Browsing by Author "Murray, Shane L"

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    Open Access
    Characterisation of a maize mutant deficient in antifungal kauralexin accumulation
    (2017) Wighard, Sara; Murray, Shane L; Korsman, Jeanne
    Fusarium verticillioides and Cercospora zeina are two economically important fungal pathogens of maize in Southern Africa. Phytoalexins are low molecular weight anti-microbial compounds produced in plants in response to pathogen infection. In maize, two classes of non-volatile terpenoid phytoalexins, viz. kauralexins and zealexins, play a role in fungal resistance. It has previously been shown that maize lines inoculated with either F. verticillioides or C. zeina induces kauralexin and zealexin accumulation. In addition, kauralexin metabolite accumulation and candidate kauralexin biosynthetic gene expression were highly correlated. In this study a mutant line with a Dissociation transposon element inserted into An2 was identified with the goal of stopping An2 from being expressed. The mutants were maintained in an inbred W22 maize line. Gene expression was compared between transposon-insertion mutants and wild type W22 at the seedling stage. A F. verticillioides and C. zeina inoculation assay was carried out on a segregating knock-down line. Phytoalexin accumulation, gene expression and disease susceptibility were subsequently examined in the mutants and wild type. F. verticillioides-inoculated mutants displayed significantly decreased kauralexin and zealexins accumulation and An2 gene expression. Fungal load and symptoms was greater in mutants than wild type controls. Kauralexin accumulation and An2 expression were negatively correlated with the quantified fungal load. C. zeina-inoculated mutants did not display significantly reduced kauralexin accumulation and An2 expression as An2 did not appear to be upregulated in the W22 maize line in response to C. zeina. This is likely due to a genetically-controlled leaf flecking phenotype in W22 leading to broad-spectrum resistance, as well as potentially impacting the jasmonic acid pathway. Lastly, an attempt was made to clone An2 towards A. thaliana transformation for overexpression analysis. Only a truncated section of An2 was able to be cloned into the expression vector.
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    Evaluation of southern African maize germplasm for phytoalexin accumulation following inoculation by Fusarium verticillioides
    (2017) Veenstra, Amy; Murray, Shane L; Rafudeen, Suhail
    Maize is a socially and economically important crop in Africa (and worldwide) that is severely affected by many fungal pathogens. The pathogen Fusarium verticillioides causes Fusarium ear rot in maize, a disease that greatly reduces quantity and quality of annual maize yields. The pathogen produces mycotoxins called fumonisins, which have been linked to adverse health effects in both humans and animals. Maize produces terpenoid phytoalexins, which are antimicrobial compounds that directly reduce the growth of many fungal pathogens including F. verticillioides. Two families of maize phytoalexins, termed kauralexins and zealexins, have been characterized. Key genes putatively involved in the biosynthetic pathway of these phytoalexins have been identified from the rice model and subsequent studies on maize. This research aimed to evaluate the correlation between phytoalexin accumulation and fungal growth in diverse southern African maize lines in response to F. verticillioides inoculation. Maize lines were inoculated with F. verticillioides using a seed soak inoculation method and grown in vitro for up to two weeks. The harvested tissue was analysed for fungal growth using quantitative PCR, putative phytoalexin biosynthetic gene expression using RT-qPCR and phytoalexin accumulation using gas-chromatography mass spectrometry. Furthermore, an endophyte growing in one of the maize lines was isolated and identified as Trichoderma asperellum. Trichoderma spp. are used as biocontrol agents against many fungal pathogens, although research on the specific antagonistic effect of T. asperellum on F. verticillioides is limited. Phytoalexin accumulation in maize containing endophytic T. asperellum was compared to maize inoculated with F. verticillioides. In vitro competition assays were performed to analyse the antagonistic effect of T. asprellum on F. verticillioides. Results from this study show that inoculation of maize lines with F. verticillioides induces the accumulation of total phytoalexins, and more specifically the accumulation of total kauralexins. Putative phytoalexin biosynthetic genes are also up-regulated in response to inoculation. Maize growing with a T. asperellum endophyte accumulated phytoalexins to the same levels as F. verticillioides, suggesting that T. asperellum induces a defence response that 'primes' the plant for further infection. In vitro competition assays between F. verticillioides and T. asperellum showed that T. asperellum significantly inhibits F. verticillioides growth. These results will aid in the identification of maize lines that can be bred with increased resistance to F. verticillioides with the goal to reduce F. verticillioides incidence in southern Africa. Furthermore, analysis of the efficacy of T. asperellum as an antagonist against F. verticillioides may provide another method for disease reduction in the field.
