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ABSTRACT: To understand early events in plant-pathogen interactions, it is necessary to explore the pathogen secretome to identify secreted proteins which help orchestrate pathology. The secretome can be obtained from pathogens grown in vitro, and then characterized using standard proteomic approaches based on protein extraction and subsequent identification of tryptic peptides by LC-MS. A subset of the secretome is composed of proteins whose presence is required to initiate infection and their removal from the secretome would result in pathogens with reduced or no virulence. We present here a comparative secretome from Fusarium graminearum. This filamentous fungus causes Fusarium Head blight on wheat and is a serious cereal diseases of many cereal growing regions. Affected grain is contaminated with mycotoxins and cannot be used for food or feed. We used label-free quantitative mass spectrometry to compare the secretomes of wild type with two non-pathogenic deletion mutants of F. graminearum, Δtri6 and Δtri10. These mutations in mycotoxin-regulating transcription factors revealed a subset of 29 proteins whose relative abundance was affected in their secretomes, as measured by spectral counting. Proteins which decreased in abundance are potential candidate virulence factors and these included cell wall-degrading enzymes, metabolic enzymes, pathogenesis-related proteins and proteins of unknown function.
Proteomics 03/2013; · 4.43 Impact Factor
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ABSTRACT: Systems biology is a scientific approach that integrates many scientific disciplines to develop a comprehensive understanding of biological phenomena, thus allowing the prediction and accurate simulation of complex biological behaviors. It may be presumptuous to write about toxin regulation at the level of systems biology, but the last decade of research is leading us closer than ever to this approach. Past research has delineated multiple levels of regulation in the pathways leading to the biosynthesis of secondary metabolites, including mycotoxins. At the top of this hierarchy, the global or master transcriptional regulators perceive various environmental cues such as climatic conditions, the availability of nutrients, and the developmental stages of the organism. Information accumulated from various inputs is integrated through a complex web of signalling networks to generate the eventual outcome. This review will focus on adapting techniques such as chemical and other genetic tools available in the model system Saccharomyces cerevisiae, to disentangle the various biological networks involved in the biosynthesis of mycotoxins in the Fusarium spp.
Toxins. 01/2013; 5(4):675-682.
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ABSTRACT: Feruloyl esterases can liberate ferulic acid (FA) from plant cell wall polymers. They are expressed by plant pathogenic fungi and could play a role in pathogenicity, although this question has not been addressed yet. The fungus Fusarium graminearum is the principal causal agent of fusarium head blight (FHB) and gibberella ear rot (GER), major diseases of wheat, barley, and maize in all temperate regions of the world. The F. graminearum genome contains seven genes with strong homology to feruloyl esterase (FAE) sequences. Phylogenetic analysis showed that these included three type B, three type C, and one type D FAE genes. Expression profiling of the seven FAE genes showed complex regulation patterns unique to each gene. In F. graminearum-infected plant tissues, the FAE genes exhibited host-specific gene expression. On wheat, FAEB1 and FAED1 were strongly expressed while FAEB2, FAEB3, and FAEC1 were expressed at more modest levels. On maize, only FAEB3, FAEC1, and FAED1 were expressed and at low levels. When growing F. graminearum in liquid culture, only FAEB1 and FAEC1 were expressed. Both genes were induced by a small group of related aromatic compounds including FA, caffeic acid, and p-coumaric acid. FAEB1 was induced by xylose, while repressed by glucose and galactose. FAEC1 was constitutively expressed at low levels in the presence of those sugars. Expression of the other five FAE genes was not detected in the culture conditions used. To determine if FAE genes were important for pathogenicity of F. graminearum, mutant strains inactivated for faeB1∆, faeD1∆ or both genes were constructed and tested on wheat plants. No statistically significant change in pathogenicity and no compensatory expression of the other FAE genes were observed in the fae gene mutants. Our results show that FAEB1 and FAED1 are not required for pathogenicity of F. graminearum on wheat.
