Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. CONCLUSION: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Detection of Maize Mold Based on a Nanocomposite Colorimetric Sensor Array under Different Substrates.

This study developed a novel nanocomposite colorimetric sensor array (CSA) to distinguish between fresh and moldy maize. First, the headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC/MS) method was used to analyze volatile organic compounds (VOCs) in fresh and moldy maize samples. Then, principal component analysis and orthogonal partial least-squares discriminant analysis (OPLS-DA) were used to identify 2-methylbutyric acid and undecane as key VOCs associated with moldy maize. Furthermore, colorimetric sensitive dyes modified with different nanoparticles were employed to enhance the dye properties used in the nanocomposite CSA analysis of key VOCs. This study focused on synthesizing four types of nanoparticles: polystyrene acrylic (PSA), porous silica nanospheres (PSNs), zeolitic imidazolate framework-8 (ZIF-8), and ZIF-8 after etching. Additionally, three types of substrates, qualitative filter paper, polyvinylidene fluoride film, and thin-layer chromatography silica gel, were comparatively used to fabricate nanocomposite CSA combining with linear discriminant analysis (LDA) and K-nearest neighbor (KNN) models for real sample detection. All moldy maize samples were correctly identified and prepared to characterize the properties of the CSA. Through initial testing and nanoenhancement of the chosen dyes, four nanocomposite colorimetric sensitive dyes were confirmed. The accuracy rates for LDA and KNN models in this study reached 100%. This work shows great potential for grain quality control using CSA methods.

The fungal pathogen Ustilago maydis targets the maize corepressor RELK2 to modulate host transcription for tumorigenesis.

Ustilago maydis is a biotrophic fungus that causes tumor formation on all aerial parts of maize. U. maydis secretes effector proteins during penetration and colonization to successfully overcome the plant immune response and reprogram host physiology to promote infection. In this study, we functionally characterized the U. maydis effector protein Topless (TPL) interacting protein 6 (Tip6). We found that Tip6 interacts with the N-terminus of RELK2 through its two Ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs. We show that the EAR motifs are essential for the virulence function of Tip6 and critical for altering the nuclear distribution pattern of RELK2. We propose that Tip6 mimics the recruitment of RELK2 by plant repressor proteins, thus disrupting host transcriptional regulation. We show that a large group of AP2/ERF B1 subfamily transcription factors are misregulated in the presence of Tip6. Our study suggests a regulatory mechanism where the U. maydis effector Tip6 utilizes repressive domains to recruit the corepressor RELK2 to disrupt the transcriptional networks of the host plant.

A conserved extracellular Ribo1 with broad-spectrum cytotoxic activity enables smut fungi to compete with host-associated bacteria.

Ribotoxins are secreted ribonucleases that specifically target and cleave the universally conserved sarcin-ricin loop sequence of rRNA, which leads to inhibition of protein biosynthesis and subsequently to cell death. We have identified and characterized a secreted Ribo1 protein of plant pathogenic smut fungi. Heterologous expression in different model systems showed that smut Ribo1 has cytotoxic activity against bacteria, yeast, host and nonhost plants. Recombinant expression of Ribo1 in Nicotiana benthamiana induced plant cell death; however, an active site mutant induced cell death only when expressed as a secreted protein. In the maize smut Ustilago maydis, transcription of Ribo1 is specifically induced in early infection stages. While a knockout mutant revealed that Ribo1 is dispensable for U. maydis virulence, the overexpression of Ribo1 in planta had a strong dominant negative effect on virulence and induced host defense responses including cell death. Our findings suggest a function of Ribo1 during the epiphytic development rather than for invasive colonization of the host. Accordingly, in the presence of the biocontrol bacteria Pantoea sp., which were isolated from maize leaves, the ribo1 knockout mutant was significantly impaired in virulence. Together, we conclude that Ribo1 enables smut fungi to compete with host-associated bacteria during epiphytic development.

Enhanced Physiological and Biochemical Performance of Mung Bean and Maize under Saline and Heavy Metal Stress through Application of Endophytic Fungal Strain SL3 and Exogenous IAA.

