No abstract

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.

A small number (10 to 20) of yeast species cause major spoilage in foods. Spoilage yeasts of soft drinks are resistant to preservatives like sorbic acid, and they are highly fermentative, generating large amounts of carbon dioxide gas. Conversely, many yeast species derive energy from respiration only, and most of these are sorbic acid sensitive and so prevented from causing spoilage. This led us to hypothesize that sorbic acid may specifically inhibit respiration. Tests with respirofermentative yeasts showed that sorbic acid was more inhibitory to both Saccharomyces cerevisiae and Zygosaccharomyces bailii during respiration (of glycerol) than during fermentation (of glucose). The respiration-only species Rhodotorula glutinis was equally sensitive when growing on either carbon source, suggesting that ability to ferment glucose specifically enables sorbic acid-resistant growth. Sorbic acid inhibited the respiration process more strongly than fermentation. We present a data set supporting a correlation between the level of fermentation and sorbic acid resistance across 191 yeast species. Other weak acids, C2 to C8, inhibited respiration in accordance with their partition coefficients, suggesting that effects on mitochondrial respiration were related to membrane localization rather than cytosolic acidification. Supporting this, we present evidence that sorbic acid causes production of reactive oxygen species, the formation of petite (mitochondrion-defective) cells, and Fe-S cluster defects. This work rationalizes why yeasts that can grow in sorbic acid-preserved foods tend to be fermentative in nature. This may inform more-targeted approaches for tackling these spoilage organisms, particularly as the industry migrates to lower-sugar drinks, which could favor respiration over fermentation in many spoilage yeasts. IMPORTANCE Spoilage by yeasts and molds is a major contributor to food and drink waste, which undermines food security. Weak acid preservatives like sorbic acid help to stop spoilage, but some yeasts, commonly associated with spoilage, are resistant to sorbic acid. Different yeasts generate energy for growth by the processes of respiration and/or fermentation. Here, we show that sorbic acid targets the process of respiration, so fermenting yeasts are more resistant. Fermentative yeasts are also those usually found in spoilage incidents. This insight helps to explain the spoilage of sorbic acid-preserved foods by yeasts and can inform new strategies for effective control. This is timely as the sugar content of products like soft drinks is being lowered, which may favor respiration over fermentation in key spoilage yeasts.

SummaryChitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.

11 12 Running title: Weak acid resistance mechanisms in A. niger conidia 13 14 15 Word count (main text, excl. legends, references and methods): 4241 16 Word count (Abstract): 234 17 18ABSTRACT 22 Propionic, sorbic and benzoic acids are organic weak acids that are widely used as food 23 preservatives, where they play a critical role in preventing microbial growth. In this study, we 24 uncovered new mechanisms of weak acid resistance in moulds. By screening a library of 401 25 transcription-factor deletion strains in Aspergillus fumigatus for sorbic acid hypersensitivity, 26 a previously uncharacterised transcription factor was identified, and named as WarA (Weak 27 Acid Resistance A). The orthologous gene in the spoilage mould Aspergillus niger was 28 identified and deleted. WarA was required for resistance to a range of weak acids, including 29 sorbic, propionic and benzoic acids. A transcriptomic analysis was performed to characterise 30 genes regulated by WarA during sorbic acid treatment in A. niger. Several genes were 31 significantly upregulated in the wild type compared with a ΔwarA mutant, including genes 32 encoding putative weak acid detoxification enzymes and transporter proteins. Among these 33 was An14g03570, a putative ABC-type transporter which we found to be required for weak 34 acid resistance in A. niger. We also show that An14g03570 is a functional homologue of the 35 Saccharomyces cerevisiae protein Pdr12p, and therefore named as PdrA. Lastly, resistance to 36 sorbic acid was found to be highly heterogeneous within genetically-uniform populations of 37 ungerminated A. niger conidia, and we demonstrate that pdrA is a determinant of this 38 heteroresistance. This study has identified novel mechanisms of weak acid resistance in A. 39 niger which could help to inform and improve future food spoilage prevention strategies. 40 41 IMPORTANCE 42Weak acids are widely used as food preservatives, as they are very effective at preventing 43 growth of most species of bacteria and fungi. However, some species of moulds can survive 44 3 and grow in the concentrations of weak acid employed in food and drink products, thereby 45 causing spoilage with resultant risks for food security and health. Current knowledge of weak 46 acid resistance mechanisms in these fungi is limited, especially in comparison to that in yeasts. 47 53 54 Microbiological spoilage of food and drinks is a serious threat to food security and human 55 health. It is estimated that 25% of global food produced annually is lost due to contamination 56 and degradation by microorganisms (1). Such spoilage also directly imperils human health, for 57 example due to the production of toxins by the microorganism. Preventing microbial spoilage 58 is therefore a key to safeguarding food supply and safety. The use of chemical food 59 preservatives to inhibit growth of bacteria and fungi is a ubiquitous and generally effective 60 strategy in reducing spoilage (2). Some of the most commonly used preservatives are weak...

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.

No abstract

A small number (10-20) of yeast species cause major spoilage in foods. Spoilage yeasts of soft drinks are resistant to preservatives like sorbic acid and they are highly fermentative, generating large amounts of carbon dioxide gas. Conversely, many yeast species derive energy from respiration only and most of these are sorbic acid-sensitive, so prevented from causing spoilage. This led us to hypothesize that sorbic acid may specifically inhibit respiration. Tests with respiro-fermentative yeasts showed that sorbic acid was more inhibitory to both Saccharomyces cerevisiae and Zygosaccharomyces bailii during respiration (of glycerol) compared with fermentation (of glucose). The respiration-only species Rhodotorula glutinis was equally sensitive when growing on either carbon source, suggesting that ability to ferment glucose specifically enables sorbic acid-resistant growth. Sorbic acid inhibited the respiration process more strongly than fermentation. We present a dataset supporting a correlation between the level of fermentation and sorbic acid resistance across 191 yeast species. Other weak acids, C2 – C8, inhibited respiration in accordance with their partition coefficients, suggesting that effects on mitochondrial respiration were related to membrane localization rather than cytosolic acidification. Supporting this, we present evidence that sorbic acid causes production of reactive oxygen species, the formation of petite (mitochondria-defective) cells, and Fe-S cluster defects. This work rationalises why yeasts that can grow in sorbic acid-preserved foods tend to be fermentative in nature. This may inform more-targeted approaches for tackling these spoilage organisms, particularly as the industry migrates to lower-sugar drinks, which could favour respiration over fermentation in many spoilage yeasts.IMPORTANCESpoilage by yeasts and moulds is a major contributor to food and drink waste, which undermines food security. Weak acid preservatives like sorbic acid help to stop spoilage but some yeasts, commonly associated with spoilage, are resistant to sorbic acid. Different yeasts generate energy for growth by the processes of respiration and/or fermentation. Here we show that sorbic acid targets the process of respiration, so fermenting yeasts are more resistant. Fermentative yeasts are also those usually found in spoilage incidents. This insight helps to explain the spoilage of sorbic acid-preserved foods by yeasts and can inform new strategies for effective control. This is timely as sugar content of products like soft drinks is being lowered, which may favour respiration over fermentation in key spoilage yeasts.