| Cassava is one of the three major potato species in the world.It is widely cultivated in tropical and subtropical regions and is the main food crop for low-income people in the tropics.Moreover,cassava is recognized as a renewable energy crop with great potential in the world.Cassava bacterial blight is a serious disease that affects the yield of cassava.So far,there are only a few genes which have been reported involved in disease resistance in cassava.Therefore,it is of great significance to develop cassava resistance-related genes to improve cassava yield.Melatonin plays multiple roles in plants,including flowering time,flowering,seed germination,root development and quantity,fruit ripening,senescence and respond to stress.In this study,we isolated melatonin synthesis-related genes at cassava,analyzed their expression profiles under different treatments,and studied their functions and signal networks.Based on our experiments,we obtained the following results.Firstly,7 melatonin synthesis genes were systematically analyzed in cassava.Quantitative real-time PCR showed that all these genes were commonly regulated by flg22 and Xanthomonas axonopodis pv.manihotis(Xam).Then,the coding regions of the 7 melatonin synthesis genes were connection into GFP in pEGAD vector.Transient expression in Nicotiana benthamiana revealed the subcellular locations and possible roles of these melatonin synthesis genes.Notably,transient expression of these genes had significant effects on the transcripts of defense-related genes,triggered the burst of callose depositions and papillae-associated plant defense and suppressed the growth of Xam,indicating the possible role of them in plant innate immunity.Moreover,we found overexpression of tryptophan decarboxylase 2(MeTDC2),N-acetylserotonin O-methyltransferase 2(MeASMT2)interacting proteins,and N-acetylserotonin O-methyltransferase 3(MeASMT3)in tobacco leaves resulted in more melatonin than when other enzymes were overexpressed.Through yeast two-hybrid,11 MeTDC2-interacting proteins,21 MeASMT2 interacting proteins,and 9 MeASMT3-complex proteins were identified.Notably,we highlighted MeWRKY20 and MeWRKY75 as common complex proteins of the 3 enzymes,as evidenced by yeast two-hybrid,and in vivo bimolecular fluorescence complementation(BiFC).Moreover,co-overexpression of MeTDC2,MeASMT2,MeASMT3 with MeWRKY20,MeWRKY75 in cassava leaf protoplasts activated the transcriptional activities of MeWRKY20 and MeWRKY75 on W-box.Additionally,we found that MeWRKY79 and heat shock transcription factor 20(MeHsf20)targeted the W-box and the heat-stress elements(HSEs)in the promoter of MeASMT2,respectively.The interaction between MeWRKY79,MeHsf20,and the MeASMT2 promoter was evidenced by the activation of promoter activity and chromatin immunoprecipitation(ChIP)in cassava protoplasts,and by an in vitro electrophoretic mobility shift assay(EMSA).The transcripts of MeWRKY79,MeHsf20,and MeASMT2 were all regulated by flg22 and Xam.In common with the phenotype of MeASMT2,transient expression of MeWRKY79 and MeHsf20 in N.benthamiana leaves conferred improved disease resistance.Through virus induced gene silencing(VIGS)in cassava,we found that MeWRKY79-and MeHsf20-silenced plants showed lower transcripts of MeASMT2 and less accumulation of melatonin,which resulted in disease sensitivity that could be reversed by exogenous melatonin.Taken together,these results indicate that MeASMT2 is a target of MeWRKY79 and MeHsf20 in plant disease resistance.Finally,gene expression assays showed that the transcripts of MeRAVs were commonly regulated by Xam and MeRAVs were specifically located in plant cell nuclei.Through virus-induced gene silencing(VIGS)in cassava,we found that MeRAV1 and MeRAV2 are essential for plant disease resistance against cassava bacterial blight.Through VIGS in cassava leaves and overexpression in cassava leave protoplasts,we found that MeRAVl and MeRAV2 positively regulated melatonin biosynthesis genes and the endogenous melatonin level.Further investigation showed that MeRAVl and MeRAV2 are direct transcriptional activators of 3 melatonin biosynthesis genes in cassava.Taken together,we identified 7 cassava melatonin biosynthesis genes and analyzed their functions in plant disease resistance.Screening the interacting proteins of some synthetic genes and identifying the upstream transcription factors will significantly extends the knowledge of melatonin signaling and complex regulation of melatonin in plant defense.The results will provide theoretical basis and candidate gene resources for the resistant varieties breeding of cassava. |
