Glutathione transferases (GSTs) represent an extended family of multifunctional proteins involved in detoxification processes and tolerance to oxidative stress. We thus anticipated that some GSTs could play an essential role in the protection of fungal necrotrophs against plant-derived toxic metabolites and reactive oxygen species that accumulate at the host-pathogen interface during infection. Mining the genome of the necrotrophic Brassica pathogen Alternaria brassicicola for glutathione transferase revealed 23 sequences, 17 of which could be clustered into the main classes previously defined for fungal GSTs and six were ‘orphans’. Five isothiocyanate-inducible GSTs from five different classes were more thoroughly investigated. Analysis of their catalytic properties revealed that two GSTs, belonging to the GSTFuA and GTT1 classes, exhibited GSH transferase activity with isothiocyanates (ITC) and peroxidase activity with cumene hydroperoxide, respectively. Mutant deficient for these two GSTs were however neither more susceptible to ITC nor less aggressive than the wild-type parental strain. By contrast mutants deficient for two other GSTs, belonging to the Ure2pB and GSTO classes, were distinguished by their hyper-susceptibility to ITC and low aggressiveness against Brassica oleracea. In particular AbGSTO1 could participate in cell tolerance to ITC due to its glutathione-dependent thioltransferase activity. The fifth ITC-inducible GST belonged to the MAPEG class and although it was not possible to produce the soluble active form of this protein in a bacterial expression system, the corresponding deficient mutant failed to develop normal symptoms on host plant tissues. Among the five ITC-inducible GSTs analyzed in this study, three were found essential for full aggressiveness of A. brassicicola on host plant. This, to our knowledge is the first evidence that GSTs might be essential virulence factors for fungal necrotrophs.

Polygalacturonases (PGs) fine-tune pectins to modulate cell wall chemistry and mechanics, impacting plant development. The large number of PGs encoded in plant genomes leads to questions on the diversity and specificity of distinct isozymes. Herein, we report the crystal structures of two Arabidopsis thaliana polygalacturonases, POLYGALACTURONASE LATERAL ROOT (PGLR) and ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2), which are co-expressed during root development. We first determined the amino acid variations and steric clashes that explain the absence of inhibition of the plant PGs by endogenous PG-Inhibiting Proteins (PGIPs). Although their beta helix folds are highly similar, PGLR and ADPG2 subsites in the substrate-binding groove are occupied by divergent amino acids. By combining molecular dynamic simulations, analysis of enzyme kinetics and hydrolysis products, we showed that these structural differences translated into distinct enzyme-substrate dynamics and enzyme processivities: ADPG2 showed greater substrate fluctuations with hydrolysis products, oligogalacturonides (OGs), with a degree of polymerization (DP) of ≤4, while the DP of OGs generated by PGLR was between 5 and 9. Using the Arabidopsis root as a developmental model, exogenous application of purified enzymes showed that the highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. This work highlights the importance of PG processivity on pectin degradation regulating plant development.

The first steps of wood degradation by fungi lead to the release of toxic compounds known as extractives. To better understand how lignolytic fungi cope with the toxicity of these molecules, a transcriptomic analysis of Phanerochaete chrysosporium genes was performed in the presence of oak acetonic extracts. It reveals that in complement to the extracellular machinery of degradation, intracellular antioxidant and detoxification systems contribute to the lignolytic capabilities of fungi, presumably by preventing cellular damages and maintaining fungal health. Focusing on these systems, a glutathione transferase ( P. chrysosporium GTT2.1 [PcGTT2.1]) has been selected for functional characterization. This enzyme, not characterized so far in basidiomycetes, has been classified first as a GTT2 compared to the Saccharomyces cerevisiae isoform. However, a deeper analysis shows that the GTT2.1 isoform has evolved functionally to reduce lipid peroxidation by recognizing high-molecular-weight peroxides as substrates. Moreover, the GTT2.1 gene has been lost in some non-wood-decay fungi. This example suggests that the intracellular detoxification system evolved concomitantly with the extracellular ligninolytic machinery in relation to the capacity of fungi to degrade wood.

Fungi and antifungal compounds are relevant to the United Nation's Sustainable Development Goals. However, the modes‐of‐action of antifungals—whether they are naturally occurring substances or anthropogenic fungicides—are often unknown or are misallocated in terms of their mechanistic category. Here, we consider the most effective approaches to identifying whether antifungal substances are cellular stressors, toxins/toxicants (that are target‐site‐specific), or have a hybrid mode‐of‐action as toxin–stressors (that induce cellular stress yet are target‐site‐specific). This newly described ‘ toxin–stressor ’ category includes some photosensitisers that target the cell membrane and, once activated by light or ultraviolet radiation, cause oxidative damage. We provide a glossary of terms and a diagrammatic representation of diverse types of stressors, toxic substances, and toxin–stressors , a classification that is pertinent to inhibitory substances not only for fungi but for all types of cellular life. A decision‐tree approach can also be used to help differentiate toxic substances from cellular stressors ( Curr Opin Biotechnol 2015 33: 228–259). For compounds that target specific sites in the cell, we evaluate the relative merits of using metabolite analyses, chemical genetics, chemoproteomics, transcriptomics, and the target‐based drug‐discovery approach (based on that used in pharmaceutical research), focusing on both ascomycete models and the less‐studied basidiomycete fungi. Chemical genetic methods to elucidate modes‐of‐action currently have limited application for fungi where molecular tools are not yet available; we discuss ways to circumvent this bottleneck. We also discuss ecologically commonplace scenarios in which multiple substances act to limit the functionality of the fungal cell and a number of as‐yet‐unresolved questions about the modes‐of‐action of antifungal compounds pertaining to the Sustainable Development Goals.

Summary: Small secreted proteins ( SSP ) have been defined as proteins containing a signal peptide and a sequence of less than 300 amino acids. In this analysis, we have compared the secretion pattern of SSP s among eight aspergilli species in the context of plant biomass degradation and have highlighted putative interesting candidates that could be involved in the degradative process or in the strategies developed by fungi to resist the associated stress that could be due to the toxicity of some aromatic compounds or reactive oxygen species released during degradation. Among these candidates, for example, some stress‐related superoxide dismutases or some hydrophobic surface binding proteins (HsbA) are specifically secreted according to the species . Since these latter proteins are able to recruit lytic enzymes to the surface of hydrophobic solid materials and promote their degradation, a synergistic action of HsbA with the degradative system may be considered and need further investigations. These SSP s could have great applications in biotechnology by optimizing the efficiency of the enzymatic systems for biomass degradation.