Junjie Yin ,Lijun Zou ,Xiobo Zhu ,Yuyn Co ,Min He ,*,Xuewei Chen ,*

a State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China,Sichuan Agricultural University at Wenjiang,Chengdu 611130,Sichuan,China

b Ecological Security and Protection Key Laboratory of Sichuan Province,Mianyang Normal University,Mianyang 621000,Sichuan,China

Keywords:Blast fungus Resistance mechanism Immunity Rice breeding

ABSTRACT Global food security is threatened by rice blast disease caused by the filamentous fungus Magnaporthe oryzae.An understanding of rice resistance mechanisms is fundamental to developing strategies for disease control.In this review,we summarize recent advances in pathogen-associated molecular patterntriggered immunity,effector-triggered immunity,defense regulator-mediated immunity,and effects of nutrient elements on rice blast resistance.We outline strategies used for breeding rice cultivars with improved disease resistance.We also present the major research challenges for rice blast disease resistance and propose approaches for future investigation.

1.Introduction

The pathogenic fungus Magnaporthe oryzae is the causal agent of rice blast disease[1],considered the severest threat to rice production worldwide.It is responsible for approximately 30%loss of rice production globally[2].Since 2013,the average annual incidence area of rice blast in China has exceeded 75 million ha(https://www.natesc.org.cn/).To prevent the outbreak of blast disease,a‘‘one-vote veto”for breeding rice with blast resistance has been implemented in rice cultivars certification in China since 2008.This means that rice cultivars showing poor blast disease resistance will not get official certificate to allow for selling as seeds to farmers for growing.It is generally accepted that identification and deployment of disease resistance genes from rice germplasm resources is the most economical and eco-friendly strategy for controlling blast disease.However,the complexity and variability of M.oryzae lead to large differences in the population structure of pathogen isolates among rice-growing regions.In consequence,even popular blastresistant cultivars are suitable for growing only in limited regions and only for a few years.

The genome sequences of rice and M.oryzae are now both available,and both species are genetically tractable.Because of these advantages,the rice blast disease has been used as a model system for studying plant-microbe interaction[3].In the past decade,great progress has been achieved in understanding how rice recognizes infection by M.oryzae and initiates resistance responses.To summarize up-to-date information in this research area,we describe recent advances in the study of molecular mechanisms of rice blast resistance and discuss breeding strategies for improving rice disease resistance.

2.Enemy in sight:Recognition of pathogen-associated molecular patterns(PAMPs)by rice membrane-associated pattern recognition receptors(PRRs)

2.1.PRRs recognize PAMPs

To defend itself against blast invasion,rice employs a twolayered innate immune system.PAMP-triggered immunity(PTI)forms the first layer of immunity,and is boosted after PAMP recognition by membrane-associated PRR on the host cell membrane[4].Chitin is an integral building block of the fungal cell wall and is a well-known type of PAMP capable of activating plant immune responses[5].Chitin from M.oryzae is recognized by rice transmembrane LysM receptor-like proteins(LysM-RLPs),including two lysin motif-containing proteins,OsLYP4 and OsLYP6,and a chitin elicitor binding protein(CEBiP)[6].When defending against M.oryzae,rice forms a receptor complex called LysM-RLPs-OsCERK1 which composes LysM-RLP(either OsLYP6,OsLYP4,orCEBiP)and Chitin Elicitor Receptor Kinase 1(OsCERK1)[7](Fig.1).This receptor complex then senses and transmits the fungal chitinderived signal.Knockdown of either CEBiP,OsLYP4,or OsLYP6 reduces blast disease resistance,demonstrating the importance of the chitin-sensing receptor complex in regulating defense response[8].Two rice Receptor like kinases(RLKs),Flagellin Sensing 2(OsFLS2)and BRI1-Associated receptor Kinase 1(OsBAK1),are also involved in PTI[9,10].

2.2.Transmission of PRR signals

After perception of fungal chitin by the LysM-RLPs-OsCERK1 receptor complex,rice disseminates the chitin-induced signal into two pathways to initiate a PTI response.One pathway is dependent on receptor-like cytoplasmic kinases(OsRLCKs),and the other is dependent on OsRacGEF1.In the OsRLCKs-dependent pathway,OsRLCK185 transmits immune signal from OsCERK1 to a mitogenactivated protein kinase(MAPK)signaling cascade by directly phosphorylating OsMAPKKK18 and OsMAPKKKε,independently promoting a OsMKK4-OsMPK3/OsMPK6 cascade to trigger a resistance response[11,12].These reports reveal that a phospho-signaling pathway in rice links intracellular activation of the MAPK cascade to cell surface chitin perception(Fig.1).Whether other OsMAPKs take part in PAMP-mediated defense activation is unknown.