Modern irrigation practices and industrial pollution can contribute to the simultaneous occurrence of salinity and heavy metal contamination in large areas of the world, resulting in significant negative effects on crop productivity and sustainability. This study aimed to investigate the growth-promoting potentials of an important endophytic fungal strain SL3 and to compare its potential with exogenous IAA (indole-3-acetic acid) in the context of salt and heavy metal stress. The strain was assessed for plant growth-promoting traits such as the production of indole-3-acetic acid, gibberellins (GA), and siderophore. We selected two important crops, mung bean and maize, and examined various physiological and biochemical characteristics under 300 mM NaCl and 2.5 mM Pb stress conditions, with and without the application of IAA and SL3. This study's results demonstrated that both IAA and SL3 positively impacted the growth and development of plants under normal and stressed conditions. In NaCl and Pb-induced stress conditions, the growth of mung bean and maize plants was significantly reduced. However, the application of IAA and SL3 helped to alleviate stress, leading to a significant increase in shoot/root length and weight compared to IAA and SL3 non-treated plants. The results revealed that photosynthetic pigments, accumulation of catalase (CAT), phenolic contents, polyphenol oxidase, and flavanols are higher in the IAA and SL3-treated plants than in the non-inoculated plants. This study's findings revealed that applying the SL3 fungal strain positively influenced various physiological and biochemical processes in tested plant species under normal and stress conditions of NaCl and Pb. These findings also suggested that SL3 could be a potential replacement for widely used IAA to promote plant growth by improving photosynthetic efficiency, reducing oxidative stress, and enhancing metabolic activities in plants, including mung and maize. Moreover, this study highlights that SL3 has synergistic effects with IAA in enhancing resilience to salt and heavy stress and offers a promising avenue for future agricultural applications in salt and heavy metal-affected regions.

Abundance and distribution of arbuscular mycorrhizal fungi associated with oil-yielding plants.

Due to their role in nutrient transmission, arbuscular mycorrhizal fungi (AMF) are widespread plant root symbionts. They may improve plant production by altering plant community structure and function. Therefore, a study was conducted in the state of Haryana to analyze the distribution pattern, diversity, and association of different AMF species with oil-yielding plants. The results of the study revealed the percentage of root colonization, sporulation, and diversity of fungal species associated with the selected 30 oil-yielding plants. The percentage root colonization ranged from 0% to 100%, the highest in Helianthus annuus (100.00 ± 0.00) and Zea mays (100.00 ± 0.00) and the least in Citrus aurantium (11.87 ± 1.43). At the same time, there was no root colonization in the Brassicaceae family. The number of AMF spores present in 50 g of soil samples varied from 17.41 ± 5.28 to 497.2 ± 8.38, with maximum spore population in Glycine max (497.2 ± 8.38) and minimum in Brassica napus (17.41 ± 5.28). Besides, the presence of several species of different genera of AMF was reported in all the studied oil-yielding plants, that is, 60 AMF belonging to six genera viz. Acaulospora, Entrophospora, Glomus, Gigaspora, Sclerocystis, and Scutellospora were observed. Overall, this study will promote AMF usage in oil-yielding plants.

Toxicogenic fungal profile, Ochratoxin A exposure and cancer risk characterization through maize (Zea mays) consumed by different age populations in the Volta region of Ghana.

