| The sexual reproduction of fungi is very important for fungal life history,and its reproductive mode has always been the focus and difficulty of research.Most of the fungi are asexually propagated,and very few have sexual types.Stemphylium spp.is an important component of the filamentous fungi,and most of Stemphylium spp.are pathogenic to farmland economic crops,which can cause a variety of diseases.Stemphylium eturmiunum is a very rare species that has sexual reproduction,which makes it a good research material.Investigating the developmental regulation process of the genus is of great significance for the prevention and treatment of diseases.There are two pheromone precursor genes(a and ?)and two pheromone receptor genes(STE2 and STE3)in most kinds of fungi.The pheromone receptor genes play key regulatory roles in sex cell recognition,mating and post-mating activities,and activate the sexual reproduction of fungal atypical cells.The receptors and the precursors of pheromone recognize each other,specifically bind,induce the expression of the downstream gene,initiate the mating signal,and cause the vegetative growth of the fungus to stagnate and the reproductive process to start.There are few studies on pheromone receptors in non-model species of filamentous fungi.This study explores the mechanism of STE2 and STE3 on the asexual growth and sexual development of S.eturmiunum.In this study,S.eturmiunum was used as experimental material to identify and clone the pheromone receptor genes(STE2 and STE3)in the whole genome of S.eturmiunum based on the reported pheromone receptor gene information and software analysis.The pheromone receptor gene was knocked out by the homologous recombination and agrobacterium-mediated genetic transformation,and single knockout and complementated transformants of STE2 and STE3 are obtained.The wild-type strain,the knockout transformants and the complementated transformants are simultaneously induced under conditions favorable for producing a sexual state.Under the conditions of this study,it is found that the wild-type hyphae grow vigorously,and the conidia grow in two weeks,and the mature asci and ascospores are produced in four weeks.DAPI staining shows uniform distribution of wild-type hyphae and ascospores.The color of the STE2 knockout transformant(?STE2)colony becomes lighter,the mycelial growth rate is reduced by 20%,almost no conidia and asci are produced.DAPI staining shows that the number of nuclei in the ?STE2 hyphae decreases and unevenly distributes;while the mycelial growth rate of STE3 knockout transformant(?STE3)does not change significantly compared with the wild type.The conidiophores are deformed and the conidia become cluster,the number of conidia is increased,the single spore becomes smaller,the number of diaphragm becomes less;and only the dysplastic asci and the reduced number of ascospores can be founded.DAPI staining shows that the number of nuclei in the ?STE3 hyphae decreases,evenly dispersed.The number of nuclei in ascospores is significantly reduced and unevenly distributed.It suggests that the pheromone receptors STE2 and STE3 affect both the asexual reproduction and its sexual development of Stemphylium eturmiunum,and the functions of STE2 and STE3 are inconsistent.Phenotypic observationnof the complemented transformants of the two genes shows that: under the condition of plate culture,the colony growth rate,mycelial growth,and fluffy recovery are consistent with the wild-type period.Microscopic observation shows that the production of conidia,asci and ascospores in the conserved transformants basically restores the growth state of the wild type.DAPI staining shows that the nuclear distributions of complemented transformants restore,which is basically consistent with WT.It confirms that the pheromone receptors STE2 and STE3 in S.eturmiunum have important effects on filamentous growth,asexual and sexual reproduction.To further explore the regulation mechanisms of pheromone receptors STE2 and STE3 in Stemphylium eturmiunum,RNA-seq analysis of wild-type vegetative growth stage,sexual reproduction stage and two single knockout transformants are performed.The results shows that the total number of differentially expressed genes between ?STE2 sample and WT vegetable stage is 2513,of which 1,724 are up-regulated and 789 are down-regulated;the total number of differentially expressed genes between ?STE3 sample and WT vegetable stage sample is 1804,of which 1115 are up-regulated and 689 are down-regulated;the total number of differentially expressed genes between ?STE2 sample and WT sexual stage is 2,890.Among them,1650 are up-regulated and 1240 are down-regulated;there are a number of differentially expressed genes between ?STE3 sample and WT sexual stage sample.The total amount is 3369,of which 1657 are up-regulated and 1712 are down-regulated.The transcriptome results show that the pheromone receptor STE2 influnces more genes in the vegetative growth stage;the pheromone receptor STE3 influnces more genes in the sexual reproduction stage.In addition,although in the sexual stage,?STE2 affects fewer genes than ?STE3,it has a greater difference in traits than wild type.It suggests that genes with significant changes in the STE2 transcriptome may contain more critical genes.From the RNA-seq results,we found 114 genes with large changes in expression that may interact with the pheromone receptor genes to regulate asexual and sexual development,and screened 5 genes with obvious changes in expression : glycoside hydrolase family 32 protein(GHF32)and heat shock protein 42(HSP42)have significant changes in both ?STE2 and ?STE3 transformants.Laccase(LAC),2-pheromone precursor protein(PPG?)and apoptosisinducing factor 2(AIF2)changes only significantly in ?STE2.These 5 genes were silenced by RNA interference to obtain transformants with a silencing efficiency of 70% or more.Silent transformants have obvious phenotypic changes,and the growth rate is affected to some extent.The HSP42 silent transformants shows obviously distorted and deformed conidiophores,the growth rate of asci slows down.The GHF32 protein silent transformants have long conidia(wild type is round),the longitudinal diaphragms are basically disappeared;the immature ascospores are produced and the ascospore spores cannot be produced.Silent transformants of laccase,PPG? and AIF2 are induced under the same conditions at the same time,there no conidia and ascus are produced.Silencing experiments show that laccase,HSP42,GHF32,PPG? and AIF2 can affect the asexual growth and sexual development of S.eturmiunum,and the effects of laccase,PPG? and AIF2 on asexual reproduction and sexual development are more pronounced and more complete than HSP42 and GHF32.To further explore the relationship between pheromone receptors and laccase,HSP42,GHF32,PPG? and AIF2,yeast double-hybrid,pull-down and MST technologies are used to verify their interactions.The experiment proves that the GHF32 and HSP42 interact with the pheromone receptors STE2 and STE3,laccase,PPG? and apoptosis inducing factor 2 interact with STE2 and do not interact with STE3.This is consistent with the trend of expression changes shown in RNA-seq results.The results shows that the GHF32 and HSP42 may cooperate with the pheromone receptors STE2 and STE3 to regulate the asexual reproduction and sexual development of S.eturmiunum,laccase,2-pheromone precursor and apoptosis inducing factor 2 only interact with the pheromone receptor STE2,and synergistically regulate the asexual reproduction and sexual development of S.eturmiunum,which is consistent with the phenotypic traits of the pheromone receptor STE2 and STE3 single knockout transformants.In summary,pheromone receptor STE2 interacts with GHF32,HSP42,laccase,2-pheromone precursor and apoptosis inducing factor 2,playing a key role in the asexual and sexual development of S.eturmiunum;the pheromone receptor STE3 interacts only with GHF32 and HSP42,playing a supporting role.These results lay a good foundation for further study on the regulation mechanism of the asexual and sexual development of filamentous fungi. |
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