A secreted fungal histidine- and alanine-rich protein regulates metal ion homeostasis and oxidative stress.

Like pathogens, beneficial endophytic fungi secrete effector proteins to promote plant colonization, for example, through perturbation of host immunity. The genome of the root endophyte Serendipita indica encodes a novel family of highly similar, small alanine- and histidine-rich proteins, whose functions remain unknown. Members of this protein family carry an N-terminal signal peptide and a conserved C-terminal DELD motif. Here we report on the functional characterization of the plant-responsive DELD family protein Dld1 using a combination of structural, biochemical, biophysical and cytological analyses. The crystal structure of Dld1 shows an unusual, monomeric histidine zipper consisting of two antiparallel coiled-coil helices. Similar to other histidine-rich proteins, Dld1 displays varying affinity to different transition metal ions and undergoes metal ion- and pH-dependent unfolding. Transient expression of mCherry-tagged Dld1 in barley leaf and root tissue suggests that Dld1 localizes to the plant cell wall and accumulates at cell wall appositions during fungal penetration. Moreover, recombinant Dld1 enhances barley root colonization by S. indica, and inhibits H2 O2 -mediated radical polymerization of 3,3'-diaminobenzidine. Our data suggest that Dld1 has the potential to enhance micronutrient accessibility for the fungus and to interfere with oxidative stress and reactive oxygen species homeostasis to facilitate host colonization.

The inconspicuous gatekeeper: endophytic Serendipita vermifera acts as extended plant protection barrier in the rhizosphere.

In nature, beneficial and pathogenic fungi often simultaneously colonise plants. Despite substantial efforts to understand the composition of natural plant-microbe communities, the mechanisms driving such multipartite interactions remain largely unknown. Here we address how the interaction between the beneficial root endophyte Serendipita vermifera and the pathogen Bipolaris sorokiniana affects fungal behaviour and determines barley host responses using a gnotobiotic soil-based split-root system. Fungal confrontation in soil resulted in induction of B. sorokiniana genes involved in secondary metabolism and a significant repression of genes encoding putative effectors. In S. vermifera, genes encoding hydrolytic enzymes were strongly induced. This antagonistic response was not activated during the tripartite interaction in barley roots. Instead, we observed a specific induction of S. vermifera genes involved in detoxification and redox homeostasis. Pathogen infection but not endophyte colonisation resulted in substantial host transcriptional reprogramming and activation of defence. In the presence of S. vermifera, pathogen infection and disease symptoms were significantly reduced despite no marked alterations of the plant transcriptional response. The activation of stress response genes and concomitant repression of putative effector gene expression in B. sorokiniana during confrontation with the endophyte suggest a reduction of the pathogen's virulence potential before host plant infection.

Serendipita indica E5'NT modulates extracellular nucleotide levels in the plant apoplast and affects fungal colonization.

Extracellular adenosine 5'-triphosphate (eATP) is an essential signaling molecule that mediates different cellular processes through its interaction with membrane-associated receptor proteins in animals and plants. eATP regulates plant growth, development, and responses to biotic and abiotic stresses. Its accumulation in the apoplast induces ROS production and cytoplasmic calcium increase mediating a defense response to invading microbes. We show here that perception of extracellular nucleotides, such as eATP, is important in plant-fungus interactions and that during colonization by the beneficial root endophyte Serendipita indica eATP accumulates in the apoplast at early symbiotic stages. Using liquid chromatography-tandem mass spectrometry, and cytological and functional analysis, we show that S. indica secrets SiE5'NT, an enzymatically active ecto-5'-nucleotidase capable of hydrolyzing nucleotides in the apoplast. Arabidopsis thaliana lines producing extracellular SiE5'NT are significantly better colonized, have reduced eATP levels, and altered responses to biotic stresses, indicating that SiE5'NT functions as a compatibility factor. Our data suggest that extracellular bioactive nucleotides and their perception play an important role in fungus-root interactions and that fungal-derived enzymes can modify apoplastic metabolites to promote fungal accommodation.

Draft genome sequencing and secretome analysis of fungal phytopathogen Ascochyta rabiei provides insight into the necrotrophic effector repertoire.

