Development Of Wheat Lines Resistant To Fusarium Head Blight Through Host Induced Gene Silencing

Wheat is one of the main food sources for human beings,and China is the largest producer of wheat.It is of great significance to ensure high quality and yield of wheat for the survival and development of the human race and to maintain world peace.However,wheat production is compromised by multiple factors including diseases and insect pests.Among diseases,FHB or wheat scab,caused by F.graminearum,is a common threat to wheat production throughout the world and in China particularly,which results in huge economic losses.F.graminearium,in the process of infecting plants,produces many kinds of mycotoxins which heavily infest the seeds.Mycotoxins do not only reduce yield and quality but also pose high risks to human and animal health even if ingested in minute quantities like parts per million(ppm)or parts per billion(ppb).This disease is favored by warm and humid weather,and in recent years,due to global warming,FHB sustainable epidemics have been reported.It has been frequently reached to epidemic levels in China since 2000.More severe and frequent recurring outbreaks,recorded from several wheat planting regions of China in the past few years are increasing concerns regarding the management of FHB.Among different methods of managing FHB,fungicide application is costly and unfriendly to the environment.Breeding for resistant varieties is so far the best technology to control fungal diseases.Commonly used methods of breeding for disease resistance include conventional selection,marker-assisted selection(MAS)and genetic engineering,but all the above-mentioned techniques bring with them certain randomness.The development of resistant varieties against FHB is nearly impossible through conventional breeding due to non-availability of completely resistant germplasm in wheat.Moreover,conventional breeding often results in low recovery of agronomic important traits in wheat.MAS is a rapid method of crop improvement.It takes 3-5 years for developing a new cultivar against 10-15 years taken by the conventional breeding and recovery of agronomic traits also good.But,it is very expensive requiring a sophisticated and well-equipped laboratory and skilled labor.For FHB control,RNAi has tremendous potential to be used as an alternative or in integration with other management strategies.The concept of host-induced gene silencing(HIGS)-the method of preparing resistant plants by introduction of respective pathogen genes into host plants-opens new avenues to control diseases.The major goals of this study are(1)identification of essential fungal genes,(2)exploitation of those essential genes to induce effective RNAi,(3)generation of transgenic wheat lines(4)evaluation of those transgenic lines for stable resistance against FHB and for agronomic performance.This study characterized Protein kinase C(Pk C)an enzyme that initiates several signaling interactive pathways,as an essential gene for growth and survival of F.graminearum.Several efforts to develop deletion mutants of F.graminearum by removing Pk C remained unsuccessful.Chs7 was also characterized as an important and essential gene of F.graminearum.It acts as chaperons which export Chs3 protein from the endoplasmic reticulum.Chs3,in turn,is responsible for the formation of chitin,which is an integral component of fungal cell walls.Secondary and three-dimensional structures of Pk C and Chs7 proteins,their multiple alignments and phylogenetic analysis also supported the importance of both genes for F.graminearum.The research was focused on the use of gene-specific tags(Gsts)from Pk C and Ch7 genes to transform Y158 and X76 wheat varieties.The Gsts,namely Pkc RNAi4 and Chs7RNAi1 were designed to produce hairpin RNA(hp RNA)inside host plants.Both sense and antisense fragments of Gst joined by an intron of pyruvate dehydrogenase kinase(Pdk)were cloned under the control of maize ubiquitin(Ubi-1)promoter and nopaline synthase(Nos)terminator.The hp RNAs are recognized by RNAi machinery of cells and are processed to produce small interfering RNAs(si RNAs).The no off-target effects and no cellular toxicity of Pkc RNAi4 and Chs7RNAi1 were ensured by using standard software of bioinformatics.In-vivo experiments designed to check off-targets by artificial inoculation of powdery mildew spores to transgenic lines expressing hp RNA from Pk CRNAi4 and Chs7RNAi1 also confirmed the results obtained through bioinformatics.Transgenic lines YP1,YP2 and YP3 were developed by co-transformation of Y158 with Pk CRNAi4-Chs7RNAi1 by particle bombardment,and 1 transgenic line,XP was recovered by the transformation of X76 with Pk CRNAi4-Chs7-RNAi1 via particle bombardment.We got YC1 transgenic line by Agrobacterium-mediated co-transformation of Y158 with Pk CRNAi4-Chs7RNAi1 and three lines,XC1,XC2,and XC3 by Agrobacterium-mediated transformation of X76 with Pk CRNAi4-Chs7RNAi1.Phosphomannose isomerase(PMI)was used as a selection marker,which diminished from transgenic lines by T4 generation.Transgenic lines were grown through successive generations and in each generation,positive plants were screened through PCR to attain homozygous lines.Seedling of transgenic lines,YP3 and XP showed 90% resistance and seedlings of YC1 and XC1 demonstrated 65% resistance.Transgenic plants of YP3 and XP showed 88-90% resistance and transgenic plants of YC1 and XC1 exhibited 50% resistance in comparison to their non-transgenic control lines.These results suggest that transgenic lines expressing Pk CRNAi4-Chs7RNAi1 exhibited high to moderate resistance.But either high or moderate,resistance was stably inherited to a minimum of three generations of homozygous lines.The q PCR results proved a strong correlation between reduced fungal transcripts and increased disease resistance.Transgenic lines demonstrated si RNAs of multiple sizes mainly 21-nt,24-nt when hybridized with Pk CRNAi4 probes.All transgenic lines were found to be highly resistant against deoxynivalenol(DON)accumulation.And no significant difference was observed in agronomic traits like plant height,number of tillers,number of seeds/spike,biomass,etc between transgenic and non-transgenic lines.Overall evaluation of the transgenic lines proved that HIGS mediated by hp RNA against FHB has great potential to be commercialized as allowed for many viral diseases.