RESUMO
We used a systems genetics approach to elucidate the molecular mechanisms of the responses of maize to grey leaf spot (GLS) disease caused by Cercospora zeina, a threat to maize production globally. Expression analysis of earleaf samples in a subtropical maize recombinant inbred line population (CML444 × SC Malawi) subjected in the field to C. zeina infection allowed detection of 20 206 expression quantitative trait loci (eQTLs). Four trans-eQTL hotspots coincided with GLS disease QTLs mapped in the same field experiment. Co-expression network analysis identified three expression modules correlated with GLS disease scores. The module (GY-s) most highly correlated with susceptibility (r = 0.71; 179 genes) was enriched for the glyoxylate pathway, lipid metabolism, diterpenoid biosynthesis and responses to pathogen molecules such as chitin. The GY-s module was enriched for genes with trans-eQTLs in hotspots on chromosomes 9 and 10, which also coincided with phenotypic QTLs for susceptibility to GLS. This transcriptional network has significant overlap with the GLS susceptibility response of maize line B73, and may reflect pathogen manipulation for nutrient acquisition and/or unsuccessful defence responses, such as kauralexin production by the diterpenoid biosynthesis pathway. The co-expression module that correlated best with resistance (TQ-r; 1498 genes) was enriched for genes with trans-eQTLs in hotspots coinciding with GLS resistance QTLs on chromosome 9. Jasmonate responses were implicated in resistance to GLS through co-expression of COI1 and enrichment of genes with the Gene Ontology term 'cullin-RING ubiquitin ligase complex' in the TQ-r module. Consistent with this, JAZ repressor expression was highly correlated with the severity of GLS disease in the GY-s susceptibility network.
Assuntos
Folhas de Planta/genética , Folhas de Planta/microbiologia , Zea mays/genética , Zea mays/microbiologia , Ascomicetos/patogenicidade , Cromossomos de Plantas/genética , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Locos de Características Quantitativas/genéticaRESUMO
Gray leaf spot (GLS), caused by the sibling species Cercospora zeina or Cercospora zeae-maydis, is cited as one of the most important diseases threatening global maize production. C. zeina fails to produce cercosporin in vitro and, in most cases, causes large coalescing lesions during maize infection, a symptom generally absent from cercosporin-deficient mutants in other Cercospora spp. Here, we describe the C. zeina cercosporin toxin biosynthetic (CTB) gene cluster. The oxidoreductase gene CTB7 contained several insertions and deletions as compared with the C. zeae-maydis ortholog. We set out to determine whether complementing the defective CTB7 gene with the full-length gene from C. zeae-maydis could confer in vitro cercosporin production. C. zeina transformants containing C. zeae-maydis CTB7 were generated by Agrobacterium tumefaciens-mediated transformation and were evaluated for in vitro cercosporin production. When grown on nitrogen-limited medium in the light-conditions conducive to cercosporin production in other Cercospora spp.-one transformant accumulated a red pigment that was confirmed to be cercosporin by the KOH assay, thin-layer chromatography, and ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Our results indicated that C. zeina has a defective CTB7, but all other necessary machinery required for synthesizing cercosporin-like molecules and, thus, C. zeina may produce a structural variant of cercosporin during maize infection.
