The Functional Research Of BZIP Transcription Factor Ss-Ada1in Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum （Lib.） de Bary is a devastating fungal plant pathogen, ithas host range which comprises at least75families and278genera including morethan450plant species, and it can cause the most serious disease on soybeans, canola,sunflower and other oil crops. bZIP transcription factors are widely exist ineukaryotes, they can regulate expression of specific genes and participate in varietyphysiological processes in vivo. bZIP transcription factor Ada-1has been found byDunlap et al when they knock-out encoding genes systematically in N.crassa.Dunlap,s study suggested that transcription factor Ada-1can regulate developmentand sexual reproduction in N.crassa. But function of this transcription factor has notyet been investigated in S. sclerotiorum. So functional research of transcription factorAda-1in S. sclerotiorum may help us improve more knowledge of developmentalmolecular mechanism in S. sclerotiorum and provide theoretical support forSclerotinia prevention.1. In this study, BlastP analysis by S. sclerotiorum genome website, amino acidsequence of a hypothetical protein（SS1G₀1411.3） in S. sclerotiorum that showed54.57%homologous with transcription factor Ada-1（NCU00499） in N.crassa hasbeen found out, named it SsAda-1. The gene SsAda-1（Transcript:SS1G₀1411T0）that encodes transcription factor SsAda-1located at chromosome1, gene size is1917bp, contains a54bp intron and it,s ORF encodes621amino acids. Proteinstructure prediction in NCBI showed that SsAda-1contains a conserved alkaline DNAbinding domain, homologous analysis with ClustalX showed that the conserveddomain of SsAda-1has95%homologous with bZIP DNA binding domain of Ada-1inN.crassa. Both of them belong to bZIP transcription factor family.2. In this study, the position of SsAda-1in genome has been found out, so upstream fragment and downstream fragment of this gene can be cloned. The1041bpupstream fragment and1488bp downstream fragment have been ligated into pXEHrespectively to construct the knock-out vector for SsAda-1. This procedure isfoundation for further functional research of transcription factor Ss-Ada1in S.sclerotiorum.3. In this study, protoplasts of S. sclerotiorum have been prepared by Lysingdecomposition of hyphae. The knock-out vector for SsAda-1then been transformedinto protoplasts of S. sclerotiorum. Identificated those transformants with PCRamplication and Southern hybridization. Biological characterization and pathogenicanalysis of the SsAda-1knock-out mutant, the resulting mutant displays seriesphenotypes that mycelial growth rate decreased significantly when compared with thewild-type, it,s development stage remained in S1stage of sclerotinia; Observation ofmycelium and infection cushion with microscope showed that mycelial branch angleof the mutant becomes larger than the wild-type, but the mutant can form normalinfection cushion. Inoculated in tomato leaives, SsAda-1knock-out mutant appeareddeclined pathogenicity. Expression analysis of Ss-Ada1in different developmentalstages of S. sclerotiorum showed a consistent result with phenotypic observation thataccumulation of Ss-Ada1reaches a maximum in S1stage of sclerotinia.This study may suggested that transcription factor Ss-Ada1in S. sclerotiorumplay a crucial role in development, especially in the formation and development ofsclerotia. Those findings are different with previous studies in N.crassa, our resultsmay indicate a new function of transcription factor Ada1in S. sclerotiorum thattranscription factor Ss-Ada1can regulate sclerotium development and maturity.