Lu Wang ,Huiting Xie ,Xiaoyuan Zheng,Jiasheng Chen,Shuai Zhang*,Jianguo Wu*

Vector-borne Virus Research Center,Fujian Province Key Laboratory of Plant Virology,State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops,Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China

Keywords:Rice antivirus R gene RNA silencing Recessive resistance Hormone

ABSTRACT Plants and viruses coexist in the natural ecosystem for extended periods of time,interacting with each other and even coevolving,maintaining a dynamic balance between plant disease resistance and virus pathogenicity.During virus-host interactions,plants often exhibit abnormal growth and development.However,plants do not passively withstand virus attacks but have evolved sophisticated and effective defense mechanisms to resist,limit,or undermine virus infections.It is widely believed that the initial stage of infection features the most intense interactions between the virus and the host and the greatest variety of activated signal transduction pathways.This review describes the most recent findings in rice antiviral research and discusses a variety of rice antiviral molecular mechanisms,including those based on R genes and recessive resistance,RNA silencing,phytohormone signaling,autophagy and WUS-mediated antiviral immunity.Finally,we discuss the challenges and future prospects of breeding rice for enhanced virus resistance.

1.Introduction

Rice is the staple crop of nearly half of the world’s population[1].Asia is the primary rice-producing area in the world,accounting for 90%of global rice production.In China,the rice planting area accounts for approximately 30%of grain crops,and the output of rice is close to half of the total grain output,which means that rice occupies a particularly important position in grain production in the country.

Rice is vulnerable to infection by a variety of pathogens,among which viruses are an important category.At least 17 rice viruses have been identified:Rice black-streaked dwarf virus(RBSDV)[2,3],Rice bunchy stunt virus(RBSV),Rice dwarf virus(RDV)[4,5],Rice gall dwarf virus(RGDV)[6],Rice grassy stunt virus(RGSV)[7],Rice giallume virus(RGV),Rice hoja blanca virus(RHBV)[8,9],Rice necrosis mosaic virus(RNMV)[10,11],Rice ragged stunt virus(RRSV)[12],Rice stripe mosaic virus(RSMV)[13],Rice stripe necrosis virus(RSNV)[14],Rice stripe virus(RSV)[15],Rice tungro bacilliform virus(RTBV)[16],Rice tungro spherical virus(RTSV)[17],Rice yellow mottle virus(RYMV)[18],Rice yellow stunt virus(RYSV)[19-21],and Southern rice black-streaked dwarf virus(SRBSDV)[22-24].Eight of these:RBSDV[25],RDV[26],RGDV[27],RGSV[28],RRSV[29],RSMV[13],RSV[30]and SRBSDV[24],are widespread in China and cause serious harm to rice production.

Viruses are obligate intracellular pathogens that manipulate and utilize the host’s molecular machinery to survive in harmful cellular environments.These intracellular parasitic properties hinder efforts to control viral diseases directly with chemical agents.In actual rice production,the control of vector insects is usually used to prevent and control viral diseases.In the host,the presence of viruses and their spread activate mechanisms that can combat viral infections at the level of single cells and entire organisms.Multiple such mechanisms have evolved during the long struggle between rice and viruses[31].

2.Multiple antiviral mechanisms of plants

Plant resistance to viruses is divided into active and passive mechanisms.Active resistance is mediated by resistance(R)gene or RNA silencing.Passive resistance is regulated primarily by the physical characteristics of the plant itself or by specific diseasesusceptibility factors[32].During virus infections,the two mechanisms act synergistically,complement each other,and coexist at allstages of the infection to counter the propagation,migration and spread of the virus in the host.

2.1.R gene-mediated antiviral immunity

During their long interaction with pathogens,plants have evolved R gene systems that can resist pathogen invasion.The plant disease resistance mechanism mediated by an R gene is highly similar to the response of the animal immune system,in that an avirulence factor(Avr)induces the corresponding R protein to respond to the infecting virus[33].R gene-mediated plant resistance is manifested as hypersensitivity(HR)and systemic acquired resistance(SAR).With HR,the plant restricts the movement of the virus in the plant system by causing programmed cell death(PCD)of the infection site;with SAR,the plant is kept away from the tissue cells of their own infection sites,thereby gaining immunity against the virus[34].

