Combined Omics Approaches Reveal Distinct Mechanisms of Resistance and/or Susceptibility in Sugar Beet Double Haploid Genotypes at Early Stages of Beet Curly Top Virus Infection.

Sugar beet is susceptible to Beet curly top virus (BCTV), which significantly reduces yield and sugar production in the semi-arid growing regions worldwide. Sources of genetic resistance to BCTV is limited and control depends upon insecticide seed treatments with neonicotinoids. Through double haploid production and genetic selection, BCTV resistant breeding lines have been developed. Using BCTV resistant (R) [KDH13; Line 13 and KDH4-9; Line 4] and susceptible (S) [KDH19-17; Line 19] lines, beet leafhopper mediated natural infection, mRNA/sRNA sequencing, and metabolite analyses, potential mechanisms of resistance against the virus and vector were identified. At early infection stages (2- and 6-days post inoculation), examples of differentially expressed genes highly up-regulated in the 'R' lines (vs. 'S') included EL10Ac5g10437 (inhibitor of trypsin and hageman factor), EL10Ac6g14635 (jasmonate-induced protein), EL10Ac3g06016 (ribosome related), EL10Ac2g02812 (probable prolyl 4-hydroxylase 10), etc. Pathway enrichment analysis showed differentially expressed genes were predominantly involved with peroxisome, amino acids metabolism, fatty acid degradation, amino/nucleotide sugar metabolism, etc. Metabolite analysis revealed significantly higher amounts of specific isoflavonoid O-glycosides, flavonoid 8-C glycosides, triterpenoid, and iridoid-O-glycosides in the leaves of the 'R' lines (vs. 'S'). These data suggest that a combination of transcriptional regulation and production of putative antiviral metabolites might contribute to BCTV resistance. In addition, genome divergence among BCTV strains differentially affects the production of small non-coding RNAs (sncRNAs) and small peptides which may potentially affect pathogenicity and disease symptom development.

Vibrio gazogenes-dependent disruption of aflatoxin biosynthesis in Aspergillus flavus: the connection with endosomal uptake and hyphal morphogenesis.

Aflatoxins, a family of fungal secondary metabolites, are toxic and carcinogenic compounds that pose an enormous threat to global food safety and agricultural sustainability. Specifically agricultural products in African, Southeast Asian and hot and humid regions of American countries suffer most damage from aflatoxin producing molds due to the ideal climate conditions promoting their growth. Our recent studies suggest that Vibrio gazogenes (Vg), an estuarine bacterium non-pathogenic to plants and humans, can significantly inhibit aflatoxin biosynthesis in the producers. In this study, we investigated the mechanism underlying Vg-dependent aflatoxin inhibition using the prominent aflatoxin producer, Aspergillus flavus. We show that aflatoxin inhibition upon Vg treatment was associated with fungal uptake of Vg-prodigiosin, a red pigment, which was consistently visible inside fungal hyphae during treatment. The association of prodigiosin with aflatoxin inhibition was further evident as Serratia marcescens, another prodigiosin producer, significantly inhibited aflatoxin, while non-producers like Escherichia coli, Staphylococcus aureus, Vibrio harveyi, and Vibrio fischeri did not. Also, pure prodigiosin significantly inhibited aflatoxin biosynthesis. Endocytosis inhibitors, filipin and natamycin, reduced the Vg-prodigiosin uptake by the fungus leading to a significant increase in aflatoxin production, suggesting that uptake is endocytosis-dependent. The Vg treatment also reduced hyphal fusion (>98% inhibition) and branching, which are both endosome-dependent processes. Our results, therefore, collectively support our theory that Vg-associated aflatoxin inhibition is mediated by an endocytosis-dependent uptake of Vg-prodigiosin, which possibly leads to a disruption of normal endosomal functions.

Leaf Bacteriome in Sugar Beet Shows Differential Response against Beet curly top virus during Resistant and Susceptible Interactions.

