Screening And Functional Characterization Of Heterologous Resistance Genes From Pinellia Ternata(Thunb.)to Enhance The Basal Resistance Level Of Nicotiana Benthamiana Domin

Non-self-recognition is one of the most important phenomena to initiate the innate immunity in multicellular organisms including plants.The present study demonstrated the effective use of non-self-recognition to introduce the durable resistance in the plants against diseases.The resistance genes from a well-known traditional Chinese medicinal herb,Pinellia ternata?Thunb.?were successfully identified and genetically introduced in Nicotiana benthamiana Domin to enhance its basal resistance against three different pathogens such as Botrytis cineria,Phytophthora capsici and tobacco mosaic virus?TMV?.The c DNA library of P.ternata was constructed in p TRV expression vector and transformed into Agrobacterium tumefaciens EHA105.49 potential pt HR?P.ternata hypersensitive response?genes were isolated using Agrobacterium mediated transient expression system.Out of initial screening,seven genes?pt HR293,pt HR759,pt HR1015,pt HR941,pt HR375,pt HR2-20 and pt HR99?were selected after checking their strong hypersensitive response?HR?in N.benthamiana,Lycopersicon esculentum Mill.and Gossypium hirsutum L.leaves.This study established that these seven pt HR genes have potential to trigger ROS?reactive oxygen species?burst,callose deposition and HR in N.benthamiana leaves.N.benthamiana leaves were infiltrated with pt HR genes and significant high expression of pathogenesis-related?PR?genes?PR-1a,PR-5,PDF-₁.2,NPR1,PAL,RBOHB and ERF-₁?in SA?Salicylic acid?and JA/ET?Jasmonic acid/ Ethylene?mediated defense pathways was obtained in upper leaves from 0-120 hours with 24 hours' time interval.These pt HR genes also expressed their ability to enhance the activity of antioxidant enzymes,Peroxidase?POD?,Polyphenol oxidase?PPO?and Superoxide dismutase?SOD?,in upper leaves from 0-168 hours with 24 hours' time interval.In next phase,selected pt HR genes were cloned into p CAMBIA3301 binary expression vector between Bgl ?-Bst E ? enzyme sites and the correct insertion was confirmed with DNA sequence analysis.The clones were introduced into A.tumefaciens EHA105 strain and seven positively transformed N.benthamiana plants?pt HR293,pt HR759,pt HR1015,pt HR941,pt HR375,pt HR2-20 and pt HR99?with clear typical phenotypic variation in leaves from non-transformed plants were successfully obtained using Agrobacterium mediated leaf disc transformation method.PCR with gene-specific primers further confirmed the positivity of target gene integration in the transformed N.benthamiana plants.The next generation of the plants was raised from the seeds of positive plants that were initially screened on MS media against bialaphos antibiotic.The expression of pt HR genes was detected up to T₃ generation of transformed N.benthamiana plants using RT-PCR and q RT-PCR.The transcriptome level of PR-genes in T₃ generation was 1.14 to 34.02 fold in transformed N.benthamiana plants compared with nontransformed N.benthamiana control and indicated the inheritance of resistance into next generations.The transformed N.benthamiana plants revealed an increase in the percentage of antioxidant enzymes?POD,PPO and SOD?with values up to 66.5%,222.3% and 134.4%,respectively.These plants also exhibited high resistance level against B.cineria?up to 69.83%?and P.capsici?up to 23.08%?.The introduction of pt HR genes in N.benthamiana revealed no side effects on seed germination,root and shoot length of the transformed plants.UPLC-QTOF-MS analysis revealed a number of bioactive compounds in the transformed plants that were proposed as potential drivers of resistance mechanism in N.benthamiana.The bioactive compounds were Ethoxzolamide,Pyrrolidine,Lignocerane,3,4-Dichloromaleimide,Sulfanilamide and 2-Undecanone in pt HR293;Oxytetracycline,Cuelure,Fluphenazine,Allantoin,Diethylstilbestrol and 1,2-Benzisothiazol-3?2H?-one in pt HR375;and Pimozide,Isoamyl salicylate,1,3-Butadiene and Citronellal in pt HR99,respectively.F-₁ hybrid plants?pt HR941-F-₁,pt HR375-F-₁,pt HR1015-F-₁,pt HR941+pt HR375-F-₁ and pt HR293+pt HR759-F-₁?were raised by crossing transformed?T₃?with non-transformed,and transformed?T₃?with another transformed?T₃?plants.These hybrid plants demonstrated a significant high expression of PR-genes?upto 20.01 fold?when compared with their respective control plants.These hybrids possessed stable immunity in a resistance anaylsis and presented inhibition up to 42.38% and 30.62% against B.cineria and P.capsici,respectivley.Collectively,it is concluded that heterologous plant genes can activate resistance against disease in another plant species and furthermore,by generating F-₁ hybrids,fresh pathogen independent plant resistance can be obtained.It is also concluded that pt HR genes play their role in SA and JA/ET defense pathways to activate the resistance against invading pathogens.