Discovery of the cryptic function of terpene cyclases as aromatic prenyltransferases.

Catalytic versatility is an inherent property of many enzymes. In nature, terpene cyclases comprise the foundation of molecular biodiversity as they generate diverse hydrocarbon scaffolds found in thousands of terpenoid natural products. Here, we report that the catalytic activity of the terpene cyclases AaTPS and FgGS can be switched from cyclase to aromatic prenyltransferase at basic pH to generate prenylindoles. The crystal structures of AaTPS and FgGS provide insights into the catalytic mechanism of this cryptic function. Moreover, aromatic prenyltransferase activity discovered in other terpene cyclases indicates that this cryptic function is broadly conserved among the greater family of terpene cyclases. We suggest that this cryptic function is chemoprotective for the cell by regulating isoprenoid diphosphate concentrations so that they are maintained below toxic thresholds.

Aaprb1, a subtilsin-like protease, required for autophagy and virulence of the tangerine pathotype of Alternaria alternata.

Subtilisin-like serine protease secreted by pathogenic fungi can facilitate the infection and acquisition of nutrients. Functions of subtilisin-like serine proteases in the phytopathogenic fungus Alternaria alternata remains unknown. In the current study, 15 subtilisin-like serine proteases were individually deleted in the citrus fungal pathogen A. alternata. Only one, designated AaPrb1, was found to be required for A. alternata pathogenesis. The AaPrb1 deficiency strain (ΔAaprb1) reduced growth, conidiation, the formation of aerial hyphae, protease production, and virulence on citrus leaves. However, biochemical analyses and bioassays revealed that ΔAaprb1 plays no role in the production of ACT toxin. Through Y2H assays, Aaprb1 was found to interact with Aapep4, a vacuole-localized proteinase A in A. alternata. Furthermore, silencing AaPep4 in A. alternata resulted in phenotypes similar with those of ΔAaprb1. Expression of AaPrb1 was found to be regulated by AaPep4. TEM showed that AaPrb1and AaPep4 were involved in the suppression of the degradation of autophagosomes. Deletion of the autophagy gene AaAtg8 in A. alternata decreased conidiation, the formation of aerial hyphae and pathogenicity similar to ΔAaprb1, implying that some phenotypes of ΔAaprb1 were due to the impairment of autophagy. Overall, this study expands our understanding of how A. alternata utilizes the subtilisin-like serine protease to achieve successful infection in the plant host.

Alternaria alternata uses two siderophore systems for iron acquisition.

Iron is one of the most abundant elements on earth and essential for life. However, Fe3+ ions are rather insoluble and microorganisms such as fungi may use siderophores as strong chelators for uptake. In addition, free cytoplasmic iron is rather toxic and intracellular siderophores are used to control the toxicity. Siderophores are also important for iron storage. We studied two siderophore systems in the plant necrotrophic fungus Alternaria alternata and show that the non-ribosomal peptide synthase, Nps2, is required for the biosynthesis of intracellular ferricrocin, whereas Nps6 is needed for the formation of extracellular coprogen and coprogen B. Whereas nps2 was dispensable for growth on iron-depleted medium, nps6 was essential under those conditions. nps2 deletion caused an increase in spore formation and reduced pathogenicity on tomato. Our results suggest that A. alternata employs an external and an internal siderophore system to adapt to low iron conditions.

Isolation and Bioactivity of Secondary Metabolites from Solid Culture of the Fungus, Alternaria sonchi.

