Widespread crop cultivation has offered more opportunity for pathogens to evolve resulting in the emergence of new virulence and lifestyle patterns. Alternate hosts surrounding a crop field can also help pathogens survive, spread and provide inoculum for subsequent growing seasons. Examining pathogen hosts under laboratory conditions can explain disease emergence mechanisms. Here, we studied four strains of Fusarium xylarioides, a soil-borne, vascular fungal pathogen that causes coffee wilt disease collected over several decades and preserved in the CABI culture collection. We observed that these fungi can colonise tomato fruits as an alternate host to coffee and used these to test the pathogenicity of historic F. xylarioides and F. oxysporum f. sp. lycopersici strains. Expression of effector genes in F. xylarioides was compared in both the primary (coffee) and an alternate plant host (tomato). We used pathogenicity assays on coffee seedlings and tomato fruits by fungal staining, diagnostic end-point PCRs and real-time quantitative PCRs to verify the infection of both plant hosts. Passaging through coffee seedlings (infection followed by re-isolation of the pathogen) resulted in increased effector gene expression and enhanced pathogenicity. These findings indicate that alternate hosts may act as reservoirs for the pathogen, with implications for disease persistence and spread. A clearer understanding of plant disease cycles is therefore essential for the development of effective management strategies aimed at mitigating impacts on global food production.

Crop protection against fungal pathogens is essential to prevent crop losses and maintain food security. Current crop protection relies heavily on chemical fungicides. However, rapid evolution of fungicide resistance, the constant appearance of new pathogens, and legislation against chemical pesticides due to concerns regarding their impact on human health and the environment, mean new crop protection strategies are urgently required. One elegant solution is double-stranded RNA-based crop protection, which aims to silence selected genes in the pathogen to reduce crop damage. This technology brings the promise of targeting specific genes, which could be chosen to maximise protection, minimize off-target effects and reduce the risk of resistance evolution. Here we discuss strategies for successful use of this novel technology based on lessons learned from fungicide resistance and recent discoveries in fungal evolution derived from genome-sequencing.

Powdery mildew fungi are serious pathogens affecting 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 powdery mildew fungus, Blumeria hordei (Bh). We identified seven putative effectors that are broadly conserved in powdery mildew species, suggesting that they are core effectors of these phytopathogens. We showed that one of these effectors, CSEP0214, interacts with the barley (Hordeum vulgare) vacuolar protein-sorting 18 (VPS18) protein, a shared component of the class C core vacuole/endosome tethering (CORVET) and homotypic fusion and protein-sorting (HOPS) endosomal tethering complexes that mediate fusion of early endosomes and multivesicular bodies, respectively, with the central vacuole. Overexpression of CSEP0214 and knockdown of either VPS18, HOPS-specific VPS41, or CORVET-specific VPS8 blocked the vacuolar pathway and the accumulation of the fluorescent vacuolar marker protein (SP)-RFP-AFVY in the endoplasmic reticulum. Moreover, CSEP0214 inhibited the interaction between VPS18 and VPS16, which are both shared components of CORVET as well as HOPS. Additionally, introducing CSEP0214 into barley leaf cells blocked the hypersensitive cell death response associated with resistance gene-mediated immunity, indicating that endomembrane trafficking is required for this process. CSEP0214 expression also prevented callose deposition in cell wall appositions at attack sites and encasements of fungal infection structures. Our results indicate that the powdery mildew core effector CSEP0214 is an essential suppressor of plant immunity.

Outbreaks of fungal diseases 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 6 decades and multiple disease outbreaks to investigate population structure and host specialisation. We found that F. xylarioides comprised at least 4 distinct lineages: 1 host-specific to Coffea arabica, 1 to C. canephora var. robusta, and 2 historic lineages isolated from various Coffea species. The presence/absence of large genomic regions across populations, the higher genetic similarities of these regions between species than expected based on genome-wide divergence and their locations in different loci in genomes across populations showed that horizontal transfers of effector genes from members of the F. oxysporum species complex contributed to host specificity. Multiple transfers into F. xylarioides populations matched different parts of the F. oxysporum mobile pathogenicity chromosome and were enriched in effector genes and transposons. Effector genes in this region and other 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 correspondence of a putative horizontally transferred regions to a Starship (large mobile element involved in horizontal gene transfers in fungi), reinforce the inference of horizontal transfers and suggest that mobile elements were involved. Our results support the hypothesis that horizontal gene transfers contributed to the repeated emergence of coffee wilt disease.

