Understanding The Role Of The PAF-AH-like And Hyd1 Gene In Trichoderma Harzianum Induced Maize Systemic Resistance To Maize Leaf Spot Pathogen Curvularia Lunata

Maize Curvularia leaf spot disease is one of the major diseases in maize producing areas in China.Using biocontrol technology to increase the maize systemic immunity is an important and green way to control and prevent from corn diseases.Trichoderma spp.are widely distributed and used in biocontrol of plant diseases.There has been no report about molecular resistance mechanism of Trichoderma applyed in seeds or roots to induce aboveground plant systemic resistance at home and abroad,especially,the molecular interactions model between Trichoderma MAMPs?microbial-associated molecular patterns?molecules and receptors in maize roots has been unclear.A PAF-AH-like gene?Platelet-activating factor acetylhydrolase like gene,PAF-AH-like gene?from the phospholipase A₂?PLA₂?enzyme superfamily of T.harzianum was successfully cloned.The PAF-AH-like protein was purified by prokaryotic expression in E.coli.The optimum enzymatic activity of PAF-AH-like occurred at pH 6 in the absence of Ca²⁺and the protein localized in the cytoplasm of the Trichoderma.The PAF-AH-like gene was closely associated with antagonism against Rhizoctonia solani and was related to secretion of the extracellular chitinase and cellulase and also to specific interaction with maize roots.Meanwhile,it demonstrated that the PAF-AH-like gene in the Trichoderma induced maize resistance against Curvularia lunata through regulating the related genes expression of the JA/ET signaling pathway in maize leaves.In order to clarify the MAMPs model of the Trichoderma inducing maize systemic resistance against leaf spot pathogen,a class II hydrophobin gene hyd1 was cloned from T.harzianum in the study.The Hyd1 was localized on the plasma membrane and the Hyd1 secreted into the extracellular to promote Trichoderma colonization on maize roots and it was also proved that the protein could be expressed on the cell membrane of the maize roots,which provided precondition that Hyd1 as MAMPs was interacted with maize roots and induced maize resistance against foliar spot pathogen.Maize B73 seeds were treated with the conidia suspension of different hyd1 transformants and grew in greenhouse until at the 4-leaf stage and the leaves were inoculated with C.lunata in vitro and in vivo.The results showed the maize leaf lesion area that treated by KO16 was larger than WT?wild type?notably,the lesion area of the overexpression treatment?OE3 and OE5?was smallest.It suggested that hyd1 had the role of inducing the plant systemic resistance.The yeast two hybrid system was used to screen the interaction protein in maize roots that was response to or recognized by Hyd1.Ubiquilin1-like protein interacted with Hyd1 was screened by yeast two hybrid method and their direct interactions were verified by BiFC and pull down.The motif of the Ubiquilin1-like protein interacted with Hyd1 was identified in yeast cells and it was also proved that the signal peptide sequence of the Hyd1 was necessary for the interactions between Hyd1 and Ubiquilin1-N,in addition,the 8cysteine amino acid of the Hyd1 was participated in the above interaction.In order to further uncover that hyd1 and ubiquilin1-like gene had a synergistic effect on increasing plant resistance,hyd1 and ubiquilin1-like genes were coexpressed in Arabidopsis thaliana by transgenic method,which significantly promoted plant resistance to Botrytis cinerea with leaf lesion area was only 50%less than the wild type.The Hyd1 and Ubiquilin1-like proteins were localized on the plasma membrane and Hyd1 promoted the stability of the Ubiquilin1-like protein in Nicotiana benthamiana.RNA-seq analysis of global genes expression in maize leaves at the 24h time point after inoculating C.lunata spores suspension showed hyd1 inducing plant systemic resistanc mainly associated with Brassinolide?brassinosteroids BR?signaling pathway as differential genes changed close to 3 times.The role of BAK1 as BR receptor in the Hyd1 induced the plant resistance was preliminarily verified in the mutant BANK1-4.Furthermore,it indicated that the genes of JA/ET pathway were also associated with hyd1.What is more,some calmodulin?CaM?and WRKY genes also changed a bit.According to the above study,MAMPs model of the Trichoderma systemically induced maize resistance against leaf spot pathogen is proposed as follows:T.harzianum secreting Hyd1 interactes with receptor like protein Ubiquilin1-like in maize roots and Ubiquilin1-like might bind to the BR receptor BAK1 and three proteins form a complex that result in BR signal transduction,meanwhile stimulate the JA/ET,calmodulin?CAM?and WRKY gene expression resulting in increasing the plant resistance.It provided an important clue for further revealing the molecular interaction mechanism between Trichoderma MAMPs hydrophobins and PRRs-like?pattern recognition receptors-like,PRRs-like?protein in the plant.