The fungus, Alternaria sonchi is considered to be a potential agent for the biocontrol of perennial sowthistle (Sonchus arvensis). A new chlorinated xanthone, methyl 8-hydroxy-3-methyl-4-chloro-9-oxo-9H-xanthene-1-carboxylate (1) and a new benzophenone derivative, 5-chloromoniliphenone (2), were isolated together with eleven structurally related compounds (3-13) from the solid culture of the fungus, which is used for the production of bioherbicidal inoculum of A. sonchi. Their structures were determined by spectroscopic (mostly by NMR and MS) methods. Alternethanoxins A and B, which were reported in A. sonchi earlier, were re-identified as moniliphenone and pinselin, respectively. The isolated compounds were tested for phytotoxic, antimicrobial, insecticidal, cytotoxic and esterase-inhibition activities. They did not demonstrate high phytotoxicity (lesions up to 2.5 mm in diameter/length at a concentration of 2 mg/mL) when tested on leaf disks/segments of perennial sowthistle (Sonchus arvensis) and couch grass (Elytrigia repens). They did not possess acute toxicity to Paramecium caudatum, and showed moderate to low cytotoxicity (IC50 > 25 µg/mL) for U937 and K562 tumor cell lines. However, chloromonilicin and methyl 3,8-dihydroxy-6-methyl-4-chloro-9-oxo-9H-xanthene-1-carboxylate (4) were shown to have antimicrobial properties with MIC 0.5-5 µg/disc. Compound 4 and chloromonilinic acid B were found to have contact insecticidal activity to wheat aphid (Schizaphis graminum) at 1 mg/mL. Compounds 2 and methyl 3,8-dihydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate displayed selective carboxylesterase inhibition activity at concentration of 100 µg/mL. Therefore, the waste solid substrate for production of A. sonchi spores can be re-utilized for the isolation of a number of valuable natural products.

Role of membrane compartment occupied by Can1 (MCC) and eisosome subdomains in plant pathogenicity of the necrotrophic fungus Alternaria brassicicola.

BACKGROUND: MCC/eisosomes are membrane microdomains that have been proposed to participate in the plasma membrane function in particular by regulating the homeostasis of lipids, promoting the recruitment of specific proteins and acting as provider of membrane reservoirs. RESULTS: Here we showed that several potential MCC/eisosomal protein encoding genes in the necrotrophic fungus A. brassicicola were overexpressed when germinated spores were exposed to antimicrobial defence compounds, osmotic and hydric stresses, which are major constraints encountered by the fungus during the plant colonization process. Mutants deficient for key MCC/eisosome components did not exhibit any enhanced susceptibility to phytoalexins and to applied stress conditions compared to the reference strain, except for a slight hypersensitivity of the ∆∆abpil1a-abpil1b strain to 2 M sorbitol. Depending on the considered mutants, we showed that the leaf and silique colonization processes were impaired by comparison to the wild-type, and assumed that these defects in aggressiveness were probably caused by a reduced appressorium formation rate. CONCLUSIONS: This is the first study on the role of MCC/eisosomes in the pathogenic process of a plant pathogenic fungus. A link between these membrane domains and the fungus ability to form functional penetration structures was shown, providing new potential directions for plant disease control strategies.

Secondary Metabolites of The Endophytic Fungus Alternaria alternata JS0515 Isolated from Vitex rotundifolia and Their Effects on Pyruvate Dehydrogenase Activity.

The fungal strain Alternaria alternata JS0515 was isolated from Vitex rotundifolia (beach vitex). Twelve secondary metabolites, including one new altenusin derivative (1), were isolated. The isolated metabolites included seven known altenusin derivatives (2-8), two isochromanones (9, 10), one perylenequinone (11), and one benzocycloalkanone (12). Their structures were determined via 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), and computational electronic circular dichroism (ECD) analysis. Compounds 3 and 11 increased pyruvate dehydrogenase (PDH) activity in AD-293 human embryonic kidney cells and significantly inhibited PDH phosphorylation. The IC50 values of 3 and 11 were 32.58 and 27.82 µM, respectively.

Enzymatic rhamnosylation of anticancer drugs by an α-L-rhamnosidase from Alternaria sp. L1 for cancer-targeting and enzyme-activated prodrug therapy.

