Browsing by Author "Ingle, Robert A"

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    Open Access
    Characterisation of phytoalexin accumulation in maize inoculated with Cercospora zeina, the causal organism of grey leaf spot disease
    (2016) Ntuli, Jean Felistas; Murray, Shane; Ingle, Robert A
    Grey Leaf Spot (GLS) is a fungal disease of Zea mays (maize) that is caused by Cercospora zeina. It thrives in sub-tropical climates and causes devastating crop losses of up to 60% in southern Africa where maize is grown as a staple food source. Phytoalexins are low molecular weight anti-microbial bio-chemicals that are synthesised in planta in response to biotic stress. Related studies have characterised many phytoalexins produced in various plants against several diseases. In maize, phytoalexins fall into to two terpenoid groups: kauralexins and zealexins. To date no studies have been carried out that examine the accumulation in maize of phytoalexins in response to C. zeina. This research project found that in maize samples inoculated with C. zeina, kauralexin accumulation significantly increased with disease development stages (T0 - 0 days post inoculation, T1 - 17 dpi, T2 - 18 dpi and T3 - 24 dpi) while zealexins did not change. Gene expression of the phytoalexin biosynthesis genes TPS6 and TPS11 (both encoding the protein terpene synthase 6/11, specific for zealexins) and CPPS2 (encoding ent-copalyl diphosphate synthase 2, specific for kauralexins) increased significantly at each time point, reaching a maximum level at T2. Infiltration of maize leaves with a chitosan elicitor to mimic fungal pathogen associated molecular pattern (PAMP), and a subsequent callose assay showed positive induction of a callose defence response. However, gene expression and phytoalexin accumulation did not change following chitosan treatment, although zealexin accumulation was higher than kauralexins. Previous studies have shown that phytoalexins accumulate transiently in seedlings. Six diverse Southern African maize lines were compared for phytoalexin accumulation at seedling stage. Zealexin accumulation was generally higher than kauralexins and there were significant differences in both zealexin and kauralexin accumulation in different lines. Gene expression analysis using Genevestigator looked at microarray files and found that expression of TPS6/11 (zealexin biosynthesis) and CPPS2 (kauralexin biosynthesis) genes to be largely co-regulated and highly expressed in response to fungal pathogens, nematodes, insect pests and abiotic stresses; Ustilago maydis, Phytophthora cinnamomi, Fusarium moniliforme, Colletotrichum graminicola, Sporisorium reilianum, Meloidogyne incognita, Ostrinia nubilalis, waterlogging and drought stress. Finally promoter region analysis showed similar cis-acting regulatory elements in the 1kb region upstream of the promoter of both genes and defence specific elements. Thus kauralexin phytoalexins are produced in response to C. zeina inoculation, chitin is not likely to be the key PAMP leading to phytoalexin accumulation, phytoalexin accumulation in seedlings is genotype-dependent and phytoalexin biosynthesis genes are expressed under different conditions suggesting a wider range of action beyond repelling fungal pathogens.
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    Characterisation of putative metal transport proteins in the nickel hyperaccumulator Senecio coronatus: investigating candidate genes for nickel tolerance and accumulation
    (2017) Cowlin, Ross Martin; Ingle, Robert A
    The accumulation of exceptionally high concentrations of heavy metals in plant tissues is an extreme phenotypic trait that has evolved independently in multiple plant taxa. The majority of research undertaken in this area has been performed on zinc/cadmium hyperaccumulators and comparatively little is known about the molecular mechanisms behind nickel accumulation. This is despite the fact that nickel hyperaccumulators constitute more than 75% of all known hyperaccumulator species. One such species is Senecio coronatus (Asteraceae), which is a useful model to study nickel hyperaccumulation - as both hyperaccumulator and non-accumulator populations have been identified on nickel-rich serpentine soils in South Africa. The nickel-transporting abilities of three proteins (ScMATE, ScVIT and ScCOP), previously shown to be constitutively over-expressed in shoot tissues of hyperaccumulating populations of S. coronatus, were investigated in order to determine if they play a role in nickel hyperaccumulation. The RNA-Seq derived nucleotide sequences of these genes were confirmed by reverse transcriptase PCR, and computational analysis suggested that the proteins encoded by these genes display identical topology to their homologues in Arabidopsis thaliana. Heterologous expression of these proteins in a metal-sensitive yeast strain was performed to determine whether they are capable of transporting nickel. Although a minor reduction in nickel sensitivity was observed in yeast expressing ScMATE, and a minor increase in ScCOP-expressing yeast, no marked changes in sensitivity to nickel were observed. C-terminal EYFP-tagged MATE and VIT fusion proteins were transiently expressed in live onion cells to determine the subcellular localization of these proteins in planta. Fluorescence microscopy indicated that MATE localises to the nucleus and VIT to the tonoplast or plasma membrane.
