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.

The identification and characterization of an oxalyl-CoA synthetase from grass pea (Lathyrus sativus L.).

Oxalic acid is a small metabolite found in many plants. It serves as protection from herbivores, a chelator of metal ions, a regulator of calcium levels, and additional tasks. However, it is also a strong di-carboxylic acid that can compromise plant viability by reducing cellular pH. Several metabolic pathways have evolved to control oxalate levels in plants by enzymatic degradation. Among them is the pathway that utilizes oxalyl-CoA synthetase (OCS, EC 6.2.1.8) and ATP to convert oxalate to oxalyl-CoA. Oxalyl-CoA can then be degraded to CO2 or utilized as a precursor for the synthesis of other compounds. In grass pea (Lathyrus sativus L.), a grain legume grown in Asia and Africa for human and animal consumption, the neurotoxic compound ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP) is synthesized from oxalyl-CoA and l-α,ß-diaminopropionic acid (l-DAPA). Here, we report on the identification and characterization of oxalyl CoA-synthetase from grass pea (LsOCS). The gene encoding LsOCS was amplified from grass pea, and then expressed and purified from E. coli cells as an untagged, monomeric protein of 56 kDa. Its catalytic efficiency with oxalate, K oxalate M = 71.5 ± 13.3 µM, V max = 8.2 ± 0.8 µmole min-1 mg-1, was similar to that of OCS homologs from Arabidopsis thaliana (AtAAE3) and Medicago truncatula (MtAAE3). The enzyme was crystalized in complex with AMP and is the first OCS whose structure was determined in the thioester-forming conformation. Finally, we propose that substituting LsOCS with an oxalate oxidase or decarboxylase may reduce the levels of ß-ODAP in grass pea.

Identification and characterization of the key enzyme in the biosynthesis of the neurotoxin ß-ODAP in grass pea.

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with ß-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme-ß-ODAP synthase (BOS). We show that BOS belongs to the benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferase. Using molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (Nicotiana benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for ß-ODAP production in vivo. The identification of BOS may pave the way toward engineering ß-ODAP-free grass pea cultivars, which are safe for human and animal consumption.

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.

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

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 ΔlaeA 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 ΔlaeA 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.

Cation-Stress-Responsive Transcription Factors SltA and CrzA Regulate Morphogenetic Processes and Pathogenicity of Colletotrichum gloeosporioides.

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 zinc-finger 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.

How Does Host Carbon Concentration Modulate the Lifestyle of Postharvest Pathogens during Colonization?

Postharvest pathogens can penetrate fruit by breaching the cuticle or directly through wounds, and they show disease symptoms only long after infection. During ripening and senescence, the fruit undergo physiological processes accompanied by a decline in antifungal compounds, which allows the pathogen to activate a mechanism of secretion of small effector molecules that modulate host environmental pH. These result in the activation of genes under their optimal pH conditions, enabling the fungus to use a specific group of pathogenicity factors at each particular pH. New research suggests that carbon availability in the environment is a key factor triggering the production and secretion of small pH-modulating molecules: ammonia and organic acids. Ammonia is secreted under limited carbon and gluconic acid under excess carbon. This mini review describes our most recent knowledge of the mechanism of activation of pH-secreted molecules and their contribution to colonization by postharvest pathogens to facilitate the transition from quiescence to necrotrophic lifestyle.

Fungal and host transcriptome analysis of pH-regulated genes during colonization of apple fruits by Penicillium expansum.

BACKGROUND: Penicillium expansum is a destructive phytopathogen that causes decay in deciduous fruits during postharvest handling and storage. During colonization the fungus secretes D-gluconic acid (GLA), which modulates environmental pH and regulates mycotoxin accumulation in colonized tissue. Till now no transcriptomic analysis has addressed the specific contribution of the pathogen's pH regulation to the P. expansum colonization process. For this purpose total RNA from the leading edge of P. expansum-colonized apple tissue of cv. 'Golden Delicious' and from fungal cultures grown under pH 4 or 7 were sequenced and their gene expression patterns were compared. RESULTS: We present a large-scale analysis of the transcriptome data of P. expansum and apple response to fungal colonization. The fungal analysis revealed nine different clusters of gene expression patterns that were divided among three major groups in which the colonized tissue showed, respectively: (i) differing transcript expression patterns between mycelial growth at pH 4 and pH 7; (ii) similar transcript expression patterns of mycelial growth at pH 4; and (iii) similar transcript expression patterns of mycelial growth at pH 7. Each group was functionally characterized in order to decipher genes that are important for pH regulation and also for colonization of apple fruits by Penicillium. Furthermore, comparison of gene expression of healthy apple tissue with that of colonized tissue showed that differentially expressed genes revealed up-regulation of the jasmonic acid and mevalonate pathways, and also down-regulation of the glycogen and starch biosynthesis pathways. CONCLUSIONS: Overall, we identified important genes and functionalities of P. expansum that were controlled by the environmental pH. Differential expression patterns of genes belonging to the same gene family suggest that genes were selectively activated according to their optimal environmental conditions (pH, in vitro or in vivo) to enable the fungus to cope with varying conditions and to make optimal use of available enzymes. Comparison between the activation of the colonized host's gene responses by alkalizing Colletotrichum gloeosporioides and acidifying P. expansum pathogens indicated similar gene response patterns, but stronger responses to P. expansum, suggesting the importance of acidification by P. expansum as a factor in its increased aggressiveness.