The synthesis of rhamnosylated compounds has gained great importance since these compounds have potential therapeutic applications. The enzymatic approaches for glycosylation of bioactive molecules have been well developed; however, the enzymatic rhamnosylation has been largely hindered by lacking of the glycosyl donor for rhamnosyltransferases. Here, we employed an α-L-rhamnosidase from Alternaria sp. L1 (RhaL1) to perform one-step rhamnosylation of anticancer drugs, including 2'-deoxy-5-fluorouridine (FUDR), cytosine arabinoside (Ara C), and hydroxyurea (Hydrea). The key synthesis conditions including substrate concentrations and reaction time were carefully optimized, and the maximum yields of each rhamnosylated drugs were 57.7 mmol for rhamnosylated Ara C, 68.6 mmol for rhamnosylated Hydrea, and 42.2 mmol for rhamnosylated FUDR. It is worth pointing out that these rhamnosylated drugs exhibit little cytotoxic effects on cancer cells, but could efficiently restore cytotoxic activity when incubated with exogenous α-L-rhamnosidase, suggesting their potential applications in the enzyme-activated prodrug system. To evaluate the cancer-targeting ability of rhamnose moiety, the rhamnose-conjugated fluorescence dye rhodamine B (Rha-RhB) was constructed. The fluorescence probe Rha-RhB displayed much higher cell affinity and cellular internalization rate of oral cancer cell KB and breast cancer cell MDA-MB-231 than that of the normal epithelial cells MCF 10A, suggesting that the rhamnose moiety could mediate the specific internalization of rhamnosylated compounds into cancer cells, which greatly facilitated their applications for cancer-targeting drug delivery.

Discovery of N-(4-fluoro-2-(phenylamino)phenyl)-pyrazole-4-carboxamides as potential succinate dehydrogenase inhibitors.

Succinate dehydrogenase (SDH), an essential component of cellular respiratory chain and tricarboxylic acid (or Krebs) cycle, has been identified as one of the most significant targets for pharmaceutical and agrochemical. Herein, with the aim of discovery of new antifungal lead structures, a class of novel N-(4-fluoro-2-(phenylamino)phenyl)-pyrazole-4-carboxamides were designed, synthesized and evaluated for their biological activities. They were bioassayed against seven phytopathogenic fungi, Rhizoctonia solani, Phytophthora infestans, Fusarium oxysporum f. sp. vasinfectum, Botryosphaeria dothidea, Gibberella zeae, Alternaria alternate and Fusarium oxysporum f. sp. niveum. The results indicated that most of the compounds displayed good antifungal activities, especially against R. solani. Among them, compounds 7 and 12 exhibited higher antifungal activities against R. solani in vitro with EC50 value of 0.034 mg/L and 0.021 mg/L, being superior to the commercially available fungicide bixafen (EC50 = 0.043 mg/L). Pot tests against R. solani showed that in vivo EC50 values of compounds 7 (2.694 mg/L) and 12 (2.331 mg/L) were higher than that of bixafen (3.724 mg/L). In addition, inhibitory activity of compound 12 against SDH indicated compound 12 (IC50 = 1.836 mg/L) showed good inhibitory activity against SDH, being close to bixafen's inhibitory activity (IC50 = 1.222 mg/L). And, molecular modeling of the SDH-compound 12 complex suggested that compound 12 could strongly bind to and interact with the binding site of the SDH. The results of the present work showed that N-(4-fluoro-2-(phenylamino)phenyl)-pyrazole-4-carboxamides were a new fungicides for discovery of SDH inhibitors and worth further study.

Transcriptome Profiles of Alternaria oxytropis Provides Insights into Swainsonine Biosynthesis.

Swainsonine (SW) is a toxic alkaloid biosynthesized by the endophytic fungus Alternaria oxytropis in Oxytropis glabra. The biosynthetic pathway of SW is poorly understood. Saccharopine reductase/dehydrogenase of fungus plays an important role in this pathway. The gene knocked out mutant M1 in A. oxytropis was constructed in our previous work. In this study, the transcriptome of wild-strain OW7.8 and M1 was firstly sequenced to understand the biosynthetic pathway and molecular mechanism of SW in A. oxytropis. A total of 45,634 Unigenes were annotated. 5 genes were up-regulated and 11,213 genes were down-regulated. 41 Unigenes possibly related to the biosynthesis of SW were identified by data analyzing. The biosynthesis pathway of SW in the fungus was speculated, including two branches of P6C and P2C. Delta1-piperidine-2-carboxylate reductase, lysine 6-dehydrogenase, and saccharopine oxidase/L-pipecolate oxidase were involved in P6C. 1-piperidine-2-carboxylate/1-pyrroline-2- carboxylate reductase [NAD(P)H] and delta1-piperidine-2-carboxylate reductase were involved in P2C. Saccharopine reductase was involved in both. In addition, 1-indolizidineone was considered to be the direct precursor in the synthesis of SW, and the hydroxymethylglutaryl-CoA lyase catalyzed the synthesis of SW. Here we analyzed details of the metabolic pathway of A. oxytropis SW, which is of great significance for the follow-up research.

