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.

An immunity-triggering effector from the Barley smut fungus Ustilago hordei resides in an Ustilaginaceae-specific cluster bearing signs of transposable element-assisted evolution.

The basidiomycete smut fungus Ustilago hordei was previously shown to comprise isolates that are avirulent on various barley host cultivars. Through genetic crosses we had revealed that a dominant avirulence locus UhAvr1 which triggers immunity in barley cultivar Hannchen harboring resistance gene Ruh1, resided within an 80-kb region. DNA sequence analysis of this genetically delimited region uncovered the presence of 7 candidate secreted effector proteins. Sequence comparison of their coding sequences among virulent and avirulent parental and field isolates could not distinguish UhAvr1 candidates. Systematic deletion and complementation analyses revealed that UhAvr1 is UHOR_10022 which codes for a small effector protein of 171 amino acids with a predicted 19 amino acid signal peptide. Virulence in the parental isolate is caused by the insertion of a fragment of 5.5 kb with similarity to a common U. hordei transposable element (TE), interrupting the promoter of UhAvr1 and thereby changing expression and hence recognition of UhAVR1p. This rearrangement is likely caused by activities of TEs and variation is seen among isolates. Using GFP-chimeric constructs we show that UhAvr1 is induced only in mated dikaryotic hyphae upon sensing and infecting barley coleoptile cells. When infecting Hannchen, UhAVR1p causes local callose deposition and the production of reactive oxygen species and necrosis indicative of the immune response. UhAvr1 does not contribute significantly to overall virulence. UhAvr1 is located in a cluster of ten effectors with several paralogs and over 50% of TEs. This cluster is syntenous with clusters in closely-related U. maydis and Sporisorium reilianum. In these corn-infecting species, these clusters harbor however more and further diversified homologous effector families but very few TEs. This increased variability may have resulted from past selection pressure by resistance genes since U. maydis is not known to trigger immunity in its corn host.

Carbohydrate profiling in seeds and seedlings of transgenic triticale modified in the expression of sucrose:sucrose-1-fructosyltransferase (1-SST) and sucrose:fructan-6-fructosyltransferase (6-SFT).

Constructs with sucrose-sucrose 1-fructosyltransferase (1-SST) from rye and or sucrose-fructan 6-fructosyltransferase (6-SFT) from wheat were placed under the control of wheat aleurone-specific promoter and expressed in triticale using biolistic and microspore transformation. Transgenic lines expressing one or both the 1-SST and the 6-SFT accumulated 50% less starch and 10-20 times more fructan, particularly 6-kestose, in the dry seed compared to the untransformed wild-type (WT) triticale; other fructans ranged in size from DP 4 to DP 15. During germination from 1 to 4 days after imbibition (dai), fructans were rapidly metabolized and only in transgenic lines expressing both 1-SST and 6-SFT were fructan contents significantly higher than in the untransformed controls after 4 days. In situ hybridization confirmed expression of 6-SFT in the aleurone layer in imbibed seeds of transformed plants. When transgenic lines were subjected to a cold stress of 4°C for 2 days, synthesis of fructan increased compared to untransformed controls during low-temperature germination. The increase of fructan in dry seed and germinating seedling was generally associated with transcript expression levels in transformed plants but total gene expression was not necessarily correlated with the time course accumulation of fructan during germination. This is the first report of transgenic modification of cereals to achieve production of fructans in cereal seeds and during seed germination.

Morphological and molecular analyses of host and nonhost interactions involving barley and wheat and the covered smut pathogen Ustilago hordei.

Ustilago hordei interactions on coleoptiles of barley host cultivars Odessa (compatible), Hannchen (incompatible, carrying the Ruh1 resistance gene), and on nonhost Neepawa wheat were studied using light and fluorescent microscopy. Autofluorescence, mainly caused by callose accumulation, was more rapidly expressed in nonhost wheat at 30 to 72 h compared with the incompatible reaction between 72 and 144 h. Microarray results demonstrated that more than half of the 893 differentially regulated genes were observed in Neepawa; of these genes, 45% fell into the defense- and stress-related classes in Neepawa compared with 25 and 37% in Odessa and Hannchen, respectively. Their expression coincided with the early morphological defense responses observed and were associated with the jasmonic acid and ethylene (JA/ET) signaling pathway. Expression patterns in Odessa and Hannchen were similar, involving fewer genes and coinciding with later morphological defense responses of these varieties. Although no visible hypersensitive response was apparent in Hannchen or Neepawa, specific upregulation of hypersensitivity-related proteins was observed, such as beta-VPE at 48 h. Expression levels of the callose synthase gene were closely associated with callose accumulation. Differential responses in defense-gene expression among disease reaction types included upregulation of PR-1.1b and downregulation of a nonspecific lipid transfer protein in the incompatible and compatible interactions, respectively. Transcript levels of EDS1 and PAD4, involved in both basal resistance and R-mediated resistance to avirulent pathogens, were up-regulated during both nonhost and Ruh1-mediated resistance. Application of methyl-jasmonate, salicylic acid and ET to leaves revealed that only PR1.1b is strongly up-regulated by all three compounds, while the majority of the defense-related genes are only slightly up-regulated by these signaling compounds.

