Barbara Kracher

Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses

Colletotrichum species are fungal pathogens that devastate crop plants worldwide. Host infection involves the differentiation of specialized cell types that are associated with penetration, growth inside living host cells (biotrophy) and tissue destruction (necrotrophy). We report here genome and transcriptome analyses of Colletotrichum higginsianum infecting Arabidopsis thaliana and Colletotrichum graminicola infecting maize. Comparative genomics showed that both fungi have large sets of pathogenicity-related genes, but families of genes encoding secreted effectors, pectin-degrading enzymes, secondary metabolism enzymes, transporters and peptidases are expanded in C. higginsianum. Genome-wide expression profiling revealed that these genes are transcribed in successive waves that are linked to pathogenic transitions: effectors and secondary metabolism enzymes are induced before penetration and during biotrophy, whereas most hydrolases and transporters are upregulated later, at the switch to necrotrophy. Our findings show that preinvasion perception of plant-derived signals substantially reprograms fungal gene expression and indicate previously unknown functions for particular fungal cell types.

Root Endophyte Colletotrichum tofieldiae Confers Plant Fitness Benefits that Are Phosphate Status Dependent.

Summary A staggering diversity of endophytic fungi associate with healthy plants in nature, but it is usually unclear whether these represent stochastic encounters or provide host fitness benefits. Although most characterized species of the fungal genus Colletotrichum are destructive pathogens, we show here that C. tofieldiae (Ct) is an endemic endophyte in natural Arabidopsis thaliana populations in central Spain. Colonization by Ct initiates in roots but can also spread systemically into shoots. Ct transfers the macronutrient phosphorus to shoots, promotes plant growth, and increases fertility only under phosphorus-deficient conditions, a nutrient status that might have facilitated the transition from pathogenic to beneficial lifestyles. The host’s phosphate starvation response (PSR) system controls Ct root colonization and is needed for plant growth promotion (PGP). PGP also requires PEN2-dependent indole glucosinolate metabolism, a component of innate immune responses, indicating a functional link between innate immunity and the PSR system during beneficial interactions with Ct.

Mosaic genome structure of the barley powdery mildew pathogen and conservation of transcriptional programs in divergent hosts

Significance Powdery mildew fungi are widespread plant pathogens with an obligate biotrophic lifestyle causing devastating damage to many crops. Blumeria graminis f. sp. hordei (Bgh) infects only barley and is engaged in an evolutionary arms race with the host immune system. Genome sequencing of Bgh isolates revealed an isolate-specific mosaic of monomorphic and polymorphic DNA blocks, suggesting a mechanism that provides a large standing genetic variation in virulence polymorphisms. Detailed Bgh transcriptome profiles during early pathogenesis on barley and immunocompromised Arabidopsis revealed a conserved Bgh transcriptional program despite ∼200 million years of reproductive isolation of these hosts. Barley powdery mildew, Blumeria graminis f. sp. hordei (Bgh), is an obligate biotrophic ascomycete fungal pathogen that can grow and reproduce only on living cells of wild or domesticated barley (Hordeum sp.). Domestication and deployment of resistant barley cultivars by humans selected for amplification of Bgh isolates with different virulence combinations. We sequenced the genomes of two European Bgh isolates, A6 and K1, for comparative analysis with the reference genome of isolate DH14. This revealed a mosaic genome structure consisting of large isolate-specific DNA blocks with either high or low SNP densities. Some of the highly polymorphic blocks likely accumulated SNPs for over 10,000 years, well before the domestication of barley. These isolate-specific blocks of alternating monomorphic and polymorphic regions imply an exceptionally large standing genetic variation in the Bgh population and might be generated and maintained by rare outbreeding and frequent clonal reproduction. RNA-sequencing experiments with isolates A6 and K1 during four early stages of compatible and incompatible interactions on leaves of partially immunocompromised Arabidopsis mutants revealed a conserved Bgh transcriptional program during pathogenesis compared with the natural host barley despite ∼200 million years of reproductive isolation of these hosts. Transcripts encoding candidate-secreted effector proteins are massively induced in successive waves. A specific decrease in candidate-secreted effector protein transcript abundance in the incompatible interaction follows extensive transcriptional reprogramming of the host transcriptome and coincides with the onset of localized host cell death, suggesting a host-inducible defense mechanism that targets fungal effector secretion or production.