Enzymatic Production and Enzymatic-Mass Spectrometric Fingerprinting Analysis of Chitosan Polymers with Different Nonrandom Patterns of Acetylation.

Chitosans, a family of ß-(1,4)-linked, partially N-acetylated polyglucosamines, are considered to be among the most versatile and most promising functional biopolymers. Chemical analysis and bioactivity studies revealed that the functionalities of chitosans strongly depend on the polymers' degree of polymerization and fraction of acetylation. More recently, the pattern of acetylation ( PA) has been proposed as another important parameter to influence functionalities of chitosans. We therefore carried out studies on the acetylation pattern of chitosan polymers produced by three recombinant fungal chitin deacetylases (CDAs) originating from different species, namely, Podospora anserina, Puccinia graminis f. sp. tritici, and Pestalotiopsis sp. We analyzed the chitosans by 1H NMR, 13C NMR, and SEC-MALS and established new methods for PA analysis based on enzymatic mass spectrometric fingerprinting and in silico simulations. Our studies strongly indicate that the different CDAs indeed produce chitosans with different PA. Finally, Zimm plot analysis revealed that enzymatically treated polymers differ with respect to their second virial coefficient and radius of gyration indicating an influence of PA on polymer-solvent interactions.

Alternaria-induced barrier dysfunction of nasal epithelial cells: role of serine protease and reactive oxygen species.

BACKGROUND: Upper airway barrier dysfunction has been associated with chronic rhinosinusitis and allergic rhinitis. Alternaria is commonly found in nasal secretion and plays a role in the pathogenesis of airway diseases. The aim of this study was to investigate the effects of Alternaria on the junctional complex of nasal epithelial cells. METHODS: Air-liquid interface nasal epithelial cultures from the inferior turbinate of septal surgery patients were stimulated with Alternaria alternate. Production of intracellular reactive oxygen species (ROS) and transepithelial resistance (TER) was measured. The expression of tight junction (TJ) and adherens junction (AJ) molecules was determined using real-time reverse transcriptase-polymerase chain reaction, Western blot analysis, and confocal microscopy. Protease activity in Alternaria was determined using protease inhibitors and heat inactivation. RESULTS: Alternaria enhanced the production of ROS and reduced the TER. Alternaria decreased the messenger RNA and protein expression of TJs (zonula occludens-1, occludin, and claudin-1), but did not influence the AJ molecule. When Alternaria was pretreated with serine protease inhibitor and heat inactivation, ROS, TER, and TJ molecule expression returned to their nonstimulated levels. CONCLUSION: Serine protease in Alternaria altered nasal epithelial barrier function. Intracellular ROS induced by Alternaria may influence the barrier function of nasal epithelial cells and enhance the inflammatory process of nasal mucosa.

Population structure and virulence analysis of Alternaria carthami isolates of India using ISSR and SSR markers.

Alternaria leaf spot caused by Alternaria carthami is one of the most devastating diseases of safflower. Diversity among 95 isolates of A. carthami was determined using virulence assays, enzyme assays, dominant (ISSR) and co-dominant (SSR) markers. Collections and isolations were made from three major safflower producing states of India. The virulence assays categorised the population into four groups based on level of virulence. Estimation of activities of cell wall degrading enzymes (CWDE) yielded concurrent results to virulence assays with maximum CWDE activities in most virulent group. Eighteen ISSR primers were used and 23 polymorphic microsatellite markers were developed to assess the genetic diversity and determine the population structure of A. carthami. Analysis of ISSR profiles revealed high genetic diversity (Nei's Genetic diversity index; h = 0.36). Microsatellite markers produced a total of 56 alleles with an average of 2.43 alleles per microsatellite marker and Nei's genetic diversity index as h = 0.43. Unweighted Neighbor-joining and population structure analysis using both the marker systems differently arranged the isolates into three clusters. Distance analysis of the marker profiles provided no evidence for geographical clustering of isolates, indicating that isolates are randomly spread across India, signifying high potential of the fungus to adapt to diverse regions. Microsatellite markers clustered the isolates in consonance to the virulence groups in the dendrogram. This implies that the fungus has a high potential to adapt to resistant cultivars or fungicides. The information can aid in the breeding and deployment of A. carthami resistant varieties, and in early blight disease management in all safflower growing regions of the world.