The OsRacGEF1-dependent pathway also functions in the intracellular dissemination of chitin-induced signal.Upon chitin elicitation,OsCERK1 rapidly activates downstream OsRacGEF1[13].Activated OsRacGEF1 then stimulates the activity of Rac/Rop GTPase (OsRac1)which associates with OsMKK4-OsMPK3/OsMPK6.The bHLH transcription factor Rac Immunity1(RAI1)is then phosphorylated by OsMPK3/OsMPK6.The activated RAI1 finally induces expression of OsWRKY19 and Phenylalanine Ammonia Lyase 1(OsPAL1)and other defense-associated genes to increase rice resistance to M.oryzae[14].In addition,activated OsRac1 can induce the generation of H2O2and biosynthesis of lignin by interacting with Respiratory burst oxidase homologue B(OsRbohB)and Cinnamoyl-CoA Reductase 1(OsCCR1),respectively,for boosting PTI responses to prevent M.oryzae infection[15](Fig.1).

3.Ultimate weapon:Rice nucleotide-binding site leucine-rich repeat(NLR)proteins launch a counterattack called effectortriggered immunity(ETI)to fight the enemy

3.1.Avirulence and NLR genes

To achieve successful infection,virulent M.oryzae isolates have evolved a strategy to secrete effectors into the rice cell for subverting PTI,leading to effector-triggered susceptibility(ETS)[16].In the ETS process,effectors such as Slp1,MoChia1,MoHTR1 and MoHTR2,interfere with host immunity and promote invasion of pathogens by changing the structure or function of host defenseassociated regulators(Fig.1).For instance,Secreted LysM protein 1(Slp1),containing two LysM domains,competes with CEBiP for binding chitin,thereby suppressing chitin-induced plant immune responses[16].Chitinase 1(MoChia1)competitively binds the rice tetratricopeptide repeat protein OsTPR1 with chitin,and then inhibits the chitin-triggered rice immune response[17].Two further effectors,MoHTR1 and MoHTR2 are delivered into the rice nucleus to abolish host immunity via transcriptional reprogramming of host immune-responsive genes[18].

To combat a blast fungus capable of subverting PTI,rice deploys nucleotide-binding site leucine-rich repeat(NLR)proteins to rec-ognize the effectors named avirulence(AVR)proteins.Several AVR proteins have been cloned,including AVR-Pita,AVR-Pi9,and Avr-Pizt[19-21].Recognition of AVR by NLR promotes strong immune responses referred to as effector-triggered immunity(ETI),which arms rice with a second layer of protection in case of disabled PTI[22](Fig.1).To date,24 AVR genes have been mapped in M.oryzae,and 12 of them have been cloned[23].Except for Avirulence Conferring Enzyme 1(ACE1),the cloned AVR genes all encode secreted proteins.Nine AVR genes have been reported to be paired with corresponding NLR genes(Table S1).

Fig.1.Rice innate immunity signaling pathways triggered by M.oryzae.(1)On the rice cell membrane,chitin perception by LysM-RLP-OsCERK1 complex initiates a receptor-like cytoplasmic kinase(OsRLCK)-dependent pathway in pathogen-associated molecular pattern(PAMP)-triggered immunity(PTI).OsRLCK185 directly interacts with and phosphorylates OsMAPKKK18 and OsMAPKKKεto activate a mitogen-activated protein kinase(MAPK)cascade.(2)Upon chitin perception,the LysM-RLP-OsCERK1 complex also elicits the OsRacGEF1-dependent pathway.Phosphorylated OsRacGEF1 activates OsRac1,which then induces the generation of H2O2 and biosynthesis of lignin by interacting with Respiratory burst oxidase homolog B(OsRbohB)and Cinnamoyl-CoA Reductase 1(OsCCR1),respectively.(3)The rice blast fungus secretes effectors,such as Chitinase 1(MoChia1)and Secreted a LysM protein 1(Slp1),into the rice cell to subvert PTI,resulting in the emergence of effector-triggered susceptibility(ETS).(4)Alternatively,secreted effectors called avirulence(AVR)proteins are recognized by rice nucleotide-binding site leucine-rich repeat(NLR)proteins,leading to a particularly strong immune response referred to as effector-triggered immunity(ETI).(5)The integrated decoy model:the interaction between NLR and AVR requires one AVR protein and two coordinated NLR proteins.(6)Pit employs its CC domain to bind to OsSPK1 for activating OsRac1 and induction of cell death.(7)In ETI,OsRac1 is required for Pb1,Pid3-mediated blast resistance.(8)NLR proteins mediate defense response by direct interaction with transcription factors.

