Klaus K. Nielsen

Characterization of a new antifungal chitin-binding peptide from sugar beet leaves

The intercellular washing fluid (IWF) from leaves of sugar beet (Beta vulgaris L.) contains a number of proteins exhibiting in vitro antifungal activity against the devastating leaf pathogen Cercospora beticola (Sacc.). Among these, a potent antifungal peptide, designated IWF4, was identified. The 30-amino-acid residue sequence of IWF4 is rich in cysteines (6) and glycines (7) and has a highly basic isoelectric point. IWF4 shows homology to the chitin-binding (hevein) domain of chitin-binding proteins, e.g. class I and IV chitinases. Accordingly, IWF4 has a strong affinity to chitin. Notably, it binds chitin more strongly than the chitin-binding chitinases. A full-length IWF4 cDNA clone was obtained that codes for a preproprotein of 76 amino acids containing an N-terminal putative signal peptide of 21 residues, followed by the mature IWF4 peptide of 30 residues, and an acidic C-terminal extension of 25 residues. IWF4 mRNA is expressed in the aerial parts of the plant only, with a constitutive expression in young and mature leaves and in young flowers. No induced expression of IWF4 protein or mRNA was detected during infection with C. beticola or after treatment with 2,6-dichloroisonicotinic acid, a well-known inducer of resistance in plants.

Photoperiodic regulation of flowering in perennial ryegrass involving a CONSTANS-like homolog

Photoperiod and vernalization are the two key environmental factors of the floral induction of perennial ryegrass (Lolium perenneL.). Transition from vegetative to reproductive growth will only occur after an extended vernalization period, followed by an increase in day length and temperature. Here we report on the isolation and characterization of a L. perennegene (LpCO) that is homologous to CONSTANS, and which is tightly coupled to the floral inductive long day signal. Like other monocot CO-like proteins, the LpCO contains a zinc finger domain with a non-conserved B-Box2. Although the B-Box2 has been demonstrated to be essential for the function of the ArabidopsisCO (AtCO), LpCO is able to complement the Arabidopsis co-2 mutant, and ectopic expression in Arabidopsis wild type leads to early flowering. The LpCO transcript exhibits diurnal oscillations and is expressed at higher levels during long days.

Frequency, type, and distribution of EST-SSRs from three genotypes of Lolium perenne, and their conservation across orthologous sequences of Festuca arundinacea, Brachypodium distachyon, and Oryza sativa

BackgroundSimple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies in several plant species. They are used for cultivar identification, variety protection, as anchor markers in genetic mapping, and in marker-assisted breeding. Currently, a limited number of SSR markers are publicly available for perennial ryegrass (Lolium perenne). We report on the exploitation of a comprehensive EST collection in L. perenne for SSR identification. The objectives of this study were 1) to analyse the frequency, type, and distribution of SSR motifs in ESTs derived from three genotypes of L. perenne, 2) to perform a comparative analysis of SSR motif polymorphisms between allelic sequences, 3) to conduct a comparative analysis of SSR motif polymorphisms between orthologous sequences of L. perenne, Festuca arundinacea, Brachypodium distachyon, and O. sativa, 4) to identify functionally associated EST-SSR markers for application in comparative genomics and breeding.ResultsFrom 25,744 ESTs, representing 8.53 megabases of nucleotide information from three genotypes of L. perenne, 1,458 ESTs (5.7%) contained one or more SSRs. Of these SSRs, 955 (3.7%) were non-redundant. Tri-nucleotide repeats were the most abundant type of repeats followed by di- and tetra-nucleotide repeats. The EST-SSRs from the three genotypes were analysed for allelic- and/or genotypic SSR motif polymorphisms. Most of the SSR motifs (97.7%) showed no polymorphisms, whereas 22 EST-SSRs showed allelic- and/or genotypic polymorphisms. All polymorphisms identified were changes in the number of repeat units. Comparative analysis of the L. perenne EST-SSRs with sequences of Festuca arundinacea, Brachypodium distachyon, and Oryza sativa identified 19 clusters of orthologous sequences between these four species. Analysis of the clusters showed that the SSR motif generally is conserved in the closely related species F. arundinacea, but often differs in length of the SSR motif. In contrast, SSR motifs are often lost in the more distant related species B. distachyon and O. sativa.ConclusionThe results indicate that the L. perenne EST-SSR markers are a valuable resource for genetic mapping, as well as evaluation of co-location between QTLs and functionally associated markers.

