Byron Puchalski

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.

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.

Control of common bunt (Tilleta tritici and T. laevis) of wheat (Triticum aestivum cv. ‘Laura’) by fumigation with acetic acid vapour

Common bunt caused by Tilletia tritici and T. laevis remains an important disease of wheat, particularly in organic production where effective fungicides are not available. Acetic acid (AA), a potential organic seed fumigant, was evaluated for control of common bunt of wheat. The highly susceptible spring wheat cultivar Laura was inoculated with bunt spores and then fumigated with 2 and 4 g kg-1 AA vapour in 23 L chambers for 1 h at 20°C. Fumigation reduced field infection levels of common bunt in trials conducted at Lethbridge, AB during 2000, 2001, and 2003. The 4 g kg-1 rate was more effective than the 2 g kg-1 rate in reducing bunt infection, although both rates were as effective as Vitavax, the standard seed-treatment fungicide treatment. Some reduction in tiller numbers was associated with the AA treatments especially at the 4 g kg-1 rate. In vitro tests on artificial growth media showed that AA significantly decreased seed-borne mold contamination caused by several species of fungi. Acetic acid fum...

DNA markers for resistance to common bunt in ‘McKenzie’ wheat

Abstract Common bunt, caused by Tilletia tritici (Bjerk.) G. Wint. in Rabenh. and T. laevis Kϋhn in Rabenh., is an economically important disease of wheat (Triticum aestivum L.) worldwide. The hexaploid wheat cultivar ‘McKenzie’ possesses effective resistance against known races of common bunt in western Canada. Understanding the nature of this resistance through DNA marker mapping will be useful for breeding. Disease reaction to T. tritici race T19 and T. laevis race L16 from field tests was analysed with simple sequence repeat markers for 174 doubled haploid lines using interval mapping. Markers Xgwm573 and Xwmc17, located on wheat chromosome 7B, were strongly associated with the resistance. The quantitative trait locus was designated QCbt.spa-7B.1. The markers were validated on two other populations in which ‘McKenzie’ was in the parentage. The markers will be useful in breeding as a tool for selecting for common bunt resistance from ‘McKenzie’ and will assist in identifying the resistance among potential new sources.

Antimicrobial peptides expressed in wheat reduce susceptibility to Fusarium head blight and powdery mildew

Badea, A., Eudes, F., Laroche, A., Graf, R., Doshi, K., Amundsen, E., Nilsson, D. and Puchalski, B. 2013. Antimicrobial peptides expressed in wheat reduce susceptibility to Fusarium head blight and powdery mildew. Can. J. Plant Sci. 93: 199-208. Antimicrobial peptides (AMPs) have a broad spectrum of action against bacteria, fungi and viruses, which makes them attractive for building plant defense against a diversity of pathogens. Peptides MsrA2 and 10R were cloned in three genetic constructs for tissue-specific expressions in wheat, using either Lem1, GstA1WIR1a, or Ltp6 and LTP6 signal peptide targeting the lemma/palea, leaves and spikes, epicarp and endomembrane system, respectively. The minimal cassettes for these three genetic constructs and for Pat marker construct were co-delivered in immature wheat scutella using biolistics, and green plantlets were regenerated in presence of 5 mg L-1 glufosinate. Molecular screening confirmed one regenerated plant carried and expressed all transgenes (AMP+): one copy of 10R driven by Ltp6 promoter, one copy of msrA2 driven by GstA1Wir1a and two copies of msrA2 driven by Lem1. Its offspring and T3 generation were challenged with Fusarium graminearum and Blumeria graminis in a contained environment. A reduction of 50% in Fusarium head blight susceptibility was observed in T1, and was inherited through T3 generation. The latter, also presented a 53% reduction in Fusarium damaged kernels and 62% reduction in deoxynivalenol accumulation compared with wild cv. Fielder and sister lines AMP-. MsrA2 and 10R producing T3 lines showed an average significant reduction of 59% in powdery mildew susceptibility compared with cv. Fielder. Synthetic MsrA2 and 10R peptides were effective as in vivo antifungal peptides in wheat. The expression of antimicrobial peptides in plant cells or tissues may have great potential to limit pathogen infection or growth protecting wheat against a diversity of fungal diseases.

Defence responses including hypersensitive cell death, oxidative burst and defence gene expression in ‘Moro’ wheat inoculated with Puccinia striiformis

Abstract This study integrated defence-related gene profiling with histological and histochemical observations to develop a detailed time-course description of Puccinia striiformis penetration and infection of susceptible ‘Fielder’ wheat and resistant ‘Moro’ that contains the Yr10 gene for stripe rust resistance. Penetration and establishment events of the pathogen within the leaf were very similar in both ‘Fielder’ and ‘Moro’ until approximately 8–12 days after inoculation (dai). Two oxidative bursts, one at 6 dai and a second at 14 dai, were recorded. Early mild up-regulation of the defence signalling genes associated with jasmonic acid pathway EDS1, PAD4 and AOS were observed as early as 4 days after inoculation (dai) in ‘Moro’ compared with ‘Fielder’. Transcripts of PR1.1, a gene associated with the salicylic acid pathway, and of a thaumatin-like protein, were differentially up-regulated from 6 to 12 dai in ‘Moro’ compared with ‘Fielder’. A toxic response in fungal tissues was observed coincident with the hypersensitive response (HR) in ‘Moro’ that terminated further growth of the pathogen. The anatomical and cytological aspects of defence expression are discussed in relation to profiling of different defence genes.

Effect of plant age on water content in crowns of fall rye and winter wheat cultivars differing in snow mold resistance

Resistance to snow molds in winter wheat increases with plant age, and older plants express higher levels of resistance than young plants. Experiments were conducted to study the effect of plant age on percent crown water content (%CWC) and dry weights in fall rye and winter wheat cultivars grown under controlled environments and in the field at Lethbridge, AB. Under controlled environments, the oldest (6 wk of pre-hardening growth at 20°C) treatments accumulated the greatest dry weights following exposure of plants to 1 to 6 wk hardening conditions at 2°C, compared with younger (1 to 4 wk pre-hardening growth) treatments. Exposure of plants to hardening temperatures had the greatest effect on %CWC values, which decreased, gradually, from 82–89% (4.95–8.67 g H2O g–1 DW) in unhardened treatments to 67–72% (2.05–2.65 g H2O g–1 DW) in plants receiving the 6 wk pre-hardening and 6 wk hardening growth. However, the oldest treatments (4 to 6 wk pre-hardening growth) always exhibited the lowest %CWC values among...