Cytotoxic activity of Nep1-like proteins on monocots.

Necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are found throughout several plant-associated microbial taxa and are typically considered to possess cytolytic activity exclusively on dicot plant species. However, cytolytic NLPs are also produced by pathogens of monocot plants such as the onion (Allium cepa) pathogen Botrytis squamosa. We determined the cytotoxic activity of B. squamosa BsNep1, as well as other previously characterized NLPs, on various monocot plant species and assessed the plant plasma membrane components required for NLP sensitivity. Leaf infiltration of NLPs showed that onion cultivars are differentially sensitive to NLPs, and analysis of their sphingolipid content revealed that the GIPC series A : series B ratio did not correlate to NLP sensitivity. A tri-hybrid population derived from a cross between onion and two wild relatives showed variation in NLP sensitivity within the population. We identified a quantitative trait locus (QTL) for NLP insensitivity that colocalized with a previously identified QTL for B. squamosa resistance and the segregating trait of NLP insensitivity correlated with the sphingolipid content. Our results demonstrate the cytotoxic activity of NLPs on several monocot plant species and legitimize their presence in monocot-specific plant pathogens.

Exocyst subunit Sec6 is positioned by microtubule overlaps in the moss phragmoplast prior to cell plate membrane arrival.

During plant cytokinesis a radially expanding membrane-enclosed cell plate is formed from fusing vesicles that compartmentalizes the cell in two. How fusion is spatially restricted to the site of cell plate formation is unknown. Aggregation of cell-plate membrane starts near regions of microtubule overlap within the bipolar phragmoplast apparatus of the moss Physcomitrella patens Since vesicle fusion generally requires coordination of vesicle tethering and subsequent fusion activity, we analyzed the subcellular localization of several subunits of the exocyst, a tethering complex active during plant cytokinesis. We found that the exocyst complex subunit Sec6 but not the Sec3 or Sec5 subunits localized to microtubule overlap regions in advance of cell plate construction in moss. Moreover, Sec6 exhibited a conserved physical interaction with an ortholog of the Sec1/Munc18 protein KEULE, an important regulator for cell-plate membrane vesicle fusion in Arabidopsis Recruitment of the P. patens protein KEULE and vesicles to the early cell plate was delayed upon Sec6 gene silencing. Our findings, thus, suggest that vesicle-vesicle fusion is, in part, enabled by a pool of exocyst subunits at microtubule overlaps, which is recruited independently of vesicle delivery.

Peeling the Onion: Towards a Better Understanding of Botrytis Diseases of Onion.

Onion is cultivated worldwide for its bulbs, but production is threatened by pathogens and pests. Three distinct diseases of onion are caused by species that belong to the fungal genus Botrytis. Leaf blight is a well-known foliar disease caused by B. squamosa that can cause serious yield losses. Neck rot is a postharvest disease that manifests in bulbs after storage and is associated with three species: B. aclada, B. allii, and B. byssoidea. The symptomless infection of onion plants in the field makes it difficult to predict the incidence of neck rot in storage, although progress on the detection of latent infection has been made. In onion cultivation for seed production, blighting of the inflorescence is caused by all four onion-specific Botrytis species plus the broad host range pathogen B. cinerea. Flower blight can reduce seed yield and contaminate seed. In this review, the long history of Botrytis diseases of onion is discussed, as well as recent and future approaches to acquire a better understanding of the biology and ecology of Botrytis spp. pathogenic on onion. New fundamental insights in the genetic, biochemical, and physiological aspects of Botrytis-onion interactions are essential to improve the breeding of Botrytis-resistant onion cultivars.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Visualization of Three Sclerotiniaceae Species Pathogenic on Onion Reveals Distinct Biology and Infection Strategies.

Botrytis squamosa, Botrytis aclada, and Sclerotium cepivorum are three fungal species of the family Sclerotiniaceae that are pathogenic on onion. Despite their close relatedness, these fungi cause very distinct diseases, respectively called leaf blight, neck rot, and white rot, which pose serious threats to onion cultivation. The infection biology of neck rot and white rot in particular is poorly understood. In this study, we used GFP-expressing transformants of all three fungi to visualize the early phases of infection. B. squamosa entered onion leaves by growing either through stomata or into anticlinal walls of onion epidermal cells. B. aclada, known to cause post-harvest rot and spoilage of onion bulbs, did not penetrate the leaf surface but instead formed superficial colonies which produced new conidia. S. cepivorum entered onion roots via infection cushions and appressorium-like structures. In the non-host tomato, S. cepivorum also produced appressorium-like structures and infection cushions, but upon prolonged contact with the non-host the infection structures died. With this study, we have gained understanding in the infection biology and strategy of each of these onion pathogens. Moreover, by comparing the infection mechanisms we were able to increase insight into how these closely related fungi can cause such different diseases.