MADS-box genes from perennial ryegrass differentially expressed during transition from vegetative to reproductive growth

In contrast to well-studied dicot plants like Arabidopsis and Antirrhinum, relatively few genes controlling the transition to flowering and flower development of agronomically important monocot species have been identified. In perennial ryegrass (Lolium perenne) the transition from vegetative to reproductive growth is triggered by an obligate vernalization period (primary induction) of at least 12 weeks at temperatures below 5 degrees C under short days, followed by increased temperature and day length (secondary induction). Here we report the isolation of nine ryegrass MADS-box (LpMADS) genes by a differential display method specific to this family of transcription factors. Three of the nine MADS-box genes show homology to the APETALA 1 (AP1) subfamily, two to the SEPALLATA (SEP) subfamily, one to the AGAMOUS-LIKE 6 (AGL6) subfamily, and three show homology to the newly identified OsMADS1 subfamily. The three AP1 homologues are up-regulated, both in the shoot apex and in leaves, in response to vernalization, while expression of the other six are increased by secondary induction during inflorescence development, although not in leaves. Differences in the sequence and hierarchy of flowering gene expression patterns indicate that the Arabidopsis-based flowering model is not completely applicable to explain the molecular events leading to the floral transition in grasses.

A hydroxyproline-containing class IV chitinase of sugar beet is glycosylated with xylose

Two acidic chitinase isoforms, SP1 and SP2, have been purified to homogeneity from leaves of sugar beet (Beta vulgaris) infected with Cercospora beticola. SP1 and SP2 are extracellular proteins with an apparent molecular mass of 35 kDa and an approximate pI of 4.2. Since the only major difference was slightly diverging Mrs, only the SP2 chitinase was further characterized. Partial amino acid sequence data for SP2 was used to generate a polymerase chain reaction (PCR) clone employed for the isolation of a cDNA clone encoding SP2. SP2 exhibits significant structural identity with the class IV chitinases from sugar beet, rapeseed, bean and maize, but differs from the other members of this class in having a longer hinge region, comprising 22 amino acid residues, with a repeated ‘TTP’ motif. Western blotting analyses, using antibody raised against SP2, demonstrated an induction of SP protein during infection with C. beticola. The induction was very local, with high protein accumulation found close to the infection site only. Amino acid compositional analysis of SP2 revealed that five out of fourteen prolines are hydroxylated. No glucosamine or galactosamine residues are present. Evidence was obtained that SP2 is glycosylated with a limited number (≤7) of xylose residues: (1) SP2 was stained with the periodic acid-Schiff (PAS) reagent, (2) electrospray mass spectrometry on SP2 gave a series of Mrs with a consistent increase between two molecular masses of 132 Da, (3) SP2 was recognized by an antibody specific for β-1,4-D-xylopyranose. The vacuolar class I chitinases A and B in tobacco have recently been shown to comprise a new class of hydroxyproline-containing proteins (Sticher et al., Science 257 (1992) 655–657). The SP2 chitinase differs from these in being glycosylated and, thus, represents a novel type of hydroxyproline-containing glycoproteins in plants.

Characterization of a new antifungal non-specific lipid transfer protein (nsLTP) from sugar beet leaves

A novel protein (IWF5) comprising 92 amino acids has been purified from the intercellular washing fluid of sugar beet leaves using cation exchange chromatography and reversed phase high performance liquid chromatography. Based on amino acid sequence homology, including the presence of eight cysteines at conserved positions, the protein can be classified as a member of the plant family of non-specific lipid transfer proteins (nsLTPs). The protein is 47% identical to IWF1, an antifungal nsLTP previously isolated from leaves of sugar beet. A potential site for N-linked glycosylation present in IWF5 (Asn-Xxx-Ser/Thr) was found not to be glycosylated. The amino acid sequence data were used to generate a polymerase chain reaction (PCR) clone, employed for the isolation of a corresponding cDNA clone. According to the cDNA clone, IWF5 is expressed as a preprotein with an N-terminal signal sequence of 26 amino acid residues. The protein shows a strong in vitro antifungal activity against Cercospora beticola (causal agent of leaf spot disease in sugar beet) and inhibits fungal growth at concentrations below 10 µg ml(-1).

New antifungal proteins from sugar beet (Beta vulgaris L.) showing homology to non-specific lipid transfer proteins