There are few studies of R genes conferring rice resistance to viruses.Zhang et al.[35]identified an RSV-resistant rice R gene,STV11,in the Indian landrace Kasalath(KAS),a cultivar with high resistance to RSV.Wang et al.[36]fine-mapped on rice chromosome 11 a major QTL from KAS,named STV11-R.Cloning and sequence analysis revealed that STV11 encodes a sulfotransferase whose main function is to catalyze the conversion of salicylic acid(SA)to sulfonated SA(SSA).Further analysis showed that the two alleles STV11-R and STV11-S are widely distributed in indica and japonica rice cultivars respectively,largely accounting for the difference in RSV resistance between these two subspecies.Stripe Disease Resistance-a(STV-a)and Stripe Disease Resistance-b(STV-b)are two loci associated with RSV resistance in Japanese upland rice[37].The first is linked to the glutinous endosperm(wx)and photosensitivity-1(Se-1)loci on chromosome 6,while the latter is located on chromosome 11,and the two loci are complementary dominant genes.Modan,an indica rice,carries a different resistance gene,Stripe Disease Resistance--b-I(STV-bi),which is an allele of STV-b and is incompletely dominant.The STV-bi gene in Modan has been mapped to a 286-kb region on chromosome 11 covered by two overlapping BAC clones[38,39].In a recent study[40],analysis of a recombinant inbred line(RIL)population of 181 lines revealed a quantitative trait locus(QTL)for resistance to RBSDV,qRBSDVD6.Association analysis of RBSDV resistance in 1070 diverse sequenced rice accessions identified the same locus repeatedly on chromosome 6.The chromosomal region showing the strongest association harbored the gene LOC_Os06g03120,encoding an aspartic protease precursor.

Rice virus disease resistance genes reported by the China Rice Data Center(http://www.ricedata.cn/index.htm)include Black Streaked Dwarf Resistance(BSV),Gray Stunt Resistance(GSV),Hoja Blanca Virus Resistance(HBV),and Tungro Spherical Resistance(TUV-aI)and(TUV-b).Most of these resistance genes have not been fine-mapped and cloned.Most resistance genes described in this review may not be classical R genes,because they lack Toll-NBSLRR(TNL)or CC-NBS-LRR(CNL)domains.

2.2.Recessive resistance mediated by eIF4Es

Viruses cannot survive independently,but rely on the replication and translation system of the host cells to multiply their own genetic material.When the host’s genes change and the expressed products cannot be recognized by the virus,the virus is not able to multiply,and the host eventually achieves the antiviral effect.In this process,plants do not need to trigger an active resistance mechanism,and the altered genes in the host are usually called recessive resistance genes[41].Among the recessive genes,some have nonsense mutations that cause protein translation to terminate prematurely and produce nonfunctional proteins,an example being the anti-TuMV recessive disease resistance gene retr02 in cabbage[42].Other missense mutations cause changes in the amino acid sequence of the encoded protein,leading to inhibition of the infection,reproduction,and spread of the virus[43].

Some recessive antiviral plant genes have been mapped and cloned,most of them encoding translation initiation factors(eIFs).They include eIF4E(the cap-binding protein)and eIF4G(a large scaffolding protein)and their isoforms eIF(iso)4E and eIF(iso)4G.To date,at least 18 eIFs have been mapped and cloned;of these members,the eIF4E is the most common,accounting for 50%,the second is IF(iso)4E with 6 members,and the remaining members include eIF4G and eIF(iso)4G[42].RYMV1(Rice Yellow Mottle Virus Resistance 1)is the first recessive gene conferring resistance to rice yellow mottle virus disease[44].RYMV is a single-stranded RNA virus that infects rice in Africa.The cultivar Gigante and Bekarosaka species(Oryza sativa),as well as the O.glaberrima cultivars Tog5681,Tog5672,Tog5674,and Tog7291,are highly resistant to RYMV[45,46].Highly resistant germplasm does not show virus disease symptoms,and virus content in inoculated plant tissues could not be detected using enzyme-linked immunosorbent assay(ELISA).The antiviral properties of these variants are determined primarily by the recessive gene RYMV1,which is located on rice chromosome 4 and consists of 9 exons and 8 introns,encoding the eukaryotic translation initiation factor isomer elF(iso)4G[47,48].The primary function of this gene is to fix the mRNA cap structure involved in the initiation of protein translation and recruitment of ribosomes[49].Four independent resistance alleles of RYMV1 have been identified:O.sativa(allele RYMV1-2)and O.glaberrima species(alleles RYMV1-3,alleles RYMV1-4 and alleles RYMV1-5),which confer antiviral properties that appear to be associated with point mutations or small deletions at amino acid sites[45,46].The second RYMV resistance gene identified is RYMV2,also known as Constitutive Expression of Pathogenesis-Related Gene 5-1(CPR5-1).RYMV2-mediated resistance is recessive and involves a nucleoporin-controlled defense mechanism conferred by a null allele identified in the O.glaberrima Tog7291 accession[50].Recently[51],RYMV3,a new resistance gene for RYMV,has been fine-mapped in African cultivated rice(O.glaberrima Tog5307),and it has been determined an NB-LRR as a very convincing candidate gene that may act in virus recognition.Screening a library of rice mutants inserted by Tos17 revealed a mutant named rim1-1 that did not exhibit typical disease symptoms after RDV infection.A cosegregation test showed that the rim1-1 mutation was caused by insertion of Tos17 in an intron of a novel NAC transcription factor.The loss of RIM1 function limits the reproduction rather than spread of RDV and does not cause the expression of defense genes or defense-related hypersensitivity reactions[52].It was accordingly suggested that RIM1-based resistance is unlikely to involve pathogen-associated molecular patterns(PAMPs)or immune responses mediated by R genes.

