The Function Of APSES Transcription Factor Gene GlSwi6 In Fungal Growth,Cell Wall Integrity And Secondary Metabolism In Ganoderma Lucidum

Ganoderma lucidum,belonging to basidiomycetes,is a well known herb medicine in China.Currently,the completed genome sequences of G lucidum have been completed.In addition,the genetic systems have been established.Moreover,the diversification of secondary metabolic pathways has been found in G lucidum.These foundations have made G lucidum to be a potential model system for studying the biosynthesis of active compounds produced by medicinal fungi.The APSES family of transcription factors is unique to fungi,and belongs to transcriptional factors of the basic helix-loop-helix class.It is found that the APSES transcription factors are involved in numerous biological processes in fungi.However,the studies regarding the role of APSES transcription factors are relatively rare in basidiomycetes.In the present study,the effects of the APSES transcription factor gene GlSwi6 on the fungal growth and development,cell wall integrity and secondary metabolism as well as other biological processes were studied systematically in G lucidum.The mainly results are as follows:1.The GlSwi6 gene was cloned in G.lucidum,and it was found that GlSwi6 was alternatively spliced into two mRNA variants GlSwi6A and GlSwi6B.Domain analysis revealed that the GlSwi6A contained a Half-pint domain involved in alternative splicing and three ANK domains which are common in APSES transcription factors,while the GlSwi6B contained a SWI/SNF domain related to DNA binding except the Half-pint and ANK domain.The amino acid sequence“-S-M-S-V-S-A-H-I-L-S-V-S-P-G-P-H-L-A" from 82th to 99th in GlSwi6A is replaced by the 82h Thr amino acid in GlSwi6B.qRT-PCR results showed that the expression of GlSwi6A during fruting body stage is 4-fold of that during mycelia stage,the expression of GlSwi6B during primordium and fruting body stage is 4-and 9-fold of that in the mycelia stage,the ratio of the expression of GlSwi6B to GlSwi6A during mycelia and fruiting body stage was 21 while was 38 during primordium stage.In addition,the results of Y2H assay showed that both GlSwi6A and GlSwi6B could interact with the MAP kinase GlSlt2 of G lucidum.2.The physiological function of GlSwi6 in G.lucidum was studied with the GlSwi6 co-silenced strains(Swi6D and Swi6E)constructed previously in our laboratory and GlSwi6A overexpression strains(OE6A-18 and OE6A-25)and GlSwi6B overexpression strains(OE6B-3 and OE6B-8)constructed in this study.Phenotype analysis showed that:1)GlSwi6 co-silencing caused decreased fungal growth and increased hyphal branching while GlSwi6B overexpression caused increased fungal growth and reduced hyphal branching.2)GlSwi6 co-silencing caused the CMCase(carboxymethyl cellulase)and Xylanase activities decreased about 40%and 50%,respectively.In addition,GlSwi6B overexpression caused the CMCase and Xylanase activities increased about 0.5-and 0.6-folder,respectively.3)Adding CaCl2 could recover the decreased activity of CMCase and Xylanase of GlSwi6 co-silenced strains,in addition,adding LaCl3 or EGTA could recover the increased activity of CMCase and Xylanase of GlSwi6B overexpression strains.4)Adding CaCl2 could recover the upregulated expression of CreA,Acel and AmyR in GlSwi6 co-silenced strains.In addition,adding LaCl3 or EGTA could recover the downregulated expression of CreA,Acel and AmyR in GlSwi6B overexpression strains.These results indicate that GlSwi6 is involved in fungal growth,hyphal branching,extracellular CMCase and Xylanase activities in G.lucidum,in addition,intracellular Ca2+ level and the expression of cellulase regulator genes is involved in the CMCase and Xylanase activities regulated by GlSwi6.3.The analysis of the function of GlSwi6 in cell wall integrity showed that:1)The expression of GlSwi6A was up-regulated by 2.1-folder under Congo red treatment while was reduced about 85%under CFW(calcofluor white)treatment,and the expression of GlSwi6B was up-regulated by 3.6-,4.5-and 2.7-folder under Congo red,SDS(sodium-dodecyl sulphate)and lysing enzyme treatment,respectively.In addition,the ratio of the expression of GlSwi6B to GlSwi6A were all signigicantly increased under cell wall stresses.2)GlSwi6 co-silencing caused increased sensivity against cell wall stresses while GlSwi6B overexpression caused decreased sensivity against cell wall stresses.3)GlSwi6 co-silencing caused chitin and β-1,3-glucan content both decreased about 22%while GlSwi6B overexpression caused chitin and β-1,3-glucan content increased about 30%and 26%,respectively.4)The results of Y1H assay and EMSA assay showed that GlSwi6B could bind to MCB element of the promoters of the chitin and β-1,3-glucan synthase related genes.These results indicate that GlSwi6 regulates cell wall integrity by affecting the synthesis of chitin and β-1,3-glucan.4.GlSwi6 co-silencing caused the ganoderic acid(GA)content decreased about 25%,and the intracellular ROS and Ca2+content reduced.GlSwi6B overexpression caused GA content increased about 45%,and the intracellular ROS and Ca2+ content increased.Further analysis showed that:1)Adding H2O2 could recover the decreased GA content of GlSwi6 co-silenced strains,in addition,adding ROS scavenger NAC could recover the increased GA content of GlSwi6B overexpression strains.2)Adding CaCl2 could recover the decreased GA content of GlSwi6 co-silenced strains.In addition,adding Ca2+antagonist LaCl3 could recover the increased GA content of GlSwi6B overexpression strains.3)Adding H2O2 could recover the decreased intracellular Ca2+ content of GlSwi6 co-silenced strains while adding CaCl2 could not recover the decreased intracellular ROS content of GlSwi6 co-silenced strains.Meanwhile,Adding ROS scavenger NAC could recover the increased intracellular Ca2+content of GlSwi6B overexpression strains while adding adding Ca2+ antagonist LaCl3 could not recover the increased intracellular ROS content of GlSwi6B overexpression strains.4)Adding H2O2+LaCl3 could not recover the decreased GA content of GlSwi6 co-silenced strains,in addition,adding NAC+CaCl2 could not recover the increased GA content of GlSwi6B overexpression strains.These results indicate that intracellular ROS and Ca2+level is involved in the GA biosynthesis regulated by GlSwi6,and ROS level may be in the upstream of Ca2+ level in the processes.In summary,we cloned the GlSwi6 gene and found that it was spliced.Gene silencing and gene overexpression were used to study the physiological role of GlSwi6 in G lucidum.The present study is helpful for understanding the function of MAPK pathway and APSES transcription factors in fungi and will provide references for other fungi.