Fast-forward genetics by radiation hybrids to saturate the locus regulating nuclear-cytoplasmic compatibility in Triticum.

The nuclear-encoded species cytoplasm specific (scs) genes control nuclear-cytoplasmic compatibility in wheat (genus Triticum). Alloplasmic cells, which have nucleus and cytoplasm derived from different species, produce vigorous and vital organisms only when the correct version of scs is present in their nucleus. In this study, bulks of in vivo radiation hybrids segregating for the scs phenotype have been genotyped by sequencing with over 1.9 million markers. The high marker saturation obtained for a critical region of chromosome 1D allowed identification of 3318 reads that mapped in close proximity of the scs. A novel in silico approach was deployed to extend these short reads to sequences of up to 70 Kb in length and identify candidate open reading frames (ORFs). Markers were developed to anchor the short contigs containing ORFs to a radiation hybrid map of 650 individuals with resolution of 288 Kb. The region containing the scs locus was narrowed to a single Bacterial Artificial Chromosome (BAC) contig of Aegilops tauschii. Its sequencing and assembly by nano-mapping allowed rapid identification of a rhomboid gene as the only ORF existing within the refined scs locus. Resequencing of this gene from multiple germplasm sources identified a single nucleotide mutation, which gives rise to a functional amino acid change. Gene expression characterization revealed that an active copy of this rhomboid exists on all homoeologous chromosomes of wheat, and depending on the specific cytoplasm each copy is preferentially expressed. Therefore, a new methodology was applied to unique genetic stocks to rapidly identify a strong candidate gene for the control of nuclear-cytoplasmic compatibility in wheat.

Emergence of a new disease as a result of interspecific virulence gene transfer.

New diseases of humans, animals and plants emerge regularly. Enhanced virulence on a new host can be facilitated by the acquisition of novel virulence factors. Interspecific gene transfer is known to be a source of such virulence factors in bacterial pathogens (often manifested as pathogenicity islands in the recipient organism) and it has been speculated that interspecific transfer of virulence factors may occur in fungal pathogens. Until now, no direct support has been available for this hypothesis. Here we present evidence that a gene encoding a critical virulence factor was transferred from one species of fungal pathogen to another. This gene transfer probably occurred just before 1941, creating a pathogen population with significantly enhanced virulence and leading to the emergence of a new damaging disease of wheat.

Accelerated evolution of the mitochondrial genome in an alloplasmic line of durum wheat.

BACKGROUND: Wheat is an excellent plant species for nuclear mitochondrial interaction studies due to availability of large collection of alloplasmic lines. These lines exhibit different vegetative and physiological properties than their parents. To investigate the level of sequence changes introduced into the mitochondrial genome under the alloplasmic condition, three mitochondrial genomes of the Triticum-Aegilops species were sequenced: 1) durum alloplasmic line with the Ae. longissima cytoplasm that carries the T. turgidum nucleus designated as (lo) durum, 2) the cytoplasmic donor line, and 3) the nuclear donor line. RESULTS: The mitochondrial genome of the T. turgidum was 451,678 bp in length with high structural and nucleotide identity to the previously characterized T. aestivum genome. The assembled mitochondrial genome of the (lo) durum and the Ae. longissima were 431,959 bp and 399,005 bp in size, respectively. The high sequence coverage for all three genomes allowed analysis of heteroplasmy within each genome. The mitochondrial genome structure in the alloplasmic line was genetically distant from both maternal and paternal genomes. The alloplasmic durum and the Ae. longissima carry the same versions of atp6, nad6, rps19-p, cob and cox2 exon 2 which are different from the T. turgidum parent. Evidence of paternal leakage was also observed by analyzing nad9 and orf359 among all three lines. Nucleotide search identified a number of open reading frames, of which 27 were specific to the (lo) durum line. CONCLUSIONS: Several heteroplasmic regions were observed within genes and intergenic regions of the mitochondrial genomes of all three lines. The number of rearrangements and nucleotide changes in the mitochondrial genome of the alloplasmic line that have occurred in less than half a century was significant considering the high sequence conservation between the T. turgidum and the T. aestivum that diverged from each other 10,000 years ago. We showed that the changes in genes were not limited to paternal leakage but were sufficiently significant to suggest that other mechanisms, such as recombination and mutation, were responsible. The newly formed ORFs, differences in gene sequences and copy numbers, heteroplasmy, and substoichiometric changes show the potential of the alloplasmic condition to accelerate evolution towards forming new mitochondrial genomes.

Analysis of ATP6 sequence diversity in the Triticum-Aegilops species group reveals the crucial role of rearrangement in mitochondrial genome evolution.

