Abstract
Plants are continuously challenged by pathogens, affecting most staple crops compromising food security. They have evolved different mechanisms to counterattack pathogen infection, including the accumulation of pathogenesis-related (PR) proteins. These proteins have been implicated in active defense, and their overexpression has led to enhanced resistance in nuclear transgenic plants, although in many cases constitutive expression resulted in lesion-mimic phenotypes. We decided to evaluate plastid transformation as an alternative to overcome limitations observed for nuclear transgenic technologies. The advantages include the possibilities to express polycistronic RNAs, to obtain higher protein expression levels, and the impeded gene flow due to the maternal inheritance of the plastome. We transformed Nicotiana tabacum plastids to co-express the tobacco PR proteins AP24 and β-1,3-glucanase. Transplastomic tobacco lines were characterized and subsequently challenged with Rhizoctonia solani, Peronospora hyoscyami f.sp. tabacina and Phytophthora nicotianae. Results showed that transplastomic plants expressing AP24 and β-1,3-glucanase are resistant to R. solani in greenhouse conditions and, furthermore, they are protected against P.hyoscyami f.sp. tabacina and P. nicotianae in field conditions under high inoculum pressure. Our results suggest that plastid co- expression of PR proteins AP24 and β-1,3-glucanase resulted in enhanced resistance against filamentous pathogens.
Highlights
IntroductionPlastid genome (plastome) transformation could overcome limitations mentioned above. This alternative approach enables the accumulation of higher levels of heterologous proteins enclosed in the organelle, with reported values of up to 51% and even 70% of total soluble protein (TSP) in Nicotiana tabacum plants[35,36]
Plastid genome transformation could overcome limitations mentioned above
AP24 and β-1,3-glucanase coding sequences were assembled to generate a dicistronic RNA, where the second cistron is preceded by the 5′-untranslated region (5′-UTR) from gene 10 of the T7 phage (T7g10)[48] (Fig. 1). putrAP24-Gluc was used to transformed N. tabacum plastid genome by particle bombardment of tobacco leaves
Summary
Plastid genome (plastome) transformation could overcome limitations mentioned above. This alternative approach enables the accumulation of higher levels of heterologous proteins enclosed in the organelle, with reported values of up to 51% and even 70% of total soluble protein (TSP) in Nicotiana tabacum plants[35,36]. Promising results regarding resistance to fungal pathogens published to date include the transformation of the chloroplast genome to accumulate MSI-99 (a synthetic antimicrobial peptide)[41,42], and the expresion of chloroperoxidase from Pseudomonas pyrrocinia[43], both cases resulting in strong inhibition of spore germination and/or growth in Aspergillus flavus, Fusarium moniliforme, and Verticillium dahliae (in vitro assays), and limitation of lesion size after inoculation of transplastomic plants with Alternaria alternata[43] or Colletotrichum destructivum[41]. There are no reports demonstrating whether plastid transformation is a suitable strategy to control oomycete pathogens This particular phylogenetic group includes Phytophthora sp., one of the most destructive genera, whose member species are responsible for severe economic losses on crops worldwide, as well as environmental damage in natural ecosystems[44,45]. Transplastomic plants expressing both AP24 and β-1,3-glucanase are highly resistant to R. solani and are protected against P. hyoscyami f. Our results indicate that co-expression of AP24 and β-1,3-glucanase in the chloroplast enhanced resistance against filamentous pathogens
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