Distinguishing Puccinia striiformis f. sp. tritici Isolates Using Genomic Sequencing: A Case Study.

We are reporting on the utilization of high-throughput sequencing and different sequencing analysis tools to delineate identification of different isolates of the stripe rust fungal pathogen Puccinia striiformis f. sp. tritici (Pst). Different approaches are shown: utilization of rDNA sequences and random sequences that may be very useful to make sure that isolates belong to Pst and to distinguished closely related isolates. Identification of unique/lost sequences could lead to the identification of effectors associated with specific isolates.

Analysis of MAPK and MAPKK gene families in wheat and related Triticeae species.

BACKGROUND: The mitogen-activated protein kinase (MAPK) family is involved in signal transduction networks that underpin many different biological processes in plants, ranging from development to biotic and abiotic stress responses. To date this class of enzymes has received little attention in Triticeae species, which include important cereal crops (wheat, barley, rye and triticale) that represent over 20% of the total protein food-source worldwide. RESULTS: The work presented here focuses on two subfamilies of Triticeae MAPKs, the MAP kinases (MPKs), and the MAPK kinases (MKKs) whose members phosphorylate the MPKs. In silico analysis of multiple Triticeae sequence databases led to the identification of 152 MAPKs belonging to these two sub-families. Some previously identified MAPKs were renamed to reflect the literature consensus on MAPK nomenclature. Two novel MPKs, MPK24 and MPK25, have been identified, including the first example of a plant MPK carrying the TGY activation loop sequence common to mammalian p38 MPKs. An EF-hand calcium-binding domain was found in members of the Triticeae MPK17 clade, a feature that appears to be specific to Triticeae species. New insights into the novel MEY activation loop identified in MPK11s are offered. When the exon-intron patterns for some MPKs and MKKs of wheat, barley and ancestors of wheat were assembled based on transcript data in GenBank, they showed deviations from the same sequence predicted in Ensembl. The functional relevance of MAPKs as derived from patterns of gene expression, MPK activation and MKK-MPK interaction is discussed. CONCLUSIONS: A comprehensive resource of accurately annotated and curated Triticeae MPK and MKK sequences has been created for wheat, barley, rye, triticale, and two ancestral wheat species, goat grass and red wild einkorn. The work we present here offers a central information resource that will resolve existing confusion in the literature and sustain expansion of MAPK research in the crucial Triticeae grains.

The stripe rust resistance gene Yr10 encodes an evolutionary-conserved and unique CC-NBS-LRR sequence in wheat.

The first seedling or all-stage resistance (R) R gene against stripe rust isolated from Moro wheat (Triticum aestivum L.) using a map-based cloning approach was identified as Yr10. Clone 4B of this gene encodes a highly evolutionary-conserved and unique CC-NBS-LRR sequence. Clone 4E, a homolog of Yr10, but lacking transcription start site (TSS) and putative TATA-box and CAAT-box, is likely a non-expressed pseudogene. Clones 4B and 4E are 84% identical and divergent in the intron and the LRR domain. Gene silencing and transgenesis were used in conjunction with inoculation with differentially avirulent and virulent stripe rust strains to demonstrate Yr10 functionality. The Yr10 CC-NBS-LRR sequence is unique among known CC-NBS-LRR R genes in wheat but highly conserved homologs (E = 0.0) were identified in Aegilops tauschii and other monocots including Hordeum vulgare and Brachypodium distachyon. Related sequences were also identified in genomic databases of maize, rice, and in sorghum. This is the first report of a CC-NBS-LRR resistance gene in plants with limited homologies in its native host, but with numerous homologous R genes in related monocots that are either host or non-hosts for stripe rust. These results represent a unique example of gene evolution and dispersion across species.

Isolation and characterization of the rye Waxy gene.

Complete genomic and cDNA sequences of the Waxy gene encoding granule-bound starch synthase I (GBSSI) were isolated from the rye genome and characterized. The full-length rye Waxy genomic DNA and cDNA are 2767 bp and 1815 bp, respectively. The genomic sequence has 11 exons interrupted by 10 introns. The rye Waxy gene is GC-rich, with a higher GC frequency in the coding region, especially in the third position of the codons. Exon regions of the rye Waxy gene are more conserved than intron regions when compared with the homologous sequences of other cereals. The mature rye GBSSI proteins share more than 95% sequence identity with their homologs in wheat and barley. A phylogenetic tree based on sequence comparisons of available plant GBSSI proteins shows the evolutionary relationship among Waxy genes from rye and other plant genomes. The identification of the rye Waxy gene will enable the manipulation of starch metabolism in rye and triticale.

Characterization and antifungal properties of wheat nonspecific lipid transfer proteins.

This study simultaneously considered the phylogeny, fatty acid binding ability, and fungal toxicity of a large number of monocot nonspecific lipid transfer proteins (ns-LTP). Nine novel full-length wheat ns-LTP1 clones, all possessing coding sequences of 348 bp, isolated from abiotic- and biotic-stressed cDNA libraries from aerial tissues, exhibited highly conserved coding regions with 78 to 99 and 71 to 100% identity at the nucleotide and amino acid levels, respectively. Phylogenetic analyses revealed two major ns-LTP families in wheat. Eight wheat ns-LTP genes from different clades were cloned into the expression vector pPICZalpha and transformed into Pichia pastoris. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and in vitro lipid binding activity assay confirmed that the eight ns-LTP were all successfully expressed and capable of in vitro binding fatty acid molecules. A comparative in vitro study on the toxicity of eight wheat ns-LTP to mycelium growth or spore germination of eight wheat pathogens and three nonwheat pathogens revealed differential toxicities among different ns-LTP. Values indicating 50% inhibition of fungal growth or spore germination of three selected ns-LTP against six fungi ranged from 1 to 7 microM. In vitro lipid-binding activity of ns-LTP was not correlated with their antifungal activity. Using the fluorescent probe SYTOX Green as an indicator of fungal membrane integrity, the in vitro toxicity of wheat ns-LTP was associated with alteration in permeability of fungal membranes.

Identification and characterization of genes differentially expressed in the resistance reaction in wheat infected with Tilletia tritici, the common bunt pathogen.

The differentially virulent race T1 of common bunt (Tilletia tritici) was used to inoculate the wheat lines Neepawa (compatible) and its sib BW553 (incompatible) that are nearly isogenic for the Bt-10 resistance gene. Inoculated crown tissues were used to construct a suppression subtractive hybridization (SSH) cDNA library. Of the 1920 clones arrayed from the SSH cDNA library, approximately 10 % were differentially regulated. A total of 168 differentially up-regulated and 25 downregulated genes were identified and sequenced; 71 % sequences had significant homology to genes of known function, of which 59 % appeared to have roles in cellular metabolism and development, 24 % in abiotic/biotic stress responses, 3 % involved in transcription and signal transduction responses. Two putative resistance genes and a transcription factor were identified among the upregulated sequences. The expression of several candidate genes including a lipase, two non-specific lipid transfer proteins (ns-LTPs), and several wheat pathogenesis-related (PR)-proteins, was evaluated following 4 to 32 days postinoculation in compatible and incompatible interactions. Results confirmed the higher overall expression of these genes in resistant BW553 compared to susceptible Neepawa, and the differential up-regulation of wheat lipase, chitinase and PR-1 proteins in the expression of the incompatible interaction.