In recent years,blast-resistance genes have been studied systematically.In rice,34 NLR genes have been cloned(Table S1).Most NLR genes in rice are constitutively expressed,but the expression of Pi5-1[24],Pi63[25],and Pb1[26]occurs in response to blast infection.NLR genes are located primarily in gene clusters.For example,the Pi2/Pi9 locus comprises five broad-spectrum resistance genes:Pi50,Pi2,Pigm,Pi9 and Piz-t[27].Clustering of NLR genes is considered to facilitate their frequent homologous recombination for evolving new NLR genes.Among the reported broad-spectrum resistance genes,Pigm is of particular interest,because it encodes a pair of antagonistic NLRs:PigmR and PigmS.Although PigmR endows rice with broad-spectrum blast resistance,it reduces grain yield[28].PigmS competitively attenuates PigmR homodimerization to suppress broad-spectrum blast resistance,but increases grain yield by promoting seed setting[28].The entire Pigm locus thus guarantees an appropriate balance between immunity and yield[28].In addition to the Pi2/Pi9 locus,Pikh[29],Pita[30],Pib[31],Pizh[32],Pi64[33],Pi1[34],and Pi5[24]separately confer broad-spectrum resistance against M.oryzae.These NLR genes have been extensively employed in breeding blast-resistant rice cultivars.

Resistance(R)genes encode typical NLR proteins in rice,with the exception of three:Pi-d2,pi21,and Ptr(Table S1).Pi-d2,cloned from the cultivar Digu as a single-copy gene,encodes aβ-lectin receptor kinase conferring race-specific resistance to M.oryzae isolate ZB15[35].A single amino acid difference at position 441 of Pi-d2 distinguishes resistant and susceptible alleles of the blast-resistance gene Pi-d2[35].pi21 is a recessive gene and encodes a proline-rich protein containing a putative heavy metal-binding domain and putative protein-protein interaction motifs[36].Ptr encodes a constitutively expressed armadillo(ARM)repeat domain protein localized mainly in the cytoplasm.It regulates broad-spectrum blast resistance mediated by the NLR gene Pi-ta and Pi-ta2[37].

3.2.Molecular mechanisms of AVR recognition by NLR protein

Three types of molecular interactions have been identified for NLR-AVR pairs.The first type is direct interaction,also known as the‘‘direct interaction model”.In rice blast resistance,the best example of the direct interaction model is the case of NLR Pi-ta and its cognate AVR effector AVR-Pita[19].Pi-ta and AVR-Pita directly interact to activate a resistance response.The leucinerich domain(LRD)of Pi-ta binds specifically to the AVR-Pita176protein.Mutation in either the AVR-Pita176protease motif or Pi-ta LRD disrupts the interaction and consequently abolishes disease resistance[38].

The second interaction type is called the‘‘integrated decoy model”[39].The most-studied model of this type is the case of the two rice NLR proteins composing RGA4 and RGA5[40](Fig.1).RGA4 and RGA5 interact to form heterodimers that can detect the M.oryzae effectors AVR-Pia or AVR-CO39[41].RGA4 is able to induce effector-dependent cell necrosis in rice,but its induction capability is inhibited by RGA5.RGA5 contains a conserved heavy metal-associated(HMA)domain,which is essential for binding two different effectors,AVR-CO39 and AVR-Pia[42].RGA5 is also a necessary component in pathogen recognition[43].The direct interaction of RGA5 with AVR-CO39 or AVR-Pia releases the inhibition of cell death mediated by RGA4,thereby triggering a resistance response[41].

