Biology subjects

Jones, D. A.

Publications and source records attributed to Jones, D. A..

6 recordsLinked to original sources

bioRxiv2024

Translocation of effector proteins into plant cells by the flax rust pathogen Melampsora lini

During infection, rust fungi secrete effector proteins into host plant cells from haustoria to aid their colonisation. How rust effectors are secreted from the haustorium and delivered into the cytoplasm of host cells remains poorly understood. We used an Agrobacterium-mediated transformation procedure to generate stable transgenic flax rust strains expressing the effectors AvrM and AvrP123 fused to yellow fluorescent protein (YFP). We showed that both AvrM-YFP and AvrP123-YFP fusion proteins were secreted by the fungus into the extrahaustorial matrix (EHMx); however only AvrM-YFP was delivered into host cells, triggering a typical resistance phenotype in plants carrying the corresponding resistance (R) gene M. The signal peptide of AvrM was sufficient to direct YFP secretion into the EHMx; however, delivery into the host cell required a larger 105 amino acid N-terminal fragment of AvrM. These results indicate that translocation of this protein into the host cell from the EHMx is a separate process from secretion into the EHMx and requires a signal present in AvrM between amino acids 34 and 105. This is in contrast to previous observations of AvrM localisation after transient expression in plants, highlighting the necessity for analysis in the natural infection system.

plant biology↗

bioRxiv2024

Evolution and inhibition of the FIKK effector kinase family in P. falciparum

Among the [~]200 Plasmodium species that infect vertebrates, six infect humans. Of these, P. falciparum causes >95% of all [~]500,000 annual fatalities. Phylogenetically, P. falciparum belongs to the Laverania subgenus, a group of Plasmodium species that infect great apes. Common to Laverania species is the family of FIKK kinases. One million years ago, a single FIKK kinase conserved in all Plasmodium species gained an export element in the Laverania subgenus and expanded into the family of [~]20 atypical FIKK kinases, most of which are exported into the host cell. The fikk genes are conserved in syntenic loci across the Laverania, arguing for a rapid expansion controlling important functions in host cell remodelling and pathogenesis. We provide evidence that the FIKK paralogues evolved specific and mutually exclusive phosphorylation motif preferences, conserved across their Laverania orthologues, in a short evolutionary timeframe. Surprisingly, we find that FIKK13 has evolved exclusive tyrosine-phosphorylation preference, which was thought to be absent in Plasmodium species. Combining a crystal structure with AlphaFold2 predictions, we identify residues that determine kinase-specificity within the FIKK family in a fast-evolving flexible loop. Finally, we show that all expressed members of the FIKK kinase family can be chemically inhibited in vitro using a single compound. Such a pan-specific inhibitor of this kinase family important for virulence could reduce the ability of the parasite to gain escape-mutations and resistance.

microbiology↗

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

Pathogenicity effector candidates and accessory genome revealed by pan-genomic analysis of Parastagonospora nodorum

The wheat pathogen Parastagonospora nodorum has emerged as a model necrotrophic fungal species with growing genomic resources. Recent population-level pan-genome studies were leveraged to provide novel insights into pathogen evolution and effector-like gene contents relevant to local crop disease outbreaks. In this study, we examined 156 isolates representing a regional population from the Western Australian (WA) wheat-belt region, and 17 internationally sourced isolates. We observed a highly diverse local population, within which were numerous small and highly similar clusters of isolates from hotter and drier regions. Pan-genome assembly and orthologous gene datasets resulted in 3579 predicted effector candidates, 2291 of which exhibited presence-absence variation (PAV) across the population, and 1362 were specific to WA isolates. There was an abundance of mutations (including repeat-induced point mutation (RIP)), distributed in hot-spots within the pan-genomic landscape that were rich in effector candidates. Three characterised effector loci (ToxA, Tox1 and Tox3) were located within sub- telomeric regions of lower diversity, but were nestled within larger high-diversity regions. RIP was widespread across the genome, but non-synonymous RIP-like mutations were strongly selected against. These improved bioinformatic resources for P. nodorum, represent progressive advancements in fungal pan-genomics, with a view towards supporting region- specific surveillance of host-pathogen interactions.

genomics↗

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↗

bioRxiv2019

Genetic risk of cholangiocarcinoma is linked to the autophagy gene ATG7

Cholangiocarcinoma is an aggressive cancer originating from the bile duct. Although cholangiocarcinoma does occur in families, to date no specific causative gene has been identified. We identified ATG7 as a cancer susceptibility gene using a joint genetic analysis of an extended pedigree with familial cholangiocarcinoma in combination with a population genetic association study. Affected family members had a germline mutation (c.2000C>T [p.Arg659*]) in the autophagy related gene, ATG7, and all of the affected individuals had cholangiocarcinoma tumors harboring somatic genomic deletions of ATG7. From a population genetic study, we identified a germline polymorphism of ATG7 (c.1591C>G [p.Asp522Glu]) associated with increased risk of cholangiocarcinoma. The autophagy substrate p62 demonstrated a higher accumulation in tumors of p.Asp522Glu carriers compared with non-carriers indicating defective autophagy. To determine whether the germline ATG7 mutation had functional consequences, we developed an ATG7-deficient cholangiocyte cell line, derived from human bile duct, to test for autophagy-mediated lipidation activity. The germline mutation from the familial cholangiocarcinoma demonstrated a lack of lipidation activity compared to the wildtype ATG7. Moreover, in zebrafish embryos depleted of atg7, a reproducible necrotic head phenotype was rescued by injection of wildtype ATG7 but not mutant ATG7. Our findings point to ATG7 as a causative genetic risk factor for cholangiocarcinoma and implicate autophagy as a novel cancer driver mechanism.

genomics↗