Regulation Of Sugar Transporter TlSTP1 On Peptaibols Biosynthesis In Trichoderma Longibrachiatum SMF2

Trichoderma spp.are widely distributed saprophytic filamentous fungi and have broad applications.Trichoderma can produce antibiotics such as peptaibols,viridin and massoilactone,and industrial enzymes such as cellulase and xylanase.In agriculture,Trichoderma is well known as biological control agents.Peptaibols are a kind of peptide antibiotics mainly produced by Trichoderma and synthesized by the Non-Ribosomal Peptide Synthetase（NRPS）.Peptaibols have broadspectrum antimicrobial activities and other biological functions including antibacterial activity,antiviral activity,induction of programmed cell death in tumor cells and elicitation of plant systemic resistance.Trichoderma longibrachiatum SMF2（hereafter strain SMF2）produces a large quantity of peptaibols,which are mainly classified into two groups,20-aa（amino acid）peptaibols（trichokonins A,TKA）and 12-aa peptaibols（trichokonins B,TKB）.TKA and TKB are synthesized by two independent synthetase genes tlx1 and tlx2,respectively.Despite the wide research of the biological functions of peptaibols,the regulatory mechanism for peptaibols biosynthesis is still unclear so far.In this dissertation,we identified a sugar transporter TlSTP1 in T.longibrachiatum SMF2,and studied its regulatory effect on the development and peptaibols biosynthesis of strain SMF2.The main research results are as follows:（1）By constructing a hexose transport system and gene knockout,TlSTP1 was shown to have no significant hexose transport activity,but affect the sugar uptake of strain SMF2.We searched the genome of strain SMF2 with the sequence of the glucose sensor RGT2 of Saccharomyces cerevisiae and the sugar transporter RCO3 of Neurospora crassa as queries and retrieved the protein SMF2FGGW₁05795,which contains a conserved sugar transporter domain,and was named TlSTP1.To ascertain the putative hexose transporter activity,TlSTP1 was expressed in S.cerevisiae EBY.VW4000 and the growth of the recombinant strain on different carbon sources was analyzed.The result indicated that TlSTP1 had no significant hexose transport activity.To further analyze the sugar-mediated functions of TlSTP1 in strain SMF2,we deleted the Tlstp1 gene from strain SMF2,and found that,after the deletion of Tlstp1,the ability of strain SMF2 to utilize carbon source was significantly decreased.These results indicate that although TlSTP1 has no significant hexose transport activity,its function is related to sugar uptake in strain SMF2.（2）TlSTPl was shown to have significant impacts on the development and the peptaibols biosynthesis of strain SMF2 by gene knockout and overexpression.Compared to the wild type（WT）SMF2,the ΔTlstp1 strain exhibited a significantly defect on both mycelia growth and conidiation,but showed an earlier and higher production of peptaibols.Moreover,the ΔTlstp1 strain had higher transcriptional levels of the synthetase genes tlx1 and tlx2,indicating that TlSTP1 regulates the biosynthesis of peptaibols by affecting the transcription of genes tlxl and tlx2.（3）The regulation of TlSTP1 was analyzed by comparing the transcriptomes of the WT SMF2 strain and the ΔTlstp1 strain.The transcriptomes of the WT SMF2 strain and the ΔTlstp1 strain were sequenced by RNA sequencing（RNA-seq）,and the regulation of TlSTP1 was analyzed by comparing the differential expression genes.The deletion of TlSTP1 caused the differential expression of a large number of genes in the SMF2 genome,including genes encoding hexose transporters,transcription factors,and epigenetic regulatory factors etc.The KEGG analysis showed that the regulation of TlSTP1 was concentrated in signal transduction,transport and catabolism,carbohydrate metabolism and amino acid metabolism.（4）A tlx1 promoter-interacting protein TlCEFR screened by yeast one-hybrid was shown to play a regulatory role in the peptaibols biosynthesis of strain SMF2.Using the 1600 bp tlx1 promoter as a bait,a C6 zinc finger transcription factor SMF2FGGW₁03735.1 was screened,which was named TlCEFR.By means of gene knockout,the differences between the WT SMF2 strain and the ΔTlcefR strain were analyzed.Compared to the WT SMF2 strain,the ΔTlcefR strain produced white mature conidia rather than green conidia,its cell wall integrity was damaged and it caused an obvious defect in the oxidative stress and osmotic pressure.Moreover,the production of peptaibols and the transcription levels of tlx1 and tlx2 were significantly reduced in the ΔTlcefR strain.These results indicate that TlCEFR plays a regulatory role in the biosynthesis of peptaibols of strain SMF2.