Comparative transcriptome profiling and co-expression network analysis uncover the key genes associated withearly-stage resistance to Aspergillus flavus in maize.

BACKGROUND: The fungus Aspergillus flavus (A. flavus) is a serious threat to maize (Zea mays) production worldwide. It causes considerable yield and economic losses, and poses a health risk to humans and livestock due to the high toxicity of aflatoxin. However, key genes and regulatory networks conferring maize resistance to A. flavus are not clear, especially at the early stage of infection. Here, we performed a comprehensive transcriptome analysis of two maize inbred lines with contrasting resistance to A. flavus infection. RESULTS: The pairwise comparisons between mock and infected kernels in each line during the first 6 h post inoculation (hpi) showed that maize resistance to A. flavus infection was specific to the genotype and infection stage, and defense pathways were strengthened in the resistant line. Further comparison of the two maize lines revealed that the infection-induced up-regulated differentially expressed genes (DEGs) in the resistant line might underlie the enhanced resistance. Gene co-expression network analysis by WGCNA (weighted gene co-expression network analysis) identified 7 modules that were significantly associated with different infection stages, and 110 hub genes of these modules. These key regulators mainly participate in the biosynthesis of fatty acid and antibiotics. In addition, 90 candidate genes for maize resistance to A. flavus infection and/or aflatoxin contamination obtained in previous studies were confirmed to be differentially expressed between the resistant and susceptible lines within the first 6 hpi. CONCLUSION: This work unveiled more A. flavus resistance genes and provided a detailed regulatory network of early-stage resistance to A. flavus in maize.

Comparative study of codon usage profiles of Zingiber officinale and its associated fungal pathogens.

Codon usage bias influences the genetic features prevalent in genomes of all the organisms. It also plays a crucial role in establishing the host-pathogen relationship. The present study elucidates the role of codon usage pattern regarding the predilection of fungal pathogens Aspergillus flavus, Aspergillus niger, Fusarium oxysporum and Colletotrichum gloeosporioides towards host plant Zingiber officinale. We found a similar trend of codon usage pattern operative in plant and fungal pathogens. This concurrence might be attributed for the colonization of fungal pathogens in Z. officinale. The transcriptome of both plant and pathogens showed bias towards GC-ending codons. Natural selection and mutational pressure seem to be accountable for shaping the codon usage pattern of host and pathogen. We also identified some distinctive preferred codons in A. flavus, F. oxysporum and Z. officinale that could be regarded as signature codons for the identification of these organisms. Knowledge of favored, avoided and unique codons will help to devise strategies for reducing spice losses due to fungal pathogens.

Proven Aspergillus flavus pulmonary aspergillosis in a COVID-19 patient: A case report and review of the literature.

Severe COVID-19 patients complicated with aspergillosis are increasingly reported. We present a histopathological proven case of fatal COVID-19-associated pulmonary aspergillosis (CAPA), due to Aspergillus flavus. This report and existing published literature indicate diagnostic challenges and poor outcomes of CAPA in ICU patients.

The AFLATOX® Project: Approaching the Development of New Generation, Natural-Based Compounds for the Containment of the Mycotoxigenic Phytopathogen Aspergillus flavus and Aflatoxin Contamination.

The control of the fungal contamination on crops is considered a priority by the sanitary authorities of an increasing number of countries, and this is also due to the fact that the geographic areas interested in mycotoxin outbreaks are widening. Among the different pre- and post-harvest strategies that may be applied to prevent fungal and/or aflatoxin contamination, fungicides still play a prominent role; however, despite of countless efforts, to date the problem of food and feed contamination remains unsolved, since the essential factors that affect aflatoxins production are various and hardly to handle as a whole. In this scenario, the exploitation of bioactive natural sources to obtain new agents presenting novel mechanisms of action may represent a successful strategy to minimize, at the same time, aflatoxin contamination and the use of toxic pesticides. The Aflatox® Project was aimed at the development of new-generation inhibitors of aflatoxigenic Aspergillus spp. proliferation and toxin production, through the modification of naturally occurring molecules: a panel of 177 compounds, belonging to the thiosemicarbazones class, have been synthesized and screened for their antifungal and anti-aflatoxigenic potential. The most effective compounds, selected as the best candidates as aflatoxin containment agents, were also evaluated in terms of cytotoxicity, genotoxicity and epi-genotoxicity to exclude potential harmful effect on the human health, the plants on which fungi grow and the whole ecosystem.

