[Show abstract][Hide abstract] ABSTRACT: The migratory locust, Locusta migratoria manilensis, is an immensely destructive agricultural pest that forms a devastating and voracious gregarious phase. The fungal insect pathogen, Metarhizium acridum, is a specialized locust pathogen that has been used as a potent mycoinsecticide for locust control. Little, however, is known about locust immune tissue, i.e. fat body and hemocyte, responses to challenge by this fungus.
RNA-seq (RNA sequencing) technology were applied to comparatively examine the different roles of locust fat body and hemocytes, the two major contributors to the insect immune response, in defense against M. acridum. According to the sequence identity to homologies of other species explored immune response genes, immune related unigenes were screened in all transcriptome wide range from locust and the differential expressed genes were identified in these two tissues, respectively.
Analysis of differentially expressed locust genes revealed 4660 and 138 up-regulated, and 1647 and 23 down-regulated transcripts in the fat body and hemocytes, respectively after inoculation with M. acridum spores. GO (Gene Ontology) enrichment analysis showed membrane biogenesis related proteins and effector proteins significantly differentially expressed in hemocytes, while the expression of energy metabolism and development related transcripts were enriched in the fat body after fungal infection. A total of 470 immune related unigenes were identified, including members of the three major insect immune pathways, i.e. Toll, Imd (immune deficiency) and JAK/STAT (janus kinase/signal transduction and activator of transcription). Of these, 58 and three were differentially expressed in the insect fat body or hemocytes after infection, respectively. Of differential expressed transcripts post challenge, 43 were found in both the fat body and hemocytes, including the LmLys4 lysozyme, representing a microbial cell wall targeting enzyme.
These data indicate that locust fat body and hemocytes adopt different strategies in response to M. acridum infection. Fat body gene expression after M. acridum challenge appears to function mainly through activation of innate immune related genes, energy metabolism and development related genes. Hemocyte responses attempt to limit fungal infection primarily through regulation of membrane related genes and activation of cellular immune responses and release of humoral immune factors.
[Show abstract][Hide abstract] ABSTRACT: Entomopathogenic fungi proliferate in insect hemolymph by using host nutrients after penetrating the cuticle. To improve the virulence of the locust specific fungus, Metarhizium acridum, we genetically modified the fungus to overexpress ATM1, an endogenous hydrolase of trehalose, which is the main carbon source in insect hemolymph. Compared with the wild-type strain, Metarhizium acridum overexpressing ATM1 gene secreted more acid trehalase into locust hemolymph. The trehalose concentrations in locusts infected with the ATM1-overexpressing strain were 5.5 and 6.1 mmol/l, lower than that in locusts infected with the wild-type strain at 3 and 5 days post-inoculation, representing 44.5 and 60.7 % reduction, respectively. Correspondingly, overexpressing ATM1 accelerated the growth of Metarhizium acridum in host hemolymph, and the dose causing 50 % mortality (LD50) of the ATM1-overexpressing strain was reduced by 8.3-fold compared with the wild-type strain, suggesting that increasing the utilization of host nutrients by pathogens could be a promising way to improve the virulence of biopesticides based on parasites of pests.
[Show abstract][Hide abstract] ABSTRACT: Background
Entomopathogenic fungi have been developed as biopesticides, but poor efficacy has blocked their application. One approach to improving virulence is by genetic manipulation. BjαIT from the venom of Buthotus judaicus is an insect-selective neurotoxin. To clarify the insecticidal potency of BjαIT as a virulence candidate in microbial biocontrol agents, the entomopathogenic fungus Metarhizium acridum was genetically modified with BjαIT and its resulting activity against locusts (Locusta migratoria manilensis) was assessed. ResultIn comparison to the wild-type strain, the engineered isolate BjαIT-102 grew significantly quicker in locust hemolymph. Correspondingly, the median lethal dose (LC50) for BjαIT-102 was 18.2-fold lower, and the median lethal times (LT50) for BjαIT-102 were reduced by 28.1% and 30.4%, respectively, after topical inoculation and injection. BjαIT-102 formed conidia on dead locusts, although the conidial yield was reduced by 1.58-fold. Moreover, there were no significant differences in germination and appressorium formation between the BjαIT-102 and wild-type strains. Conclusion
Expression of BjαIT in M. acridum significantly increased virulence against locusts by shortening the in vivo infection period without affecting conidia formation on the carcasses. This study demonstrated that engineering entomopathogenic fungi to incorporate BjαIT offers great potential for increasing their virulence.