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    Increased resistance to biotrophic pathogens in the Arabidopsis constitutive induced resistance 1 mutant is EDS1 and PAD4-dependent and modulated by environmental temperature
    (Public Library of Science, 2014) Carstens, Maryke; McCrindle, Tyronne K; Adams, Nicolette; Diener, Anastashia; Guzha, Delroy T; Murray, Shane L; Parker, Jane E; Denby, Katherine J; Ingle, Robert A
    The Arabidopsis constitutive induced resistance 1 ( cir1 ) mutant displays salicylic acid (SA)-dependent constitutive expression of defence genes and enhanced resistance to biotrophic pathogens. To further characterise the role of CIR1 in plant immunity we conducted epistasis analyses with two key components of the SA-signalling branch of the defence network, ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4). We demonstrate that the constitutive defence phenotypes of cir1 require both EDS1 and PAD4, indicating that CIR1 lies upstream of the EDS1-PAD4 regulatory node in the immune signalling network. In light of this finding we examined EDS1 expression in cir1 and observed increased protein, but not mRNA levels in this mutant, suggesting that CIR1 might act as a negative regulator of EDS1 via a post-transcriptional mechanism. Finally, as environmental temperature is known to influence the outcome of plant-pathogen interactions, we analysed cir1 plants grown at 18, 22 or 25°C. We found that susceptibility to Pseudomonas syringae pv. tomato ( Pst ) DC3000 is modulated by temperature in cir1 . Greatest resistance to this pathogen (relative to PR-1:LUC control plants) was observed at 18°C, while at 25°C no difference in susceptibility between cir1 and control plants was apparent. The increase in resistance to Pst DC3000 at 18°C correlated with a stunted growth phenotype, suggesting that activation of defence responses may be enhanced at lower temperatures in the cir1 mutant.
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    RNA-Seq analysis of resistant and susceptible sub-tropical maize lines reveals a role for kauralexins in resistance to grey leaf spot disease, caused by Cercospora zeina
    (BioMed Central, 2017-11-13) Meyer, Jacqueline; Berger, Dave K; Christensen, Shawn A; Murray, Shane L
    Background: Cercospora zeina is a foliar pathogen responsible for maize grey leaf spot in southern Africa that negatively impacts maize production. Plants use a variety of chemical and structural mechanisms to defend themselves against invading pathogens such as C. zeina, including the production of secondary metabolites with antimicrobial properties. In maize, a variety of biotic and abiotic stressors induce the accumulation of the terpenoid phytoalexins, zealexins and kauralexins. Results: C. zeina-susceptible line displayed pervasive rectangular grey leaf spot lesions, running parallel with the leaf veins in contrast to C. zeina-resistant line that had restricted disease symptoms. Analysis of the transcriptome of both lines indicated that genes involved in primary and secondary metabolism were up-regualted, and although different pathways were prioritized in each line, production of terpenoid compounds were common to both. Targeted phytoalexin analysis revealed that C. zeina-inoculated leaves accumulated zealexins and kauralexins. The resistant line shows a propensity toward accumulation of the kauralexin B series metabolites in response to infection, which contrasts with the susceptible line that preferentially accumulates the kauralexin A series. Kauralexin accumulation was correlated to expression of the kauralexin biosynthetic gene, ZmAn2 and a candidate biosynthetic gene, ZmKSL2. We report the expression of a putative copalyl diphosphate synthase gene that is induced by C. zeina in the resistant line exclusively. Discussion: This study shows that zealexins and kauralexins, and expression of their biosynthetic genes, are induced by C. zeina in both resistant and susceptible germplasm adapted to the southern African climate. The data presented here indicates that different forms of kauralexins accumulate in the resistant and susceptible maize lines in response to C. zeina, with the accumulation of kauralexin B compounds in a resistant maize line and kauralexin A compounds accumulating in the susceptible line.