Thermodynamics enhancement of Alternaria tenuissima KM651985 laccase by covalent coupling to polysaccharides and its applications.

This study full filed in enhancement of catalytic, thermodynamics and storage stability of Alternaria tenuissima KM651985 laccase by conjugation to sodium periodate oxidized starch. The starch conjugated A. tenuissima KM651985 laccase was active over a wide range of temperatures and pHs with the highest activity at 60 °C and 4, respectively. The thermal stability of conjugated A. tenuissima KM651985 laccase was indicated by, high T1/2 values (half life) 1076.16, 382.42 and 191.23 min at 50, 60 and 70 °C, respectively, low Kd (denaturation rate constant) 6.44 × 10-4, 18.13 × 10-4 and 36.25 × 10-4 min-1 at the same temperatures, high D-values (decimal reduction time) 3575.56, 1270.61 and 635.38 min at the same temperatures. Also, the thermal stability of conjugated A. tenuissima KM651985 laccase was emphasized by high ΔHd (enthalpy), high ΔGd (free energy) and low ΔSd (entropy). The conjugated A. tenuissima KM651985 laccase showed high effectiveness in dyes decolorization of Remazol Brilliant Blue R (RBBR) and Malachite Green (MG). Moreover, the addition of conjugated A. tenuissima KM651985 laccase with hemicellulolytic enzymes cocktail improved the saccharification of corn cobs, rice straw, corn cobs leaves and water hyacinth with the highest reducing sugar production 847.44 ±â€¯19.17 mg from corn cops.

Purification and characterization of an intracellular α-l-rhamnosidase from a newly isolated strain, Alternaria alternata SK37.001.

A strain, Alternaria alternata SK37.001, which produces an intracellular α-l-rhamnosidase, was newly isolated from citrus orchard soil. The molecular mass of the enzyme was 66 kDa, as evaluated by SDS-PAGE and 135 kDa, as determined by gel filtration, which indicated that the enzyme is a dimer. The enzyme had a specific activity of 21.7 U mg-1 after step-by-step purification. The optimal pH and temperature were 5.5 and 60 °C, respectively. The enzyme was relatively stable at a pH of 4.0-8.0 and a temperature between 30 and 50 °C compared with other pH levels and temperatures investigated. The enzyme activity was accelerated by Ba2+ and Al3+ but inhibited by Ni2+, Cu2+ and Co2+, especially Ni2+. The kinetic parameters of Km and Vmax were 4.84 mM and 53.1 µmol mg-1 min-1, respectively. The α-l-rhamnosidase could hydrolyze quercitrin, naringin and neohesperidin, hesperidin and rutin rhamnose-containing glycosides but could not hydrolyze ginsenoside Rg2 or saiko-saponin C.

Secreted protein extract analyses present the plant pathogen Alternaria alternata as a suitable industrial enzyme toolbox.

