Carbon regulation of environmental pH by secreted small molecules that modulate pathogenicity in phytopathogenic fungi

[EN]Fruit pathogens can contribute to the acidification or alkalinization of the host environment. This capability has been used to divide fungal pathogens into acidifying and/or alkalinizing classes. Here, we show that diverse classes of fungal pathogens—Colletotrichum gloeosporioides, Penicillium expansum, Aspergillus nidulans and Fusarium oxysporum—secrete small pH-affecting molecules. These molecules modify the environmental pH, which dictates acidic or alkaline colonizing strategies, and induce the expression of PACC-dependent genes. We show that, in many organisms, acidification is induced under carbon excess, i.e. 175 mm sucrose (the most abundant sugar in fruits). In contrast, alkalinization occurs under conditions of carbon deprivation, i.e. less than 15 mm sucrose. The carbon source is metabolized by glucose oxidase (gox2) to gluconic acid, contributing to medium acidification, whereas catalysed deamination of non-preferred carbon sources, such as the amino acid glutamate, by glutamate dehydrogenase 2 (gdh2), results in the secretion of ammonia

Cation-stress-responsive transcription factors SltA and CrzA regulate morphogenetic processes and pathogenicity of Colletotrichum gloeosporioide

27 p.-12 fig.Growth of Colletotrichum gloeosporioides in the presence of cation salts NaCl and KCl inhibited fungal growth and anthracnose symptom of colonization. Previous reports indicate that adaptation of Aspergillus nidulans to salt- and osmotic-stress conditions revealed the role of zinc-finger transcription factors SltA and CrzA in cation homeostasis. Homologs of A. nidulans SltA and CrzA were identified in C. gloeosporioides. The C. gloeosporioides CrzA homolog is a 682-amino acid protein, which contains a C2H2 zinc finger DNA-binding domain that is highly conserved among CrzA proteins from yeast and filamentous fungi. The C. gloeosporioides SltA homolog encodes a 775-amino acid protein with strong similarity to A. nidulans SltA and Trichoderma reesei ACE1, and highest conservation in the three zincfinger regions with almost no changes compared to ACE1 sequences. Knockout of C. gloeosporioides crzA (ΔcrzA) resulted in a phenotype with inhibited growth, sporulation, germination and appressorium formation, indicating the importance of this calciu006D-activated transcription factor in regulating these morphogenetic processes. In contrast, knockout of C. gloeosporioides sltA (ΔsltA) mainly inhibited appressorium formation. Both mutants had reduced pathogenicity on mango and avocado fruit. Inhibition of the different morphogenetic stages in the ΔcrzA mutant was accompanied by drastic inhibition of chitin synthase A and B and glucan synthase, which was partially restored with Ca2+ supplementation. Inhibition of appressorium formation in ΔsltA mutants was accompanied by downregulation of the MAP kinase pmk1 and carnitine acetyl transferase (cat1), genes involved in appressorium formation and colonization, which was restored by Ca2+ supplementation. Furthermore, exposure of C. gloeosporioides ΔcrzA or ΔsltA mutants to cations such as Na+, K+ and Li+ at concentrations that the wild type C. gloeosporioides is not affected had further adverse morphogenetic effects on C. gloeosporioides which were partially or fully restored by Ca2+. Overall results suggest that both genes modulating alkali cation homeostasis have significant morphogenetic effects that reduce C. gloeosporioides colonization.This work was supported by the US/Israel Binational Agricultural Research Fund(ISBARD), grant no. IS-4773-14, the Ministerio de EconomõÂa y Competitividad and Fondo Europeo de Desarrollo Regional (FEDER), grant nos. BFU2012-33142 and BFU2015-66806R.Peer reviewe

Does the Host Contribute to Modulation of Mycotoxin Production by Fruit Pathogens?

