Elucidation of defence response mechanisms in pearl millet

Pearl millet is a staple food source for millions of African families living in semi-arid regions of the continent. Yet, despite its importance and ability to provide consistent yields, very little research and resources have been directed towards understanding mechanisms governing this crop’s resilience to biotic and abiotic stresses. The research outlined in this thesis therefore aimed to elucidate defence response mechanisms in pearl millet, a non-model cereal crop. This was accomplished through the construction and characterisation of a pearl millet defence response cDNA library, which was subsequently utilised in large scale gene expression studies to profile pearl millet’s response to the defence signalling compounds nitric oxide (NO), methyl jasmonate (MeJA) and salicylic acid (SA), and to the biotrophic rust fungus Puccinia substriata var. indica. A pearl millet cDNA library was constructed by treating pearl millet plants with the defence elicitors chitin and flagellin, and by wounding the plants. Suppression subtractive hybridisation (SSH) was employed to enrich the library for defence response transcripts. In order to characterise the cDNA libraries, a quantitative cDNA microarray-based screening method was developed that enabled identification of false positive transcripts, as well as clones that represented rare or abundant transcripts. Based on this screening method, a number of clones were selected for sequence analysis, and their identity ascertained through homology searches with previously sequenced genes. This revealed a number of genes known to play important roles during pathogen attack. The pearl millet SSH defence response library, consisting of 1920 cDNAs either up- or down regulated in defence response, was spotted onto a glass slide microarray and used in transcript profiling studies to examine pearl millet’s response to the defence signalling molecules NO, MeJA and SA. Whilst only 45 cDNAs responded significantly to NO treatment, 279 and 224 cDNAs responded to MeJA and SA sprays, respectively. Closer examination of MeJA and SA responsive genes revealed that many of the induced transcripts were common to both signalling pathways, demonstrating that a substantial network of regulatory interactions exists between the salicylate and jasmonate pathways, which were previously believed to act in an antagonistic manner. Pathology studies indicated that pretreatment of pearl millet with SA conferred resistance to a virulent isolate of P. substriata var. indica, whereas MeJA application did not significantly reduce subsequent infection levels. Transcript profiling of a susceptible pearl millet line in response to virulent rust infection revealed that genes common to both the jasmonate and salicylate pathways were induced, suggesting that the plant adopts elements from a number of defence signalling pathways in an attempt to ward off infection by the virulent rust fungus. However, in view of results obtained from pearl millet defence signalling molecule pretreatments, it is probably genes that are significantly induced in response to SA, but to a lesser extent by MeJA that actually confer resistance to an avirulent rust isolate. Treatment of pearl millet plants with an avirulent P. substriata strain and subsequent microarray analysis would answer this hypothesis by revealing whether an incompatible reaction elicits more elements of the salicylate defence response pathway.Thesis (PhD (Botany))--University of Pretoria, 2009.Plant Scienceunrestricte

Quantitative detection of Exserohilum turcicum in northern leaf blight diseased sorghum and maize leaves

Exserohilum turcicum is the causal agent of northern leaf blight (NLB) disease in sorghum and maize. Early detection of this economically important pathogen is essential for effective disease management to limit yield losses. Here we present a real-time quantitative PCR (qPCR) assay specific for E. turcicum detection and biomass quantification in sorghum and maize. In planta fungal quantification was achieved through amplification of a cytochrome P450 oxidoreductase (cpr1) gene fragment and subsequent normalisation to the host glutathione S-transferase III gene (gst3). The assay could specifically detect E. turcicum in sorghum and maize, but the cpr1 gene fragment was not amplified in non-target fungal pathogens. Application of the assay with NLB diseased sorghum and maize leaf material revealed a significant increase in E. turcicum DNA in leaves with lesion symptoms when compared to leaves with early stage chlorotic fleck symptoms in both hosts. Furthermore, E. turcicum was detected at levels as low as 1 pg in infected sorghum and maize leaves. The assay enables rapid detection and quantification of E. turcicum in sorghum and maize and has useful applications in crop breeding programmes and disease management where cultivar selection and early detection of the pathogen are essential to limit disease spread.The National Research Foundation of South Africahttp://link.springer.com/journal/13313hj2021Forestry and Agricultural Biotechnology Institute (FABI)Plant Production and Soil Scienc

