Functional Identification Of ZmFAH,One Of Interactors Of ZmFBL41 For Banded Leaf And Sheath Blight Resistance In Maize

Previously,a resistance gene ZmFBL41 had been cloned from 318 maize inbred lines with genome-wide association analysis.In inbred line B73,ZmFBL41 encodes a F-box protein which negatively regulates the maize resistance to banded leaf and sheath blight?BLSB?.ZmFBL41 is a key components of E3 ubiquitin ligase complex?Skpl-Cullins-F-box,SCF?which mediates the degradation of cinnamyl alcohol dehydrogenase of ZmCAD in susceptible maize B73,subsequently blocks the synthesis of lignin to decrease its accumulation.However,in resistant maize inbred lines,such as Chang7-2,ZmFBL41^Chang7-2,has two key amino acid substitutions which makes it lost the targeting ability to the specific substrate and the degradation of ZmCAD,resulted in failed blocking the synthesis of lignin and the accumulation of lignin to enhance disease resistance.Meanwhile,during the screening the SCF^FBL41 substrate protein with LRR of ZmFBL41^B73 as bait by using yeast two hybrid?Y2H?,in addition to the ZmCAD identified above,other candidate interaction proteins,such as a fumarylacetoacetate hydrolase?ZmFAH?,have been also screened out.In order to fully analyze the mechanism of ZmFBL41-mediated BLSB resistance,we have further identified the protein interaction,the degradation and biological function of ZmFAH in the process of resistance to BLSB in this study.The main results was obtained as the following listed.1.The interaction between the LRR domain of ZmFBL41 and ZmFAH have been validated by using point-to-point experiments of Y2H,pull down and co-immunocoprecipitation.2.In vitro and in vivo degradation experiments showed that ZmFBL41 can mediate the degradation of ZmFAH protein.And this degradation can be inhibited by the ubiquitin inhibitor MG132.Different from the interaction of ZmFBL41-ZmCAD,the resistant allele ZmFBL41^Chang7-2 still interacts with ZmFAH.3.Homozygote test and disease resistance analysis were carried out on the mutant zmfah of Uniform Mu transposon insertion line.Compared to wild type W22,the zmfah was more susceptible after inoculated with YWK196 for 3 days,suggesting that ZmFAH might positively regulate the resistance to BLSB.4.There was no significant difference in the expression of ZmFAH before and after inoculation,indicating that the expression of ZmFAH was not induced by BLSB pathogens.Overall,we have identified an novel interaction protein and ubiquitination substrate of ZmFBL41 protein that was enlarged the ZmFBL41-mediated BLSB resistance mechanism in this study.Furthermore,ZmFAH may still be degraded by ZmFBL41^Chang7-2hang7-2 in resistant materials to negatively regulate the disease resistance,implying that it is possible to further enhance the BLSB resistance by editing the ZmFAH in maize.