Characterization of a Stress-Enhanced Promoter from the Grass Halophyte, Spartina alterniflora L.

Stress-inducible promoters are vital for the desirable expression of genes, especially transcription factors, which could otherwise compromise growth and development when constitutively overexpressed in plants. Here, we report on the characterization of the promoter region of a stressresponsive gene SaAsr1 from monocot halophyte cordgrass (Spartina alterniflora). Several cis-acting elements, such as ABRE (ABA-responsive element), DRE-CRT (dehydration responsive-element/CRepeat), LTRE (low temperature-responsive element), ERE (ethylene-responsive element), LRE (light-responsive element), etc. contributed at varying degrees to salt-, drought- and ABA-enhanced expression of gusA reporter gene in Arabidopsis thaliana under the full-length promoter, pAsr11875 and its deletion derivatives with an assortment of cis-regulatory motifs. The smallest promoter, pAsr1491, with three cis-acting elements (a CCAAT box-heat responsive, an LRE, and a copper responsive element) conferred drought-enhanced expression of gusA; pAsr1755 (with an ABRE and a DRE) presented the highest expression in ABA and drought; and pAsr1994 with seven ABREs and two DREs conferred optimal induction of gusA, especially under drought and ABA. Arabidopsis transgenics expressing a known abiotic stress-responsive gene, SaADF2 (actin depolymerization factor 2), under both pAsr11875 and p35S promoters outperformed the wild type (WT) with enhanced drought and salt tolerance contributed by higher relative water content and membrane stability with no significant difference between pAsr11875:SaADF2 or p35S:SaADF2 lines. However, pAsr11875:SaADF2 lines produced healthy plants with robust shoot systems under salt stress and control compared to slightly stunted growth of the p35S:SaADF2 plants. This reestablished the evidence that transgene expression under a stress-inducible promoter is a better strategy for the genetic manipulation of crops

Expression of an Antimicrobial Peptide via the Chloroplast Genome to Control Phytopathogenic Bacteria and Fungi

The antimicrobial peptide MSI-99, an analog of magainin 2, was expressed via the chloroplast genome to obtain high levels of expression in transgenic tobacco (Nicotiana tabacum var. Petit Havana) plants. Polymerase chain reaction products and Southern blots confirmed integration of MSI-99 into the chloroplast genome and achievement of homoplasmy, whereas northern blots confirmed transcription. Contrary to previous predictions, accumulation of MSI-99 in transgenic chloroplasts did not affect normal growth and development of the transgenic plants. This may be due to differences in the lipid composition of plastid membranes compared with the membranes of susceptible target microbes. In vitro assays with protein extracts from T1 and T2 plants confirmed that MSI-99 was expressed at high levels to provide 88% (T1) and 96% (T2) inhibition of growth against Pseudomonas syringae pv tabaci, a major plant pathogen. When germinated in the absence of spectinomycin selection, leaf extracts from T2 generation plants showed 96% inhibition of growth against P. syringae pv tabaci. In addition, leaf extracts from transgenic plants (T1) inhibited the growth of pregerminated spores of three fungal species, Aspergillus flavus, Fusarium moniliforme, and Verticillium dahliae, by more than 95% compared with non-transformed control plant extracts. In planta assays with the bacterial pathogen P. syringae pv tabaci resulted in areas of necrosis around the point of inoculation in control leaves, whereas transformed leaves showed no signs of necrosis, demonstrating high-dose release of the peptide at the site of infection by chloroplast lysis. In planta assays with the fungal pathogen, Colletotrichum destructivum, showed necrotic anthracnose lesions in non-transformed control leaves, whereas transformed leaves showed no lesions. Genetically engineering crop plants for disease resistance via the chloroplast genome instead of the nuclear genome is desirable to achieve high levels of expression and to prevent pollen-mediated escape of transgenes

Disease Resistance Conferred by the Expression of a Gene Encoding a Synthetic Peptide in Transgenic Cotton (Gossypium hirsutum L.) Plants

Fertile, transgenic cotton plants expressing the synthetic antimicrobial peptide, D4E1, were produced through Agrobacterium-mediated transformation. PCR products and Southern blots confirmed integration of the D4E1 gene, while RT-PCR of cotton RNA confirmed the presence of D4E1 transcripts. In vitro assays with crude leaf protein extracts from T0 and T1 plants confirmed that D4E1 was expressed at sufficient levels to inhibit the growth of Fusarium verticillioides and Verticillium dahliae compared to extracts from negative control plants transformed with pBI-d35SΩ-uidA-nos (CGUS). Although in vitro assays did not show control of pre-germinated spores of Aspergillus flavus, bioassays with cotton seeds in situ or in planta, inoculated with a GFP-expressing A. flavus, indicated that the transgenic cotton seeds inhibited extensive colonization and spread by the fungus in cotyledons and seed coats. In planta assays with the fungal pathogen,Thielaviopsis basicola, which causes black root rot in cotton, showed typical symptoms such as black discoloration and constriction on hypocotyls, reduced branching of roots in CGUS negative control T1 seedlings, while transgenic T1 seedlings showed a significant reduction in disease symptoms and increased seedling fresh weight, demonstrating tolerance to the fungal pathogen. Significant advantages of synthetic peptides in developing transgenic crop plants that are resistant to diseases and mycotoxin-causing fungal pathogens are highlighted in this report

