Cloning Of Transcription Factor PibHLH From Penicillium Italicum And Its Mechanism On Regulating Prochloraz Resistance

Penicillium italicum is a species of pathogenic fungus which infects post-harvest citrus and leads to blue mold disease.Citrus decay by blue mold during storage and transportation has caused large economic losses.Currently,chemical drugs are predominantly applied to control various citrus pathogenic fungi including P.italicum.14α-demethylase inhibitor(DMI)class fungicides such as prochloraz and imazalil are widely exploited to control these pathogens.DMI-class fungicides kill fungi by inhibiting sterol biosynthesis and impairing cell membrane function.However,long-period use of these chemical drugs led to more and more frequent emergence of fungicide-resistant isolates.The mechanisms underlying such fungicide-resistance,especially for P.italicum strains,have become research hotspots.Basic helix-loop-helix(bHLH)transcription factor family members are widely involved in fungal physiology regulation,including growth,development,sporulation,virulence,and resistance to drugs or environmental stressors.Sterol regulatory element binding proteins(SREBP)such as SreA and SrbA containing bHLH motif have been identified as transcription factors that regulate drug resistance of P.digitatum and Aspergillus fumigatus.However,the function of P.italicum bHLH-like transcription factor has not been studied yet.In this work,a bHLH-like transcription factor,named PibHLH,was cloned from highly prochloraz-resistant P.italicum strain YN1,and sequence analysis and phylogenetic classification were performed.The effect of PibHLH on the fungal growth,sporulation,virulence and drug resistance was investigated using Pib HLH-knockout and-complemented strains.The mechanisms underlying PibHLH regulation were further studied by transcriptome analysis and real-time quantitative PCR(RT-qPCR)validation.The main results are listed below.1.PibHLH gene cloning,sequence analysis and phylogenetic classification.The present study cloned a PibHLH gene with 1129-bp full-length and a 55-bp intron sequence in the middle.The PibHLH gene has an open reading frame(ORF)of 1074 bp which encodes a protein containing 357 aa residues with predicted molecular weight～39 kDa.The secondary structure prediction showed only one bHLH motif at the middle of PibHLH amino acid(aa)sequence(146-197aa).Phylogenetic analysis indicated>90%similarity of PibHLH bHLH motif to its homologous sequences from P.chrysogenum and A.fumigatus.They all belong to F5 group of fungal bHLH family.2.Construction of PibHLH-knockout and-complemented strains to investigate the role of PibHLH in regulating P.italicum growth,sporulation,virulence and fungicide-resistance.The present study designed a homologous knockout-cassette based on the PibHLH gene sequence and constructed PibHLH-knockout strain by protoplast-mediated transformation.This work also constructed a complemention vector to obtain a PibHLH-complemented strain by agrobacterium-mediated transformation.Phenotypic analysis of PibHLH-knockout and-complemented strains demonstrated that PibHLH deletion had no obvious effect on P.italicum growth,sporulation and virulence,but did affect fungal resistance to DMI fungicides.The PibHLH-knockout strain showed lower resistance to prochloraz and imazalil,and the independent-sample t-test verified extremely-significant or significant difference in EC50 values between wild-type and PibHLH-knockout strains.In contrast,the deletion of PibHLH did not affect YN1 resistance to thiabendazole and fludioxonil.3.RT-qPCR analysis of prochloraz resistance-related gene expression in the transcription factor PibHLH regulation.The present study applied RT-qPCR to compare expression patterns of 15 DMI-resistance genes between wild-type and PibHLH-knockout strains at prochloraz treatments.The PibHLH gene knockout significantly deceased prochloraz-induced expression folds of several key enzyme genes in sterol biosynthesis pathway(e.g.,CYP51A,ERG2,ERG6)and drug pump genes(e.g.,MFS1 and MFS4),suggesting that these genes might be involved in PibHLH regulation of P.italicum fungicide-resistance.In addition,the PibHLH-knockout strain showed a typical metabolic compensation effect with higher induced up-regulated expression of drug pump genes ABC1,ABC2,MFS2,MFS3,and mitogen-activated protein kinase(MAPK)signaling pathway genes Bck1,Mkk1,and Slt2.This may be the reason why prochloraz resistance of P.italicum strain YN1 did not decreased too much as initially expected.4.Transcriptom analysis of PibHLH regulation mechanisms underlying P.italicum prochloraz-resistance.Transcriptomic techniques were applied to compare the gene expression patterns of wild-type YN1 no-induced(NI-WT)and prochloraz-induced(I-WT),and PibHLH-knockout strain no-induced(NI-ΔPibHLH)and prochloraz-induced(I-ΔPibHLH),and PibHLH knockout strain no-induced(NI-APibHLH)and wild-type strain YN1 no-induced(NI-WT),and PibHLH knockout strain prochloraz-induced(I-ΔPibHLH)and wild-type strain YN1 prochloraz-induced(I-WT).Volcano plot in combination with Venn diagram analysis selected 815 differentially expressed genes as potential prochloraz-resistance genes.They were enriched into 36 GO terms,and the most significantly enriched terms were oxidation-reduction process(GO:0055114),transmembrane transport(GO:0055085),integral component of membrane(GO:0016021),transporter activity(GO:0005215),and transmembrane transporter activity(GO:0022857).KEGG enrichment analysis showed 70 metabolic pathways associated with PibHLH regulation of prochloraz-resistance.Amongest them,the most significantly enriched pathways were amino acid metabolism(ko00350),fatty acid degradation(ko00071)and peroxisome pathway(ko04146).Totally,GO and KEGG enrichments revealed 24 candidate genes involved in the PibHLH regulation of prochloraz-resistance.RT-qPCR demonstrated that 10 genes were consistent with transcriptome results in their expression profiles,including 5 MFS genes,2 nitrogen metabolism-related genes,and 3 MAPK signaling pathway genes.These results indicated that these genes might be invloved in the regulation of transcription factor PibHLH on P.italicum prochloraz-resistance.In summary,the present study cloned a transcription factor gene PibHLH from P.italicum,and phylogenetically classified it into F5 group in the fungal bHLH family;PibHLH-knockout and-complemented analysis demonstrated that PibHLH was involved in the regulation of P.italicum prochloraz-and imazalil-resistance;transcriptome analysis suggested that the PibHLH regulation of P.italicum prochloraz-resistance was associated with MFS drug-pumps,nitrogen metabolism and MAPK signaling pathway.The obtained results provide theoretical cues for further study on the role(s)of bHLH transcription factor(s)in fungicide resistance regulation.