Functional Studies On Rice Stripe Virus(RSV) And Pepper Mild Mottle Virus(PMMoV) Coat Proteins

Plant viral diseases cause severe economic losses in agricultural production.Plant virus disease is a persistent disease among plant diseases,causing widespread damage and causing a reduction of many crops worldwide.Plant virus particle,also known as a virion,is an extremely small infectious agent.Virion consists of an inner core of nucleic acid(either ribonucleic acid[RNA]or deoxyribonucleic acid[DNA])surrounded by coat protein(referred to as the capsid).In addition to providing a protective shell for viral genomic nucleic acids,the coat(capsid)proteins(CPs)can regulate virus infectivity,pathogenicity,symptom expression,interactions with the host factors,virus movement,vector transmission,host range.In this paper,pepper mild mottle virus(positive-sense single-stranded RNA virus,(+)ssRNA virus)and rice stripe virus(multi-component negative-sense single-stranded virus,(-)ssRNA virus)are taken as model to explore the role of coat proteins in their viral infection cycle and pathogenicity.Rice stripe virus(RSV),genus Tenuivirus,is one of the most devastating pathogens of rice(Oryza sativa)in rice-growing regions of East Asia.Upon RSV infection,JA content was increased and the expression level of JA biosynthetic genes were upregulated.In addition,methyl jasmonate-treated plants showed resistance to RSV and reduced the accumulation of RSV.SHAM-treated plants were more susceptible to RSV,showing more severe symptom and higher RSV RNAs.In addition,we used tobacco rattle virus(TRV)system to silence COI1,a key component of JA signaling,and then inoculated RSV.Compared to TRV:00,more serious RSV symptoms appeared on the systemically infected leaves of COI1-silenced plants.Blotting analysis showed that viral RNAs and CP accumulation in leaves systemically infected with RSV was nearly 50%higher on COI1-silenced plants than on the controls.These results indicate that JA pathway plays crucial roles in plant defense against RSV.To detect which RSV protein was responsible for induction of the JA pathway,we expressed p2,p3,p4,pc4 and CP in leaves of N.benthamiana transiently by agroinfiltration and detected their ability to induce JA.Overexpression of CP up-regulated JA synthetic genes and increased JA accumulation.The result was further validated by using CP-transgenic rice.qRT-PCR analysis of JA synthetic gene and LC-MS/MS detection of JA content showed that JA pathway was activated in CP-transgenic rice.The above results show that CP was the major viral component responsible for inducing the JA pathway.RSV is transmitted by small brown planthoppers(Laodelphax striatellus)in a persistent and circulative-propagative manner.Interestingly,methyl jasmonate treatment attracted SBPH to feed on rice plants while a JA-deficient mutant(L145)was less attractive than wild type rice.Moreover,SBPH showed an obvious preference for feeding on transgenic rice lines expressing RSV CP.These results indicated that JA accumulation induced by RSV infection was helpful to attract the SBPH to feed.Taken together,our results demonstrated that CP is an inducer of the JA pathway that activates the plant defense against RSV while also attracting SBPH to feed and benefitting viral transmission.To investigate whether RSV CP contains a specific motif for JA induction,we expressed ΔCPI,ΔCP2,ΔCP3,ΔCP4 in leaves of N.benthamiana transiently by agroinfiltration and detected their ability to induce JA.Results showed a motif at N-terminus of CP(named JIM)was responsible for inducing JA pathway.In addition,we noticed that the expression of JIM caused the cell death with the accumulation of H2O2 and the significant up-regulation of HR marker gene Hin1.Cell death induced by JIM was alleviated in SHAM-treated or COI1-silienced plants.This demonstrated that JIM-induced cell death is JA-dependent.Mutant analysis showed that regions responsible for the self-interaction and nuclear localization of the CP were necessary for JIM to induce JA and cause cell death.Furthmore,we found C-terminus of CP(ΔCP4)compromise induction of JA pathway and cell death induced by JIM.Pepper mild mottle virus(PMMoV),genus Tobamovirus,causes serious economic losses in pepper production in China.In a survey for viral diseases on pepper,two PMMoV isolates(named PMMoV-ZJ1 and PMMoV-ZJ2)were identified with different symptoms in Zhejiang province.The PMMoV-ZJ1 causes slight wrinkling in pepper leaves,while PMMoV-ZJ2 causes yellowing.The full-length genomic sequences of PMMoV-ZJ1(MN616926)and PMMoV-ZJ2(MN616927)were first determined by RT-PCR and 5’ and 3’ RACE(rapid amplification of cDNA ends).Sequence alignment analysis suggested there were only four nucleotide differences between the isolates:U854G,A2256G,G3559A and G5742A.The mutations of the four nucleotides cause the differences of corresponding amino acid,of which there are four amino acid differences between the isolates:Val262Gly,Ile629Met and Ala1164Thr in the replicase,and Asp20Asn in the coat protein.Infectious cDNA clones of both isolates were constructed and shown to cause distinctive symptoms.Chlorosis symptoms appeared only on PMMoV-ZJ2-infected plants.To identify which mutation at the above four positions as the key site for symptom formation,we therefore produced mutated clones of PMMoV-ZJ1 individually replacing the nucleotides at each of the variable sites:PMMoV-ZJ1-1(T854G),PMMoV-ZJ1-2(A2256G),PMMoV-ZJ1-3(G3559A),and PMMoV-ZJ1-4(G5742A).Result of virus inoculation and symptom observation indicted that the Asp20Asn substitution in the CP was responsible for the induction of chlorosis.Confocal assays revealed that the subcellular localization pattern of the two CPs was different,CP20Asp was mainly located at the cell periphery,whereas most CP20Asn located in the chloroplast.The above results suggest that the 20th amino acid of PMMoV CP determines the location of CP,and then affects the normal function of chloroplast,which leads to the chlorosis symptom,indicating the function of CP in viral pathogenicity.To sum up,we have studied the functions and possible mechanisms of RSV and PMMoV CP in their infection process.Our research further extends the multifunctional roles of viral CP,provides us a new comprehension of pathogenicity,symptom and transmission mechanism of RSV or PMMoV,and suggests a novel approach to control the viral diseases.