Two novel, nearly identical antifungal proteins, IWF1 and IWF2, were isolated from the intercellular washing fluid (IWF) of sugar beet leaves. The proteins were purified to homogeneity and their amino acid sequences were determined. They are basic, monomeric proteins of 91 amino acid residues, 89 of which are identical. Both proteins show strongin vitro antifungal activity againstCercospora beticola, the casual agent of leaf spot disease in sugar beet. Based on primary sequence homology, including the presence of 8 conserved cysteine residues, IWF1 and IWF2 are related to the family of plant non-specific lipid transfer proteins (nsLTPs). Antibodies were raised against IWF2 after conjugation to diphtheria toxoid. The amino acid sequence data was used to generate a polymerase chain reaction (PCR) clone, employed for the isolation of a cDNA clone encoding a closely related isoform IWFA, which differs from IWF1 by two amino acid substitutions only. The induction and subcellular localization of these proteins were studied by western and northern blotting analyses after treatment with 2,6-dichloroisonicotinic acid (INA), a compound capable of inducing resistance againstC. beticola, and after fungal infection. The following observations were made: (1) the proteins were present in leaves of non-INA-treated and uninfected control plants, (2) they were only slightly induced by INA treatment and during infection withC. beticola, and (3) they were present both intra- and extracellularly. However, their strong antifungal potentials together with immunohistological investigations, the proteins accumulating in contact with the fungus and in autolysing cells, suggested a role of these proteins in plant defence. Finally, immunohistology revealed a remarkable expression pattern of the IWF1 and IWF2 proteins, or serologically related proteins, in sugar beet styles, in that single or a few scattered papillae and a few cells in the lower transmitting tissue strongly and specifically reacted with the antibody.

A high-throughput Agrobacterium-mediated transformation system for the grass model species Brachypodium distachyon L.

In the ongoing process of developing Brachypodium distachyon as a model plant for temperate cereals and forage grasses, we have developed a high-throughput Agrobacterium-mediated transformation system for a diploid accession. Embryogenic callus, derived from immature embryos of the accession BDR018, were transformed with Agrobacterium tumefaciens strain AGL1 carrying two T-DNA plasmids, pDM805 and pWBV-Ds-Ubi-bar-Ds. Transient and stable transformation efficiencies were optimised by varying the pre-cultivation period, which had a strong effect on stable transformation efficiency. On average 55% of 17-day-old calli co-inoculated with Agrobacterium regenerated stable transgenic plants. Stable transformation frequencies of up to 80%, which to our knowledge is the highest transformation efficiency reported in graminaceous species, were observed. In a study of 177 transgenic lines transformed with pDM805, all of the regenerated transgenic lines were resistant to BASTA®, while the gusA gene was expressed in 88% of the transgenic lines. Southern blot analysis revealed that 35% of the tested plants had a single T-DNA integration. Segregation analysis performed on progenies of ten selected T0 plants indicated simple Mendelian inheritance of the two transgenes. Furthermore, the presence of two selection marker genes, bar and hpt, on the T-DNA of pWBV-Ds-Ubi-bar-Ds allowed us to characterize the developed transformation protocol with respect to full-length integration rate. Even when not selected for, full-length integration occurred in 97% of the transformants when using bialaphos as selection agent.

Floral inhibition in red fescue (Festuca rubra L.) through expression of a heterologous flowering repressor from Lolium

Extension of the vegetative growth phase through delay of flowering is an important goal in todays breeding programs of both forage and turf grasses. In forage grasses, the stem and inflorescence production comprise a significant reduction in the digestibility, nutritional value and productivity of the crop, and in turf grasses the stems that start to emerge during the growth season suppress the formation of new shoots and affect the quality, density and persistence of the sward. We have tested the potential of the strong floral repressor LpTFL1 from perennial ryegrass (Lolium perenne L.) to manipulate the transition to flowering in red fescue (Festuca rubra L.), a cool-season turf grass. Expression of LpTFL1 from the constitutive maize ubiquitin promoter represses flowering in red fescue, and the flowering repression phenotype correlates well with the level of LpTFL expression. Transgenic lines showing low to intermediate expression of LpTFL1 flowered approximately two weeks later than the controls, and transgenic lines showing very high LpTFL1 expression levels still remained non-flowering after exposure to natural vernalization conditions (Danish winter) in two successive years. There were no other phenotypic effects associated with the LpTFL transgene expression during vegetative growth. However, there was a tendency towards an LpTFL1-mediated reduction in stem length among the flowering lines. Expression of a truncated LpTFL, caused by transgene rearrangements during the transformation, lead to increased flowering and stem production and a decrease in panicle size. This is to our knowledge the first report on full inhibition of floral development in a commercially important grass species.

Expressed sequence tag-derived microsatellite markers of perennial ryegrass (Lolium perenne L.)

An expressed sequence tag (EST) library of the key grassland species perennial ryegrass (Lolium perenne L.) has been exploited as a resource for microsatellite marker development. Out of 955 simple sequence repeat (SSR) containing ESTs, 744 were used for primer design. Primer amplification was tested in eight genotypes of L. perenne and L. multiflorum representing (grand-) parents of four mapping populations and resulted in 464 successfully amplified EST-SSRs. Three hundred and six primer pairs successfully amplified products in the mapping population VrnA derived from two of the eight genotypes included in the original screening and revealed SSR polymorphisms for 143 ESTs. Here, we report on 464 EST-derived SSR primer sequences of perennial ryegrass established in laboratory assays, providing a dedicated tool for marker assisted breeding and comparative mapping within and among forage and turf grasses.