon [88] demonstrated that Jaburetox-expressing tobacco plants also

produced high mortality and a pronounced reduction in the leaf consumption by the lepidopteran *H. armigera.* These authors started the development of gene stacking strategy, with plants expressing simultaneously Jaburetox and a double-stranded RNA complementary to the *rieske* gene, which has the advantage of diminishing the possibility of resistance development events [87]. The use of plants overexpressing Soyuretox or Jaburetox would, in principle, be a way to avoid harming beneficial or innocuous insects, since only those species that fed on the plants would be affected. Nevertheless, since off-target effects could be an issue related to the broad insecticidal activity of Jaburetox/Soyuretox, transgenic crops can be improved using tissue-specific or damage-induced promoters [124,125].

These latest studies [87,88,123] are a proof of concept that IDPs in general, and the urease-derived peptides in particular, are very attractive pesticides that can be engineered for use as an effective and environmental-friendly strategy, alone or in combination with other IDPs or toxic molecules. The multidisciplinary research approaches employed by our group and collaborators improved significantly the understanding of the structural and biological aspects of these IDPs and encourage us to pursue a full comprehension of their mechanism of action that would, ultimately, facilitate their application in the field.

**Author Contributions:** All authors wrote the original draft. M.V.C.G., F.C.L., A.H.S.M. and L.L.F. designed the tables and figures. C.R.C. and L.L.F. revised the manuscript. C.R.C. acquired funding. All authors have read and agreed to the published version of the manuscript.

**Funding:** Work in C.R.C. laboratory is supported by the Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [proj. 44.6052/2014-1 and Edital Jovem Talento–BJT 40.0189/2014-3, Science Without Borders]; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) [Finance Code 001, Edital Toxinologia 63/2010 proj. 1205/2011 and Portal de Periódicos]; Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, Edital PRONEX 008/2009, proj. 10/0014-2). M.V.C.G. received a CAPES Ph. D. fellowship. F.C.L. received a Ph. D. fellowship from CAPES and is a post-doctoral fellow from CAPES (PNPD). A.H.S.M. received Ph. D. fellowship from CNPq and a post-doctoral fellowship from CAPES. C.R.C. is a Research Productivity Fellow from CNPq. L.L.F. is a member of the National Research Council (CONICET, Argentina).

**Acknowledgments:** The authors thank Rodrigo Ligabue-Braun (Departamento de Farmacociências, UFCSPA, Porto Alegre, RS, Brazil) for assistance with bioinformatics and conceptual discussions.

**Conflicts of Interest:** The authors declare no conflict of interest.
