Biology subjects

Outram, M. A.

Publications and source records attributed to Outram, M. A..

9 recordsLinked to original sources

bioRxiv2025

Virulence on Pm4 kinase-based resistance is determined by two divergent wheat powdery mildew effectors

The wheat resistance gene Pm4 encodes a kinase fusion protein and has gained particular attention as it confers race-specific resistance against two major wheat pathogens: powdery mildew and blast. Here, we describe the identification of AvrPm4, the mildew avirulence effector recognised by Pm4, using UV- mutagenesis, and its functional validation in wheat protoplasts. We show that AvrPm4 directly interacts with and is phosphorylated by Pm4. Using genetic association and QTL mapping, we furthermore demonstrate that evasion of Pm4 resistance by virulent mildew isolates relies on a second fungal component, SvrPm4, which suppresses AvrPm4-induced cell death. Surprisingly, SvrPm4 was previously described as AvrPm1a. We show that SvrPm4, but not its inactive variant svrPm4, is recognised by the NLR immune receptor Pm1a. These multiple roles of a single effector provide a new perspective on fungal (a)virulence proteins and their combinatorial interactions with different types of immune receptors.

genetics↗

bioRxiv2024

Revisiting the evolution and function of NIP2 parologs in the Rhynchosporium spp. complex

The fungus Rhynchosporium commune, the causal agent of barley scald disease, contains a paralogous effector gene family called Necrosis-Inducing Protein 2 (NIP2) and NIP2-like protein (NLP). However, the function and full genomic context of these paralogs remains uncharacterised. Here we present a highly contiguous long-read assembly of R. commune WAI453. Using this assembly, we show that the duplication of the NIP2 and NLP gene families is distributed throughout the genome and pre-dates the speciation of R. commune from its sister species. Some NIP2 paralogs have subsequently been lost or are absent in the sister species. The diversity of these paralogs was examined from R. commune global populations and their expression was analysed during in planta and in vitro growth to analyse the importance of these genes during infection. The majority of NIP2 and NLP paralogs in WAI453 genome were significantly upregulated during plant infection suggesting that the NIP2 and NLP genes harbour virulence roles. An attempt to further characterise function of NIP2.1 by infiltrating purified protein into barley leaves did not induce necrosis questioning its previously reported role as an inducer of host cell death. Together these results suggest that the NIP2 effector family does play a role during infection of barley, however the exact function of NIP2, like many effectors, remains uncharacterised.

plant biology↗

bioRxiv2024

A wheat tandem kinase sensor activates an NLR helper to trigger immunity

Most plant resistance genes encode membrane-anchored receptor-like proteins or intracellular nucleotide-binding and leucine-rich repeat (NLR) receptors. In wheat and barley, tandem kinases (TKs) have emerged as a new class of resistance determinants. To understand the modus operandi of the wheat stem rust resistance protein Sr62TK, we identified two genetic interactors-- a host gene required for Sr62TK function and the corresponding fungal AvrSr62 effector. We discovered that the SR62 locus consists of a digenic module encoding Sr62TK and an NLR (Sr62NLR). AvrSr62 binds to the N-terminal kinase of Sr62TK. This triggers displacement of the C-terminal kinase allowing it to recruit Sr62NLR for activation of immune responses. Understanding the mechanism of this two-component resistance complex will help engineering and breeding for durable resistance.

plant biology↗

bioRxiv2023

AvrSr27 is a zinc-bound effector with a modular structure important for immune recognition

Stem rust, caused by the fungal pathogen Puccinia graminis f. sp.tritici (Pgt) is a major threat for wheat production and global food security. Central to the success of Pgt is the secretion of proteinaceous effectors that promote infection and colonisation, while immunity in wheat is driven by receptor-mediated recognition of these effectors resulting in pathogen avirulence. Here, we report the crystal structure of the cysteine-rich effector AvrSr27, the third experimentally derived structure of a Pgt effector. The AvrSr27 structure reveals a novel {beta}-strand rich modular fold consisting of two structurally similar domains and confirms the poor prediction we obtained from the AlphaFold2-derived model. The highly prevalent cysteine residues within the protein facilitate the co-ordination of 4 zinc molecules. Utilising the structure, we show that the N-terminal domain of AvrSr27 is sufficient for immune recognition and interaction by Sr27. The 7-cys motif sequence in each AvrSr27 domain, which facilitates zinc binding, was also found in two haustorially-expressed, structurally homologous Pgt proteins. Remarkably, despite relatively low sequence identity, we show that these proteins can associate with Sr27 and trigger cell death in heterologous systems and wheat protoplasts, albeit weaker than AvrSr27. Collectively, our findings have important implications for the field embarking on bespoke engineering of immunity receptors as solutions to plant disease.

plant biology↗

bioRxiv2023

Cyanobacterial α-carboxysome carbonic anhydrase is allosterically regulated by the Rubisco substrate RuBP

Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally significant proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in [a]-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present the first structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate (RuBP), and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial [a]-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of -carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage. One-Sentence SummaryThe carboxysomal carbonic anhydrase, CsoSCA, is allosterically activated by the Rubisco substrate RuBP, revealing a novel mechanism controlling key enzyme activity in cyanobacterial -carboxysomes.

molecular biology↗

bioRxiv2022

A pathogen-induced putative NAC transcription factor mediates leaf rust resistance in barley

Leaf rust, caused by Puccinia hordei, is one of the most widespread and damaging foliar diseases affecting barley (Hordeum spp.). The barley leaf rust resistance locus Rph7, located on the short arm of chromosome 3H, confers defence at all growth stages and was previously shown to have unusually high sequence and haplotype divergence. Earlier, four candidate genes for Rph7 were reported and, despite an in-depth comparative sequence analysis and haplotypic characterisation, the causal gene could not be resolved. Here, we successfully cloned Rph7 utilising a fine mapping approach in combination with an RNA-Seq based expression analysis. We identified three up-regulated and pathogen-induced genes with presence/absence variation (PAV) at this locus. Sequence analysis of chemically induced Rph7 knockout mutant lines identified multiple independent non-synonymous variants, including a premature stop codon in a single non-canonical resistance gene that encodes a 302-amino acid protein. Progeny from four independent transgenic lines segregated for the expected avirulent Rph7 infection type in response to several avirulent P. hordei pathotypes, however, all plants were susceptible to a single virulent pathotype confirming the specificity. Structural analysis using an AlphaFold2 protein model suggests that Rph7 encodes a putative NAC transcription factor, as it shares structural similarity to ANAC019 from Arabidopsis, with a C-terminal BED domain. A global gene expression analysis suggests Rph7 is involved in the activation of basal defence.

genetics↗

bioRxiv2021

The structural repertoire of Fusarium oxysporum f. sp. lycopersici effectors revealed by experimental and computational studies

Plant pathogens secrete proteins, known as effectors, that function in the apoplast or inside plant cells to promote virulence. Effector recognition by cell-surface or cytosolic receptors results in the activation of defence pathways and plant immunity. Despite their importance, our general understanding of fungal effector function and recognition by immunity receptors remains poor. One complication often associated with effectors is their high sequence diversity and lack of identifiable sequence motifs precluding prediction of structure or function. In recent years, several studies have demonstrated that fungal effectors can be grouped into structural classes, despite significant sequence variation and existence across taxonomic groups. Using protein x-ray crystallography, we identify a new structural class of effectors hidden within the secreted in xylem (SIX) effectors from Fusarium oxysporum f. sp. lycopersici (Fol). The recognised effectors Avr1 (SIX4) and Avr3 (SIX1) represent the founding members of the Fol dual-domain (FOLD) effector class, with members containing two distinct domains. Using AlphaFold2, we predicted the full SIX effector repertoire of Fol and show that SIX6 and SIX13 are also FOLD effectors, which we validated experimentally for SIX6. Based on structural prediction and comparisons, we show that FOLD effectors are present within three divisions of fungi and are expanded in pathogens and symbionts. Further structural comparisons demonstrate that Fol secretes effectors that adopt a limited number of structural folds during infection of tomato. This analysis also revealed a structural relationship between transcriptionally co-regulated effector pairs. We make use of the Avr1 structure to understand its recognition by the I receptor, which leads to disease resistance in tomato. This study represents an important advance in our understanding of Fol-tomato, and by extension plant-fungal interactions, which will assist the development of novel control and engineering strategies to combat plant pathogens.

plant biology↗

bioRxiv2021

Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be useful when investigating the function and recognition of a broad range of disulfide-bond containing effectors.

biochemistry↗

bioRxiv2020

The crystal structure of SnTox3 from the necrotrophic fungus Parastagonospora nodorum reveals a unique effector fold and insights into Kex2 protease processing of fungal effectors

O_LIPlant pathogens cause disease through secreted effector proteins, which act to modulate host physiology and promote infection. Typically, the sequences of effectors provide little functional information and further targeted experimentation is required. Here, we utilised a structure/function approach to study SnTox3, an effector from the necrotrophic fungal pathogen Parastagonospora nodorum, which causes cell death in wheat-lines carrying the sensitivity gene Snn3. C_LIO_LIWe developed a workflow for the production of SnTox3 in a heterologous host that enabled crystal structure determination. We show this approach can be successfully applied to effectors from other pathogenic fungi. Complementing this, an in-silico study uncovered the prevalence of an expanded subclass of effectors from fungi. C_LIO_LIThe {beta}-barrel fold of SnTox3 is a novel fold among fungal effectors. We demonstrate that SnTox3 is a pre-pro-protein and that the protease Kex2 removes the pro-domain. Our in-silico studies suggest that Kex2-processed pro-domain (designated here as K2PP) effectors are common in fungi, and we demonstrate this experimentally for effectors from Fusarium oxysporum f sp. lycopersici. C_LIO_LIWe propose that K2PP effectors are highly prevalent among fungal effectors. The identification and classification of K2PP effectors has broad implications for the approaches used to study their function in fungal virulence. C_LI

plant biology↗