A chromosome-level genome assembly of Zasmidium syzygii isolated from banana leaves.

Accurate taxonomic classification of samples from infected host material is essential for disease diagnostics and genome analyses. Despite the importance, diagnosis of fungal pathogens causing banana leaf diseases remains challenging. Foliar diseases of bananas are mainly caused by 3 Pseudocercospora species, of which the most predominant causal agent is Pseudocercospora fijiensis. Here, we sequenced and assembled four fungal isolates obtained from necrotic banana leaves in Bohol (Philippines) and obtained a high-quality genome assembly for one of these isolates. The samples were initially identified as P. fijiensis using PCR diagnostics; however, the assembly size was consistently 30 Mb smaller than expected. Based on the internal transcribed spacer (ITS) sequences, we identified the samples as Zasmidium syzygii (98.7% identity). The high-quality Zasmidium syzygii assembly is 42.5 Mb in size, comprising 16 contigs, of which 11 are most likely complete chromosomes. The genome contains 98.6% of the expected single-copy BUSCO genes and contains 14,789 genes and 10.3% repeats. The 3 short-read assemblies are less continuous but have similar genome sizes (40.4-42.4 Mb) and contain between 96.5 and 98.4% BUSCO genes. All 4 isolates have identical ITS sequences and are distinct from Zasmidium isolates that were previously sampled from banana leaves. We thus report the first continuous genome assembly of a member of the Zasmidium genus, forming an essential resource for further analysis to enhance our understanding of the diversity of pathogenic fungal isolates as well as fungal diversity.

Analysis of plant cell death-inducing proteins of the necrotrophic fungal pathogens Botrytis squamosa and Botrytis elliptica.

Fungal plant pathogens secrete proteins that manipulate the host in order to facilitate colonization. Necrotrophs have evolved specialized proteins that actively induce plant cell death by co-opting the programmed cell death machinery of the host. Besides the broad host range pathogen Botrytis cinerea, most other species within the genus Botrytis are restricted to a single host species or a group of closely related hosts. Here, we focused on Botrytis squamosa and B. elliptica, host specific pathogens of onion (Allium cepa) and lily (Lilium spp.), respectively. Despite their occurrence on different hosts, the two fungal species are each other's closest relatives. Therefore, we hypothesize that they share a considerable number of proteins to induce cell death on their respective hosts. In this study, we first confirmed the host-specificity of B. squamosa and B. elliptica. Then we sequenced and assembled high quality genomes. The alignment of these two genomes revealed a high level of synteny with few balanced structural chromosomal arrangements. To assess the cell death-inducing capacity of their secreted proteins, we produced culture filtrates of B. squamosa and B. elliptica that induced cell death responses upon infiltration in host leaves. Protein composition of the culture filtrate was analysed by mass spectrometry, and we identified orthologous proteins that were present in both samples. Subsequently, the expression of the corresponding genes during host infection was compared. RNAseq analysis showed that the majority of the orthogroups of the two sister species display similar expression patterns during infection of their respective host. The analysis of cell death-inducing proteins of B. squamosa and B. elliptica provides insights in the mechanisms used by these two Botrytis species to infect their respective hosts.

Dynamics in Secondary Metabolite Gene Clusters in Otherwise Highly Syntenic and Stable Genomes in the Fungal Genus Botrytis.

Fungi of the genus Botrytis infect >1,400 plant species and cause losses in many crops. Besides the broad host range pathogen Botrytis cinerea, most other species are restricted to a single host. Long-read technology was used to sequence genomes of eight Botrytis species, mostly pathogenic on Allium species, and the related onion white rot fungus, Sclerotium cepivorum. Most assemblies contained <100 contigs, with the Botrytis aclada genome assembled in 16 gapless chromosomes. The core genome and pan-genome of 16 Botrytis species were defined and the secretome, effector, and secondary metabolite repertoires analyzed. Among those genes, none is shared among all Allium pathogens and absent from non-Allium pathogens. The genome of each of the Allium pathogens contains 8-39 predicted effector genes that are unique for that single species, none stood out as potential determinant for host specificity. Chromosome configurations of common ancestors of the genus Botrytis and family Sclerotiniaceae were reconstructed. The genomes of B. cinerea and B. aclada were highly syntenic with only 19 rearrangements between them. Genomes of Allium pathogens were compared with ten other Botrytis species (nonpathogenic on Allium) and with 25 Leotiomycetes for their repertoire of secondary metabolite gene clusters. The pattern was complex, with several clusters displaying patchy distribution. Two clusters involved in the synthesis of phytotoxic metabolites are at distinct genomic locations in different Botrytis species. We provide evidence that the clusters for botcinic acid production in B. cinerea and Botrytis sinoallii were acquired by horizontal transfer from taxa within the same genus.