Lignocellulosic plant biomass is the most abundant carbon source in the planet, which makes it a potential substrate for biorefinery. It consists of polysaccharides and other molecules with applications in pharmaceutical, food and feed, cosmetics, paper and textile industries. The exploitation of these resources requires the hydrolysis of the plant cell wall, which is a complex process. Aiming to discover novel fungal natural isolates with lignocellulolytic capacities, a screening for feruloyl esterase activity was performed in samples taken from different metal surfaces. An extracellular enzyme extract from the most promising candidate, the natural isolate Alternaria alternata PDA1, was analyzed. The feruloyl esterase activity of the enzyme extract was characterized, determining the pH and temperature optima (pH 5.0 and 55-60 °C, respectively), thermal stability and kinetic parameters, among others. Proteomic analyses derived from two-dimensional gels allowed the identification and classification of 97 protein spots from the extracellular proteome. Most of the identified proteins belonged to the carbohydrates metabolism group, particularly plant cell wall degradation. Enzymatic activities of the identified proteins (ß-glucosidase, cellobiohydrolase, endoglucanase, ß-xylosidase and xylanase) of the extract were also measured. These findings confirm A. alternata PDA1 as a promising lignocellulolytic enzyme producer. SIGNIFICANCE: Although plant biomass is an abundant material that can be potentially utilized by several industries, the effective hydrolysis of the recalcitrant plant cell wall is not a straightforward process. As this hydrolysis occurs in nature relying almost solely on microbial enzymatic systems, it is reasonable to infer that further studies on lignocellulolytic enzymes will discover new sustainable industrial solutions. The results included in this paper provide a promising fungal candidate for biotechnological processes to obtain added value from plant byproducts and analogous substrates. Moreover, the proteomic analysis of the secretome of a natural isolate of Alternaria sp. grown in the presence of one of the most used vegetal substrates on the biofuels industry (sugar beet pulp) sheds light on the extracellular enzymatic machinery of this fungal plant pathogen, and can be potentially applied to developing new industrial enzymatic tools. This work is, to our knowledge, the first to analyze in depth the secreted enzyme extract of the plant pathogen Alternaria when grown on a lignocellulosic substrate, identifying its proteins by means of MALDI-TOF/TOF mass spectrometry and characterizing its feruloyl esterase, cellulase and xylanolytic activities.

Genome-Wide Analysis of Microsatellites in Alternaria arborescens and Elucidation of the Function of Polyketide Synthase (PksJ).

Microsatellites or simple sequence repeats (SSRs) have been the most widely applied class of molecular markers used in genetic studies, having applications in genetic conservation, population studies, as well as diagnostics of fungi. Mining and analysis of SSRs of the whole genome sequence have been carried out in this study for the fungus Alternaria arborescens causing early blight of tomato and well known for producing mycotoxins like alternariol (AOH), alternariol monomethyl ether (AME), etc. A total of 4097 microsatellites were identified in A. Arborescens genome. Contig 1 was identified as the most SSR-rich region which was further analyzed to correlate the presence of SSRs with different biological processes. A total of 246 putative genes were predicted in this study and KEGG pathway analysis of 155 predicted genes indicated that SSRs can be linked with important metabolic pathways, molecular functioning, signal transduction, and cellular processes. The prediction of fungal mycotoxin inducer gene Polyketide synthase (PksJ) linked with SSR in this study may be a potential candidate participating in oncogenic signal transduction in human. Our study is the first report of PksJ gene in A. arborescens, a precursor of AOH and AME.

A Destructive Leaf Spot and Blight Caused by Alternaria kareliniae sp. nov. on a Sand-Stabilizing Plant, Caspian Sea Karelinia.

Leaf spots and stem lesions causing widespread mortality of Caspian Sea karelinia (Karelinia caspia) were observed in desert regions of Xinjiang Uyghur Autonomous Region, China. Fifteen samples were collected from five widely distributed counties of Tarim and Junggar Basins in 2016. The pathogen was identified using morphological observations and phylogenetic analyses based on combined partial sequences from seven genes (Alt a 1, ATPase, calmodulin, glyceraldehyde 3-phosphate dehydrogenase, internal transcribed spacer, RNA polymerase II, and translation elongation factor 1), and placed as a new species: Alternaria kareliniae sp. nov. in section Dianthicola. The fungus has a small conidium (24.3 to) 29.1 to 64.8 (to 75.8) by (9.3 to) 12.4 to 16.5 (to 21.7) µm with a long beak (130 to) 183.9 to 350.4 (to 378.2) µm, as well as four to eight transverse septa, which differs significantly from other species of Alternaria section Dianthicola. On potato carrot agar, it grew significantly more slowly than others of this section. Pathogenicity tests showed that the fungus could infect leaves and stems of K. caspia and cause the same symptoms as those observed in the field. The fungus was reisolated from inoculated leaves and stems of the host. The disease in desert regions appears to be increasing, and it may have future negative implications for desert ecology in these areas. Future research should concentrate on elucidating the disease cycle and disease management alternatives.

