Exploring the utility of Brachypodium distachyon as a model pathosystem for the wheat pathogen Zymoseptoria tritici

peer-reviewedBackground Zymoseptoria tritici, the causative organism of Septoria tritici blotch disease is a prevalent biotic stressor of wheat production, exerting substantial economic constraints on farmers, requiring intensive chemical control to protect yields. A hemibiotrophic pathogen with a long asymptomless phase of up to 11 days post inoculation (dpi) before a rapid switch to necrotrophy; a deficit exists in our understanding of the events occurring within the host during the two phases of infection. Brachypodium distachyon has demonstrated its potential as a model species for the investigation of fungal disease resistance in cereal and grass species. The aim of this study was to assess the physical interaction between Z. tritici (strain IPO323) and B. distachyon and examine its potential as a model pathosystem for Z. tritici. Results Septoria tritici blotch symptoms developed on the wheat cultivar Riband from 12 dpi with pycnidial formation abundant by 20 dpi. Symptoms on B. distachyon ecotype Bd21-1 were visible from 1 dpi: characteristic pale, water soaked lesions which developed into blotch-like lesions by 4 dpi. These lesions then became necrotic with chlorotic regions expanding up to 7 dpi. Sporulation on B. distachyon tissues was not observed and no evidence of fungal penetration could be obtained, indicating that Z. tritici was unable to complete its life cycle within B. distachyon ecotypes. However, observation of host responses to the Z. tritici strain IPO323 in five B. distachyon ecotypes revealed a variation in resistance responses, ranging from immunity to a chlorotic/necrotic phenotype. Conclusions The observed interactions suggest that B. distachyon is an incompatible host for Z. tritici infection, with STB symptom development on B. distachyon comparable to that observed during the early infection stages on the natural host, wheat. However first visible symptoms occurred more rapidly on B. distachyon; from 1 dpi in comparison to 12 dpi in wheat. Consequently, we propose that the interaction between B. distachyon and Z. tritici as observed in this study could serve as a suitable model pathosystem with which to investigate mechanisms underpinning an incompatible host response to Z. tritici.Teagasc Walsh Fellowship Programm

Molecular techniques for studying <i>Fusarium</i> ear blight of wheat

This work has compared polymerase chain reaction (PCR) assays and traditional visual disease assessment to evaluate the severity of Fusarium ear blight (FEB) ofwheat under field and glasshouse conditions. In a field trial, PCR analysis highlighted the problem of diagnosis of FEB of wheat based on visual disease assessment where natural inoculum was present. PCR-based analysis detected F. poae predominantly in the glumes and Microdochium nivale sub-species were predominantly found in the rachis component of ears. M. nivale var. majus was more frequently observed than var. nivale (64 and 36 %, respectively). Quantitative PCR analysis and conventional visual disease assessment were used to evaluate fungicide efficacy against FEB of wheat caused by F. culmorum and F. poae in three glasshouse trials (1994/5-1996/7). Prochloraz and tebuconazole significantly decreased both visual symptoms of FEB and fungal DNA content of F. culmorum and F. poae ear blight of wheat. Overall, both fungicides appeared equally effective, although the efficacy ofthese fungicides was consistently greater as measured by PCR analysis rather than by visual disease assessment. Inoculation with F. culmorum significantly reduced yield (1000 grain weight) whereas inoculation with F. poae had no significant effect on yield. Fusarium culmorum was successfully transformed with the GUS reporter gene. GUS activity levels of transformants varied, but transformation did not affect pathogenicity on wheat seedlings. A GUS transformant was used to study the effectiveness of two fungicides against F. culmorum foot rot of wheat. Primers to the Tri5 gene were used to develop a PCR-based assay for the specific detection of potential trichothecene-producing Fusarium species. These primers were also used to develop an RT-PCR-based assay for the detection and semiquantification of F. culmorum Tri5 gene expression 'relative to p-tubulin gene expression' in RNA extracts from F. culmorum. This assay was used to show that time and fungicides can affect Tri5 gene expression in liquid culture

