| Poplar leaf blight caused by Alternaria alternata leading to the early leaf fall of diseased trees and affects their normal growth.Seriously,it may cause seedling death and premature aging of trees,resulting in economic losses.However,the molecular mechanism of poplar response to Alternaria infection is unclear,which hinders the process of tree disease resistance genetic engineering breeding.In this study,the transcriptome of poplar in response to A.alternata infection were constructed,the main signal pathways and metabolic pathways of poplar in response to A.alternata infection were identified,the genes involved in the resistance of A.alternata infection were also screened.Then,the function of transcription factor PdbMYB85 was analyzed through transgenic and various molecular biotechnology.The results are helpful to further reveal the molecular mechanism of poplar resistance to leaf blight,and provide a theoretical reference for the application of forest disease resistance genetic engineering.The transcriptome of Populus davidiana × P.bolleana in response to A.alternata infection was constructed,a total of 5,930 differentially expressed genes(DEGs)were obtained.GO analysis showed that these DEGs were mainly involved in "secondary metabolic process","defense response to fungus","plant-type cell wall","integral component of plasma membrane","beta-glucosidase activity" and "glucosidase activity".KEGG pathway analysis showed that DEGs were mainly concentrated in "plant hormone signal transduction" pathway(ko04075),"phenylpropane biosynthesis" pathway(ko00940)and "MAPK signal transduction" pathway(ko04016).In addition,genes encoding defense related proteins and related to reactive oxygen species(ROS)metabolism and a large number of transcription factor(IF)genes,such as b HLH,WRKY and MYB families,were also differentially expressed by A.alternata.All above suggesting that,these pathways and genes maybe involved in poplar in response to pathogen infection.Genes involved in jasmonic acid(JA)biosynthesis and signal,were continuously upregulated in response to Alternaria infection,such as LOX,MYC and COI.Among which the expressions of PdbLOX were induced during the infection,suggesting that it maybe involved in poplar in response to pathogen infection.Therefore,the function of PdbLOX was further analyzed by loss-and gain-function of it.The phenotypes,physiological and biochemical indexes of wild-type(WT)and transgenic plants after inoculation with A.alternata were analyzed.The results showed that disease spots on the leaves of over-expressed PdbLOX plants were significantly smaller than those of WT,while the disease spots on inhibiting PdbLOX plants were significantly larger than those of WT.Meanwhile,over-expressed PdbLOX in poplar increased contents of JA,expressions of JA related gene,accumulation of ROS and activities of antioxidant enzymes.In conclusion,PdbLOX gene is involved in the resistance of P.davidiana×P.bolleana to A.alternata infection.The promoter sequence of PdbLOX was cloned and analyzed.The results showed that the promoter sequence of PdbLOX gene contained a variety of cis elements related to plant disease resistance.The upstream TF PdbMYB85,which may regulate PdbLOX gene,was caught by yeast-one hybrid(Y1H).The regulatory relationship between PdbMYB85 and PdbLOX was further verified by Y1 H,dual-luciferase reporter assay(LUC)and chromatin immunoprecipitation assay(Ch IP).The 831 bp PdbMYB85,coding 276 aa,was R2R3 type MYB TF。The PdbMYB85 was located in the nucleus and had transcriptional activation activity.RT-q PCR showed that PdbMYB85 was induced by exogenous Me JA treatment and A.alternata infection.The transgenic plants of PdbMYB85 were obtained,and phenotypes,physiological and biochemical indexes of WT and transgenic plants inoculated with A.alternata were analyzed.The results showed that after inoculation with A.alternata,the lesion area on the leaves of plants overexpressed PdbMYB85 gene were significantly smaller than that of WT,while the lesion area on the leaves of plants inhibiting PdbMYB85 were larger than those of WT.Meanwhile,overexpressed of PdbMYB85 induced the expression of PdbLOX and genes related to JA signal,enhanced LOX enzyme activity,increased JA content.However,these physiological indexes were the opposite in inhibiting PdbMYB85 plants.In conclusion,PdbMYB85 participates in the resistance of P.davidiana×P.bolleana to A.alternata infection by positively regulates the expression of PdbLOX,as well as activates the expression of JA related pathway genes.In brief,the mainly ways of poplar in response to A.alternata infection,were phenylpropane biosynthesis and JA/ET signal transduction pathway.And the PdbMYB85 participates in the resistance of P.davidiana × P.bolleana to A.alternata infection by positively regulates the expression of PdbLOX,which then activates the expression of JA signal pathway genes. |
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