Barley (Hordeum vulgare) is an important cereal crop, and its development, defence, and stress responses are modulated by different hormones including jasmonates (JAs) and the antagonistic gibberellins (GAs). Barley productivity is severely affected by the foliar biotrophic fungal pathogen Blumeria hordei. In this study, primary leaves were used to examine the molecular processes regulating responses to methyl-jasmonate (MeJA) and GA to B. hordei infection along the leaf axis. Flow cytometry, microscopy, and spatiotemporal expression patterns of genes associated with JA, GA, defence, and the cell cycle provided insights on cell cycle progression and on the gradient of susceptibility to B. hordei observed along the leaf. Notably, the combination of B. hordei with MeJA or GA pre-treatment had a different effect on the expression patterns of the analysed genes compared to individual treatments. MeJA reduced susceptibility to B. hordei in the proximal part of the leaf blade. Overall, distinctive spatiotemporal gene expression patterns correlated with different degrees of cell proliferation, growth capacity, responses to hormones, and B. hordei infection along the leaf. Our results highlight the need to further investigate differential spatial and temporal responses to pathogens at the organ, tissue, and cell levels in order to devise effective disease control strategies in crops.

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.### Competing Interest StatementThe authors have declared no competing interest.

According to current textbooks, the principal task of transfer and ribosomal RNAs (tRNAs and rRNAs, respectively) is synthesizing proteins. During the last decade, additional cellular roles for precisely processed tRNA and rRNAs fragments have become evident in all kingdoms of life. These RNA fragments were originally overlooked in transcriptome datasets or regarded as unspecific degradation products. Upon closer inspection, they were found to engage in a variety of cellular processes, in particular the modulation of translation and the regulation of gene expression by sequence complementarity- and Argonaute protein-dependent gene silencing. More recently, the presence of tRNA and rRNA fragments has also been recognized in the context of plant-microbe interactions, both on the plant and the microbial side. While most of these fragments are likely to affect endogenous processes, there is increasing evidence for their transfer across kingdoms in the course of such interactions; these processes may involve mutual exchange in association with extracellular vesicles. Here, we summarize the state-of-the-art understanding of tRNA and rRNA fragment's roles in the context of plant-microbe interactions, their potential biogenesis, presumed delivery routes, and presumptive modes of action.

The establishment of host-microbe interactions requires molecular communication between both partners, which may involve the mutual transfer of noncoding small RNAs. Previous evidence suggests that this is also true for powdery mildew disease in barley, which is caused by the fungal pathogen Blumeria hordei. However, previous studies lacked spatial resolution regarding the accumulation of small RNAs upon host infection by B. hordei. Here, we analysed site-specific small RNA repertoires in the context of the barley-B. hordei interaction. To this end, we dissected infected leaves into separate fractions representing different sites that are key to the pathogenic process: epiphytic fungal mycelium, infected plant epidermis, isolated haustoria, a vesicle-enriched fraction from infected epidermis, and extracellular vesicles. Unexpectedly, we discovered enrichment of specific 31-33-base 5 '-terminal fragments of barley 5.8S ribosomal RNA in extracellular vesicles and infected epidermis, as well as particular B. hordei transfer RNA fragments in haustoria. We describe canonical small RNAs from both the plant host and the fungal pathogen that may confer cross-kingdom RNA interference activity. Interestingly, we found first evidence of phased small interfering RNAs in B. hordei, a feature usually attributed to plants, which may be associated with the posttranscriptional control of fungal coding genes, pseudogenes, and transposable elements. Our data suggest a key and possibly site-specific role for cross-kingdom RNA interference and noncoding RNA fragments in the host-pathogen communication between B. hordei and its host barley.

The powdery mildew fungi (Erysiphaceae) are globally distributed plant pathogens with a range of more than 10,000 plant hosts. In this review, we discuss the long- and short-term evolution of these obligate biotrophic fungi and outline their diversity with respect to morphology, lifestyle, and host range. We highlight their remarkable ability to rapidly overcome plant immunity, evolve fungicide resistance, and broaden their host range, for example, through adaptation and hybridization. Recent advances in genomics and proteomics, particularly in cereal powdery mildews (genus Blumeria), provided first insights into mechanisms of genomic adaptation in these fungi. Transposable elements play key roles in shaping their genomes, where even close relatives exhibit diversified patterns of recent and ongoing transposon activity. These transposons are ubiquitously distributed in the powdery mildew genomes, resulting in a highly adaptive genome architecture lacking obvious regions of conserved gene space. Transposons can also be neofunctionalized to encode novel virulence factors, particularly candidate secreted effector proteins, which may undermine the plant immune system. In cereals like barley and wheat, some of these effectors are recognized by plant immune receptors encoded by resistance genes with numerous allelic variants. These effectors determine incompatibility ("avirulence") and evolve rapidly through sequence diversification and copy number variation. Altogether, powdery mildew fungi possess plastic genomes that enable their fast evolutionary adaptation towards overcoming plant immunity, host barriers, and chemical stress such as fungicides, foreshadowing future outbreaks, host range shifts and expansions as well as potential pandemics by these pathogens.