Beet curly top virus (BCTV) significantly reduces sugar beet yield in semi-arid production areas. Genetic resistance to BCTV is limited; therefore, identification of additional resistance-associated factors is highly desired. Using 16S rRNA sequencing and BCTV resistant (R) genotypes (KDH13, KDH4-9) along with a susceptible (S) genotype (KDH19-17), we investigated leaf bacteriome changes during BCTV post inoculation (pi). At day 6 (~6-week-old plants), Cyanobacteria were predominant (~90%); whereas, at week 4 (~10-week-old plants) Firmicutes (11-66%), Bacteroidetes (17-26%), and Verrucomicrobia (12-29%) were predominant phyla and genotype dependent. Both Bacteroidetes and Verrucomicrobia, increased post infection only in the R lines. The bacterial genera Brevibacillus increased at 6 dpi, and Akkermansia and Bacteroides at 4 wkpi in the R lines. Linear discriminant analysis effect size (LEfSe) identified potential biomarkers in the R vs. S lines. Functional profiling revealed bacterial enrichment associated with the TCA cycle, polyisoprenoid, and L-methionine biosynthesis pathways only in KDH4-9 at 6 dpi. At 4 wkpi, bacteria associated with tryptophan and palmitate biosynthesis in the R lines, and uridine monophosphate, phosphatidyl glycerol, and phospholipid biosynthesis in the S line, were enriched. Future characterization of bacterial genera with antiviral properties will help establish their use as biocontrol agents/biomarkers against BCTV.

Cell-Wall-Degrading Enzymes-Related Genes Originating from Rhizoctonia solani Increase Sugar Beet Root Damage in the Presence of Leuconostoc mesenteroides.

Sugar beet crown and root rot caused by Rhizoctonia solani is a major yield constraint. Root rot is highly increased when R. solani and Leuconostoc mesenteroides co-infect roots. We hypothesized that the absence of plant cell-wall-degrading enzymes in L. mesenteroides and their supply by R. solani during close contact, causes increased damage. In planta root inoculation with or without cell-wall-degrading enzymes showed greater rot when L. mesenteroides was combined with cellulase (22 mm rot), polygalacturonase (47 mm), and pectin lyase (57 mm) versus these enzymes (0-26 mm), R. solani (20 mm), and L. mesenteroides (13 mm) individually. Carbohydrate analysis revealed increased simpler carbohydrates (namely glucose + galactose, and fructose) in the infected roots versus mock control, possibly due to the degradation of complex cell wall carbohydrates. Expression of R. solani cellulase, polygalacturonase, and pectin lyase genes during root infection corroborated well with the enzyme data. Global mRNAseq analysis identified candidate genes and highly co-expressed gene modules in all three organisms that might be critical in host plant defense and pathogenesis. Targeting R. solani cell-wall-degrading enzymes in the future could be an effective strategy to mitigate root damage during its interaction with L. mesenteroides.

Changes in Bacterial Endophyte Community Following Aspergillus flavus Infection in Resistant and Susceptible Maize Kernels.

Aspergillus flavus (A. flavus)-mediated aflatoxin contamination in maize is a major global economic and health concern. As A. flavus is an opportunistic seed pathogen, the identification of factors contributing to kernel resistance will be of great importance in the development of novel mitigation strategies. Using V3-V4 bacterial rRNA sequencing and seeds of A. flavus-resistant maize breeding lines TZAR102 and MI82 and a susceptible line, SC212, we investigated kernel-specific changes in bacterial endophytes during infection. A total of 81 bacterial genera belonging to 10 phyla were detected. Bacteria belonging to the phylum Tenericutes comprised 86-99% of the detected phyla, followed by Proteobacteria (14%) and others (<5%) that changed with treatments and/or genotypes. Higher basal levels (without infection) of Streptomyces and Microbacterium in TZAR102 and increases in the abundance of Stenotrophomonas and Sphingomonas in MI82 following infection may suggest their role in resistance. Functional profiling of bacteria using 16S rRNA sequencing data revealed the presence of bacteria associated with the production of putative type II polyketides and sesquiterpenoids in the resistant vs. susceptible lines. Future characterization of endophytes predicted to possess antifungal/ anti-aflatoxigenic properties will aid in their development as effective biocontrol agents or microbiome markers for maize aflatoxin resistance.

Regulatory Roles of Small Non-coding RNAs in Sugar Beet Resistance Against Beet curly top virus.

