Defining the signalling network linking pathogen infection and asparagine accumulation in wheat grain

This project arises from discoveries that an amino acid called asparagine accumulates in wheat grain in response to disease and that the plant's response to floral infection by a disease-causing fungus called Fusarium graminearum (Fg) involves a protein called SnRK1. SnRK1 is a master regulator of plant metabolism and it controls the activity of genes encoding an enzyme called asparagine synthetase that is responsible for making asparagine. The project will involve a multidisciplinary team from Rothamsted Research, with collaboration from a team from University College Dublin (not eligible for BBSRC funding but fully involved in the project through the sharing of resources, expertise and data analyses). It will define what we are calling a signalling hub (a control point within a network) involving SnRK1 and partner proteins that links pathogen infection with asparagine synthesis and accumulation in wheat grain. We believe that the increase in asparagine concentration induced through the activation of this hub upon Fg infection is an important part of how plants defend themselves when under attack from disease-causing organisms.

Fg causes Fusarium head blight disease, which reduces yield and grain quality, and contaminates grain with toxic compounds called mycotoxins, of which the most common is called deoxynivalenol (DON). SnRK1 is involved in the regulation of defence mechanisms when wheat is infected by Fg and a protein that partners with SnRK1, called TaFROG, has also been shown to contribute to Fg and DON resistance. Recent work has shown that Fg infection and DON treatment both affect SnRK1 but in different ways, with Fg infection causing the SnRK1 protein to be divided into smaller proteins in a way not seen with DON on its own. Subsequently, a protein that is secreted by the fungus, called OSP24, has been shown to partner with SnRK1 and to cause SnRK1 to be broken down. TaFROG, on the other hand, competes with OSP24 for partnering with SnRK1 and protects SnRK1 from degradation. These fascinating discoveries mean that this project can focus directly on the signalling hub and its relationships to other proteins in SnRK1's wider network. That network likely includes several proteins called bZIP transcription factors. These proteins control the activity of some target genes, possibly including asparagine synthetase genes involved in asparagine synthesis, and have characteristics suggesting that they could be controlled by SnRK1.

We aim to identify all the components of this signalling hub linking Fg infection with asparagine accumulation in wheat grain. We will dissect the hub in different types of wheat using different strains of Fg, such as strains that do not make DON and/or the OSP24 protein. We will use a technique called RNA-seq that will enable us to identify all of the genes affected by infection by Fg or treatment with DON, looking in particular for those that could be involved in making or breaking down asparagine. We will use protein-based studies to identify additional hub components, and find out if the bZIP transcription factors we are interested in do control the activity of asparagine synthetase genes. Finally, we will see if the ability of different Fg strains to cause disease is linked to their effect on the signalling hub and asparagine. These experiments will enable us to model the signalling hub and perform further experiments to target the genes involved in the hub so that we can test whether our model is correct.

SnRK1 has been implicated in other plant defence mechanisms, including those against herbivores, viruses and bacteria, as well as other fungi. In addition, the amount of asparagine in wheat grain has implications for food safety because asparagine can be converted into a cancer-causing contaminant called acrylamide during baking. This means that the project, while focussed on basic science, will have potential impact for a range of stakeholders in the agrifood sector.

This project arises from discoveries that free asparagine (Asn) accumulates in wheat grain in response to pathogenic fungi, and that responses invoked by Fusarium graminearum (Fg) and its mycotoxin, DON, involve the protein kinase, SnRK1, a regulator of plant metabolism that controls Asn synthetase gene expression. Another protein, TaFROG, interacts with SnRK1, contributing to Fg/DON resistance. SnRK1 becomes more active in response to DON treatment, and wheat becomes more sensitive to DON when SnRK1 is silenced. Fg infection (but not DON on its own) causes a loss of SnRK1 protein integrity. A fungal secreted protein, OSP24, promotes degradation of SnRK1, with TaFROG protecting SnRK1 by competing with OSP24 for binding. Furthermore, several bZIP transcription factors (TFs) are potential targets for SnRK1, and Asn synthetase gene promoters contain bZIP binding sites.

This project will define this signalling hub linking Fg infection with Asn synthesis. It will identify hub components, separate the effects of Fg and DON on the hub, and characterise the hub's targets and interactions with wider signalling systems. It will identify SnRK1 breakdown proteins induced by Fg, use RNA-seq, phosphoproteomics and TurboID to identify additional hub components and investigate the interaction of different TFs with Asn synthetase genes. The ability of Fg to cause disease will be linked to its interactions with the hub, and a model of how the hub works will be produced and validated by knocking components out using VIGS.

The project will involve teams from Rothamsted and University College Dublin. It will test the hypothesis that the increase in Asn concentration in wheat grain upon Fg infection is part of the plant's defence against the disease. It will also enable us to assess the potential for targeting the hub for Fg control and the prevention of Asn accumulation. This has food safety implications because Asn can be converted to the carcinogen, acrylamide, during baking.