Molecular Mechanism Of Metarhizium Rileyi Evading Host Immune Responses

Entomopathogenic fungi infect their host insects via penetrating the insect’s cuticle.Once entomopathogenic fungi reach the insect hemocoel,they will face the defense of the host’s efficient and powerful immune system.The insect immune response begins with the recognition and differentiation of nonself components by pattern recognition receptors（PRRs）.Once conserved pathogen-associated molecular patterns（PAMPs）on the pathogen are recognized by the insect PRRs,then the innate immune system,which consists of humoral and cellular immunity,is activated and rapidly eliminates the pathogen.Entomopathogenic fungi,after entering the host insect hemocoel after infection,will switch to yeastlike hyphal bodies,which use this special cell morphology to suppress or evade the immune response of the host.The suppression of immune response is mainly achieved by secreting secondary metabolites and proteins with immunosuppressive activities into the hemolymph of the host,Whlie the immune evasion response is achieved by reducing or hiding PAMPs on the cell wall surface of the hyphal bodies to avoid recognition by the host immune system.Up to date,there are many researches on entomopathogenic fungi inhibiting host immune response,and the mechanism is well understood.However,there are few researches on the immune response of evasion,and the mechanism of this aspect is not very clear.In this paper,Metarhizium rileyi,an entomopathogenic fungus that mainly parasitises lepidopterans,especially noctuid pests,was studied from many aspects to explore the strategies of M.rileyi to evade host insect immune response,focusing on revealing the molecular mechanism of M.rileyi to evade host immunity during the parasitism process.Main results are described as follows:1.Hyphal bodies escape host’s cell immune responses.Entomopathogenic fungi evade host immune defense in the form of hyphal bodies.In order to explore the mechanism of hyphal bodies immune evasion,we compared the cellular immune response of Helicoverpa armigera to M.rileyi conidia and hyphal bodies and the recognition ability of PRRs of H.armigera to M.rileyi.The result showed that the lethal medium concentration(LC₅₀)of the hyphal bodies was significantly lower than that of the conidia.The phagocytosis rate and nodule number of hyphal bodies by H.armigera were significantly lower than conidia,indicating that hyphal bodies had a strong ability to evade host cell immunity.Western blot results showed that fiveβ-1,3-glucan recognition protein（r HaβGRPs）and one C-type lectin 3（r Ha CTL3）could bind to conidia,but r HaβGRP2b and r HaβGRP4 could not bind to the hyphal bodies.Although the other four r Ha PRRs were able to bind to hyphal bodies,they were weaker than conidia.Immunocytochemical results showed that the fluorescence intensity of six r Ha PRRs to hyphal bodies was significantly weaker than that of conidia.These results suggest that hyphal bodies can evade the recognition of Ha PRRs.Transmission electron microscopy（TEM）showed that highly ordered brush-like structures existed only on the outermost layer of the cell wall of hyphal bodies,and the thickness of the cell wall of hyphal bodies was significantly thinner than that of the conidia.Based on the above experimental results,we hypothesized that the mechanism of hyphal bodies escaping host cell immune responses was due to two aspects,on the one hand,the composition or quantity of PAMPs of the hyphal bodies is reduced,on the other hand,the cell wall of hyphal bodies is covered with a substance that masks PAMPs,allowing the hyphal bodies to evade the host immune response.2.MrCWP camouflages PAMPs on cell wall,allowing hyphal bodies to escape from host recognition and cellular immune responseThere were highly ordered brush-like structures on the outermost layer of the cell wall of hyphal bodies of M.rileyi.Seven surface proteins of the cell wall of M.rileyi were identified after treatment with dithithreitol（DTT）.We investigated one of the cell wall proteins,known as MrCWP.In order to further explore the biological function of MrCWP,the knockout strainΔMrCWP and the overexpression strain MrCWP^OE were constructed,and the recombinant protein r MrCWP was purified by prokaryotic expression.We found that knocking out MrCWP gene resulted in thinning of cell walls of both conidia and hyphal bodies,slow proliferation of hyphal bodies in hemocoel of H.armigera,and decreased virulence of the strain.On the contrary,the overexpression of MrCWP gene resulted in the thickening of cell walls of both conidia and hyphal bodies,the acceleration of proliferation of hyphal bodies and the enhancement of virulence of the strain.In addition,the expression of HaβGRPs in hemocytes of H.armigera increased after injection ofΔMrCWP,and the phagocytosis and nodulation ofΔMrCWP conidia and hyphal bodies by H.armigera were enhanced.On the contrary,the expression of HaβGRPs of H.armigera decreased after injection of MrCWP^OE hyphal bodies,and the phagocytosis and nodulation of MrCWP^OE conidia and hyphal bodies were weakened,suggesting that MrCWP promoted M.rileyi to escape from host cellular immunity.The binding of r MrCWP to hyphal bodies ofΔMrCWP was enhanced,but the binding of r MrCWP to hyphal bodies of MrCWP^OE was not detected.The binding of r Ha CTL3 to hyphal