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    Characterisation of the AT4G11100 gene, a negative regulator of disease resistance in Arabidopsis thaliana
    (2015) McCrindle, Tyronne K; Ingle, Robert A
    Plants have evolved a complex system of defence to prevent pathogen establishment. The Arabidopsis thaliana cir1 (constitutively induced resistance 1) mutant displays enhanced resistance to infection by the virulent bacterial pathogen Pseudomonas syringae and constitutively expresses a number of defence genes. Evidence suggests that CIR1 is a negative regulator of plant immunity important in the absence of pathogen attack. Genetic mapping experiments indicate that cir1 is located on the lower arm of chromosome 4 of A. thaliana and may be one of 8 known genes in the region. Analysis of T-DNA knockouts of these 8 genes suggests that AT4G11100 is the mostly likely candidate for CIR1. This project established that the disease resistance phenotype of cir1 is temperature dependent and linked to reduced plant growth. Genetic crosses between cir1 and at4g11100 T-DNA knockout mutants revealed that the mutants complement and therefore AT4G11100 is not CIR1. However, like cir1, the at4g11100 T-DNA knockout mutants display enhanced disease resistance. Over expression of AT4G11100 leads to increased susceptibility to infection by Pseudomonas syringae (Pst) and reduced induction of the salicylic acid defence gene PR2 following Pst infection, suggesting that AT4G11100 may too be a negative regulator of immunity. Additionally, a plant line with exceptionally high AT4G11100 expression levels displayed distinct leaf morphology, possibly implicating AT4G11100 in leaf development.
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    Defence responses of Arabidopsis thaliana to infection by Pseudomonas syringae are regulated by the circadian clock
    (Public Library of Science, 2011) Bhardwaj, Vaibhav; Meier, Stuart; Petersen, Lindsay N; Ingle, Robert A; Roden, Laura C
    The circadian clock allows plants to anticipate predictable daily changes in abiotic stimuli, such as light; however, whether the clock similarly allows plants to anticipate interactions with other organisms is unknown. Here we show that Arabidopsis thaliana (Arabidopsis) has circadian clock-mediated variation in resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 ( Pst DC3000), with plants being least susceptible to infection in the subjective morning. We suggest that the increased resistance to Pst DC3000 observed in the morning in Col-0 plants results from clock-mediated modulation of pathogen associated molecular pattern (PAMP)-triggered immunity. Analysis of publicly available microarray data revealed that a large number of Arabidopsis defence-related genes showed both diurnal- and circadian-regulation, including genes involved in the perception of the PAMP flagellin which exhibit a peak in expression in the morning. Accordingly, we observed that PAMP-triggered callose deposition was significantly higher in wild-type plants inoculated with Pst DC3000 hrpA in the subjective morning than in the evening, while no such temporal difference was evident in arrhythmic plants. Our results suggest that PAMP-triggered immune responses are modulated by the circadian clock and that temporal regulation allows plants to anticipate and respond more effectively to pathogen challenges in the daytime.