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, 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 of 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 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 and increases in the abundance of Stenotrophomonas and Sphingomonas in the resistant lines 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 antifungal type II polyketides, 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

Developing Resistance to Aflatoxin in Maize and Cottonseed

At this time, no “magic bullet” for solving the aflatoxin contamination problem in maize and cottonseed has been identified, so several strategies must be utilized simultaneously to ensure a healthy crop, free of aflatoxins. The most widely explored strategy for the control of aflatoxin contamination is the development of preharvest host resistance. This is because A. flavus infects and produces aflatoxins in susceptible crops prior to harvest. In maize production, the host resistance strategy has gained prominence because of advances in the identification of natural resistance traits. However, native resistance in maize to aflatoxin contamination is polygenic and complex and, therefore, markers need to be identified to facilitate the transfer of resistance traits into agronomically viable genetic backgrounds while limiting the transfer of undesirable traits. Unlike maize, there are no known cotton varieties that demonstrate enhanced resistance to A. flavus infection and aflatoxin contamination. For this reason, transgenic approaches are being undertaken in cotton that utilize genes encoding antifungal/anti-aflatoxin factors from maize and other sources to counter fungal infection and toxin production. This review will present information on preharvest control strategies that utilize both breeding and native resistance identification approaches in maize as well as transgenic approaches in cotton

Microbiota of maize kernels as influenced by Aspergillus flavus infection in susceptible and resistant inbreds

BackgroundNearly everything on Earth harbors a microbiome. A microbiome is a community of microbes (bacteria, fungi, and viruses) with potential to form complex networks that involve mutualistic and antagonistic interactions. Resident microbiota on/in an organism are determined by the external environment, both biotic and abiotic, and the intrinsic adaptability of each organism. Although the maize microbiome has been characterized, community changes that result from the application of fungal biocontrol strains, such as non-aflatoxigenic Aspergillus flavus, have not.MethodsWe silk channel inoculated field-grown maize separately with a non-aflatoxigenic biocontrol strain (K49), a highly toxigenic strain (Tox4), and a combination of both A. flavus strains. Two maize inbreds were treated, A. flavus-susceptible B73 and A. flavus-resistant CML322. We then assessed the impacts of A. flavus introduction on the epibiota and endobiota of their maize kernels.ResultsWe found that the native microbial communities were significantly affected, irrespective of genotype or sampled tissue. Overall, bacteriomes exhibited greater diversity of genera than mycobiomes. The abundance of certain genera was unchanged by treatment, including genera of bacteria (e.g., Enterobacter, Pantoea) and fungi (e.g., Sarocladium, Meyerozyma) that are known to be beneficial, antagonistic, or both on plant growth and health.ConclusionBeneficial microbes like Sarocladium that responded well to A. flavus biocontrol strains are expected to enhance biocontrol efficacy, while also displacing/antagonizing harmful microbes

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

Dynamic geospatial modeling of mycotoxin contamination of corn in Illinois: unveiling critical factors and predictive insights with machine learning

Mycotoxin contamination of corn is a pervasive problem that negatively impacts human and animal health and causes economic losses to the agricultural industry worldwide. Historical aflatoxin (AFL) and fumonisin (FUM) mycotoxin contamination data of corn, daily weather data, satellite data, dynamic geospatial soil properties, and land usage parameters were modeled to identify factors significantly contributing to the outbreaks of mycotoxin contamination of corn grown in Illinois (IL), AFL &gt;20 ppb, and FUM &gt;5 ppm. Two methods were used: a gradient boosting machine (GBM) and a neural network (NN). Both the GBM and NN models were dynamic at a state-county geospatial level because they used GPS coordinates of the counties linked to soil properties. GBM identified temperature and precipitation prior to sowing as significant influential factors contributing to high AFL and FUM contamination. AFL-GBM showed that a higher aflatoxin risk index (ARI) in January, March, July, and November led to higher AFL contamination in the southern regions of IL. Higher values of corn-specific normalized difference vegetation index (NDVI) in July led to lower AFL contamination in Central and Southern IL, while higher wheat-specific NDVI values in February led to higher AFL. FUM-GBM showed that temperature in July and October, precipitation in February, and NDVI values in March are positively correlated with high contamination throughout IL. Furthermore, the dynamic geospatial models showed that soil characteristics were correlated with AFL and FUM contamination. Greater calcium carbonate content in soil was negatively correlated with AFL contamination, which was noticeable in Southern IL. Greater soil moisture and available water-holding capacity throughout Southern IL were positively correlated with high FUM contamination. The higher clay percentage in the northeastern areas of IL negatively correlated with FUM contamination. NN models showed high class-specific performance for 1-year predictive validation for AFL (73%) and FUM (85%), highlighting their accuracy for annual mycotoxin prediction. Our models revealed that soil, NDVI, year-specific weekly average precipitation, and temperature were the most important factors that correlated with mycotoxin contamination. These findings serve as reliable guidelines for future modeling efforts to identify novel data inputs for the prediction of AFL and FUM outbreaks and potential farm-level management practices