Functional Study Of 9-LOXs Pathway Mediated Resistance To Stalk Rot/Ear Rot In Maize

Maize has become the No.1 crop species in China since 2012.However,the planting area and yield have to be reduced due to the overstock and the reformation of supply-side structure,thus it becomes more important to improve efficiently the yield and quality of maize in the future.On the other hand,however,ear rot and stem rot caused by Fusarium graminearum and the ear rot caused by Aspergillus spp.have become one of the major disease problems that result in signifcant yield loss in maize production and quality reduction in recent years;besides,both F.graminearum and A.flavus also produces various mycotoxins that are one of the most threatened agents to human and livestock health.Thus,screening and identifying the new genetic resources and exploring the novel genes involved in the disease resistance will not only help us to understand the molecular mechanism underlying the resistance gene-mediated immunity to maize stalk rot and ear rot,but also facilitate the molecular breeding of maize varieties with enhanced resistance,thereby the higher yield and quality.It has been shown that lipoxygenases(LOXs)pathway and the oxylipin pathway were significantly activated by F.graminearum in the resistant varieties of wheat;meanwhile,there were numerous studies that had shown the ultimate product of 13-LOXs pathway,jasmonates(JAs),plays essential role in regulating the plant resistance to Fusarium spp.,yet,very little is known about the function of 9-LOXs pathway and 9-oxylipins in the resistance to Fusarium spp..Despite of this,it has been found that the 9-LOX gene ZmLOX3 might be hijacked by F.verticilliodes to facilitate their pathogenicity,thus functioning as a negative regulator in maize immunity,suggesting that 9-LOXs pathway might also play an important role in maize resistance to stem rot and ear rot caused by Fusarium spp..To address whether 9-LOXs pathway indeed play roles in the resistance to Fusarium spp.,also to understand 9-LOXs and 9-oxylipin pathways-mediated maize resistance to stem rot and ear rot caused by F.graminearum,a combination of genetic,biochemical,transcriptomic and bioinformatic approaches were deployed in this study to investigate the response of 9-lox mutants to infection of F.graminearum,the differential gene expression between WT and 9-lox mutants upon infection of the fungi,as well as identification of the interacting proteins of 9-LOX,ZmLOX5.The results showed that maize genome encodes 13 LOX genes,which are distributed differentially on six chromosomes,each of which proteins has three functional domains.Upon infection,9-lox mutants including lox3-4,lox5-3,and lox4-10 displayed enhanced resitance to both stem rot caused by F.graminearum compared to WT,However,9-lox mutant kernels displayed contrast response to the ear rot caused by F.graminearum and Aspergillus flavus,respectively.Moreover,transcriptomics analysis by RNA-seq in B73 and 9-lox mutant lox5-3 showed that large set of differentially expressed gene(DEG)are identified,some of which might be involved in the resistance to F.graminearum;GO cluster analysis of those DEGs indicated that these genes are mainly involved in plant stress response,lipid metabolism,defense response,and cell formation,etc..Finally,protein-preotein interaction study by yeast-2-hybrid(Y2H)provided numberous candiate genes that might interact with ZmLOX5.Interaction of several candidate genes,scarecrow-like protein 8(GRMZM2G043737),acid phosphatase 1(GRMZM2G103526)and Membrane-binding protein(GRMZM5G882228)with ZmLOX5 were confirmed by BiFC.In summary,our findings showed that maize 9-LOXs pathway might play a negative role in the resistance of maize to stem rot and ear rot caused by Fusarium graminearum,which could be achieved through regulating diverse resistance genes and signaling pathways.However,more extensive experiments are required to address the exact molecular mechanisms mediated by 9-LOXs pathways.