Overexpression of two bZIP transcription factors in rice reduced symptoms of RTBV infection[53].Two host transcription factors,RF2a and RF2b,activated RTBV promoter transcription by interacting with a cis-acting regulatory element,Box II.Rice cultivar Utri Merah is resistant to RTSV and is dominated by one or more genes[54,55].Correlation analysis of the antiviral traits and the phenotypic variation in backcross populations derived from Utri Merah and Taichung Native 1(TN1)showed that the resistance to RTSV in Utri Merah was controlled by a recessive gene(TSV1),which was mapped to a 200-kb(22.05 to 22.25 Mb)region of chromosome 7.TSV1 encodes a putative translation initiation factor 4G,and RTSV resistance is conferred by a single-nucleotide polymorphism(SNP)or codon deletion of Val1060-1061in exon 9 of the gene[56].RBDSV can infect maize and cause maize rough dwarf disease(MRDD)syndrome,which includes such symptoms as severe plant stunting,shortened internodes,and malformed tassels and ears.The P7-1 protein encoded by RBSDV hijacks the host susceptibility factor RabGDIαto form a potential trafficking complex to target viral plasmodesmata,promoting virus movement and spread from cell to cell and thereby accelerating virus infection[57].Inserting Helitron TE into ZmGDIαinduces alternative splicing to form a recessive resistance allele ZmGDIα-hel,and P7-1 has a weak binding affinity to it,resulting in low efficiency of the P7-1/RabGDIαhel transport complex.The P7-1/RabGDIα-hel complex confines RBSDV to the infected cells,thereby restraining the movement of the virus in the plant.This work identified the naturally occurring recessive gene ZmGDIα-hel,which directly targets virus transport,thereby conferring passive resistance to MRDD in maize[57].

2.3.Plant antiviral RNA silencing

RNA interference(RNAi)-mediated antiviral defense is one of the most conserved active defense mechanisms in plants[58-60]and plays a vital role in plant biological processes,including developmental time and pattern,transposon control,DNA methylation,and chromatin modification.When viruses invade plants,plants rely on viral double-stranded RNA (dsRNA) or selfcomplementary RNA to trigger a series of host antiviral immune processes.The host Dicer-like enzyme(DCL)recognizes the viral dsRNA and then cleaves it into 21-24-nucleotide(nt)small interfering(si)RNA or virus-derived siRNA(vsiRNA)[61].These siRNAs and vsiRNAs guide the assembly of a multiprotein effector complex,the RNA-induced silencing complex(RISC)[62,63].RISC mediates RNAi and inhibits the expression of target RNA.Argonautes(AGOs)are the catalytic components of RISC,binding to vsiRNA and mediating the transcription or posttranscriptional repression of target viral DNA or RNA through a sequence homology-dependent mechanism[64-66].vsiRNA or abnormal viral RNA can enter the RNA-Dependent RNA Polymerase(RDR)-mediated amplification cycle to enhance the antiviral silencing response[67-73].DCL,AGO,and RDR proteins are the core elements in the process of RNA silencing and play important roles in plant antiviral activity.There are four DCLs in Arabidopsis:DCL1,DCL2,DCL3,and DCL4[74].In contrast,8 DCLs have been identified in rice:DCL1a,DCL1b,DCL1c,DCL2a,DCL2b,DCL3a,DCL3b,and DCL4[75].The increased amount of virus-derived small interfering RNA(vsiRNA)in OsDCL2-knockdown plants leads to unstable maintenance of Oryza sativa endornavirus(OsEV)during cell division[76].Knockdown plants of rice DCL(DCL1,-2,-3a,-3b,and-4)are more susceptible to RSV infection than are wild-type plants,accumulating more RSV genomic RNA,coat protein(CP)gene transcripts,and RSV-driven vsiRNA[77].This finding indicates that DCL plays a key role in rice antiviral defense.In addition to cleaving target mRNA,AGOs inhibit translation and mediate DNA methylation.Ten AGOs have been identified in Arabidopsis.Among them,AGO1 is the primary antiviral effector in the plant RNA-silencing pathway.AGO2 and AGO7 participate in the antiviral process[71,78],and AGO3,AGO5,and AGO10 have weak antiviral function[79-81].In rice there are 19 AGO genes.AGO18 has been identified as having an antiviral function depending on its chelating effect on miR168,which can alleviate the inhibitory role played by AGO1 in antiviral RNAi[82].After RSV infects rice,AGO18 also isolates miR528 from AGO1,leading to the accumulation of miR528′s target gene L-a scorbate oxidase(AO)and initiating reactive oxygen species(ROS)-mediated resistance to RSV[83].Rice AGO1 is an effector of vsiRNA against RSV or RDV infection[83].AGO2 was also induced in rice plants infected with RSV[84],suggesting that these proteins function in rice antiviral defense.In the process of antiviral RNA silencing,host RDR contributes to the production of second-generation vsiRNA[85].The model plant Arabidopsis has six RDR genes and rice has five.The antiviral function of Arabidopsis RDRs(including RDR1,RDR2,and RDR6)has been demonstrated[86],but related research in rice is still in the early stages.Rice RDR1 and RDR6 support the defense response against RSV and RDV infection[87-89],and RDR6 expression was also induced by RNMV and Cucumber mosaic virus(CMV)infection[90].

