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
Source: PubMed


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.

Download full-text


Available from: David L Joly, Jul 24, 2014
  • Source
    • "tritici, the flax rust Melampsora lini and the coffee rust Hemileia vastatrix (Cantu et al., 2011, 2013; Duplessis et al., 2011a; Zheng et al., 2013; Cristancho et al., 2014; Nemri et al., 2014). Secretomes of rust fungi have been determined based on the presence of predicted N-terminal signal peptides in proteins (Cantu et al., 2011, 2013; Duplessis et al., 2011a; Fernandez et al., 2012; Hacquard et al., 2012; Saunders et al., 2012; Bruce et al., 2013; Garnica et al., 2013; Zheng et al., 2013; Link et al., 2014; Nemri et al., 2014). Signal peptides can be defined using predictors available online (Emanuelsson et al., 2007). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The poplar leaf rust fungus, Melampsora larici-populina has been established as a tree-microbe interaction model. Understanding the molecular mechanisms controlling infection by pathogens appears essential for durable management of tree plantations. In biotrophic plant-parasites, effectors are known to condition host cell colonization. Thus, investigation of candidate secreted effector proteins (CSEPs) is a major goal in the poplar–poplar rust interaction. Unlike oomycetes, fungal effectors do not share conserved motifs and candidate prediction relies on a set of a priori criteria established from reported bona fide effectors. Secretome prediction, genome-wide analysis of gene families and transcriptomics of M. larici-populina have led to catalogs of more than a thousand secreted proteins. Automatized effector-mining pipelines hold great promise for rapid and systematic identification and prioritization of CSEPs for functional characterization. In this review, we report on and discuss the current status of the poplar rust fungus secretome and prediction of candidate effectors from this species.
    Full-text · Article · Dec 2015 · Frontiers in Plant Science
  • Source
    • "The RNA samples were sent to Cofactor Genomics (St. Louis, MO) for sequencing. RNA Sequencing and In Silico Subtraction Cofactor Genomics (St. Louis, MO) used the total RNA for sequencing as previously summarized by Bruce et al. (2014). In brief, the cDNA was sequenced using Illumina 60-bp paired-end reads (Cofactor). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Leaf rust, caused by Puccinia triticina Eriks., is one of the most widespread diseases of wheat and breeding for resistance is one of the most effective methods of control. Lr16 is a wheat leaf rust resistance gene (R-gene) that provides resistance at both the seedling and adult stages. Simple-sequence repeat (SSR) markers have been used to map Lr16 to the distal end of chromosome 2B. The objectives of this study were to use RNA sequencing (RNA-seq) and in silico subtraction to identify new R-gene analogs (RGAs) and use them as Lr16 markers. RNA was isolated from the susceptible wheat cultivar Thatcher (Tc) and the resistant Tc isolines TcLr10, TcLr16, TcLr21, and sequenced using Illumina technology. Using in silico subtraction, sequences from the resistant Tc isolines were aligned to a Tc reference expressed sequence tag (EST) set. Sequences not aligning to the Tc reference were assembled into contigs and analyzed using BLASTx to determine putative gene functions. Primer pairs were designed for 181 RGA sequences, of which, 137 amplified in at least one of the parents. A mapping population was developed with 165 F2 lines from a cross between the rust-susceptible cultivar Chinese Spring (CS) and TcLr16. Two RGA markers XTaLr16_RGA266585 and XTaLr16_RGA22128 were identified that mapped proximally 1.2 and 23.8 cM from Lr16, respectively. Three SSR markers Xwmc764, Xwmc661, and Xbarc35 mapped between these two RGA markers at distances of 5.0, 10.9, and 16.1 cM from Lr16, respectively. In silico subtraction is an effective technique for isolating RGAs linked to R-genes of interest.
    Preview · Article · Jul 2015 · The Plant Genome
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Aug 2014 · Frontiers in Plant Science
Show more