Beet curly top virus (BCTV) mediated yield loss in sugar beets is a major problem worldwide. The circular single-stranded DNA virus is transmitted by the beet leafhopper. Genetic sources of BCTV resistance in sugar beet are limited and commercial cultivars rely on chemical treatments versus durable genetic resistance. Phenotypic selection and double haploid production have resulted in sugar beet germplasm (KDH13; 13 and KDH4-9; 4) that are highly resistant to BCTV. The molecular mechanism of resistance to the virus is unknown, especially the role of small non-coding RNAs (sncRNAs) during early plant-viral interaction. Using the resistant lines along with a susceptible line (KDH19-17; 19), we demonstrate the role of sugar beet microRNAs (miRNAs) in BCTV resistance during early infection stages when symptoms are not yet visible. The differentially expressed miRNAs altered the expression of their corresponding target genes such as pyruvate dehydrogenase (EL10Ac1g02046), carboxylesterase (EL10Ac1g01087), serine/threonine protein phosphatase (EL10Ac1g01374), and leucine-rich repeats (LRR) receptor-like (EL10Ac7g17778), that were highly expressed in the resistant lines versus susceptible lines. Pathway enrichment analysis of the miRNA target genes showed an enrichment of genes involved in glycolysis/gluconeogenesis, galactose metabolism, starch, and sucrose metabolism to name a few. Carbohydrate analysis revealed altered glucose, galactose, fructose, and sucrose concentrations in the infected leaves of resistant versus susceptible lines. We also demonstrate differential regulation of BCTV derived sncRNAs in the resistant versus susceptible lines that target sugar beet genes such as LRR (EL10Ac1g01206), 7-deoxyloganetic acid glucosyltransferase (EL10Ac5g12605), and transmembrane emp24 domain containing (EL10Ac6g14074) and altered their expression. In response to viral infection, we found that plant derived miRNAs targeted BCTV capsid protein/replication related genes and showed differences in expression among resistant and susceptible lines. The data presented here demonstrate the contribution of miRNA mediated regulation of metabolic pathways and cross-kingdom RNA interference (RNAi) in sugar beet BCTV resistance.

Contribution of Maize Polyamine and Amino Acid Metabolism Toward Resistance Against Aspergillus flavus Infection and Aflatoxin Production.

Polyamines (PAs) are ubiquitous polycations found in plants and other organisms that are essential for growth, development, and resistance against abiotic and biotic stresses. The role of PAs in plant disease resistance depends on the relative abundance of higher PAs [spermidine (Spd), spermine (Spm)] vs. the diamine putrescine (Put) and PA catabolism. With respect to the pathogen, PAs are required to achieve successful pathogenesis of the host. Maize is an important food and feed crop, which is highly susceptible to Aspergillus flavus infection. Upon infection, the fungus produces carcinogenic aflatoxins and numerous other toxic secondary metabolites that adversely affect human health and crop value worldwide. To evaluate the role of PAs in aflatoxin resistance in maize, in vitro kernel infection assays were performed using maize lines that are susceptible (SC212) or resistant (TZAR102, MI82) to aflatoxin production. Results indicated significant induction of both PA biosynthetic and catabolic genes upon A. flavus infection. As compared to the susceptible line, the resistant maize lines showed higher basal expression of PA metabolism genes in mock-inoculated kernels that increased upon fungal infection. In general, increased biosynthesis and conversion of Put to Spd and Spm along with their increased catabolism was evident in the resistant lines vs. the susceptible line SC212. There were higher concentrations of amino acids such as glutamate (Glu), glutamine (Gln) and γ-aminobutyric acid (GABA) in SC212. The resistant lines were significantly lower in fungal load and aflatoxin production as compared to the susceptible line. The data presented here demonstrate an important role of PA metabolism in the resistance of maize to A. flavus colonization and aflatoxin contamination. These results provide future direction for the manipulation of PA metabolism in susceptible maize genotypes to improve aflatoxin resistance and overall stress tolerance.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the α-amylase Inhibitor from Lablab purpureus L.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are potent carcinogens that substantially reduce crop value in maize and other oil rich crops like peanut besides posing serious threat to human and animal health. Different approaches, including traditional breeding, transgenic expression of resistance associated proteins, and RNA interference (RNAi)-based host-induced gene silencing of critical A. flavus gene targets, are being evaluated to increase aflatoxin resistance in susceptible crops. Past studies have shown an important role of α-amylase in A. flavus pathogenesis and aflatoxin production, suggesting this gene/enzyme is a potential target to reduce both A. flavus growth and aflatoxin production. In this regard, the current study was undertaken to evaluate heterologous expression (under control of the constitutive CaMV 35S promoter) of a Lablab purpureus L. α-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is a competitive inhibitor of A. flavus α-amylase enzyme and belongs to the lectin-arcelin-α-amylase inhibitor protein family in common bean. In vitro studies prior to the current work had demonstrated the role of AILP in inhibition of A. flavus α-amylase activity and fungal growth. Fungal growth and aflatoxin production in mature kernels were monitored in real time using a GFP-expressing A. flavus strain. This kernel screening assay (KSA) is very simple to set up and provides reliable and reproducible data on infection and the extent of spread that could be quantified for evaluation of germplasm and transgenic lines. The fluorescence from the GFP strain is closely correlated to fungal growth and, by extension, it is well-correlated to aflatoxin values.  The goal of the current work was to implement this previous knowledge in a commercially important crop like maize to increase aflatoxin resistance. Our results show a 35%-72% reduction in A. flavus growth in AILP-expressing transgenic maize kernels which, in turn, translated into a 62%-88% reduction in aflatoxin levels.

