Automated cell annotation in multi-cell images using an improved CRF_ID algorithm.

Cell identification is an important yet difficult process in data analysis of biological images. Previously, we developed an automated cell identification method called CRF_ID and demonstrated its high performance in C. elegans whole-brain images (Chaudhary et al, 2021). However, because the method was optimized for whole-brain imaging, comparable performance could not be guaranteed for application in commonly used C. elegans multi-cell images that display a subpopulation of cells. Here, we present an advance CRF_ID 2.0 that expands the generalizability of the method to multi-cell imaging beyond whole-brain imaging. To illustrate the application of the advance, we show the characterization of CRF_ID 2.0 in multi-cell imaging and cell-specific gene expression analysis in C. elegans. This work demonstrates that high accuracy automated cell annotation in multi-cell imaging can expedite cell identification and reduce its subjectivity in C. elegans and potentially other biological images of various origins.

Pathogenic bacteria modulate pheromone response to promote mating.

Pathogens generate ubiquitous selective pressures and host-pathogen interactions alter social behaviours in many animals1-4. However, very little is known about the neuronal mechanisms underlying pathogen-induced changes in social behaviour. Here we show that in adult Caenorhabditis elegans hermaphrodites, exposure to a bacterial pathogen (Pseudomonas aeruginosa) modulates sensory responses to pheromones by inducing the expression of the chemoreceptor STR-44 to promote mating. Under standard conditions, C. elegans hermaphrodites avoid a mixture of ascaroside pheromones to facilitate dispersal5-13. We find that exposure to the pathogenic Pseudomonas bacteria enables pheromone responses in AWA sensory neurons, which mediate attractive chemotaxis, to suppress the avoidance. Pathogen exposure induces str-44 expression in AWA neurons, a process regulated by a transcription factor zip-5 that also displays a pathogen-induced increase in expression in AWA. STR-44 acts as a pheromone receptor and its function in AWA neurons is required for pathogen-induced AWA pheromone response and suppression of pheromone avoidance. Furthermore, we show that C. elegans hermaphrodites, which reproduce mainly through self-fertilization, increase the rate of mating with males after pathogen exposure and that this increase requires str-44 in AWA neurons. Thus, our results uncover a causal mechanism for pathogen-induced social behaviour plasticity, which can promote genetic diversity and facilitate adaptation of the host animals.

Spermidine Is Critical for Growth, Development, Environmental Adaptation, and Virulence in Fusarium graminearum.

Putrescine, spermidine, and spermine are the most common natural polyamines. Polyamines are ubiquitous organic cations of low molecular weight and have been well characterized for the cell function and development processes of organisms. However, the physiological functions of polyamines remain largely obscure in plant pathogenic fungi. Fusarium graminearum causes Fusarium head blight (FHB) and leads to devastating yield losses and quality reduction by producing various kinds of mycotoxins. Herein, we genetically analyzed the gene function of the polyamine biosynthesis pathway and evaluated the role of the endogenous polyamines in the growth, development, and virulence of F. graminearum. Our results found that deletion of spermidine biosynthesis gene FgSPE3 caused serious growth defects, reduced asexual and sexual reproduction, and increased sensitivity to various stresses. More importantly, ΔFgspe3 exhibited significantly decreased mycotoxin deoxynivalenol (DON) production and weak virulence in host plants. Additionally, the growth and virulence defects of ΔFgspe3 could be rescued by exogenous application of 5 mM spermidine. Furthermore, RNA-seq displayed that FgSpe3 participated in many essential biological pathways including DNA, RNA, and ribosome synthetic process. To our knowledge, these results indicate that spermidine is essential for growth, development, DON production, and virulence in Fusarium species, which provides a potential target to control FHB.

Microfluidic Pneumatic Printed Sandwiched Microdroplet Array for High-Throughput Enzymatic Reaction and Screening.

High-throughput enzyme screening for desired functionality is highly demanded. This paper utilizes a newly developed microfluidic pneumatic printing platform for high-throughput enzyme screening applications. The novel printing platform can achieve distinct features including a disposable cartridge, which avoids crosstalk; a flexible cartridge design, allowing for integration of multiple channels; and fast printing speed with submicroliter spot size. Moreover, a polydimethylsiloxane (PDMS)-based sandwich structure has been proposed and used during the printing and imaging, which can lead to better results, including reduced evaporation as well as a uniform light path during imaging. Using this microfluidic pneumatic printed PDMS sandwiched microdroplet array platform, we have demonstrated the capability of high-throughput generation of a combinatorial droplet array with concentration and volume gradients. Furthermore, the potential for enzymatic study has been validated by quantified cellulose reaction implemented with the printing platform.

Capping proteins regulate fungal development, DON-toxisome formation and virulence in Fusarium graminearum.

Deoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in organized endoplasmic reticulum structures called toxisomes. However, the mechanism for toxisome formation and the components of toxisomes are not yet fully understood. In a previous study, we found that myosin I (FgMyo1)-actin cytoskeleton participated in toxisome formation. In the current study, we identified two new components of toxisomes, the actin capping proteins (CAPs) FgCapA and FgCapB. These two CAPs form a heterodimer in F. graminearum, and physically interact with FgMyo1 and Tri1. The deletion mutants ΔFgcapA and ΔFgcapB and the double deletion mutant ΔΔFgcapA/B dramatically reduced hyphal growth, asexual and sexual reproduction and endocytosis. More importantly, the deletion mutants markedly disrupted toxisome formation and DON production, and attenuated virulence in planta. Collectively, these results suggest that the actin CAPs are associated with toxisome formation and contribute to the virulence and development of F. graminearum.

The b-ZIP transcription factor FgTfmI is required for the fungicide phenamacril tolerance and pathogenecity in Fusarium graminearum.

