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Using transcription of six Puccinia triticina races to identify the effective secretome during infection of wheat

Frontiers in Plant Science (Impact Factor: 3.95). 01/2014; 4:520. DOI: 10.3389/fpls.2013.00520
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ABSTRACT Wheat leaf rust, caused by the basidiomycete Puccinia triticina, can cause yield losses of up to 20% in wheat producing regions. During infection, the fungus forms haustoria that secrete proteins into the plant cell and effect changes in plant transcription, metabolism, and defense. It is hypothesized that new races emerge as a result of overcoming plant resistance via changes in the secreted effector proteins. To understand gene expression during infection and find genetic differences associated with races, RNA from wheat leaves infected with six different rust races, at 6 days post inoculation, was sequenced using Illumina. As P. triticina is an obligate biotroph, RNA from both the host and fungi were present and separated by alignment to the P. triticina genome and a wheat EST reference. A total of 222,571 rust contigs were assembled from 165 million reads. An examination of the resulting contigs revealed 532 predicted secreted proteins among the transcripts. Of these, 456 were found in all races. Fifteen genes were found with amino acid changes, corresponding to putative avirulence effectors potentially recognized by 11 different leaf rust resistance (Lr) genes. Twelve of the potential avirulence effectors have no homology to known genes. One gene had significant similarity to cerato-platanin, a known fungal elicitor, and another showed similarity to fungal tyrosinase, an enzyme involved in melanin synthesis. Temporal expression profiles were developed for these genes by qRT-PCR and show that the genes expression patterns were consistent between races from infection initiation to just prior to spore eruption.

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Available from: David L Joly, Jul 24, 2014
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    • "Moreover, such temporal succession of expression patterns has been confirmed in other rust species such as Hemileia vastatrix (Fernandez et al., 2012), P. striiformis f. sp. tritici (Cantu et al., 2013), and Puccinia triticina (Bruce et al., 2014). This highlights the need for a better understanding of expression regulation in rust fungi, whether by transcription factors or via epigenetic control, such as reported in Phytophthora infestans or in Leptosphaeria maculans (Judelson, 2012; Soyer et al., 2014). "
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    ABSTRACT: 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.
    Frontiers in Plant Science 08/2014; 5:416. DOI:10.3389/fpls.2014.00416 · 3.95 Impact Factor
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    • "In addition, expression analysis can be informative, since secreted proteins with specific expression profiles during infection are stronger effector candidates (Ellis et al., 2009). Time-course experiments have been successful in other rust pathogen systems to elucidate the effector-like proteins involved in multiple or highly specific stages during infection (Joly et al., 2010; Duplessis et al., 2011a; Bruce et al., 2014). Also, resequencing of closely related rust pathogens such as Cronartium ribicola, C. flaccidum, and Peridermium harknessii (Vogler and Bruns, 1998) would improve precision of gene family delineations and identification of true singleton Cqf effectors, which are likely to be more newly evolved than effectors in families, and may therefore be products of highly-specific host-Cqf coevolution. "
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    ABSTRACT: Rust fungi are a group of fungal pathogens that cause some of the world's most destructive diseases of trees and crops. A shared characteristic among rust fungi is obligate biotrophy, the inability to complete a lifecycle without a host. This dependence on a host species likely affects patterns of gene expansion, contraction, and innovation within rust pathogen genomes. The establishment of disease by biotrophic pathogens is reliant upon effector proteins that are encoded in the fungal genome and secreted from the pathogen into the host's cell apoplast or within the cells. This study uses a comparative genomic approach to elucidate putative effectors and determine their evolutionary histories. We used OrthoMCL to identify nearly 20,000 gene families in proteomes of 16 diverse fungal species, which include 15 basidiomycetes and one ascomycete. We inferred patterns of duplication and loss for each gene family and identified families with distinctive patterns of expansion/contraction associated with the evolution of rust fungal genomes. To recognize potential contributors for the unique features of rust pathogens, we identified families harboring secreted proteins that: (i) arose or expanded in rust pathogens relative to other fungi, or (ii) contracted or were lost in rust fungal genomes. While the origin of rust fungi appears to be associated with considerable gene loss, there are many gene duplications associated with each sampled rust fungal genome. We also highlight two putative effector gene families that have expanded in Cqf that we hypothesize have roles in pathogenicity.
    Frontiers in Plant Science 06/2014; 5:299. DOI:10.3389/fpls.2014.00299 · 3.95 Impact Factor
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    • "Consequently, an alternative strategy based on transcriptome sequencing was initiated. Transcriptome sequencing has emerged as a powerful tool for the identification of (candidate) Avr genes from phytopathogenic fungi (Bruce et al. 2014; Cantu et al. 2013; de Jonge et al. 2012; van de Wouw et al. 2013 "
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    ABSTRACT: The Cf-5 gene of tomato confers resistance to strains of the fungal pathogen Cladosporium fulvum carrying the avirulence gene Avr5. Although Cf-5 has been cloned, Avr5 has remained elusive. We report the cloning of Avr5 using a combined bioinformatic and transcriptome sequencing approach. RNA-Seq was performed on the sequenced race 0 strain (0WU; carries Avr5), as well as a race 5 strain (IPO 1979; lacks a functional Avr5 gene), during infection of susceptible tomato. Forty-four in planta-induced C. fulvum Candidate Effector (CfCE) genes of 0WU were identified that putatively encode a secreted, small cysteine-rich protein. An expressed transcript sequence comparison between strains revealed two polymorphic CfCE genes in IPO 1979. One of these conferred avirulence to IPO 1979 on Cf-5 tomato following complementation with the corresponding 0WU allele, confirming identification of Avr5. Complementation also led to increased fungal biomass during infection of susceptible tomato, signifying a role for Avr5 in virulence. Seven of eight race 5 strains investigated escape Cf-5-mediated resistance through deletion of the Avr5 gene. Avr5 is heavily flanked by repetitive elements, suggesting that repeat instability, in combination with Cf-5-mediated selection pressure, has led to the emergence of race 5 strains deleted for the Avr5 gene.
    Molecular Plant-Microbe Interactions 03/2014; 27(8):846-857. DOI:10.1094/MPMI-02-14-0050-R · 4.46 Impact Factor
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