Identification and functional analysis of the aspergillic acid gene cluster in Aspergillus flavus.

Aspergillus flavus can colonize important food staples and produce aflatoxins, a group of toxic and carcinogenic secondary metabolites. Previous in silico analysis of the A. flavus genome revealed 56 gene clusters predicted to be involved in the biosynthesis of secondary metabolites. A. flavus secondary metabolites produced during infection of maize seed are of particular interest, especially with respect to their roles in the biology of the fungus. A predicted nonribosomal peptide synthetase-like (NRPS-like) gene, designated asaC (AFLA_023020), present in the uncharacterized A. flavus secondary metabolite gene cluster 11 was previously shown to be expressed during the earliest stages of maize kernel infection. Cluster 11 is composed of six additional genes encoding a number of putative decorating enzymes as well as a transporter and transcription factor. We generated knock-out mutants of the seven predicted cluster 11 genes. LC-MS analysis of extracts from knockout mutants of these genes showed that they were responsible for the synthesis of the previously characterized antimicrobial mycotoxin aspergillic acid. Extracts of the asaC mutant showed no production of aspergillic acid or its precursors. Knockout of the cluster 11 P450 oxidoreductase afforded a pyrazinone metabolite, the aspergillic acid precursor deoxyaspergillic acid. The formation of hydroxyaspergillic acid was abolished in a desaturase/hydroxylase mutant. The hydroxamic acid functional group in aspergillic acid allows the molecule to bind to iron resulting in the production of a red pigment in A. flavus identified previously as ferriaspergillin. A reduction of aflatoxin B1 and cyclopiazonic acid that correlated with reduced fungal growth was observed in maize kernel infection assays when aspergillic acid biosynthesis in A. flavus is halted.

The Aspergillus flavus Spermidine Synthase (spds) Gene, Is Required for Normal Development, Aflatoxin Production, and Pathogenesis During Infection of Maize Kernels.

Aspergillus flavus is a soil-borne saprophyte and an opportunistic pathogen of both humans and plants. This fungus not only causes disease in important food and feed crops such as maize, peanut, cottonseed, and tree nuts but also produces the toxic and carcinogenic secondary metabolites (SMs) known as aflatoxins. Polyamines (PAs) are ubiquitous polycations that influence normal growth, development, and stress responses in living organisms and have been shown to play a significant role in fungal pathogenesis. Biosynthesis of spermidine (Spd) is critical for cell growth as it is required for hypusination-mediated activation of eukaryotic translation initiation factor 5A (eIF5A), and other biochemical functions. The tri-amine Spd is synthesized from the diamine putrescine (Put) by the enzyme spermidine synthase (Spds). Inactivation of spds resulted in a total loss of growth and sporulation in vitro which could be partially restored by addition of exogenous Spd. Complementation of the Δspds mutant with a wild type (WT) A. flavus spds gene restored the WT phenotype. In WT A. flavus, exogenous supply of Spd (in vitro) significantly increased the production of sclerotia and SMs. Infection of maize kernels with the Δspds mutant resulted in a significant reduction in fungal growth, sporulation, and aflatoxin production compared to controls. Quantitative PCR of Δspds mutant infected seeds showed down-regulation of aflatoxin biosynthetic genes in the mutant compared to WT A. flavus infected seeds. Expression analyses of PA metabolism/transport genes during A. flavus-maize interaction showed significant increase in the expression of arginine decarboxylase (Adc) and S-adenosylmethionine decarboxylase (Samdc) genes in the maize host and PA uptake transporters in the fungus. The results presented here demonstrate that Spd biosynthesis is critical for normal development and pathogenesis of A. flavus and pre-treatment of a Δspds mutant with Spd or Spd uptake from the host plant, are insufficient to restore WT levels of pathogenesis and aflatoxin production during seed infection. The data presented here suggest that future studies targeting spermidine biosynthesis in A. flavus, using RNA interference-based host-induced gene silencing approaches, may be an effective strategy to reduce aflatoxin contamination in maize and possibly in other susceptible crops.

RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels.

MAIN CONCLUSION: Expressing an RNAi construct in maize kernels that targets the gene for alpha-amylase in Aspergillus flavus resulted in suppression of alpha-amylase (amy1) gene expression and decreased fungal growth during in situ infection resulting in decreased aflatoxin production. Aspergillus flavus is a saprophytic fungus and pathogen to several important food and feed crops, including maize. Once the fungus colonizes lipid-rich seed tissues, it has the potential to produce toxic secondary metabolites, the most dangerous of which is aflatoxin. The pre-harvest control of A. flavus contamination and aflatoxin production is an area of intense research, which includes breeding strategies, biological control, and the use of genetically-modified crops. Host-induced gene silencing, whereby the host crop produces siRNA molecules targeting crucial genes in the invading fungus and targeting the gene for degradation, has shown to be promising in its ability to inhibit fungal growth and decrease aflatoxin contamination. Here, we demonstrate that maize inbred B104 expressing an RNAi construct targeting the A. flavus alpha-amylase gene amy1 effectively reduces amy1 gene expression resulting in decreased fungal colonization and aflatoxin accumulation in kernels. This work contributes to the development of a promising technology for reducing the negative economic and health impacts of A. flavus growth and aflatoxin contamination in food and feed crops.

Control of Aspergillus flavus growth and aflatoxin production in transgenic maize kernels expressing a tachyplesin-derived synthetic peptide, AGM182.

Aspergillus flavus is an opportunistic, saprophytic fungus that infects maize and other fatty acid-rich food and feed crops and produces toxic and carcinogenic secondary metabolites known as aflatoxins. Contamination of maize with aflatoxin poses a serious threat to human health in addition to reducing the crop value leading to a substantial economic loss. Here we report designing a tachyplesin1-derived synthetic peptide AGM182 and testing its antifungal activity both in vitro and in planta. In vitro studies showed a five-fold increase in antifungal activity of AGM182 (vs. tachyplesin1) against A. flavus. Transgenic maize plants expressing AGM182 under maize Ubiquitin-1 promoter were produced through Agrobacterium-mediated transformation. PCR products confirmed integration of the AGM182 gene, while RT-PCR of maize RNA confirmed the presence of AGM182 transcripts. Maize kernel screening assay using a highly aflatoxigenic A. flavus strain (AF70) showed up to 72% reduction in fungal growth in the transgenic AGM182 seeds compared to isogenic negative control seeds. Reduced fungal growth in the AGM182 transgenic seeds resulted in a significant reduction in aflatoxin levels (76-98%). The results presented here show the power of computational and synthetic biology to rationally design and synthesize an antimicrobial peptide against A. flavus that is effective in reducing fungal growth and aflatoxin contamination in an economically important food and feed crop such as maize.

Aspergillus flavus Secondary Metabolites: More than Just Aflatoxins.

Aspergillus flavus is best known for producing the family of potent carcinogenic secondary metabolites known as aflatoxins. However, this opportunistic plant and animal pathogen also produces numerous other secondary metabolites, many of which have also been shown to be toxic. While about forty of these secondary metabolites have been identified from A. flavus cultures, analysis of the genome has predicted the existence of at least 56 secondary metabolite gene clusters. Many of these gene clusters are not expressed during growth of the fungus on standard laboratory media. This presents researchers with a major challenge of devising novel strategies to manipulate the fungus and its genome so as to activate secondary metabolite gene expression and allow identification of associated cluster metabolites. In this review, we discuss the genetic, biochemical and bioinformatic methods that are being used to identify previously uncharacterized secondary metabolite gene clusters and their associated metabolites. It is important to identify as many of these compounds as possible to determine their bioactivity with respect to fungal development, survival, virulence and especially with respect to any potential synergistic toxic effects with aflatoxin.