bodies ofΔMrCWP was enhanced,while the binding of of r Ha CTL3 to hyphal bodies of MrCWP^OE was weakened.Wild-type（WT）conidia preincubated with r MrCWP reduced the activation of HaβGRPs in the host,promoted the proliferation and colonization of hyphal bodies in the host hemocoel,accelerated the death of the host,and enhanced virulence.Moreover,r MrCWP promoted the escape of WT andΔMrCWP strains from host phagocytosis and nodule action.These results suggest that MrCWP can evade host recognition and immunity by masking PAMPs includingβ-1 and 3-glucan,promote proliferation and colonization of the hyphal bodies in the host hemocoel,accelerate the death of the host,and enhance pathogenicity.3.MrGA degradesβ-1,3-glucan on the cell wall,allowing hyphal bodies to escape from host recognition and cellular immune responseAnother cell wall surface protein,beta-1,3-endoglucanase（MrGA）,was identified by DTT treatment.We found that the cell walls of conidia and hyphal bodies ofΔMrGA were thickened,which is speculated that the deletion of MrGA gene causes the inadequacy of the decomposition ofβ-1,3-glucan,which leads to the accumulation ofβ-1,3-glucan on the cell wall.The median lethal time（LT50）ofΔMrGA was significantly prolonged and virulence was decreased in both injection and natural infection,the virulence ofΔMrGA was decreased,and the proliferation rate of hyphal bodies ofΔMrGA in the host hemocoel was slowed down.The phagocytosis and nodulation of H.armigera onΔMrGA conidia and hyphal bodies were enhanced.It was demonstrated that MrGA gene helps M.rileyi evade host cell immunity.Preincubating WT conidia with r MrGA reduced the activation of HaβGRPs in the host,promoted the proliferation and colonization of hyphal bodies in the host hemocoel,accelerated the death of the host,and enhanced virulence.In addition,the phagocytosis and nodulation of H.armigera on r MrGA treated hyphal bodies were significantly decreased.These results suggest that MrGA,as aβ-1,3-glucanase,degradesβ-1,3-glucan on cell wall,reduces the recognition of hostβGRPs by reducing the content ofβ-1,3-glucan,enables hyphal bodies to evade host immunity,promotes the proliferation and colonization of hyphal bodies in the host hemocoel,accelerates host death,and enhances the pathogenicity..The results provide new insight into the strategies used by entomopathogenic fungi to evade insect immune responses.4.Mr Hog regulates hyphal bodies to evade host cell immune responseMAPK signaling pathway regulates the synthesis and distribution of carbohydrate and other components in fungal cell walls,and we speculate that this pathway may also be involved in the regulation escape host immunity of hyphal bodies of M.rileyi.To verify this hypothesis,we studied the Mr Hog gene,a member of the MAPK pathway of M.rileyi.The results showed that knockout Mr Hog gene resulted in a significant acceleration of conidial germination and decreased conidial production.Knockout of Mr Hog gene resulted in prolonged LT₅₀ and decreased virulence in both natural and injectable infections,as well as decreased proliferation rate of hyphal bodies.The phagocytosis and nodulation of H.armigera onΔMr Hog conidia and hyphal bodies were significantly increased.These results indicated that Mr Hog gene promoted the production of conidia,promoted the escape of insect cell immunity,enhanced the proliferation and colonization of hyphal bodies in the host hemocoel,and enhanced the pathogenicity.5.Mr Wet negatively regulates hyphal bodies to evade host cellular immunityThe central developmental pathway（CDP）,Brl A-Aba A-Wet A,regulates the synthesis of fungal cell wall components.We speculate that this pathway may also be involved in the regulation escape host immunity of hyphal bodies of M.rileyi.To verify this hypothesis,we investigated the function of Mr Wet,the gene at the end of this pathway in M.rileyi.The results showed that after Mr Wet gene knockout,the conidial cell wall was thickened,conidial germination and colony growth rate were accelerated,sporulation was increased,the proliferation rate of hyphal bodies in the host hemocoel was accelerated,and virulence was enhanced.Overexpression of Mr Wet gene resulted in thinning of conidial cell wall,delayed conidial germination,slow down of colony growth,slow down of hyphal bodies proliferation and reduced virulence.The nodules ofΔMr Wet conidia and hyphal bodies by H.armigera decreased,and the phagocytosis and nodules of Mr Wet^OE conidia and hyphal bodies by H.armigera increased.These results indicate that Mr Wet gene plays a negative regulatory role in conidial production,conidial germination,escape of insect cell immune response,proliferation and colonization of hyphal bodies in the host hemocoel,and pathogenicity.In conclusion,M.rileyi evades the recognition of the host immune system by covering PAMPs on the cell wall surface of the hyphal bodies and reducing or altering the content or distribution of PAMPs on the hyphal bodies cell wall,so that the hyphal bodies can evade the host immune response.The results of this study comprehensively reveal the molecular mechanism of entomopathogenic fungi to escape the immune response of host insects,deepen our understanding of the pathogenic mechanism of entomopathogenic fungi,and provide theoretical support for better use of entomopathogenic fungi in pest control in production practice.