Study Of Proteinase Inhibitor NbSIPI6 Resistance Against PVY

Viruses is a main limitation for plant growth and agricultural production,and the damage for crops is destructive and hard to cure and prevent.After infection,the organelles of host cell are hired by virus to produce the components for its replication and movement.Thus,how to intercept the replication and movement of viruses are the key steps to prevent and cure plant virus.Plant protease inhibitor is one of the main plant defence proteins,which could increase the plant resistance to diseases,pests and weeds etc.However,researches of PPI against viruses have rarely been reported.NbSIPI6 is a member of soybean trypsin inhibitor families that is induced by various stress.Previous researches showed that NbSIPI6 could suppress the long distance movement of PVY and increase the plant resistance to this virus.In this study,the transgenic lines carryed the binary vectors NbSIPI6-GFP and NbSIPI6m-GFP were used for analyzing virus PVY resistance,subcellular localization and immunoprecipitation of NbSIPI6.The expression patterns of NbSIPI6 in responding to viruses and SA were also analyzed.The main results are as follows:1.The binary vectors NbSIPI6-GFP and NbSIPI6m-GFP(mutated the key amino acid of NbSIPI6)were transformed to N.benthamiana.Detecting GFP genes in the plants that could survive under the treatment of antibiotic Hyg,there were three positive plants that containing the vector with GFP on the C-termination of NbSIPI6 and seven positive plants that containg the vector with GFP on the C-termination of NbSIPI6 m.Comparing the quantity of NbSIPI6 gene in transgenic plants with wild type plants by qPCR,the result showed that the expression of transgenic plants was extremely high.2.The proper folding is required for the functions of proteins.While,sometimes altering these proteins could interfere the proper folding of target protein and make them lose functions.Therefore,the anti-virus functions were tested to ensure the availability of these transgenic plants.The T1 generation transgenic plants were separately inoculated with PVX-INF1 and PVY,the results showed that overexpressed N.benthamiana significantly inhibited the movement of the virus and improved the plants anti-virus capability,comparing with the mutants and wild type plants,the results of real-time PCR showed that the expression of NbSIPI6 in overexpressed lines was significantly higher than that in the wild type,and the phenotype after virus treatment was positively correlated with the expression of NbSIPI6.These results suggested that the fusing protein didn't influence to suppress functions of NbSIPI6 against the vural movement.Moreover,the inhibition of NbSIPI6 was not only limited to PVX,but also improved the resistance to PVY.3.In the cells,the correct subcellular localization of protein is required for protein function.In order to clarify the subcellular localization of NbSIPI6,mCherry,plasma membrane Marker,was transiently expressed in leaves of transgenic plant and results showed that partial areas appeared yellow on the laser confocal microscope,indicating that GFP overlapped with mCherry,however,the clear green color could be observed in other areas of the cell,implying NbSIPI6 might not uniquely exist in cell membrane.In addition,the expression of NbSIPI6 was significantly higher than that of the control after treatment with salicylic acid or PVX,indicating that its expression was the characteristics of responding to salicylic acid and PVX.4.Gene silencing is an important way for plants to inhibit viruses,on the contrary,viruses employ silencing suppressors,which inhibit gene silencing and is benefit for viral replication and movement.Therefore,to explore whether NbSIPI6 has the function to inhibit virus silence suppressors,the overexpression vectors,such as NbSIPI6-1300 or NbSIPI6m-1300,were combined with silencing suppressors Hc-Pro and P19 respectively,and then they were co-injected with GFP-1300 into 16 c.The effect of NbSIPI6 on silencing suppressors was evaluated by detecting the presence and intensity of green fluorescence.The result showed that the green fluorescence intensity in the group of NbSIPI6 and HC-Pro or the group of NbSIPI6 and P19 was similar to the control groups,which suggested that NbSIPI6 may not have the function of inhibiting virus silencing suppressors.