Research On The Response Of Entomopathogenic Fungi Hirsutella Satumaensis To Two Insect Hormones

As an important part of the biological pesticide industry,the fungal insecticides are entomogenous fungi with a broad range of host and could permeate the insect cuticle directly to kill insects.However,the entomogenous fungi are sensitive to adverse environment factors,which limit their widespread use in the field seriously.Thus,a comprehensive understanding of the molecular mechanism involved in the process of infestation to insects is essential for commercial development and genetic characters improvement of these biocontrol fungi.Currently,it has showed that insect hormones can affect the immune system of insects,but it is not clear whether there is a relationship between insect hormones and entomogenous fungi on the environmental adaptation and colonization process in insect haemocoele directly.Our previous studies found that induction culture of ecdysterone and juvenile hormone can cause changes in the phenotype of the entomogenous fungus Hirsutella satumaensis,which is manifested by significant increase in the production of fungal pigment and the thickness of conida mucilage,but the detailed mechanism is not clear.In order to explain the phenomenon that insect hormone regulates the mucilage production,this paper aim to reveal the response molecular mechanism of H.satumaensis to two insect hormones through the transcriptome and the mucilage proteomics analysis.Except for the secreted proteins,some insect hormone metabolism genes in H.satumaensis are also involved in the response process.For the first time,an ecdysone inactivated enzyme gene Hsegt was isolated and identified from the 20E induced group,and the function of this gene was further studied.In addition,the lectin proteins were also involved in the response process.Hscal1,a lectin-like protein,was isolated and identified from the 20E treatment group.Functional studies showed that this protein could bind Ca²⁺to play an active role and identify trehalose specifically.The main results showed that there was a direct interaction between the two insect hormones and the fungi,which developed a specific adaptive strategies to cope with the physiological stress caused by hormones.One goals of this paper is to provide a new clue for exploring the mechanism of entomopathogenic fungi in response to insect hormones,and another is to expect to achieve a new breakthrough on understanding the mechanisms of entomopathogenic fungi in adapting to the internal environment of insect haemocoele.The main results are as follows:1.Molecular basis of the response of H.satumaensis to two insect hormonesThere is a direct interaction relationship between the H.satumaensis and ecdysterone and juvenile hormone.Referring to the hormones concentration level in silkworm,400 ng/mL concentration of 20E and 250 ng/mL of JH were used to induce the culture of H.satumaensis respectively and then analyzed by transcriptome and proteome.Our results showed that most of the differentially expressed genes induced by hormones interrelated with the PHI,indicating that response to hormones is one of the most important processes in the colonization of fungal insects.The response of the fungi to 20E was mainly focused on the initiation of host infection mechanism and the metabolism of amino sugars and other important biological processes.The differentially expressed proteins in the JH treatment group were mainly involved in the process of ribosome anabolism and protein transcription and translation.In addition,exocrine proteins were also involved in the response,and 45 proteins were co-expressed in the two treatment groups and most of them were up-regulated,which was consistent with the phenotypic phenomenon of increased mucilage thickness.There are specific strategies for the response of H.satumaensis to insect hormones.After hormone induction,it was found that the secretory Fibrohexamerin with high homology to the host silkworm was significantly highly expressed.This protein could also be identified in mucilage,which indicating that this kind of protein responded to insect hormone interaction.Firstly,two insect hormone metabolic pathways were identified from the secondary metabolic pathways of the differentially expressed genes and four insect hormone metabolization-related genes were screened out from fungi,including ecdysin inactivation enzyme and juvenile hormone synthesis pathway key enzyme,which were induced and highly expressed by the two insect hormones respectively.The discovery of these genes further confirmed that there are a direct relationship between entomogenous fungi and insect hormones.2.Composition of extracellular mucilage of H.satumaensis and its effect on spore surface propertiesThe mucilage significantly affected adhesion and toxicity of conidia.Extracellular mucilage of H.satumaensis was extracted with a mild method and the extraction conditions were optimized.The extraction method as follows:the spore concentration was 5×10⁷ conidia/mL,2mM DTT was extracted at a low temperature for 24 h.After mucilage extraction,the hydrophobicity and adhesion rate of conidia cells surface were significantly decreased.This may be due to the destruction of adhesion recognition protein,appressorium morphogenetic proteins and insect cuticle degrading enzymes in mucilage,resulting in a decrease in virulence.The composition of mucilage is closely related to its function.Using Label-free protein spectrum method,the composition of conidia mucilage was identified at normal growth condition.After associated with the transcriptome data,we found that there are 474 proteins of extracellular mucilage in H.satumaensis were identified,including 7 adhesion related proteins,13 insect cuticle degrading and appressorium formation enzymes,7 proteins directly involved in the defense mechanism and 2 insect hormone metabolism related enzymes.Mucilage also contains sugars,including neutral,alkaline,and alkaline insoluble sugars,which may be present in the form of sugar-protein binding to improve protein stability and environmental resistance.Mucilage contains lectin-like proteins.Non-denaturing method was used to extract the extracellular mucilage of the conidia of H.satumaensis.After further separation and purification,a component Sl with high agglutination activity?128 unit?was obtained from Sephades g-100 molecular sieve chromatography.SDS-PAGE detected that the molecular weight of the component was 32 KDa.A legume-like lectin?HSL?protein was identified by mass spectrometry finally.Lectin can be involved in cell recognition and adhesion,which indicating that the mucilage is also involved in host recognition.3.The biological function of ecdysone inactivating enzyme Hsegt and lectin protein Hscal1.The genetic transformation system of H.satumaensis was established.The herbicide Phosphinothricin?PPT?and Chlorimuron ethyl?sur?were used as the resistance markers for H.satumaensis.After screening experiment,the optimal inhibitory concentration of PPT was 0.5?L/mL and Sur was 150?g/mL.The method of agrobacterium-mediated transformation of H.satumaensis was optimized,and the over-expression vector of green fluorescent protein and HtA virulently protein was successfully transferred into the wild type strain respectively and expressed stably.Based on above work,the genetic transformation system of H.satumaensis was constructed successfully.Function analysis of Hsegt and Hscal1.The important response gene Hsegt was annotated as a kind of ecdysone inactivation of enzyme?Ecdysteroid UDP-glucosyl transferase?,which belongs to egt superfamily.Based on the genetic transformation system of H.satumaensis,we have obtained Hsegt gene disruption??Hsegt?,over-expression?OE-Hsegt?and reverse complementation?Comp?strains respectivly.Biological function analysis of the gene showed that Hsegt gene had no effect on the virulence of the strain after disrupted,but the virulence of the strain was enhanced after the gene overexpression.It is showed that Hsegt gene has the inactivation effect on 20E,and the up-regulated expression of this gene in the host can inhibit the concentration of 20E in the worm,as well as the up-regulated expression of insect-related anti-fungal genes and the activity of phenol oxidase.Thus the host's immune response was weakened,which further causing the imbalance of bacterial growth,mass reproduction and accelerating the death of the host.In addition,lectin-like protein Hscal1,one protein that specifically responds to insect ecdysone weighted with 45.21 kDa molecular,was expressed in vitro and identified as Concanavalin A-like lectin/glucanase,belonging to the C-type lectin family.This protein has clotting activity,Ca²⁺binding activity and trehalose binding activity,which may be involved in the nutrition recognition and metabolism of fungi in insect haemocoele.