Viral infection differentially affects the accumulation of small RNA(sRNA),thereby changing the pathogenicity of the virus or the resistance of the host[91].For example,RSV infection can induce the expression of miR444,-168,-394,-395,-398,-399,-160*,-171*,and-1425*and simultaneously cause the downregulation of miR528,-396,-156,-166,-167,and-171[92].These miRNAs participate to varying degrees in the interactions between rice and viruses.Viral infection leads to increased expression of miR168.Elevated miR168 expression appears to be a common mechanism of antiviral defense[84,93].The nonstructural protein 3(NS3)encoded by RSV interacts with DsRNA-binding protein 1(OsDRB1),a key component of the miRNA-processing complex.When NS3 interacts with OsDRB1,NS3 acts as a scaffold between OsDRB1 and primary miRNA(pri-miRNA)and facilitates pri-miRNA biogenesis.Taken together,these findings indicate a previously unknown mechanism by which the viral protein hijacks the pri-miRNA processing complex,leading to miRNA biogenesis and increasing viral infection and pathogenesis in rice[94].When RSV infects rice,AGO18 competes with AGO1 to bind miR168.This competition inhibits the autoregulation of AGO1 mediated by miR168,thereby increasing the accumulation of AGO1,enhancing host defense capabilities,and subsequently achieving antiviral function[82].In addition to miR168,AGO18 recruits several other miRNAs,including miR528,miR159a,and miR159b,in RSV-infected rice.miR528 reduces AO-mediated accumulation of reactive oxygen species by cleaving AO messenger RNA and negatively regulates rice virus resistance.After virus infection,AGO18 preferentially binds to miR528 and blocks its cleavage of target genes,resulting in increased AO activity,and the accumulation of basic ROS strengthens the antiviral defense[83].SPL9 showed high-affinity binding to specific motifs in the miR528 promoter region and activated the expression of the miR528 gene in vivo[95].SPL9-mediated transcriptional activation of miR528 represents a new regulatory layer in the miR528-AO antiviral defense pathway.In the absence of viral infection,the expression of OsRDR1 is inhibited by target genes of miR444.After virus infection,the expression of miR444 is upregulated,reducing the expression of its target genes.The OsRDR1-dependent RNA silencing pathway is then activated to protect rice from virus infection by silencing viral RNA and enhancing host gene defense[89].Transgenic rice plants overexpressing miR319a and miR319b exhibited disease-like phenotypes and increased sensitivity to RRSV.Further research[96]revealed that Teosinte branched/Cycloidea/PCF 21(TCP21)regulated by miR319 is a regulator of plant development and RRSV sensitivity in rice,and the increased sensitivity of rice to RRSV is due to the dynamic manipulation of the jasmonic acid(JA)pathway by miR319/TCP21.The overexpression of miR393 in transgenic rice has been observed[97]to inhibit the expression of auxin receptor transport inhibitor response 1(TIR1),thereby inhibiting the JA antiviral pathway activated by auxin and increasing the sensitivity of rice to RBSDV.Very recently[98,99],it was reported that the P3 protein encoded by RGSV can recruit an inducible E3 ubiquitin ligase,called P3 inducible protein 1(P3IP1),which targets and degrades host RNA polymerase IV in a UPS-dependent pathway,revealing a new virulence mechanism underlying rice and virus interaction.In rice and tobacco(Nicotiana benthamiana),ubiquitin like protein 5(UBL5)mediates the degradation of RSV P3 protein through the 26S proteasome,conferring resistance to RSV in plants[100].

2.4.Phytohormone signaling-mediated antiviral immunity

Phytohormones are plant signaling molecules[101].Auxin,ethylene,gibberellin(GA),cytokinin(CK),abscisic acid(ABA),brassinosteroid(BR),JA,SA,and strigolactones(SL)have been identified as primary plant hormones.These hormones are synthesized via different pathways and are sensed by receptor proteins,and subsequently initiate intracellular signal transduction[102].The complex network of plant hormone signals enables plants to activate effective defenses against viruses,thereby balancing the tradeoff between plant defense responses and plant growth.The most common symptoms of plant viruses infecting rice include severe stunting,dwarfing,excess tillering,and chlorosis.Although these types of symptoms have long been known to be associated with disturbances in plant hormone biosynthesis,accumulation,and perception,our understanding of the roles and mechanism of action of plant hormones in the formation of viral symptoms remains limited.Phytohormones are central in the plant’s defense against viruses,and multiple recent studies highlight emerging roles for hormones or their signaling crosstalk in rice-virus interactions.

