Method for RNA extraction and transcriptomic analysis of single fungal spores

Transcriptomic analysis of single cells has been increasingly in demand in recent years, thanks to technological and methodological advances as well as growing recognition of the importance of individuals in biological systems. However, the majority of these studies have been performed in mammalian cells, due to their ease of lysis and high RNA content. No single cell transcriptomic analysis has yet been applied to microbial spores, even though it is known that heterogeneity at the phenotype level exists among individual spores. Transcriptomic analysis of single spores is challenging, in part due to the physically robust nature of the spore wall. This precludes the use of methods commonly used for mammalian cells. Here, we describe a simple method for extraction and amplification of transcripts from single fungal conidia (asexual spores), and its application in single-cell transcriptomics studies. The method can also be used for studies of small numbers of fungal conidia, which may be necessary in the case of limited sample availability, low-abundance transcripts or interest in small subpopulations of conidia.• The method allows detection of transcripts from single conidia of Aspergillus niger• The method allows detection of genomic DNA from single conidia of Aspergillus nige

Weak Acid Resistance A (WarA), a Novel Transcription Factor Required for Regulation of Weak-Acid Resistance and Spore-Spore Heterogeneity in Aspergillus niger

Copyright © 2020 Geoghegan et al. Propionic, sorbic, and benzoic acids are organic weak acids that are widely used as food preservatives, where they play a critical role in preventing microbial growth. In this study, we uncovered new mechanisms of weak-acid resistance in molds. By screening a library of 401 transcription factor deletion strains in Aspergillus fumigatus for sorbic acid hypersensitivity, a previously uncharacterized transcription factor was identified and named weak acid resistance A (WarA). The orthologous gene in the spoilage mold Aspergillus niger was identified and deleted. WarA was required for resistance to a range of weak acids, including sorbic, propionic, and benzoic acids. A transcriptomic analysis was performed to characterize genes regulated by WarA during sorbic acid treatment in A. niger Several genes were significantly upregulated in the wild type compared with a ΔwarA mutant, including genes encoding putative weak-acid detoxification enzymes and transporter proteins. Among these was An14g03570, a putative ABC-type transporter which we found to be required for weak-acid resistance in A. niger We also show that An14g03570 is a functional homologue of the Saccharomyces cerevisiae protein Pdr12p and we therefore name it PdrA. Last, resistance to sorbic acid was found to be highly heterogeneous within genetically uniform populations of ungerminated A. niger conidia, and we demonstrate that pdrA is a determinant of this heteroresistance. This study has identified novel mechanisms of weak-acid resistance in A. niger which could help inform and improve future food spoilage prevention strategies.IMPORTANCE Weak acids are widely used as food preservatives, as they are very effective at preventing the growth of most species of bacteria and fungi. However, some species of molds can survive and grow in the concentrations of weak acid employed in food and drink products, thereby causing spoilage with resultant risks for food security and health. Current knowledge of weak-acid resistance mechanisms in these fungi is limited, especially in comparison to that in yeasts. We characterized gene functions in the spoilage mold species Aspergillus niger which are important for survival and growth in the presence of weak-acid preservatives. Such identification of weak-acid resistance mechanisms in spoilage molds will help in the design of new strategies to reduce food spoilage in the future

Chitosan Mediates Germling Adhesion in Magnaporthe oryzae and Is Required for Surface Sensing and Germling Morphogenesis.

The fungal cell wall not only plays a critical role in maintaining cellular integrity, but also forms the interface between fungi and their environment. The composition of the cell wall can therefore influence the interactions of fungi with their physical and biological environments. Chitin, one of the main polysaccharide components of the wall, can be chemically modified by deacetylation. This reaction is catalyzed by a family of enzymes known as chitin deacetylases (CDAs), and results in the formation of chitosan, a polymer of β1,4-glucosamine. Chitosan has previously been shown to accumulate in the cell wall of infection structures in phytopathogenic fungi. Here, it has long been hypothesized to act as a 'stealth' molecule, necessary for full pathogenesis. In this study, we used the crop pathogen and model organism Magnaporthe oryzae to test this hypothesis. We first confirmed that chitosan localizes to the germ tube and appressorium, then deleted CDA genes on the basis of their elevated transcript levels during appressorium differentiation. Germlings of the deletion strains showed loss of chitin deacetylation, and were compromised in their ability to adhere and form appressoria on artificial hydrophobic surfaces. Surprisingly, the addition of exogenous chitosan fully restored germling adhesion and appressorium development. Despite the lack of appressorium development on artificial surfaces, pathogenicity was unaffected in the mutant strains. Further analyses demonstrated that cuticular waxes are sufficient to over-ride the requirement for chitosan during appressorium development on the plant surface. Thus, chitosan does not have a role as a 'stealth' molecule, but instead mediates the adhesion of germlings to surfaces, thereby allowing the perception of the physical stimuli necessary to promote appressorium development. This study thus reveals a novel role for chitosan in phytopathogenic fungi, and gives further insight into the mechanisms governing appressorium development in M.oryzae

Localization of mCherry tagged Cbp1 and Cda2.

