As biotrophic fungi, rusts infect and keep host tissues alive while extracting nutrients to complete their infection cycles. Rust fungi develop a specialized structure called the haustorium for uptake of cytoplasmic nutrients while suppressing host defence responses (Garnica et al., 2013). The major roles of haustoria have been suggested by interpretation of transcriptomic and proteomic data from isolated haustoria (Garnica et al., 2013; Mendgen et al., 2000; Voegele et al., 2001). Haustorial transcriptomics from wheat stripe rust, broad bean rust, stem rust and soybean rust fungi (see Box 1 in Chapter 1 for common and scientific fungal names) has revealed the up-regulation of genes coding for amino acid transporters, carbohydrate transporters and ATPases, suggesting active transport of nutrients (Garnica et al., 2013; Hahn and Mendgen, 1997; Link et al., 2014). The haustorial proteome of Puccinia triticina revealed that 33% of all peptides identified proteins related to energy metabolism, including ATPases, ATP synthases, and glycerol metabolism, among others (Song et al., 2011). Additionally, a fungal invertase involved in hexose uptake was immunolocalised to Uromyces fabae haustoria (Voegele et al., 2006). Studies in the non-rust biotrophs barley and pea powdery mildew suggested that their haustoria take up host sugars. In Blumeria graminis f. sp. hordei haustoria, the electric potential
CHAPTER 2
Effects of host growth stage and
metabolism on the development of
suggesting active transport (Mendgen and Nass, 1988). In Erysiphe pisi, 14C labelled
photosynthates were found in haustoria by electron microscopy (Spencer-Phillips and Gay, 1980). Overall specific roles for haustoria are apparent, but little has been studied directly due to the difficulty in working with these specialised cells.
Sugar alcohols (or polyols) are commonly present during fungal infections. Polyols are storage molecules that are utilised later for germination. Fungi also turn sugars into polyols, reducing their internal sugar concentrations and thus maintaining a favourable concentration gradient toward the fungus (Clancy and Coffey, 1980; Meena et al., 2015; Reisener et al., 1962; Voegele et al., 2005). Polyols accumulate in haustoria and hyphae, and are then probably translocated to the spores for storage (Maclean and Scott, 1976; Voegele et al., 2005).Other metabolites important for germination include abundant lipids which have a high capacity to store energy (Solomon et al., 2003). During germination of P. striiformis f. sp. tritici and U. appendiculatus
spores, lipids are broken down by the β-oxidation pathway and the glyoxylate cycle for the ultimate production of sugars and amino acids (Cooper et al., 2007; Zhao et al., 2015). Most of the data available to help understand the metabolic transition from dormancy to germination is derived from early metabolic studies and proteomics. The existing metabolomics data are from
Melampsora lini and P. graminis f. sp. tritici (Daly et al., 1967; Jackson and Frear, 1967; Reisener et al., 1962). In both fungi, the lipid composition of urediniospores changed in the first 8 hours of germination, while esterified fatty acids, including triglycerides, decreased in Pgt within the first hour of germination with a consequent increased in glycerol due to the lipases activity. However more accurate and modern techniques are now available that might help to improve our knowledge of spore metabolism. Therefore, the first part of this Chapter describes experiments to measure the abundance and changes in metabolites present in Pst
urediniospores and germinated spores, and compares these observations with the literature.
The nutrient demands of biotrophic fungi changes the metabolism of infected host cells. Several studies have reported the metabolic reprogramming of infected tissue to increase its sink capacity and hence divert photosynthate to the fungus (Bethenod et al., 2005; Carretero et al., 2011; Swarbrick et al., 2006; Voegele, 2006). Sink metabolism can also be induced by plant developmental and environmental conditions, and some studies have shown that these conditions affect how plant respond to fungal infection. For example, soybean leaves grown under reduced light (20% of normal) are more susceptible to Phakopsora pachyrhizi during the early stages of infection (Dias et al., 2010), while external application of nitrogen seems to enhance the development of fungal diseases (Devadas et al.; Neumann et al., 2004; Solomon and Oliver, 2001).Some resistance genes expressed only in adult plants (APR genes; adult plant
resistance) reduce growth and sporulation by rust fungi and Blumeria graminis f. sp. tritici
(Dakouri et al., 2013; Griffey, 1993; Herrera-Foessel et al., 2014). The underlying resistance mechanism for one of these, Yr46, is thought to be the reduction of carbon nutrients to the pathogen (Dodds and Lagudah, 2016).
Overall the available data suggest that the growth of rust fungi is sensitive to plant nutrient status, which can change due to environmental and developmental conditions. However, while older studies focussed on metabolism during rust infections, the aim of most recent studies has been to understand host resistance mechanisms and suppression of defence responses by the fungus (Cantu et al., 2011; de Carvalho et al., 2016; Dobon et al., 2016). Consequently, the interaction between fungal and host biology is still not well understood. To expand the study of the metabolomics of the Pst-wheat interaction, I manipulated plant growth conditions to modify photosynthesis and hence nutrient availability, which should impact on fungal growth. This is an indirect way to understand fungal metabolism, since direct manipulation of the fungus is still not possible without affecting the plant. I took different approaches to alter the content of nutrients in Pst-susceptible wheat leaves. After standardizing the infection methods, I manipulated the growth of plants expressing two different APR genes which code for a sugar transporter and an ATP-binding cassette (ABC) transporter, respectively. The results showed a direct correlation between nutrient availability and fungal growth in susceptible plants, while in resistant plants changes in nutrient distribution increased susceptibility. The results suggest alternative strategies for the control of fungal infection.