Studies On Molecular Mechanisms For Response To The Phytotoxin Oxalic Acid In The Mycoparasite Coniothyrium Minitans

Sclerotinia sclerotiorum?Lib.?de Bary is a notorious plant pathogenic fungus,which can cause sclerotinia rot of many economic crops,resulting in severe economic losses.Coniothyrium minitans is an important biocontrol agent,which is an obligate mycoparasite of S.sclerotiorum.Oxalic acid is a key virulence factor of S.sclerotiorum,with the characteristics of acidity,reducibility and chelating calcium ion.Previous studies showed that,in the process of interaction between S.sclerotiorum and C.minitans,oxalic acid secreted by S.sclerotiorum can induce C.minitans to produce oxalate decarboxylase Cm Oxdc1 and antifungal substances to suppress S.sclerotiorum.After degradation of oxalic acid by Cm Oxdc1,the ambient p H increased to weakly acidic and neutral p H,which significantly up-regulated gene expression of mycoparasitic related enzymes?such as chitinase,glucanase,and protease?by Cmpac C signaling pathway.These results suggested that oxalic acid play a crucial role in the interaction between C.minitans and S.sclerotiorum.However,the molecular mechanism of C.minitans in response to oxalic acid stress is still unknown.Therefore,a study was conducted to analyze the transcriptome of C.minitans response to oxalic acid stress,to determine the function of a new oxalate decarboxylase gene Cm Oxdc3 and a calcineurin-responsive zinc finger transcription factor gene Cm CRZ,and to analyze the b ZIP gene family which regulates the redox balance.Results are described as follows:1.The transcriptome result of C.minitans in response to 24 m M oxalic acid at different time points?0 h,20 min,1 h,3 h,6 h,9 h?showed that 92 genes were significantly down-regulated at 5 time points after exposure to oxalic acid,while 165genes were significantly up-regulated.These differentially expressed genes were mainly enriched in the pathway of redox balance,Ca²⁺signaling,cell proliferation and organic metabolism,which means these genes may play important roles in response to oxalic acid stress.2.Based on transcriptome result,a new oxalate decarboxylase gene Cm Oxdc3 was selected for further study.The expression patterns and gene functions of Cm Oxdc3 and Cm Oxdc1 were compared.RT-q PCR results showed that Cm Oxdc3 was only up-regulated by oxalic acid,malonic acid and hydrochloric acid,while Cm Oxdc1 was induced by a variety of acids.The results of oxalic acid degradation assay suggested that Cm Oxdc3 played a key role in the degradation of high concentration of oxalic acid?24m M?,but Cm Oxdc1 had a major effect on the degradation of low concentration of oxalic acid?12 m M?.Deletion of Cm Oxdc3 in C.minitans reduced its ability to parasitize the sclerotia of S.sclerotiorum.Subcellular localization showed that Cm Oxdc3 was localized in cytoplasm.After oxalic acid stimulation,Cm Oxdc3 moved to vacuole.The signal peptide of Cm Oxdc1 was confirmed by yeast secretion system.These results indicated that Cm Oxdc3 may degrade oxalic acid in the cytoplasm,while Cm Oxdc1 may degrade oxalic acid in extracellular.3.Because oxalic acid can capture and chelate Ca²⁺,the function of the calcineurin-responsive zinc finger transcription factor gene Cm CRZ were analyzed.Deletion of Cm CRZ caused serious defects in mycelial growth,colony morphology,conidiation,melanin deposition and mycoparasitism.Cm CRZ deletion mutant was more tolerant to oxalic acid stress,and low concentration oxalic acid?8 m M?can restore the defects in growth and conidiation.Acid-base sensitivity test found that Cm CRZ deletion mutant was more tolerant to acidic environment,but more sensitive to alkaline environment.The knockout of Cm CRZ resulted in a significant decrease in the expression of ROS scavenging related genes,a significant increase in intracellular H₂O₂ level,enhanced tolerance to oxidants?H₂O₂ and VK₃?,and increased secretion of extracellular oxidants.Besides,the Cm CRZ deletion mutant displayed inhibitory effect on mycelial growth of S.Sclerotiorum.The inhibition zone can be reduced by adding antioxidant?Vitamin C?,and disappeared when adjusted to acidic environment?p H 4?,suggesting that the inhibition zone may be caused by large amount of the oxidative substances secreted by Cm CRZ deletion mutant.The metal ion sensitivity test showed that Cm CRZ deletion mutant was more sensitive to Na⁺?Ca²⁺and Mn²⁺,while Mg²⁺could restore the growth and conidiation of the mutant.These results suggested that Cm CRZ play an important role in growth,conidiation,redox balance,p H sensing,Ca²⁺signaling,antibiosis and mycoparasitism in C.minitans.4.Oxalic acid is mainly used as a reducing agent,which may disturb the intracellular redox balance of C.minitans.We analysed the b ZIP transcription factor gene family in C.minitans,which regulates the redox balance.A total of 34 b ZIP genes were identified from the C.minitans genome.The intron position in b ZIP domain was highly conserved.There were significant expression differences of Cmb ZIP genes under oxalic acid stress,other abiotic stresses,and conidiation.Four Cmb ZIP genes were knocked out,but three of them?Cmb ZIP07,Cmb ZIP09,Cmb ZIP13?had no effect on the growth,conidiation,and sensitivity to oxalic acid.Only Cmb ZIP16 deletion mutant showed more sensitive to H₂O₂,suggesting that Cmb ZIP16 played an important role in response to oxidative stress.In conclusion,the function of the oxalate decarboxylase gene Cm Oxdc3 was identified in this study.We found that Cm Oxdc3 was mainly responsible for degrading oxalic acid in the cytoplasm.The calcineurin-responsive zinc finger transcription factor gene Cm CRZ has been proved to play an important role in the interaction between C.minitans and S.sclerotiorum.The b ZIP gene family in C.minitans was analysis,and Cmb ZIP16 was involved in oxidative stress response.Taken together,these findings enhance our understanding of molecular mechanism of C.minitans response to oxalic acid,and lay a foundation for revealing the molecular mechanisms of the interaction between C.minitans and S.sclerotiorum.