Biology subjects

Spanu, P. D.

Publications and source records attributed to Spanu, P. D..

6 recordsLinked to original sources

bioRxiv2024

A powdery mildew core effector protein targets host endosome tethering complexes HOPS and CORVET

Powdery mildew fungi are serious pathogens of many plant species. Their genomes encode extensive repertoires of secreted effector proteins that suppress host immunity. Here, we revised and analyzed the candidate secreted effector protein (CSEP) effectome of the barley powdery mildew fungus, Blumeria hordei (Bh) and identified seven CSEPs that are broadly conserved in powdery mildew fungal species, rendering them core effectors of these phytopathogens. We show that one of these, CSEP0214, interacts with the barley VPS18 protein, a core component of the CORVET and HOPS endosomal tethering complexes, mediating fusions of early endosomes and multivesicular bodies with the central vacuole, respectively. Overexpression of CSEP0214 and knockdown of either VPS18, HOPS-specific VPS41 or CORVET-specific VPS8 caused a block of the vacuolar pathway and the accumulation of the fluorescent vacuolar marker protein (SP)-RFP-AFVY in the endoplasmic reticulum. Co-immunoprecipitation and yeast two-hybrid experiments suggest that CSEP0214 blocks the interaction of VPS18 and VPS16, which are both core components of CORVET as well as HOPS. Additionally, expression of CSEP0214 blocked the hypersensitive cell death response associated with resistance gene-mediated immunity in barley, indicating that endomembrane traffic is required for this process. It also prevented callose deposition in cell wall appositions at attack sites and encasements of fungal infection structures. Our results indicate that this powdery mildew core effector is an essential immunity suppressor. One sentence summaryThe Blumeria hordei effector protein CSEP0214 interacts with barley VPS18, a core component of the CORVET and HOPS endosomal tethering complexes, thereby interfering with host endomembrane trafficking.

plant biology↗

bioRxiv2024

Barley powdery mildew invasion coincides with the dynamic accumulation of leaf apoplastic extracellular vesicles that are associated with host stress response proteins

The mutual exchange of extracellular vesicles across kingdom borders is a feature of many plant-microbe interactions. The occurrence and cargos of extracellular vesicles has been studied in several instances, but their dynamics in the course of infection have remained elusive. Here we used two different procedures, differential high-speed centrifugation and polymer-based enrichment, to collect extracellular vesicles from the apoplastic wash fluid of barley (Hordeum vulgare) leaves challenged by its fungal powdery mildew pathogen, Blumeria hordei. Both methods yielded extracellular vesicles of similar quality and morphological characteristics, though the polymer approach was associated with higher reproducibility. We noted that extracellular vesicles derived from the apoplastic wash fluid constitute polydisperse populations that are selectively responsive to leaf infection by B. hordei. Extracellular vesicles of [~]100 nm - 300 nm diameter became progressively more abundant, in particular from 72 hours post inoculation onwards, resulting in a major peak late during fungal infection. Vesicles of [~]300 nm - 500 nm showed similar accumulation dynamics but reached much lower levels, suggesting they might constitute a separate population. Proteome analysis uncovered an enrichment of biotic stress response proteins associated with the extracellular vesicles. The barley t-SNARE protein Ror2, the ortholog of the PEN1 marker protein of extracellular vesicles in Arabidopsis thaliana, accumulates in extracellular vesicles during powdery mildew infection, hence also qualifying as a potential marker protein. Our study serves as a starting point for investigating the role of extracellular vesicles at different stages of plant-microbe interactions.

plant biology↗

bioRxiv2023

Horizontal transfers between fungal Fusarium species contributed to successive outbreaks of coffee wilt disease