Assuntos
Ascomicetos/genética , Proteínas Fúngicas/genética , Teste de Complementação Genética , Perileno/análogos & derivados , Zea mays/microbiologia , Processamento Alternativo/genética , Sequência de Aminoácidos , Ascomicetos/isolamento & purificação , Sequência de Bases , Vias Biossintéticas/genética , Simulação por Computador , Sequência Conservada/genética , DNA Fúngico/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Íntrons/genética , Espectrometria de Massas , Família Multigênica , Oxirredutases/metabolismo , Perileno/metabolismo , Transcrição Gênica , Transformação GenéticaRESUMO
South Africa is one of the leading maize-producing countries in sub-Saharan Africa. Since the 1980s, Cercospora zeina, a causal agent of gray leaf spot of maize, has become endemic in South Africa, and is responsible for substantial yield reductions. To assess genetic diversity and population structure of C. zeina in South Africa, 369 isolates were collected from commercial maize farms in three provinces (KwaZulu-Natal, Mpumalanga, and North West). These isolates were evaluated with 14 microsatellite markers and species-specific mating type markers that were designed from draft genome sequences of C. zeina isolates from Africa (CMW 25467) and the United States (USPA-4). Sixty alleles were identified across 14 loci, and gene diversity values within each province ranged from 0.18 to 0.35. High levels of gene flow were observed (Nm = 5.51), and in a few cases, identical multilocus haplotypes were found in different provinces. Overall, 242 unique multilocus haplotypes were identified with a low clonal fraction of 34%. No distinct population clusters were identified using STRUCTURE, principal coordinate analysis, or Weir's theta θ statistic. The lack of population differentiation was supported by analysis of molecular variance tests, which indicated that only 2% of the variation was attributed to variability between populations from each province. Mating type ratios of MAT1-1 and MAT1-2 idiomorphs from 335 isolates were not significantly different from a 1:1 ratio in all provinces, which provided evidence for sexual reproduction. The draft genome of C. zeina CMW 25467 exhibited a complete genomic copy of the MAT1-1 idiomorph as well as exonic fragments of MAT genes from both idiomorphs. The high level of gene diversity, shared haplotypes at different geographical locations within South Africa, and presence of both MAT idiomorphs at all sites indicates widespread dispersal of C. zeina between maize fields in the country as well as evidence for sexual recombination. The outcomes of this genome-enabled study are important for disease management since the high diversity has implications for dispersal of fungicide resistance should it emerge and the need for diversified resistance breeding.
Assuntos
Ascomicetos/genética , Variação Genética , Genética Populacional , Genoma Fúngico/genética , Doenças das Plantas/microbiologia , Zea mays/microbiologia , Ascomicetos/isolamento & purificação , Fluxo Gênico , Geografia , Repetições de Microssatélites/genética , Análise de Sequência de DNA , África do SulRESUMO
The aim of any breeding process is to fully express the targeted, superior/desirable parent characteristic in the progeny. Hybrids are often used in this dynamic, and complex process for which homozygous parents-which may require up to eight generations of back crossing and selection-are required. Doubled haploid (DH) technologies can facilitate the production of true breeding lines faster and in a more efficient manner than the traditional back crossing and selection strategies. Sunflower is the third most important oilseed crop in the world and has no available double haploid induction procedure/technique that can be efficiently used in breeding programs. A reproducible and efficient doubled haploid induction method would be a valuable tool in accelerating the breeding of new elite sunflower varieties. Although several attempts have been made, the establishment of a sunflower doubled haploid induction protocol has remained a challenge owing recalcitrance to in vitro culture regeneration. Approaches for haploid development in other crops are often cultivar specific, difficult to reproduce, and rely on available tissue culture protocols-which on their own are also cultivar and/or species specific. As an out-crossing crop, the lack of a double haploid system limits sunflower breeding and associated improvement processes, thereby delaying new hybrid and trait developments. Significant molecular advances targeting genes, such as the centromeric histone 3 (CenH3) and Matrilineal (MTL) gene with CRISPR/Cas9, and the successful use of viral vectors for the delivery of CRISPR/Cas9 components into plant cells eliminating the in vitro culture bottleneck, have the potential to improve double haploid technology in sunflower. In this review, the different strategies, their challenges, and opportunities for achieving doubled haploids in sunflower are explored.