Generating Phenotypic Diversity in a Fungal Biocatalyst to Investigate Alcohol Stress Tolerance Encountered during Microbial Cellulosic Biofuel Production

peer-reviewedConsolidated bioprocessing (CBP) of lignocellulosic biomass offers an alternative route to renewable energy. The crop pathogen Fusarium oxysporum is a promising fungal biocatalyst because of its broad host range and innate ability to co-saccharify and ferment lignocellulose to bioethanol. A major challenge for cellulolytic CBP-enabling microbes is alcohol inhibition. This research tested the hypothesis that Agrobacterium tumefaciens - mediated transformation (ATMT) could be exploited as a tool to generate phenotypic diversity in F. oxysporum to investigate alcohol stress tolerance encountered during CBP. A random mutagenesis library of gene disruption transformants (n=1,563) was constructed and screened for alcohol tolerance in order to isolate alcohol sensitive or tolerant phenotypes. Following three rounds of screening, exposure of select transformants to 6% ethanol and 0.75% n-butanol resulted respectively in increased (≥11.74%) and decreased (≤43.01%) growth compared to the wild –type (WT). Principal component analysis (PCA) quantified the level of phenotypic diversity across the population of genetically transformed individuals and isolated candidate strains for analysis. Characterisation of one strain, Tr. 259, ascertained a reduced growth phenotype under alcohol stress relative to WT and indicated the disruption of a coding region homologous to a putative sugar transporter (FOXG_09625). Quantitative PCR (RT-PCR) showed FOXG_09625 was differentially expressed in Tr. 259 compared to WT during alcohol-induced stress (P<0.05). Phylogenetic analysis of putative sugar transporters suggests diverse functional roles in F. oxysporum and other filamentous fungi compared to yeast for which sugar transporters form part of a relatively conserved family. This study has confirmed the potential of ATMT coupled with a phenotypic screening program to select for genetic variation induced in response to alcohol stress. This research represents a first step in the investigation of alcohol tolerance in F. oxysporum and has resulted in the identification of several novel strains, which will be of benefit to future biofuel research.Funding provided through the Irish Department of Agriculture and Food's Research Stimulus Fund (Project Code RSF 07 513

Ensifer-mediated Arabidopsis thaliana Root Transformation (E-ART): A Protocol to Analyse the Factors that Support Ensifer-mediated Transformation (EMT) of Plant Cells

peer-reviewedEnsifer adhaerens OV14, a soil borne alpha-proteobacteria of the Rhizobiaceae family, fortifies the novel plant transformation technology platform termed ‘Ensifer-mediated transformation’ (EMT). EMT can stably transform both monocot and dicot species, and the host range of EMT is continuously expanding across a diverse range of crop species. In this protocol, we adapted a previously published account that describes the use of Arabidopsis thaliana roots to investigate the interaction of A. thaliana and Agrobacterium tumefaciens. In our laboratory, we routinely use A. thaliana root explants to examine the factors that enhance the utility of EMT. In addition, the E-ART protocol can be used to study the transcriptional response of E. adhaerens and host plant following exposure to explant tissue, the transformability of different Ensifer adhaerens strains/mutants as well as testing the susceptibility of A. thaliana mutant lines as a means to decipher the mechanisms underpinning EMT

Insights into the transcriptomic response of the plant engineering bacterium Ensifer adhaerens OV14 during transformation

peer-reviewedThe ability to engineer plant genomes has been primarily driven by the soil bacterium Agrobacterium tumefaciens but recently the potential of alternative rhizobia such as Rhizobium etli and Ensifer adhaerens OV14, the latter of which supports Ensifer Mediated Transformation (EMT) has been reported. Surprisingly, a knowledge deficit exists in regards to understanding the whole genome processes underway in plant transforming bacteria, irrespective of the species. To begin to address the issue, we undertook a temporal RNAseq-based profiling study of E. adhaerens OV14 in the presence/absence of Arabidopsis thaliana tissues. Following co-cultivation with root tissues, 2333 differentially expressed genes (DEGs) were noted. Meta-analysis of the RNAseq data sets identified a clear shift from plasmid-derived gene expression to chromosomal-based transcription within the early stages of bacterium-plant co-cultivation. During this time, the number of differentially expressed prokaryotic genes increased steadily out to 7 days co-cultivation, a time at which optimum rates of transformation were observed. Gene ontology evaluations indicated a role for both chromosomal and plasmid-based gene families linked specifically with quorum sensing, flagellin production and biofilm formation in the process of EMT. Transcriptional evaluation of vir genes, housed on the pCAMBIA 5105 plasmid in E. adhaerens OV14 confirmed the ability of E. adhaerens OV14 to perceive and activate its transcriptome in response to the presence of 200 µM of acetosyringone. Significantly, this is the first study to characterise the whole transcriptomic response of a plant engineering bacterium in the presence of plant tissues and provides a novel insight into prokaryotic genetic processes that support T-DNA transfer

