Isolation and Characterization of Extracellular Vesicles from the Fungal Phytopathogen Colletotrichum higginsianum

Fungal phytopathogens secrete extracellular vesicles (EVs) associated with enzymes and phytotoxic metabolites. While these vesicles are thought to promote infection, defining the true contents and functions of fungal EVs, as well as suitable protein markers, is an ongoing process. To expand our understanding of fungal EVs and their possible roles during infection, we purified EVs from the hemibiotrophic phytopathogen Colletotrichum higginsianum, the causative agent of anthracnose disease in multiple plant species, including Arabidopsis thaliana. EVs were purified in large numbers from the supernatant of protoplasts but not the supernatant of intact mycelial cultures. We purified two separate populations of EVs, each associated with over 700 detected proteins, including proteins involved in vesicle transport, cell wall biogenesis and the synthesis of secondary metabolites. We selected two SNARE proteins (Snc1 and Sso2) and one 14-3-3 protein (Bmh1) as potential EV markers and generated transgenic lines expressing fluorescent fusions. Each marker was confirmed to be protected inside EVs. Fluorescence microscopy was used to examine the localization of each marker during infection on Arabidopsis leaves. These findings further our understanding of EVs in fungal phytopathogens and will help build an experimental system to study EV inter-kingdom communication between plants and fungi.

Seasonal Differences in Structural and Genetic Control of Digestibility in Perennial Ryegrass

Perennial ryegrass is an important forage crop in dairy farming, either for grazing or haying purposes. To further optimise the forage use, this study focused on understanding forage digestibility in the two most important cuts of perennial ryegrass, the spring cut at heading and the autumn cut. In a highly diverse collection of 592 Lolium perenne genotypes, the organic matter digestibility (OMD) and underlying traits such as cell wall digestibility (NDFD) and cell wall components (cellulose, hemicellulose, and lignin) were investigated for 2 years. A high genotype × season interaction was found for OMD and NDFD, indicating differences in genetic control of these forage quality traits in spring versus autumn. OMD could be explained by both the quantity of cell wall content (NDF) and the quality of the cell wall content (NDFD). The variability in NDFD in spring was mainly explained by differences in hemicellulose. A 1% increase of the hemicellulose content in the cell wall (HC.NDF) resulted in an increase of 0.81% of NDFD. In autumn, it was mainly explained by the lignin content in the cell wall (ADL.NDF). A 0.1% decrease of ADL.NDF resulted in an increase of 0.41% of NDFD. The seasonal traits were highly heritable and showed a higher variation in autumn versus spring, indicating the potential to select for forage quality in the autumn cut. In a candidate gene association mapping approach, in which 503 genes involved in cell wall biogenesis, plant architecture, and phytohormone biosynthesis and signalling, identified significant quantitative trait loci (QTLs) which could explain from 29 to 52% of the phenotypic variance in the forage quality traits OMD and NDFD, with small effects of each marker taken individually (ranging from 1 to 7%). No identical QTLs were identified between seasons, but within a season, some QTLs were in common between digestibility traits and cell wall composition traits confirming the importance of hemicellulose concentration for spring digestibility and lignin concentration in NDF for autumn digestibility.

Comparative Transcriptomic Analysis of Two Cucumis Melo Var. Saccharinus Germplasms Differing in Fruit Physical and Chemical Characteristics

Background: Hami melon (Cucumis melo var. saccharinus) is a popular fruit in China noted for its excellent taste, which is largely determined by its physical and chemical characteristics, including flesh texture, sugar content, aroma, and nutrient composition. However, the mechanisms through which the associated with these characteristics are regulated have not yet been sufficiently determined. In this study, we monitored changes in the fruits of two germplasms differing in physical and chemical characteristics throughout the period of fruit development. Results: Ripe fruit of the bred variety ‘Guimi’ had significantly higher soluble sugar contents than the fruit of the common variety ‘Yaolong’, whereas differences in fruit shape and color between these two germplasms were observed during the course of development. Comparative transcriptome analysis, conducted to identify regulators and pathways underlying the observed differences at corresponding stages of development, revealed a higher number of differentially expressed genes (DEGs) in Guimi than in Yaolong. Moreover, most of the DEGs detected during early fruit development of Guimi were associated with cell wall biogenesis. Temporal analysis of the identified DEGs revealed similar trends in the enrichment of downregulated genes in both germplasms, although there were certain differences in the enrichment trends of upregulated genes. Further analyses revealed trends in the differential changes of multiple genes involved in cell wall biogenesis and sugar metabolism during fruit ripening.Conclusions: We were thus able to identify a number of genes associated with the ripening of Hami melon, which will accordingly provide novel insights into the molecular mechanisms underlying the development of fruit characteristics in these melons.