<p>Confocal fluorescence microscopy images of germlings expressing Cbp1:mCherry, showing fluorescence in conidia, germ tubes and appressoria at 0, 2 and 4hpi on hydrophobic glass (<b>A-C</b>). <b>D</b>) Successful complementation of <i>CBP1</i>:<i>mCherry</i> fusion construct. Appressorium development was fully restored in the complemented strain at 8 hpi. Error bars show SD n = 3. <b>E</b> & <b>F)</b> Confocal fluorescence microscopy images of germlings expressing Cda2:mCherry at 2 hpi (<b>E</b>) and 4 hpi (<b>F</b>). Scale bars: 10 μm.</p

Chitosan is a component of the cell wall in germ tubes and appressoria.

<p>Germlings, labelled with <b>A</b>) monoclonal anti-chitosan antibody mAbG7, <b>B</b>) polyclonal anti-chitosan antibody (staining with antibodies was performed at 16 hpi) and <b>C</b>) The anti-chitosan probe OGA488 which localized chitosan in live cells at different stages of appressorium development (2–16 hpi, as indicated). Germlings were inoculated onto a hydrophobic glass surface (inductive to appressorium formation). Scale bars: 10 μm.</p

Waxes can partially restore appressorium development in the <i>cda2/cbp1/cda3</i> mutant.

<p>Percentage of germ tubes demonstrating appressorium development in presence of 1-octacosanol and/or 1,16 hexadecanediol, comparing the WT and <i>cda2/cbp1/cda3</i> strain at 24 hpi.</p

Restored pathogenic development on plant surfaces is not accompanied by restored chitin deacetylation.

<p>Fluorescent labelling of chitosan with OGA488 in WT and <i>cda2/cbp1/cda3</i> germlings germinated on <b>A</b>) rice leaf sheaths or onion epidermis for 24 hr, showing complete absence of chitosan in the <i>cda2/cbp1/cda3</i> strain. <b>B</b>) Fluorescent labelling of chitosan in WT and <i>cda2/cbp1/cda3</i> germlings germinated on onion epidermis for 4 hr, showing absence of chitosan in unmelanized appressoria of the deletion strain. Scale bars: as stated in pictures.</p

Pathogenicity is unaffected in the <i>cda</i> mutants.

<p><b>A</b>) Pathogenicity of the WT and <i>cda2/cbp1/cda3</i> strains on whole rice plants. Rice plants were inoculated with conidia of the WT or <i>cda2/cbp1/cda3</i> strain at 3 different spore concentrations, and incubated for 4 days. The experiment was repeated 3 times, representative leaves are shown. <b>B</b>) Appressorium development in the WT and <i>cda2/cbp1/cda3</i> strains on rice leaf sheath and on onion epidermis. Restored appressorium development and successful penetration of the plant cells was observed in <i>cda2/cbp1/cda3</i>. Scale bars: 20 μm.</p

Combinations of chemical inducers are required to restore appressorium development in <i>cda2/cbp1/cda3</i>.

<p>Induction of appressorium development in the <i>cda2/cbp1/cda3</i> strain by chemical inducers, on <b>A</b>) hydrophobic and <b>C</b>) hydrophilic glass surfaces. Appressorium development could be partially restored in the triple deletion strain (grey bars) after a 24 hr incubation with IBMX, although chitosan staining of these germlings (<b>B</b>) showed that the appressorium development was associated with an increase in chitosan in the germlings of the <i>cda2/cbp1/cda3</i> mutant. <b>C</b>) Appressorium development on hydrophilic surfaces could be restored to levels similar to the WT strain by combinations of 0.01% chitosan (w/v) and 2.5mM IBMX or with 200 μm 1,16 hexadecanediol (HDD). White bars: WT, grey bars: <i>cda2/cbp1/cda3</i>. Error bars: SD, n = 3.</p

<i>M</i>.<i>oryzae</i> has 10 putative chitin deacetylases.

<p><b>A</b>) Table of the 10 putative chitin deacetylases in <i>M</i>.<i>oryzae</i>, showing gene numbers, given gene names and protein domain architectures (as defined by Pfam). <b>B</b>) Expression of the chitin deacetylases during appressorium development on rice leaves. Leaves of the susceptible cultivar CO-39 were inoculated with conidia of <i>M</i>.<i>oryzae</i>, and incubated for 5 hr, to induce appressorium development. Expression levels of the <i>CDA</i>s were analyzed by qRT-PCR and normalized to <i>Actin</i> (<i>MGG_03982</i>). Error bars show standard deviation, n = 3.</p