Quantification of In Planta Zymoseptoria tritici Progression Through Different Infection Phases and Related Association with Components of Aggressiveness

peer-reviewedIn planta growth of Zymoseptoria tritici, causal agent of Septoria tritici blotch of wheat, during the infection process has remained an understudied topic due to the long symptomless latent period before the emergence of fruiting bodies. In this study, we attempted to understand the relationship between in planta growth of Z. tritici relative to the primary components of aggressiveness, i.e., latent period and pycnidia coverage in regard to contrasting host resistance. We tested isolates collected from Ireland against the susceptible cultivar Gallant and cultivar Stigg, which has strong partial resistance. A clear isolate−host interaction effect (F = 3.018; P = 0.005, and F = 6.008; P < 0.001) for latent period and pycnidia coverage, respectively, was identified. Furthermore, during the early infection phase of latency from 5 to 11 days postinoculation (dpi), in planta growth rate of fungal biomass was significantly (F = 30.06; P < 0.001) more affected by host resistance than isolate specificity (F = 1.27; P = 0.27), indicating the importance of host resistance in the early infection phase. In planta Z. tritici growth rates in cultivar Gallant spiked between 11 and 16 dpi followed by a continuous fall onward, whereas in cultivar Stigg it was slowly progressive in nature. From correlation and regression analysis, we found that the in planta growth rate preceding the average latent period of cultivar Gallant has more influence on latency duration and pycnidia production. Likewise, correlation between component of aggressiveness and in planta growth rate of pathogen supports our understanding of aggressiveness to be driven by the pathogen’s multiplication capacity within host tissue.H2020 Marie Skłodowska-Curie Action

Genome sequence of Ensifer adhaerens OV14 provides insights into its ability as a novel vector for the genetic transformation of plant genomes

Background: Recently it has been shown that Ensifer adhaerens can be used as a plant transformation technology, transferring genes into several plant genomes when equipped with a Ti plasmid. For this study, we have sequenced the genome of Ensifer adhaerens OV14 (OV14) and compared it with those of Agrobacterium tumefaciens C58 (C58) and Sinorhizobium meliloti 1021 (1021); the latter of which has also demonstrated a capacity to genetically transform crop genomes, albeit at significantly reduced frequencies. Results: The 7.7 Mb OV14 genome comprises two chromosomes and two plasmids. All protein coding regions in the OV14 genome were functionally grouped based on an eggNOG database. No genes homologous to the A. tumefaciens Ti plasmid vir genes appeared to be present in the OV14 genome. Unexpectedly, OV14 and 1021 were found to possess homologs to chromosomal based genes cited as essential to A. tumefaciens T-DNA transfer. Of significance, genes that are non-essential but exert a positive influence on virulence and the ability to genetically transform host genomes were identified in OV14 but were absent from the 1021 genome. Conclusions: This study reveals the presence of homologs to chromosomally based Agrobacterium genes that support T-DNA transfer within the genome of OV14 and other alphaproteobacteria. The sequencing and analysis of the OV14 genome increases our understanding of T-DNA transfer by non-Agrobacterium species and creates a platform for the continued improvement of Ensifer-mediated transformation (EMT).Science Foundation Irelan

Exploiting the inter-strain divergence of Fusarium oxysporum for microbial bioprocessing of lignocellulose to bioethanol