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    ERF5 and ERF6 play redundant roles as positive regulators of JA/Et-mediated defense against Botrytis cinerea in Arabidopsis
    (Public Library of Science, 2012) Moffat, Caroline S; Ingle, Robert A; Wathugala, Deepthi L; Saunders, Nigel J; Knight, Heather; Knight, Marc R
    The ethylene response factor (ERF) family in Arabidopsis thaliana comprises 122 members in 12 groups, yet the biological functions of the majority remain unknown. Of the group IX ERFs, the IXc subgroup has been studied the most, and includes ERF1, ERF14 and ORA59, which play roles in plant innate immunity. Here we investigate the biological functions of two members of the less studied IXb subgroup: ERF5 and ERF6. In order to identify potential targets of these transcription factors, microarray analyses were performed on plants constitutively expressing either ERF5 or ERF6 . Expression of defense genes, JA/Et-responsive genes and genes containing the GCC box promoter motif were significantly upregulated in both ERF5 and ERF6 transgenic plants, suggesting that ERF5 and ERF6 may act as positive regulators of JA-mediated defense and potentially overlap in their function. Since defense against necrotrophic pathogens is generally mediated through JA/Et-signalling, resistance against the fungal necrotroph Botrytis cinerea was examined. Constitutive expression of ERF5 or ERF6 resulted in significantly increased resistance. Although no significant difference in susceptibility to B. cinerea was observed in either erf5 or erf6 mutants, the erf5 erf6 double mutant showed a significant increase in susceptibility, which was likely due to compromised JA-mediated gene expression, since JA-induced gene expression was reduced in the double mutant. Taken together these data suggest that ERF5 and ERF6 play positive but redundant roles in defense against B. cinerea . Since mutual antagonism between JA/Et and salicylic acid (SA) signalling is well known, the UV-C inducibility of an SA-inducible gene, PR-1 , was examined. Reduced inducibilty in both ERF5 and ERF6 constitutive overexepressors was consistent with suppression of SA-mediated signalling, as was an increased susceptibility to avirulent Pseudomonas syringae . These data suggest that ERF5 and ERF6 may also play a role in the antagonistic crosstalk between the JA/Et and SA signalling pathways.
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    Functional characterisation of ScIRT1 and ScIREG2 transport proteins in the nickel hyperaccumulator, Senecio coronatus
    (2020) van der Pas, Llewelyn; Ingle, Robert A
    Nickel hyperaccumulation is a unique plant adaption that has led to roughly 390 plant taxa being able to not only withstand the toxicity associated with Ni but actively translocate it to aerial tissues. However, the underlining molecular mechanisms that drive Ni hyperaccumulation remain unclear. Senecio coronatus, a Ni hyperaccumulator, is a novel species as both hyperaccumulating and non-accumulating populations can be found on the serpentine soils of the Barberton Greenstone Belt, South Africa. A comparative RNA-seq analysis on these populations of S. coronatus revealed that ScIRT1 and ScIREG2 , putative homologues of the Arabidopsis transporters, AtIRT1 and AtIREG2 which are capable of transporting Ni, showed much higher expression in the hyperaccumulating populations compared to the non-hyperaccumulating populations, suggesting a potential role in Ni hyperaccumulation. It was thus necessary to investigate whether ScIRT1 and ScIREG2 encode functional homologues of these Arabidopsis transporters. To accomplish this, irt1 and ireg2 mutants were obtained from a T-DNA insertion seed collection and their homozygosity was then determined by PCR genotyping. Since a lack of iron induces IRT1 and IREG2 expression, loss of gene expression of homozygous irt1 and ireg2 mutants by means of reverse transcriptase PCR on plant roots grown hydroponically in the absence of Fe was then done to establish full knock-out status. From this, homozygous mutants were identified, however, absence of gene expression for irt1 and ireg2 mutants was not clear. In addition to validating homozygosity, phenotypic characterisation, with the aim of developing reliable assays to be used in complementation analysis, was done by growing homozygous mutants and Col-0 in hydroponic media deficient in Fe and supplemented with Ni. The assays revealed that under Fe-deficient and Ni-supplemented conditions, a reduction in root biomass was a more reliable phenotypic characteristic for ireg2 mutants than root length or shoot biomass. In contrast, for irt1, no observable phenotype was established under Fe-deficiency conditions. In parallel, Gateway cloning was employed to create expression clones where ScIRT1 and ScIREG2 protein coding expression was to be driven by native Arabidopsis promoters pAtIRT1 and pAtIREG2 (i.e. pAtIRT1:ScIRT1 and pAtIREG2:ScIREG2) respectively for complementation of the Arabidopsis irt1 and ireg2 mutants. The open reading frames of the S. coronatus genes and the Arabidopsis promoters were PCR amplified, cloned into appropriate pDONR221 vectors, and sequence verified. The ScIREG2 clone however, revealed point mutations and could not be used. pAtIRT1 was successfully recombined with ScIRT1 to generate a two-fragment expression clone which was verified by DNA sequencing. Thus herein, the foundations for ScIRT1 and ScIREG2 complementation experiments have been established.