Outbreaks of fungal disease have devastated plants and animals throughout history. Over the past century, the repeated emergence of coffee wilt disease caused by the fungal pathogen Fusarium xylarioides severely impacted coffee production across sub-Saharan Africa. To improve the disease management of such pathogens, it is crucial to understand their genetic structure and evolutionary potential. We compared the genomes of 13 historic strains spanning six decades and multiple disease outbreaks to investigate population structure and host specialisation. We found F. xylarioides comprises at least four distinct lineages: one host-specific to Coffea arabica, one to C. canephora var. robusta, and two historic lineages isolated from various Coffea species. Mapping variation onto a new long-read reference genome showed that host-specificity appears to be acquired through horizontal transfer of effector genes from members of the F. oxysporum species complex. This species complex is known to cause wilt disease in over 100 plant species. Multiple transfers into the F. xylarioides populations matched to different parts of the F. oxysporum mobile pathogenicity chromosome and were enriched in effector genes and transposons. Effector genes in this region and other horizontally transferred carbohydrate-active enzymes important in the breakdown of plant cell walls were shown by transcriptomics to be highly expressed during infection of C. arabica by the fungal arabica strains. Widespread sharing of specific transposons between F. xylarioides and F. oxysporum, and the presence of large Starship elements, indicate that transposons were involved in horizontal transfers. Our results support the hypothesis that horizontal gene transfers contributed to the repeated emergence of this fungal disease.

evolutionary biology↗

bioRxiv2018

A fungal ribonuclease-like effector protein inhibits plant host ribosomal RNA degradation

The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. Proteins with a predicted ribonuclease (RNase)-like fold (termed RALPHs) comprise the largest set of secreted effector candidates within the B. graminis f. sp. hordei genome. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in monocotyledenous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with five host proteins: two translation elongation factors (eEF1 and eEF1{gamma}), two pathogenesis-related proteins (PR5 and PR10) and a glutathione-S-transferase. We present the first crystal structure of a RALPH, CSEP0064/BEC1054, demonstrating it has an RNase-like fold. The protein interacts with total RNA and weakly with DNA. Methyl jasmonate levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose that CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins that would otherwise lead to host cell death, an unviable interaction and demise of the fungus.

plant biology↗

bioRxiv2018

Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen

Powdery mildews are biotrophic pathogenic fungi infecting a number of economically important plants. The grass powdery mildew, Blumeria graminis, has become a model organism to study host specialization of obligate biotrophic fungal pathogens. We resolved the large-scale genomic architecture of B. graminis forma specialis hordei (Bgh) to explore the potential influence of its genome organization on the co-evolutionary process with its host plant, barley (Hordeum vulgare). The near-chromosome level assemblies of the Bgh reference isolate DH14 and one of the most diversified isolates, RACE1, enabled a comparative analysis of these haploid genomes, which are highly enriched with transposable elements (TEs). We found largely retained genome synteny and gene repertoires, yet detected copy number variation (CNV) of secretion signal peptide-containing protein-coding genes (SPs) and locally disrupted synteny blocks. Genes coding for sequence-related SPs are often locally clustered, but neither the SP clusters nor TEs are enriched in specific genomic regions. Extended comparative analysis with different host-specific B. graminis formae speciales revealed the existence of a core suite of SPs, but also isolate-specific SP sets as well as congruence of SP CNV and phylogenetic relationship. We further detected evidence for a recent, lineage-specific expansion of TEs in the Bgh genome. The characteristics of the Bgh genome (largely retained synteny, CNV of SP genes, recently proliferated TEs and a lack of compartmentalization) are consistent with a \"one-speed\" genome that differs in its architecture and (co-)evolutionary pattern from the \"two-speed\" genomes reported for several other filamentous phytopathogens.

genomics↗

bioRxiv2018

Yeast transformation efficiency is enhanced by TORC1- and eisosome-dependent signalling

Transformation of bakers yeast (Saccharomyces cerevisiae) plays a key role in several experimental techniques, yet the molecular mechanisms underpinning transformation are still unclear. Addition of amino acids to the growth and transformation medium increases transformation efficiency. Here, we show that target of rapamycin complex 1 (TORC1) activated by amino acids enhances transformation via ubiquitin-mediated endocytosis. We created mutants of the TORC1 pathway, -arrestins, and eisosome-related genes. Our results demonstrate that the TORC1-Npr1-Art1/Rsp5 pathway regulates yeast transformation. Based on our previous study, activation of this pathway results in a 13-fold increase in transformation efficiency, or greater. Additionally, we suggest DNA is taken up by domains at the membrane compartment of Can1 (MCC) in the plasma membrane formed by eisosomes. Yeast studies on transformation could be used as a platform to understand the mechanism of DNA uptake in mammalian systems, which is clinically relevant to optimise gene therapy.

microbiology↗