Mechanism Of The SsFkh1 Coordinated MAPK Protein Kinase-mediated Cell Wall Integrity Pathway Regulating The Development And Pathogenicity In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum is a homonymic vegetative plant pathogenic fungus.Sclerotinia which can infect more than 600 plants,is distributed all over the world and causes huge economic losses.Sclerotia is a dormant body rich in melanin,which can survive in the environment for a long time,which makes it more difficult to control.FKH is a transcription factor of Forkhead family.As a transcription factor with many biological functions in eukaryotes,there is little research on the role of FKH protein in phytopathogenic fungi.Previous studies found that Ss Fkh1 positively regulated sclerotium formation.Therefore,in order to reveal the sclerotium formation mechanism of S.sclerotiorum,it is of great scientific significance and application value to study Ss Fkh1's regulation pathway and regulation network for sclerotium formation in S.sclerotiorum.And the following important research results are obtained.1.Ss Fkh1 is involved in cell wall integrity pathway that regulate sclerotium development and pathogenicity.Compared with wild-type and backfeeding mutants,Ss Fkh1 gene knockout mutant(?Ssfkh1)has fewer sclerotia,lower dry weight,fewer infection cushion and weaker pathogenicity.And?Ssfkh1 is more sensitive to high concentration H₂O₂,osmotic stress Na Cl and KCl,and cell wall stimulation.These results indicated that the deletion of Ss Fkh1 affected the growth and pathogenicity of S.sclerotiorum.In addition,the cell wall integrity of?Ssfkh1 strain was destroyed,which indicated that ssfkh1 might be involved in the cell wall integrity pathway.2.Differential expression analysis of Ss Fkh1.In order to further explore the mechanism of transcription factor Ss Fkh1 in regulating the development of infection cushion and sclerotia in S.sclerotiorum,transcriptome sequencing analysis was performed on the hyphae of?Ssfkh1 and wild-type strains at different development stages.GO and KEGG enrichment analyses revealed a large number of cell wall-related components that were enriched for MAPK pathways.The MAPK kinase-mediated cell wall integrity pathway was found in the analysis of differential gene metabolic pathways.The pathway was mainly composed of four key protein kinases,namely,PKC1,BCK1,MKK1,and SLT2(SMK3).Interaction analysis between Ss Fkh1 and four proteins in this pathway revealed that Ss Fkh1 interacted with Ss Mkk1.Based on the above results,considering that the Ss Fkh1 knockout mutant was extremely sensitive to cell wall stimulation and oxidative stress,we took this as the breakthrough point to study the relationship between Ss Fkh1 and MAPK pathway and further explore the functions of genes related to MAPK cascade,especially those related to the conservative cell wall integrity pathway in S.sclerotiorum.3.Knockout CWIPs-related genes affected the hyphal growth and sclerotia development.Except that the SMkk1 knockout mutant(?Ssmkk1)showed slower hyphal growth,the mycelial growth rate of other knockout mutants has no obvious difference compared with that of wild type.Interestingly,?Ssmkk1 strain did not cover the entire PDA after three weeks of growth,but the mycelium became more and thicker.Microscopically,the mycelia of?Ssmkk1 and?Sssmk3 strains grew curved.It is speculated that this phenomenon may affect the growth rate of mycelia.The curved mycelia lead to the retardation and thickening of mycelia of?Ssmkk1 strain.In addition,compared with the wild-type,the distribution of chitin in the hypha tip of?Ssmkk1 was not uniform,indicating that the knocking out of Ss Mkk1 gene affected the distribution of chitin in the hypha tip of the mutant.The chitin content in the hypha of the mutant was decreased,resulting in the slow hypha growth of the mutant.In addition,the CWIPs-related gene knockout mutant strain produced less melanin than the wild-type strain,and the expression of genes related to melanin formation was decreased in the CWIPs-related gene knockout mutant strain,especially in the?Ssmkk1 strain.Phenotypic observation showed that?Ssmkk1 strain did not produce sclerotia,and the number