Compounds targeting the inflammasome-caspase-1 pathway could be of use for the treatment of inflammation and inflammatory diseases. Previous caspase-1 inhibitors were in great majority covalent inhibitors and failed in clinical trials. Using a mixed modelling, computational screening, synthesis and in vitro testing approach, we identified a novel class of non-covalent caspase-1 non cytotoxic inhibitors which are able to inhibit IL-1β release in activated macrophages in the low μM range, in line with the best activities observed for the known covalent inhibitors. Our compounds could form the basis of further optimization towards potent drugs for the treatment of inflammation and inflammatory disorders including also dysregulated inflammation in Covid 19.

Growth defects associated with disruption of the mildew susceptibility gene MLO are rescued in bread wheat and Arabidopsis by transcriptional activation of a proximal monosaccharide transporter

Plant microbiomes and immune responses have coevolved through history, and this applies just as much to the phyllosphere microbiome and defense phytohormone signaling. When in homeostasis, the phyllosphere microbiome confers benefits to its host. However, the phyllosphere is also dynamic and subject to stochastic events that can modulate community assembly. Investigations into the impact of defense phytohormone signaling on the microbiome have so far been limited to culture-dependent studies; or focused on the rhizosphere. In this study, the impact of the foliar phytohormone salicylic acid (SA) on the structure and composition of the phyllosphere microbiome was investigated. 16S rRNA amplicons were sequenced from aerial tissues of two Arabidopsis mutants that exhibit elevated SA signaling through different mechanisms. SA signaling was shown to increase community diversity and to result in the colonization of rare, satellite taxa in the phyllosphere. However, a stable core community remained in high abundance. Therefore, we propose that SA signaling acts as a source of intermediate disturbance in the phyllosphere. Predictive metagenomics revealed that the SA-mediated microbiome was enriched for antibiotic biosynthesis and the degradation of a diverse range of xenobiotics. Core taxa were predicted to be more motile, biofilm-forming and were enriched for traits associated with microbe-microbe communication; offering potential mechanistic explanation of their success despite SA-mediated phyllospheric disturbance.

Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein (RIP) from barley (Hordeum vulgare) and is involved in the plant immune response dependent on jasmonate hormones. Here, we demonstrate in Nicotiana benthamiana that transient expression of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163-185) is removed, initiates cell death, leading to extensive necrosis of leaf tissues. We used structure prediction of JIP60 to identify potential catalytic amino acids in the active site and tested these by mutagenesis and in planta assays of necrosis induction by expression in N. benthamiana, as well as through an in vitro translation-inactivation assay. We found that Tyr 96, Glu 201, Arg 204, and Trp 234 in the presumptive active site of JIP60 are conserved in 815 plant RIPs in the Pfam database that were identified by HUMMR as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose that the role of these amino acids in JIP60 activity is to depurinate adenosine in ribosomes. This study provides insight into the catalytic mechanism of JIP60.

There is a growing evidence of the relation between an excessive release of pro-inflammatory IL-1beta triggered by the inflammasome-caspase 1 complex, and various immune and inflammatory disorders, including CNS diseases such as Alzheimer's (AD), Parkinson's (PD) and Huntington's (HD) diseases, amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). Moreover, there is also a growing evidence that the pathway inflammasome NLRP3/caspase-1 is overactivated by the SARS-Cov-2 and may be responsible for the high mortality observed in the COVID-19 patients due to the inflammatory internal organs collapse driven by the cytokine storm induced by the virus. It is now quite clear that an effective pharmacological approach to Covid-19 must comprise an antiviral drug in combination with an inflammation modulator able to regulate the innate immunity system response preserving its regular activity but quenching its overactivation. For all of these reasons a new caspase-1 inhibitor drug could be a useful tool to treat inflammation driven diseases including Covid-19.For this purpose, we have designed a new class of non-covalent, non-peptidic, small molecule caspase-1 inhibitors through structure-based drug design. The multicomponent reaction approach allows the straightforward synthesis of the desired compounds, offering a significant advantage over conventional linear step synthesis by permitting the one pot assembly of very complex structures. This new class of inhibitors shows an IC50 in the low micromolar range, and could be further optimized towards new inflammasome/caspase-1 pathway inhibitors characterized by high bioavailability, potency and reduced toxicity.