Plant Growth Promotion Study And Genetic Characterization Of Hydrophobin Family Genes Within Trichoderma NJAU 4742

Trichoderma guizhouense NJAU 4742 is a plant-beneficial fungal strain,originally isolated from Chinese medcine residues,which has been now commercially available in China.It is known as a plant growth stimulator and also used for biocontrol of several phytopathogenic diseases.Hydrophobins(HFBs)are the small secreted cysteine-rich proteins uniquely found in filamentous fungi.They have a distinct ability to self-assemble into stable amphiphilic layers at hydrophobic-hydrophilic interfaces and/or attach to various surfaces modulating their hydrophobicity.Thus,they can be involved in a diversity of functions related to the development of the fungus,its resistance to biotic and abiotic stress factors and in interactions with other organisms in the environment.In order to know more about this important Trichoderma strain,the present work has proved the oppotunistic colonization ability of T.guizhouense NJAU 4742 and its significant effect on plant growth promotion via pot experiment and field trials.Then,the methods of genome mining,transcriptional assay with RT-qPCR,protein hetero-expression,gene knock out and knock in were uased to study the functional genetics of the important protein family----HFB in Trichoderma.The main results obtained in this study are listed as follows:1.T.guizhouense NJAU 4742 strain is able to colonize in tomato roots(rhizosphere soils)and able to stimulate tomato seedling growth observably,shown by the significant(>20%)increase of plant biomass compared to the un-inoculated control in both greenhouse and field conditions as well as the increased tomato yield(10%)in field condition.Moreover,a phytohormone-like metabolite----Harzianolide was isolated from the PDA fermenting broth of T.guizhouense NJAU 4742,which has shown significant stimulating effect on tomato root growth and root tips.2.The analysis of the genome of T.guizhouense NJAU 4742(Harzianum Clade)revealed at least 10 hfb encoding genes,what is essentially higher compared to other Trichoderma species and to any other ascomycete fungi investigated so far.Thus,a phylogenetic tree was constructed to identify the new genes,including hfb8,hfb10,hfb11,hfb12,hfb14,hfb15 and hfb16.Meanwhile,a RT-qPCR assay was performed to study the transcriptional mode of the whole hfb family members(HFBome)in two conditions,i.e.submerged culture and plate culture.The resultes showed that hfb4,hfb10,hfb8 and hfb3 are the dominant transcriptional genes during the hyphal growth,and hfb4 is the only transcriptional one during the conidiation.And co-cultivation with tomato seedlings did not give a difference of hfbs transcription except hffb4,which was significantly down-regulated in the co-cultivated Trichoderma(with tomato seedlings)compared to the solo-cultivated ones,indicating a potential role of HFB4 in Trichoderma-host interactions.3.Pichia(P.pastories)cell factory system was used to hetero-express HFBs,including HFB4,HFB8 and HFB15(encoding genes were originally from T guizhouense NJAU 4742),and their fluorescent-labled proteins HFB4::GFPuv and HFB8::mRFP.Also,it was found that HFBs are able to attach to the surfaces of plant roots and glass beads.4.An improved gene editing method fusing PEG-mediating transformation,overlapping PCR and fungal direct colony PCR was made and used to knock out/in hfb4 gene in T.guizhouense NJAU 4742 and the international standard Harzianum strain T.harzianum CBS 226.95.Four and three hfb4-deleted mutants were obtained for T.guizhouense NJAU 4742 and T.harzianum CBS 226.95,respectively,as well as three hfb4-overpression mutants for each.An extra copy of hfb4 gene in all the hfb4-overpression mutants was determined by qPCR.5.The biological funtion of hfb4 gene was investigaed by the compatison study of phenotypes,including biomass,growth rate,aerial hypha formation,hydrophobicity of hyphae and spores,spore disepersion by air,response to mechanical injury,anti-fungicides,anti-oxidation,colonization on roots and antagonism against phytopathogens,beween the mutants and the wild types as well as between the two sibling species.In conclusion,HFB4 is most likely the key member of the HFB family in T.guizhouense NJAU 4742 strain.It is directly involved in the process of filamentous fungi using HFBs to decrease the medium surface tension in order to form aerial hypha.After that,it stays at the cell wall of aerial hypha together with the other 3 HFBs(HFB3,HFB8 and HFB 10)to support the cell surface hydrophobicity,self protection and attachment to environment/host,till to conidiation.At the end,it forms an only-HFB4 protein film at the spore surfaces to gain its hydrophobicity which is remarkably needed for the dispersion of spores by air.HFBs like HFB4 are able to protect the fungal cells from fungicides(Fluconazole)and oxidants(Menadione)like a raincoat for fungal cells but work like"anchors" for Amphotericin to gain the stress.Also,HFB4 is the responsive protein of T.guizhouense NJAU 4742 to mechanical injury,which can be induced to express by injury.The results was also supported by the dull response of hfb4-deleted mutants to mechanical injury.The opposite phenotypes found in T.harzianum CBS 226.95 can be explained by the different regulation network of hfbs expression between species.The corresponding functions of HFB4 may.be replaced by HFB10 in the sibling strain of T.harzianum CBS 226.95.Moreover,HFB4 is involved in Trichoderma-plant-phytopathogen interactions since it is able to change the hyphal surface hydrophobicity(attachment to different hosts).Thus,HFB4 is also an important biological factor for Trichoderma antagonism against other fungi and for the colonization on plant roots.