of sclerotia produced by strains?Ssbck1,?Sspkc1 and?Sssmk3 was less than that of wild type.In addition,the number of sclerotia produced by?Ssbck1 strain was small,but the sclerotia size was slightly larger than that of the complement mutant,but the overall mass was lighter than that of the wild-type and complement mutant strains.4.The results of knockout CWIPs-related genes delayed development of the infection cushion.The observation of infection cushion showed that compared with the wild-type and the complement mutant,the infection cushion of the?Ssbck1,?Sspkc1,?Ssmkk1 and?Sssmk3 strains developed more slowly,did not form mature infection cushion,and the number of infection cushion was significantly reduced.In particularly,no infection structure capable of effectively infecting plants was formed at the hyphal tip of?Ssmkk1 strain.And that expression level of the gene related to infection cushion formation in the?Ssmkk1 strain is obviously reduce.These results indicated that the knock-out of Ss Mkk1 gene deprived S.sclerotiorum of the ability to form infection cushion,and the knockout of Ss Pkc1,Ss Bck1 and Ss Smk3 genes resulted in the development delay of infection cushion.5.Knockout of CWIPs-related genes weakened the virulence of S.sclerotiorum.In the pathogenicity experiment,the strain?Ssmkk1 could not infect the unwound tomato leaves but could infect the wound tomato leaves,indicating that the virulence of SMkk1 was mainly affected by the infection cushion.After Ss Mkk1 was knocked out,infection structure was produced,so the hypha could not penetrate the epidermis of tomato leaf surface,thus reducing the toxicity.?Ssbck1,?Sspkc1,and?Sssmk3 could infect the wound and unwound tomato leaves,but the lesion area was smaller than that of wild type,which was also related to the less infection cushion produced by knockout mutant.In addition,CWIPs-related gene knockout mutant strains showed varying degrees of oxalic acid content reduction and oxalic acid synthesis related gene expression significantly decreased.These results indicated that the decrease of Ss Pkc1,Ss Bck1,Ss Mkk1 and Ss Smk3 gene infection structure reduced the virulence of S.sclerotiorum;on the other hand,and the decrease of oxalic acid content,an important virulence factor,also weakened the virulence in S.sclerotiorum.6.CWIPs-related gene knockout mutant was sensitive to H₂O₂,osmotic stress cell wall stimulation.The?Ssbck1,?Sspkc1,?Ssmkk1 and?Sssmk3 strains were all sensitive to oxygen stress,osmotic stress,and cell wall irritation.The Ss Mkk1 knockout mutant was the most sensitive to H₂O₂.?Sspkc1 and?Sssmk3 were more sensitive to CFW,while?Ssbck1was more sensitive to CR.These results indicated that the cell walls of CWIPs-related gene knockout mutants were damaged.In summary,the transcription factor Ss Fkh1 is involved in cell wall integrity pathways that regulate the growth,development and pathogenicity of S.sclerotiorum.The protein interaction analysis of Ss Fkh1 and differential gene expression analysis indicated that Ss Fkh1 was related to the MAPK-mediated cell wall integrity pathway.Analysis of the function of key protein kinases in the MAPK-mediated cell wall integrity pathway in S.sclerotiorum revealed that the phenotype of the four major protein CWIPs-related gene knockout mutants in this pathway was highly consistent with that of the transcription factor Ss Fkh1 knockout mutant.Therefore,we speculated that the transcription factor Ss Fkh1 cooperated with MAPK kinase-mediated cell wall integrity pathway to regulate the growth,development and pathogenicity of S.sclerotiorum.At present,there is no report about the regulation of growth,development and pathogenicity by MAPK-mediated cell wall integrity pathway in S.sclerotiorum and few studies on transcription factor Ss Fkh1.This study has resolved the role of transcription factor Ss Fkh1 of S.sclerotiorum in the formation of regulation pathways and regulation networks to a certain extent,which provides an important theoretical basis for drugs targeting the forkhead protein regulation pathway in S.sclerotiorum and has important scientific significance and economic value for the prevention and treatment of S.sclerotiorum.