Annual Progress Report on Malting Barley Research March, 2002

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156 DIRECTED EXPRESSION OF ANTIFUNGAL GENES IN BARLEY AND INFLUENCES OF NATIVE ANTIFUNGAL SEED PROTEINS ON MALTING QUALITY Ronald W. Skadsen USDA, Agricultural Research Service, Cereal Crops Research Unit; 501 N. Walnut St., Madison, WI 53705 INTRODUCTION AND OBJECTIVES The objective of this research is to produce Fusarium-resistant barley through genetic transformation with specifically targeted native antifungal protein genes. Tissue-specific promoters will ultimately be needed to target antifungal protein gene expression to spike tissues. Our studies with a strain of F. graminearum expressing the green fluorescent protein gene (gfp) point to the lemma/palea and pericarp (particularly the ovary epithelial hairs) as important targets for antifungal gene expression. The targeted expression of antifungal proteins will reduce metabolic burdens on the plant and minimize pressures that select for resistant pathogen strains. We have focused on the cloning and expression of two antifungal proteins found in barley endosperm, permatin and hordothionin (HTH). HTH is a highly basic, sulfur-rich low molecular weight protein. Barley leaves and roots produce thionins related to seed HTH. These can be induced by fungi and have antifungal properties in vitro. Despite these properties, the non-seed thionins apparently do not protect existing barley cultivars from F. graminearum. Although we have demonstrated the effectiveness of HTH against Fusarium, HTH is found only in the starchy endosperm and, as normally expressed, is not available to prevent colonization by Fusarium on outer floret tissues. The same is true of permatin. Permatins are thaumatin-like proteins (TLPs) found in many cereals and are homologous with PR-5 proteins. Permatin has been purified from maize and found to have antifungal properties. However, purified permatin has not been produced and tested against Fusarium. Because thionin is the only protein shown to be effective against Fusarium, barley transformation efforts having been concentrated on expressing this particular gene. However, even though Hth can be expressed at the mRNA in transformants, there are considerable barriers in leafy tissues to its expression at the protein level (see Results). In addition, besides their antifungal properties, the effects of permatin and HTH on malting quality have never been analyzed. METHODOLOGY Tissue-specific promoters - In order to develop gene promoters to target antifungal proteins to specific tissues, genes expressed only in the lemma/palea, and not in leaves, were cloned. This effort was also extended to the pericarp. The differential display screening procedure used to select these genes was described in the 1999 AMBA <strong>Annual</strong> <strong>Progress</strong> <strong>Report</strong>. We have now adopted the PCR-based suppressive subtractive hybridization (SSH) method to detect tissue-specific genes. The corresponding promoter sequences were derived from BAC clones and analyzed for tissue-specificity. Ultimately, vectors will be constructed to express an HTH gene under the control of tissue-specific promoters, and transformants will be tested for Fusarium resistance.
157 Tissue-specific gene products from the above differential display PCRs were used by Dr. Andris Kleinhofs (Washington State U.) as probes to find the corresponding Morex genomic clones in a BAC library. We have also adopted additional options, the inverse PCR procedure and PCR-based gene walking. Before the promoter region can be subcloned from a BAC genomic clone, it is necessary to determine the 5’ end of the mRNA. Determination of the 5' (and, where needed, 3') end sequences of mRNAs are performed by Rapid Amplification of cDNA ends (RACE) using the GeneRacer TM kit (InVitrogen), as described by the supplier. These techniques were described in the 2001 AMBA progress report. Molecular cloning and vector construction - Vectors for stable expression of antifungal genes were prepared from the Ubi/GUS+Ubi/BAR vector pAHC25 by replacing the GUS gene. For stable tissue-specific expression of Hth, a barley promoter/Hth fusion will replace the Ubi/GUS fusion in pAHC25. Transient expression studies of promoter activity are conducted with the pAHC17 vector, which contains the Ubi promoter but has no selectable marker. We have modified this vector so that the Gfp gene occurs downstream from the Ubi promoter, allowing constitutive expression of Gfp (pAHCSGFP vector). To test our promoters, the Ubi promoter is removed and replaced with lemma/palea or pericarp promoter candidates. The modified vectors are used to transform barley through the biolistic (gene gun) approach. The particle delivery and tissue culture is performed essentially as described by Wan and Lemaux (1994). Screening of putative transformants was conducted using PCR on leaf DNA extracted by the CTAB procedure. Transient expression analysis of promoters and antifungal testing - The putative promoter regions typically include up to 1500 bp of upstream sequence (with respect to the mRNA 5' end). In addition to the upstream region, regulatory sequences are sometimes found in the mRNA 5' UTR and in the first intron. Initially, the upstream region are subcloned from BAC clones or from Gene Walker PCR products and ligated in front of the Gfp gene in our pAHCSGFP vector (above). Particle bombardments of spikelets from emerged spikes, pericarps of intact seeds (after removing lemma and palea), and leaves are conducted and monitored for up to 72 h with short-wave blue light. Sequencing of cDNAs and transcribed regions of BAC clones is used to determine the location of introns. We have now encountered two genes, Lem2 and EpiLTP, in which introns near the 5’ end may influence expression. In these cases, additional tests are conducted by joining 5' UTR and/or first intron sequences with the upstream sequence, in which the first intron is ligated between the promoter region originally tested and the Gfp coding sequence. In order to detect Hth gene expression in transformants, northern blots and RT-PCR were conducted. Total RNA was isolated from leaves by the GT procedure. First strand cDNA was made by priming a reverse transcriptase reaction with oligo d(T). For RT- PCRs, reactions were then conducted with an upstream Hth primer and a downstream NOS primer. As an internal control, actin primers were used simultaneously. Since much more mRNA was produced in Hth2 transformants, northern blots were used to analyze expression.
