Degradation Of Viral RNA By Rhp-PSP Protein Of Rhodopseudomonas Palustris And Its Key Amino Acids Sites

Plant viral disease is a kind of viral disease occurring in plants,also known as plant cancer.At present,there is no effective control method.Tobacco mosaic virus?TMV?is a common plant virus.Its virus particles are straight rod-like stru cture,mainl y composed of nucleic acid and coat protein.Plant diseases caused by TMV mainl y show symptoms such as deformity,mosaic and poor growth.Protein pesticides,as a new biological pesticide,belong to protein elicitors.As a new biological pestic ide,protein pesticide is expected to become a new way to control plant viral diseases efficiently and green.The antiviral Rhp-PSP protein isolated from Rhodopseudomonas palustris showed by mass spectrometry that Rhp-PSP protein belongs to the YjgF/YER057c/uK114 family,whose important function is the degradation of nucleic acid.Previous studies have confirmed that the antiviral protein Rhp-PSP has an in vitro passivation effect on TMV virus particles,and the inhibitory effect is related to the effect o f the protein on viral nucleic acid.Therefore,it is speculated that Rhp-PSP may target viral RNA and inhibit virus proliferation by interfering with its normal function.The secondary struct ure alignment of the ami no acid sequences of Rhp-PSP protein and its family homologous proteins showed that there were five key sites in the structure of Rhp-PSP protein:Tyrosine at position 23(Tyr^{2 3}),aspartic acid at 72(Asn^{7 2}),phenylalanine at position 120(Phe^{1 2 0}),arginine at position 129(Arg^{1 2 9}),and glutamate at position 143(Gln^{1 4 3}).In order to further elucidate the degradation of TMV CP mRNA by Rhp-PSP protein and the mechanism of degradation of TMV CP mRNA by protein after mutation of key amino acid sites,Tyr^{2 3}?tyrosine?,Arg^{1 2 9}?arginine?and Gln^{1 4 3}?glutamic acid?were selected for mutation,and construction of prokaryotic expression vector of mutant protein.The degradation of TMV CP mRNA by Rhp-PSP protein and mutant protein was detected b y Northern blot technique.The results showed that Rhp-PSP protein cou ld degrade TMV CP mRNA.After mutation of Arg^{1 2 9},the mutant protein did not degrade nucleic acid;but mutation of Tyr^{2 3} and Gln^{1 4 3},the mutant protein still degraded TMV CP mRNA.This indicates that the key site for Rhp-PSP protein to degrade nucleic acid is 129arginine.The inhibition of Rhp-PSP protein and mutant protein on TMV virus particles and TMV-RNA was verified by half-leaf inoculation of Nicotiana glutinosa.The results were consistent with those of Northern blot in detecting the degradation of TMV CP mRNA by Rhp-PSP protein and mutant protein.It was concluded that Rhp-PSP protein degraded nucleic acid,and the key site of Rhp-PSP protein degraded nucleic acid was 129 arginine on the?fold near the C-terminal.Rhp-PSP proteins bind viral RNA via Arg^{1 2 9} and participate in its subsequent degradation to achieve inhibition of viral proliferation.