Transcriptomic profiling of Alternaria longipes invasion in tobacco reveals pathogenesis regulated by AlHK1, a group III histidine kinase.

Tobacco brown spot, caused by Alternaria species, is a devastating tobacco disease. To explore the role of a group III histidine kinase (AlHK1) on A. longipes pathogenesis, the invasion progress of A. longipes was monitored. We found that the wild-type strain C-00 invaded faster than the AlHK1-disrupted strain HK∆4 in the early and middle infection stages and the reverse trend occurred in the late infection stage. Then, eight invasion transcriptomes were performed using RNA-Seq and 205 shared, 505 C-00 and 222 HK∆4 specific differentially expressed genes (DEGs) were identified. The annotation results showed seven antioxidant activity genes were specifically identified in the HKΔ4 DEGs. A subsequent experiment confirmed that HKΔ4 was more resistant to low concentrations oxidative stress than C-00. In addition, the results from 1) statistics for the number of DEGs, GO enriched terms, DEGs in clusters with rising trends, and 2) analyses of the expression patterns of some DEGs relevant for osmoadaptation and virulence showed that changes in C-00 infection existed mainly in the early and middle stages, while HKΔ4 infection arose mainly in the late stage. Our results reveal firstly the pathogenesis of A. longipes regulated by AlHK1 and provide useful insights into the fungal-plant interactions.

Chitinosanase: A fungal chitosan hydrolyzing enzyme with a new and unusually specific cleavage pattern.

The biological activities of partially acetylated chitosan oligosaccharides (paCOS) depend on their degree of polymerization (DP), fraction of acetylation (FA), and potentially their pattern of acetylation (PA). Therefore, analyzing structure-function relationships require fully defined paCOS, but these are currently unavailable. A promising approach for obtaining at least partially defined paCOS is using chitosanolytic enzymes. Here we purified and characterized a novel chitosan-hydrolyzing enzyme from the fungus Alternaria alternata possessing an absolute cleavage specificity, yielding fully defined paCOS. It cleaves specifically after GlcN-GlcNAc pairs and is most active towards moderately acetylated chitosans, but shows no activity against fully acetylated or fully deacetylated substrates. These unique properties match neither those of chitinases nor chitosanases. Therefore, the enzyme represents the first member of a new class of chitosanolytic enzymes that will allow for the production of fully defined paCOS. Additionally, it represents a highly valuable tool for fingerprinting analyses of chitosan polymers.

Establishment of CRISPR/Cas9 in Alternaria alternata.

The filamentous fungus Alternaria alternata is a potent producer of many secondary metabolites, some of which like alternariol or alternariol-methyl ether are toxic and/or cancerogenic. Many Alternaria species do not only cause post-harvest losses of food and feed, but are aggressive plant pathogens. Despite the great economic importance and the large number of research groups working with the fungus, the molecular toolbox is rather underdeveloped. Gene deletions often result in heterokaryotic strains and therefore, gene-function analyses are rather tedious. In addition, A. alternata lacks a sexual cycle and classical genetic approaches cannot be combined with molecular biological methods. Here, we show that CRISPR/Cas9 can be efficiently used for gene inactivation. Two genes of the melanin biosynthesis pathway, pksA and brm2, were chosen as targets. Several white mutants were obtained after several rounds of strain purification through protoplast regeneration or spore inoculation. Mutation of the genes was due to deletions from 1bp to 1.5kbp. The CRISPR/Cas9 system was also used to inactivate the orotidine-5-phosphate decarboxylase gene pyrG to create a uracil-auxotrophic strain. The strain was counter-selected with fluor-orotic acid and could be re-transformed with pyrG from Aspergillus fumigatus and pyr-4 from Neurospora crassa. In order to test the functioning of GFP, the fluorescent protein was fused to a nuclear localization signal derived from the StuA transcription factor of Aspergillus nidulans. After transformation bright nuclei were visible.