A Cell Wall Integrity-Related MAP Kinase Kinase Kinase AflBck1 Is Required for Growth and Virulence in Fungus Aspergillus flavus.

Aspergillus flavus represents an important fungal pathogen, causing severe economic losses in crops. The mitogen-activated protein (MAP) kinase signaling pathway contributes to many physiological processes, but its precise role in A. flavus is not yet fully understood. In this study, we focused on the AflBck1 gene, which encodes a MAP kinase kinase kinase of the Slt2-MAPK pathway. Targeted deletion of AflBck1 led to a significant defect in growth and development, and a AflBck1-deleted mutant (∆AflBck1) showed higher sensitivity to cell-wall stress than wild type (WT). Importantly, we observed that ∆AflBck1 displayed an enhanced ability to produce aflatoxin, a potential carcinogenic mycotoxin. However, the pathogenicity of the ∆AflBck1 mutant was markedly reduced in peanut seeds. We also presented evidence that AflBck1 was genetically epistatic to AflMkk2 in the Slt2-MAPK pathway. Finally, we found that loss of the proline-rich region at the N terminus of AflBck1 affected the reproduction of A. flavus. Collectively, this study not only extended the understanding that the MAPK pathway regulated A. flavus pathogenicity but also provided a possible strategy to control A. flavus contamination.

MAPK pathway-related tyrosine phosphatases regulate development, secondary metabolism and pathogenicity in fungus Aspergillus flavus.

Mitogen-activated protein kinase (MAPK) cascades are highly conserved in eukaryotic cells and are known to play crucial roles in the regulation of various cellular processes. However, compared with kinase-mediated phosphorylation, dephosphorylation catalysed by phosphatases has not been well characterized in filamentous fungi. In this study, we identified five MAPK pathway-related phosphatases (Msg5, Yvh1, Ptp1, Ptp2 and Oca2) and characterized their functions in Aspergillus flavus, which produces aflatoxin B1 (AFB1 ), one of the most toxic and carcinogenic secondary metabolites. These five phosphatases were identified as negative regulators of MAPK (Slt2, Fus3 and Hog1) pathways. Deletion of Msg5 and Yvh1 resulted in significant defects in conidiation, sclerotia formation, aflatoxin production and crop infection. Additionally, double knockout mutants (ΔMsg5/ΔPtp1, ΔMsg5/ΔPtp2 and ΔMsg5/ΔOca2) displayed similar defects to those observed in the ΔMsg5 single mutant, indicating that Msg5 plays a major role in the regulation of development and pathogenicity in A. flavus. Importantly, we found that the active site at C439 is essential for the function of the Msg5 phosphatase. Furthermore, the MAP kinase Fus3 was found to be involved in the regulation of development, aflatoxin biosynthesis and pathogenicity, and its conserved phosphorylation residues (Thr and Tyr) were critical for the full range of its functions in A. flavus. Overall, our results reveal that MAPK related tyrosine phosphatases play important roles in the regulation of development, secondary metabolism and pathogenicity in A. flavus, and could be developed as potential targets for preventing damage caused by this fungal pathogen.

Antioxidant-related catalase CTA1 regulates development, aflatoxin biosynthesis, and virulence in pathogenic fungus Aspergillus flavus.

