Rapid in vivo analysis of synthetic promoters for plant pathogen phytosensing

Abstract

BackgroundWe aimed to engineer transgenic plants for the purpose of early detection of plant pathogen infection, which was accomplished by employing synthetic pathogen inducible promoters fused to reporter genes for altered phenotypes in response to the pathogen infection. Toward this end, a number of synthetic promoters consisting of inducible regulatory elements fused to a red fluorescent protein (RFP) reporter were constructed for use in phytosensing.ResultsFor rapid analysis, an Agrobacterium-mediated transient expression assay was evaluated, then utilized to assess the inducibility of each synthetic promoter construct in vivo. Tobacco (Nicotiana tabacum cv. Xanthi) leaves were infiltrated with Agrobacterium harboring the individual synthetic promoter-reporter constructs. The infiltrated tobacco leaves were re-infiltrated with biotic (bacterial pathogens) or abiotic (plant defense signal molecules salicylic acid, ethylene and methyl jasmonate) agents 24 and 48 hours after initial agroinfiltration, followed by RFP measurements at relevant time points after treatment. These analyses indicated that the synthetic promoter constructs were capable of conferring the inducibility of the RFP reporter in response to appropriate phytohormones and bacterial pathogens, accordingly.ConclusionsThese observations demonstrate that the Agrobacterium-mediated transient expression is an efficient method for in vivo assays of promoter constructs in less than one week. Our results provide the opportunity to gain further insights into the versatility of the expression system as a potential tool for high-throughput in planta expression screening prior to generating stably transgenic plants for pathogen phytosensing. This system could also be utilized for temporary phytosensing; e.g., not requiring stably transgenic plants.

Evening Expression of Arabidopsis GIGANTEA Is Controlled by Combinatorial Interactions among Evolutionarily Conserved Regulatory Motifs

“…Our work on GI defines combinations of motifs that confer temporally regulated light induction, and together with other recent applications of related methods contributes to the definition of a plant circadian transcriptional code (Harmer and Kay, 2005;Michael et al, 2008;Spensley et al, 2009;Helfer et al, 2011;Li et al, 2011). In addition, the observation that light inducibility and circadian control are contributed by short, defined, separable modules fuels the design of synthetic promoters constructed by combining such fragments from different promoters and thereby conferring complex transcriptional patterns (Rushton et al, 2002;Liu et al, 2011). (A) Phylogenetic shadowing analysis of the FKF1 promoter.…”

Synthetic Botany

“…Examples include a light-inducible gene expression system activated by red light and switched off by far-red light (Müller et al 2014) as well as a range of in vivo biosensors to monitor levels of cytokinin (Müller and Sheen 2008;Zurcher et al 2013), auxin (Wend et al 2013), plant pathogens (Liu et al 2011;Fethe et al 2014), and elicitors (Koschmann et al 2012). These are examples of synthetic circuits that have been constructed in plants using methods for DNA manipulation, transgene regulation, transformation, and analysis to yield basic systems for chassis engineering and genetic network design in plant synthetic biology.…”

“…Traits of agricultural importance successfully introduced to plants using recombinant DNA technology include herbicide resistance (Comai et al 1985), drought resistance (Kumar et al 2014), pest resistance (Bates et al 2005), pathogen resistance (Brunner et al 2011;Horvath et al 2012;Jones et al 2014), abiotic stress resistance (Jaglo-Ottosen et al 1998), enhanced photosynthetic capacity (Ku et al 2001), im-proved nitrogen use efficiency (Yanagisawa et al 2004), and added nutritional value (Ye et al 2000). However, although plants hold a unique promise for bioproduction at the gigatonne scale, efforts in genetic engineering of plants are lagging compared with microbial systems (Antunes et al 2009(Antunes et al , 2011Liu et al 2011Liu et al , 2013Koschmann et al 2012;Wend et al 2013;Zurcher et al 2013;Fethe et al 2014;Mül-ler et al 2014).…”

Cis-regulatory elements used to control gene expression in plants

“…For example Liu et al (2011) integrated cis elements induced by phytohormones, produced during pathogen infection, with a minimal promoter CaMV 35S . The obtained regulatory cassette was applied to control a gene of red fluorescence protein pporRFP (Porites porites red fluorescent protein) in rice.…”