Molecular Researches Of The ClvelB And Clt-1 Genes In Regulating The Toxin Biosynthesis In Curvularia Lunata

The Curvularia leaf spot caused by Curvularia lunata is one of the most widely distributed maize leaf diseases.The 5-(hydroxymethyl)furan-2-carboxylate(M5HF2C)toxin produced by the fungus in infected maize leaves brings the large economic losses in China.However,very little has been conducted on the understanding of control mode and synthesis mechanism of this toxin.The clvelB gene from C.lunata was successfully cloned and shown to be closely associated with sporulation,M5HF2 C toxin synthesis,and virulence.Meanwhile,it demonstrated that clvelB affected the expression of clt-1 gene which was related to the toxin synthesis.To study the regulation mechanism of clt-1 in toxin synthesis,the yeast two-hybrid(Y2H)cDNA library from C.lunata CX-3 was constructed.Y2 H,bimolecular fluorescence complementation and GST Pull-down assays were conducted to verify the interaction of Clt-1 with full-length ClXyn24 and ClAxe43.Furthermore,a Y2 H study revealed that Clt-1 physically interacted with ClXyn24 and ClAxe43 through its BTB domain.In addition,the 3 cysteine amino acid of Clt-1 were participated in the above interaction.The capability of mutants to colonize the surrounding host tissue was impaired.The amount of M5HF2 C toxin produced by mutants was significantly declined compared with wild-type(WT)strain.However,the capabilities of M5HF2 C production were increased in all strains when using xylose powder instead of xylan.In addition,?Clt-1,?ClXyn24,?ClAxe43,and ?ClXyn24&ClAxe43 displayed lower production of acetyl-CoA and malonyl-CoA than WT.Our results showed that Clt-1 physically interacted with ClXyn24 and ClAxe43 through its BTB domain,which acts upstream of xylose metabolism.The utilization of xylan provides acetyl-CoA and energy for M5HF2 C toxin synthesis.We performed transcriptome analysis by comparing the Clt-1 mutant(T806),and wild type(CX-3)using RNA sequencing.The pks18 gene was significantly downregulated in T806 compared with WT,and was proved to be responsible for M5HF2 C toxin synthesis.It was found by Blastp of differential genes against the C.lunata CX-3 genome that 13 flanking genes of pks18 in genome were simultaneously downregulated in T806.pks18 and its 13 flanking genes are found clustered in a region spanning 57.89 kb on scaffold 9 of the C.lunata CX-3 genome.Genes being involved in the biosynthesis of secondary metabolites were usually clustered,and the combination of pks18 and its 13 flanking genes was like a cercosporin-associated gene cluster of Cercospora nicotianae.Therefore,this cluster in C.lunata is a putative gene cluster for M5HF2 C toxin biosynthesis.Above all,it was inferred that the clvelB and clt-1 genes synergistically regulated the synthesis of M5HF2 C toxin in C.lunata.ClVelB affected the expression of Clt-1,which physically interacted with ClXyn24 and ClAxe43 to degrade xylan.And the utilization of xylan provides acetyl-CoA and energy for M5HF2 C toxin synthesis.This study will lay the groundwork for the research of synthetic mechanism and control mode of M5HF2 C toxin,and provide target sites for designing new fungicide to manage C.lunata.