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Annual Pro
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-i- INTRODUCTION The March 2002 <st
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Barley Transformation G.J. Muehlbau
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1 2000/2001 WINTER NURSERY: BARLEY
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3 2001 Barley Stripe Rust Screening
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5 Triumph/Tyra//Arupo, a selection
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7 Washington (Fossum Cereals and Wa
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9 This project is supported by the
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11 ‘Triumph’, ‘Valier’, ‘
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13 Table 2. Agronomic data for sele
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15 Table 4. Agronomic data for sele
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17 Table 8. Malting quality data* f
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19 Six-rowed barley selections of c
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21 Table 14. Agronomic data for sel
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23 Table 16. Agronomic data for win
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25 Table 19. Malting quality data*
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Garnet/98Ab11865 Garnet/98Ab12895 9
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29 rust resistant NSGC accessions o
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31 DEVELOPMENT OF SIX-ROWED MALTING
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33 backcrossing. The selfed lines w
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35 MINNESOTA BARLEY IMPROVEMENT PRO
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37 ADVANCED LINE EVALUATION Varieti
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39 OTHER BARLEY DISEASES In 2001, r
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41 Wingbermuehle, W. J., K.M. Belin
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43 In 2001 winter/spring, over 300
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45 assessments were made by countin
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47 IDENTIFICATION OF NOVEL DISEASE
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49 Unfortunately, disease levels in
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51 BARLEY TRANSFORMATION G.J. Muehl
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53 We have available the sugarcane
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55 Secondly, we tried to grow F. gr
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57 References: Issac, S. and Jennin
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59 Huntley-dryland) with two replic
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61 Table 2. 1992 thru 2001 Overall
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63 performance for Montana farmers.
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65 Blake, T,. Hensleigh P., Boss D.
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67 settling tower cannot accommodat
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Table 3. Seedling reaction to strip
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71 TWO-ROWED BARLEY IMPROVEMENT PRO
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Table 1. Agronomic trait comparison
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75 mutants reduce plant vigor and g
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77 SIX-ROWED BARLEY IMPROVEMENT PRO
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79 this variety has decreased over
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Table 7. Malt quality comparisons o
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83 • Management studies for malt
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85 STUDIES ON BARLEY DISEASES AND T
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87 With funding from the US Wheat a
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89 MALTING AND BREWING QUALITY OF B
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91 β-glucans, it might be most ins
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10 9 8 7 6 5 4 3 2 1 0 93 Figure 1.
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95 Bolin, P., Schwarz, P., Jones, B
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97 Results Results from field and g
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99 DEVELOPING BARLEY TISSUE CULTURE
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101 trials of plants regenerated fr
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103 Table 2. Agronomic performance
Page 113 and 114: 105 BSMV spread between entries. Ho
Page 115 and 116: 107 involving two susceptible backg
Page 117 and 118: 109 THE OREGON BARLEY IMPROVEMENT P
Page 119 and 120: 111 for traits that are difficult/e
Page 121 and 122: Germplasm Development: 113 Crosses:
Page 123 and 124: 115 • All lines in spring yield t
Page 125 and 126: 117 Table 2. Agronomic data for Ore
Page 127 and 128: 119 Table 6. Malting quality of win
Page 129 and 130: 121 Table 7. Across location summar
Page 131 and 132: 123 MOLECULAR MARKER ASSISTED MODIF
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Page 135 and 136: 127 exchanges. Spring and winter ba
Page 137 and 138: 129 Table 2. 2000-2001 Agronomic Pe
Page 139 and 140: 131 Table 7. Spring 2- and 6-Row #
Page 141 and 142: 133 2. Direct Seeding Cropping Syst
Page 143 and 144: 135 D.M. Wesenberg - spring and win
Page 145 and 146: 137 A STUDY OF THE MALTING QUALITY
Page 147 and 148: 139 Methods. Our malting schedule.
Page 149 and 150: 141 modification measures indicated
Page 151 and 152: 143 CHARACTERIZATION OF THE PROTEIN
Page 153 and 154: 145 Purification of a serine endope
Page 155 and 156: 147 STUDIES ON THE PROTEINASES THAT
Page 157 and 158: 149 with the proteinase T will be s
Page 159 and 160: 151 10°C increase in thermostabili
Page 161 and 162: 153 Sissons MJ and MacGregor AW (19
Page 163: µM Carbohydrate / Unit of α-Gluco
Page 167 and 168: 159 The promoters were subcloned fr
Page 169: 161 Fig. 3. Expression of Trx-Hth f
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Annual Progress Report on Malting Barley Research March, 2002

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