Polyamines in the life of Arabidopsis: profiling the expression of S-adenosylmethionine decarboxylase (SAMDC) gene family during its life cycle.

BACKGROUND: Arabidopsis has 5 paralogs of the S-adenosylmethionine decarboxylase (SAMDC) gene. Neither their specific role in development nor the role of positive/purifying selection in genetic divergence of this gene family is known. While some data are available on organ-specific expression of AtSAMDC1, AtSAMDC2, AtSAMDC3 and AtSAMDC4, not much is known about their promoters including AtSAMDC5, which is believed to be non-functional. RESULTS: (1) Phylogenetic analysis of the five AtSAMDC genes shows similar divergence pattern for promoters and coding sequences (CDSs), whereas, genetic divergence of 5'UTRs and 3'UTRs was independent of the promoters and CDSs; (2) while AtSAMDC1 and AtSAMDC4 promoters exhibit high activity (constitutive in the former), promoter activities of AtSAMDC2, AtSAMDC3 and AtSAMDC5 are moderate to low in seedlings (depending upon translational or transcriptional fusions), and are localized mainly in the vascular tissues and reproductive organs in mature plants; (3) based on promoter activity, it appears that AtSAMDC5 is both transcriptionally and translationally active, but based on it's coding sequence it seems to produce a non-functional protein; (4) though 5'-UTR based regulation of AtSAMDC expression through upstream open reading frames (uORFs) in the 5'UTR is well known, no such uORFs are present in AtSAMDC4 and AtSAMDC5; (5) the promoter regions of all five AtSAMDC genes contain common stress-responsive elements and hormone-responsive elements; (6) at the organ level, the activity of AtSAMDC enzyme does not correlate with the expression of specific AtSAMDC genes or with the contents of spermidine and spermine. CONCLUSIONS: Differential roles of positive/purifying selection were observed in genetic divergence of the AtSAMDC gene family. All tissues express one or more AtSAMDC gene with significant redundancy, and concurrently, there is cell/tissue-specificity of gene expression, particularly in mature organs. This study provides valuable information about AtSAMDC promoters, which could be useful in future manipulation of crop plants for nutritive purposes, stress tolerance or bioenergy needs. The AtSAMDC1 core promoter might serve the need of a strong constitutive promoter, and its high expression in the gametophytic cells could be exploited, where strong male/female gametophyte-specific expression is desired; e.g. in transgenic modification of crop varieties.

The Pathogenesis-Related Maize Seed (PRms) Gene Plays a Role in Resistance to Aspergillus flavus Infection and Aflatoxin Contamination.

Aspergillus flavus is an opportunistic plant pathogen that colonizes and produces the toxic and carcinogenic secondary metabolites, aflatoxins, in oil-rich crops such as maize (Zea mays ssp. mays L.). Pathogenesis-related (PR) proteins serve as an important defense mechanism against invading pathogens by conferring systemic acquired resistance in plants. Among these, production of the PR maize seed protein, ZmPRms (AC205274.3_FG001), has been speculated to be involved in resistance to infection by A. flavus and other pathogens. To better understand the relative contribution of ZmPRms to A. flavus resistance and aflatoxin production, a seed-specific RNA interference (RNAi)-based gene silencing approach was used to develop transgenic maize lines expressing hairpin RNAs to target ZmPRms. Downregulation of ZmPRms in transgenic kernels resulted in a ∼250-350% increase in A. flavus infection accompanied by a ∼4.5-7.5-fold higher accumulation of aflatoxins than control plants. Gene co-expression network analysis of RNA-seq data during the A. flavus-maize interaction identified ZmPRms as a network hub possibly responsible for regulating several downstream candidate genes associated with disease resistance and other biochemical functions. Expression analysis of these candidate genes in the ZmPRms-RNAi lines demonstrated downregulation (vs. control) of a majority of these ZmPRms-regulated genes during A. flavus infection. These results are consistent with a key role of ZmPRms in resistance to A. flavus infection and aflatoxin accumulation in maize kernels.

The Aspergillus flavus Homeobox Gene, hbx1, is Required for Development and Aflatoxin Production.

