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Developing transgenic wheat to encounter rusts and powdery mildew by overexpressing barley chi26 gene for fungal resistance.

ABSTRACT: BACKGROUND:The main aim of this study was to improve fungal resistance in bread wheat via transgenesis. Transgenic wheat plants harboring barley chitinase (chi26) gene, driven by maize ubi promoter, were obtained using biolistic bombardment, whereas the herbicide resistance gene, bar, driven by the CaMV 35S promoter was used as a selectable marker. RESULTS:Molecular analysis confirmed the integration, copy number, and the level of expression of the chi26 gene in four independent transgenic events. Chitinase enzyme activity was detected using a standard enzymatic assay. The expression levels of chi26 gene in the different transgenic lines, compared to their respective controls, were determined using qRT-PCR. The transgene was silenced in some transgenic families across generations. Gene silencing in the present study seemed to be random and irreversible. The homozygous transgenic plants of T4, T5, T6, T8, and T9 generations were tested in the field for five growing seasons to evaluate their resistance against rusts and powdery mildew. The results indicated high chitinase activity at T0 and high transgene expression levels in few transgenic families. This resulted in high resistance against wheat rusts and powdery mildew under field conditions. It was indicated by proximate and chemical analyses that one of the transgenic families and the non-transgenic line were substantially equivalent. CONCLUSION:Transgenic wheat with barley chi26 was found to be resistant even after five generations under artificial fungal infection conditions. One transgenic line was proved to be substantially equivalent as compared to the non-transgenic control.

SUBMITTER: Eissa HF

PROVIDER: S-EPMC5441082 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Developing transgenic wheat to encounter rusts and powdery mildew by overexpressing barley <i>chi26</i> gene for fungal resistance.

Eissa Hala F HF Hassanien Sameh E SE Ramadan Ahmed M AM El-Shamy Moustafa M MM Saleh Osama M OM Shokry Ahmed M AM Abdelsattar Mohamed M Morsy Yasser B YB El-Maghraby Maher A MA Alameldin Hussien F HF Hassan Sabah M SM Osman Gamal H GH Mahfouz Hesham T HT Gad El-Karim Gharib A GA Madkour Magdy A MA Bahieldin Ahmed A

Plant methods 20170522

<h4>Background</h4>The main aim of this study was to improve fungal resistance in bread wheat via transgenesis. Transgenic wheat plants harboring barley <i>chitinase</i> (<i>chi26</i>) gene, driven by maize <i>ubi</i> promoter, were obtained using biolistic bombardment, whereas the herbicide resistance gene, <i>bar,</i> driven by the <i>CaMV 35S</i> promoter was used as a selectable marker.<h4>Results</h4>Molecular analysis confirmed the integration, copy number, and the level of expression of t ...[more]

PMID: 28539970

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Project description:Genetically modified (GM) plants offer an ideal model system to study the influence of single genes that confer constitutive resistance to pathogens on the ecological behaviour of plants. We used phytometers to study competitive interactions between GM lines of spring wheat Triticum aestivum carrying such genes and control lines. We hypothesized that competitive performance of GM lines would be reduced due to enhanced transgene expression under pathogen levels typically encountered in the field. The transgenes pm3b from wheat (resistance against powdery mildew Blumeria graminis) or chitinase and glucanase genes from barley (resistance against fungi in general) were introduced with the ubiquitin promoter from maize (pm3b and chitinase genes) or the actin promoter from rice (glucanase gene). Phytometers of 15 transgenic and non-transgenic wheat lines were transplanted as seedlings into plots sown with the same 15 lines as competitive environments and subject to two soil nutrient levels. Pm3b lines had reduced mildew incidence compared with control lines. Chitinase and chitinase/glucanase lines showed the same high resistance to mildew as their control in low-nutrient treatment and slightly lower mildew rates than the control in high-nutrient environment. Pm3b lines were weaker competitors than control lines. This resulted in reduced yield and seed number. The Pm3b line with the highest transgene expression had 53.2% lower yield than the control whereas the Pm3b line which segregated in resistance and had higher mildew rates showed only minor costs under competition. The line expressing both chitinase and glucanase genes also showed reduced yield and seed number under competition compared with its control. Our results suggest that single transgenes conferring constitutive resistance to pathogens can have ecological costs and can weaken plant competitiveness even in the presence of the pathogen. The magnitude of these costs appears related to the degree of expression of the transgenes.

Project description:Aegilops markgrafii (Greuter) Hammer is an important source of genes for resistance to abiotic stresses and diseases in wheat (Triticum aestivum L.). A series of six wheat 'Alcedo'-Ae. markgrafii chromosome disomic addition lines, designated as AI(B), AII(C), AIII(D), AV(E), AIV(F), and AVIII(G) carrying the Ae. markgrafii chromosomes B, C, D, E, F, and G, respectively, were tested with SSR markers to establish homoeologous relationships to wheat and identify markers useful in chromosome engineering. The addition lines were evaluated for resistance to rust and powdery mildew diseases. The parents Alcedo and Ae. markgrafii accession 'S740-69' were tested with 1500 SSR primer pairs and 935 polymorphic markers were identified. After selecting for robust markers and confirming the polymorphisms on the addition lines, 132 markers were considered useful for engineering and establishing homoeologous relationships. Based on the marker analysis, we concluded that the chromosomes B, C, D, E, F, and G belong to wheat homoeologous groups 2, 5, 6, 7, 3, and 4, respectively. Also, we observed chromosomal rearrangements in several addition lines. When tested with 20 isolates of powdery mildew pathogen (Blumeria graminis f. sp. tritici) from five geographic regions of the United States, four addition lines [AIII(D), AV(E), AIV(F), and AVIII(G)] showed resistance to some isolates, with addition line AV(E) being resistant to 19 of 20 isolates. The addition lines were tested with two races (TDBJ and TNBJ) of the leaf rust pathogen (Puccinia triticina), and only addition line AI(B) exhibited resistance at a level comparable to the Ae. markgrafii parent. Addition lines AII(C) and AIII(D) had been previously identified as resistant to the Ug99 race group of the stem rust pathogen (Puccinia graminis f. sp. tritici). The addition lines were also tested for resistance to six United States races (PSTv-4, PSTv-14, PSTv-37, PSTv-40, PSTv-51, and PSTv-198) of the stripe rust pathogen (Puccinia striiformis f. sp. tritici); we found no resistance either in Alcedo or any of the addition lines. The homoeologous relationships of the chromosomes in the addition lines, molecular markers located on each chromosome, and disease resistance associated with each chromosome will allow for chromosome engineering of the resistance genes.

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Dataset Information

Developing transgenic wheat to encounter rusts and powdery mildew by overexpressing barley chi26 gene for fungal resistance.

Publications

Developing transgenic wheat to encounter rusts and powdery mildew by overexpressing barley <i>chi26</i> gene for fungal resistance.

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