One-sentence summary:?TaWRKY19 transcriptionally represses the expression ofTaNOX10, impairing the host ROS burst and resulting in wheat susceptibility to stripe rust.
1 introduction
ROS
1）Rapid ROS accumulation in plant tissues in response to avirulent pathogens triggers an early defense response – the hypersensitive response – which causes localized cell necrosis in the vicinity of the infection site that can inhibit pathogen growth.
2) ROS initiate:?
pathogen-associated molecular patterns (PAMPs) : bacterial protein flagellin and fungus derived polygalacturonase interaction between a pathogen avirulence (Avr) gene and a host resistance (R) gene?
3) The role in plant immunity
inducing the hypersensitive response, ROS act as signaling molecules that regulate the expression of genes involved in plant immunity, such as those encoding antimicrobial peptides and those that close stomata and other points through which pathogens might invade
4)Production
ROS are produced mainly by NADPH oxidases (NOXs) at the plasma membrane and organelle internal membranes
In plants, NOXs are also known as respiratory burst oxidase homologs (RBOHs)
5)TaNOX
There are 15 NOX/RBOH genes in the wheat genome, most of which are expressed specifically during rooting, anthesis, and seed germination, with the exception of TaNOX10 which is expressed throughout plant development
6)?
Many host interactions with incompatible pathogens (avirulent pathogens), including fungi, viruses and bacteria, involve a burst of ROS production that results in resistance, whereas compatible (virulent) pathogens elicit no such ROS burst
?Puccinia striiformis?f. sp. tritici (Pst)
The causal agent for stripe rust, Puccinia striiformis f. sp. tritici (Pst), is a biotrophic fungal pathogen of wheat
2 Results
1 Silencing of BdWRKY67 confers resistance to Puccinia brachypodii
2?BdWRKY67 binds to the BdRBOHD promoter to repress Its expression
3?Silencing the BdWRKY67 homolog TaWRKY19 in wheat enhances ROS accumulation and resistance to Pst
4?Three homeoalleles of TaWRKY19 in wheat play redundant roles in regulation of ROS production and Pst resistance.
Extracellular ROS accumulation detection and quantification?
Preparation of the apoplastic fluid from wheat leaves was performed as described (vander Linde et al., 2012) with some modifications. Briefly, 5 g of wheat leaves were collected and cut into about 4-cm segments and placed in a beaker filled with Tris buffered EDTA (TBS) at pH 7.5. Leaves were vacuum-infiltrated using a vacuum pump for 3 x 20 min at 400 mbar. The leaves were then transferred into a 20-mL syringe and the syringe was placed into a 50-mL falcon tube and centrifuged for 15 min at 2500 g and 4oC to collect the apoplastic fluid. The apoplastic fluid ROS contents were quantified by using the Plant reactive oxygen species (ROS) ELISA kit (Trust Specialty Zeal Biological trade, USA) according to the manufacturer’s instructions. In detail, ROS were extracted from plant leaves (> 50 mg) in 1 mg·μL–1 PBS (KH2PO4 0.24 g·L–1, Na2HPO4 1.44 g·L–1, NaCl 8 g·L–1, KCl 0.2 g·L–1, pH = 7.4); 50 μL samples were then incubated in the wells of the 96-well plate provided with the kit for 45 min at 37oC. Each well was washed five times with 200 μL wash buffer 200, after which 50 μL biotinylated anti-IgG was added to each well and incubated for 30 min at 37oC. After washing as above, 50 μL streptavidin-HRP was added into the plate and incubated at 37oC for 15 min. Finally, chromogen solution was added, followed by the stop solution (provided with the kit) as per manufacturer’s methods. The optical density (OD) at 450 nm was then measured with a Multiskan Spectrum plate reader (Tecan, M?nnerdorf, Switzerland). Each experiment comprised three independent biological replicates.
