Transcriptome Analysis Of Photoreceptor Gene And Functional Identification Of EaWC1 In Elsino(?) Arachidis

Elsinoe arachidis is an important fungal disease in peanut producing areas in China.It produces the photosensitive mycotoxin Elsinochrome（ESC）,which has strong cytotoxicity and is an important virulence factor.Previous studies have found that ESC biosynthesis is significantly regulated by light,revealing the photoregulation and molecular mechanism of toxin biosynthesis,which is of great significance to explore the infection process,pathogenesis and regulatory network of this pathogen,and can provide scientific theoretical support for the efficient formulation of disease control strategies.This paper explored the effects of light quality on pathogen growth and development,ESC toxin production and related gene expression.Transcriptome sequencing and photoreceptor gene analysis were performed on samples cultured by light quality,and the blue light receptor Ea WC1 gene was cloned and bioinformatics analysis was performed.The gene knockout vector was constructed and the function of blue light receptor Ea WC1 gene was preliminally verified.The research results aim to provide important molecular evidence for the analysis of the pathogenesis of E.arachidis and the exploration of the photoregulatory network,and lay E.arachidis scientific theoretical foundation for the formulation of comprehensive disease control strategies.1.The effects of light quality on growth and toxin biosynthesis of Elsinoe arachidis were determined.Light quality plays an important role in the regulation of fungal growth and development,ESC biosynthesis and related gene expression.By systematically studying the effects of light quality（dark,white,blue and red）on colony morphology,growth and development,toxin content,virulence and toxin synthesis gene expression of E.arachidis（LN-JH-C01,LN-FT H01）,the results showed that,Light quality regulates the growth and development of pathogens and the expression of ESC-toxin biosynthesis and toxin synthesis genes.Light treatment had no significant difference in colony growth,and the growth rate was 0.78-0.88 mm/d.The conidia yield reached 1.33×10⁴-2.17×10^4/m L under dark and red light conditions,and only 0.17×10⁴-0.50×10⁴/m L under white and blue light conditions.The toxin content of the two strains increased significantly under blue light treatment,and the ESC content of the two strains was 70.62 nmol plug^-1 and 41.23 nmol plug^-1,respectively.No toxin was detected under red light and dark condition.There were differences in the pathogenicity of the pathogen under light quality treatment.Under white light condition,the pathogen spots were produced in the leaves inoculated by peanut.Under blue light condition,the pathogen spots were stronger,while under dark and red light condition,there was no pathogenicity.The expression of ESC gene cluster 12 was regulated differently by light quality.Ea PKS1,Ea Om Fm1,Ea MFS1,Ea Omf1,Ea Ps TF1 and Ea CYP1 genes showed the same trend with the accumulation of ESC toxin.The relative expression levels of Ea PKS1,Ea Omf1,Ea Ps TF1 and Ea CYP1 genes were high in blue light culture,which were 4.65,3.70,1.93,4.20,2.19 and 1.48 times of those in dark culture.The expression patterns of Ea FAD1,Ea DH1,Ea NP1,Ea HP1,Ea HP2 and Ea HP3 genes were negatively regulated by light,and the relative expression levels of Ea FAD1,Ea DH1,Ea NP1,Ea HP1,Ea HP2 and Ea HP3 genes were 0.25,0.03,0.31,0.46,0.19 and 0.14 times of those in the dark condition,respectively.Studies showed that light quality regulated the growth and development of E.arachidis and toxin biosynthesis,blue light significantly regulated it,but the interspecific effects of Elsinoe were significantly different.2.Genes such as photoreceptors regulated by light were screened.On the basis of genome sequencing,transcriptomic sequencing was performed on the fungi treated with different light quality by high-throughput sequencing technology.89.96 Gb of Clean data were obtained,with GC content of 54%and Q30 base percentage above 94.02%,indicating good sequencing quality.Compared with darkness,5926,6344 and 4507 differentially expressed genes（DEGs）were obtained in white light,blue light and red light conditions,respectively.GO classification and KEGG enrichment analysis showed that the differentially expressed genes were significantly enriched in metabolic pathway and genetic information processing pathway.474 genes,including 11 putative photoreceptor genes,269 transcription factors,81 genes involved in photosignaling pathways,and 352 genes related to secondary metabolism,were identified by gene function annotation.Five blue light receptor genes（EVM0003940 is a WC1 homolog gene）,two green light receptor genes,one red light receptor gene,four global regulatory factors,five polyketosynthase genes and 12 secondary metabolism related genes were screened by Blast comparison,phylogenetic tree and domain analysis.The results showed that the expression patterns of these genes were basically consistent with the transcriptome expression patterns,and the light-quality induced blue light receptor gene expression regulated the biosynthesis of coelisin,which provided a research basis for further analysis.3.The blue light receptor gene of E.arachidis was cloned and its expression pattern was analyzed.Specific primers were designed for PCR amplification according to the sequence of blue light receptor Ea WC1 gene obtained in the genome,and the full length of Ea WC1 gene was 3261bp.Bioinformatics prediction analysis of Ea WC1 gene using bioinformatics online analysis tool showed that Ea WC1 gene has a complete open reading frame,with one exon,no intron,coding 1086 amino acid protein.The physical and chemical properties of the protein encoded by this gene were predicted.It was an unstable hydrophilic protein and had a conserved domain of blue light receptor encoding gene WC-1,namely one LOV domain,two PAS domains and one ZNF domain.There is no signal peptide and transmembrane region.α-helix and random curl were the main secondary structure of the protein,and the tertiary structure model was established.There are multiple phosphorylation sites,with subcellular predictive localization to the nucleus.Homology cluster analysis showed that Ea WC1 had a high homology with the blue light receptor WC1 of E.arachidis.The expression of Ea WC1 gene was up-regulated under both white and blue light conditions,and the expression of Ea WC1 gene under blue light culture condition was 2.15 times of that under white light culture condition.The expression pattern of Ea WC1 gene under blue light culture condition was the same as the trend of ESC toxin content,which proved that the biosynthesis of ESC was regulated by Ea WC1 gene.4.The function of Ea WC1 gene in E.arachidis was preliminarily verified.The Ea WC1gene knockout vector with hygromycin resistance and Ea WC1 gene homologous sequence was constructed by homologous recombination technique.The protoplasts of E.arachidis were transformed by the established genetic transformation system of E.arachidis.The Ea WC1 gene knockout mutant of E.arachidis was obtained by screening the hygromycin resistance medium.PCR results showed that△Ea WC1-4 and△Ea WC1-5 were mutant strains.Biological verification of the mutant strain showed that Ea WC1 gene regulated the biosynthesis of toxin.Compared with the WT toxin content of 16.26 nmol/plug,the mutant strain showed white to yellowish white colonies,no red pigment,no absorption peak at460nm,and no ESC toxin was detected.The mutant grew slowly,with growth rates of 0.68mm/d and 0.70 mm/d,respectively.Conidia production was not affected,and the conidia quantity was 0.5×10⁴-0.67×10⁴/m L.Pathogenicity test showed that the mutant did not produce disease spots and had weak pathogenicity.Studies have shown that the destruction of Ea WC1 gene inhibits the growth and regulates the biosynthesis of ESC toxin in E.arachidis,which is necessary in the pathogenic process.