Mutation and chromosomal rearrangements are the two main forces of increasing genetic diversity for natural selection to act upon, and ultimately drive the evolutionary process. Although genome evolution is a function of both forces, simultaneously, the ratio of each can be varied among different genomes and genomic regions. It is believed that in plant mitochondrial genome, rearrangements play a more important role than point mutations, but relatively few studies have directly addressed this phenomenon. To address this issue, we isolated and sequenced the ATP6-1 and ATP6-2 genes from 46 different euplasmic and alloplasmic wheat lines. Four different ATP6-1 orthologs were detected, two of them reported for the first time. Expression analysis revealed that all four orthologs are transcriptionally active. Results also indicated that both point mutation and genomic rearrangement are involved in the evolution of ATP6. However, rearrangement is the predominant force that triggers drastic variation. Data also indicated that speciation of domesticated wheat cultivars were simultaneous with the duplication of this gene. These results directly support the notion that rearrangement plays a significant role in driving plant mitochondrial genome evolution.

A unique wheat disease resistance-like gene governs effector-triggered susceptibility to necrotrophic pathogens.

Plant disease resistance is often conferred by genes with nucleotide binding site (NBS) and leucine-rich repeat (LRR) or serine/threonine protein kinase (S/TPK) domains. Much less is known about mechanisms of susceptibility, particularly to necrotrophic fungal pathogens. The pathogens that cause the diseases tan spot and Stagonospora nodorum blotch on wheat produce effectors (host-selective toxins) that induce susceptibility in wheat lines harboring corresponding toxin sensitivity genes. The effector ToxA is produced by both pathogens, and sensitivity to ToxA is governed by the Tsn1 gene on wheat chromosome arm 5BL. Here, we report the cloning of Tsn1, which was found to have disease resistance gene-like features, including S/TPK and NBS-LRR domains. Mutagenesis revealed that all three domains are required for ToxA sensitivity, and hence disease susceptibility. Tsn1 is unique to ToxA-sensitive genotypes, and insensitive genotypes are null. Sequencing and phylogenetic analysis indicated that Tsn1 arose in the B-genome diploid progenitor of polyploid wheat through a gene-fusion event that gave rise to its unique structure. Although Tsn1 is necessary to mediate ToxA recognition, yeast two-hybrid experiments suggested that the Tsn1 protein does not interact directly with ToxA. Tsn1 transcription is tightly regulated by the circadian clock and light, providing further evidence that Tsn1-ToxA interactions are associated with photosynthesis pathways. This work suggests that these necrotrophic pathogens may thrive by subverting the resistance mechanisms acquired by plants to combat other pathogens.

A Two-Step Molecular Detection Method for Pyrenophora tritici-repentis Isolates Insensitive to QoI Fungicides.

Tan spot, caused by Pyrenophora tritici-repentis, is an important disease of wheat worldwide. To manage tan spot, quinone outside inhibitor (QoI) fungicides such as azoxystrobin and pyraclostrobin have been applied in many countries. QoI fungicides target the cytochrome b (cyt b) site in complex III of mitochondria and, thus, pose a serious risk for resistance development. The resistance mechanism to QoI fungicides is mainly due to point mutations in the cyt b gene. The objective of this study was to develop a molecular detection method for the four currently known mutations responsible for shifts in sensitivity toward QoI fungicides in P. tritici-repentis. Twelve specific primers were designed based on sequences from the National Center for Biotechnology Information accessions AAXI01000704 and DQ919068 and used to generate a fragment of the cyt b gene which possesses four known single-nucleotide polymorphisms (SNPs). These mutant clones served as positive controls because QoI-insensitive and -reduced-sensitive isolates of P. tritici-repentis have not yet been reported in the United States. The partial cyt b gene clones were sequenced to identify the SNPs at sites G143A and F129L. Genomic DNA of the mutated partial cyt b gene clones and the European QoI-insensitive and -reduced-sensitive isolates of P. tritici-repentis possessing G143A (GCT) and F129L (TTA, TTG, and CTC) mutations were amplified by polymerase chain reaction (PCR) using two specific primer pairs and were further digested with three specific restriction enzymes (BsaJI, Fnu4HI, and MnlI). The results of the digested PCR product from genomic DNA of known QoI-insensitive and -reduced-sensitive isolates of P. tritici-repentis had DNA bands consistent with the mutation GCT at G143A and the mutations TTA, TTG, and CTC at F129L. The amplified region at the F129 site also had 99% sequence similarity with P. teres, the net blotch pathogen of barley. To validate mutations, we further tested two isolates of P. teres known to have reduced sensitivity to QoI fungicides possessing the mutations TTA and CTC at F129L. After PCR amplification and restriction digestion, DNA bands identical to those observed for the partial cyt b mutant clones were detected. These results suggest that this newly developed two-step molecular detection method is rapid, robust, and specific to monitor QoI-insensitive and -reduce-dsensitive isolates of P. tritici-repentis.

Tsn1-mediated host responses to ToxA from Pyrenophora tritici-repentis.

