Introduction Fusarium graminearum Gibberella zeae Triticum aestivum Hordeum vulgare 1982 1997 2004 Fusarium 1994 1982 1990 2001 2001 1991 2000 2001 2001 2002 2005 2005 2005 2006 2006 F. graminearum F. graminearum 2000 2001 2002 2003 F. culmorum Fusarium 2003 1999 2001 2003 1999 2003 2003 Arabidopsis Arabidopsis F. oxysporum 1997 Arabidopsis NPR1 2006 As part of our effort to increase variation for genetic resistance to FHB and to understand the relationship between defense response gene expression and FHB resistance, we produced wheat plants carrying a wheat α-1-purothionin, a barley tlp-1, or a barley β-1,3-glucanase transgene. We evaluated these plants against FHB under greenhouse and field conditions. Our results show that the overexpression of α-1-purothionin, tlp-1, or β-1,3-glucanase in wheat results in enhanced resistance to FHB. Materials and methods Plant materials 1996 Plant transformation plasmids pAHC25 1 1992 uidA bar uidA bar Fig. 1 nos Agrobacterium tumefaciens uidA Escherichia coli bar Streptomyces hygroscopicus  pKM1 Bam Bgl Bgl − Bgl − 1992 Bgl − Bgl bar pAHCBarPR5 Xho Bam 1992 pUBKBarGluc-3 1991 Eco Bgl Bam bar Bgl − Wheat transformation 2006 RNA isolation and transcript analysis RNA was isolated from leaf tissue using the Trizol reagent (Invitrogen, Carlsbad, CA) as per manufacturers’ instructions. RNA was subjected to RT-PCR based on the protocol accompanying the Calypso RT-PCR kit (GenSys Ltd., Farnborough, UK) using primers synthesized by Integrated DNA Technologies Inc. (Coralville, IA). The 5′ sense primer was a maize ubiquitin promoter sequence (5′-GATGCATATACATGATGGCATATGCAG-3′) and the 3′ antisense primers were oligonucleotides that corresponded to defense response gene coding sequences for α-1-purothionin (5′-GTTACAGAAATTGACACAAGCATCGCC-3′), tlp-1 (5′-GACAGAAGGTGATCTGGTAGTTATTATT-3′) and β-1,3-glucanase (5′-GATGTTCACGGCAGGGTAGT-3′ and 5′-GCCACGTCCGTCATGTAGGCGTTC-3′). A wheat actin gene (GenBank accession number AB181991) with the primer sequences 5′-GCCACACTGTTCCAATCTATGA-3′ and 5′-TGATGGAATTGTATGTCGCTTC-3’ was used as a positive control. Sizes for the amplified products from the α-1-purothionin and tlp-1 transgenes were 600 and 805, respectively. Sizes for the two primers for the β-1,3-glucanase transgene were 577 and 777 bp. Size for the wheat actin gene was 369 bp. Fusarium graminearum Fusarium graminearum 2000 Fusarium graminearum 6 7 F. graminearum 5 2 FHB incidence and severity were evaluated visually 21 days after the initial inoculation. Incidence was recorded as the percentage of spikes with symptomatic spikelets and severity as the percentage of symptomatic spikelets in 20 spikes of primary tillers arbitrarily selected per plot. 1999 1998 Western blot analysis 2 4 Samples were subjected to SDS-PAGE using 12% gels, transferred to PVDF-PLUS transfer membrane (Micron Separations Inc., Westborough, MA) and cross reacted with an affinity-purified polyclonal antibody (1:1000 dilution of supplied material). The tlp-1 and β-1,3-glucanase antibodies were provided by Quality Controlled Biochemicals Inc., Hopkinton, MA. For tlp-1, two peptides (QAYQHPNDVATHAC and CINVPAG TQAGRIWAR) were used