Chitin-induced systemic disease resistance in rice requires both OsCERK1 and OsCEBiP and is mediated via perturbation of cell-wall biogenesis in leaves

SummaryChitin is a well-known elicitor of disease resistance whose recognition by plants is crucial to perceive fungal infections. Chitin can induce both a local immune response and a systemic disease resistance when provided as a supplement in soils. Unlike local immune responses, how chitin-induced systemic disease resistance is deployed has not been studied in detail.In this study, we evaluated systemic disease resistance against the fungal pathogen Bipolaris oryzae by performing a transcriptome analysis and monitoring cell-wall composition in rice plants grown in chitin-supplemented soils. We also examined the local immune response to chitin by measuring the production of reactive oxygen species in leaves.Chitins induced both local immune response and systemic disease resistance with differing requirements for the receptors OsCERK1 and OsCEBiP. Transcriptome analysis suggested that a perturbation in cell-wall biogenesis is involved in the induction of systemic disease resistance, an idea which was supported by the induction of disease resistance by treatment with a cellulose biosynthesis inhibitor and alterations of cell-wall composition.These findings suggest that chitin-induced systemic disease resistance in rice is caused by a perturbation of cell-wall biogenesis in leaves through long-distance signalling after recognition of chitins by OsCERK1 and OsCEBiP.

Integrated multi-omics analysis uncovers roles of mdm-miR164b-MdORE1 in strigolactone mediated inhibition of adventitious root formation in apple

Adventitious root (AR) formation is important for the vegetative propagation. The effects of strigolactones (SLs) on AR formation have been rarely reported, especially in woody plants. In this study, we first verified the inhibitory effects of SLs on AR formation in apple materials. Transcriptome analysis identified 12,051 differentially expressed genes over the course of AR formation, with functions related to organogenesis, cell wall biogenesis or plant senescence. WGCNA suggests SLs might inhibit AR formation through repressing the expression of two core hub genes, MdLAC3 and MdORE1. We further verified that enhanced cell wall formation and accelerated senescence were involved in the AR inhibition caused by SLs. Combining small RNA and degradome sequencing, as well as a dual-luciferase sensor system, we identified and validated three negatively correlated miRNA–mRNA pairs, including mdm-miR397–MdLAC3 involved in secondary cell wall formation, and mdm-miR164a/b–MdORE1 involved in senescence. Finally, we have experimentally demonstrated the role of mdm-miR164b–MdORE1 in SLs-mediated inhibition of AR formation. Overall, our findings not only propose a comprehensive regulatory network for the function of SLs on AR formation, but also provide novel candidate genes for the potential genetic improvement of AR formation in woody plants using transgenic or CRISPR technology.

Multi-omics Analysis of the Symbiotic Green Algae, Chlorella Variabilis, Revealing the Genetic Basis of the Obligate Endosymbiotic Lifestyle

Background: Photosynthetic eukaryotes have evolved through the acquisition of plastids by secondary endosymbiosis, a process that requires several steps. Immediately before plastid acquisition, the genome of the symbiont is known to be dramatically reduced, but few studies have focused on the genomic changes in the symbiont at the early stages of secondary endosymbiosis. Methods: To investigate the genetic basis of the transition from facultative to obligate endosymbiosis, we compared the genomes of Chlorella variabilis, a representative symbiotic alga, with that of Paramecium bursaria, to compare closely related free-living species and transcriptomes between organisms in symbiotic and non-symbiotic conditions. Results: We found that the non-reduced genome of C. variabilis and its genes play a crucial role in endosymbiosis, being involved in cell wall biogenesis and degradation, and metabolic exchanges with the host. Our results suggest that the genetic mechanism underlying the enhancement of photosynthesis under symbiosis is the increasing light absorption efficiency and carbon fixation capacity of the endosymbiont, resulting in an increase in the supply of maltose to P. bursaria.