Fungal Biology 04/2012; 116(4):478-88. · 1.43 Impact Factor
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ABSTRACT: This study presents a high-throughput proteomic analysis of phosphopeptides from Fusarium graminearum strain DAOM 233423 grown in vitro without nutritional limitation. Using a combination of strong cation exchange (SCX) and immobilized metal affinity chromatography (IMAC) followed by LC-MS, we identified 2902 putative phosphopeptides with homologous matches to 1496 different proteins. Functional classification of the annotated protein set revealed that phosphopeptides from nuclear proteins with ATP-binding function were the most abundant. There are indications that phosphorylation sites from well-characterized phosphoproteins representing diverse biological processes are conserved in F. graminearum: sequences of three phosphopeptides from known phosphoproteins (transcription elongation factor 1β, acidic ribosomal proteins, and glycogen synthase) revealed phosphorylation site conservation.
Proteomics 04/2012; 12(7):1002-5. · 4.43 Impact Factor
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ABSTRACT: In F. graminearum, the transcriptional regulator Tri6 is encoded within the trichothecene gene cluster and regulates genes involved in the biosynthesis of the secondary metabolite deoxynivalenol (DON). The Tri6 protein with its Cys₂His₂ zinc-finger may also conform to the class of global transcription regulators. This class of global transcriptional regulators mediate various environmental cues and generally responds to the demands of cellular metabolism. To address this issue directly, we sought to find gene targets of Tri6 in F. graminearum grown in optimal nutrient conditions. Chromatin immunoprecipitation followed by Illumina sequencing (ChIP-Seq) revealed that in addition to identifying six genes within the trichothecene gene cluster, Tri1, Tri3, Tri6, Tri7, Tri12 and Tri14, the ChIP-Seq also identified 192 additional targets potentially regulated by Tri6. Functional classification revealed that, among the annotated genes, ∼40% are associated with cellular metabolism and transport and the rest of the target genes fall into the category of signal transduction and gene expression regulation. ChIP-Seq data also revealed Tri6 has the highest affinity toward its own promoter, suggesting that this gene could be subject to self-regulation. Electro mobility shift assays (EMSA) performed on the promoter of Tri6 with purified Tri6 protein identified a minimum binding motif of GTGA repeats as a consensus sequence. Finally, expression profiling of F. graminearum grown under nitrogen-limiting conditions revealed that 49 out of 198 target genes are differentially regulated by Tri6. The identification of potential new targets together with deciphering novel binding sites for Tri6, casts new light into the role of this transcriptional regulator in the overall growth and development of F. graminearum.
PLoS Pathogens 09/2011; 7(9):e1002266. · 9.13 Impact Factor
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ABSTRACT: Despite the tremendous economic impact of cereal crop pathogens such as the fungus Fusarium graminearum, the development of strategies for enhanced crop protection is hampered by complex host genetics and difficulties in performing high-throughput analyses. To bypass these challenges, we have developed an assay in which the interaction between F. graminearum and the model plant Arabidopsis thaliana is monitored in liquid media in 96-well plates. In this assay, fungal infection is associated with the development of dark lesion-like spots on the cotyledons of Arabidopsis seedlings by 4 days postinoculation. These symptoms can be alleviated by the application of known defense-activating small molecules and in previously described resistant host genetic backgrounds. Based on this infection phenotype, we conducted a small-scale chemical screen to identify small molecules that protect Arabidopsis seedlings from infection by F. graminearum. We identified sulfamethoxazole and the indole alkaloid gramine as compounds with strong protective activity in the liquid assay. Remarkably, these two chemicals also significantly reduced the severity of F. graminearum infection in wheat. As such, the Arabidopsis-based liquid assay represents a biologically relevant surrogate system for high-throughput studies of agriculturally important plant-pathogen interactions.
Molecular Plant-Microbe Interactions 02/2011; 24(6):640-8. · 4.43 Impact Factor
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ABSTRACT: Plant immunity can be induced by two major classes of pathogen-associated molecules. Pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) are conserved molecular components of microbes that serve as "non-self" features to induce PAMP-triggered immunity (PTI). Pathogen effector proteins used to promote virulence can also be recognized as "non-self" features or induce a "modified-self" state that can induce effector-triggered immunity (ETI). The Arabidopsis protein RIN4 plays an important role in both branches of plant immunity. Three unrelated type III secretion effector (TTSE) proteins from the phytopathogen Pseudomonas syringae, AvrRpm1, AvrRpt2, and AvrB, target RIN4, resulting in ETI that effectively restricts pathogen growth. However, no pathogenic advantage has been demonstrated for RIN4 manipulation by these TTSEs. Here, we show that the TTSE HopF2(Pto) also targets Arabidopsis RIN4. Transgenic plants conditionally expressing HopF2(Pto) were compromised for AvrRpt2-induced RIN4 modification and associated ETI. HopF2(Pto) interfered with AvrRpt2-induced RIN4 modification in vitro but not with AvrRpt2 activation, suggestive of RIN4 targeting by HopF2(Pto). In support of this hypothesis, HopF2 (Pto) interacted with RIN4 in vitro and in vivo. Unlike AvrRpm1, AvrRpt2, and AvrB, HopF2(Pto) did not induce ETI and instead promoted P. syringae growth in Arabidopsis. This virulence activity was not observed in plants genetically lacking RIN4. These data provide evidence that RIN4 is a major virulence target of HopF2(Pto) and that a pathogenic advantage can be conveyed by TTSEs that target RIN4.