Li Yi’s research group at Peking University discovered a new mechanism by which JA signaling and RNA silencing synergistically enhance rice antiviral defense[82,83].RSV CP triggers JA accumulation and upregulates JA in response to the transcription factor JAMYB to initiate host defense mechanisms.In the downstream network of JA signaling,JAMYB directly targets the promoter of AGO18 to activate AGO18 transcription.Excessive AGO18 can not only isolate miR168 to reduce the expression of AGO1[82]but also isolate miR528 to reduce AO expression[83].By these two paths,the resistance of plants to viruses is enhanced.As the initiator of JA-AGO18 signaling,RSV CP connects JA signaling and RNA silencing in tandem,highlighting a novel and important function of JA in balancing defense and growth in rice.The P5-1 protein encoded by RBSDV inhibits the ubiquitination activity of SCF E3 ligase by interacting with COP9 signalosome 5A(OsCSN5A),thereby hindering the deRUBylation of Cullin 1(CUL1),leading to inhibition of genes involved in JA signal transduction rather than biosynthesis,and ultimately enhancing RBSDV infection in rice[103].

The BR and JA signaling pathways engage in extensive and direct crosstalk during plant antiviral defense.Rice Glycogen synthase kinase 2(OsGSK2)is a key inhibitor of BR signal transduction.OsGSK2 interacts with OsJAZ4,a negative regulator of JA signaling and antiviral defense,recruits it as a substrate,and phosphorylates it.But OsGSK2 also disrupts the OsJAZ4-OsNINJA complex and OsJAZ4-OsJAZ11 dimerization by competitively binding to the ZIM domain.The dual effect of OsGSK2 on OsJAZ4 helps to promote the degradation of OsJAZ4 by the 26S proteasome and activates JA signaling to enhance the defense of plants against RBSDV[104].A high-throughput sequencing approach comparing the global gene expression of RBSDV-infected rice with that of healthy plants revealed that many defense-or stress-associated genes were upregulated,and the transcript numbers of the JA and BR pathways changed in opposite directions.The JA pathway was induced,whereas the BR pathway was inhibited.JA-mediated defense inhibited BR-mediated sensitivity to RBSDV infection,and this inhibitory effect depended on the JA cosensor OsCOI1[105].In another study[106],BR-mediated sensitivity prevented peroxidase-mediated oxidative bursts and suppressed the signals of plant defense hormones,such as JA and SA.Both BR and JA can positively regulate RSV resistance,and RSV infection inhibits the BR signaling pathway and increases the accumulation of OsGSK2[107].OsGSK2 interacts with OsMYC2(a key positive regulator of JA response)and phosphorylates it,thereby labeling it for proteasome-mediated degradation,which,in turn,reduces JA-mediated defense and promotes viral infection.This effect implies that BR-mediated RSV resistance requires a complete and active JA pathway.These results help to elucidate the crosstalk between plant hormones and viral hosts and deepen our understanding of RSV resistance mechanisms[107].

The role of ABA in plant immunity is divided into two stages.In the early stage of infection,ABA can positively regulate plant defense capabilities by inducing stomatal closure and callose deposition,while in the later stage of infection,it can impair plant immunity by antagonizing SA or JA signal transduction[108].Relatively few studies have investigated the function of ABA in mediating plant defense against viruses.The application of ABA increased callose deposition at plasmodesmata in tobacco and restricted the movement of viruses from cell to cell,thereby increasing resistance to Tobacco mosaic virus(TMV)[109,110].ABA-mediated resistance of Arabidopsis to Bamboo mosaic virus(BaMV)is achieved mainly by induction of the expression of AGO2 and AGO3[111].In RBSDV-infected rice plants,the expression of ABA pathway genes was significantly reduced.Exogenous hormone therapy and viral vaccination showed that ABA suppressed JA signaling.ABA also inhibits the accumulation of ROS by inducing the expression of peroxidase dismutase and catalase genes.In short,ABA inhibits the JA pathway and precisely controls the accumulation of ROS,thereby increasing the sensitivity of rice to RBSDV[112].

Auxin signaling plays a vital role in almost every aspect of plant growth and development.The P2 capsid protein encoded by RDV interacts with the rice Aux/IAA protein OsIAA10,increases the protein stability of IAA10,abolishes auxin signal transduction,and causes infected plants to show typical RDV symptoms,including dwarfing,excessive tillering,and stunted crown roots.This work indicates a new mechanism by which RDV reprograms auxin signaling,leading to increased viral infection[113].Most recently,Qin et al.[114]reported that RDV infection intensified auxin synthesis and accumulation,and exogenous auxin treatment reduced OsIAA10 protein abundance,thereby releasing its interacting protein OsARFs to activate downstream genes.However,the functions of these OsARFs in regulating rice antiviral defense are utterly different.This work clarifies a novel auxin-OsIAA10-ARFs-mediated signaling mechanism in the tradeoffs between rice and RDV for defense and counter defense responses[114].Manipulation of auxin signals by viral proteins is a common pathogenic strategy of plant RNA viruses.Both SP8 of SRBSDV and P2 of RSV interact with OsARF17 to promote viral infection,although in different ways.SRBSDV P8 interferes with auxin signaling by inhibiting the transcriptional activation activity of OsARF17 and interfering with OsARF17-OsARF17 dimerization.RSV P2 interacts with OsARF17 through the DNA-binding domain(DBD)and hinders the DNA binding of OsARF17.The RSMV Mprotein can also interact specifically with OsARF17 through the MR domain of OsARF17[115].These studies reveal that different plant viruses use independently evolved viral proteins to target key factors in auxin signaling with different strategies and manipulate auxin signals to achieve the goal of benefiting the virus itself.