The toxin sensitivity gene Tsn1 interacts with Ptr ToxA (ToxA), a host-selective toxin produced by the necrotrophic fungus Pyrenophora tritici-repentis. The molecular mechanisms associated with cell death in sensitive wheat cultivars following ToxA application are not well understood. To address this question, we used the Affymetrix GeneChip Wheat Genome Array to compare gene expression in a sensitive wheat cultivar possessing the Tsn1 gene with the insensitive wheat cv. Nec103, which lacks the Tsn1 gene. This analysis was performed at early timepoints after infiltration with ToxA (e.g., 0.5 to 12 h postinfiltration [hpi]); at this time, ToxA is known to internalize into mesophyll cells without visible cell death symptoms. Gene expression also was monitored at later timepoints (24 to 48 hpi), when ToxA causes extensive damage in cellular compartments and visible cell death. At both early and late timepoints, numerous defense-related genes were induced (2- to 197-fold increases) and included genes involved in the phenylpropanoid pathway, lignification, and the production of reactive oxygen species (ROS). Furthermore, a subset of host genes functioning in signal transduction, metabolism, and as transcription factors was induced as a consequence of the Tsn1-ToxA interaction. Nine genes known to be involved in the host defense response and signaling pathways were selected for analysis by quantitative real-time polymerase chain reaction, and the expression profiles of these genes confirmed the results obtained in microarray experiments. Histochemical analyses of a sensitive wheat cultivar showed that H(2)O(2) was present in leaves undergoing cell death, indicating that ROS signaling is a major event involved in ToxA-mediated cell death. The results suggest that recognition of ToxA via Tsn1 triggers transcriptional reprogramming events similar to those reported for avirulence-resistance gene interactions, and that host-derived genes play an important role in the modulation of susceptibility to P. tritici-repentis.

Detection and quantitation of immunogenic epitopes related to celiac disease in historical and modern hard red spring wheat cultivars.

Celiac disease (CD) develops in genetically susceptible individuals as a result of ingesting gluten-forming proteins found in cereals, such as wheat (Triticum aestivum L.), rye (Secale cereale L.) and barley (Hordeum sativum L.). There are claims that breeding practices have changed wheat protein chemistry over the years and this has resulted in modern wheat being more antigenic in terms of CD as opposed to historical wheat. The aim of this study was to detect and quantify celiac-disease-initiating peptides of α-gliadin proteins in historical and modern spring wheat cultivars. The results indicate that immunogenic epitopes are detected in both historical and modern spring wheat cultivars irrespective of release year. Quantitation indicated that the amount of immunogenic epitopes glia-α9 (PFPQPQLPY) and glia-α20 (FRPQQPYPQ), and total α-gliadin varied randomly across the cultivars that were analyzed, suggesting there is no association between cultivar release year and amounts of immunogenic epitopes and α-gliadin.

Isolation and characterization of two endoxylanases from Fusarium graminearum.

This paper reports the first isolation from cultures of two endoxylanases secreted by Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schweinitz) Petch]. When F. graminearum is grown on wheat bran hydrated with a modified synthetic medium, high xylanase activity can be extracted. The two endoxylanases were identified by LC-MS/MS as the products of genes FGSG_6445 (Genbank gene id 2788192 ) (xylanase 1) and FGSG_3624 (GenBank accession no. AJ863566 ) (xylanase 2) with 61 and 51% sequence coverage, respectively. Both enzymes showed a pH optimum at pH 6, with xylanase 1 exhibiting a wider active pH range (5.5-9) than xlylanase 2 (5.5-7.5). Their temperature dependences were similar, >60% between 35 and 60 °C, with optimal temperatures of 45 °C for xylanase 1 and 50 °C for xylanase 2. Kinetic studies found that both enzymes had a lower K(m) for linear beachwood xylan than arabinoxylan. For xylanase 2, the V(max) increased with arabinoxylan, but decreased for xylanase 1.

Gene cloning using degenerate primers and genome walking.

Gene cloning is the first step of targeted gene replacement for functional studies, discovery of gene alleles, and gene expression among other applications. In this chapter, we will describe a cloning technique suitable for fungal species where the genomic information and sequences available are limited. This strategy involves obtaining protein sequences of the gene of interest from various organisms to identify at least two conserved regions. Degenerate primers are designed from these two conserved regions and the resulting PCR products are sequenced. The sequence of the PCR products can be analyzed using suitable databases to determine their similarity to the gene/protein of interest. In cases where the entire gene cannot be cloned directly using these primers, this initial nucleotide sequence can be used as a template for further primer design and genome walking in both directions for either the cloning of a longer fragment or even the cloning of the complete gene. Here, we describe the partial cloning of a reducing polyketide synthase gene from the fungal plant pathogen Ascochyta rabiei using this strategy.