Reactive oxygen species (ROS) induce the synthesis of a myriad of secondary metabolites, including aflatoxins. It raises significant concern as it is a potent environmental contaminant. In Aspergillus flavus., antioxidant enzymes link ROS stress response with coordinated gene regulation of aflatoxin biosynthesis. In this study, we characterized the function of a core component of the antioxidant enzyme catalase (CTA1) of A. flavus. Firstly, we verified the presence of cta1 corresponding protein (CTA1) by Western blot analysis and mass-spectrometry based analysis. Then, the functional study revealed that the growth, sporulation and sclerotia formation significantly increased, while aflatoxins production and virulence were decreased in the cta1 deletion mutant as compared with the WT and complementary strains. Furthermore, the absence of the cta1 gene resulted in a significant rise in the intracellular ROS level, which in turn added to the oxidative stress level of cells. A further quantitative proteomics investigation hinted that in vivo, CTA1 might maintain the ROS level to facilitate the aflatoxin synthesis. All in all, the pleiotropic phenotype of A. flavus CTA1 deletion mutant revealed that the antioxidant system plays a crucial role in fungal development, aflatoxins biosynthesis and virulence.

The role of the VosA-repressed dnjA gene in development and metabolism in Aspergillus species.

The DnaJ family of proteins (or J-proteins) are molecular chaperones that govern protein folding, degradation, and translocation in many organisms. Although J-proteins play key roles in eukaryotic and prokaryotic biology, the role of J-proteins in Aspergillus species is currently unknown. In this study, we characterized the dnjA gene, which encodes a putative DnaJ protein, in two Aspergillus species: Aspergillus nidulans and Aspergillus flavus. Expression of the dnjA gene is inhibited by the velvet regulator VosA, which plays a pivotal role in spore survival and metabolism in Aspergillus. The deletion of dnjA decreased the number of asexual spores (conidia), produced abnormal conidiophores, and reduced sexual fruiting bodies (cleistothecia) or sclerotia. In addition, the absence of dnjA caused increased sterigmatocystin or aflatoxin production in A. nidulans and A. flavus, respectively. These results suggest that DnjA plays a conserved role in asexual and sexual development and mycotoxin production in Aspergillus species. However, DnjA also plays a species-specific role; AniDnjA but not AflDnjA, affects conidial viability, trehalose contents, and thermal tolerance of conidia. In plant virulence assay, the infection ability of the ΔAfldnjA mutant decreased in the kernels, suggesting that DnjA plays a crucial role in the pathogenicity of A. flavus. Taken together, these results demonstrate that DnjA is multifunctional in Aspergillus species; it is involved in diverse biological processes, including fungal differentiation and secondary metabolism.

High-density SNP map facilitates fine mapping of QTLs and candidate genes discovery for Aspergillus flavus resistance in peanut (Arachis hypogaea).

KEY MESSAGE: Two novel resistant QTLs mapped and candidate genes identified for Aspergillus flavus resistance in cultivated peanut using SLAF-seq. Aflatoxin contamination in peanuts caused by Aspergillus flavus is a serious food safety issue for human health around the world. Host plant resistance to fungal infection and reduction in aflatoxin are crucial for mitigating this problem. Identification of the resistance-linked markers can be used in marker-assisted breeding for varietal development. Here we report construction of two high-density genetic linkage maps with 1975 SNP loci and 5022 SNP loci, respectively. Two consistent quantitative trait loci (QTL) were identified as qRAF-3-1 and qRAF-14-1, which located on chromosomes A03 and B04, respectively. QTL qRAF-3-1 was mapped within 1.67 cM and had more than 19% phenotypic variance explained (PVE), while qRAF-14-1 was located within 1.34 cM with 5.15% PVE. While comparing with the reference genome, the mapped QTLs, qRAF-3-1 and qRAF-14-1, were located within a physical distance of 1.44 Megabase pair (Mbp) and 2.22 Mbp, harboring 67 and 137 genes, respectively. Among the identified candidate genes, six genes with the same function were found within both QTLs regions. In addition, putative disease resistance RPP13-like protein 1 (RPP13), lipoxygenase (Lox), WRKY transcription factor (WRKY) and cytochrome P450 71B34 genes were also identified. Using microarray analysis, genes responded to A. flavus infection included coding for RPP13, pentatricopeptide repeat-containing-like protein, and Lox which may be possible candidate genes for resistance to A. flavus. The QTLs and candidate genes will further facilitate marker development and validation of genes for deployment in the molecular breeding programs against A. flavus in peanuts.