There are relatively few studies of the involvement of ethylene in plant response to viral infections,and findings on the mechanisms are also scarce.Some studies[116-122]have found that ethylene can positively and negatively regulate host defences.A recent study[123]found that the Pns11 protein encoded by RDV interacts specifically with rice S-adenosine-L-methionine synthase 1(SAMS1),a key component of the ethylene synthesis pathway,stimulates and enhances the enzymatic activity of SAMS to trigger the accumulation of SAM,ACC and ethylene,increasing the susceptibility of virus-infected rice to RDV.This discovery indicates a new mechanism by which RDV manipulates ethylene biosynthesis in host plants to achieve effective infection.The RDV P2 proteininteracts with ent-kaurene oxidase(OsKO)in vivo,resulting in a decrease in the accumulation of endogenous active GA and causing RDV-infected rice plants to exhibit a dwarf phenotype[124].Rice OsRFPH2-10 is a Ring-H2 finger E3 ubiquitin ligase whose function is to interact with and degrade P2 protein through the 26S proteasome pathway and to raise an antiviral defense in the early stages of RDV infection[125].

The SA pathway plays an essential role in the basic resistance of plants to viruses and pathogens.The SA signal transduction pathway is crucial for the resistance of rice to RSV caused by overexpressing Hypersensitive Induced Reaction 3(OsHIR3)[126].The identified and obtained RSV-resistant rice R gene STV11,which encodes a sulfotransferase,catalyzes the conversion of SA to SSA[36].Thus,SA functions in antiviral defense in rice,but it is unknown how SA accumulation increases RSV resistance.

2.5.Autophagy-mediated antiviral immunity

Autophagy is,a conserved mechanism in eukaryotes,degrades cytoplasmic components and damaged organelles via the lysosomal pathway.Autophagy is essential for the survival,differentiation,development and homeostasis of animals and plants.There are 33 autophagy(ATG)genes in the rice genome[127],the expression of these genes is strictly regulated by hormones,abiotic or biotic stress,and nutrient restriction.The initiation of autophagy in plants is usually associated with increased ATG transcription,altered plant hormone content,or ROS accumulation[128,129].As a conserved antiviral mechanism,autophagy plays a very important role in the process of plant immunity.On the one hand,autophagy can activate the plant immune system and hinder virus proliferation,and autophagy-related factors NBR1,ATG6(Beclin-1)and ATG8f can act as cargo receptors and adaptor proteins that directly interact with the virus-encoded protein and mediate its degradation to achieve disease resistance[129-131].On the other hand,viruses can also block autophagy or induce incomplete autophagy to promote their own replication and increase pathogenicity[132,133].The tobacco calmodulin-like protein rgs-CaM can bind to the silencing suppressor protein 2b of CMV to transport it to autolysosomes for proteolysis[134].The host sacrifices rgs-CaM via the autophagy pathway to counterattack viral RNA silencing suppressors.Among the 11 viral proteins encoded by TuMV,viral genome-linked protein(VPg)is the only one to induce degradation of suppressor of gene silencing 3(SGS3)and its close partner RDR6 via the autophagy pathway.This finding indicates that the virus sabotages host antiviral RNA silencing to increase its own pathogenicity[135].Recently,Jiang et al.[136]screened the interacting protein of RSV silencing suppressor P3 in tobacco,identified an unknown function plant protein,and named it NtP3IP.Using the tobacco transient expression system,they found that NbP3IP harbors a special ability to target p3 to autophagy and degrade it by interacting with NbATG8f,thereby disrupting the RSV RNA silencing-suppression strategy.A similar mechanism is observed in rice.OsP3IP,a homolog of NbP3IP,also mediates the degradation of P3 and interacts with OsATG8b and P3.This work indicates a novel mechanism by which autophagy plays an antiviral role against negative single-stranded RNA viruses.The RSV movement protein NSvc4 binds to and interferes with the S-acetylation of group 1 remorin(REM1),thereby preventing its correct targeting to the plasma membrane and plasmodesmata,but stays in the endoplasmic reticulum(ER),triggering its autophagous turnover and thereby preventing the REM1-mediated negative regulation of viral cell-to-cell movement[137].

