John P. Fellers

Molecular characterization of the major wheat domestication gene Q.

The Q gene is largely responsible for the widespread cultivation of wheat because it confers the free-threshing character. It also pleiotropically influences many other domestication-related traits such as glume shape and tenacity, rachis fragility, spike length, plant height, and spike emergence time. We isolated the Q gene and verified its identity by analysis of knockout mutants and transformation. The Q gene has a high degree of similarity to members of the AP2 family of transcription factors. The Q allele is more abundantly transcribed than q, and the two alleles differ for a single amino acid. An isoleucine at position 329 in the Q protein leads to an abundance of homodimer formation in yeast cells, whereas a valine in the q protein appears to limit homodimer formation. Ectopic expression analysis allowed us to observe both silencing and overexpression effects of Q. Rachis fragility, glume shape, and glume tenacity mimicked the q phenotype in transgenic plants exhibiting post-transcriptional silencing of the transgene and the endogenous Q gene. Variation in spike compactness and plant height were associated with the level of transgene transcription due to the dosage effects of Q. The q allele is the more primitive, and the mutation that gave rise to Q occurred only once leading to the worlds cultivated wheats.

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.

BAC-FISH in wheat identifies chromosome landmarks consisting of different types of transposable elements

Fluorescence in situ hybridization (FISH) has been widely used in the physical mapping of genes and chromosome landmarks in plants and animals. Bacterial artificial chromosomes (BACs) contain large inserts making them amenable for FISH mapping. We used BAC-FISH to study genome organization and evolution in hexaploid wheat and its relatives. We selected 56 restriction fragment length polymorphism (RFLP) locus-specific BAC clones from libraries of Aegilops tauschii (the D-genome donor of hexaploid wheat) and A-genome diploid Triticum monococcum. Different types of repetitive sequences were identified using BAC-FISH. Two BAC clones gave FISH patterns similar to the repetitive DNA family pSc119; one BAC clone gave a FISH pattern similar to the repetitive DNA family pAs1. In addition, we identified several novel classes of repetitive sequences: one BAC clone hybridized to the centromeric regions of wheat and other cereal species, except rice; one BAC clone hybridized to all subtelomeric chromosome regions in wheat, rye, barley and oat; one BAC clone contained a localized tandem repeat and hybridized to five D-genome chromosome pairs in wheat; and four BAC clones hybridized only to a proximal region in the long arm of chromosome 4A of hexaploid wheat. These repeats are valuable markers for defined chromosome regions and can also be used for chromosome identification. Sequencing results revealed that all these repeats are transposable elements (TEs), indicating the important role of TEs, especially retrotransposons, in genome evolution of wheat.

Identification of the wheat curl mite as the vector of Triticum mosaic virus.

Triticum mosaic virus (TriMV) is a newly discovered virus found infecting wheat (Triticum aestivum) in Kansas. This study was conducted to determine if the wheat curl mite (WCM, Aceria tosichella) and the bird cherry oat aphid (Rhopalosiphum padi) could transmit TriMV. Using different sources of WCM and two different isolates of TriMV, we were able to show the WCM is the vector of TriMV. Field analysis by enzyme-linked immunosorbent assay (ELISA) demonstrated natural infection patterns of wheat infected with TriMV, Wheat streak mosaic virus (WSMV), or both TriMV and WSMV, putatively infected by viruliferous WCM from a volunteer source growing adjacent to the wheat. Moreover, by single WCM transfers using WCM obtained from different wheat plants naturally infected with TriMV and WSMV and naturally infested with WCM, we showed that these WCM also transmitted TriMV only to wheat or transmitted both TriMV and WSMV to wheat. The infection rates of wheat with TriMV only using WCM transmission was low in both laboratory and field analyses. However, field analyses by ELISA showed that levels of infection of wheat by both TriMV and WSMV were high. No transmission of TriMV to wheat by R. padi occurred in our studies.

