| The development of the basidiomycete fruiting body is a highly complex process that requires coordination between genetic,environmental,and physiological factors,In the model mushroom Coprinopsis cinerea,upon nutritional depletion,single hyphae locally undergo intense branching to form primary hyphal knots in the dark.When continuously kept in the dark,these primary hyphal knots develop into sclerotia.Under a 12 h light/12 h dark rhythm,the radial growth of primary hyphal knots and hyphal interactions lead to the formation of secondary hyphal knots.The secondary hyphal knots differentiates into a tiny fruiting body primordium with distinct cap and stipe tissues.As the primordium gradually enlarges and matures,karyogamy and meiosis occur successively in basidia in the hymenium of the gills.At the same time,the stipe elongates,and the cap expands in parallel,giving rise to a mature fruiting body.Previously,our laboratory found that glucanase and chitinase can cooperate to participate in the elongation growth of the stipe.However,the mechanisms that regulate the transcription of glucanase and chitinase are still unclear.It has been reported that the transcription factor NsdD inAspergillus nidulans regulates the endo-1,3-?-glucanase egl C involved in cell wall remodeling.The purpose of this study is to explore whether there is such a NsdD transcription factor in C.cinerea that affects cell wall extension of the stipe by regulating the transcription of chitinase and glucanase.Two genes nsdD1 and nsdD2,which are homologous toA.nidulans nsdD.q RTPCR showed that the expression of genes nsdD1 and nsdD2 at the apical of the stipe was higher than that at the basal.NsdD1 or NsdD2 knockdown strains were constructed.The single knockdown of NsdD1 and NsdD2 did not exhibited any apparent phenotype associated with growth and development of the strain.Therefore the double knockdown of NsdD1 and NsdD2 transformant was constructed,and showed impaired mycelial growth and defects in fruiting bodies.q RT-PCR analysis showed that both nsdD1 and nsdD2 were strongly downregulated in the NsdD1/NsdD2-RNAi transformant.After 4d of dark cultivation mock transformant or 5d of dark cultivation for NsdD1/NsdD2-RNAi transformant until the mycelia covered the entire agar medium surface,none of the mycelia formed primary hyphal knots.After 1d of 12 h light/12 h dark rhythm cultivation or dark cultivation,all mycelia in the mock transformant,and NsdD1/NsdD2-RNAi transformant only formed similar small hyphal knots.After 2d of12 h light/12 h dark rhythm cultivation,the primary hyphal knots of the mock transformant differentiated into large secondary hyphal knots,while the primary hyphal knots of the NsdD1/NsdD2-RNAi transformant did not differentiate into secondary hyphal knots but still remained a small hyphal knot status.in contrast,after 2d of dark cultivation,all mycelia of the mock transformant and NsdD1/NsdD2-RNAi transformant produced only both primary hyphal knots and sclerotia.After 4d of 12 h light/12 h dark rhythm cultivation,the secondary hyphal knots of the mock transformant further developed to fruiting body primordia,while primary hyphal knots of NsdD1/NsdD2-RNAi transformant only developed into sclerotia.in contrast,after 4d of constant dark cultivation,all of the mock transformant and NsdD1/NsdD2-RNAi transformant only produced smaller hyphal knots and sclerotia.These data indicated that the knockdown of NsdD1/NsdD2 led to the differentiation of primary hyphal knots into sclerotia under 12 h light/12 h dark rhythm conditions.RNA-seq showed that some genes encoding predicted proteins related to hyphal knots and primordium formation were downregulated.For example,all three cyclopropane-fatty-acyl-phospholipid synthase genes cfs1-3;all three galectin coding genes cgl1-3,two S-adenosylmethionine-dependent methyltransferase genes ich1 and ich2,three of 34 hydrophobin genes hyd1-3,and three of 17 laccase genes lcc12,lcc16 and lcc7.However,three reported photoreceptor protein genes dst1,dst2 and wc-2,showed inconsistent expression changes.Three genes reported to be involved in primary hyphal knot formation,Cc.rmt1,Cc.snf5 and Cc.ubc2 did not show significant differential expression.In addition,the GH18 family chitinases and GH16 family ?