Functional Analysis Of Transcription Factors PsCZF1, PsHSF1 And PsMAD1 In Phytophthora Sojae

Phytophthora sojae (Kaufman & Gerdman), an Oomycete causing one of the most destructive diseases on soybean worldwide,has become an important agricultural pathogen. P. sojae has been developed as a model species for the genus, having in place excellent genetic and genomics resources (including genetic maps, BAC libraries,and EST sequences),being established gene-silencing for funcational study, as well as having a well organized community of researchers.Transcription factors are key proteins which regulate gene expression and are of great value for study. Studying the molecular mechanism of transcription factors underlyging growth, development, and pathogenicity of P. sojae will help research to find novel targets of chemical control and provide new sight and good idea for designing comprehensive management of Phytophthora disaeases.Bioinformatics analysis of C2H2 zinc finger transcription factors, heat shock transcription factors and MADS-box transcription factors in genome of Phytophthora soja:The completion of genome sequencing of P. sojae has provided novel insights and platform to find and study TFs.In order to comprehensively functional study of TFs during growth, development, and pathogenisis, we used known TFs protein sequence from fungi and plants as query sequences with BLAST algorithm to explore genome-wide inventory of three classes of TFs,such as C2H2 zinc finger transcription factors, heat shock transcription factors and MADS-box transcription factors, and analyze motif or domain organization and their distribution in genome in details.The results showed that some genes of the same family often clustered in genome.All these bioinformatics analysis provided a useful clue to characterize TFs in P. sojae.PsCZFl gene encoding a C2H2 zinc finger protein is required for growth, development and pathogenesis in Phytophthora sojae:C2H2 zinc finger proteins form one of the largest families of transcriptional regulators in eukaryotes.We identified a Phytophthora sojae C2H2 Zinc Finger (PsCZF1),which was highly conserved in the sequenced oomycete pathogens.In the transformants of P. sojae containing the PsCZF1 promoter fused to theβ-glucuronidase (GUS)reporter gene, GUS activity was highly induced in the P. sojae oospores stage and upregulated by the infection. To elucidate the function, expression of PsCZF1 was silenced by introducing antisense constructs into P sojae. PsCZFl silencing transformants exhibited none alternation in the cell size and morphology of sporangia and hyphae; however, the hyphal growth rate was around 50% reduced in the mutants. PsCZF1-deficient mutants were also impaired in the production of oospores,swimming zoospores and the germinating cysts, indicating that the gene was involved in the various stages of the life cycles.Furthermore, we found PsCZF1-deficient mutants completely lost the virulence on the host soybeans.Our results suggested that this oomycete specific C2H2-type zinc finger protein played the important roles in the growth, development and pathogenesis,therefore, PsCZF1 might be an attractive oomycete-specific target for chemical fungicides screening.A heat shock transcription factor PsHSFl is involved in oxidative stress, heat shock and pathogenicity in Phytophthora sojae:Analysis of heat shock transcription factors (HSF) in genome of Phytophthora sojae revealed one gene PsHSF1 encoding HSF, which was clustered together with other highly homologous HSF in P. sojae genome. Bioinformatics analysis showed that there hardly existed high orthologs in other oomycete pathogens.In this paper, RT-PCR was used to analyze expression of PsHSF1 during asexual life stage and oxidative stress mediated with hydrogen peroxidase.These results indicated that PsHSF1 is uniquely expressed in cysts and germinated cysts, and obviously upregulated in latter stage; PsHSF1 also was induced by oxidative stress mediated with hydrogen peroxidase. To elucidate its molecular function, expression of PsHSF1 was silenced by introducing antisense constructs into P. sojae.Silencing of PsHSF1 did not disturb hyphal growth or sporulating, but severely affected hyphal growth on media containing hydrogen peroxidase or inoculated at 30℃. Furthermore, PsHSF1-silenced mutants showed decreased germination ratio.Finally, the pathogenicity of PsHSFl-silenced mutants were depressed. All the results revealed that PsHSFs would play important roles in antioxidative function and pathogenicity in P. sojae.Study on MADS-box transcription factor of P. sojae:MADS-box transcription factors play important roles in reproductive and vegetative development, and in signal transduction. Previously, in silico exploration in genome of soybean root rot pathogen, P. sojae,a gene PsMADl encoding MADS-box transcription factor has been identified. At the same time, the orthologs of other Phytophthora were identified.Phylogenetic analysis revealed that Phytophthora MADS-box genes are divided into animal subgroup of myocyte enhancer factor clade.Using RT-PCR and GUS reporter gen, this study reveled that PsMAD1 constitutively expressed during life stage, highly upregulated in sporulating hyphae,cysts and germinated cysts, furthermore, it was induced by early infection. Finally, examination of promoter region of PsMADl and its Phytophthora orthologs showed that their promoters contain two TATA-like element and one conserved 19-nt core promoter structure of oomycetes.These results indicated that PsMAD1 may play important roles during asexual life cycles and infection as a putative transcription factor.