Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used Arabidopsis thaliana mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus Botrytis cinerea, which infects a variety of hosts, with the Brassicaceae-specific fungus Alternaria brassicicola. B. cinerea isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while A. brassicicola was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that B. cinerea stimulates the accumulation of glucosinolates to a greater extent than A. brassicicola. In our work with A. brassicicola, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, in vivo, hydrolysis products of indole glucosinolates were found to be involved in defense against B. cinerea, but not in the host response to A. brassicicola. We suggest that the Brassicaceae-specialist A. brassicicola has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist B. cinerea are more sensitive to these phytochemicals.

Glucosinolates accumulate mainly in cruciferous plants and their hydrolysis-derived products play important roles in plant resistance against pathogens. The pathogen Botrytis cinerea has variable sensitivity to glucosinolates, but the mechanisms by which it responds to them are mostly unknown. Exposure of B. cinerea to glucosinolate-breakdown products induces expression of the Major Facilitator Superfamily transporter, mfsG, which functions in fungitoxic compound efflux. Inoculation of B. cinerea on wild-type Arabidopsis thaliana plants induces mfsG expression to higher levels than on glucosinolate-deficient A. thaliana mutants. A B. cinerea strain lacking functional mfsG transporter is deficient in efflux ability. It accumulates more isothiocyanates (ITCs) and is therefore more sensitive to this compound in vitro; it is also less virulent to glucosinolates-containing plants. Moreover, mfsG mediates ITC efflux in Saccharomyces cerevisiae cells, thereby conferring tolerance to ITCs in the yeast. These findings suggest that mfsG transporter is a virulence factor that increases tolerance to glucosinolates.

Trypanosomatid genomes encode for numerous proteins containing an RNA recognition motif (RRM), but the function of most of these proteins in mRNA metabolism is currently unknown. Here, we report the function of two such proteins that we have named PTB1 and PTB2, which resemble the mammalian polypyrimidine tract binding proteins (PTB). RNAi silencing of these factors indicates that both are essential for life. PTB1 and PTB2 reside mostly in the nucleus, but are found in the cytoplasm, as well. Microarray analysis performed on PTB1 and PTB2 RNAi silenced cells indicates that each of these factors differentially affects the transcriptome, thus regulating a different subset of mRNAs. PTB1 and PTB2 substrates were categorized bioinformatically, based on the presence of PTB binding sites in their 5′ and 3′ flanking sequences. Both proteins were shown to regulate mRNA stability. Interestingly, PTB proteins are essential for trans-splicing of genes containing C-rich polypyrimidine tracts. PTB1, but not PTB2, also affects cis-splicing. The specificity of binding of PTB1 was established in vivo and in vitro using a model substrate. This study demonstrates for the first time that trans-splicing of only certain substrates requires specific factors such as PTB proteins for their splicing. The trypanosome PTB proteins, like their mammalian homologs, represent multivalent RNA binding proteins that regulate mRNAs from their synthesis to degradation.

Pathogenic fungi must respond effectively to changes in environmental pH for successful host colonization, virulence and toxin production. Aspergillus carbonarius is a mycotoxigenic pathogen with the ability to colonize many plant hosts and secrete ochratoxin A (OTA). In this study, we characterized the functions and addressed the role of PacC-mediated pH signaling in A. carbonarius pathogenicity using designed pacC gene knockout mutant. ΔAcpacC mutant displayed an acidity-mimicking phenotype, which resulted in impaired fungal growth at neutral/alkaline pH, accompanied by reduced sporulation and conidial germination compared to the wild type (WT) strain. The ΔAcpacC mutant was unable to efficiently acidify the growth media as a direct result of diminished gluconic and citric acid production. Furthermore, loss of AcpacC resulted in a complete inhibition of OTA production at pH 7.0. Additionally, ΔAcpacC mutant exhibited attenuated virulence compared to the WT toward grapes and nectarine fruits. Reintroduction of pacC gene into ΔAcpacC mutant restored the WT phenotype. Our results demonstrate important roles of PacC of A. carbonarius in OTA biosynthesis and in pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.

Penicillium expansum is one of the most harmful post-harvest pathogens of pomaceous fruits and the causal agent of blue rot disease. During infection, P. expansum produces the toxic secondary metabolites patulin and citrinin that can impact virulence and, further, render the fruit inedible. Several studies have shown that epigenetic machinery controls synthesis of secondary metabolites in fungi. In this regard, the epigenetic reader, SntB, has been reported to govern the production of multiple toxins in Aspergillus species, and impact virulence of plant pathogenic fungi. Here we show that deletion of sntB in P. expansum results in several phenotypic changes in the fungus including stunted vegetative growth, reduced conidiation, but enhanced germination rates as well as decreased virulence on Golden Delicious apples. In addition, a decrease in both patulin and citrinin biosynthesis in vitro and patulin in apples, was observed. SntB positively regulates expression of three global regulators of virulence and secondary metabolism (LaeA, CreA, and PacC) which may explain in part some of the phenotypic and virulence defects of the PeΔsntB strain. Lastly, results from this study revealed that the controlled environmental factors (low temperatures and high CO₂ levels) to which P. expansum is commonly exposed during fruit storage, resulted in a significant reduction of sntB expression and consequent patulin and citrinin reduction. These data identify the epigenetic reader SntB as critical factor regulated in post-harvest pathogens under storage conditions and a potential target to control fungal colonization and decaying of stored fruit.