Ammonia activates pacC and patulin accumulation in an acidic environment during apple colonization by Penicillium expansum.

Penicillium expansum, the causal agent of blue mould rot, causes severe post-harvest fruit maceration simultaneously with the secretion of d-gluconic acid (GLA) and the mycotoxin patulin in colonized tissue. The factor(s) inducing patulin biosynthesis during colonization of the host acidic environment is unclear. During the colonization of apple fruit in vivo and growth in culture, P. expansum secretes pH-modulating GLA and ammonia. Although patulin and its possible opportunistic precursor GLA accumulate together during fungal development, ammonia is detected on the colonized tissue's leading edge and after extended culture, close to patulin accumulation. Here, we demonstrate ammonia-induced transcript activation of the global pH modulator PacC and patulin accumulation in the presence of GLA by: (i) direct exogenous treatment of P. expansum growing on solid medium; (ii) direct exogenous treatment on colonized apple tissue; (iii) growth under self-ammonia production conditions with limited carbon; and (iv) analysis of the transcriptional response to ammonia of the patulin biosynthesis cluster. Ammonia induced patulin accumulation concurrently with the transcript activation of pacC and patulin biosynthesis cluster genes, indicating the regulatory effect of ammonia on pacC transcript expression under acidic conditions. Electrophoretic mobility shift assays using P. expansum PacC and antibodies to the different cleaved proteins showed that PacC is not protected against proteolytic signalling at pH 4.5 relative to pH 7.0, but NH4 addition did not further enhance its proteolytic cleavage. Ammonia enhanced the activation of palF transcript in the Pal pathway under acidic conditions. Ammonia accumulation in the host environment by the pathogen under acidic pH may be a regulatory cue for pacC activation, towards the accumulation of secondary metabolites, such as patulin.

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

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. Functional analyses of Δgdh2 mutants showed reduced alkalinization and pathogenicity during growth under carbon deprivation, but not in high-carbon medium or on fruit rich in sugar, whereas analysis of Δgox2 mutants showed reduced acidification and pathogencity under conditions of excess carbon. The induction pattern of gdh2 was negatively correlated with the expression of the zinc finger global carbon catabolite repressor creA. The present results indicate that differential pH modulation by fruit fungal pathogens is a host-dependent mechanism, affected by host sugar content, that modulates environmental pH to enhance fruit colonization.

Accumulation of the mycotoxin patulin in the presence of gluconic acid contributes to pathogenicity of Penicillium expansum.

Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest fruit maceration through secretion of D-gluconic acid (GLA) and secondary metabolites such as the mycotoxin patulin in colonized tissue. GLA involvement in pathogenicity has been suggested but the mechanism of patulin accumulation and its contribution to P. expansum pathogenicity remain unclear. The roles of GLA and patulin accumulation in P. expansum pathogenicity were studied using i) glucose oxidase GOX2-RNAi mutants exhibiting decreased GOX2 expression, GLA accumulation, and reduced pathogenicity; ii) IDH-RNAi mutants exhibiting downregulation of IDH (the last gene in patulin biosynthesis), reduced patulin accumulation, and no effect on GLA level; and iii) PACC-RNAi mutants exhibiting downregulation of both GOX2 and IDH that reduced GLA and patulin production. Present results indicate that conditions enhancing the decrease in GLA accumulation by GOX2-RNAi and PACC-RNAi mutants, and not low pH, affected patulin accumulation, suggesting GLA production as the driving force for further patulin accumulation. Thus, it is suggested that GLA accumulation may modulate patulin synthesis as a direct precursor under dynamic pH conditions modulating the activation of the transcription factor PACC and the consequent pathogenicity factors, which contribute to host-tissue colonization by P. expansum.

A Penicillium expansum glucose oxidase-encoding gene, GOX2, is essential for gluconic acid production and acidification during colonization of deciduous fruit.

Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest maceration of fruit through secretion of total, d-gluconic acid (GLA). Two P. expansum glucose oxidase (GOX)-encoding genes, GOX1 and GOX2, were analyzed. GOX activity and GLA accumulation were strongly related to GOX2 expression, which increased with pH to a maximum at pH 7.0, whereas GOX1 was expressed at pH 4.0, where no GOX activity or extracellular GLA were detected. This differential expression was also observed at the leading edge of the decaying tissue, where GOX2 expression was dominant. The roles of the GOX genes in pathogenicity were further studied through i) development of P. expansum goxRNAi mutants exhibiting differential downregulation of GOX2, ii) heterologous expression of the P. expansum GOX2 gene in the nondeciduous fruit-pathogen P. chrysogenum, and iii) modulation of GLA production by FeSO(4) chelation. Interestingly, in P. expansum, pH and GLA production elicited opposite effects on germination and biomass accumulation: 26% of spores germinated at pH 7.0 when GOX activity and GLA were highest whereas, in P. chrysogenum at the same pH, when GLA did not accumulate, 72% of spores germinated. Moreover, heterologous expression of P. expansum GOX2 in P. chrysogenum resulted in enhanced GLA production and reduced germination, suggesting negative regulation of spore germination and GLA production. These results demonstrate that pH modulation, mediated by GLA accumulation, is an important factor in generating the initial signal or signals for fungal development leading to host-tissue colonization by P. expansum.