Function And Mechanism Of BrERF11b In Defense To Spodoptera Litura And Myzus Persicae

Spodoptera litura is a species of leaf-chewing insect with the characteristics of polyphagia and gluttony,and aphids are sap-sucking insects.They both cause substantial damage to many crops worldwide.ERF transcription factors function in plants to regulate their growth and development,resistance to biotic stresses and resistance to abiotic stresses.ERF transcription factors that enhance plant resistance to insects are few,and the molecular mechanism is rarely studied.There is no report on ERF transcription factor which enhances plant resistance to sap-sucking and leaf-chewing insects.ERF11-like was obtained from Chinese cabbage via RT-PCR and was termed BrERFl1b.Its function in defense to Spodoptera litura and Myzus persicae was studied and its molecular mechanism was analyzed.The plasmid pBI121-BrERF1lb was constructed and transformed into tobacco plants(Nicotiana tabacum cv.K326 and N.benthamiana)by the Agrobacterium-mediated transformation method.The function of the BrERF11b gene in the resistance of the transgenic plants to Myzus persicae and S.litura was assayed.The results showed that the weights of the S.litura larvae fed the transgenic plant line were significantly reduced from 28 to 76%compared to those of the S.litura larvae fed the control plants(wild type),and the transgenic plant line KLine12 significantly reduced the survival rate of S.litura by 62.5%.The colony growth of the apteral aphids fed the transgenic plant line was significantly reduced from 22.5 to 63.4%compared to that of the apteral aphids fed the control plants.Compared to control plants,the transgenic plant line significantly reduced the survival rate of the aphids from 46 to 55%.These results first indicate that the BrERF11b gene significantly enhances plant resistance to S.litura and M.persicae.By transcriptome sequencing,RT-qPCR verification and plant hormone level analysis,we found that the transcription factor BrERF11b promoted JA synthesis by activating the transcription of JA synthetase genes,and activated JA/ET signaling pathway.They jointly regulate the expression of protease inhibitor genes,thus enhancing the resistance of plants to the leaf-chewing pest Spodoptera litura.On the other hand,BrERF11b positively regulates the concentrations of jasmonate(JA),salicylate(SA)and ethylene,activates the expression of Harpin-induced protein gene Hin1 and MYB transcription factor gene NbMYB4,and inhibits the expression of β-1,3-glucanase,which is related to callose deposition in phloem.These results suggest that BrERF11b may positively regulate resistance of plants to aphids in a way similar to that induced by Harpins protein HrpNEa.By conducting extensive ChIP-seq,bioinformatic analyses,integrative analysis of ChIP-seq and RNA-seq data and EMSA,we revealed the genome-wide binding landscape of BrERF11b in Nicotiana benthamian.BrERF11b showed a binding preference for the vicinityof the transcription start sites(TSSs),which is expected for a transcription factor.We found that BrERF11b regulates its downstream genes through preferentially binding to the GCCbox-like motif(VCGCCGCC).We designed the probes according to sequences of peak summits to conduct EMSA,and demonstrated NbNIMIN2,NbTAF15b and NbERF4 were indeed the target genes of BrERF11b.The two target genes,NbNIMIN2 and NbTAF15b,are related with plant insect resistance,and are potential insect resistant genes.NbERF4 is not only a direct target gene of BrERF11b,but also belongs to the subfamily Ⅷa(B-la)of ERF family,and has an EAR(ERF associated inhibitory expression)motif.In JASPAR database,the consensus binding motif of BrERF11b has the highest similarity with that of Arabidopsis AtERF4,suggesting that maybe NbERF4 could bind to its own transcriptional regulatory region,self-regulating its own gene in negative or positive feedback control.By virus-induced gene silence(VIGS)technique,we investigated the influence of NbERF4 silencing on the resistance of N.benthamiana to M.persicae,and the result showed that the NbERF4 silencing had a significant inhibitory effect on the resistance of N.benthamiana to M.persicae.In conclusion,BrERF11b is the first reported ERF transcription factor that enhances the resistance of plants to chewing insects and sap-sucking insects.NbNIMIN2,NbTAF15b and NbERF4 are the target genes of BrERF11b.BrERF11b may regulate NPR1 activity by directly inhibiting the expression of NbNIMIN2 in tobacco plants,and then enhance the resistance of tobacco plants to the leaf-chewing insect Spodoptera litura by positively regulating the expression of JA synthase genes and protease inhibitor genes.BrERF11b may directly activate the expression of target genes NbTAF15b and NbERF4,and then up regulate the expression of Harpin inducible protein gene Hin1,MYB transcription factor gene NbMYB4 and WRKY,and down regulate the expression of callose degrading enzyme gene β1,3-glucanase to enhance the resistance of plants to Myzus persicae.