The third interaction type is an indirect model that involves signaling molecules such as Piz-t and AvrPiz-t.Twelve AvrPiz-tinteracting proteins(APIPs)have been shown to associate with AvrPiz-t[44].Among these APIPs,the Bowman-Birk trypsin inhibitor(BBI)protein APIP4 can independently bind to either AvrPiz-t or Piz-t.When binding with AvrPiz-t,APIP4 is inhibited in its trypsin activity to facilitate infection by M.oryzae.The binding of APIP4 with Piz-t potentially promotes the activity of APIP4 in increasing rice immunity[45].APIP5 interacts with AvrPiz-t via its bZip DNA-binding domain,and is important for the stability and accumulation of Piz-t[46].The rice APIP6 encodes a RING E3 ubiquitin ligase.AvrPiz-t can block ubiquitin ligase activity of APIP6 to suppress rice PTI[44].

Several other APIPs are involved in regulation of Piz-tdependent immunity.APIP10 promotes the degeneration of Piz-t via the 26S proteasome system,and knockdown of its expression results in accumulation of Piz-t and autoimmune cell death[47].APIP10 also interacts with two transcription factors:Vascular plant One-Zinc finger 1(OsVOZ1)and OsVOZ2,to promote their proteasomal degradation[48].OsVOZ1 has transcriptional inhibitory activity,whereas OsVOZ2 has transcriptional activation activity.OsVOZ1 has the potential to trigger the transcriptional activation activity of OsVOZ2[48].OsVOZ1 and OsVOZ2 synergistically negatively regulate rice cell death and basal resistance,but positively regulate ETI response mediated by the NLR protein Piz-t[48].The rice APIP12 takes part in basal resistance against M.oryzae and is a virulence target of AvrPiz-t[49].OsAKT1,a rice plasmamembrane-localized K+channel protein,also interacts with AvrPiz-t to suppress OsAKT1-mediated K+currents.Loss of function of OsAKT1 reduces K+content and resistance against M.oryzae[50].

3.3.Intracellular signal transmission after NLR recognition of AVR

Despite the cloning of several NLR and AVR genes,it remains largely unknown how NLR proteins transduce defense signals to downstream NLR.Recent studies suggest that the N-terminal coiled-coil(CC)domains of NLR function as a platform for relaying defense signals by interacting with downstream elements.For example,the transcriptional activator RAI1 can associate with the CC domain of NLR protein PID3 to modulate blast resistance[51].In Pit-mediated immunity,a DOCK-family GEF protein OsSPK1 serves as a direct signaling target of Pit.Because of its ability to bind OsSPK1,the CC domain of Pit is required for OsRac1 activation and induction of cell death[52](Fig.1).The CC domain of PigmR also interacts with a RNA-recognition motif(RRM)transcription factor,PigmR-Interacting and Blast resistance Protein 1(PIBP1)[53].The majority of rice NLR proteins belong to the CC-NLR category.This fact,together with the similar molecular mechanism of PID3,Pit,and PigmR,suggests that rice NLR proteins have evolved a conserved mechanism whereby they employ CC domains to associate with downstream regulators for transmitting defense signals and initiating blast disease resistance.

For blast resistance mediated by NLR protein,the small GTPase OsRac1 functions in the signaling pathway by associating with different NLR proteins to form an immune complex[54].For example,OsRac1 is required for Pia-mediated blast resistance[55].It also participates in Pit-mediated disease resistance by functioning as a downstream molecular switch of Pit to control the generation of reactive oxygen species(ROS)as well as progression of cell death[56].PID3 also induces RAI1 expression via OsRac1 to confer blast resistance[51](Fig.1).

Upon the perception of effectors by rice NLRs,rapid activation of immune signaling events occurs to inhibit invasion by thepathogen.Many transcription factors are involved in this process[57].For example,PIBP1 is capable of directly activating expression of rice defense genes OsWAK14 and OsPAL1 after interaction with the NLR protein PigmR,thereby establishing a link between transcriptional activation of immune responses and pathogen perception mediated by NLR [53].The NLR protein RPM1-Like Resistance gene 1(OsRLR1)also mediates defense response by direct interaction with the transcription factor OsWRKY19 in the nucleus[58](Fig.1).

4.Combat heroes:Defense regulators(DR)guarantee effective signal transduction to ensure rice plant survival of blast attack

Rice disease resistance conferred by NLR genes is often overcome within three to five years,owing to the fast-evolving and highly variable nature of the blast fungus[59].In contrast to NLR genes,DR genes usually confer partial but durable resistance to multiple M.oryzae isolates.

