Identification Of Genes Differentially Expressed By Metarhizium Anisopliae Infect Locusta Migratoria And Construction Of CDNA Library During Colonization Of Hemolymph

Metarhizium anisopliae is an economically important insect pathogenic fungus widely used as an insect biocontrol agent. Mycoinsecticides have many advantages compared with chemical insecticides, including low toxicity to non-targeted organisms, high insect specificity, and low environmental impact, but the slow killing speed has been considered a potential drawback which prevents the utilization of these fungi against insects. M. anisopliae adheres to insects cuticle and germinates to form short germ tubes, then elaborates a specialized infection cell called appressorium that forms a thin penetration peg to initiate penetration. M. anisopliae produces a mumber of cuticle-degrading proteases to facilitate host penetration. The formation of appressorium paly an important role in fungal pathogenesis. M. anisopliae traverses insect cuticle and enters host hemolymph, then colonizes the host hemolymph. It utilizes the nutrients available in hemolymph, and produces insecticidal secondary metabolites, which cause insect death. Identification genes differentilly expressed by M. anisopliae during appressorium formation and colonization of host hemolymph would reveal the mechanisms of fungal pathogenesis.EST analysis and microarray approaches are useful and successful for studying insect fungal pathogenesis. Many researchers adopted these strategies to study the developmental and transcriptional events during M. anisopliae infection. The methodologies of their studies are that M. anisopliae were cultured in liquid minimum medium supplemented with various inducer, followed by cDNA library construction and ESTs analysis. The life of the fungus cultured in liquid medium is saprophytic, while the life of the fungus infecting insect is parasitical. There are large difference between the life of the fungus cultured in vitro with the fact that M. anisopliae naturally infect insects. It is impossible to elucidate the mechanism underlying fungal pathogenesis and adaptation to host hemolymph, furthermore to understand the host-pathogen interactions.In this study, Metarhizium anisopliae var. acridum strain CQMa102 and Locusta migratoria were used, we prepared the hyphal bodies from infected insect hemolymph without host nucleic acid contamination, and employed a reagent called RNA fragmentation buffer to degrade the locust wing nucleic acid before inoculation with M. anisopliae conidia. This study was aimed at identifying genes were up regulated during colonization of hemolymph when hyphal bodies occur, and during appressorium formation on locust wings. A normalized cDNA library of M. anisopliae during colonization of host hemolymph was successfully constructed utilizing DSN (duplex-specific nuclease) normalization method combined with SMART (switching mechanism at 5′end of RNA transcript) library construction method, ESTs analysis of cDNA library revealed new insight on the host-pathogen interactions.The main results were as follows:1. The process of M. anisopliae strain CQMa102 infect locust L. migratoria was observed with a digital microscope at experimental conditions. M. anisopliae enters and colonizes the host hemolymph within 24 h post inoculation, hyphal bodies (short hyphal lengths and yeast-like blastospores) had attached to hemocytes and appeared to stimulate hemocytes aggregation. At the time point 3-6 days, hyphal bodies become phagocytosed and encapsulated by hemocytes. Before time 24-48 h of insect death, hyphal bodies have an increase of the concentration accompanied by reduction in hemocyte counts, and freely floating in the hemolymph.2. We perfectly establish a method to lyse host hemocytes for isolating hyphal body by utilization of proteinase K and SDS according to the different cell structure between fungus and host hemocyte. The procedure takes about 40 min, and RT-PCR analysis indicates that endogenous RNases that released from membrane-bound organelles upon disruption and sequent simple washing steps could rapidly remove locust nucleic acid before fungal mRNA extraction. This assertion was further confirmed by sequent EST analysis.3. Suppression subtractive hybridization was performed here using cDNA generated from purified hyphal bodies in hemolymph as tester, and cDNA generated during conidia germination and appressorium formation on locust wing as driver. 350 positive clones were obtained by cDNA array dot blotting, sequenced, resulted in 120 unique expressed sequence tags (ESTs) that were up-regulated during colonization of hemolymph, including 100 singletons and 20 contigs. Among these 120 ESTs, 29 (24.17% of total) were significantly similar to known proteins involved in various cell and molecular processes, such as general metabolism, energy production, maintenance of cell function and structure, cell cycle and control, stress response etc.4. To confirm the reliability of SSH and dot blotting, the expression levels of five randomly chosen genes in different stages of pathogenesis were analyzed using semi-quantitative RT-PCR analysis. These ESTs included those with homology to α-1,2-mannosyltransferase,α-enolase, pisatin demethylase, prenyl transferase and choline dehydrogenase. All the five genes confirmed by RT-PCR analysis were up regulated during colonization of host hemolymph.5. To identify the genes were up-regulated during appressorium formation, suppression subtractive hybridization was also performed here using cDNA generated during conidia germination and appressorium formation on locust wing as tester and cDNA generated from purified hyphal bodies in hemolymph as driver. 460 positive clones were obtained by cDNA array dot blotting, and sequenced, resulted in 329 high quality ESTs which represented 78 uniESTs. Among these 78 ESTs, 46 with significant similarity to NCBI hypothetical proteins or known proteins, 24 ESTs represented a broad spectrum of biological functions, including protein metabolism proteins, cell metabolism proteins, cell sturctrue and function proteins and stress response proteins, etc.6. A total of 12 ESTs predicted to be up regulated expressed by SSH were tested by semi-quantitative reverse transcription PCR. For time-course experiments, total RNA were extracted from the fungi collected after 8 h, 15 h, 24 h, 30 h from locust wings respectively and the mixture of host hemocytes and hyphal bodies ( 2 days, 3 days, 4 days, 5 days, 6 days, 7 days). Total RNA were treated by DNAseⅠbefore RT-PCR. The glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was selected as the endogenous control. The results demonstrated that these genes all were up-regulated during appressorium formation on locust wing.7. A normalized cDNA library of M. anisopliae during colonization of host hemolymph was successfully constructed utilizing DSN (duplex-specific nuclease) normalization method combined with SMART (switching mechanism at 5′end of RNA transcript) library construction method. 5222 clones were sequenced, and a total of 4606 ESTs of high quality was obtained, resulted in 1863 unique ESTs, including 1237 singletons and 626 contigs. The ESTs had significant similarity to known proteins involved in various cell and molecular processes, such as general metabolism, energy production, maintenance of cell function and structure, cell cycle and control, stress response etc.8. A full length cDNA library of M. anisopliae during colonization of host hemolymph was successfully constructed utilizing SMART (switching mechanism at 5′end of RNA transcript) library construction method. Clones were sequenced, and a total of 187 unique ESTs was obtained, which represented the high abundant genes during the colonization of host hemolymph.