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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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    Regulation of desiccation tolerance in Xerophyta seedlings and leaves
    (2016) Lyall, Rafe; Illing, Nicola; Ingle, Robert A
    A small, diverse group of angiosperms known as resurrection plants display vegetative desiccation tolerance and can survive loss of up to 95% of cellular water, a feat only seen in the seeds and pollen of other angiosperms. Xerophyta humilis is a resurrection plant native to Southern Africa that has been the target of previous transcriptomic and proteomic studies into the mechanisms of plant desiccation tolerance. The aim of this study was to investigate the hypothesis that vegetative desiccation tolerance is derived from the networks that control desiccation tolerance in seeds and germinating seedlings in angiosperms, particularly the epigenetically silenced seed maturation genes. Germinating seedlings of X. humilis and the related resurrection plant X. viscosa were found to be VDT from the earliest stages of germination, and exhibited the characteristic vegetative trait of poikilochlorophylly as seen in mature leaves. The X. humilis desiccation transcriptome comprising 76,768 distinct gene clusters was successfully assembled from sequencing samples at five relative water contents (100%, 80%, 60%, 40% and 5%) to identify the networks activated in response to water loss. Desiccation was associated with successive waves of transcription factor induction, as well as widespread down-regulation of histone modification enzymes. Many seed-specific genes, such as late embryogenesis abundant (LEA) proteins, seed storage proteins and oleosins, were induced in vegetative tissue. LEA transcripts in particular were highly up-regulated during desiccation, and the large number of distinct LEA transcripts (over 150) suggests possible LEA gene expansion in Xerophyta compared to desiccation-sensitive plants. Components of the PYL/SnRK2/ABF ABA-signalling pathway were also induced, although the ABF transcription factors activated in response to desiccation were most similar to those induced by drought in A. thaliana rather than seed maturation. Of the canonical seed master regulators (such as the LEC1/ABI3/FUS3/LEC2 network and ABI5) only three ABI3 transcripts were expressed, all of which encoded proteins lacking the seed motif-binding B3-domain. The results of this study suggest that vegetative desiccation tolerance in X. humilis is not associated with re-activation of seed master regulators in vegetative tissue, but may instead involve activation of seed genes by vegetative drought response regulators.
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    The role of citrate in plant-pathogen interactions
    (2016) Hendry, Tia Lynne; Ingle, Robert A
    Bacterial plant pathogens have evolved a wide range of mechanisms to suppress the immune response that they trigger in their hosts, including the production of effectors and phytotoxins. The tri-carboxylic acid citrate, which is secreted into the apoplast by both bacterial pathogens and plant hosts has previously been shown to increase the virulence of the gram negative pathogen Pseudomonas syringae DC3000 (Pst DC3000), by acting both as a chemoattractant and as an inducer of genes associated with the type III secretion system (T3SS) and phytotoxin production. The effect of citrate on the host is less clear, though microarray analysis of Arabidopsis thaliana has demonstrated that application of exogenous citrate leads to the differential expression of 1876 genes suggesting that it might act as a metabolic signal for transcriptional reprogramming. In this study, functional enrichment analysis revealed statistically significant enrichment for gene ontology terms associated with defence in both citrate up-regulated and down-regulated gene sets. Furthermore this project demonstrated that exogenous citrate can increase the success of virulent Pst DC3000 infection in Arabidopsis; bacterial titres in plants pre-treated with citrate 24 hours prior to infection were significantly higher than those in control plants. This phenomenon was also observed in plants pre-treated with a non-metabolisable citrate analogue but not in plants pre-treated with another TCA cycle intermediate, malate, suggesting that it is citrate specific. However, it remains unclear whether the increased apoplastic citrate concentrations lead to increased bacterial titres through a suppressive effect on the host immune response, an enhanced induction of the T3SS system in Pst DC3000, or a combination of both.