4.1.Signaling transduction

DR genes can activate various signaling pathways,such as MAPK cascades and the ubiquitination-mediated pathway,as well as hormonal signaling.Upon activation by extracellular stimuli,MAPKs transmit signals from the cell membrane to the nucleus,acting in defense against M.oryzae[60].For instance,by controlling the expression of PR genes and the accumulation of ROS,both OsMAPK5 and OsMAPK15 influence rice blast resistance[61,62].Other protein kinases,such as RLKs and calcium-dependent protein kinase (CDPKs),are also involved in shielding rice from M.oryzae infection.Two rice cell wall-associated kinases,OsWAK25 and OsWAK91,are necessary for broad-spectrum resistance against M.oryzae[63,64](Fig.2).OsCPK4 negatively regulates innate immunity of rice.Activation of the OsCPK4-OsRLCK176 phosphorylation circuit increases plant immunity by disabling OsRLCK176 degradation machinery[65].

A key role in blast disease resistance is played by the rice ubiquitination-mediated pathway[66].The E3 ubiquitin ligase Enhanced Blight and Blast Resistance 1(EBR1)directly targets Bcl-2-Associated athanoGene 4 (OsBAG4)for ubiquitinationmediated degradation.The EBR1 and OsBAG4 module orchestrates innate immune homeostasis and balances growth and defense in rice[67].The rice U-box/ARM E3 ubiquitin ligase OsPUB15 undergoes direct interaction with the receptor-like kinase PID2 and positively regulates programmed cell death to increase blast disease resistance[68].Another two E3 ubiquitin ligases,Spotted Leaf 11(SPL11),and Cullin3a(OsCUL3a),also confer broad-spectrum resistance against M.oryzae [69,70](Fig.2).

Transcription factors(TFs)are also involved in defense against infection by M.oryzae.Of particular interest are broad-spectrum resistance Digu 1(bsr-d1)and Ideal Plant Architecture 1(IPA1).The gene bsr-d1 was identified in the durably resistant cultivar Digu,and encodes a C2H2-type TF.Compared to susceptible rice cultivars,Digu contains a single-nucleotide substitution in the bsr-d1 promoter that confers broad-spectrum blast resistance without significant yield penalty.Another TF,MYBS1,acts as a repressor of bsr-d1 expression and is able to bind to the bsr-d1 promoter.This binding reduces the expression of bsr-d1 and peroxidase genes,thereby inhibiting H2O2degradation to increase resistance against blast disease[71](Fig.2).

IPA1 is the first TF identified as increasing both blast resistance and yield of rice.In response to blast infection,IPA1 phosphorylation occurs rapidly and alters its DNA-binding specificity.Phosphorylated IPA1 preferentially binds to promoter of the defense gene WRKY45 and activates its expression to increase disease resistance.Within 48 h of infection,IPA1 returns to a non-phosphorylated state,resuming support for the growth required for high yield[72](Fig.2).Several WRKY TFs,including OsWRKY45,OsWRKY30,OsWRKY62,and OsWRKY76,also regulate blast resistance in rice[73-75].In a recent study[76],blast resistance mediated by bsr-d1 was reported to associate with a MYB TF,OsMYB30.Binding of OsMYB30 to promoters of either 4-coumarate:coenzyme A ligase 5(Os4CL5)or Os4CL3 activates their expression,elevating the accumulation of lignin subunits G and S.This in turn thickens sclerenchyma cells near the epidermis,hindering the penetration of M.oryzae and increasing disease resistance.

4.2.Downstream immune responses:ROS burst and accumulation of antimicrobial compounds and hormones

4.2.1.ROS burst

Lesion-mimic mutant(LMM)genes are the main DR genes capable of activating immune responses such as ROS bursts.Lesionmimic mutants,including spl30-1[77],spl33[78],spl35[79],lmm24[80],and spl-D[81],usually show increased disease resistance.Several other DR genes can confer similar blast resistance by initiating ROS bursts.For example,SPL11 cell-Death Suppressor 2(SDS2)is a ubiquitination substrate of SPL11(an E3 ubiquitin ligase comprising an armadillo repeat domain and a U-box domain).SDS2 interacts with OsRLCK118/176 and phosphorylates OsRbohB,and then induces a ROS burst,resulting in increased resistance to M.oryzae[82].