RESUMO
BACKGROUND: Anti-malarial drug resistance threatens to undermine efforts to eliminate this deadly disease. The resulting omnipresent requirement for drugs with novel modes of action prompted a national consortium initiative to discover new anti-plasmodial agents from South African medicinal plants. One of the plants selected for investigation was Dicoma anomala subsp. gerrardii, based on its ethnomedicinal profile. METHODS: Standard phytochemical analysis techniques, including solvent-solvent extraction, thin-layer- and column chromatography, were used to isolate the main active constituent of Dicoma anomala subsp. gerrardii. The crystallized pure compound was identified using nuclear magnetic resonance spectroscopy, mass spectrometry and X-ray crystallography. The compound was tested in vitro on Plasmodium falciparum cultures using the parasite lactate dehydrogenase (pLDH) assay and was found to have anti-malarial activity. To determine the functional groups responsible for the activity, a small collection of synthetic analogues was generated - the aim being to vary features proposed as likely to be related to the anti-malarial activity and to quantify the effect of the modifications in vitro using the pLDH assay. The effects of the pure compound on the P. falciparum transcriptome were subsequently investigated by treating ring-stage parasites (alongside untreated controls), followed by oligonucleotide microarray- and data analysis. RESULTS: The main active constituent was identified as dehydrobrachylaenolide, a eudesmanolide-type sesquiterpene lactone. The compound demonstrated an in vitro IC50 of 1.865 µM against a chloroquine-sensitive strain (D10) of P. falciparum. Synthetic analogues of the compound confirmed an absolute requirement that the α-methylene lactone be present in the eudesmanolide before significant anti-malarial activity was observed. This feature is absent in the artemisinins and suggests a different mode of action. Microarray data analysis identified 572 unique genes that were differentially expressed as a result of the treatment and gene ontology analysis identified various biological processes and molecular functions that were significantly affected. Comparison of the dehydrobrachylaenolide treatment transcriptional dataset with a published artesunate (also a sesquiterpene lactone) dataset revealed little overlap. These results strengthen the notion that the isolated compound and the artemisinins have differentiated modes of action. CONCLUSIONS: The novel mode of action of dehydrobrachylaenolide, detected during these studies, will play an ongoing role in advancing anti-plasmodial drug discovery efforts.
Assuntos
Antimaláricos/farmacologia , Asteraceae/química , Extratos Vegetais/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Sesquiterpenos/farmacologia , Relação Estrutura-Atividade , Antimaláricos/química , Antimaláricos/isolamento & purificação , Antimaláricos/metabolismo , Asteraceae/genética , Asteraceae/metabolismo , Fracionamento Químico , Cromatografia , Cristalografia por Raios X , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Análise em Microsséries , Extratos Vegetais/isolamento & purificação , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/metabolismo , África do SulRESUMO
BACKGROUND: Plasmodium falciparum, 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 P. falciparum spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level. RESULTS: Morphological analysis of P. falciparum 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. CONCLUSIONS: 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 P. falciparum, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Espermidina Sintase/antagonistas & inibidores , Cicloexilaminas/farmacologia , Perfilação da Expressão Gênica , Redes e Vias Metabólicas , Plasmodium falciparum/enzimologia , Poliaminas/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
Phytophthora cinnamomi Rands (Pc) is a hemibiotrophic oomycete and the causal agent of Phytophthora root rot (PRR) of the commercially important fruit crop avocado (Persea americana Mill.). Plant defense against pathogens is modulated by phytohormone signaling pathways such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET), auxin and abscisic acid. The role of specific signaling pathways induced and regulated during hemibiotroph-plant interactions has been widely debated. Some studies report SA mediated defense while others hypothesize that JA responses restrict the spread of pathogens. This study aimed to identify the role of SA- and JA- associated genes in the defense strategy of a resistant avocado rootstock, Dusa in response to Pc infection. Transcripts associated with SA-mediated defense pathways and lignin biosynthesis were upregulated at 6 hours post-inoculation (hpi). Results suggest that auxin, reactive oxygen species (ROS) and Ca2+ signaling was also important during this early time point, while JA signaling was absent. Both SA and JA defense responses were shown to play a role during defense at 18 hpi. Induction of genes associated with ROS detoxification and cell wall digestion (ß-1-3-glucanase) was also observed. Most genes induced at 24 hpi were linked to JA responses. Other processes at play in avocado at 24 hpi include cell wall strengthening, the formation of phenolics and induction of arabinogalactan, a gene linked to Pc zoospore immobility. This study represents the first transcriptome wide analysis of a resistant avocado rootstock treated with SA and JA compared to Pc infection. The results provide evidence of a biphasic defense response against the hemibiotroph, which initially involves SA-mediated gene expression followed by the enrichment of JA-mediated defense from 18 to 24 hpi. Genes and molecular pathways linked to Pc resistance are highlighted and may serve as future targets for manipulation in the development of PRR resistant avocado rootstocks.