-1,3-glucanases related to stipe elongation were not significantly downregulated in the NsdD1/NsdD2-RNAi transformant compared to the mock transformant.q RT-PCR analysis confirmed that the three cyclopropane-fatty-acyl-phospholipid synthase genes cfs-3,three galectin-coding genes cgl1-3,two S-adenosylmethionine-dependent methyltransferase genes ich1 and ich2,three hydrophobin genes hyd1-3,and three laccase genes lcc12,lcc16 and lcc7 were downregulated in the NsdD1/NsdD2-RNAi transformant compared to the mock transformant.The Ch IP-seq experiment revealed that NsdD2 can directly regulate the related genes cfs1,cfs2,cgl1,cgl3,and hyd1 involved in the formation of hyphal knots.MEME analysis of 1-kb 5'-upstream regions of NsdD2-target genes from C.cinerea identified a highly conserved GATC motif localized approximately-995 to-143 nt upstream from the translation start point,and this result is confirmed by EMSA.These findings supported the conclusion that these genes related to hyphal knots and primordium formation,cfs1,cfs2,cgl1,cgl3 and hyd1,were the target genes of NsdD2.after 4d of constant dark cultivation,in the mycelia of theAmut Bmut strain grown under a 12 h light/12 h dark rhythm for extra 1d the expression level of nsdD2 increased by 90.20 % and the NsdD2-target hyphal knots formation genes,cfs1,cfs2,cgl1,cgl3 and hyd1,also were correspondingly upregulated,while the expression level of nsdD1 showed little change compared with mycelia grown in constant darkness for 4d;in contrast,the expression level of nsdD1 and nsdD2 in the mycelia grown in the darkness for extra 1d decreased by 23.71 % and 23.10 %,respectively,compared with mycelia grown in constant dark for 4d.However,when the mycelia of theAmut Bmut stain were grown continuously in the constant darkness for extra 2d after 4d of constant dark cultivation,the expression level of nsdD1 and nsdD2 were also upregulated and even higher than that of the mycelia grown under light/dark rhythm for 2d after 4d of dark cultivation,and cfs1,cfs2,cgl1 and cgl3 but not hyd1,were also upregulated.This indicates that only first day of darkness or the light/dark thythm cultivation after 4d of constant dark cultivation resulted in differential expression of NsdD2 and its target genes in mycelia.To understand effect of the first day of the dark or light/dark rhythm cultivation condition after 4d of continuous dark cultivation on the mycelium differentiation,after 4d of dark cultivation,theAmut Bmut mycelia was continuously incubated in the darkness for extra 1d and then grown under 12 h light/12 h dark rhythm for 1-4d,they only formed hyphal knots and sclerotia on the first day of 12 h light/12 h dark rhythm cultivation;however,they did not further produce more sclerotia but started to form some secondary hyphal knots on the second day of 12 h light/12 h dark rhythm cultivation,which further developed into fruiting body primordia after 4d of 12 h light/12 h dark rhythm cultivation.In contrast,after 4d of constant darkness cultivation,if the culture was first grown under a 12 h light/12 h dark rhythm for 1d and then transferred to constant darkness to continuously grow for 1-4d,Amut Bmut mycelia still formed secondary hyphal knots,but the secondary hyphal knots finally developed etiolated stipes.This indicated that after 4d of dark cultivation,upon depletion of nutrients,the followed one day of dark or light/dark cultivation was vital to determination of the developmental fate of C.cinerea primary hyphal knots.In this study,overexpressing strains of NsdD1 or NsdD2 were constructed.The results showed that the overexpressing of NsdD1 did not show any apparent phenotype compared to the mock strain.more primary hyphal knots,fruiting body initiation and fruiting body appeared in the overexpression of NsdD2.However,under the condition of continuous darkness,the overexpressed strains of NsdD2 did not form secondary hyphal knots and primordia;instead,they only formed more primary hyphal knots and sclerotia.The q RT-PCR results revealed that,compared with the mock transformant,the NsdD2-OE transformant showed the high expression of nsdD2 and the apparent upregulation of its target genes cfs1,cfs2,cgl1,cgl3 and hyd1.The results showed that NsdD2 could promote the formation of secondary hyphal knots.In addition,it was also found that NsdD was involved in the formation of asexual oidia.The amount of oidia produced by the knockdown of NsdD1 and NsdD2 decreased by 43 % compared with the control,while the amount of oidia produced by overexpression of NsdD2 was 1.73 times higher than that of the mock strains.The overexpression of NsdD1 has no effect on the production of oidia,indicating only the NsdD2 is regulating the production of oidia.RNA-seq and qRT-PCR analysis showed that abaA were downregulated in the NsdD1/NsdD2-RNAi transformant.Ch IP-seq and EMSA confirmed that NsdD2 can specifically bind to the GATC motif in the promoter region of the abaA.Firstly,the complement strain ofAbaA was constructed in this study.It was found thatAbaA could compensate for the defects of NsdD1/NsdD2-RNAi strain,such as slow mycelial growth,absence of fruiting body and reduced production of asexual spores.The q RTPCR showed the expression levels of nsdD1,nsdD2,cfs1,cfs2,cgl1,cgl3 and hyd1 were all up-regulated in the complementary strains,suggesting that abaA is the downstream target gene of NsdD2 and that theAbaA may be regulating nsdD2,and thus affecting the development process of C.cinerea.This result was confirmed by DAP-seq. |
PDF Full Text Request