The light-harvesting complex II protein LHCB5 is important for broad-spectrum resistance of rice.Its phosphorylation increases resistance via accumulation of ROS within the chloroplast[83].MicroRNAs(miRNAs),a category of 20-24-nucleotide small noncoding RNAs,also function in rice blast defense response by regulating ROS signaling[84].For example,miR398b fine-tunes ROS production via four superoxide dismutase(SOD)members to mediate rice immunity[85].miR164a targets OsNAC60 and negatively regulates rice blast defense response.Suppressing miR164a expression results in the induction of defense responses including ROS accumulation[86](Fig.2).miR168 controls immunity by targeting Argonaute 1(AGO1),a major component of the RNA-induced silencing complex[87].The miR168-AGO1 module regulates the expression of miR1320 and miR164 to improve immunity.The RNaseIII enzyme Dicer-like 1(OsDCL1)also acts in rice blast disease resistance by processing microRNA biogenesis.There is a negative feedback loop between OsDCL1 and miR162a in rice.Silencing of OsDCL1 leads to accumulation of H2O2and cell death,consequently activating basal resistance against rice blast[88].

4.2.2.Hormone-mediated defense responses

Hormones are another class of regulators involved in rice blast defense response.Suppressor of Salicylic acid Insensitivity-2(OsSSI2),OsSec3a(a principal subunit of the exocyst complex in rice),OsAAA-ATPase 1 all mediate resistance by modulating salicylic acid(SA)signaling[89-91].JA-resistant 1(OsJAR1)and JA-responsive MYB(OsJAMyb)are associated with jasmonic acid(JA)signaling,and determine rice blast disease resistance[92,93].Numerous rice DR genes control blast resistance by regulating both SA and JA signaling,including rice Class III acyl-CoA-binding proteins-5(OsACBP5),OsALDH2B1,and Sucrose non-fermenting-1-related protein kinase-1(OsSnRK1a)[94-96].

Indole-3-acetic acid(IAA),ethylene(ET),and gibberellin(GA)participate in rice resistance against the blast fungus.GH3-2,encoding an IAA-amido synthetase,controls defense responses bysuppressing pathogen-induced IAA accumulation[97].The ethylene response factor (ERF) gene OsERF83 and the 1-aminocyclopropane-1-carboxylic acid synthase gene OsACS2 confer blast resistance by affecting ET signaling[98,99].GA 20-oxidase-3(OsGA20ox3)mediates resistance by affecting GA biosynthesis[100].The Abscisic Acid 2(ABA2)mutation in rice leads to increased disease resistance and cell death[101].

Fig.2.Defense regulator-mediated immunity.(1)Rice cell wall-associated kinases(OsWAKs)on the rice plasma membrane are involved in triggering defense response.(2)Various E3 ubiquitin ligases regulate defense response by modifying specific substrates with ubiquitination for proteasomal degradation.(3)miRNAs regulate rice defense response via ROS signaling.(4)Phosphorylated Ideal Plant Architecture 1(IPA1)binds to the WRKY45 promoter and increases blast resistance.MYBS1 binds tightly to the broad-spectrum resistance Digu 1(bsr-d1)promoter containing a natural G allele,whereby the expression of bsr-d1 is inhibited to impede expression of peroxidase genes for increasing ROS accumulation and broad-spectrum blast resistance.(5)Lesion resembling disease 6-6(Lrd6-6)regulates the expression of phytoalexin biosynthetic genes or the accumulation of antimicrobial compounds to inhibit infection by pathogens.(6)Phosphorylated light-harvesting complex II protein LHCB5 increases resistance by promoting ROS within the chloroplast.(7)Other defense regulators(DR),such as Cell Death and Susceptible to blast 1(CDS1)and Broad-spectrum resistance kitaake-1(Bsr-k1),also confer resistance to rice blast.

4.2.3.Accumulation of antimicrobial compounds

In the early stage of infection by pathogens,rice accumulates antimicrobial compounds as a defense response.For example,cyanide contributes to rice resistance by restricting fungal growth[102].Bayogenin 3-O-cellobioside confers cultivar-nonspecific defense against the rice blast fungus[103].Diterpenoids are a major group of antimicrobial phytoalexins in rice,and their role in rice disease resistance has been indicated[104]by functional analysis of a diterpenoid gene cluster(DGC7)located on rice chromosome 7.