Maize (Zea mays) is an important staple food crop for the majority of Ghanaians. Maize is mostly contaminated by fungal species and particularly mycotoxins. This work aimed to identify and quantify the incidence of fungal infection and exposure to Ochratoxin A (OTA) as well as the health risk characterization in different age populations due to maize consumption in the Volta region. Maize samples were plated on Dichloran Rose Bengal Chloramphenicol (DRBC) agar, and Oxytetracycline Glucose Yeast Extract (OGYE) agar. All media were prepared in accordance with the manufacturers' instructions. The plates were incubated at 28 ± 2 °C for 5-7 days. High-Performance Liquid Chromatography connected to a fluorescence detector (HPLC-FLD) was used to analyze the ochratoxin A (OTA) levels in maize. Cancer risk assessments were also conducted using models prescribed by the Joint FAO/WHO Expert Committee on Additives (JECFA). The maize samples collected from the Volta region contained fungal population between the range of 3.08-4.58 log10 CFU/g. Eight (8) genera were recorded belonging to Aspergillus, Trichoderma, Penicillium, Fusarium, Saccharomyces, Mucor, Rhodotorula and Rhizopus. The species diversity includes A. flavus, A. niger, T. harzianum, P. verrucosum, F. oxysporum, Yeast, F. verticillioides, Rhodotorulla sp, A. fumigatus, R. stolonifer, M. racemosus species. Additionally, the ochratoxins level contained in the samples were very noteworthy and ranged from 1.22 to 28.17 µg/kg. Cancer risk assessments of OTA produced outcomes also ranged between 2.15 and 524.54 ng/kg bw/day, 0.03-8.31, 0.0323, and 0.07-16.94 for cases/100,000 person/yr for Estimated Daily Intake (EDI), Margin of Exposure (MOE), Average Potency, and Cancer Risks respectively for all age categories investigated. There was very high mycoflora load on the maize sampled from the Volta region, likewise the range of mycotoxins present in the maize grains, suggesting the potential to pose some adverse health effects with the populace of the Volta region.

Maize Antifungal Protein AFP1 Elevates Fungal Chitin Levels by Targeting Chitin Deacetylases and Other Glycoproteins.

Pathogenic fungi convert chitin to chitosan to evade plant perception and disarm chitin-triggered immune responses. Whether plants have evolved factors to counteract this evasion mechanism remains obscure. Here, we decipher the mechanism underlying the antifungal activity of maize secretory mannose-binding cysteine-rich receptor-like secreted protein (CRRSP), antifungal protein 1 (AFP1). AFP1 binds to multiple sites on the surface of sporidial cells, filaments, and germinated spores of the biotrophic fungus Ustilago maydis. It inhibits cell growth and budding, as well as spore germination. AFP1 promiscuously interacts with most chitin deacetylases (CDAs) by recognizing the conserved NodB domain to interfere with the enzyme activity. Deletion of O-mannosyltransferase 4 decreases protein mannosylation, which correlates with reduced AFP1 binding and antifungal activity, suggesting that AFP1 interacts with mannosylated proteins to exhibit an inhibitory effect. AFP1 also has extended inhibitory activity against Saccharomyces cerevisiae; however, AFP1 did not reduce binding to the double ΔΔcda1,2 mutant, suggesting the targets of AFP1 have expanded to other cell surface glycoproteins, probably facilitated by its mannose-binding property. Increasing chitin levels by modulating the activity of cell surface glycoproteins is a universal feature of AFP1 interacting with a broad spectrum of fungi to inhibit their growth. IMPORTANCE Plants alert immune systems by recognizing the fungal pathogen cell wall component chitin via pattern recognition cell surface receptors. Successful fungal pathogens escape the perception by deacetylating chitin to chitosan, which is also necessary for fungal cell development and virulence. Targeting glycoproteins that are associated with regulating chitin metabolism and maintaining cell wall morphogenesis presents an effective strategy to combat fungal pathogens by simultaneously altering cell wall plasticity, activating chitin-triggered immunity, and impairing fungal viability. Our study provides molecular insights into a plant DUF26 domain-containing secretory protein in warding off a broad range of fungal pathogens by acting on more than one glycoprotein target.

Chemical Constituents and Bioactive Principles from the Mexican Truffle and Fermented Products of the Derived Fungus Ustilago maydis MZ496986.