Pest Management Science 01/2015; 71(1). DOI:10.1002/ps.3762 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The protein kinase sucrose non-fermenting-1(Snf1) regulates the derepression of glucose-repressible genes and plays a major role in carbon source utilization. In this study, MaSnf1, a sucrose non-fermenting protein kinase gene, has been identified from the entomopathogenic fungus Metarhizium acridum, which has a great potential as a biocontrol agent. The functions of MaSnf1 were characterized using gene disruption and complementation strategies. Disruption of MaSnf1 reduced the conidial yield and delayed the conidial germination on potato dextrose agar (PDA) medium. MaSnf1 is also important for response to ultraviolet radiation and heat shock stress and carbon source utilization in M. acridum. Bioassays by topical inoculation and intrahemocoel injection showed that the MaSnf1 deletion mutant exhibited greatly reduced pathogenicity. The reduced expression level of chitinase gene (Chi) and protease gene (Pr1A) in MaSnf1-disruption transformant (ΔMaSnf1) most likely affects the initial penetration into its host. Additionally, the reduced expression level of acidic trehalase gene (ATM1) probably causes a decline in growth rate in insect hemolymph. Inactivation of MaSnf1 led to a significant decrease in virulence, probably owing to reduction in conidial germination, and appressorium formation, impairment in penetration, and decrease in growth rate in insect hemolymph.
[Show abstract][Hide abstract] ABSTRACT: LqhIT2 is an insect-specific neurotoxin from the venom of scorpion. In this study, the LqhIT2 gene was introduced into the entomopathogenic fungus, Metarhizium acridum. The virulence of the genetically modified strain MaLqhIT2 was then evaluated against locusts (Locusta migratoria manilensis). Compared with the wild-type strain, the median lethal cell density (LC50) for MaLqhIT2 was a 22.6-fold lower, and the median times to death (LT50) for MaLqhIT2 were reduced by 30.3 and 29.6 %, respectively, after topical inoculation and injection. MaLqhIT2 also grew significantly faster in the hemolymph than wild-type strain. There were no significant differences in germination, appressorium formation and sporulation in locust carcasses between the MaLqhIT2 and wild-type strain. These results indicate that LqhIT2 increased the virulence of M. acridum towards locusts by shortening the in vivo infection period, without affecting cuticle penetration or conidia formation in the carcasses. LqhIT2 thus shows considerable potential for increasing fungal virulence against locusts.
Journal of Industrial Microbiology and Biotechnology 08/2014; 41(11). DOI:10.1007/s10295-014-1497-1 · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calcineurin is highly conserved and regulates growth, conidiation, stress response, and pathogenicity in fungi. However, the functions of calcineurin and its regulatory network in entomopathogenic fungi are not clear. In this study, calcineurin was functionally analyzed by deleting the catalytic subunit MaCnA from the entomopathogenic fungus Metarhizium acridum. The ΔMaCnA mutant had aberrant, compact colonies and blunt, shortened hyphae. Conidia production was reduced, and phialide differentiation into conidiogenous cells was impaired in the ΔMaCnA mutant. ΔMaCnA had thinner cell walls and greatly reduced chitin and β-1,3-glucan content compared to the wild type. The ΔMaCnA mutant was more tolerant to cell wall-perturbing agents and elevated or decreased exogenous calcium but less tolerant to heat, ultraviolet irradiation, and caspofungin than the wild type. Bioassays showed that ΔMaCnA had decreased virulence. Digital gene expression profiling revealed that genes involved in cell wall construction, conidiation, stress tolerance, cell cycle control, and calcium transport were downregulated in ΔMaCnA. Calcineurin affected some components of small G proteins, mitogen-activated protein kinase, and cyclic AMP (cAMP)-protein kinase A signaling pathways in M. acridum. In conclusion, our results gave a global survey of the genes downstream of calcineurin in M. acridum, providing molecular explanations for the changes in phenotypes observed when calcineurin was deleted.
[Show abstract][Hide abstract] ABSTRACT: Entomopathogenic fungi have great potential for development as insecticides. However, large-scale use of mycoinsecticides is partially limited by poor efficiency. In many fungal pathogens, the yeast and fungal extracellular signal-regulated kinase (YERK1) subfamily is crucial to the fungal pathogenicity. In this study, a Fus3/Kss1-type mitogen-activated protein kinase (MAPK) gene MaMk1 (GenBank accession no. EFY93607) was identified in Metarhizium acridum, which encodes a member of the YERK1 subfamily. Targeted gene disruption was used to analyze the function of MaMk1 in fungal growth, conidial yield and virulence. Growth assays showed that MaMk1 disruption did not affect fungal growth and conidial yield on potato dextrose agar (PDA) plates. Bioassays by topical inoculation showed that a MaMk1-disruption mutant entirely lost its pathogenicity for the locusts, likely because of failure to penetrate the insect cuticle, which might have been caused by inability to form appressoria during infection. However, bioassays by injection showed no significant difference in virulence among the wild type (WT), ΔMaMk1 mutant and complementary transformant. ΔMaMk1 mutant failed to penetrate the cuticle outwards and sporulate on the locust cadaver. These results suggest that MaMk1 is required for penetration of the insect cuticle both into the hemocele and outside from the hemocele, but is dispensable for fungal growth in insect hemolymph. Gene expression pattern analysis showed that MaMk1 disruption downregulated expression of Mad1 and Mpl1, but did not reduce expression of Pr1 in M. acridum.
