Molecular Mechanism Of BZIP63.5 Regulating Trichoderma Harzianum Against Poplar Leaf Blight Pathogen

The poplar leaf blight affects the growth of poplar trees and causes great economic loss to forestry production.Trichoderma spp.are widely recognized as biological control agents for plant fungal diseases and have broad spectrum resistance to various pathogenic fungi.Currently,studies on the mechanism of Trichoderma against pathogenic fungus mainly focus on single gene and omics level,while there are few systematic studies on it from the transcriptional regulation level.Revealing the molecular mechanism of Trichoderma against poplar leaf blight pathogen at the regulatory level can provide theoretical basis for further understanding of the biocontrol mechanism of Trichoderma.bZIP（basic leucine zipper）transcription factors are widely present in eukaryotes,and there is little research on the mechanism of how bZIP transcription factors regulate biocontrol fungi against pathogens.This study identified 32 bZIP transcription factors from the genome of T.harzianum Tha739,among which bZIP63.5 strongly responded to the stress of secondary metabolites from poplar leaf blight pathogen.Plate confrontation showed that,compared with wild type,the inhibition rates of bZIP63.5 overexpression T.harzianum strains Tha-OE1 and Tha-OE3 on poplar leaf blight pathogen increased by 50.48%and 20.60%at 6 d,respectively;the inhibition rates of bZIP63.5 suppression expression T.harzianum Tha-IN1and Tha-IN2 on poplar leaf blight pathogen decreased by 19.70%and 15.00%,respectively,indicating that bZIP63.5 played a positive regulatory role in the antagonistic ability of T.harzianum against poplar leaf blight pathogen.In addition,under the stress of secondary metabolites from poplar leaf blight pathogen,compared with wild type,at 48 h,the CAT activities of Tha-OE1 and Tha-OE3 increased by 46.58%and 24.81%;POD activities increased by 15.52%and 11.58%;GST activities increased by 29.96%and 18.68%,respectively,indicating that bZIP63.5 could regulate the CAT,POD,and GST activities to enhance antioxidant and detoxification abilities of T.harzianum.Transcriptome analysis showed that,under the stress of secondary metabolites from poplar leaf blight pathogen,compared with wild type,bZIP63.5 overexpression T.harzianum strain had 989 and 1136 up-regulated genes,and 1080 and 675 down-regulated genes at 6 h and 24 h,respectively.bZIP63.5 regulated the expression of transcription factor genes（20Zn₂Cys₆ZF,17 C₂H₂ZF,2 GATA ZF,1 ZZ ZF,4 MYB,and 5 b HLH）,detoxification related genes（23 ABC,38 CYP,9 GST,and 7 MDR）,antioxidant enzyme genes（4 CAT and 4 POD）,and heat shock protein genes（15 HSP）in response to secondary metabolite stress of poplar leaf blight pathogen.bZIP63.5 could also regulate the expression of polyketide synthase genes（10 PKS）,β-1,3-glucanase genes（4 Gluc）,and chitinase genes（10 Chit）to resist poplar leaf blight pathogen.TF-Centered Y1H and Yeast one-hybrid were used to identify the cis-acting elements bound by bZIP63.5.The results showed that bZIP63.5 could not only bind to cis-acting elements TGTCACACMCUUMISIN,BIHD1OS,GTGANTG10,and WRKY71OS,but also bind to the four core sequences（TACG,CGGA,and GGAC）of unknown element TACGGAC.It was proved that bZIP63.5 could bind to the four core sequences of unknown element by EMSA.To identify the target genes directly regulated by bZIP63.5,combining with the transcriptome analysis result and the cis-acting elements bound by bZIP63.5,Ch IP identified 29 genes directly regulated by bZIP63.5,including 10 transcription factor genes（1MYB,1 b HLH,4 C₂H₂ZF,3 Zn₂Cys₆ZF,and 1 ZZ ZF）,9 detoxification related genes（4 ABC,4 P450,and 1 GST）,2 antioxidase genes（CAT-4 and POD-1）,4 heat shock protein genes HSP and 4 polyketide synthase genes PKS.RT-q PCR analysis of directly regulated genes showed that bZIP63.5 positively regulated the expression of the above 29 genes.These results indicated that bZIP63.5 could directly regulate the expression of these genes to resist the stress of poplar leaf blight pathogen.Transcriptional self-activation assay showed that bZIP63.5 has transcriptional activation activity,containing two activation regions located at the N-terminal（51-100 amino acid）and C-terminal（388-488 amino acid）,respectively.After removing the self-activating region,five proteins interacting with bZIP63.5 were identified through yeast two-hybrid,namely bZIP45.3,adapter protein,histone H4,heat shock protein HSP70,and an unknown protein.In summary,study on bZIP63.5 regulating T.harzianum against to poplar leaf blight pathogen provides theoretical guidance for the biological control of poplar leaf blight.The study on cis-acting elements,downstream target genes,and interacting proteins bound by bZIP63.5 reveals the molecular mechanism of Trichoderma against pathogenic fungi at the regulatory level,providing a theoretical basis for controlling plant diseases.