To look in-depth into the traditional Mexican truffle, this study investigated the phytochemical and pharmacological properties of field-collected corn galls and the fermentate of its pathogen Ustilago maydis MZ496986. Here, we established the chemical profiles of both materials via the gradient HPLC-UV method and successfully identified six previously unreported chemical entities, ustilagols A-F (1-6), and 17 known components. Compounds 3, 5, and 9 exhibited potent nitric oxide production inhibitory activities in murine brain microglial BV-2 cells (IC50 = 6.7 ± 0.5, 5.8 ± 0.9, and 3.9 ± 0.1 µM) without cytotoxic effects. DIMBOA (9) also attenuates lipopolysaccharide (LPS)-stimulated NF-κB activation in RAW 264.7 macrophages (IC50 = 58.1 ± 7.2 µM). Ustilagol G (7) showed potent antiplatelet aggregation in U46619-stimulated human platelets (IC50 = 16.5 ± 5.3 µM). These findings highlighted the potential of corn galls and U. maydis MZ496986 fermentate as functional foods for improving inflammation-related discomforts and vascular obstruction.

Plant-based natural flavonoids show strong inhibition of aflatoxin production and related gene expressions correlated with chemical structure.

Aflatoxins are strong carcinogenic and mutagenic fungal metabolites, and aflatoxin contamination is a critical issue in agriculture and food production. Natural flavonoids can suppress aflatoxin biosynthesis; however, the structure-activity relationship remains unclear. In the present study, a total of 36 structurally related natural flavonoids were tested against the aflatoxigenic Aspergillus flavus, both in-vitro and in-situ (on maize kernels), to investigate their structure-activity relationship and biological activity. Aflatoxin production (IC50 values: 10.85-20.09 µg/mL) and the expression of related genes (aflD, aflK, aflQ, and aflR) were found to be strongly inhibited. Structure-activity relationship studies revealed that the [-OH] or [-O-CH3] groups at position 6 of ring A and position 4' of ring B were closely associated with antifungal and antiaflatoxigenic activities. These findings provide valuable information for the development of clean and safe methods to prevent aflatoxin contamination in food.

Efficacy of the antifungal metabolites of Streptomyces philanthi RL-1-178 on aflatoxin degradation with its application to prevent aflatoxigenic fungi in stored maize grains and identification of the bioactive compound.

Aflatoxin B1 is a potent carcinogen produced by Aspergillus flavus (A. flavus) and Aspergillus. parasiticus (A. parasiticus), mainly during grain storage. The efficacy of the freeze-dried culture filtrate of Streptomyces philanthi (S. philanthi) strain RL-1-178 (DCF) on degradation of aflatoxin B1 (AFB1) were evaluated and its bioactive compounds were identified. The DCF at a concentration of 9.0% (w/v) completely inhibited growth and AFB1 production of A. parasiticus TISTR 3276 and A. flavus PSRDC-4 after 7 days tested in yeast-extract sucrose (YES) medium and on stored maize grains after 28 and 14 days incubation, respectively. This indicated the more tolerance of A. parasiticus over A. flavus. The DCF and bacterial cells of S. philanthi were capable to degrade AFB1 by 85.0% and 100% for 72 h and 8 days, respectively. This confirmed the higher efficacy of the DCF over the cells. After separation of the DCF on thin-layer chromatography (TLC) plate by bioautography bioassay, each active band was identified by liquid chromatography-quadrupole time of flight mass spectrometer (LC-Q-TOF MS/MS). The results revealed two compounds which were identified as azithromycin and an unknown based on mass ions of both ESI+ and ESI- modes. The antifungal metabolites in the culture filtrate of S. philanthi were proved to degrade aflatoxin B1. It could be concluded that the DCF may be applied to prevent the growth of the two aflatoxin-producing fungi as well as the occurrence of aflatoxin in the stored maize grains.

Neobacillus rhizosphaerae sp. nov., isolated from the rhizosphere, and reclassification of Bacillus dielmonensis as Neobacillus dielmonensis comb. nov.

