Studies On The Application Of Pinellia Ternata Genes Which Induce Hypersensitive Response In Nicotiana Benthamiana

Pinellia ternata(Araceae)is one of the main components in many decoctions in traditional Chinese medicine that has been applied since ancient times for anti-emetic,anti-tussive,sedative and anti-inflammatory purposes.Hypersensitive response(HR)is a typical phenotype of plant and pathogen incompatible interaction to avoid the spread of disease.Previous studies in our lab have shown that some P.ternata genes can trigger HR syptoms in Nicotiana benthamiana.In this study,we have performed some basic experiments to evaluate the mechanism of hypersensitive response triggered by these test genes.P.ternate c DNA library was already constructed by using p BE-S vector and Bacillus subtilis(WB800 strain).By using transient expression system method,it was found that some colonies of B.subtilis can trigger HR symptoms in N.benthamiana.By using agrobacterium mediated transient expression system,it has been found that 49 newly identified P.ternata genes cloned in p TRV2 Ex vector can trigger HR symptoms in N.benthamiana,but these genes couldn’t show any response on P.ternata itself.These newly identified genes were named as pt HR(Pinella ternata hypersensitive response)genes.Same study was also performed on Lycopersicon esculentum Mill.and Gossypium hirsutum to confirm the activity of these genes.So it is considered that this HR activity is due to plant nonself recognization meachanism to avoid the invasion of pathogen.It is also evaluated that pt HR 99 and pt HR 2-20 can induce ROS accumulation,callose depositon,defense-related enzymes activities and also systemic acquired resistance(SAR).RT-q PCR was performed to evaluate the relative expression of some defense-related genes viz.PR-1a,PR-5,PDF1.2,NPR1,PAL,RBOHB and ERF1,which play vital role in different defense-related pathways.It was found that pt HR genes play a significant role in the upregulation of defense-related genes of N.benthamiana.By using agrobacterium mediated gene transformation,pt HR 99,pt HR 375,pt HR941 and pt HR 2-20 were transformed in N.benthamiana.Obtained positive transformants were used for analysis of defense-related enzymes activities and systemic acquired resistance.