Fungal-plant interactions
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Frontiers: Effector proteins of rust fungi (2014)

Frontiers: Effector proteins of rust fungi (2014) | Fungal-plant interactions | Scoop.it

Rust fungi include many species that are devastating crop pathogens. To develop resistant plants, a better understanding of rust virulence factors, or effector proteins, is needed. Thus far, only six rust effector proteins have been described: AvrP123, AvrP4, AvrL567, AvrM, RTP1 and PGTAUSPE-10-1. Although some are well established model proteins used to investigate mechanisms of immune receptor activation (avirulence activities) or entry into plant cells, how they work inside host tissues to promote fungal growth remains unknown. The genome sequences of four rust fungi (two Melampsoraceae and two Pucciniaceae) have been analyzed so far. Genome-wide analyses of these species, as well as transcriptomics performed on a broader range of rust fungi, revealed hundreds of small secreted proteins considered as rust candidate secreted effector proteins (CSEPs). The rust community now needs high-throughput approaches (effectoromics) to accelerate effector discovery/characterization and to better understand how they function in planta. However, this task is challenging due to the non-amenability of rust pathosystems (obligate biotrophs infecting crop plants) to traditional molecular genetic approaches mainly due to difficulties in culturing these species in vitro. The use of heterologous approaches should be promoted in the future.


Via Kamoun Lab @ TSL
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Plant Cell: Colletotrichum orbiculare Secretes Virulence Effectors to a Biotrophic Interface at the Primary Hyphal Neck via Exocytosis Coupled with SEC22-Mediated Traffic (2014)

Plant Cell: Colletotrichum orbiculare Secretes Virulence Effectors to a Biotrophic Interface at the Primary Hyphal Neck via Exocytosis Coupled with SEC22-Mediated Traffic (2014) | Fungal-plant interactions | Scoop.it

The hemibiotrophic pathogen Colletotrichum orbiculare develops biotrophic hyphae inside cucumber (Cucumis sativus) cells via appressorial penetration; later, the pathogen switches to necrotrophy. C. orbiculare also expresses specific effectors at different stages. Here, we found that virulence-related effectors of C. orbiculare accumulate in a pathogen–host biotrophic interface. Fluorescence-tagged effectors accumulated in a ring-like region around the neck of the biotrophic primary hyphae. Fluorescence imaging of cellular components and transmission electron microscopy showed that the ring-like signals of the effectors localized at the pathogen–plant interface. Effector accumulation at the interface required induction of its expression during the early biotrophic phase, suggesting that transcriptional regulation may link to effector localization. We also investigated the route of effector secretion to the interface. An exocytosis-related component, the Rab GTPase SEC4, localized to the necks of biotrophic primary hyphae adjacent to the interface, thereby suggesting focal effector secretion. Disruption of SEC4 in C. orbiculare reduced virulence and impaired effector delivery to the ring signal interface. Disruption of the v-SNARE SEC22also reduced effector delivery. These findings suggest that biotrophy-expressed effectors are secreted, via the endoplasmic reticulum-to-Golgi route and subsequent exocytosis, toward the interface generated between C. orbiculare and the host cell.


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Frontiers | The genome sequence and effector complement of the flax rust pathogen Melampsora lini | Plant-Microbe Interaction

Frontiers | The genome sequence and effector complement of the flax rust pathogen Melampsora lini | Plant-Microbe Interaction | Fungal-plant interactions | Scoop.it
Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimise parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analysed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their hosts.
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A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize

A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize | Fungal-plant interactions | Scoop.it

The biotrophic fungus Ustilago maydis causes smut disease in maize with characteristic tumor formation and anthocyanin induction. Here, we show that anthocyanin biosynthesis is induced by the virulence promoting secreted effector protein Tin2. Tin2 protein functions inside plant cells where it interacts with maize protein kinase ZmTTK1. Tin2 masks a ubiquitin–proteasome degradation motif in ZmTTK1, thus stabilizing the active kinase. Active ZmTTK1 controls activation of genes in the anthocyanin biosynthesis pathway. Without Tin2, enhanced lignin biosynthesis is observed in infected tissue and vascular bundles show strong lignification. This is presumably limiting access of fungal hyphae to nutrients needed for massive proliferation. Consistent with this assertion, we observe that maize brown midrib mutants affected in lignin biosynthesis are hypersensitive to U. maydis infection. We speculate that Tin2 rewires metabolites into the anthocyanin pathway to lower their availability for other defense responses.