Storage of freshly harvested fruit is a key factor in modulating their supply for several months after harvest; however, their quality can be reduced by pathogen attack. Fruit pathogens may infect their host through damaged surfaces, such as mechanical injuries occurring during growing, harvesting, and packing, leading to increased colonization as the fruit ripens. Of particular concern are fungal pathogens that not only macerate the host tissue but also secrete significant amounts of mycotoxins. Many studies have described the importance of physiological factors, including stage of fruit development, biochemical factors (ripening, C and N content), and environmental factors (humidity, temperature, water deficit) on the occurrence of mycotoxins. However, those factors usually show a correlative effect on fungal growth and mycotoxin accumulation. Recent reports have suggested that host factors can induce fungal metabolism, leading to the synthesis and accumulation of mycotoxins. This review describes the new vision of host-factor impact on the regulation of mycotoxin biosynthetic gene clusters underlying the complex regulation of mycotoxin accumulation in ripening fruit

gUMI-BEAR, a modular, unsupervised population barcoding method to track variants and evolution at high resolution.

Cellular lineage tracking provides a means to observe population makeup at the clonal level, allowing exploration of heterogeneity, evolutionary and developmental processes and individual clones' relative fitness. It has thus contributed significantly to understanding microbial evolution, organ differentiation and cancer heterogeneity, among others. Its use, however, is limited because existing methods are highly specific, expensive, labour-intensive, and, critically, do not allow the repetition of experiments. To address these issues, we developed gUMI-BEAR (genomic Unique Molecular Identifier Barcoded Enriched Associated Regions), a modular, cost-effective method for tracking populations at high resolution. We first demonstrate the system's application and resolution by applying it to track tens of thousands of Saccharomyces cerevisiae lineages growing together under varying environmental conditions applied across multiple generations, revealing fitness differences and lineage-specific adaptations. Then, we demonstrate how gUMI-BEAR can be used to perform parallel screening of a huge number of randomly generated variants of the Hsp82 gene. We further show how our method allows isolation of variants, even if their frequency in the population is low, thus enabling unsupervised identification of modifications that lead to a behaviour of interest

Differential gene expression in tomato fruit and Colletotrichum gloeosporioides during colonization of the RNAi–SlPH tomato line with reduced fruit acidity and higher pH

Abstract Background The destructive phytopathogen Colletotrichum gloeosporioides causes anthracnose disease in fruit. During host colonization, it secretes ammonia, which modulates environmental pH and regulates gene expression, contributing to pathogenicity. However, the effect of host pH environment on pathogen colonization has never been evaluated. Development of an isogenic tomato line with reduced expression of the gene for acidity, SlPH (Solyc10g074790.1.1), enabled this analysis. Total RNA from C. gloeosporioides colonizing wild-type (WT) and RNAi–SlPH tomato lines was sequenced and gene-expression patterns were compared. Results C. gloeosporioides inoculation of the RNAi–SlPH line with pH 5.96 compared to the WT line with pH 4.2 showed 30% higher colonization and reduced ammonia accumulation. Large-scale comparative transcriptome analysis of the colonized RNAi–SlPH and WT lines revealed their different mechanisms of colonization-pattern activation: whereas the WT tomato upregulated 13-LOX (lipoxygenase), jasmonic acid and glutamate biosynthesis pathways, it downregulated processes related to chlorogenic acid biosynthesis II, phenylpropanoid biosynthesis and hydroxycinnamic acid tyramine amide biosynthesis; the RNAi–SlPH line upregulated UDP-D-galacturonate biosynthesis I and free phenylpropanoid acid biosynthesis, but mainly downregulated pathways related to sugar metabolism, such as the glyoxylate cycle and L-arabinose degradation II. Comparison of C. gloeosporioides gene expression during colonization of the WT and RNAi–SlPH lines showed that the fungus upregulates ammonia and nitrogen transport and the gamma-aminobutyric acid metabolic process during colonization of the WT, while on the RNAi–SlPH tomato, it mainly upregulates the nitrate metabolic process. Conclusions Modulation of tomato acidity and pH had significant phenotypic effects on C. gloeosporioides development. The fungus showed increased colonization on the neutral RNAi–SlPH fruit, and limited colonization on the WT acidic fruit. The change in environmental pH resulted in different defense responses for the two tomato lines. Interestingly, the WT line showed upregulation of jasmonate pathways and glutamate accumulation, supporting the reduced symptom development and increased ammonia accumulation, as the fungus might utilize glutamate to accumulate ammonia and increase environmental pH for better expression of pathogenicity factors. This was not found in the RNAi–SlPH line which downregulated sugar metabolism and upregulated the phenylpropanoid pathway, leading to host susceptibility