Some DR genes contribute to inhibiting pathogenic infection by regulating the expression of phytoalexin biosynthetic genes or the accumulation of antimicrobial compounds.For example,rice ETHYLENE INSENSITIVE 2(OsEIN2)likely activates phytoalexin production upon M.oryzae infection to promote resistance[105].A rice homolog of mammalian Selenium-Binding Proteins(OsSBP)positively regulates defense gene expression and enables phytoalexin accumulation[106].Lesion resembling disease 6-6(Lrd6-6)encodes an AAA-type ATPase and regulates the MVB-mediated trafficking pathway[107].Owing to inhibition of this pathway,the lrd6-6 mutant exhibits elevated biosynthesis of antimicrobial compounds and is therefore increased in resistance against M.oryzae[107](Fig.2).

4.3.Other DR genes

Many other DR genes confer rice resistance to blast disease.Broad-spectrum resistance kitaake-1(Bsr-k1),encoding a tetratricopeptide repeat(TPR)-containing protein,negatively regulates broad-spectrum disease resistance.Loss of function of the Bsr-k1 gene promotes accumulation of OsPAL1-7 mRNAs and increases basal immunity,thereby improving resistance against diverse isolates of M.oryzae[108].Cell Death and Susceptible to blast 1(CDS1),encoding a cyclic nucleotide-gated channel protein OsCNGC9,mediates PAMP-induced Ca2+influx and positively regulates rice blast disease resistance[109](Fig.2).Syntaxin of Plants 121 protein(OsSYP121)is accumulated at fungal penetration sites and functions in rice blast resistance[110].Rice copine genes OsBON1 and OsBON3 act as negative regulators of blast disease resistance.The expression levels of OsBON1 and OsBON3 and their protein subcellular localization likely contribute to balancing immunity and agronomic traits[111].

5.Effects of nutrient elements on rice blast resistance

Excessive or deficient supply of nutrients,such as nitrogen,phosphate,potassium,and silica,affects stress response and can potentially influence rice disease resistance.For instance,nitrogen partially breaks down rice blast resistance triggered by the Pi1 gene[112],and excessive nitrogen increases rice susceptibility to pathogen infection.Phosphate Transporters-8(OsPT8)modulates transduction of Pi signaling for negatively regulating rice immunity[113].As a result of miR399f overexpression,Pi content in rice leaves increases,but the expression of defense genes decreases,leading to weakened resistance to M.oryzae[114].Potassiumis also associated with rice blast resistance[115].For example,M.oryzae can disrupt rice immune response by regulating host K+channels[50].

Silicon nutrition can mitigate various biotic stresses[116].On one hand,silicon acts as a physical barrier against plant disease.On the other hand,silicon boosts the plant’s defense by functioning as a biological inducer[117].For instance,silicon-induced defense response and cell silicification of leaves both contribute to rice blast disease resistance[118].In M.oryzae-infected rice plants,silicon-enhanced blast disease resistance is also associated with an increase in photochemical efficiency and adjustment of mineral nutrient absorption[119].Despite the known effect of silicon on rice resistance against M.oryzae,the molecular mechanisms remain poorly understood.

6.Resistance breeding

In the past few years,great progress has been made in the conceptual science of the rice blast disease resistance.The conceptual discoveries have helped breed many new rice cultivars with excellent resistance,such like F-you498,Yixiangyou 2115,Zhongke 804,and Longliangyou 3189[28,120-122].Owing to R genes’strong effects and their ease of selection,rice breeders have relied largely on transferring R genes.However,loss of R gene-mediated rice resistance is frequently observed after several years of planting,limiting the application of single R genes in breeding.To overcome this limitation,breeding new cultivars with broad-spectrum resistance is needed.

Pyramiding multiple R genes by DNA marker-assisted selection is one of the strategies for achieving broad-spectrum resistance.Tian et al.[123]introduced three rice blast resistance genes(Pi-33,Pi-2,and Pi-1)from the indica cultivar Zhongjin 23 into Kongyu 131 by this approach.The resulting Kongyu 131 cultivar carrying all three blast resistance genes displays better resistance to different blast isolates than cultivars carrying only one or two.A combination of R genes and quantitative trait loci(QTL)is another strategy for obtaining durable resistance.A rice line with multiple blast-resistance QTLs,including pi21,Pi34,qBR4-2,and qBR12-1,shows broad-spectrum and stable resistance against M.oryzae[124].