Constant evolutionary pressure acting on pathogens refines their molecular strategies to attain successful pathogenesis. Recent studies have shown that pathogenicity mechanisms of necrotrophic fungi are far more intricate than earlier evaluated. However, only a few studies have explored necrotrophic fungal pathogens. Ascochyta rabiei is a necrotrophic fungus that causes devastating blight disease of chickpea (Cicer arietinum). Here, we report a 34.6 megabase draft genome assembly of A. rabiei. The genome assembly covered more than 99% of the gene space and 4,259 simple sequence repeats were identified in the assembly. A total of 10,596 high confidence protein-coding genes were predicted which includes a large and diverse inventory of secretory proteins, transporters and primary and secondary metabolism enzymes reflecting the necrotrophic lifestyle of A. rabiei. A wide range of genes encoding carbohydrate-active enzymes capable for degradation of complex polysaccharides were also identified. Comprehensive analysis predicted a set of 758 secretory proteins including both classical and non-classical secreted proteins. Several of these predicted secretory proteins showed high cysteine content and numerous tandem repeats. Together, our analyses would broadly expand our knowledge and offer insights into the pathogenesis and necrotrophic lifestyle of fungal phytopathogens.

Comparative structural modeling of six old yellow enzymes (OYEs) from the necrotrophic fungus Ascochyta rabiei: insight into novel OYE classes with differences in cofactor binding, organization of active site residues and stereopreferences.

Old Yellow Enzyme (OYE1) was the first flavin-dependent enzyme identified and characterized in detail by the entire range of physical techniques. Irrespective of this scrutiny, true physiological role of the enzyme remains a mystery. In a recent study, we systematically identified OYE proteins from various fungi and classified them into three classes viz. Class I, II and III. However, there is no information about the structural organization of Class III OYEs, eukaryotic Class II OYEs and Class I OYEs of filamentous fungi. Ascochyta rabiei, a filamentous phytopathogen which causes Ascochyta blight (AB) in chickpea possesses six OYEs (ArOYE1-6) belonging to the three OYE classes. Here we carried out comparative homology modeling of six ArOYEs representing all the three classes to get an in depth idea of structural and functional aspects of fungal OYEs. The predicted 3D structures of A. rabiei OYEs were refined and evaluated using various validation tools for their structural integrity. Analysis of FMN binding environment of Class III OYE revealed novel residues involved in interaction. The ligand para-hydroxybenzaldehyde (PHB) was docked into the active site of the enzymes and interacting residues were analyzed. We observed a unique active site organization of Class III OYE in comparison to Class I and II OYEs. Subsequently, analysis of stereopreference through structural features of ArOYEs was carried out, suggesting differences in R/S selectivity of these proteins. Therefore, our comparative modeling study provides insights into the FMN binding, active site organization and stereopreference of different classes of ArOYEs and indicates towards functional differences of these enzymes. This study provides the basis for future investigations towards the biochemical and functional characterization of these enigmatic enzymes.

Comprehensive genome-wide analysis reveals different classes of enigmatic old yellow enzyme in fungi.

In this study, we systematically identify Old Yellow Enzymes (OYEs) from a diverse range of economically important fungi representing different ecology and lifestyle. Using active site residues and sequence alignments, we present a classification for these proteins into three distinct classes including a novel class (Class III) and assign names to sequences. Our in-depth phylogenetic analysis suggests a complex history of lineage-specific expansion and contraction for the OYE gene family in fungi. Comparative analyses reveal remarkable diversity in the number and classes of OYE among fungi. Quantitative real-time PCR (qRT-PCR) of Ascochyta rabiei OYEs indicates differential expression of OYE genes during oxidative stress and plant infection. This study shows relationship of OYE with fungal ecology and lifestyle, and provides a foundation for future functional analysis and characterization of OYE gene family.

Comparative transcriptome analysis of the necrotrophic fungus Ascochyta rabiei during oxidative stress: insight for fungal survival in the host plant.