Assuntos
Regulação da Expressão Gênica de Plantas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Persea/imunologia , Phytophthora/patogenicidade , Doenças das Plantas/imunologia , Ácido Abscísico/imunologia , Ácido Abscísico/metabolismo , Ciclopentanos/imunologia , Ciclopentanos/metabolismo , Etilenos/imunologia , Etilenos/metabolismo , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Ácidos Indolacéticos/imunologia , Ácidos Indolacéticos/metabolismo , Oxilipinas/imunologia , Oxilipinas/metabolismo , Persea/genética , Persea/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/imunologia , Raízes de Plantas/microbiologia , Ácido Salicílico/imunologia , Ácido Salicílico/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
A quantitative screening method was developed to evaluate the quality of cDNA libraries constructed by suppression subtraction hybridization (SSH) or other enrichment techniques. The SSH technique was adapted to facilitate screening of the resultant library on a small number of glass slide microarrays. A simple data analysis pipeline named SSHscreen using "linear models for microarray data" (limma) functions in the R computing environment was developed to identify clones in the cDNA libraries that are significantly differentially expressed, and to determine if they were rare or abundant in the original treated sample. This approach facilitates the choice of clones from the cDNA library for further analysis, such as DNA sequencing, Northern blotting, RT-PCR, or detailed expression profiling using a custom cDNA microarray. Furthermore, this strategy is particularly useful for studies of nonmodel organisms for which there is little genome sequence information.
Assuntos
Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Vidro/química , Hibridização de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Pennisetum/genética , Proteínas de Plantas/genética , RNA de Plantas/genética , Clonagem Molecular , Pennisetum/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas/metabolismo , Supressão Genética/genéticaRESUMO
The genomes of Cercospora zeina, Fusarium pininemorale, Hawksworthiomyces lignivorus, Huntiella decipiens, and Ophiostoma ips are presented in this genome announcement. Three of these genomes are from plant pathogens and otherwise economically important fungal species. Fusarium pininemorale and H. decipiens are not known to cause significant disease but are closely related to species of economic importance. The genome sizes range from 25.99 Mb in the case of O. ips to 4.82 Mb for H. lignivorus. These genomes include the first reports of a genome from the genus Hawksworthiomyces. The availability of these genome data will allow the resolution of longstanding questions regarding the taxonomy of these species. In addition these genome sequences through comparative studies with closely related organisms will increase our understanding of how these species or close relatives cause disease.
RESUMO
Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression subtractive hybridization (SSH). The methodology was applied to two independent SSHs from pearl millet and banana. Following two-color cyanin dye labeling and hybridization of subtracted tester with either unsubtracted driver or unsubtracted tester cDNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). Graphical representation of ER1 and ER2 enabled the identification of clones that were likely to represent up-regulated transcripts. Normalization of each clone by the SSH process was determined from the ER2 values, thereby indicating whether clones represented rare or abundant transcripts. Differential expression of pearl millet and banana clones identified from both libraries by this quantitative approach was verified by inverse Northern blot analysis.
Assuntos
DNA de Plantas/genética , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Hibridização Genética/genética , Musa/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Pennisetum/genética , Proteínas de Plantas/genética , Musa/metabolismo , Pennisetum/metabolismo , Proteínas de Plantas/metabolismo , Supressão Genética/genéticaRESUMO
Studies were undertaken to assess the induction of defence response pathways in pearl millet (Pennisetum glaucum) in response to infection with the leaf rust fungus Puccinia substriata. Pretreatment of pearl millet with salicylic acid (SA) conferred resistance to a virulent isolate of the rust fungus, whereas methyl jasmonate (MeJA) did not significantly reduce infection levels. These results suggest that the SA defence pathway is involved in rust resistance. In order to identify pearl millet genes that are specifically regulated in response to SA and not MeJA, and thus could play a role in resistance to P. substriata, gene expression profiling was performed. Substantial overlap in gene expression responses between the treatments was observed, with MeJA and SA treatments exhibiting 17% co-regulated transcripts. However, 34% of transcripts were differentially expressed in response to SA treatment, but not in response to MeJA treatment. SA-responsive transcripts represented genes involved in SA metabolism, defence response, signal transduction, protection from oxidative stress and photosynthesis. The expression profiles of pearl millet plants after treatment with SA or MeJA were more similar to one another than to the response during a compatible infection with P. substriata. However, some SA-responsive genes were repressed during P. substriata infection, indicating possible manipulation of host responses by the pathogen.