BACKGROUND: Fusarium head blight (FHB) is a devastating disease of cereal crops worldwide mainly caused by Fusarium graminearum. Due to the unavailability of FHB-resistant wheat cultivars, chemical fungicide application is currently the most effective approach for controlling FHB now. In the last few years, a novel cyanoacrylate fungicide, phenamacril, has been widely used in China for FHB disease management. In previous studies, we identified that myosin I (FgMyo1) is the target of phenamacril and is essential for mycotoxin deoxynivalenol (DON) biosynthesis and fungal growth. However, the regulation of FgMYO1 gene expression is still largely unknown. RESULTS: In this study, we identified a b-ZIP transcription factor, FgTfmI, which regulates the mRNA expression of FgMYO1 upon phenamacril treatment. The FgTfmI directly binds to the promoter region of FgMYO1, and is required for the upregulation of FgMYO1 in response to phenamacril treatment. The deletion mutant of FgTFMI (ΔFgTfmI) displayed a slight growth defect, while it showed hypersensitivity to phenamacril, but not to other tested fungicides. FgTfmI also contributed to DON biosynthesis and the infection process in planta. CONCLUSIONS: The transcription factor FgTfmI plays an important role in regulating transcription of the genes involved in phenamacril tolerance, DON biosynthesis and virulence in F. graminearum. © 2019 Society of Chemical Industry.

Whole genome deep sequencing revealed host impact on population structure, variation and evolution of Rice stripe virus.

High-throughput deep sequencing and variant detection showed that variations of Rice stripe virus (RSV) populations obtained from small brown planthopper-transmitted rice plants and sap-inoculated N. benthamiana plants were single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels). The SNPs were more uniform across RSV genome, but InDels occurred mainly in the intergenic regions (IRs) and in the 5' or 3' noncoding regions. There were no clear patterns of InDels, although the inserted sequences were all from virus itself. Six, one, and one non-synonymous substitutions were respectively observed in the RdRP ORF, IR and the movement protein ORF. These non-synonymous substitutions were found to be stable, resulting in new consensus sequences in the NBL11 RSV population. Furthermore, the numbers of SNPs and InDels in RSV genome from N. benthamiana plants were much higher than that from O. sativa plants. These differences are likely caused by selection pressures generated by different host plants.

Effects of two kinds of imidazolium-based ionic liquids on the characteristics of steroid-transformation Arthrobacter simplex.

BACKGROUND: Ionic liquids (ILs) are a promising alternative for organic solvents because these liquids exhibit unique properties and enhanced steroid 1-dehydrogenation biotransformation caused by Arthrobacter simplex CPCC 140451 (ASP). However, the effect of ILs on the whole cell itself remains poorly understood and must be further investigated. RESULTS: A comparative investigation was performed to determine the effect of imidazolium-based ILs, namely, hydrophobic [PrMIm]PF6, and hydrophilic [PrMIm]BF4, on the steroid conversion, activity, permeability, and material basis of ASP cells. Both ILs weakened permeability barriers, enhanced steroid transformation, whereas reduced the activity of cells. The influence of [PrMIm]PF6 on the steroid conversion, permeability and activity of cells is more serious than that of [PrMIm]BF4 Transmission electron microscopy micrographs directly showed wrinkles, gross creases, and several small pores in ILs-treated cells surface. The total lipid content of [PrMIm]BF4-treated cells reduced by 8.3 %, while that of [PrMIm]PF6-treated cells reduced twice more, among which the content of long-chain fatty acids was decreased, whereas the content of unsaturated fatty acids was increased. The protein profile of LC-MS/MS revealed that the reduced proteins of cells treated with the two ILs were mainly located in the cytoplasm and plasma membrane, 19.27 % of reduced proteins were located on the cell membrane for [PrMIm]PF6-pretreated cells, whereas only 12.8 % for [PrMIm]BF4-pretreated cells. It suggests that most reduced proteins functioned in energy production and conversion, material transport and metabolism, signal recognition and transmission, transcription, and translation and posttranslational modification. In particular, the identified differential proteins functioned in the pentose phosphate pathway, synthesis of purines and pyrimidines, and oxidative phosphorylation and fatty acid pathway. CONCLUSION: Treatment with ILs improved permeability at the molecular level and exerted significant positive effects on steroid conversion. This study provides a material basis and elucidates the mechanisms underlying cellular changes that enhanced conversion rate.

Hydroxypropyl-ß-cyclodextrin-mediated alterations in cell permeability, lipid and protein profiles of steroid-transforming Arthrobacter simplex.

Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) enhances steroid 1-dehydrogenation biotransformation by Arthrobacter simplex. In this work, HP-ß-CD-induced improvement of A. simplex CPCC 140451 cell envelope permeability which had positive effects on the steroid bioconversion was confirmed by a comparative investigation which showed a lower dehydrogenase activity and higher cell permeability of the cells after being incubated with HP-ß-CD. Atomic force microscopy and transmission electron microscopy micrographs showed that HP-ß-CD altered the size, sharpness, and surface structure of the cell envelope. The analysis of lipid composition revealed that the proportion of extractable lipids decreased and the fatty acids profile was considerably altered. The contents of unsaturated fatty acids and long-chain fatty acids were reduced by 11.77 and 14.98%, respectively. The total leakage of protein level increased to 8%. Proteins belonging to the ATP-binding cassette superfamily and major facilitator superfamily were observed outside the cell. These alterations can explain the change of permeability on the molecular level under HP-ß-CD treatment. Results showed the material basis and mechanisms underlying the cellular changes, thus most likely contributing to the conversion rate in addition to cyclodextrins known effects on substrate solubility.