Crustacean Meal Elicits Expression of Growth and Defense-Related Genes in Roots of Lettuce and Tomato

Powdered crab and lobster shells (crustacean meal) obtained from fisheries are used as soil amendments to promote plant health and defense. In this study, a commercial crustacean meal amendment used to promote health of lettuce, tomato, and some other crop plants was applied to roots of lettuce and tomato seedlings. Gene expression profiling of the treated roots was assessed by RNA sequencing (RNA-seq) at 24 h after application relative to a 0 h time point. The RNA-seq analyses revealed upregulation of different types of genes in both tomato and lettuce roots at 24 h. Gene ontology analyses revealed increased expression of genes associated with oxidoreductases/metal ion binding in tomato at 24 h, while there was predominantly increased expression of genes associated with cell wall organization, lyases, and hydrolases in lettuce roots at 24 h. The types of defense-related genes expressed was also markedly different. In tomato, the most highly induced gene (Log₂ fold change 13.84, P = <0.001) encoded a defense associated miraculin-like protein, but transcripts of a similar gene were not induced in lettuce roots. Interestingly, phenylpropanoid pathway genes relating to cell wall biogenesis and lignification were significantly upregulated in both lettuce and tomato roots, suggesting that strengthening of plant cell walls is a common response to crustacean meal application. This research provides insight into gene expression patterns in the roots of lettuce and tomato in response to crustacean meal, improving our understanding of how this amendment may aid in plant health.

Structural studies on cell wall biogenesis-I: A method to produce high-quality protoplasts from alga Micrasterias denticulata, an emerging model in plant biology

Micrasterias denticulate is a freshwater unicellular green alga emerging as a model system in plant cell biology. This is an algae that has been examined in the context of cell wall research from early 1970s. Protoplast production from such a model system is important for many downstream physiological and cell biological studies. The algae produce intact protoplast in a straight two-step protocol involving 5% mannitol, 2% cellulysin, 4mM calcium chloride under a temperature ramping strategy. The process of protoplast induction and behavior of protoplast was examined by light microscopy and reported in this study.

Plant Transcriptome Reprograming and Bacterial Extracellular Metabolites Underlying Tomato Drought Resistance Triggered by a Beneficial Soil Bacteria

Water deficit is one of the major constraints to crop production and food security worldwide. Some plant growth-promoting rhizobacteria (PGPR) strains are capable of increasing plant drought resistance. Knowledge about the mechanisms underlying bacteria-induced plant drought resistance is important for PGPR applications in agriculture. In this study, we show the drought stress-mitigating effects on tomato plants by the Bacillus megaterium strain TG1-E1, followed by the profiling of plant transcriptomic responses to TG1-E1 and the profiling of bacterial extracellular metabolites. Comparison between the transcriptomes of drought-stressed plants with and without TG1-E1 inoculation revealed bacteria-induced transcriptome reprograming, with highlights on differentially expressed genes belonging to the functional categories including transcription factors, signal transduction, and cell wall biogenesis and organization. Mass spectrometry-based analysis identified over 40 bacterial extracellular metabolites, including several important regulators or osmoprotectant precursors for increasing plant drought resistance. These results demonstrate the importance of plant transcriptional regulation and bacterial metabolites in PGPR-induced plant drought resistance.

The wall-less bacterium Spiroplasma poulsonii builds a polymeric cytoskeleton composed of interacting MreB isoforms

A rigid cell wall defines the morphology of most bacteria. MreB, a bacterial homologue of actin, plays a major role in coordinating cell wall biogenesis and defining cell shape. In contrast with most bacteria, the Mollicutes family is devoid of cell wall. As a consequence, many Mollicutes have undefined morphologies. Spiroplasma species are an exception as they robustly grow with a characteristic helical shape, but how they maintain their morphology remains unclear. Paradoxal to their lack of cell wall, the genome of Spiroplasma contains five homologues of MreB (SpMreBs). Since MreB is a homolog of actin and that short MreB filaments participate in its function, we hypothesize that SpMreBs form a polymeric cytoskeleton. Here, we investigate the function of SpMreB in forming a polymeric cytoskeleton by focusing on the Drosophila endosymbiont Spiroplasma poulsonii. We found that in vivo, Spiroplasma maintain a high concentration of all five MreB isoforms. By leveraging a heterologous expression system that bypasses the poor genetic tractability of Spiroplasma, we found that strong intracellular levels of SpMreb systematically produced polymeric filaments of various morphologies. Using co-immunoprecipitation and co-expression of fluorescent fusions, we characterized an interaction network between isoforms that regulate the filaments formation. Our results point to a sub-functionalization of each isoform which, when all combined in vivo, form a complex inner polymeric network that shapes the cell in a wall-independent manner. Our work therefore supports the hypothesis where MreB mechanically supports the cell membrane, thus forming a cytoskeleton.