[Show abstract][Hide abstract] ABSTRACT: In most eukaryotes, tetraspanins regulate cellular activities by associating with other membrane components. In phytopathogenic fungi, the tetraspanin Pls1 controls appressorium-mediated penetration. However, regulation of Pls1 and its associated signalling pathways are not clear. In this study, the MaPls1 gene from the entomopathogenic fungus Metarhizium acridum was functionally characterized. MaPls1 was highly expressed in mycelium and appressorium, and accumulated on the plasma membrane or in the cytoplasm. Compared with a wild-type strain, the deletion mutant ΔMaPls1 had delayed germination and appressorium formation and impaired turgor pressure on locust wings, but normal germination on medium and non-host insect matrices. Bioassays showed that ΔMaPls1 had decreased virulence and hyphal body formation in haemolymph when topically inoculated, but was not different from wild type when the insect cuticle was bypassed. Moreover, the ability to grow out of the cuticle was impaired in ΔMaPls1. Digital gene expression profiling revealed that genes involved in hydrolysing host cuticle and cell wall synthesis and remodelling were downregulated in ΔMaPls1. MaPls1 participated in crosstalk with signalling pathways such as the cyclic adenosine monophosphate-dependent protein kinase A and calmodulin-dependent pathways. Taken together, these results demonstrated the important roles of MaPls1 at the early stage of infection-associated development in M. acridum.
[Show abstract][Hide abstract] ABSTRACT: To infect its insect host, the entomopathogenic fungus Metarhizium acridum has to breach the physical barrier of the host cuticle to gain entry into insect tissue. To identify virulence-associated genes to improve the biocontrol efficacy of M. acridum, it is necessary to understand the genes expressed by M. acridum during its infection of the insect host cuticle. In this study, we performed a large scale gene expression analysis of M. acridum during locust cuticle infection. We report the identification of 4,739 unique expressed sequence tags (ESTs), of which 80 % have not been previously identified. Analysis of the ESTs provided evidence that M. acridum may employ several different adaptation strategies to infect locust cuticle, such as metabolism reprogramming, protein secretion, endocytosis, cell-cycle-regulated morphogenesis and effector-dependent suppression of host immune responses. By searching against the pathogen-host interaction database, several genes associated with different adaptation processes were found to potentially encode important virulence genes. Gene expression pattern analysis by semi-quantitative RT-PCR revealed that M. acridum differentially regulates subsets of genes in response to different infection niches between the host cuticle and hemolymph. The present study provides novel candidate genes to investigate the biology of insect infection by M. acridum.
Current Genetics 10/2012; 58(5). DOI:10.1007/s00294-012-0382-6 · 2.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The efficacy of entomopathogenic fungi in pest control is mainly affected by various adverse environmental factors, such as heat shock and UV-B radiation, and by responses of the host insect, such as oxidative stress, osmotic stress and fever. In this study, an adenylate cyclase gene (MaAC) was cloned from the locust-specific entomopathogenic fungus, Metarhizium acridum, which is homologous to various fungal adenylate cyclase genes. RNA silencing was adapted to analyze the role of MaAC in virulence and tolerance to adverse environmental and host insect factors.
Compared with the wild type, the vegetative growth of the RNAi mutant was decreased in PD (potato dextrose medium), Czapek-dox and PDA plates, respectively, demonstrating that MaAC affected vegetative growth. The cAMP levels were also reduced in PD liquid culture, and exogenous cAMP restored the growth of RNAi mutants. These findings suggested that MaAC is involved in cAMP synthesis. The knockdown of MaAC by RNAi led to a reduction in virulence after injection or topical inoculation. Furthermore, the RNAi mutant grew much slower than the wild type in the haemolymph of locust in vitro and in vivo, thus demonstrating that MaAC affects the virulence of M. acridum via fungal growth inside the host locust. A plate assay indicated that the tolerances of the MaAC RNAi mutant under oxidative stress, osmotic stress, heat shock and UV-B radiation was decreased compared with the wild type.
