Transplastomic Nicotiana benthamiana plants expressing multiple defence genes encoding protease inhibitors and chitinase display broad‐spectrum resistance against insects, pathogens and abiotic stresses

Abstract

SummaryPlastid engineering provides several advantages for the next generation of transgenic technology, including the convenient use of transgene stacking and the generation of high expression levels of foreign proteins. With the goal of generating transplastomic plants with multiresistance against both phytopathogens and insects, a construct containing a monocistronic patterned gene stack was transformed into Nicotiana benthamiana plastids harbouring sweet potato sporamin, taro cystatin and chitinase from Paecilomyces javanicus. Transplastomic lines were screened and characterized by Southern/Northern/Western blot analysis for the confirmation of transgene integration and respective expression level. Immunogold localization analyses confirmed the high level of accumulation proteins that were specifically expressed in leaf and root plastids. Subsequent functional bioassays confirmed that the gene stacks conferred a high level of resistance against both insects and phytopathogens. Specifically, larva of Spodoptera litura and Spodoptera exigua either died or exhibited growth retardation after ingesting transplastomic plant leaves. In addition, the inhibitory effects on both leaf spot diseases caused by Alternaria alternata and soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum were markedly observed. Moreover, tolerance to abiotic stresses such as salt/osmotic stress was highly enhanced. The results confirmed that the simultaneous expression of sporamin, cystatin and chitinase conferred a broad spectrum of resistance. Conversely, the expression of single transgenes was not capable of conferring such resistance. To the best of our knowledge, this is the first study to demonstrate an efficacious stacked combination of plastidexpressed defence genes which resulted in an engineered tolerance to various abiotic and biotic stresses.

Review: The future of cystatin engineering

“…An excellent example exists within Nicotiana benthamiana plants where expressing multiple defense genes, such as sporamin, a taro cystatin and chitinase, was achieved. Such plants have broad-spectrum resistance against insects, pathogens and abiotic stresses not achievable with single transgene expression [49]. Another excellent example of a functional engineered multi-protease inhibitor is the coupling of a cysteine and a serine protease inhibitor.…”

Transplastomic plants for innovations in agriculture. A review

“…Recently, in order to generate plants with multiresistance against phytopathogens as well as insects, a construct with aadA as marker gene and a gene stack harboring sweet potato sporamin, taro cystatin (CeCPI), and chitinase from Paecilomyces javanicus has been introduced into Nicotiana benthamiana (Chen et al 2014). Surprisingly, the transplastomic plants conferred a broad spectrum of resistance not only against different pests and diseases (Table 2) but also against abiotic (salt, osmotic and oxidative) stressors (Table 3), and the transgenes were effectively expressed both in leaf and root plastids.…”

“…For instance, several transgenic crops with herbicide resistance encoded in the nucleus are commercialized. Herbicides can also be used as selective agents (Table 1); therefore, much effort has been devoted to develop transplastomic plants with resistance to different herbicides such as glyphosate (Daniell et al 1998;Ye et al 2001Ye et al , 2003Chin et al 2003;Roudsari et al 2009), phosphinothricin/ glufosinate ammonium (Iamtham and Day 2000;Lutz et al 2001;Kang et al 2003;Ye et al 2003), sulcotrione (Falk et al 2005), isoxaflutole/diketonitrile (Dufourmantel et al 2007), chlorophenylthio-triethylamine (CPTA) (Wurbs et al 2007), pyrimidinylcarboxylate, imidazolinone and sulfonylurea (Shimizu et al 2008), and paraquat (methyl-viologen) (Le Poage et al 2011;Chen et al 2014). Herbicide resistance is achieved (1) by the insertion of a bacterial marker gene (such as bar) encoding an enzyme that inactivates the herbicide (phosphinothricin/glufosinate ammonium-Iamthan and Day 2000; Lutz et al 2001Lutz et al , 2006, (2) by overexpression of the genes of plastidial metabolic enzymes that are the targets of herbicides (e.g., EPSPS: glyphosate- Daniell et al 1998; hppd: sulcotrione and the isoxaflutole derivative, diketonitrile -Falk et al 2005 andDufourmantel et al 2007, respectively), (3) by expression of the genes of mutant, herbicide-resistant plant enzymes (CP4: glyphosate- Ye et al 2001Ye et al , 2003Roudsari et al 2009; mALS: pyrimidinylcarboxylate, imidazolinone, and sulfonylurea- Shimizu et al 2008), or (4) by expression of enzyme genes involved in antioxidant defense, minimizing this way the metabolic impact of the herbicides via the generation of ROS (DHAR, GST, gor: paraquat/methyl-viologen-Le MnSOD: paraquat/methyl-viologen-Poage et al 2011).…”

Oligosaccharides from Botrytis cinerea and Elicitation of Grapevine Defense

“…Chitinases play a direct role in plant defense by degrading chitin, a major component of fungal cell walls, and thus inhibit hyphal growth (Collinge et al 1993). An increased pathogen resistance was observed in transgenic plants overexpressing chitinases (Grison et al 1996;Prasad et al 2013;Chen et al 2014). Moreover, chitinolytic breakdown products induce the production of phytoalexins and systemic acquired resistance (Brunner et al 1998;Van Loon and Van Strien 1999).…”