Localized cell death, known as the hypersensitive response (HR), is an important defense mechanism for neutralizing phytopathogens. The hallmark of the HR is an oxidative burst produced by the host plant. We aimed to identify genes of the necrotrophic chickpea blight fungus Ascochyta rabiei that are involved in counteracting oxidative stress. A subtractive cDNA library was constructed after menadione treatment, which resulted in the isolation of 128 unigenes. A reverse northern blot was used to compare transcript profiles after H(2)O(2), menadione and sodium nitroprusside treatments. A total of 70 unigenes were found to be upregulated by more than two-fold following menadione treatment at different time intervals. A large number of genes not previously associated with oxidative stress were identified, along with many stress-responsive genes. Differential expression patterns of several genes were validated by quantitative real-time PCR (qRT-PCR) and northern blotting. In planta qRT-PCR of several selected genes also showed differential expression patterns during infection and disease progression. These data shed light on the molecular responses of the phytopathogen A. rabiei to overcome oxidative and nitrosative stresses and advance the understanding of necrotrophic fungal pathogen survival mechanisms.

A highly efficient Agrobacterium mediated transformation system for chickpea wilt pathogen Fusarium oxysporum f. sp. ciceri using DsRed-Express to follow root colonisation.

The soil-borne fungus Fusarium oxysporum f. sp. ciceri (Foc) causes vascular wilt of chickpea (Cicer arietinum L.), resulting in substantial yield losses worldwide. Agrobacterium tumefaciens mediated transformation (ATMT) has served as a resourceful tool for plant-pathogen interaction studies and offers a number of advantages over conventional transformation systems. Here, we developed a highly efficient A. tumefaciens mediated transformation system for Foc. In addition, a binary vector for constitutive expression of red fluorescent protein (DsRed-Express) was used to study developmental stages and host-pathogen interactions. Southern hybridisation was performed to confirm the transformation event and the presence of T-DNA in selected hygromycin resistant transformants. Most of the transformants showed single copy integrations at random positions. Microscopic studies revealed significant levels of fluorescent protein, both in conidia and mycelia. Confocal microscopy of chickpea roots infected with the transformed Foc showed rapid colonisation. These studies will allow us to develop strategies to determine the mechanisms of Foc-chickpea interaction in greater detail and to apply functional genomics for the characterisation of involved genes at the molecular level either by insertional mutagenesis or gene knock-out.

High reliability transformation of the wheat pathogen Bipolaris sorokiniana using Agrobacterium tumefaciens.

Bipolaris sorokiniana, the causal agent of spot blotch of wheat, significantly reduces grain yield worldwide. In order to study pathogenic mechanisms of the fungus, conditions for efficient transformation using Agrobacterium-mediated transformation were investigated. To study different stages of hyphal fusion and pathogenic mechanisms of the fungus, two fluorescence markers viz. the red fluorescent protein (DsRed-Express) and the green fluorescent protein (EGFP1) were constitutively expressed. Southern hybridizations confirmed the presence of T-DNA in all hygromycin B or geneticin resistant transformants, and also showed random and single copy integration. Fluorescence microscopy suggested the high level expression of both DsRed and EGFP fluorescent proteins in spores and mycelia. The results signify that DsRed and EGFP can be used as efficient reporter gene for monitoring B. sorokiniana hyphal fusion as well as colonization in the host tissues. This work will be useful to develop methodologies for understanding the mechanisms of Bipolaris-wheat interaction and functional genomics of B. sorokiniana for various applications including insertional mutagenesis, targeted disruption of specific genes, ectopic complementation of loss-of-function strains and over-expression.

Expression of the fluorescent proteins DsRed and EGFP to visualize early events of colonization of the chickpea blight fungus Ascochyta rabiei.

Ascochyta blight caused by the ascomycete fungus Ascochyta rabiei, is a major biotic constraint of chickpea (Cicer arietinum L.), resulting in disastrous crop losses worldwide. To study early stages of development and pathogenic mechanisms of the fungus, two binary vectors for the constitutive expression of the red fluorescent protein (DsRed-Express) and the green fluorescent protein (EGFP1) were constructed. Furthermore, we have developed an improved and highly reproducible Agrobacterium tumefaciens-mediated transformation protocol for A. rabiei. Transformation events were confirmed through Southern hybridizations that suggest single-copy integration of reporter genes in majority of the transformants. High level expression of both DsRed and EGFP proteins was obtained both in spores and in mycelia as detected by fluorescence microscopy. Intense fluorescence was used as a highly efficient vital marker to visualize early developmental changes of the fungus. The formation of infection structures like appressoria and germ tubes were observed both in vitro and in planta. This work will be useful to develop methodologies for understanding the mechanisms of Ascochyta-chickpea interaction and functional genomics of A. rabiei towards the isolation of virulence genes.