Abstract
Real-time systems that provide evidence of pathogen contamination in crops can be an important new line of early defense in agricultural centers. Plants possess defense mechanisms to protect against pathogen attack. Inducible plant defense is controlled by signal transduction pathways, inducible promoters and cis-regulatory elements corresponding to key genes involved in defense, and pathogen-specific responses. Identified inducible promoters and cis-acting elements could be utilized in plant sentinels, or 'phytosensors', by fusing these to reporter genes to produce plants with altered phenotypes in response to the presence of pathogens. Here, we have employed cis-acting elements from promoter regions of pathogen inducible genes as well as those responsive to the plant defense signal molecules salicylic acid, jasmonic acid, and ethylene. Synthetic promoters were constructed by combining various regulatory elements supplemented with the enhancer elements from the Cauliflower mosaic virus (CaMV) 35S promoter to increase basal level of the GUS expression. The inducibility of each synthetic promoter was first assessed in transient expression assays using Arabidopsis thaliana protoplasts and then examined for efficacy in stably transgenic Arabidopsis and tobacco plants. Histochemical and fluorometric GUS expression analyses showed that both transgenic Arabidopsis and tobacco plants responded to elicitor and phytohormone treatments with increased GUS expression when compared to untreated plants. Pathogen-inducible phytosensor studies were initiated by analyzing the sensitivity of the synthetic promoters against virus infection. Transgenic tobacco plants infected with Alfalfa mosaic virus showed an increase in GUS expression when compared to mock-inoculated control plants, whereas Tobacco mosaic virus infection caused no changes in GUS expression. Further research, using these transgenic plants against a range of different pathogens with the regulation of detectable reporter gene could provide biological evidence to define the functional differences between pathogens, and provide new technology and applications for transgenic plants as phytosensors.
Highlights
The need for assurance of plant biosecurity is at an all-time high
We performed research to design and screen synthetic promoterreporter gene constructs using inducible regulatory elements based upon published information
Pathogen inducible regulatory elements were grouped according to their responsiveness to plant signal defense molecules: salicylic acid, jasmonic acid and ethylene responsive elements, or classified in accordance to core sequence(s) (e.g., GCC-like boxes, W-like boxes)
Summary
The need for assurance of plant biosecurity is at an all-time high. is there a risk of natural outbreaks of emerging pathogens, but intentional releases of plant disease-causing agents as a terrorist act is a real threat [1]. Real-time systems that provide evidence of intentional or natural pathogen contamination in crops are needed [2]. Plants possess defense mechanisms to protect against pathogen attack. These defense systems are highly regulated on the transcriptional level, and can be induced by chemical elicitors produced by pathogens. Variation in host resistance is often controlled by the segregation of single resistance (R) genes, the products of which directly or indirectly interact with specific elicitors produced by the pathogen and coded for by avirulence (avr) genes [7,8]. Defense mechanisms deployed range from the hypersensitive response (HR), a rapid death of cells at the infection site [8] to systemic acquired resistance (SAR) and induced systemic resistance (ISR) through distinct and coordinated signaling pathways [11,12,13,14]. Bacterial, and viral pathogens contain species that are specific pathogens to economically important crops
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