Time-course RNAseq reveals exserohilum turcicum effectors and pathogenicity determinants

Exserohilum turcicum (sexual stage Setosphaeria turcica) is the hemibiotrophic causal agent of northern leaf blight ofmaize and sorghum. This study aimed to identify the genes involved in host colonization during the biotrophic and necrotrophic phases of infection. It also aimed to identify race-specific differences in gene expression. RNAseq of maize seedlings inoculated with a race 13N or 23N E. turcicum isolate was conducted before inoculation and at 2, 5, 7, and 13 days post-inoculation (dpi). Biological replicates were pooled per time point for each race and sequenced. A bioinformatics pipeline was used to identify candidate effectors, and expression was validated for selected candidates. Fungal biomass was positively correlated with the percentages of E. turcicum reads mapped, which were low at early time points (2–7 dpi) with a significant increase at 13 dpi, indicating a lifestyle switch from biotrophy to necrotrophy between 7 and 13 dpi. AVRHt1 is the putative E. turcicum effector recognized by the maize resistance gene Ht1. Consistent with this, AVRHt1 was expressed in planta by race 23N, but transcripts were absent in race 13N. In addition, specific transposable elements were expressed in 23N only. Genes encoding the virulence-associated peptidases leupeptin-inhibiting protein 1 and fungalysin were expressed in planta. Transcriptional profiles of genes involved in secondary metabolite synthesis or cell wall degradation revealed the importance of these genes during late stages of infection (13 dpi). A total of 346 expressed candidate effectors were identified, including Ecp6 and proteins similar to the secreted in xylem (SIX) effectors common to formae speciales of Fusarium oxysporum, SIX13 and SIX5. Expression profiling of Ecp6 and SIX13-like indicated a peak in expression at 5 and 7 dpi compared to 2 and 13 dpi. Sequencing of SIX13-like from diverse isolates of E. turcicum revealed host-specific polymorphisms that were mostly non-synonymous, resulting in two groups of SIX13-like proteins that corresponded to the maize or sorghum origin of each isolate. This study suggests putative mechanisms whereby E. turcicum causes disease. Identification of the candidate effector SIX13-like is consistent with the infection mode of E. turcicum through the xylem of susceptible hosts.The National Research Foundation of South Africahttp://www.frontiersin.org/Microbiologyam2020Forestry and Agricultural Biotechnology Institute (FABI)Plant Production and Soil Scienc

Microsatellite and mating type primers for the maize and sorghum pathogen, Exserohilum turcicum

Northern corn leaf blight (NCLB) is a destructive foliar disease of maize that results from infection with the fungal pathogen, Exserohilum turcicum. Annual yield losses incurred from NCLB in South Africa may exceed 50 % when environmental conditions optimal for disease development prevail. In order to study the genetic diversity of E. turcicum, 13 microsatellite markers and mating type PCR primers were developed. Thirty-two primer pairs were designed from the E. turcicum genome sequence to flank microsatellite regions. A multiplex PCR assay amplifying both mating type idiomorphs was designed from the MAT1-1 and MAT1-2 gene sequences, and a protocol for multiplex PCR amplification of MAT loci was optimized. Initial screening identified 13 microsatellite regions that were polymorphic in 9 isolates of E. turcicum. To test the efficacy of the markers, 26 isolates of E. turcicum from 6 South African provinces, including 2 isolates from sorghum, were genotyped. A total of 90 alleles across 13 loci were obtained and the gene diversity ranged from 0.074 to 0.929. Cross-species amplification withE. rostratum was obtained for one SSR marker (SSR27). The MAT markers were specific to E. turcicum and could be used to differentiate isolates of E. turcicum and E. rostratum. The markers developed in this study will be useful to elucidate the population genetic structure, genetic diversity and mode of reproduction of E. turcicum on maize and sorghum.Research supported in part by the National Research Foundation of South Africa (grant specific unique reference number (UID) 85076), and by the Maize Trust and the University of Pretoria’s Research and Development Programme.http://link.springer.com/journal/133132015-09-30hb201