A Gram-stain-positive, facultative anaerobic endospore-forming bacterium, which originated from roots/rhizosphere of maize (Zea mays), was investigated for its taxonomic position. On the basis of 16S rRNA gene sequence similarities, strain JJ-3T was grouped together with Neobacillus species showing the highest similarities to Neobacillus bataviensis (98.8 %) and the three species Neobacillus dendrensis, Neobacillus soli and Neobacillus cucumis (all 98.6 %). The 16S rRNA gene sequence similarities to the sequences of the type strains of other Neobacillus species were lower than 98.5 %. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between the JJ-3T genome assembly and those of the other Neobacillus type strains were <83, <85 and <27 %, respectively. Chemotaxonomic features supported the grouping of the strain to the genus Neobacillus, e.g. the major fatty acids were C15 : 0 anteiso and C15 : 0 iso, the polar lipid profile contained the major components diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, and the major quinone was menaquinone MK-7. Physiological and biochemical test results were slightly different from those of the most closely related species. For this reason, JJ-3T represents a novel species of the genus Neobacillus, for which we propose the name Neobacillus rhizosphaerae sp. nov., with JJ-3T (= CIP 111895T=LMG 32087T=DSM 111784T=CCM 9084T) as the type strain. We also propose to reclassify Bacillus dielmonensis as Neobacillus dielmonensis comb. nov. based mainly on the results of phylogenomic and conserved signature indel analyses.

Comparative Analysis of Multiple GWAS Results Identifies Metabolic Pathways Associated with Resistance to A. flavus Infection and Aflatoxin Accumulation in Maize.

Aflatoxins are carcinogenic secondary metabolites produced by several species of Aspergillus, including Aspergillus flavus, an important ear rot pathogen in maize. Most commercial corn hybrids are susceptible to infection by A. flavus, and aflatoxin contaminated grain causes economic damage to farmers. The creation of inbred lines resistant to Aspergillus fungal infection or the accumulation of aflatoxins would be aided by knowing the pertinent alleles and metabolites associated with resistance in corn lines. Multiple Quantitative Trait Loci (QTL) and association mapping studies have uncovered several dozen potential genes, but each with a small effect on resistance. Metabolic pathway analysis, using the Pathway Association Study Tool (PAST), was performed on aflatoxin accumulation resistance using data from four Genome-wide Association Studies (GWAS). The present research compares the outputs of these pathway analyses and seeks common metabolic mechanisms underlying each. Genes, pathways, metabolites, and mechanisms highlighted here can contribute to improving phenotypic selection of resistant lines via measurement of more specific and highly heritable resistance-related traits and genetic gain via marker assisted or genomic selection with multiple SNPs linked to resistance-related pathways.

Aflatoxin Contamination of Maize, Groundnut, and Sorghum Grown in Burkina Faso, Mali, and Niger and Aflatoxin Exposure Assessment.

Aflatoxin contamination of staple crops by Aspergillus flavus and closely related fungi is common across the Sahel region of Africa. Aflatoxins in maize, groundnut, and sorghum collected at harvest or from farmers' stores within two weeks of harvest from Burkina Faso, Mali, and Niger were quantified. Thereafter, aflatoxin exposure values were assessed using per capita consumption rates of those crops. Mean aflatoxin concentrations in maize were high, 128, 517, and 659 µg/kg in Mali, Burkina Faso, and Niger, respectively. The estimated probable daily intake (PDI) of aflatoxins from maize ranged from 6 to 69, 29 to 432, and 310 to 2100 ng/kg bw/day in Mali, Burkina Faso, and Niger, respectively. Similarly, mean aflatoxin concentrations in sorghum were high, 76 and 259 µg/kg in Mali and Niger, respectively, with an estimated PDI of 2-133 and 706-2221. For groundnut, mean aflatoxin concentrations were 115, 277, and 628 µg/kg in Mali, Burkina Faso, and Niger, respectively. Aflatoxin exposure values were high with an estimated 9, 28, and 126 liver cancer cases/100,000 persons/year in Mali, Burkina Faso, and Niger, respectively. Several samples were extremely unsafe, exceeding manyfold regulatory levels of diverse countries (up to 2000 times more). Urgent attention is needed across the Sahel for integrated aflatoxin management for public health protection, food and nutrition security, and access to trade opportunities.

Asymmetrical lineage introgression and recombination in populations of Aspergillus flavus: Implications for biological control.