to raise the antibody. For β-1,3-glucanase, one peptide (CGLFNPDKSPAYNIQF) was used to raise the antibody. Protein was visualized using an ECF Western Blotting Reagent Pack (rabbit) (Amersham Biosciences, Piscataway, NJ), and fluorescence detection was carried out using a Storm 840 (Molecular Dynamics, Sunnyvale, CA). Specificity of β-1,3-glucanase and tlp-1 antibodies was confirmed through cross-reacting the antibodies with the peptides on Western blots. Southern blot analysis 1996 Statistical analysis t ® Results Generation of transgenic wheat plants 1 uidA bar uidA bar 2006 0 1 Table 1 Production of transgenic wheat plants Transgene Number of embryos bombarded a Transformation (%) Wheat α-1-purothionin 1787 25 1.4 Barley thaumatin-like protein-1 825 25 3.0 Barley β-1,3-glucanase 1079 31 2.9 a 2 1 0 1 2 Greenhouse evaluation of transgenic plants for response to Fusarium head blight 2 F. graminearum 2 2 3 2 2 P P P Table 2 Percent Fusarium head blight severity in greenhouse evaluations of seven wheat lines carrying wheat α-1-purothionin, barley thaumatin-like protein 1, or barley β-1,3-glucanase that were selected in initial tests and three wheat varieties used as disease checks  a b T 2 T 2 2 T 3 3 3 3 4 CM17 38 * (9) 44 ** (8) c 38 ** (10) 74 (14) - - * CM21 39 * (12) 38 (4) *** 89 (11) – – * – CM23 37 * (14) 55 (14) ** 41 * (12) – – – – CM27 38 * (12) 34 ** (12) – – * – – – CM28 26 ** (10) 37 * (10) – – ** – – – CM30 19 *** (8) 36 * (11) – – 51 (4) – – – CM33 33 (8) 48 * (11) * – – * – – Bobwhite 63 (31) 71 (28) 78 (28) 71 (28) 78 (28) 64 (18) 73 (33) 54 (36) Wheaton 70 (33) 85 (45) – 85 (45) – 91 (21) 94 (57) 99 (60) Sumai 3 26 (22) 9 (25) 10 (78) 9 (25) 10 (78) 21 (16) 16 (46) 7 (61) Numbers in parenthesis represent the number of plants in the screen. a 2 3 2 3 b c * ** *** Molecular characterization of transgenic plants 2 Fig. 2 a c a Eco b Hin c Xho  3 4 Fig. 3 RT-PCR analysis of transgenic wheat lines carrying the wheat α-1-purothionin (CM17), barley tlp-1 (CM21 and CM 23), and barley β-1,3-glucanase (CM27, CM28, CM30, and CM33) transgenes. The fragment sizes for the α-1-purothionin, barley tlp-1, and barley β-1,3-glucanase amplified the expected products of 600, 805, and 577 bp, respectively. The wheat actin gene was used as a positive control and it exhibited the expected size of 369 bp  Fig. 4 a b a b  Field evaluation of transgenic plants for response to Fusarium head blight 3 Table 3 Percent Fusarium head blight (FHB) severity, deoxynivalenol (DON) concentration, and percent visual scabby kernels (VSK) in transgenic wheat plants carrying wheat α-1-purothionin, barley thaumatin-like protein l, and barley β-1,3-glucanase and check wheat varieties evaluated in the field in 2004 and 2005 a FHB severity (%) b VSK (%) Bobwhite 65.1 16.3 29.6 Alsen *** *** *** Wheaton *** *** *** Wheaton (noninoculated) 64.3 17.8 32.2 Roblin 70.9 18.8 * 2375 ** *** *** CM17 * 15.7 24.7 CM21 55.1 *** 19.8* CM23 57.2 13.4* 21.0 CM27 *** *** 17.7* CM28 58.2 17.6 25.8 CM30 *** ** 34.3 CM33 *** 14.3 20.3 a 6 7 b * ** *** Discussion 2001 2003 F. graminearum 1999 2000 2001 2001 2002 2005 2005 2006 1988 1998 1991 2003 2 1998 2003 1995 1999