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Clustering of Pattern Recognition Receptors for Fungal Detection

Clustering of Pattern Recognition Receptors for Fungal Detection | Fungal-plant interactions | Scoop.it

 

The innate immune system is the first line of defense against invading pathogens. Innate cells recognize microbes via pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs); initiate innate immune responses; and eventually trigger adaptive immunity. Association of heterologous PRRs synergistically enhances their signal intensity. Such PRR cluster formation is essential for fungal detection in generating antifungal immunity. In this review, PRR cluster formation to detect fungi and to initiate innate immune responses is discussed.

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MPMI: Comparative and functional analysis of the widely occurring family of Nep1-like proteins (2014)

MPMI: Comparative and functional analysis of the widely occurring family of Nep1-like proteins (2014) | Fungal-plant interactions | Scoop.it

Nep-1 Like Proteins (NLPs) are best known for their cytotoxic activity in dicot plants. NLPs are taxonomically widespread among microbes with very different lifestyles. To learn more about this enigmatic protein family we analyzed more than 500 available NLP protein sequences from fungi, oomycetes, and bacteria. Phylogenetic clustering showed that, besides the previously documented two types, an additional more divergent third NLP type could be distinguished. By closely examining the three NLP types, we identified a non-cytotoxic subgroup of type 1 NLPs (designated type 1a), which have substitutions in amino acids making up a cation-binding pocket that is required for cytotoxicity. Type 2 NLPs were found to contain a putative calcium-binding motif, which was shown to be required for cytotoxicity. Members of both type 1 and type 2 NLPs were found to possess additional cysteine residues that, based on their predicted proximity, make up potential disulfide bridges that could provide additional stability to these secreted proteins. Type 1 and type 2 NLPs, although both cytotoxic to plant cells, differ in their ability to induce necrosis when artificially targeted to different cellular compartments in planta, suggesting they have different mechanisms of cytotoxicity.


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N-Glycosylation of Effector Proteins by an α-1,3-Mannosyltransferase Is Required for the Rice Blast Fungus to Evade Host Innate Immunity

N-Glycosylation of Effector Proteins by an α-1,3-Mannosyltransferase Is Required for the Rice Blast Fungus to Evade Host Innate Immunity | Fungal-plant interactions | Scoop.it

Plant pathogenic fungi deploy secreted effectors to suppress plant immunity responses. These effectors operate either in the apoplast or within host cells, so they are putatively glycosylated, but the posttranslational regulation of their activities has not been explored. In this study, the ASPARAGINE-LINKED GLYCOSYLATION3 (ALG3)-mediated N-glycosylation of the effector, Secreted LysM Protein1 (Slp1), was found to be essential for its activity in the rice blast fungus Magnaporthe oryzae. ALG3 encodes an α-1,3-mannosyltransferase for protein N-glycosylation. Deletion of ALG3 resulted in the arrest of secondary infection hyphae and a significant reduction in virulence. We observed that Δalg3 mutants induced massive production of reactive oxygen species in host cells, in a similar manner to Δslp1 mutants, which is a key factor responsible for arresting infection hyphae of the mutants. Slp1 sequesters chitin oligosaccharides to avoid their recognition by the rice (Oryza sativa) chitin elicitor binding protein CEBiP and the induction of innate immune responses, including reactive oxygen species production. We demonstrate that Slp1 has three N-glycosylation sites and that simultaneous Alg3-mediated N-glycosylation of each site is required to maintain protein stability and the chitin binding activity of Slp1, which are essential for its effector function. These results indicate that Alg3-mediated N-glycosylation of Slp1 is required to evade host innate immunity.

Wheat Pathogenomics's insight:

Ecp6 homologue in Magnaporthe requires N-glycosylation for efficient binding to chitin.

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Effectors and Effector Delivery in Magnaporthe oryzae

Effectors and Effector Delivery in Magnaporthe oryzae | Fungal-plant interactions | Scoop.it

 

Two distinct effector secretion systems have been identified in M. oryzae. The apoplastic effectors (◊) utilize the conventional ER-Golgi secretion pathway for secretion. Disruption of the cytoskeleton by benzimidazoleand LatA treatments interferes with the secretion of apoplastic effectors. In contract, the secretion of cytoplasmic effectors (♦) into the BIC is independent of the Golgi bodies and cytoskeleton but involves the exocyst complex and t-SNAREs.

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Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector

Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector | Fungal-plant interactions | Scoop.it

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.

 

TMV graphic from http://cronodon.com

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