In planta induced genes of Puccinia triticina

SUMMARY Wheat leaf rust disease, caused by the biotrophic fungus Puccinia triticina, is a result of complex interactions requiring the coordinated activities of the two organisms involved. In an effort to understand the molecular basis of wheat-rust interactions, we isolated and characterized cDNA corresponding to in planta induced fungal genes (PIGs) from susceptible wheat leaves infected with P. triticina by using suppression subtractive hybridization to construct a cDNA library. 350 clones were sequenced, of which 104 were unique. Forty-four cDNA clones encode ribosomal proteins, comprising the single largest category of clones isolated. Twenty-five of these ribosomal protein genes are likely to be of fungal origin, as was suggested by sequence homology. Hybridization of 56 selected non-ribosomal protein clones to rust germling cDNA or genomic DNA probes showed that at least 44 were of fungal origin, demonstrating that the library was highly enriched for fungal cDNA. Differential expression analysis identified 26 non-ribosomal protein genes that were induced in rust-infected leaves. At least 21 of the induced genes were from the rust fungus, indicating that the majority of the induced genes were rust PIGs that are likely to play a role in parasitism. Some of the induced genes share homology to known PIGs or virulence genes in other fungi, suggesting similarities in parasitism among different fungi. Eight clones correspond to novel PIGs that have not been reported in any organism. This paper reports, for the first time, the isolation of P. triticina PIGs and discusses the use of total rust genomic DNA to identify the source of genes.

Triticum mosaic virus: A New Virus Isolated from Wheat in Kansas

In 2006, a mechanically-transmissible and previously uncharacterized virus was isolated in Kansas from wheat plants with mosaic symptoms. The physiochemical properties of the virus were examined by purification on cesium chloride density gradients, electron microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), sequencing of the nucleotides and amino acids of the coat protein, and immunological reactivity. Purified preparations contained flexuous, rod-shaped particles that resembled potyviruses. The coat protein was estimated from SDS-PAGE to have a mass of approximately 35 kDa. Its amino acid sequence, as deduced from DNA sequencing of cloned, reverse-transcribed viral RNA and separately determined by time-of-flight mass spectrometry, was most closely related (49% similarity) to Sugarcane streak mosaic virus, a member of the Tritimovirus genus of the family Potyviridae. The virus gave strong positive reactions during enzyme-linked immunosorbent assays using polyclonal antibodies raised against purified preparations of the cognate virus but gave consistent negative reactions against antibodies to Wheat streak mosaic virus (WSMV), other wheat potyviruses, and the High Plains virus. When the virus was inoculated on the WSMV-resistant wheat cv. RonL, systemic symptoms appeared and plant growth was diminished significantly in contrast with WSMV-inoculated RonL. Taken together, the data support consideration of this virus as a new potyvirus, and the name Triticum mosaic virus (TriMV) is proposed.

A group of related cDNAs encoding secreted proteins from Hessian fly [Mayetiola destructor (Say)] salivary glands

A group of cDNAs has been isolated and characterized from Hessian fly [Mayetiola destructor (Say)] salivary glands. Members in this group appear to encode proteins with secretion signal peptides at the N‐terminals. The mature putative proteins are small, basic proteins with calculated molecular weights that ranged from 8.5 to 10 kDa, and isoelectric points from 9.92 to 10.90. Sequence analysis indicated a strong selection for mutations that generate amino acid changes within the coding region. Northern blot analysis revealed that these genes are expressed only in the first instar larvae, a critical stage that determines if the interaction between a specific Hessian fly biotype and a specific wheat cultivar is compatible. Genomic analysis demonstrated that multiple copies of similar genes are clustered within a short region on chromosome 2A. This is the same arm in which two avirulence genes have been mapped.

A strategy for mapping bicoid on the phylogenetic tree

SJB and RED thank Barbara Van Slyke for expert technical assistance. This work was supported by NIH and NSF (SJB and RED), and by the Max-Planck-Gesellschaft and the Deutsche Forschungsgemeinschaft (US-O).

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

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.

Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi

BackgroundRust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology.ResultsTo support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt), we have generated Expressed Sequence Tags (ESTs) by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores) and asexual (germinated urediniospores) stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum), 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with 25,558 previously generated ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs). Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt) and stripe rust, P. striiformis f. sp. tritici (Pst), and poplar leaf rust Melampsora species, and the corn smut fungus, Ustilago maydis (Um). While extensive homologies were found, many genes appeared novel and species-specific; over 40% of genes did not match any known sequence in existing databases. Focusing on spore stages, direct comparison to Um identified potential functional homologs, possibly allowing heterologous functional analysis in that model fungus. Many potentially secreted protein genes were identified by similarity searches against genes and proteins of Pgt and Melampsora spp., revealing apparent orthologs.ConclusionsThe current set of Pt unigenes contributes to gene discovery in this major cereal pathogen and will be invaluable for gene model verification in the genome sequence.