Proceedings of the National Academy of Sciences 02/2010; 107(5):2349-54. · 9.68 Impact Factor
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ABSTRACT: Fusarium graminearum grown under stress, such as nutrient deprivation, activates, among others, the trichothecene pathway that produces the mycotoxin deoxynivalenol and its derivatives. The kinase inhibitor staurosporine reduced the production of trichothecenes by 39% compared with control in vitro. On the other hand, phosphatase inhibitor okadaic acid increased the amount by 72% compared with the control in vitro. This suggests that phosphorylation events are involved in the signalling pathway, leading to the activation of the trichothecene pathway. Three approaches were used to study the phosphoproteome of F. graminearum under nitrogen-limiting conditions: 2-DE (2-DE: IEFxSDS-PAGE) in combination with MS protein identification; SDS-PAGE in combination with off-line IMAC and TiO(2) enrichment and gel electrophoresis LC-MS analysis; and a gel-free approach using strong anion exchange chromatography, IMAC and LC-MS. A total of 348 phosphorylation sites localized in 301 peptides from 241 proteins were identified. By 2-DE, 20 phosphoproteins were identified, nine of which underwent changes during the time course examined. Using gel electrophoresis LC-MS 231 phosphopeptides were identified from three samples (ten gel slices each) at time points of nitrogen starvation t=0, 6, and 12 h. The gel-free analysis added 70 peptides from 65 proteins to the total. Proteins of unknown function and enzymes of known function comprised the largest groups overall. Ten protein kinases and seven transcription factors were identified. This is the first reported phosphoproteome of F. graminearum.
Proteomics 11/2009; 10(1):124-40. · 4.43 Impact Factor
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ABSTRACT: Pollination triggers not only embryo development but also the differentiation of the ovule integuments to form a specialized seed coat. The mucilage secretory cells of the Arabidopsis thaliana seed coat undergo a complex differentiation process in which cell growth is followed by the synthesis and secretion of pectinaceous mucilage. A number of genes have been identified affecting mucilage secretory cell differentiation, including MUCILAGE-MODIFIED4 (MUM4). mum4 mutants produce a reduced amount of mucilage and cloning of MUM4 revealed that it encodes a UDP-L-rhamnose synthase that is developmentally up-regulated to provide rhamnose for mucilage pectin synthesis. To identify additional genes acting in mucilage synthesis and secretion, a screen for enhancers of the mum4 phenotype was performed. Eight mum enhancers (men) have been identified, two of which result from defects in known mucilage secretory cell genes (MUM2 and MYB61). Our results show that, in a mum4 background, mutations in MEN1, MEN4, and MEN5 lead to further reductions in mucilage compared to mum4 single mutants, suggesting that they are involved in mucilage synthesis or secretion. Conversely, mutations in MEN2 and MEN6 appear to affect mucilage release rather than quantity. With the exception of men4, whose single mutant exhibits reduced mucilage, none of these genes have a single mutant phenotype, suggesting that they would not have been identified outside the compromised mum4 background.
Journal of Experimental Botany 05/2009; 60(9):2601-12. · 5.36 Impact Factor
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ABSTRACT: Overexpression (OE) was used to study the role of the Arabidopsis Golden2-like (GLK1) transcriptional activator in regulating gene expression. Affymetrix Gene Chip and RT-PCR analyses indicated that GLK1 OE in Arabidopsis reprogrammed gene expression networks to enhance a high constitutive expression of genes encoding disease defense related proteins. These include PR10, isochorismate synthase, antimicrobial peptides, glycosyl hydrolases, MATE efflux and other genes associated with pathogen response and detoxification. However, PR1, an indicator of systemic acquired resistance (SAR), was downregulated in GLK1 OE. GLK1 OE in Arabidopsis confers resistance to Fusarium graminearum, a broad host pathogen responsible for major losses in cereal crops. This is the first identification of the GLK1 'regulon' and a novel role for GLK1 in plant defense, suggesting its potential use for providing disease resistance in crop plants.