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    The role of Iron Regulated 2 and Iron Regulated Transporter 1 in nickel hyperaccumulation traits in Senecio coronatus
    (2017) Mackay, Angus; Ingle, Robert A
    Metal hyperaccumulating plants accumulate exceptionally high concentrations of metal ions in their above ground tissues and are defined as containing 1000 μg/g dry mass Co, Cu, Cr, Pb, Zn or Ni. This is remarkable because plants typically only require small amounts of these metals for survival, such as 0.004 μg/g Ni and 15-20 μg/g Zn. Scientific investigation has sought to understand the mechanisms underpinning hyperaccumulation in order to apply them in the phyto-technological processes of phytoremediation (removal of metal pollutants from the environment) and phytomining. However, little is known about the molecular mechanisms underlying Ni hyperaccumulation despite the fact that Ni hyperaccumulators account for almost three quarters of all known hyperaccumulating species. A comparative RNA-Seq experiment carried out on Ni accumulating and non-accumulating populations of the South African Ni hyperaccumulator Senecio coronatus (Asteraceae) identified a number of putative transport proteins that are constitutively upregulated in the hyperaccumulator plants. This MSc project focused on two of these, iron regulated 2 (ScIREG2) and iron regulated transporter 1 (ScIRT1), and aimed to validate the RNA-Seq derived nucleotide sequences, test for Ni transport activity and determine their sub-cellular localisation. Full-length ScIREG2 and ScIRT1 protein coding sequences were obtained using RT-PCR and conformed to the predicted sequences derived from the RNA-Seq data. Heterologous expression of ScIRT1 in yeast consistently conferred an increased Ni resistance phenotype to yeast across a variety of experimental conditions, suggesting that this protein is capable of transporting Ni, and may function as a Ni export protein in yeast. In contrast, the results obtained from heterologous expression of ScIREG2 were variable and thus inconclusive. An attempt was made to determine the subcellular localization of ScIRT1 using transient expression of an ScIRT1-YFP fusion protein in onion cells. While inconclusive, a YFP signal was detected in these cells, and appeared to localise to the plasma membrane. The work conducted serves as a pilot study to optimize the experimental systems necessary to identify Ni transporters from S. coronatus. These experimental systems can now be applied to characterise the remaining transport proteins identified in the RNA-Seq analysis.
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    The window of desiccation tolerance shown by early-stage germinating seedlings remains open in the resurrection plant, Xerophyta viscosa
    (Public Library of Science, 2014) Lyall, Rafe; Ingle, Robert A; Illing, Nicola
    Resurrection plants are renowned for their vegetative desiccation tolerance (DT). While DT in vegetative tissues is rare in angiosperms, it is ubiquitous in mature orthodox seeds. During germination, seedlings gradually lose DT until they pass a point of no return, after which they can no longer survive dehydration. Here we investigate whether seedlings of the resurrection plant Xerophyta viscosa ever lose the capacity to establish DT. Seedlings from different stages of germination were dehydrated for 48 hours and assessed for their ability to recover upon rehydration. While a transient decline in the ability of X. viscosa seedlings to survive dehydration was observed, at no point during germination was the ability to re-establish DT completely lost in all seedlings. Pre-treatment of seedlings with PEG or sucrose reduced this transient decline, and improved the survival rate at all stages of germination. Additionally, we observed that the trait of poikilochlorophylly (or loss of chlorophyll) observed in adult X. viscosa leaves can be induced throughout seedling development. These results suggest that the window of DT seen in germinating orthodox seeds remains open in X. viscosa seedlings and that vegetative DT in Xerophyta species may have evolved from the ability to retain this program through to adulthood.