| As the largest grain crop in China,maize plays an important role in the national economy and people’s livelihood.Plant diseases often reduce maize yield and affect maize quality.Therefore,it is very important to identify effective maize resistant genes and cultivate maize varieties that are resistant to disease and with high yield.Nucleotide binding,leucine-rich-repeat(NLR)protein is one of the major classes of resistance(R)proteins for plants to defend against pathogen infection.The recognition between NLR proteins and their cognate pathogen effectors usually triggers a rapid localized cell death at the pathogen infection site,termed hypersensitive response(HR).Maize Rpl-D21 is an NLR protein,which is produced by the other two NLR proteins Rpl-D and Rp1-dp2 through the intragenic recombination.Rpl-D21 is an automatic immune mutant,and its transient expression in tobacco can also generate HR.In addition,the phenotype of Rp1-D21 is influenced by genetic background,temperature and light.In the early stage,multiple genes regulating Rpl-D21-mediated HR,such as HCT and CCoAOMT,were found by GW AS(Genome-wide association study)and RNA-seq analysis,and the functions of multiple genes were verified by functional analysis.Therefore,Rpl-D21 can be used as an effective tool to explore maize resistance defense response genes and study its molecular mechanism.2-oxoglutarate-dependent dioxygenases(2OGDs)are a class of enzymes,which involved in secondary metabolism and signaling molecule synthesis in plants.2OGDs are extensively distributed in land plants.It is a superfamily,depending on the enzyme activity,it can be divided into flavonols synthase(FLS),anthocyanins synthetase(ANS),flavone synthase(FNS),Flavanone 3-hydroxylase(F3H)and others.Flavone synthase Ⅰ(FNSI)is one of the key enzymes in flavonoid biosynthesis pathway,which catalyzes the formation of flavanones into flavones.There are two types of flavone synthase.Flavone synthase Ⅰ(FNSI)is a soluble ferrous ion/2ketoglutarate-dependent dioxygenase,while Flavone synthase Ⅱ(FNSII)is an oxygen and NADPH-dependent cytochrome P450 monooxygenase.We major in the type Ⅰflavone synthase(ZmFNSIs)of maize.Its homologous gene AtDMR6/AtS5H in arabidopsis thaliana also showed the activity of salicylic acid 5-hydroxylase(S5H).A close homolog of AtDMR6/AtS5H,AtS3H,has SA 3-hydroxylase(S3H)activity.AtDMR6/AtS5H and AtS3H play important roles in plant innate immunity.However,the underlying molecular mechanism and the relationship between FNSI/S5H and S3H with the NLR-mediated HR are not known in any plant species.In this study,we identified three genes encoding ZmFNSI-1,ZmFNSI-2 and ZmS3H that are significantly up-regulated in an autoactive NLR Rp1-D21 mutant.Functional analysis showed that ZmFNSI-1 and ZmFNSI-2,but not ZmS3H suppress Rpl-D21-and its signaling domain CCD21-mediated HR when transiently expressed in N.benthamiana,however,they have no obvious effect on cell death caused by other elicitors.ZmFNSI-1 and ZmFNSI-2 physically interact with CCD21 by coimmunoprecipitation and yeast two hybrid assays.Furthermore,we showed that ZmFNSI-1 and ZmFNSI-2 interact with HCT,HCT,a key enzyme in lignin biosynthesis pathway,which can also suppress Rpl-D21-mediated HR,suggesting that ZmFNSI may form complexes with HCT and play a role in Rpl-D21-mediated HR.To investigate the function of ZmFNSIs in maize disease resistance,we obtained their mutants and over-expressed lines and inoculated them with Fusarium gramineae and Fusarium verticillioides.The preliminary results indicated that ZmFNSIs as negative regulators to play a role in resistance to maize stem rot.This provides new genetic resources for maize disease resistance breeding in the future. |
