Generation Of Transgenic Wheat Plants Expressing Defense-Related Genes And Analysis Of Their Resistance To Fusarium Head Blight

Fusarium head blight(FHB) and seedling blight(FSB), caused by Fusarium species, are devastating diseases of wheat(Triticum aestivum L.) and other small grain cereals worldwide. Due to the change of global climate and tillage management, these diseases frequently reach epidemic levels and cause huge yield losses across millions of hectares in global wheat production regions. In addition, Fusarium pathogens produce various mycotoxins in grains that are highly toxic to both humans and domestic animals.The best control strategy against Fusarium pathogens is the prevention of infection in fields through the cultivation of genetically resistant varieties. Due to the complex nature of wheat-Fusarium interactions and limited availability of naturally resistant germplasm, the development of resistant wheat varieties with acceptable agronomic traits via conventional approaches is a challenge. Therefore, the introduction of alien defense-related genes into the wheat genome via transgenic approaches has been proposed as an important strategy to protect plants against Fusarium pathogens.In this study, the minimal cassettes of defense-related genes with different sources and resistance mechanisms were introduced into elite wheat varieties Zhengmai9023, Yangmai158, and Yangmai15 by particle bombardment. The main results were as follows:1. An antibody fusion comprised of the Fusarium spp.-specific recombinant antibody gene CWP2 derived from chicken, and the endochitinase gene Ech42 from the biocontrol fungus Trichoderma atroviride was introduced into the elite wheat variety Zhengmai9023. Expression of this fusion gene was regulated by the tissue-specific promoter Lem2 derived from barley; its expression was confirmed as lemma/palea-specific in transgenic wheat. By single-floret injection and natural infection assays, the two independent transgenic wheat lines Z1 and Z4 from T3 to T6 generations revealed significant resistance to fungal spreading and initial infection under field test conditions. Gas chromatography–mass spectrometry(GC-MS) analysis revealed marked reduction of mycotoxins in the grains of the transgenic wheat lines. Progenies of crosses between the transgenic lines and the FHB-susceptible cultivar Huamai13 also showed significantly enhanced FHB resistance. q RT-PCR analysis revealed that the tissue-specific expression of the antibody fusion was induced by salicylic acid drenching and induced to a greater extent by F. graminearum(Fg) infection. Histochemical analysis showed substantial restriction of mycelial growth in the lemma tissues of the transgenic plants. Thus, the combined tissue-specific and pathogen-inducible expression of this Fusarium-specific antibody fusion can effectively protect wheat against Fusarium pathogens.2. Three defense-related genes containing a maize guanylyl cyclase-like gene Zm GC, an anti-fungal peptide gene ACE from onion seeds, and an endochitinase gene Ech42 from Trichoderma atroviride were co-transferred into the elite wheat variety Yangmai158. The transgenes stability of inheritance and expression were confirmed by PCR, RT-PCR and Southern blot analyses during several generations. As transformed with plasmids reported previously, unpredictable transgene instability and silencing were also observed in this study using transformation with minimal cassettes lacking vector backbone sequences, especially within T3 generation. One of genetically stable transgenic wheat lines Y3b-1 and its progeny carrying the three multiple transgenes was screened and challenged with Fusarium pathogens, this transgenic line from T4 to T7 generations showed a signi?cantly enhanced FHB resistance. Our results implicated that it is important to identification and screen of transgenes stability and expression within low generations for transgenic crop improvement programmes; and overexpression of different defense-related genes with a complementary and synergistic activity could be used as an effective strategy for the protection of crops against fungal pathogens.3. Two genetically stable transgenic wheat lines CZI and SZP were obtained by introduction of defense-related genes into the elite wheat variety Zhengmai9023. The line CZI was co-inherited with two defense-related genes Hv Chi CWP2 and UEch42. The other line SZP was inherited with one defense-related gene UEch42. The antibody fusion gene Hv Chi CWP2 was comprised of a barley chitinase gene Hv Chi, and the Fusarium-specific antibody gene CWP2. UEch42 was a codon-optimized Ech42 gene. These two defense-related genes were both driven by Lem2 promoter. PCR analyses were confirmed during several generations. The line SZP showed no segregation in the T4 generation; however, the line CZI displayed an abnormal inheritance phenomenon, with segregation ratios(positive: null) of 1:3.5, 1:3.2, 1:3.0 and 1:2.4 in the T1 to T4 generation, respectively. Single-floret inoculation of the two transgenic wheat lines in T4 generation revealed that the line CZI showed significant resistance to fungal spreading; however, there was no obvious difference between the line SZP and the non-transgenic control.4. Two defense-related genes AFP2CWP2 and D4E1 were co-transferred into the elite wheat variety Yangmai15. The antibody fusion gene AFP2CWP2 was comprised of an anti-fungal peptide gene AFP2 from radish seed, and the Fusarium-specific antibody gene CWP2. D4E1 was a fungicidal synthetic peptide. These two defense-related genes were both driven by Ubi promoter. The genetically stable transgenic wheat line SYP was confirmed by PCR analyses during several generations, and it also displayed an abnormal inheritance phenomenon, with segregation ratios(positive: null) of 1:1.7, 1:1.5, 1:1.5 and 1:1.2 in the T1 to T4 generation, respectively. By seedling inoculation and single-floret injection, the T4 transgenic wheat line SYP showed significant resistance against Fusarium pathogens. Unexpectedly, this line displayed abnormal spike morphology, and the cause required further research.5. The expression of RNAi sequences derived from an essential Fg virulence gene, chitin synthase(Chs) 3b, was proposed as a method to enhance resistance of wheat against Fusarium pathogens. Three most effective hairpin RNAi constructs(designated as Chs3 b RNAi-1,-3 and-5) corresponding to the different regions of Chs3 b c DNA sequence were co-transferred into the elite wheat variety Yangmai15. When challenged with Fusarium pathogens, the Chs3 b RNAi transgenic lines L1 and L3 conferred highly stable and consistent resistance to both FHB and FSB throughout the T3 to T5 generations. Presence of the correspoding specific ~21 nt si RNAs in transgenic wheat plants was confirmed by Northern blot, and these si RNAs efficiently down-regulated Chs3 b in the Fg-infected transgenic wheat seedlings and spikes. Confocal microscopy revealed profoundly restricted mycelia in the Fg-infected transgenic wheat plants. Our results demonstrated that host-induced gene silencing(HIGS) of an essential fungal chitin synthase gene is an effective approach for controlling fungal pathogens. Due to the high speci?city with pathogenic fungal-derived sequence, HIGS provides a promising strategy for generating ‘user-friendly’ transgenic crops for diseases management.