Aspergillus flavus is an agriculturally important fungus that causes ear rot of maize and produces aflatoxins, of which B1 is the most carcinogenic naturally-produced compound. In the US, the management of aflatoxins includes the deployment of biological control agents that comprise two nonaflatoxigenic A. flavus strains, either Afla-Guard (member of lineage IB) or AF36 (lineage IC). We used genotyping-by-sequencing to examine the influence of both biocontrol agents on native populations of A. flavus in cornfields in Texas, North Carolina, Arkansas, and Indiana. This study examined up to 27,529 single-nucleotide polymorphisms (SNPs) in a total of 815 A. flavus isolates, and 353 genome-wide haplotypes sampled before biocontrol application, three months after biocontrol application, and up to three years after initial application. Here, we report that the two distinct A. flavus evolutionary lineages IB and IC differ significantly in their frequency distributions across states. We provide evidence of increased unidirectional gene flow from lineage IB into IC, inferred to be due to the applied Afla-Guard biocontrol strain. Genetic exchange and recombination of biocontrol strains with native strains was detected in as little as three months after biocontrol application and up to one and three years later. There was limited inter-lineage migration in the untreated fields. These findings suggest that biocontrol products that include strains from lineage IB offer the greatest potential for sustained reductions in aflatoxin levels over several years. This knowledge has important implications for developing new biocontrol strategies.

Sutcliffiella rhizosphaerae sp. nov. isolated from roots.

An aerobic, Gram-staining-positive, endospore-forming bacterium, isolated from the rhizosphere of roots of maize (Zea mays), was taxonomically studied. On the basis of 16S rRNA gene sequence similarity comparisons, strain JJ-125T clustered together with species of the genus Sutcliffiella and showed the highest similarities with Sutcliffiella zhanjiangensis (98.7 %). The 16S rRNA gene sequence similarities to the sequences of the type strains of other species of the genus Sutcliffiella were <98.4 %. The genome sequence of JJ-125T was 4 516 360 bp long and had a DNA G+C content of 37.3 %. A DNA-DNA hybridization with the type strain of S. zhanjiangensis DSM 23010T resulted in values of 42.3 and 43.9 % (reciprocal). The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between the JJ-125T genome assembly and those of the other type strains of species of the genus Sutcliffiella were <75%, <80 % and <21 %, respectively. Chemotaxonomic features supported the grouping of the strain with the genus Sutcliffiella, e.g. the major fatty acids included iso-C15 : 0, iso-C17 : 1 ω10c and iso-C17 : 0, the polar lipid profile contained the major components diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, the only quinone was menaquinone MK-7 and the characteristic diamino acid was meso-diaminopimelic acid. Physiological and biochemical test results were also different from those of the most closely related species. As a consequence, JJ-125T represents a novel species of the genus Sutcliffiella, for which we propose the name Sutcliffiella rhizosphaerae sp. nov., with JJ-125T (= CIP 111883T = LMG 32156T = CCM 9046T) as the type strain.

Mycotoxin risks are lower in biotech corn.

Mycotoxins are food contaminants that occur when toxigenic fungi colonize crops. Unfortunately, corn, a major staple crop worldwide, is highly susceptible to mycotoxin contamination. Some mycotoxins, most notably aflatoxin, cause human cancer and other harmful effects such as immunotoxicity and growth impairment. Hence, many nations have set food-safety standards on mycotoxins. Aside from regulations, good agricultural and manufacturing practices lower mycotoxin risks. Agricultural biotechnology has made notable advances in reducing mycotoxins recently. While transgenic Bt corn has been known for years to reduce the mycotoxin fumonisin, new studies have shown its benefit in reducing aflatoxin as well. Other transgenic and RNA-interference corn hybrids target mycotoxin reduction specifically, and gene editing through clustered regularly interspaced short palindromic repeat systems has focused on preventing mycotoxin biosynthesis.

Transcriptome analysis of maize pathogen Fusarium verticillioides revealed FvLcp1, a secreted protein with type-D fungal LysM and chitin-binding domains, that plays important roles in pathogenesis and mycotoxin production.