Aspergillosis in albatrosses.

Aspergillosis is a respiratory fungal disease of importance in captive marine birds. The aim of this study was to describe the occurrence of aspergillosis in Thalassarche melanophris during rehabilitation events and to identify the etiological agent. All the albatrosses that were received for rehabilitation and died within a 2-year period were included in the study. The proportionate mortality rate caused by aspergillosis was 21.4% (3/14). One of the etiological agents was Aspergillus flavus/oryzae lineage, and the other was A. fumigatus sensu stricto. Our study suggests that aspergillosis can act as a limiting factor in the rehabilitation of albatrosses.

Aspergillus flavus Exploits Maize Kernels Using an "Orphan" Secondary Metabolite Cluster.

Aspergillus flavus is a saprophytic cosmopolitan fungus, capable of infecting crops both pre- and post-harvest and exploiting different secondary metabolites, including aflatoxins. Aflatoxins are known carcinogens to animals and humans, but display no clear effect in host plants such as maize. In a previous study, we mined the genome of A. flavus to identify secondary metabolite clusters putatively involving the pathogenesis process in maize. We now focus on cluster 32, encoding for fungal effectors such as salicylate hydroxylase (SalOH), and necrosis- and ethylene-inducing proteins (npp1 domain protein) whose expression is triggered upon kernel contact. In order to understand the role of this genetic cluster in maize kernel infection, mutants of A. flavus, impaired or enhanced in specific functions (e.g., cluster 32 overexpression), were studied for their ability to cause disease. Within this frame, we conducted histological and histochemical experiments to verify the expression of specific genes within the cluster (e.g., SalOH, npp1), the production of salicylate, and the presence of its dehydroxylated form. Results suggest that the initial phase of fungal infection (2 days) of the living tissues of maize kernels (e.g., aleuron) coincides with a significant increase of fungal effectors such as SalOH and Npp1 that appear to be instrumental in eluding host defences and colonising the starch-enriched tissues, and therefore suggest a role of cluster 32 to the onset of infection.

The HosA Histone Deacetylase Regulates Aflatoxin Biosynthesis Through Direct Regulation of Aflatoxin Cluster Genes.

Histone deacetylases (HDACs) always function as corepressors and sometimes as coactivators in the regulation of fungal development and secondary metabolite production. However, the mechanism through which HDACs play positive roles in secondary metabolite production is still unknown. Here, classical HDAC enzymes were identified and analyzed in Aspergillus flavus, a fungus that produces one of the most carcinogenic secondary metabolites, aflatoxin B1 (AFB1). Characterization of the HDACs revealed that a class I family HDAC, HosA, played crucial roles in growth, reproduction, the oxidative stress response, AFB1 biosynthesis, and pathogenicity. To a lesser extent, a class II family HDAC, HdaA, was also involved in sclerotia formation and AFB1 biosynthesis. An in vitro analysis of HosA revealed that its HDAC activity was considerably diminished at nanomolar concentrations of trichostatin A. Notably, chromatin immunoprecipitation experiments indicated that HosA bound directly to AFB1 biosynthesis cluster genes to regulate their expression. Finally, we found that a transcriptional regulator, SinA, interacts with HosA to regulate fungal development and AFB1 biosynthesis. Overall, our results reveal a novel mechanism by which classical HDACs mediate the induction of secondary metabolite genes in fungi.

HexA is required for growth, aflatoxin biosynthesis and virulence in Aspergillus flavus.

