Abstract
Pm3 from wheat encodes a nucleotide-binding leucine-rich repeat type of receptor and confers resistance to powdery mildew caused by the fungal pathogen Blumeria graminis f.sp. tritici (Bgt). Each of the 17 functional Pm3 alleles identified so far confers resistance to a distinct spectrum of Bgt isolates. Variant Pm3e has been found in wheat donor line W150 and differs only by two amino acids from the non-functional variant Pm3CS. In order to evaluate the capability of Pm3e to provide powdery mildew field resistance, we generated transgenic Pm3e lines by biolistic transformation of the powdery mildew susceptible spring wheat cultivar Bobwhite. Field trials conducted during four field seasons in Switzerland showed significant and strong powdery mildew resistance of the Pm3e transgenic lines, whereas the corresponding biological sister lines, not containing the transgene, were severely powdery mildew infected. Thus Pm3e alone is responsible for the strong resistance phenotype. The field grown transgenic lines showed high transgene expression and Pm3e protein accumulation with no fitness costs on plant development and yield associated with Pm3e abundance. Line E#1 as well as sister line E#1 showed delayed flowering due to somaclonal variation. The study shows the capability of Pm3e in providing strong powdery mildew field resistance, making its use in wheat breeding programs very promising.
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Acknowledgements
We would like to thank the staff from Agroscope in Zurich-Reckenholz and Helen Zbinden, Esther Jung and Linda Lüthi from UZH for help with the field trial and Dr. Simone Oberhänsli for helpful advice on statistical analyses. Funding was provided by a grant from the Swiss National Science Foundation 310030_163260 and by Forschungskredit Grant No. FK-15-098 from the University of Zurich to TK.
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Supplemental Figure 1
Gene cassettes used for biolistic transformation of wheat cultivar Bobwhite. (a) Plasmid containing Pm3e-HA under maize Ubi-1 promoter. Prior to transformation the gene cassette was cut out from the plasmid using restriction enzyme NotI. The hybridization site on Pm3e for the Southern blot probe is indicated. (b) Plasmid containing the selection marker mannose-6-phosphate-isomerase (manA) gene from E.coli under the maize Ubi-1 promoter. Prior to transformation the gene cassette was cut out from the plasmid using restriction enzymes HindIII and NotI. Other features included in the plasmid maps: bla (ampR): beta-lactamase gene (= ampicillin resistance gene), NOS: nopalin synthase terminator sequence, BamHI and DraI restriction enzyme sites. (TIFF 22950 kb)
Supplemental Figure 2
(a) Southern blot analyses of transgenic lines to determine full-length Pm3e insertion. Genomic DNA of the indicated lines was digested with BamHI (Pm3e-HA is flanked by BamHI restriction enzyme sites) and hybridized with a Pm3 specific probe (forward primer 5′-CAGCACGTCCTTCTATC-3′ and reverse primer 5′-ACTGCACATACCACAAG-3′). Full-length copy of Pm3e is visible at 4.5 kb (arrow). The additional incomplete copies in line and sister line E#4 are marked with an asterisk. Genomic DNA from non-transgenic cultivar W150 (Pm3e) not containing the BamHI restriction enzyme sites flanking Pm3e and genomic DNA of non-transformed Bobwhite (BW) served as negative controls, genomic DNA from Bobwhite mixed with plasmid pUbi:Pm3e-HA served as positive control. (b) Southern blot analyses of transgenic lines to determine copy number of Pm3e insertion. Genomic DNA of the indicated lines was digested with DraI and hybridized with the Pm3 specific probe. Stars indicate Pm3e transgenes. Incomplete Pm3e copy is visible in line and sister line E#4. Genomic DNA of non-transformed Bobwhite (BW) served as negative control. (c) Absence/presence detection of unwanted transformation plasmid backbone in the transgenic lines and sister lines. Genomic DNA was amplified using primers specific for the ampicillin resistance gene beta-lactamase, bla from the transformation plasmid backbone and primers for the Mlo gene as positive control for DNA quality. Primer sequences: bla_F 5′-TTTCCGTGTCGCCCTTATTC-3′, bla_R 5′-CAGTGAGGCACCTATCTCAG-3′, Mlo_F 5′-TTCTCCTTGGCCGTGTTC-3′, Mlo_R 5′-CCCAGCAACAAGTTCTTC-3′ (TIFF 3540 kb)
Supplemental Figure 3
Heat map of yield scores from field trial 2015. (a) Triticale plots. (b) Wheat plots containing the test lines. (AI 263 kb)
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Koller, T., Brunner, S., Herren, G. et al. Field grown transgenic Pm3e wheat lines show powdery mildew resistance and no fitness costs associated with high transgene expression. Transgenic Res 28, 9–20 (2019). https://doi.org/10.1007/s11248-018-0099-5
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DOI: https://doi.org/10.1007/s11248-018-0099-5
Keywords
- Wheat
- Powdery mildew
- Genetic engineering (GE)
- Field trial
- Disease resistance