The Function Of Transcription Factor CgHapX In Colletotrichum Graminicola

Colletotrichum gramincola is a hemibiotrophic plant pathogen of maize.This fungus employs complicated pathogenic strategies in different lifestyles.Besides as a vital nutrient metal for organisms’ growth and a cofactor of enzymes for cellular progresses,iron is considered to regulate pathogenicity of pathogens in different lifestyles.Transcription factor Hap X plays a crucial role in iron metabolism,but the function of Cg Hap X in C.graminicola is still not clear.In this study,Cghap X was selected to be target gene,Agrobacterium tumefacien mediated transformation(ATMT)method was used to get the ΔCg Hap X of C.graminicola.The function of Cg Hap X was studied about its influences on both iron metabolism and pathogenicity.The main results are as following:1.Cg Hap X gene was cloned,and the physical and chemical properties of Cg Hap X were analyzed by bioinformatic methods.Based on the nucleotide sequences in C.graminicola genome database,Cglac(encoding laccase)and Cg Hap X genes(encoding Cghap X)were cloned by PCR.The physical and chemical properties of Cg Hap X were analyzed by bioinformatic methods.The full length of Cg Hap X was 1914 bp,including one open reading frame which encoded 637 amino acids.The Cg Hap X was predicted to be a homodimer protein located in nucleus.Phylogenetic analysis suggested that Cg Hap X was closest to Hap X of Colletotrichum higginsianum.2.Cg Hap X made effects on growth,resistance and pathogenicity of C.graminicolaBinary vector p PZP100-Cg Hap X was constructed by infusion cloning then transformed into A.tumefacien strain AGL-1 by electroporation.The strains were cocultured with conidia of C.graminicola.Candidate transformants were screened and verified by PCR.The ΔCg Hap X strain was obtained.The color of ΔCg Hap X colony was red-brown.The growth rate of mycelium,the amount and generation of conidia decreased significantly.The formation rate of appressoria also decreased.It indicated that Cg Hap X influenced on biological characteristics and formation of infection structures.After the treatments of cell integrity stress,ion stress and oxidative stress,the growth rate of ΔCg Hap X decreased significantly.It indicated that Cg Hap X play a role in stress resistance.Compare the inoculation of wild-type strain,the pathogenicity of ΔCg Hap X reduced obviously in maize cultivar Zhengdan 958.3.Cghap X was corresponded to iron concentration and regulates genes related to iron hemostasis and genes related to pathogenicity in different lifestyles.The results of quantitative Real-time PCR showed that Cg Hap X upregulated in iron starvation.During iron starvation,Cg Hap X-deficiency caused NRPS2 and NRPS6 genes downregulated significantly,which encoded crucial synthases in siderophore biosynthesis pathway,while CCCA upregulated significantly,which regulated vacuolar iron transportation.In contrast,during iron excess,Cg Hap X-deficiency caused CCCA downregulated significantly.The above results indicated that,Cg Hap X balanced iron homeostasis corresponding to change of iron concentration.The results of relative expressions of genes related to pathogenicity in different lifestyles in Cg Hap X-deficiency were analyzed.During iron starvation,Cg Hap X-deficiency caused AAR downregulated significantly,which regulated the formation of appressoria in biotrophic stage.Cg Hap X-deficiency caused PKS upregulated significantly,which encoding polyketide synthase and regulated secondary metabolism in necrotrophic stage.In contrast,during iron excess,AAR of ΔCg Hap X upregulated significantly,while PKS of ΔCg Hap X downregulated significantly.The above results showed indicated that Cg Hap X regulated pathogenicity in different lifestyle stages of C.graminicola.