Homeobox proteins, a class of well conserved transcription factors, regulate the expression of targeted genes, especially those involved in development. In filamentous fungi, homeobox genes are required for normal conidiogenesis and fruiting body formation. In the present study, we identified eight homeobox (hbx) genes in the aflatoxin-producing ascomycete, Aspergillus flavus, and determined their respective role in growth, conidiation and sclerotial production. Disruption of seven of the eight genes had little to no effect on fungal growth and development. However, disruption of the homeobox gene AFLA_069100, designated as hbx1, in two morphologically different A. flavus strains, CA14 and AF70, resulted in complete loss of production of conidia and sclerotia as well as aflatoxins B1 and B2, cyclopiazonic acid and aflatrem. Microscopic examination showed that the Δhbx1 mutants did not produce conidiophores. The inability of Δhbx1 mutants to produce conidia was related to downregulation of brlA (bristle) and abaA (abacus), regulatory genes for conidiophore development. These mutants also had significant downregulation of the aflatoxin pathway biosynthetic genes aflC, aflD, aflM and the cluster-specific regulatory gene, aflR. Our results demonstrate that hbx1 not only plays a significant role in controlling A. flavus development but is also critical for the production of secondary metabolites, such as aflatoxins.

RNA Interference (RNAi) as a Potential Tool for Control of Mycotoxin Contamination in Crop Plants: Concepts and Considerations.

Mycotoxin contamination in food and feed crops is a major concern worldwide. Fungal pathogens of the genera Aspergillus. Fusarium, and Penicillium are a major threat to food and feed crops due to production of mycotoxins such as aflatoxins, 4-deoxynivalenol, patulin, and numerous other toxic secondary metabolites that substantially reduce the value of the crop. While host resistance genes are frequently used to introgress disease resistance into elite germplasm, either through traditional breeding or transgenic approaches, such resistance is often compromised by the evolving pathogen over time. RNAi-based host-induced gene silencing of key genes required by the pathogen for optimal growth, virulence and/or toxin production, can serve as an alternative, pre-harvest approach for disease control. RNAi represents a robust and efficient tool that can be used in a highly targeted, tissue specific manner to combat mycotoxigenic fungi infecting crop plants. Successful transgenic RNAi implementation depends on several factors including (1) designing vectors to produce double-stranded RNAs (dsRNAs) that will generate small interfering RNA (siRNA) species for optimal gene silencing and reduced potential for off-target effects; (2) availability of ample target siRNAs at the infection site; (3) efficient uptake of siRNAs by the fungus; (4) siRNA half-life and (5) amplification of the silencing effect. This review provides a critical and comprehensive evaluation of the published literature on the use of RNAi-based approaches to control mycotoxin contamination in crop plants. It also examines experimental strategies used to better understand the mode of action of RNAi with the aim of eliminating mycotoxin contamination, thereby improving food and feed safety.

Glutamate, Ornithine, Arginine, Proline, and Polyamine Metabolic Interactions: The Pathway Is Regulated at the Post-Transcriptional Level.

The metabolism of glutamate into ornithine, arginine, proline, and polyamines is a major network of nitrogen-metabolizing pathways in plants, which also produces intermediates like nitric oxide, and γ-aminobutyric acid (GABA) that play critical roles in plant development and stress. While the accumulations of intermediates and the products of this network depend primarily on nitrogen assimilation, the overall regulation of the interacting sub-pathways is not well understood. We tested the hypothesis that diversion of ornithine into polyamine biosynthesis (by transgenic approach) not only plays a role in regulating its own biosynthesis from glutamate but also affects arginine and proline biosynthesis. Using two high putrescine producing lines of Arabidopsis thaliana (containing a transgenic mouse ornithine decarboxylase gene), we studied the: (1) effects of exogenous supply of carbon and nitrogen on polyamines and pools of soluble amino acids; and, (2) expression of genes encoding key enzymes in the interactive pathways of arginine, proline and GABA biosynthesis as well as the catabolism of polyamines. Our findings suggest that: (1) the overall conversion of glutamate to arginine and polyamines is enhanced by increased utilization of ornithine for polyamine biosynthesis by the transgene product; (2) proline and arginine biosynthesis are regulated independently of polyamines and GABA biosynthesis; (3) the expression of most genes (28 that were studied) that encode enzymes of the interacting sub-pathways of arginine and GABA biosynthesis does not change even though overall biosynthesis of Orn from glutamate is increased several fold; and (4) increased polyamine biosynthesis results in increased assimilation of both nitrogen and carbon by the cells.

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids.

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic-active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane-localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element-companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.