BACKGROUND: Woronin bodies are fungal-specific organelles whose formation is derived from peroxisomes. The former are believed to be involved in the regulation of mycotoxins biosynthesis, but not in their damage repair function. The hexagonal peroxisome protein (HexA or Hex1) encoded by hexA gene in Aspergillus is the main and the essential component of the Woronin body. However, little is known about HexA in Aspergillus flavus. RESULTS: In this study, hexA knock-out mutant (ΔhexA) and complementation strain (ΔhexAC) were produced using homologous recombination. The results showed that, ΔhexA and ΔhexAC were successfully constructed. And the data analysis indicated that the colony diameter, stress sensitivity and the sclerotia formation of A. flavus were nearly not affected by the absence of HexA. Yet, the deletion of hexA gene reduced the production of asexual spores and lessened virulence on peanuts and maize seeds markedly. In addition, it was also found that there was a significant decrease of Aflatoxin B1 production in deletion mutant, when compared to wild type. CONCLUSIONS: Therefore, it suggested that the hexA gene has an essential function in conidia production and secondary metabolism in A. flavus. The gene is also believed to be playing an important role in the invasion of A. flavus to the host.

Lysine acetylation contributes to development, aflatoxin biosynthesis and pathogenicity in Aspergillus flavus.

Aspergillus flavus is a pathogenic fungus that produces carcinogenic aflatoxins, posing a great threat to crops, animals and humans. Lysine acetylation is one of the most important reversible post-translational modifications and plays a vital regulatory role in various cellular processes. However, current information on the extent and function of lysine acetylation and aflatoxin biosynthesis in A. flavus is limited. Here, a global acetylome analysis of A. flavus was performed by peptide pre-fractionation, pan-acetylation antibody enrichment and liquid chromatography-mass spectrometry. A total of 1313 high-confidence acetylation sites in 727 acetylated proteins were identified in A. flavus. These acetylation proteins are widely involved in glycolysis/gluconeogenesis, pentose phosphate pathway, citric acid cycle and aflatoxin biosynthesis. AflO (O-methyltransferase), a key enzyme in aflatoxin biosynthesis, was found to be acetylated at K241 and K384. Deletion of aflO not only impaired conidial and sclerotial developments, but also dramatically suppressed aflatoxin production and pathogenicity of A. flavus. Further site-specific mutations showed that lysine acetylation of AflO could also result in defects in development, aflatoxin production and pathogenicity, suggesting that acetylation plays a vital role in the regulation of the enzymatic activity of AflO in A. flavus. Our findings provide evidence for the involvement of lysine acetylation in various biological processes in A. flavus and facilitating in the elucidation of metabolic networks.

The Aspergillus flavus rtfA Gene Regulates Plant and Animal Pathogenesis and Secondary Metabolism.

Aspergillus flavus is an opportunistic fungal plant and human pathogen and a producer of mycotoxins, including aflatoxin B1 (AFB1). As part of our ongoing studies to elucidate the biological functions of the A. flavusrtfA gene, we examined its role in the pathogenicity of both plant and animal model systems. rtfA encodes a putative RNA polymerase II (Pol II) transcription elongation factor previously characterized in Saccharomyces cerevisiae, Aspergillus nidulans, and Aspergillus fumigatus, where it was shown to regulate several important cellular processes, including morphogenesis and secondary metabolism. In addition, an initial study in A. flavus indicated that rtfA also influences development and production of AFB1; however, its effect on virulence is unknown. The current study reveals that the rtfA gene is indispensable for normal pathogenicity in plants when using peanut seed as an infection model, as well as in animals, as shown in the Galleria mellonella infection model. Interestingly, rtfA positively regulates several processes known to be necessary for successful fungal invasion and colonization of host tissue, such as adhesion to surfaces, protease and lipase activity, cell wall composition and integrity, and tolerance to oxidative stress. In addition, metabolomic analysis revealed that A. flavusrtfA affects the production of several secondary metabolites, including AFB1, aflatrem, leporins, aspirochlorine, ditryptophenaline, and aflavinines, supporting a role of rtfA as a global regulator of secondary metabolism. Heterologous complementation of an A. flavusrtfA deletion strain with rtfA homologs from A. nidulans or S. cerevisiae fully rescued the wild-type phenotype, indicating that these rtfA homologs are functionally conserved among these three species.IMPORTANCE In this study, the epigenetic global regulator rtfA, which encodes a putative RNA-Pol II transcription elongation factor-like protein, was characterized in the mycotoxigenic and opportunistic pathogen A. flavus Specifically, its involvement in A. flavus pathogenesis in plant and animal models was studied. Here, we show that rtfA positively regulates A. flavus virulence in both models. Furthermore, rtfA-dependent effects on factors necessary for successful invasion and colonization of host tissue by A. flavus were also assessed. Our study indicates that rtfA plays a role in A. flavus adherence to surfaces, hydrolytic activity, normal cell wall formation, and response to oxidative stress. This study also revealed a profound effect of rtfA on the metabolome of A. flavus, including the production of potent mycotoxins.