The Stagonospora nodorum-wheat pathosystem involves multiple proteinaceous host-selective toxins and corresponding host sensitivity genes that interact in an inverse gene-for-gene manner.

We recently showed that the wheat pathogen Stagonospora nodorum produces proteinaceous host-selective toxins (HSTs). These toxins include SnTox1 as well as SnToxA, a HST first identified from Pyrenophora tritici-repentis that was implicated in a very recent horizontal gene transfer event from S. nodorum to P. tritici-repentis. Compelling evidence implicating SnToxA and SnTox1 in disease development has been obtained. Here, we report the partial purification and characterization of a third HST designated SnTox2, as well as the genetic characterization of the corresponding host-sensitivity gene. SnTox2 was protease sensitive and is estimated between 7 and 10 kDa in size. Sensitivity to SnTox2 was conferred by a single dominant gene designated Snn2, which mapped to the short arm of wheat chromosome 2D. Genetic analysis of reaction to conidial inoculations in a segregating wheat population indicated that both the Snn2-SnTox2 and the Tsn1-SnToxA interactions were involved in disease development, and together they accounted for the majority of the phenotypic variation. Therefore, S. nodorum produces multiple toxins that rely on specific interactions with host gene products to cause disease. The identification of multiple HST-host gene interactions important for disease development and the availability of the S. nodorum whole genome sequence indicate the potential for this pathosystem to serve as a toxin-based, inverse gene-for-gene model.

The Tsn1-ToxA interaction in the wheat-Stagonospora nodorum pathosystem parallels that of the wheat-tan spot system.

The wheat tan spot fungus (Pyrenophora tritici-repentis) produces a well-characterized host-selective toxin (HST) known as Ptr ToxA, which induces necrosis in genotypes that harbor the Tsn1 gene on chromosome 5B. In previous work, we showed that the Stagonospora nodorum isolate Sn2000 produces at least 2 HSTs (SnTox1 and SnToxA). Sensitivity to SnTox1 is governed by the Snn1 gene on chromosome 1B in wheat. SnToxA is encoded by a gene with a high degree of similarity to the Ptr ToxA gene. Here, we evaluate toxin sensitivity and resistance to S. nodorum blotch (SNB) caused by Sn2000 in a recombinant inbred population that does not segregate for Snn1. Sensitivity to the Sn2000 toxin preparation cosegregated with sensitivity to Ptr ToxA at the Tsn1 locus. Tsn1-disrupted mutants were insensitive to both Ptr ToxA and SnToxA, suggesting that the 2 toxins are functionally similar, because they recognize the same locus in the host to induce necrosis. The locus harboring the tsn1 allele underlies a major quantitative trait locus (QTL) for resistance to SNB caused by Sn2000, and explains 62% of the phenotypic variation, indicating that the toxin is an important virulence factor for this fungus. The Tsn1 locus and several minor QTLs together explained 77% of the phenotypic variation. Therefore, the Tsn1-ToxA interaction in the wheat-S. nodorum pathosystem parallels that of the wheat-tan spot system, and the wheat Tsn1 gene serves as a major determinant for susceptibility to both SNB and tan spot.

Role of the arginyl-glycyl-aspartic motif in the action of Ptr ToxA produced by Pyrenophora tritici-repentis.

A fundamental problem of plant science is to understand the biochemical basis of plant/pathogen interactions. The foliar disease tan spot of wheat (Triticum aestivum), caused by Pyrenophora tritici-repentis, involves Ptr ToxA, a proteinaceous host-selective toxin that causes host cell death. The fungal gene ToxA encodes a 17.2-kD pre-pro-protein that is processed to produce the mature 13.2-kD toxin. Amino acids 140 to 142 of the pre-pro-protein form an arginyl-glycyl-aspartic (RGD) sequence, a motif involved in the binding of some animal proteins and pathogens to transmembrane receptor proteins called integrins. Integrin-like proteins have been identified in plants recently, but their role in plant biology is unclear. Our model for Ptr ToxA action predicts that toxin interacts with a putative host receptor through the RGD motif. Mutant clones of a ToxA cDNA, created by polymerase chain reaction such that the RGD in the pro-toxin was changed to arginyl-alanyl-aspartic or to arginyl-glycyl-glutamic, were expressed in Escherichia coli. Extracts containing mutated forms of toxin failed to cause host cell death, but extracts from E. coli expressing both a wild-type pro-protein cDNA and a control mutation away from RGD were active in cell death development. In competition experiments, 2 mM RGD tripeptide reduced the level of electrolyte leakage from wheat leaves by 63% when co-infiltrated with purified Ptr ToxA (15 microg mL(-1)) obtained from the fungus, but the control peptide arginyl-glycyl-glutamyl-serine provided no protection. These experiments indicate that the RGD motif of Ptr ToxA is involved with toxin action, possibly by interacting with a putative integrin-like receptor in the host.