Starch synthesis and programmed cell death during endosperm development in triticale (x Triticosecale Wittmack).

Triticale (x Triticosecale Wittmack) grains synthesize and accumulate starch as their main energy source. Starch accumulation rate and synthesis activities of ADP-glucose pyrophosphorylase, soluble starch synthases, granule-bound starch synthase and starch-branching enzyme showed similar pattern of unimodal curves during endosperm development. There was no significant difference in activity of the starch granule-bound protein isolated from total and separated starch granules at different developmental stages after anthesis in triticale. Evans Blue staining and analysis of DNA fragmentation indicated that cells of triticale endosperm undergo programmed cell death during its development. Dead cells within the endosperm were detected at 6 d post anthesis (DPA), and evidence of DNA fragmentation was first observed at 21 DPA. The period between initial detection of PCD to its rapid increase overlapped with the key stages of rapid starch accumulation during endosperm development. Cell death occurred stochastically throughout the whole endosperm, meanwhile, the activities of starch biosynthetic enzymes and the starch accumulation rate decreased in the late stages of grain filling. These results suggested that the timing and progression of PCD in triticale endosperm may interfere with starch synthesis and accumulation.

Adult plant resistance-related gene expression in 'Camp Remy' wheat inoculated with Puccinia striiformis.

The French wheat variety 'Camp Remy' (CR) possesses a durable, adult plant resistance to yellow rust (YR), caused by the pathogen Puccinia striiformis. Using cDNA-AFLP on different sets of heterogeneous inbred families (HIFs) derived from the cross CR x Récital, we compared gene expression profiles during one seedling and two adult developmental stages following inoculation with P. striiformis. Transcripts differentially expressed in response to YR infection were isolated and cloned. Sequence analysis of the resultant clones revealed several classes of putative genes, including those related to resistance/defence responses, transcription and signal transduction, and primary metabolism. The expression profiles of seven selected genes were obtained using real-time PCR in CR leaves at the same three stages of development. The results confirmed the stage-specific expression of the genes at one or two specific stages in response to P. striiformis infection and demonstrated that CR modifies the expression of some resistance/defence-related genes during its transition from the seedling to adult growth stages. These results provided the first clue to understand the molecular basis of quantitative trait loci for adult plant resistance to YR and connect it with durability.

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.

Compatible and Incompatible Interactions in Wheat Involving the Bt-10 Gene for Resistance to Tilletia tritici, the Common Bunt Pathogen.

ABSTRACT The infection of wheat lines Neepawa (susceptible), and its sib BW553 that is nearly isogenic for the Bt-10 resistance gene by differentially virulent races T1 and T27 of common bunt (Tilletia tritici), was followed for 21 days following seeding (dfs) using fluorescence and confocal microscopy. Spore germination was nonsynchronous and all spore stages including germination were observed 5 to 21 dfs. Initial host perception of pathogen invasion, based on autofluorescence in epidermal cells adjacent to the appressoria, was similar in both compatible and incompatible interactions, and occurred as early as 5 to 6 dfs. The total number of sites on a 1-cm segment of coleoptile adjacent to the seed that exhibited autofluorescence was similar in both the compatible and incompatible interactions and rose to a maximum of 35 to 40 per 1 cm length of coleoptile following 17 dfs, although new infection events were observed as late as 21 dfs. In the compatible interaction, the autofluorescence became more diffuse 10 to 12 dfs, emanating in all directions in association with fungal spread. In the incompatible interaction, autofluorescence remained restricted to a small area surrounding the penetration site. Two different reaction zones that extended further in tissues surrounding the penetration point in the incompatible interaction compared with the compatible interaction were identified. The accumulation of callose around invading fungal hyphae was observed during both the compatible and incompatible interactions from 8 to 21 dfs. While callose accumulation was more extensive and widespread in the incompatible interaction, it was clearly present in compatible interactions, particularly in treatments involving BW553. These results were confirmed by expression of callose synthase transcripts that were more abundant in BW553 than in Neepawa and were upregulated during pathogen infection in both compatible and incompatible interactions.

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.