LaeA regulation of secondary metabolism modulates virulence in Penicillium expansum and is mediated by sucrose

Penicillium expansum, the causal agent of blue mould rot, is a critical health concern because of the production of the mycotoxin patulin in colonized apple fruit tissue. Although patulin is produced by many Penicillium species, the factor(s) activating its biosynthesis are not clear. Sucrose, a key sugar component of apple fruit, was found to modulate patulin accumulation in a dose-responsive pattern. An increase in sucrose culture amendment from 15 to 175 mm decreased both patulin accumulation and expression of the global regulator laeA by 175- and five-fold, respectively, whilst increasing expression of the carbon catabolite repressor creA. LaeA was found to regulate several secondary metabolite genes, including the patulin gene cluster and concomitant patulin synthesis in vitro. Virulence studies of laeA mutants of two geographically distant P. expansum isolates (Pe-21 from Israel and Pe-T01 from China) showed differential reduction in disease severity in freshly harvested fruit, ranging from no reduction for Ch-Pe-T01 strains to 15%-25% reduction for both strains in mature fruit, with the laeA strains of Is-Pe-21 always showing a greater loss in virulence. The results suggest the importance of abiotic factors in LaeA regulation of patulin and other secondary metabolites that contribute to pathogenicity

Westland ecosystems of Sokolov regoin and their using in school practise

"The wetland ecosystems of Sokolov region and their using in school practice" The topic of the thesis is to monitor the biodiversity of wetland ecosystems, with a focus on protected species at selected localities of Sokolov region. Localities that were studied - Nadlesí, Stará Ovčárna and pískovna Erika, have been selected with regard to their accessibility for schools. The objective of the thesis was also to propose the possibility of the didactic use of localities for the natural science and biology at primary schools, therefore, for each a site excursion was propose; with a follow up creation of worksheets. The theoretical part contains the definition of the term wetlands, Sokolov region characteristics and it also summarizes the previous inventory research conducted at the given localities. In the practical part, the floristic and zoological inventory is determined along with methodology and didactic applications, i.e. the descriptions of excursions, the real-life testing of the excursions in school practice and the worksheets

Germination and appressorium formation by spores of the <i>C</i>. <i>gloeosporioides</i> wild-type (WT), <i>ΔsltA10A</i> and <i>ΔsltA21C</i> strains in the presence of water or 10 mM CaCl₂.

<p>Five drops of spores of the different strains were placed on a single glass slide and incubated in a humid petri dish at 24°C. Microscopic evaluation of germination and appressorium formation was evaluated 20 h later. a-appressorium, c-conidia, g-germinating tube.</p

Relative expression of <i>ena1</i> (A) and <i>vcxA</i> (B) of wild-type (WT) and <i>ΔsltA</i> strains of <i>C</i>. <i>gloeosporioides</i>.

<p>RT-PCR values were normalized against <i>18S</i> rRNA and compared to the lowest expression value in the respective treatments, which was set to 1. Experiments were repeated three times and results of a single representative experiment are shown. Average of three technical replications is presented and asterisks marked columns are significantly different at P ≤ 0.05 according to the Student’s t-test. One asterisk represents lower expression and two asterisks represent higher expression. NS represents non-significant difference.</p