Fig.3.Multiple strategies for improving rice blast disease resistance.There are two main strategies for effective control of blast disease resistance:breeding elite rice varieties and developing suitable measures for cultivation and management.These two strategies are interdependent and mutually beneficial,in a way resembling the Chinese Yin-Yang or Tai-Chi diagram.For breeding elite rice varieties,it is necessary to survey rice germplasm resources,clone broad-spectrum resistance(BSR)genes,dissect the genetic basis of resistant accessions,characterize the crosstalk between resistance and other physiological processes,and design approaches for molecular breeding.For developing measures of cultivation and management,it is necessary to monitor the dynamics of M.oryzae population structures for rationally deploying rice cultivars with corresponding resistance genes,study effects of nutrition and light on disease resistance,exploit efficient cultivation measures,and identify eco-friendly antimicrobial compounds.

A cross-species transgenic approach can be exploited to achieve broad-spectrum resistance.Overexpressing the Arabidopsis NLR genes RPS2 and RPM1 in rice improved broad-spectrum resistance to M.oryzae[125].Host-induced gene silencing has also been employed to increase rice broad-spectrum blast resistance,as shown by the increased resistance of a rice line expressing RNAhairpins targeted to knock down the M.oryzae transcription factor MoAP1[126].Genetic editing of DR genes is useful for broadspectrum resistance against M.oryzae,as shown by the genetic editing of pi21 leading to rice broad-spectrum blast resistance[127].

7.Concluding remarks and perspectives

Upon invasion by M.oryzae,rice PRRs initiate PTI defense response by recognizing PAMPs,while NLRs initiate ETI by recognizing avirulence effectors.A series of defense-regulator genes also function in transduction of immunity-associated signal.Despite these achievements,there is still a large gap between the theoretical discoveries and the practical needs for controlling rice blast disease.At present,we still have limited understanding of the mechanism of interaction between NLR and AVR.Our understanding of the manner in which NLR activates ETI downstream resistance response upon sensing pathogens remains limited.We have yet to understand the dynamic changes in the dominant pathogenic isolates of M.oryzae and to elucidate the molecular mechanism of durable broad-spectrum rice blast resistance.

The main challenges are as follows:(1)systematic identification of resistant rice accessions by study of worldwide collections from diverse climates and elucidation of the genetic basis of their resistance.(2)systematic study of the molecular mechanisms of disease resistance,including the coordinated regulation of disease resistance by PTI and ETI,and crosstalk between disease resistance and physiological processes such as dynamic cell wall reconstruction,nutrient adaption,circadian clock,and light response;(3)application of modern biotechnologies including CRISPR for rational and precise improvement of disease resistance;(4)development of eco-friendly antimicrobial compounds for rice blast disease control.

To meet these challenges,we suggest intensifying blast resistance research on these topics(Fig.3).First:survey and create new germplasm with broad-spectrum disease resistance and clone the corresponding resistance genes.It has been confirmed that high immunity can coexist with high yield in rice.To accelerate breeding rice with high disease resistance and yield,it is desirable to identify or create more genetic resources,to clone new disease resistance genes or genetic loci from these resources.Second:characterize the mechanisms regulating broad-spectrum disease resistance and the crosstalk between disease resistance and other physiological processes,thereby establishing a theoretical framework for prevention and control of rice blast.Third:investigate the dynamic population distribution and changes of rice blast fungus prevalence in diverse rice growing regions,and optimize the corresponding combinations of rice resistance genes in breeding by precise control of resistance gene expression using modern gene editing technology.Fourth:characterize the nutritional demands and light adaptation features of rice,design cultivation measures suitable for diverse ecological areas and rice cultivars,and identify environment-friendly antimicrobial compounds as ecologically safe measures for controlling rice blast disease.

CRediT authorship contribution statement

Min HeandXuewei Chenconceived the article and revised the manuscript.The original draft was prepared byJunjie YinandLijuan Zou,Xiaobo ZhuandYuyan Caodrew figures and collected data for tables.All authors read and approved the final manuscript.

Declaration of competing interest

Authors declare that there are no conflicts of interest.

Acknowledgments

We thank Prof.James C.Nelson from Kansas State University for kindly revising the manuscript.This work was supported by the National Natural Science Foundation of China(NSFC)(32072041)to J.Yin;the NSFC(31825022)to X.Chen;the NSFC(31871920)to M.He;the NSFC(32072407)to X.Zhu;and the NSFC(31972258)to L.Zou.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.03.009.