Network modeling of signal transduction: establishing the global view

Embryonic development and adult tissue homeostasis are controlled through activation of intracellular signal transduction pathways by extracellular growth factors. In the past, signal transduction has largely been regarded as a linear process. However, more recent data from large‐scale and high‐throughput experiments indicate that there is extensive cross‐talk between individual signaling cascades leading to the notion of a signaling network. The behavior of such complex networks cannot be predicted by simple intuitive approaches but requires sophisticated models and computational simulations. The purpose of such models is to generate experimentally testable hypotheses and to find explanations for unexpected experimental results. Here, we discuss the need for, and the future impact of, mathematical models for exploring signal transduction in different biological contexts such as for example development. BioEssays 30:1110–1125, 2008.

Survival trade-offs in plant roots during colonization by closely related beneficial and pathogenic fungi

The sessile nature of plants forced them to evolve mechanisms to prioritize their responses to simultaneous stresses, including colonization by microbes or nutrient starvation. Here, we compare the genomes of a beneficial root endophyte, Colletotrichum tofieldiae and its pathogenic relative C. incanum, and examine the transcriptomes of both fungi and their plant host Arabidopsis during phosphate starvation. Although the two species diverged only 8.8 million years ago and have similar gene arsenals, we identify genomic signatures indicative of an evolutionary transition from pathogenic to beneficial lifestyles, including a narrowed repertoire of secreted effector proteins, expanded families of chitin-binding and secondary metabolism-related proteins, and limited activation of pathogenicity-related genes in planta. We show that beneficial responses are prioritized in C. tofieldiae-colonized roots under phosphate-deficient conditions, whereas defense responses are activated under phosphate-sufficient conditions. These immune responses are retained in phosphate-starved roots colonized by pathogenic C. incanum, illustrating the ability of plants to maximize survival in response to conflicting stresses.

Conservation of NLR-triggered immunity across plant lineages

The nucleotide-binding domain and leucine-rich repeat (NLR) family of plant receptors detects pathogen-derived molecules, designated effectors, inside host cells and mediates innate immune responses to pathogenic invaders. Genetic evidence revealed species-specific coevolution of many NLRs with effectors from host-adapted pathogens, suggesting that the specificity of these NLRs is restricted to the host or closely related plant species. However, we report that an NLR immune receptor (MLA1) from monocotyledonous barley is fully functional in partially immunocompromised dicotyledonous Arabidopsis thaliana against the barley powdery mildew fungus, Blumeria graminis f. sp. hordei. This implies ∼200 million years of evolutionary conservation of the underlying immune mechanism. A time-course RNA-seq analysis in transgenic Arabidopsis lines detected sustained expression of a large MLA1-dependent gene cluster. This cluster is greatly enriched in genes known to respond to the fungal cell wall-derived microbe-associated molecular pattern chitin. The MLA1-dependent sustained transcript accumulation could define a conserved function of the nuclear pool of MLA1 detected in barley and Arabidopsis. We also found that MLA1-triggered immunity was fully retained in mutant plants that are simultaneously depleted of ethylene, jasmonic acid, and salicylic acid signaling. This points to the existence of an evolutionarily conserved and phytohormone-independent MLA1-mediated resistance mechanism. This also suggests a conserved mechanism for internalization of B. graminis f. sp. hordei effectors into host cells of flowering plants. Furthermore, the deduced connectivity of the NLR to multiple branches of immune signaling pathways likely confers increased robustness against pathogen effector-mediated interception of host immune signaling and could have contributed to the evolutionary preservation of the immune mechanism.

Negative regulation of ABA signaling by WRKY33 is critical for Arabidopsis immunity towards Botrytis cinerea 2100

The Arabidopsis mutant wrky33 is highly susceptible to Botrytis cinerea. We identified >1680 Botrytis-induced WRKY33 binding sites associated with 1576 Arabidopsis genes. Transcriptional profiling defined 318 functional direct target genes at 14 hr post inoculation. Comparative analyses revealed that WRKY33 possesses dual functionality acting either as a repressor or as an activator in a promoter-context dependent manner. We confirmed known WRKY33 targets involved in hormone signaling and phytoalexin biosynthesis, but also uncovered a novel negative role of abscisic acid (ABA) in resistance towards B. cinerea 2100. The ABA biosynthesis genes NCED3 and NCED5 were identified as direct targets required for WRKY33-mediated resistance. Loss-of-WRKY33 function resulted in elevated ABA levels and genetic studies confirmed that WRKY33 acts upstream of NCED3/NCED5 to negatively regulate ABA biosynthesis. This study provides the first detailed view of the genome-wide contribution of a specific plant transcription factor in modulating the transcriptional network associated with plant immunity. DOI: http://dx.doi.org/10.7554/eLife.07295.001

