Functional Analysis Of Maize Lipoxygenase ZmLOX9 In Defense Response Mediated By Rp1-D21

As one of the three major food crops in the world,maize is an indispensable source of food for humans,but the invasion of pathogens leads to a decline in maize yield and quality.Therefore,understanding the molecular mechanism of plant disease resistance and cultivating new varieties with high disease resistance have become an important way to solve global food security problems.The plant disease resistance mechanisms are mainly divided into two major levels,pathogen-associated molecular pattern(PAMP)-triggered immunity(PTI)and effector-triggered immunity(ETI).As the major type of R proteins,NLR(Nucleotide binding leucine rich repeat)proteins play important roles in plant disease resistance.Maize NLR protein Rpl-D21 is derived from an intragenic recombination between two NLRs,Rpl-D and Rpl-dp2.Rpl-D21 confers auto-active ’lesion mimic ’phenotype in maize.And it can induce HR(hypersensitive response)when transiently expressed in N.benthamiana.By crossing Rp1-D21 into the maize nested association mapping population,a genome-wide association study identified 44 single-nucleotide polymorphisms(SNPs)locus highly associated with variation in the severity of Rpl-D21-induced HR.One SNP located at 16578218 of chromosome 1 is located in the third intron of a gene coding maize lipoxygenase ZmLOX9.From our previous RNA-seq analysis,we identified that ZmLOX9 was significantly up-regulated in Rp1-D21 mutant compared to cognate wild type.Therefore,we investigated the funciont of ZmLOX9 in Rpl-D21-mediated HR and also in disease resistance to different pathogens.First,we transiently co-expressed agrobacteria containing ZmLOX9 and Rpl-D21 in the leaves of N.benthamiana,and found that ZmLOX9 enhanced Rpl-D21-mediated HR,but had no effect on Rpl-D or Rpl-dp2 to induce HR.ZmLOX9 itself was not capable of producing autonomous HR.ZmLOX9 did not enhance the HR phenotypes mediated by other autoactive NLRs MLA10(D502V)and RPM1(D505V),and it also had no obvious effect on INF-or BAX-mediated HR.This indicated that ZmLOX9 might specifically enhance Rpl-D21-mediated HR.ZmLOX9 belongs to the lipoxygenase class,and mutation of the amino acids responsible for its putative enzyme activity did not change the phenotype of enhancing Rp 1-D21-mediated HR,suggesting that the enzyme activity may be independent of its function of enhancing Rp1-D21-mediated HR.ZmLOX9 contains two important domains;N-terminal LH2 and C-terminal LOX domains.We further found that the LH2 domain enhanced Rpl-D21-mediated HR while the LOX domain had no obvious phenotype.The enzyme activity center of ZmLOX9 is located in the LOX domain,so we further verified that the enzyme activity may be independent of its ability to enhance Rpl-D21-mediated HR.To investigate where ZmLOX9 function in plant cells,we performed the subcellular localization experiments.We found that the full length of ZmLOX9 and the LH2 domain were mainly localized in chloroplast,while the LOX domain was mainly localized in the cytoplasm.Removing the predicted chloroplast transitpeptide(cTP)from the LH2 domain(LH2ΔcTP),it was localized in the cytoplasm and nucleus,and not in the chloroplast.LH2 ΔcTP did not enhance Rpl-D21-mediated HR.Therefore,the function of ZmLOX9 to enhance HR may be related to its chloroplast localization.To investigate whether ZmLOX9 is involved in maize disease resistance,we inoculated ZmLOX9 mutants with the corn smut pathogen Ustilago maydis physiological race SG200 and the stem rot pathogen Fusarium verticillioides.The results showed that the ZmLOX9 mutants had no obvious disease-resistant phenotype for corn smut,but they were more sensitive to F.verticillioides compared to wild type.This suggests that ZmLOX9 might play important roles in resistance to stalk rot in maize.Based on the above results,we concluded that ZmLOX9 played a positive role in enhancing Rpl-D21-mediated HR and its chloroplast localization is important for its function.ZmLOX9 also acted in resistance to F.verticillioides which caused stalk rot in maize.Therefore,ZmLOX9 is a potential candidate gene for maize diseae resistance.