Pumpkin (Cucurbita moschata) against Aspergillus flavus and aflatoxin B1 induced lung cyto-morphological damage in rats.

The protective efficacy of pumpkin (Cucurbita moschata) fruit aqueous extract against either aflatoxin B1 (AFB1) toxicity and Aspergillus flavus fungus infection induced lung histolomorphological damage in rats was investigated. AFB1 and A. flavus were administered intraperitoneally (0.2mg/Kg body weight) for 15 successive days. The result demonstrated that intoxication of rats with AFB1 induced lung damage as observed by alveolar hyperplasia, pulmonary hemorrhage and fibrosis. Infection with A. flavus also showed damaging impact on the rat lung as observed by fibrosis of bronchioles and alveolar hyperplasia. Oral co-administration of aqueous extract of pumpkin fruits (1.0 mg / kg of body weight) to either rat groups intoxicated with AFB1 or infected with A. flavus for 20 consecutive days showed more or less normal histological structure of rat lungs. In conclusion, aqueous extract of pumpkin fruits has a protective role against AFB1 or A. flavus induced lung damage which may be related to the antioxidant constituents of the plant extract.

Carbon catabolite repression gene creA regulates morphology, aflatoxin biosynthesis and virulence in Aspergillus flavus.

Carbon catabolite repression (CCR) is a very important mechanism employed in the utilization of carbon as an energy source, required for the regulation of growth, development and secondary metabolite production in fungi. Despite the wide study of this mechanism in fungi, little is known about the major CCR gene creA in A. flavus. Hence, we report identification of A. flavus carbon catabolite repression gene creA, which is responsible for the repression of secondary carbon sources. Gene deletion and over-expression was employed to explicate the role of creA in the morphology, pathogenicity, and secondary metabolite production in A. flavus. We investigated these factors using three carbon sources including glucose, sucrose and maltose. Gene deletion mutant (ΔcreA) had a significant growth defect on complete medium and minimal medium containing maltose. Conidia production in ΔcreA was significantly impaired irrespective of the carbon source available, while sclerotia production was significantly increased, compared to wild type (WT) and over-expression strain (OE::creA). Importantly, ΔcreA produced insignificant amount of aflatoxin in complete medium, and its ability to colonize hosts was also impaired. Concisely, we showed that creA played an important role in the morphology, pathogenicity and secondary metabolite production of A. flavus.

The epigenetic reader SntB regulates secondary metabolism, development and global histone modifications in Aspergillus flavus.