2.6.Wuschel(WUS)-mediated antiviral immunity

When plants are infected by viruses,most plant tissues lose resistance.However,there is one exception:the shoot apex meristem.Meristem tip culture can eliminate viruses in infected plants and is widely used to develop virus-free cultivars.Although this is the only approach in animals and plants that can completely eliminate viruses in living cells,the broad-spectrum antiviral mechanism covered by this biological phenomenon has long puzzled researchers.Very recently,Wu et al.[138]found that the detoxification area of the stem tip tissue is consistent with the distribution of stem cells.Stem cells in plants continuously supply daughter cells to form new organs,and may have unknown functions in protecting cells from biological invasion.Arabidopsis stem cells can protect the apical meristem and its new progeny cells from CMV infection.The stem cell regulator WUS responds to CMV infection and inhibits virus accumulation in the center and surrounding area of the meristem.WUS inhibits the synthesis of viral proteins by inhibiting the expression of plant S-adenosyl-L-methionine-dependent methyltransferase(SAM-Mtase),which is involved in ribosomal RNA processing and ribosomal stability.This discovery solves a problem that has plagued researchers for many years and provides a molecular basis for the WUS-mediated broadspectrum inherent antiviral immunity of plants.

3.Rice breeding strategy for virus resistance

To cut the chain of virus transmission,insect control is still the main strategy for controlling viruses in rice production,but chemical methods to control viruses are not sustainable strategies.The most economical,efficient,and environmentally friendly method is to improve the defense system of rice itself and develop and popularize superior virus-resistant cultivars.Here we summarize the latest advances in the interaction between rice and virus in the anti-virus defense network(Fig.1;Table 1),and provide gene reserves and new ideas for rice anti-virus breeding.With the further development of genetic engineering and sequencing technologies,some resistance breeding strategies can gradually be realized.1)Wild rice germplasm resources have long been grown in natural environments and contain high genetic diversity,which is the material basis for identifying and deploying desirable genes.This strategy involves screening,identifying,and evaluating wild rice germplasm resources,identify anti-virus genes,sequence existing identified but not yet fine-mapped or cloned resistance genes,and use biotechnology to create new rice germplasm.2)Based on RNAi strategy,one approach is to fine-tune the nucleotide sequence of miRNA and another is to guide virus-derived single or multiple tandem dsRNAs into rice to interfere with the replication of viruses in plants and generate new rice germplasm with stable inheritance of multi-virus resistance.3)This strategy uses vector-insect antiviral genes to conduct genetic engineering research,interfere with the replication and transmission of viruses in insects,and cultivate antiviral vector insects.4)A new antiviral strategy is to feed insects with artificially synthesized virus dsRNA to induce RNAi-mediated silencing,thereby depriving insects of their ability to carry and transmit viruses.5)In Arabidopsis,WUS-mediated stem cell virus immunity has a broad spectrum of resistance.Because it interferes with the processing of rRNA and the stability of ribosomes,the virus cannot use plant cells to complete its own protein translation as well as replication and assembly,inhibiting its proliferation and spread.As a conservative and broad-spectrum antiviral strategy,WUS-mediated antiviral mechanism is promising[139].In rice,WUS can be transformedinto a virus-inducible protein expressed specifically in the phloem that directly halts virus replication in the early stage of virus infection,providing an important gene reserve for the cultivation of new virus-resistant germplasm.

Fig.1.Schematic diagram of the regulatory network of rice and virus interaction.

Table 1Summary of host-virus interactions in rice.

This review describes multiple antiviral mechanisms that have evolved during the long struggle between rice and viruses,including R gene-,recessive gene-,RNAi-,hormone-,autophagy-,and WUS-mediated antiviral pathways.The detailed regulatory route is described in the main body of the review.CMV,Cucumber mosaic virus;RBSDV,Rice black streaked dwarf virus;RDV,Rice dwarf virus;RGSV,Rice grassy stunt virus;RHBV,Rice hoja blanca virus;RRSV,Rice ragged stunt virus;RSMV,Rice stripe mosaic virus;RSV,Rice stripe virus;RTBV,Rice tungro bacilliform virus;RTSV,Rice tungro spherical virus;RYMV,Rice yellow mottle virus;SRBSDV,Southern rice black-streaked dwarf virus;ABA,abscisic acid;BR,brassinosteroid;ETH,ethylene;GA,gibberellins;JA,jasmonic acid;SA,salicylic acid;SSA,sulfonated SA;AO,L-ascorbate oxidase;ARF17,auxin response factor 17;ATG8,autophagy 8;BSV,black streaked dwarf resistance;DRB1,DsRNA-binding protein 1;GSK2,glycogen synthase kinase 2;GSV,gray stunt resistance;HBV,hoja blanca virus resistance;IAA10,indoleacetic acid-induced protein 10;JAZ,jasmonate-ZIM-domain protein;KOs,ent-kaurene oxidase;MTase,methyltransferase;NRPD1a,nuclear RNA polymerase D1a;P3IP1,P3 inducible protein 1;RDR1/6,RNA-dependent RNA polymerase 1/6;RIM1,rice dwarf virus multiplication 1;ROS,reactive oxygen species;RYMV1/2/3,rice yellow mottle virus resistance 1/2/3;SAMS1,S-adenosine-L-methionine synthase 1;SPL9,squamosa promoter binding protein-like 9;STV-a,stripe disease resistance-a;STV-b,tripe disease resistance-b;STV-bi,stripe disease resistance-b-I;TCP21,TCP domain protein 21;TIR1,transport inhibitor response 1;TUV-a/b,tungro resistance-a/b;WUS,Wuschel.

