Pathogenic fungi must extend filamentous hyphae across solid surfaces to cause diseases of plants. However, the full inventory of genes which support this is incomplete and many may be currently concealed due to their essentiality for the hyphal growth form. During a random T-DNA mutagenesis screen performed on the pleomorphic wheat (Triticum aestivum) pathogen Zymoseptoria tritici, we acquired a mutant unable to extend hyphae specifically when on solid surfaces. In contrast “yeast-like” growth, and all other growth forms, were unaffected. The inability to extend surface hyphae resulted in a complete loss of virulence on plants. The affected gene encoded a predicted type 2 glycosyltransferase (ZtGT2). Analysis of >800 genomes from taxonomically diverse fungi highlighted a generally widespread, but discontinuous, distribution of ZtGT2 orthologues, and a complete absence of any similar proteins in non-filamentous ascomycete yeasts. Deletion mutants of the ZtGT2 orthologue in the taxonomically un-related fungus Fusarium graminearum were also severely impaired in hyphal growth and non-pathogenic on wheat ears. ZtGT2 expression increased during filamentous growth and electron microscopy on deletion mutants (ΔZtGT2) suggested the protein functions to maintain the outermost surface of the fungal cell wall. Despite this, adhesion to leaf surfaces was unaffected in ΔZtGT2 mutants and global RNAseq-based gene expression profiling highlighted that surface-sensing and protein secretion was also largely unaffected. However, ΔZtGT2 mutants constitutively overexpressed several transmembrane and secreted proteins, including an important LysM-domain chitin-binding virulence effector, Zt3LysM. ZtGT2 likely functions in the synthesis of a currently unknown, potentially minor but widespread, extracellular or outer cell wall polysaccharide which plays a key role in facilitating many interactions between plants and fungi by enabling hyphal growth on solid matrices.

Highly specific rabbit polyclonal antibodies for the obligate sugar-beet root parasite, Polymyxa betae, were produced using a novel recombinant DNA approach. Parasite cDNA was selectively isolated from infected roots, expressed in vitro, and the purified protein used to raise antibodies. This produced clean, precisely targeted antibodies, and allowed for rigorous screening of candidate genes and their products at the molecular level prior to animal immunization. This approach selects for genes whose products are highly expressed by the parasite in planta, and five such candidate genes from Polymyxa betae were identified and cloned. Polyclonal antiserum developed using the product of one such gene was found to react specifically with P. betae in sugar-beet roots and with the closely related Polymyxa graminis in barley roots, and to cross-react with Plasmodiophora brassicae in cabbage roots, without the need for further purification. No cross-reaction was detected with protein extracts from potato roots infected by the plasmodiophoromycete Spongospora subterranea. In all cases, there was no interaction with proteins from host plants, or from other microorganisms found in association with uninoculated sugar-beet, barley, cabbage and potato roots.

Pea seeds are widely consumed in their immature form, known as garden peas and petit pois, mostly after preservation by freezing or canning. Mature dry peas are rich in iron in the form of ferritin, but little is known about the content, form or bioavailability of iron in immature stages of seed development. Using specific antibodies and in-gel iron staining, we show that ferritin loaded with iron accumulated gradually during seed development. Immunolocalization and high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that iron-loaded ferritin was located at the surface of starch-containing plastids. Standard cooking procedures destabilized monomeric ferritin and the iron-loaded form. Iron uptake studies using Caco-2 cells showed that the iron in microwaved immature peas was more bioavailable than in boiled mature peas, despite similar levels of soluble iron in the digestates. By manipulating the levels of phytic acid in the digestates we demonstrate that phytic acid is the main inhibitor of iron uptake from mature peas in vitro. Taken together, our data show that immature peas and mature dry peas contain similar levels of ferritin-iron, which is destabilized during cooking. However, iron from immature peas is more bioavailable because of lower phytic acid levels compared to mature peas.

Fusarium Head Blight (FHB) is the number one floral disease of cereals and poses a serious health hazard by contaminating grain with the harmful mycotoxin deoxynivalenol (DON). Fungi adapt to fluctuations in their environment, coordinating development and metabolism accordingly. G-protein coupled receptors (GPCRs) communicate changes in the environment to intracellular G-proteins that direct the appropriate biological response, suggesting that fungal GPCR signalling may be key to virulence. Here we describe the expansion of non-classical GPCRs in the FHB causing pathogen, Fusarium graminearum , and show that class X receptors are highly expressed during wheat infection. We identify class X receptors that are required for FHB disease on wheat, and show that the absence of a GPCR can cause an enhanced host response that restricts the progression of infection. Specific receptor sub-domains are required for virulence. These non-classical receptors physically interact with intracellular G-proteins and are therefore bona fide GPCRs. Disrupting a class X receptor is shown to dysregulate the transcriptional coordination of virulence traits during infection. This amounts to enhanced wheat defensive responses, including chitinase and plant cell wall biosynthesis, resulting in apoplastic and vascular occlusions that impede infection. Our results show that GPCR signalling is important to FHB disease establishment.

Wheat contains abundant xylan in cell walls of all tissues, but in endosperm, there is an unusual form of xylan substituted only by arabinose (arabinoxylan; AX) that has long chains and low levels of feruloylation, a fraction of which is extractable in water (WE-AX). WE-AX acts as soluble dietary fibre but also gives rise to viscous extracts from grain, a detrimental trait for some nonfood uses of wheat. Here, we show that a glycosyl transferase family 43 wheat gene abundantly expressed in endosperm complements the Arabidopsis irx9 mutant and so name the three homoeologous genes TaIRX9b. We generated wheat lines with a constitutive knockout of TaIRX9b by stacking loss-of-function alleles for these homeologues from a mutagenized hexaploid wheat population resulting in decreases in grain extract viscosity of 50%-80%. The amount and chain length of WE-AX molecules from grain of these triple-stack lines was decreased accounting for the changes in extract viscosity. Imaging of immature wheat grain sections of triple-stacks showed abolition of immunolabelling in endosperm with LM11 antibody that recognizes epitopes in AX, but also showed apparently normal cell size and shape in all cell types, including endosperm. We identified differentially expressed genes from endosperm of triple-stacks suggesting that compensatory changes occur to maintain this endosperm cell wall integrity. Consistent with this, we observed increased ferulate dimerization and increased crosslinking of WE-AX molecules in triple-stacks. These novel wheat lines lacking functional TaIRX9b therefore provide insight into control of wheat endosperm cell walls.