Due to the role, both beneficial and harmful, that fungal secondary metabolites play in society, the study of their regulation is of great importance. Genes for any one secondary metabolite are contiguously arranged in a biosynthetic gene cluster (BGC) and subject to regulation through the remodeling of chromatin. Histone modifying enzymes can place or remove post translational modifications (PTM) on histone tails which influences how tight or relaxed the chromatin is, impacting transcription of BGCs. In a recent forward genetic screen, the epigenetic reader SntB was identified as a transcriptional regulator of the sterigmatocystin BGC in A. nidulans, and regulated the related metabolite aflatoxin in A. flavus. In this study we investigate the role of SntB in the plant pathogen A. flavus by analyzing both ΔsntB and overexpression sntB genetic mutants. Deletion of sntB increased global levels of H3K9K14 acetylation and impaired several developmental processes including sclerotia formation, heterokaryon compatibility, secondary metabolite synthesis, and ability to colonize host seeds; in contrast the overexpression strain displayed fewer phenotypes. ΔsntB developmental phenotypes were linked with SntB regulation of NosA, a transcription factor regulating the A. flavus cell fusion cascade.

Effect of Aspergillus flavus on the mortality and activity of antioxidant enzymes of Spodoptera litura Fab. (Lepidoptera: Noctuidae) larvae.

Insects have developed tolerance against mycoses caused by entomopathogenic fungi through several humoral and cellular mechanisms. Antioxidant enzymes such as superoxide dismutase, lipid peroxidase, and peroxidase can play a role in defense against mycosis, but the physiological interactions between the fungus and the insect are not well characterized. In this study, the effects of infection by entomopathogenic fungus, Aspergillus flavus on the antioxidant defense system of Spodoptera litura, were investigated. The fungi, A. flavus exposure resulted in modification of the levels of antioxidant enzymes, as well as significant decline in phenoloxidase titers and the total hemocyte count 48 h post exposure. A significant increase was observed in detoxifying enzymes. All these results suggest that A. flavus infects S. litura by directly acting on the immune system, resulting in decreased immune function. Bioassay results showed that A. flavus affects third and fourth instar larvae of S. litura. This report supports the importance of A. flavus as a candidate for biological control of S. litura.

Cyclopiazonic Acid Is a Pathogenicity Factor for Aspergillus flavus and a Promising Target for Screening Germplasm for Ear Rot Resistance.

Aspergillus flavus, an opportunistic pathogen, contaminates maize and other key crops with carcinogenic aflatoxins (AFs). Besides AFs, A. flavus makes many more secondary metabolites (SMs) whose toxicity in insects or vertebrates has been studied. However, the role of SMs in the invasion of plant hosts by A. flavus remains to be investigated. Cyclopiazonic acid (CPA), a neurotoxic SM made by A. flavus, is a nanomolar inhibitor of endoplasmic reticulum calcium ATPases (ECAs) and a potent inducer of cell death in plants. We hypothesized that CPA, by virtue of its cytotoxicity, may serve as a key pathogenicity factor that kills plant cells and supports the saprophytic life style of the fungus while compromising the host defense response. This proposal was tested by two complementary approaches. A comparison of CPA levels among A. flavus isolates indicated that CPA may be a determinant of niche adaptation, i.e., isolates that colonize maize make more CPA than those restricted only to the soil. Further, mutants in the CPA biosynthetic pathway are less virulent in causing ear rot than their wild-type parent in field inoculation assays. Additionally, genes encoding ECAs are expressed in developing maize seeds and are induced by A. flavus infection. Building on these results, we developed a seedling assay in which maize roots were exposed to CPA, and cell death was measured as Evans Blue uptake. Among >40 maize inbreds screened for CPA tolerance, inbreds with proven susceptibility to ear rot were also highly CPA sensitive. The publicly available data on resistance to silk colonization or AF contamination for many of the lines was also broadly correlated with their CPA sensitivity. In summary, our studies show that i) CPA serves as a key pathogenicity factor that enables the saprophytic life style of A. flavus and ii) maize inbreds are diverse in their tolerance to CPA. Taking advantage of this natural variation, we are currently pursuing both genome-wide and candidate gene approaches to identify novel components of maize resistance to Aspergillus ear rot.