Functional Analysis Of Adenylate Cyclase Gene From Magnaporthe Oryzae In Thermomyces Lanuginosus

M.oryzae is one of the most important diseases on rice,it seriously affects the yield and quality of rice,causing serious economic losses and threatening global food security every year.It is of great significance to study rice blast.Adenylate cyclase-related protein is a key enzyme in the cAMP pathway.It converts ATP to cAMP,and cAMP is the second intracellular messenger,which is critical in signal transduction.Adenylate cyclase?CAP?in M.oryzae is reported to be involved in mycelial growth,sporulation,and attachment cell formation and also regulates cAMP levels intracellular.Preliminary research in the laboratory found that there are two homologous proteins in the Colletotrichum higginsianum that are similar to the structure of the M.oryzae MoCAP protein.When the two C.higginsianum genes were added back to the CAP mutant of T.lanuginosus,it was found that the two genes regulate different functions,and it is guessed that there are two segments with different functions in MoCAP.With the advantages of rapid growth,efficient spore production and mature transformation system of T.lanuginosus,we hope to study the function of different functional segments by replenishing the entire segment and two different segments of M.oryzae MoCAP gene into the CAP mutant of T.lanuginosus.To study the function of different functional sections,and the functional difference of MoCAP in the T.lanuginosus.Adenylate cyclase-related protein is a key enzyme in the cAMP pathway.It converts ATP into cAMP and plays an important role in the signal transmission process of cell perception.Adenylate cyclase?CAP?in M.oryzae is reported to be involved in mycelial growth,sporulation,and attachment cell formation and also regulates cAMP levels intracellular.Preliminary research in the laboratory found that there are two homologous gene in the Colletotrichum higginsianum that are similar in structure to the rice blast fungus MoCAP.At the same time,the CAP gene in T.lanuginosus is knocked out.When two segments of C.higginsianum genes were added back into T.lanuginosus CAP mutant??CAP?,they can restore the function of?CAP to varying degrees,Two genes were found to regulate different functions.It is guessed that there are two different functional segments in MoCAP.With the advantages of rapid growth?efficient spore production and mature transformation system of T.lanuginosus,we hope to study the genes and different functional sections of the M.oryzae by replenishing the entire segment and sub-segment of the MoCAP gene in M.oryzae into T.lanuginosus.Through the early acquisition of CAP knockout mutants of T.lanuginosus,their own genes were supplemented to obtain?CAP/CAP.By comparing with the analysis of two proteins of C.higginsianum to M.oryzae,the N-terminal gene?amino acid sequence 1-347,hereinafter referred to MoCAP-N?and the carbon-terminal gene?amino acid sequence 347-533,hereinafter referred to MoCAP-C?of M.grisea are supplemented into?CAP mutant.Then two complementary mutants?CAP/MoCAP-N and?CAP/MoCAP-C are obtained.As a control,the full length of the M.grisea MoCAP gene was replenished into the?CAP mutant to obtain?CAP/MoCAP.Comparative phenotypic analysis show that when the MoCAP gene of M.oryzae replenishing into the?CAP mutant,the defects?growth rate,spore production and lifespan?of the?CAP were restored,which again showed the functional similarity between the homologous CAP genes?MoCAP supplementation to Saccharomyces cerevisiae CAP mutants can also restore their defects?.At the same time,the segment complementary mutants?CAP/MoCAP-N and?CAP/MoCAP-C can partially recover the vegetative growth defects of?CAP.However,in terms of sporulation,the two sections of M.oryzae showed significant functional differences.The spore production of?CAP/MoCAP-N is greatly reduced,even lower than that of T.lanuginosus mutant?CAP,while the spore production of?CAP/MoCAP-C has increased,reaching closely the level of wild-type sporulation,showing that the N-terminal segment of the MoCAP gene has a negative regulatory effect on sporulation,while the carbon-terminal segment of MoCAP has a positive regulatory effect.Under the same nutritional conditions,the formation of a large number of spores will inevitably lead to defects in vegetative growth.So this study also compared the lifespan of each mutant,studies have confirmed that the growth rate of?CAP/MoCAP-C is significantly reduced under continuous culture conditions,close to stagnation,show that the life span of it is reduced.On the other hand,the lifespan of?CAP/MoCAP-N increased slightly,which was higher than that of?CAP knockout mutant,which confirmed the negative correlation between mycelial growth and sporulation.In addition,exogenous cAMP can restore the spore production of?CAP/MoCAP-N to a certain extent,but it cannot fully achieve the wild-type production.It is speculated that the MoCAP-C gene in M.oryzae does play a role in the production of cAMP.Adversity experiment analysis showed that each mutant had similar growth restriction as the wild type,indicating that MoCAP and its segments were not sensitive to H₂O₂in the T.lanuginosus and had nothing to do with cell wall integrity.In summary,MoCAP of M.oryzae is functionally similar to CAP in T.lanuginosus.M.oryzae MoCAP and its two segments are related to vegetative growth;the MoCAP-N functional segment?amino acid sequence 1-347?is a negative regulatory segment of spore formation and increases the lifespan of mycelium,while MoCAP-C functional segment?amino acid sequence 374-531?is a positive regulation fragment of spore formation and reduces the lifespan of mycelium.The two fragments play an integration effect in the full-length MoCAP gene and participate in the hypha growth and development spore formation and aging process in T.lanuginosus;MoCAP is related to cAMP synthesis and regulates intracellular cAMP levels in T.lanuginosus.