Biochemical and Biophysical Research Communications 08/2007; 359(2):234-8. · 2.48 Impact Factor
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ABSTRACT: Most plant disease resistance (R) proteins contain a series of leucine-rich repeats (LRRs), a nucleotide-binding site (NBS), and a putative amino-terminal signaling domain. They are termed NBS-LRR proteins. The LRRs of a wide variety of proteins from many organisms serve as protein interaction platforms, and as regulatory modules of protein activation. Genetically, the LRRs of plant R proteins are determinants of response specificity, and their action can lead to plant cell death in the form of the familiar hypersensitive response (HR). A total of 149 R genes are potentially expressed in the Arabidopsis genome, and plant cells must deal with the difficult task of assembling many of the proteins encoded by these genes into functional signaling complexes. Eukaryotic cells utilize several strategies to deal with this problem. First, proteins are spatially restricted to their sub-cellular site of function, thus improving the probability that they will interact with their proper partners. Second, these interactions are architecturally organized to avoid inappropriate signaling events and to maintain the fidelity and efficiency of the response when it is initiated. Recent results provide new insights into how the signaling potential of R proteins might be created, managed and held in check until specific stimulation following infection. Nevertheless, the roles of the R protein partners in these regulatory events that have been defined to date are unclear.
Current Opinion in Plant Biology 09/2004; 7(4):391-9. · 9.27 Impact Factor
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ABSTRACT: Transcriptional reprogramming is critical for plant disease resistance responses; its global control is not well understood. Salicylic acid (SA) can induce plant defense gene expression and a long-lasting disease resistance state called systemic acquired resistance (SAR). Plant-specific "Whirly" DNA binding proteins were previously implicated in defense gene regulation. We demonstrate that the potato StWhy1 protein is a transcriptional activator of genes containing the PBF2 binding PB promoter element. DNA binding activity of AtWhy1, the Arabidopsis StWhy1 ortholog, is induced by SA and is required for both SA-dependent disease resistance and SA-induced expression of an SAR response gene. AtWhy1 is required for both full basal and specific disease resistance responses. The transcription factor-associated protein NPR1 is also required for SAR. Surprisingly, AtWhy1 activation by SA is NPR1 independent, suggesting that AtWhy1 works in conjunction with NPR1 to transduce the SA signal. Our analysis of AtWhy1 adds a critical component to the SA-dependent plant disease resistance response.
Developmental Cell 03/2004; 6(2):229-40. · 14.03 Impact Factor
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ABSTRACT: The pathogenesis-related gene PR-7Oa (formerly STH-2) is induced in various organs of potato after wounding, elicitor treatment, or infection by Phytophthora infestam. Deletion analysis of the promoter of the PR-lOa gene enabled us to identify a 50-bp region, located between positions -155 and -105, necessary for the elicitor responsiveness of the P-glucuronidase reporter gene in transgenic potato plants. Within this region, a 30-bp sequence, located between positions -135 and -105, was necessary for the activation of the promoter by the elicitor. However, strong promoter activity after elicitor treatment required the presence of a 20-bp sequence located between positions -155 and -135. The region between -135 and -105 was specifically recognized by two nuclear factors, PBF-1 (PR-lOa Binding Eactor 1) and PEF-2, and binding of PBF-1 ,was coordinated with the accumulation of the PR-lOa mRNA. Gel shift assays using nuclear extracts pretreated with sodium deoxycholate or alkaline phosphatase suggested that PBM is a multimeric factor in which at least one of the constituent proteins can be phosphorylated. Treatment with alkaline phosphatase also indicated that binding of PBF-1 is positively regulated by phosphorylation and that it is phosphorylated only in tissues in which PR-7Oa is expressed. The use of protein phosphatase and kinase inhibitors in vivo provided additional evidence that wounding and elicitor treatment induce the phosphorylation of PBF-1 and that this phosphorylation is associated with gene activation.