The CRISPR/Cas gene editing system,which was born in recent years,has the advantages of simple operation and high efficiency and has become one of the most advanced systems for crop genome engineering.A combination of modern breeding methods will play an important role in the improvement of major crops,as follows:1)CRISPR/Cas technology can reduce the time and cost of breeding new cultivars and accelerate the breeding process through gene knockout,knock-in,substitution,point mutation,tuning of gene expression,and mutation of multiple genes in the same transformation event.It can also be used for antiviral breeding and high-throughput mutant library construction;2)CRISPR/Cas can yield non-GMO mutant plants in the first generation and can also be used for precise excision of selectable marker genes in transgenic rice,eliminating biological safety concerns and improving breeding technology;3)CRISPR/Cas-mediated gene knockout technology can produce male-sterile lines and speed the process of hybrid breeding;4)The CRISPR/Cas system provides a defense mechanism that can lyse invading bacteria and DNA and RNA viruses and endow plants with antiviral properties;5)The CRISPR/Cas system accelerates the domestication of wild rice,thereby fostering new domesticated rice crops with stronger tolerance.Considering the rapid development and iterative updating of CRISPR/Cas technology,it is expected that the field of rice antivirus research will also be expanded,also bringing revolutionary changes to rice anti-virus breeding.

4.Conclusions and prospects

During the interaction between virus and host,rice usually shows severe growth defects,such as dwarfing,tillering,and sterility.The arms race between the host and the virus is an evolvingprocess involving multiple layers of interaction,in which miRNA biogenesis and hormonal signals are the main targets for the immune mechanism competition between the virus and the rice.sRNA acts as an important regulator of plant growth by controlling the expression of key developmental and stress-related genes.Virus infection changes the accumulation of sRNA in the host plant,causing the proportions of different sRNAs to change.RSV,RBSDV,and RRSV promote virus pathogenicity by affecting miRNA biogenesis.The host can also change the content of miR444,miR528,miR168,and vsiRNA to resist RSV and RDV infection.For hormone signals,it acts as a chemical messenger during the whole life process of plants from germination to senescence.RDV,RSV,SRBSDV,RSMV manipulate auxin signals to achieve the goal of benefiting the virus itself.RDV can also manipulate ethylene and GA signals to increase virus pathogenicity.However,plants do not sit still waiting for death.The antiviral immunity of plants can regulate JA,BR,and SA signals to resist RSV and RDV infection.Viruses have different infection patterns and reproduction methods than other pathogens,and the antiviral mechanisms of plants have their own peculiarities.Here,we have discussed recent advances and emerging trends in multiple antiviral defense networking(Fig.1;Table 1),and summarized identified R genes and recessive genes with antiviral functions in rice(Table 2).Research on plant antiviral mechanisms can not only deepen our understanding of the interaction between plants and viruses but also help to elucidate the entire plant signal transduction system,a goal with potential application value.Given China’s abundant rice seed resources,the country has clear advantages in research on rice antiviral mechanisms.Especially in recent years,with the rapid development of life sciences and genome sequencing technology,many R genes in rice have been identified,and many novel antiviral mechanisms have been discovered,but many basic questions still have not been answered.These questions include the following:1)Many R genes in rice have repetitive sequences,but their function and evolutionary significance are unclear.In fact,many antiviral R genes in rice have been located within a certain range of the genome interval,but because the corresponding varieties have no genome resequencing,it is difficult to isolate these antiviral genes.2)The nature and mechanism of intermittent outbreaks of rice virus disease warrant further research.3)RNA silencing signal moves between cells,from the local infection site to the remote tissue,which is beneficial from the sRNA shuttle.sRNAs are short in length and small in size,making them flexible and diffusible.The sRNAs in plants are derived not only from the host itself but also from the interacting species.However,the mechanism by which sRNAs move between cells,tissues,and species is unknown,and we need to investigate multiple sRNA interaction networks to identify their potential functions;4)Rice virus cannot infect rice offspring through seeds.Whether there is a powerful antiviral mechanism in the seed,similar to the wus-mediated antiviral network,remains to be investigated in the future.6)To date,no virus receptors have been found in rice.This fact may be the key to the realization that viruses do not rely on vector insects to infect rice,and awaits research attention.

Table 2List of identified R genes and recessive genes in rice.

CRediT authorship contribution statement

Lu WangandShuai Zhangwrote the manuscript;Huiting Xie,Xiaoyuan ZhengandJiasheng Chengathered data;Jianguo Wuconceptualized,supervised and financed this work,and edited the manuscript.All authors contributed to the revisions.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Natural Science Foundation of China(32025031,U1905203,31772128,and 32072381),the Fok Ying Tung Education Foundation(161024),and the Outstanding Youth Research Program of Fujian Agriculture and Forestry University(xjq202003).