Inferring Boolean network structure via correlation

MOTIVATION Accurate, context-specific regulation of gene expression is essential for all organisms. Accordingly, it is very important to understand the complex relations within cellular gene regulatory networks. A tool to describe and analyze the behavior of such networks are Boolean models. The reconstruction of a Boolean network from biological data requires identification of dependencies within the network. This task becomes increasingly computationally demanding with large amounts of data created by recent high-throughput technologies. Thus, we developed a method that is especially suited for network structure reconstruction from large-scale data. In our approach, we took advantage of the fact that a specific transcription factor often will consistently either activate or inhibit a specific target gene, and this kind of regulatory behavior can be modeled using monotone functions. RESULTS To detect regulatory dependencies in a network, we examined how the expression of different genes correlates to successive network states. For this purpose, we used Pearson correlation as an elementary correlation measure. Given a Boolean network containing only monotone Boolean functions, we prove that the correlation of successive states can identify the dependencies in the network. This method not only finds dependencies in randomly created artificial networks to very high percentage, but also reconstructed large fractions of both a published Escherichia coli regulatory network from simulated data and a yeast cell cycle network from real microarray data.

Induced Genome-Wide Binding of Three Arabidopsis WRKY Transcription Factors during Early MAMP-Triggered Immunity

Genome-wide analysis reveals the in vivo binding sites of Arabidopsis WRKY18, WRKY33, and WRKY40 transcription factors during early MTI and the consequences of WRKY18 and WRKY40 binding on transcriptional output. During microbial-associated molecular pattern-triggered immunity (MTI), molecules derived from microbes are perceived by cell surface receptors and upon signaling to the nucleus initiate a massive transcriptional reprogramming critical to mount an appropriate host defense response. WRKY transcription factors play an important role in regulating these transcriptional processes. Here, we determined on a genome-wide scale the flg22-induced in vivo DNA binding dynamics of three of the most prominent WRKY factors, WRKY18, WRKY40, and WRKY33. The three WRKY factors each bound to more than 1000 gene loci predominantly at W-box elements, the known WRKY binding motif. Binding occurred mainly in the 500-bp promoter regions of these genes. Many of the targeted genes are involved in signal perception and transduction not only during MTI but also upon damage-associated molecular pattern-triggered immunity, providing a mechanistic link between these functionally interconnected basal defense pathways. Among the additional targets were genes involved in the production of indolic secondary metabolites and in modulating distinct plant hormone pathways. Importantly, among the targeted genes were numerous transcription factors, encoding predominantly ethylene response factors, active during early MTI, and WRKY factors, supporting the previously hypothesized existence of a WRKY subregulatory network. Transcriptional analysis revealed that WRKY18 and WRKY40 function redundantly as negative regulators of flg22-induced genes often to prevent exaggerated defense responses.

A core function of EDS1 with PAD4 is to protect the salicylic acid defense sector in Arabidopsis immunity

Plant defenses induced by salicylic acid (SA) are vital for resistance against biotrophic pathogens. In basal and receptor-triggered immunity, SA accumulation is promoted by Enhanced Disease Susceptibility1 with its co-regulator Phytoalexin Deficient4 (EDS1/PAD4). Current models position EDS1/PAD4 upstream of SA but their functional relationship remains unclear. In a genetic and transcriptomic analysis of Arabidopsis autoimmunity caused by constitutive or conditional EDS1/PAD4 overexpression, intrinsic EDS1/PAD4 signaling properties and their relation to SA were uncovered. A core EDS1/PAD4 pathway works in parallel with SA in basal and effector-triggered bacterial immunity. It protects against disabled SA-regulated gene expression and pathogen resistance, and is distinct from a known SA-compensatory route involving MAPK signaling. Results help to explain previously identified EDS1/PAD4 regulated SA-dependent and SA-independent gene expression sectors. Plants have evolved an alternative route for preserving SA-regulated defenses against pathogen or genetic perturbations. In a proposed signaling framework, EDS1 with PAD4, besides promoting SA biosynthesis, maintains important SA-related resistance programs, thereby increasing robustness of the innate immune system.