Denis A. Gaudet

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.

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.

Plant development affects the cold-induced expression of plant defence-related transcripts in winter wheat

Abstract In winter cereals, low temperature hardening, plant age and genotype are known to influence the expression of resistance to snow mould diseases. A study was undertaken to determine the effects of genotype, plant age and duration of cold hardening on the temporal expression of the PR-protein and other defence-related protein transcripts under controlled environment conditions, and in the field during autumn, winter, and early spring. The plant defence-related proteins studied were classified into two groups based on their patterns of expression. The first group consisted of γ-thionin, chitinase, lipid transfer protein (LTP) and phenylalanine ammonia lyase (PAL) whereby transcript levels increased to maximum levels during November–December, but decreased gradually throughout the winter and early spring. Higher transcript levels were observed in late seeded treatments compared with early seeded treatments. Under controlled conditions, transcript levels accumulated rapidly in response to hardening, reaching maximum levels following 1-week and generally maintained levels throughout the hardening treatments. The second group consisted of β-1,3-glucanase, peroxidase and PR-1a whose transcripts increased to maximum levels in the field during November–December, then fluctuated throughout the winter and early spring. Higher transcript levels were observed in early seeded treatments compared to later seeded treatments. Under controlled environment conditions, these transcripts gradually accumulated in response to hardening at 2°C and reached maximum levels after 6 weeks. PR-1a, PAL, and LTP transcript levels were generally higher in the snow mould resistant cultivars PI181268 and D+ throughout the winter and early spring compared to the cold-hardy, snow mould susceptible cultivar, Norstar. Conversely the chitinase and γ-thionin transcripts were expressed at higher levels in the susceptible cultivar Norstar. These results demonstrate that the temporal expression of cold-induced, plant defence-related transcripts in winter wheat is differentially regulated among genotypes and during different plant development stages, and are the first to implicate LTPs in the expression of genotypic-based snow mould resistance in wheat. Potential plant defence signalling pathways involved in snow mould resistance induced at low temperatures during natural acclimation of winter wheat are discussed.

Low temperature induced defence gene expression in winter wheat in relation to resistance to snow moulds and other wheat diseases.

Cold hardening of winter wheat at 2 °C for 1-6 wks increased resistance to the snow mould pathogens LTB, Typhula incarnata, and Microdochium nivale as well as to powdery mildew (Blumaria graminis f. sp. graminis) and stripe rust (Puccinia striiformis). Using microarrays and hardening of winter wheat for 0.25, 0.5, 1, 7, 21 and 49 d, an upregulation of a wide range of stress-response genes that include defence-related and abiotic stress-related genes, transcription factors including several lipoxygenases and ethylene responsive factors, and WRKY genes was observed. For the majority of these genes, the upregulation occurred later in the 21-49 d hardening treatments and coincided with the highest expression levels of snow mould resistance. Defence-related sequences were upregulated to a greater extent and were more numerous in the snow mould resistant line CI14106 compared to cold hardy DH+268. Transcript profiling of candidate defence and other stress-related genes under prolonged conditions at -3 °C with or without snow mould infection showed that there was a decline in transcripts of the defence-related genes PR1.1b and NPR3 during the 12wks incubation. Additionally, 14 d hardening was insufficient to permit full expression of the jasmonic acid synthesis gene, allene oxide synthase (AOS) and the fructan degrading enzyme β-fructofuranosidase compared the 42 d hardening treatment. The snow mould resistant line CI14106 was able to maintain higher transcript levels of AOS for longer conditions compared to the susceptible line Norstar under artificial snow mould conditions. These results explain the nature of cold-induced resistance to snow moulds and provide direction on establishing selection criteria for improving resistance and cold tolerance in winter wheat.

Anthocyanin expression in marker free transgenic wheat and triticale embryos

Wheat and triticale plants were transformed by bombardment of isolated scutella with a genetic construct consisting of the two anthocyanin biosynthesis regulatory genes, C1 and Bperu, each under the control of the Ltp1 embryo-specific promoter. Transgenic plants were obtained in the absence of selective pressure and selectable marker gene at a transformation frequency of 0.93% and 1.55% in triticale and wheat, respectively. Initial screening of T0 lines was performed by polymerase chain reaction (PCR), and further confirmation of PCR positives was done using real-time PCR and by phenotypic observation. In this study, quantitative real-time PCR (qRT-PCR) was developed to determine the transgene copy number in transgenic wheat and triticale. A conserved wheat housekeeping gene, puroindoline-b, was used as an internal control to calculate the transgene copy number in wheat and the SYBR green detection method with a standard curve, constructed on the basis of serially diluted plasmid, was used to calculate the transgene copy in triticale. Estimated transgene copies varied from 3 to 8 in wheat and 4 to 7 in triticale lines. The presence of anthocyanin regulatory genes, promoter, and termination sequences was detected in six wheat lines and four triticale lines. However, anthocyanin-pigmented embryos were only observed visually in mature T1 seeds of two transgenic wheat lines and a single triticale line. Multisite insertion and reorganization of transgenes was likely the explanation for the failure of expression for the anthocyanin genes in the remaining wheat and triticale transgenic lines.

Phenotypic and marker-assisted evaluation of spring and winter wheat germplasm for resistance to fusarium head blight

Fusarium head blight (FHB) caused by Fusarium species, is among the most devastating wheat diseases, causing losses in numerous sectors of the grain industry through yield and quality reduction, and the accumulation of poisonous mycotoxins. A germplasm collection of spring and winter wheat, including nine reference cultivars, was tested for Type II FHB resistance and deoxynivalenol (DON) content. Genetic diversity was evaluated on the basis of Simple Sequence Repeat (SSR) markers linked to FHB resistance quantitative trait loci (QTLs) and Diversity Arrays Technology (DArT) markers. The allele size of the SSR markers linked to FHB resistance QTLs from known resistance sources was compared to a germplasm collection to determine the presence of these QTLs and to identify potentially novel sources of resistance. Forty-two accessions were identified as resistant or moderately resistant to Fusarium spread, and two also had very low DON concentrations. Genetic relationships among wheat accessions were generally consistent with their geographic distribution and pedigree. SSR analysis revealed that several resistant accessions carried up to four of the tested QTLs. Resistant and moderately resistant lines without any known QTLs are considered to be novel sources of resistance that could be used for further genetic studies.

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.

Transient embryo-specific expression of anthocyanin in wheat

SummaryThree barley tissue-specific promoters (Dhn12, Itr1, and Ltp1) were transcriptionally fused to the C1 and/or the B-peru genes involved in anthocyanin biosynthesis. Transient expression of these constructs was examined in different wheat tissues. Constructs with tissue-specific promoters were only active in embryos. A green fluorescent protein (GFP) driven by the actin promoter was used as an internal standard to monitor the effectiveness of each bombardment. Further, normalization of the transient expression assay using the GFP reference significantly reduced the variability between separate bombardments, and the intensity of anthocyanin pigmentation quantified by image analysis allowed for a rapid and accurate evaluation of different promoters. Compared to CaMV35S promoter, tissue-specific promoters were more effective in directing anthocyanin production, specifically in wheat embryos, and the C1 gene was more effective than B-peru. Among the tissue-specific promoters, the Ltp1 was superior to ltr1 and Dhn12 in combination with C1 and/or B-peru gene(s). Analysis of nucleotide sequences of all three tissue-specific promoters revealed the presence of G-box, E-box, and RY elements, which might trigger embryo-specific expression in wheat.

Starch Synthesis and Programmed Cell Death during Endosperm Development in Triticale (×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.

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.