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

Phenotypes of WRKY-RNAi plants

Identification of Agrobacterium-mediated transgenic plants and their growth phenotypes

The Dex-induced lines exhibited fewer hyphal branches and shorter hyphae at 24 and 48 hpi

the infected areas were smaller at 120 hpi compared to wild-type and non-induced controls

Relative BdWRKY67 transcript levels in BdWRKY67-RNAi transgenic plants

the ratio of fungal/B. distachyon DNA and measured by qPCR at 12dpi

2 BdWRKY67 binds to the BdRBOHD promoter to repress Its expression

BdWRKY67 expression of in the compatible and incompatible interaction between B. distachyon and rust fungi

BdWRKY67 suppresses transcription of BdRBOHD (A) Heatmap of differentially regulated genes of the plant–pathogen interaction pathway in three plants of the L2-7 transgenic line expressing the BdWRKY67-RNAi construct and induced with dexamethasone(RNAi Dex 48) and three wild-type plants treated with water (WT 48). (B) Binding of recombinant GST-BdWRKY67 protein to the BdRBOHD promoter, analyzed byelectrophoretic mobility shift assay (EMSA) with titration. (C) RPKM (Reads Per Kilobase Million) and relative expression of BdWRKY67 (top) and BdRBOHD(bottom) in WT and BdWRKY67-silenced plants, as determined by digital RNA-seq (left) and RT-qPCR(right) 48 hpi.

3 Silencing the BdWRKY67 homolog TaWRKY19 in wheat enhances ROS accumulation and resistance to Pst

(A) Amino acid alignment of the protein sequences of TaWRKY19 and BdWRKY67. The blocks outlined in red indicate the WRKY domains

(B) WRKY domains of TaWRKY19 and BdWRKY67 in the N-terminal (NT) and C-terminal (CT) domains.NT and CT sequences were derived from the sequences of the Arabidopsis WRKY transcription factorfamily by using GENEDOC.

Subcellular localization of TaWRKY19 and BdWRKY67 in Nicotianabenthamiana epidermal cells

Subcellular localization of TaWRKY19 and BdWRKY67 in wheat protoplasts.

TaWRKY19 contributes to wheat susceptibility to virulent Pst CYR31 (A) Schematic diagram of the specific gene fragments targeted for silencing in the TaWRKY19 gene. (B) Wheat leaves were either treated with 1×FES buffer (Mock) or infected with BSMV-γ as negativecontrols, or they were infected with BSMV-TaPDS (conferring a photobleaching phenotype as positivecontrol for silencing).

Histological observation of Pst growth and development inTaWRKY19-silencing wheat leaves inoculated with Pst CYR31

4 Three homeoalleles of TaWRKY19 in wheat play redundant roles in regulation of ROS production and Pst resistance.

Three homeoalleles of TaWRKY19 in wheat play redundant roles in regulation of ROS production and Pst resistance

TaWRKY19 directly binds the TaNOX10 promoter

TaNOX10 plays a positive role in ROS production and wheat resistance to Pst.

Elevated expression of TaWRKY19 represses RBOH-mediated ROS production

Loss of TaWRKY19 confers resistance to Pst.

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, Männerdorf, Switzerland). Each experiment comprised three independent biological replicates.