Microbial bioprocessing of lignocellulose to bioethanol still poses challenges in terms of substrate catabolism. A targeted evolution-based study was undertaken to determine if inter-strain microbial variability could be exploited for bioprocessing of lignocellulose to bioethanol. The microorganism studied was Fusarium oxysporum because of its capacity to both saccharify and ferment lignocellulose. Strains of F. oxysporum were isolated and assessed for their genetic variability. Using optimised solid-state straw culture conditions, experiments were conducted that compared fungal strains in terms of their growth, enzyme activities (cellulases, xylanase and alcohol dehydrogenase) and yield of bioethanol and the undesirable by-products acetic acid and xylitol. Significant inter-strain divergence was recorded in regards to the capacity of studied F. oxysporum strains to produce alcohol from untreated straw. No correlation was observed between bioethanol synthesis and either the biomass production or microbial enzyme activity. A strong correlation was observed between both acetic acid and xylitol production and bioethanol yield. The level of diversity recorded in the alcohol production capacity among closely-related microorganism means that a targeted screening of populations of selected microbial species could greatly improve bioprocessing yields, in terms of providing both new host strains and candidate genes for the bioethanol industry

A Pathogen-Responsive Leucine Rich Receptor Like Kinase Contributes to Fusarium Resistance in Cereals

peer-reviewedReceptor-like kinases form the largest family of receptors in plants and play an important role in recognizing pathogen-associated molecular patterns and modulating the plant immune responses to invasive fungi, including cereal defenses against fungal diseases. But hitherto, none have been shown to modulate the wheat response to the economically important Fusarium head blight (FHB) disease of small-grain cereals. Homologous genes were identified on barley chromosome 6H (HvLRRK-6H) and wheat chromosome 6DL (TaLRRK-6D), which encode the characteristic domains of surface-localized receptor like kinases. Gene expression studies validated that the wheat TaLRRK-6D is highly induced in heads as an early response to both the causal pathogen of FHB disease, Fusarium graminearum, and its’ mycotoxic virulence factor deoxynivalenol. The transcription of other wheat homeologs of this gene, located on chromosomes 6A and 6B, was also up-regulated in response to F. graminearum. Virus-induced gene silencing (VIGS) of the barley HvLRRK-6H compromised leaf defense against F. graminearum. VIGS of TaLRRK-6D in two wheat cultivars, CM82036 (resistant to FHB disease) and cv. Remus (susceptible to FHB), confirmed that TaLRRK-6D contributes to basal resistance to FHB disease in both genotypes. Although the effect of VIGS did not generally reduce grain losses due to FHB, this experiment did reveal that TaLRRK-6D positively contributes to grain development. Further gene expression studies in wheat cv. Remus indicated that VIGS of TaLRRK-6D suppressed the expression of genes involved in salicylic acid signaling, which is a key hormonal pathway involved in defense. Thus, this study provides the first evidence of receptor like kinases as an important component of cereal defense against Fusarium and highlights this gene as a target for enhancing cereal resistance to FHB disease.This work was supported by Department of Agriculture, Food and the Marine Research Stimulus Project Wheatenhance (11/S/103) and by Science Foundation Ireland research projects 10/IN.1/B3028 and 14IA2508

Fungal-mediated consolidated bioprocessing: the potential of Fusarium oxysporum for the lignocellulosic ethanol industry

peer-reviewedMicrobial bioprocessing of lignocellulose to bioethanol still poses challenges in terms of substrate catabolism. The most important challenge is to overcome substrate recalcitrance and to thus reduce the number of steps needed to biorefine lignocellulose. Conventionally, conversion involves chemical pretreatment of lignocellulose, followed by hydrolysis of biomass to monomer sugars that are subsequently fermented into bioethanol. Consolidated bioprocessing (CBP) has been suggested as an efficient and economical method of manufacturing bioethanol from lignocellulose. CBP integrates the hydrolysis and fermentation steps into a single process, thereby significantly reducing the amount of steps in the biorefining process. Filamentous fungi are remarkable organisms that are naturally specialised in deconstructing plant biomass and thus they have tremendous potential as components of CBP. The fungus Fusarium oxysporum has potential for CBP of lignocellulose to bioethanol. Here we discuss the complexity and potential of CBP, the bottlenecks in the process, and the potential influence of fungal genetic diversity, substrate complexity and new technologies on the efficacy of CPB of lignocellulose, with a focus on F. oxysporum