Fusarium verticillioides is a key maize pathogen and produces fumonisins, a group of mycotoxins detrimental to humans and animals. Unfortunately, our understanding on how this fungus interacts with maize to trigger mycotoxin biosynthesis is limited. We performed a systematic computational network-based analysis of large-scale F. verticillioides RNA-seq datasets to identify gene subnetwork modules associated with virulence and fumonisin regulation. F. verticillioides was inoculated on two different maize lines, moderately resistant line hybrid 33K44 and highly susceptible line maize inbred line B73, to generate time-course RNA-Seq data. Among the highly discriminative subnetwork modules, we identified a putative hub gene FvLCP1, which encodes a putative a type-D fungal LysM protein with a signal peptide, three LysM domains, and two chitin binding domains. FvLcp1 is a unique protein that harbors these domains amongst five representative Fusarium species. FvLcp1 is a secreted protein important for fumonisin production with the LysM domain playing a critical role. The chitin-binding domain was essential for in vitro chitin binding. Using Magnaporthe oryzae, we learned that FvLcp1 accumulates in appressoria, suggesting that FvLcp1 is involved in host recognition and infection. Full length FvLcp1 suppressed BAX-triggered plant cell death in Nicotiana benthamiana. This unique type-D LysM secreted protein with a chitin-binding domain in F. verticillioides was shown to be potentially involved in suppressing host cell death and promoting fumonisin biosynthesis while the pathogen colonizes maize kernels.

Candidate Effector Proteins from the Maize Tar Spot Pathogen Phyllachora maydis Localize to Diverse Plant Cell Compartments.

Most fungal pathogens secrete effector proteins into host cells to modulate their immune responses, thereby promoting pathogenesis and fungal growth. One such fungal pathogen is the ascomycete Phyllachora maydis, which causes tar spot disease on leaves of maize (Zea mays). Sequencing of the P. maydis genome revealed 462 putatively secreted proteins, of which 40 contain expected effector-like sequence characteristics. However, the subcellular compartments targeted by P. maydis effector candidate (PmEC) proteins remain unknown, and it will be important to prioritize them for further functional characterization. To test the hypothesis that PmECs target diverse subcellular compartments, cellular locations of super yellow fluorescent protein-tagged PmEC proteins were identified using a Nicotiana benthamiana-based heterologous expression system. Immunoblot analyses showed that most of the PmEC-fluorescent protein fusions accumulated protein in N. benthamiana, indicating that the candidate effectors could be expressed in dicot leaf cells. Laser-scanning confocal microscopy of N. benthamiana epidermal cells revealed that most of the P. maydis putative effectors localized to the nucleus and cytosol. One candidate effector, PmEC01597, localized to multiple subcellular compartments including the nucleus, nucleolus, and plasma membrane, whereas an additional putative effector, PmEC03792, preferentially labelled both the nucleus and nucleolus. Intriguingly, one candidate effector, PmEC04573, consistently localized to the stroma of chloroplasts as well as stroma-containing tubules (stromules). Collectively, these data suggest that effector candidate proteins from P. maydis target diverse cellular organelles and could thus provide valuable insights into their putative functions, as well as host processes potentially manipulated by this fungal pathogen.

Organic acids and glucose prime late-stage fungal biotrophy in maize.

Many plant-associated fungi are obligate biotrophs that depend on living hosts to proliferate. However, little is known about the molecular basis of the biotrophic lifestyle, despite the impact of fungi on the environment and food security. In this work, we show that combinations of organic acids and glucose trigger phenotypes that are associated with the late stage of biotrophy for the maize pathogen Ustilago maydis. These phenotypes include the expression of a set of effectors normally observed only during biotrophic development, as well as the formation of melanin associated with sporulation in plant tumors. U. maydis and other hemibiotrophic fungi also respond to a combination of carbon sources with enhanced proliferation. Thus, the response to combinations of nutrients from the host may be a conserved feature of fungal biotrophy.