Quantitative phenotyping of grey leaf spot disease in maize using real-time PCR

Grey leaf spot is an important maize foliar disease caused by the fungal pathogens Cercospora zeae-maydis and Cercospora zeina. Although methods exist to detect these Cercospora species in maize, current techniques do not allow quantification of the fungi in planta. We developed a real-time SYBR® Green PCR assay for quantification of grey leaf spot disease in maize based on the amplification of a fragment of a cytochrome P450 reductase (cpr1) gene. In planta fungal DNA content was normalised to a maize glutathione S-transferase III gene (gst3) to yield values was specific to the two Cercospora spp., and we observed no amplification of the cpr1 fragment in non-target maize leaf pathogens or saprophytes. The assay was employed to quantify C. zeina in glasshouse inoculated maize plants and grey leaf spot infected field plants of resistant and susceptible maize lines. In both instances, C. zeina DNA content correlated with symptomatic leaf lesion area, and the susceptible maize line contained significantly more C. zeina DNA than the resistant line. Sequence differences between the C. zeina and C. zeae-maydis cpr1 amplicons enabled us to perform melt curve analyses to identify the Cercospora species causing grey leaf spot at a particular location. This assay has application in the early detection and quantification of Cercospora spp., both of which are important tools in grey leaf spot disease management and maize breeding programmes.The Technology Innovation Agency (TIA), the National Research Foundation (NRF) and the University of Pretoria (UP), South Africa.http://www.springerlink.com /content/100265/nf201

Expression of a β-1,3-glucanase from a biocontrol fungus in transgenic pearl millet

Sclerospora graminicola is an oomycete (heterotrophic Stramenopiles), fungal-like obligate phytopathogen, the causal agent of downy mildew in pearl millet (Pennisetum glaucum [L.] R. Br.), and a major constraint in the production of this cereal crop. In this study a hydrolytic enzyme, β-1,3-glucanase (gluc78), from the biocontrol fungus Trichoderma atroviride, was introduced into the genome of a pearl millet breeding line, 842B, by particle bombardment. Constructs were prepared containing the gluc78 gene, encoding the 78 kDa β-1,3-glucanase protein, downstream of either the constitutive ubiquitin promoter or the wound inducible potato proteinase inhibitor IIK gene promoter (pin2). The positive selectable marker gene, manA, encoding mannose-6-phosphate isomerase (phosphomannose isomerase) under the control of the ubiquitin promoter, was used for co-transformation. Transgenic plants were obtained harbouring the manA selectable marker gene and the antifungal gene gluc78 downstream of either the ubiquitin or pin2 promoter. Full constructs or minimal transgene expression cassettes containing the genes of interest were successfully introduced into the genome of pearl millet. Progeny of stably transformed plants, harbouring the gluc78 transgene which is driven by the pin2 promoter and followed by the rice Act1 intron sequences, was subjected to pathogenicity trials. One transgenic event exhibited a reduction of 58% in the incidence of S. graminicola infection, however other transgenic pearl millet events showed no resistance to this phytopathogen. The event conferring decreased susceptibility to S. graminicola had high levels of the glucanase transcript especially in transgenic plants showing higher levels of downy mildew infection.The European Commission contract number: ERBIC18 CT98 0316. Some results presented in this document are outputs from several research projects of the Plant Sciences Research Programme funded by the UK Department for International Development (DFID) and administered by the Centre for Arid Zone Studies (CAZS) for the benefit of developing countries.http://www.elsevier.com/locate/sajbnf201

Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum</it>, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting <it>P. falciparum </it>spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level.</p> <p>Results</p> <p>Morphological analysis of <it>P. falciparum </it>3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was also observed. Most notably, uridine phosphorylase, adenosine deaminase, lysine decarboxylase (LDC) and S-adenosylmethionine synthetase were differentially expressed at the transcript and/or protein level. Several genes in associated metabolic pathways (purine metabolism and various methyltransferases) were also affected. The specific nature of the perturbation was additionally reflected by changes in polyamine metabolite levels.</p> <p>Conclusions</p> <p>This study details the malaria parasite's response to PfSpdSyn inhibition on the transcriptomic, proteomic and metabolic levels. The results corroborate and significantly expand previous functional genomics studies relating to polyamine depletion in this parasite. Moreover, they confirm the role of transcriptional regulation in <it>P. falciparum</it>, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies.</p

Physiological responses of selected African sorghum landraces to progressive water stress and re-watering

Sorghumis particularly drought tolerant compared with other cereal crops and is favoured for subsistence farming in water scarce regions of the world. This study was conducted to identify South African sorghum landraces with superior drought tolerance compared with a drought-tolerant breeding line (P898012). Seedlings of 14 South African sorghumlandrace accessions were initially screened for drought tolerance by assessing percentage leaf water content (LWC) during progressive water deficit. Four landraces (designated LR5, LR6, LR35, and LR36) recorded higher LWC than P898012. These were subsequently evaluated with P898012 during the reproductive growth stage, for their physiological responses to mild (4 days) and severe (6 days)water stress treatments and a moderate re-watered treatment on day 7. Plant height, soil moisture, and LWC were measured during harvests. Chlorophyll, carotenoid, and proline contents were quantified. All five genotypes maintained LWC above 80% during mild and severe stress treatments. For LR35 and LR36, LWC were recorded within 8% less in comparison to their well-watered controls following the moderate re-watered treatment. Significantly higher chlorophyll and carotenoid contents were recorded for both LR6 and LR35 in comparison to P898012 during severe stress. When LWC was reduced in LR36 (to 73.68%) and LR35 (to 73.51%), their proline content significantly increased by 14- and 16-fold, respectively. In this study,we have identified four previously uncharacterised sorghum genotypes exhibiting drought tolerance and described their physiological responses during water deficit and moderate re-watering. Aside from their application to breeding, these landraces are valuable resources to elucidate genetic mechanisms that enable drought tolerance in South African sorghum.Council for Scientific and Industrial Research (CSIR) - Biosciences, Pretoria and the Gauteng Department of Agriculture and Rural Development (GDARD), South Africa. Ms Natrisha Devnarain was awarded a Professional Development Programme (PDP) PhD Scholarship from DST-NRF to work on this project.http://www.elsevier.com/locate/sajb2017-03-31hb201

Extract from Ceratonia siliqua exhibits depigmentation properties

Skin hyper-pigmentation is a condition initiated by the overproduction of melanin existing in the melanocytes. Melanin pigment is responsible for the colour of skin in humans. It is formed through a series of oxidative reactions involving the amino acid tyrosine in the presence of the key enzyme tyrosinase. In continuation with our efforts to identify tyrosinase inhibitors from plants sources, the methanol extract from leaf, bark and fruit of Ceratonia siliqua were screened for tyrosinase inhibition and diphenolase activity. The bark extract exhibited significant inhibition on mushroom tyrosinase using L-tyrosine as a substrate and showed diphenolase activity. The extract further significantly inhibited tyrosinase mRNA levels in B16-F10 mouse melanocytes. Bioassay-guided fractionation led to the isolation of six compounds. Compounds (-)-epicatechin-3-O-gallate, 1,2,3,6-tetra-O-galloyl-ß-D-glucose and Gallocatechin-3-O-gallate showed tyrosinase inhibitions with the IC50 values of 27.52, 83.30 and 28.30 μg/mL, respectively. These compounds also exhibited L-DOPA activities with IC50 values of >200, 150 and 200 μg/mL, respectively. A clinical study was conducted using 20 volunteers in a patch testing trial for irritancy potential and skin depigmentation. The clinical results showed the sample to be non-irritant with irritancy potential of -34.21 and depigmentation trial showed an improvement in the even skin tone of UV induced pigmentation at 3% after 28 days of application.University of Pretoria and National Research Foundation (NRF).http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1099-15732016-11-30hb2016GeneticsPlant Scienc