MaAC is required for virulence and tolerance to oxidative stress, osmotic stress, heat shock and UV-B radiation. MaAC affects fungal virulence via vegetative growth inside the insect and tolerance against oxidative stress, osmotic stress and locust fever.
[Show abstract][Hide abstract] ABSTRACT: Appressorium is a specialized infection structure of filamentous pathogenic fungi and plays an important role in establishing a pathogenic relationship with the host. The Egh16/Egh16H family members are involved in appressorium formation and pathogenesis in pathogenic filamentous fungi. In this study, a homolog of Egh16H, Magas1, was identified from an entomopathogenic fungus, Metarhizium acridum. The Magas1 protein shared a number of conserved motifs with other Egh16/Egh16H family members and specifically expressed during the appressorium development period. Magas1-EGFP fusion expression showed that Magas1 protein was not localized inside the cell. Deletion of the Magas1 gene had no impact on vegetative growth, conidiation and appressorium formation, but resulted in a decreased mortality of host insect when topically inoculated. However, the mortality was not significant between the Magas1 deletion mutant and wild-type treatment when the cuticle was bypassed by injecting conidia directly into the hemocoel. Our results suggested that Magas1 may influence virulence by affecting the penetration of the insects' cuticle.
Journal of Microbiology and Biotechnology 07/2012; 22(7):889-93. DOI:10.4014/jmb.1111.11055 · 1.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract The shortage of available genomic and transcriptomic data hampers the molecular study on the migratory locust, Locusta migratoria manilensis (L.) (Orthoptera: Acrididae) central nervous system (CNS). In this study, locust CNS RNA was sequenced by deep sequencing. 41,179 unigenes were obtained with an average length of 570 bp, and 5,519 unigenes were longer than 1,000 bp. Compared with an EST database of another locust species Schistocerca gregaria Forsskåi, 9,069 unigenes were found conserved, while 32,110 unigenes were differentially expressed. A total of 15,895 unigenes were identified, including 644 nervous system relevant unigenes. Among the 25,284 unknown unigenes, 9,482 were found to be specific to the CNS by filtering out the previous ESTs acquired from locust organs without CNS's. The locust CNS showed the most matches (18%) with Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) sequences. Comprehensive assessment reveals that the database generated in this study is broadly representative of the CNS of adult locust, providing comprehensive gene information at the transcriptional level that could facilitate research of the locust CNS, including various physiological aspects and pesticide target finding.
[Show abstract][Hide abstract] ABSTRACT: The rice blast fungus Magnaporthe oryzae elaborates a specialized infection structure called an appressorium to breach the rice leaf surface and gain access to plant tissue. Appressorium development is controlled by cell cycle progression, and a single round of nuclear division occurs prior to appressorium formation. Mitosis is always followed by programmed cell death of the spore from which the appressorium develops. Nuclear degeneration in the spore is known to be essential for plant infection, but the precise mechanism by which it occurs is not known.
In yeast, nuclear breakdown requires a specific form of autophagy, known as piecemeal microautophagy of the nucleus (PMN), and we therefore investigated whether this process occurs in the rice blast fungus. Here, we report that M. oryzae possesses two conserved components of a putative PMN pathway, MoVac8 and MoTsc13, but that both are dispensable for nuclear breakdown during plant infection. MoVAC8 encodes a vacuolar membrane protein and MoTSC13 a peri-nuclear and peripheral ER protein.
We show that MoVAC8 is necessary for caffeine resistance, but dispensable for pathogenicity of M. oryzae, while MoTSC13 is involved in cell wall stress responses and is an important virulence determinant. By functional analysis of ΔMoatg1 and ΔMoatg4 mutants, we demonstrate that infection-associated nuclear degeneration in M. oryzae instead occurs by non-selective macroautophagy, which is necessary for rice blast disease.
PLoS ONE 03/2012; 7(3):e33270. DOI:10.1371/journal.pone.0033270 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adverse conditions, including low humidity, UV irradiation, and high temperature, appreciably affect the efficacy of mycoinsecticides. Oil formulation increased the virulence of Metarhizium
anisopliae var. acridum (Ascomycota: Hypocreales) against locusts and grasshoppers by reducing the dependence on saturated water. A mycoinsecticide diluent (a water-in-oil emulsion) has been widely used to dilute the oil formulation of M. anisopliae in China. The aim of our study was to elucidate the mechanism by which the mycoinsecticide diluent improves the virulence of M.
anisopliae. We investigated the effects of the mycoinsecticide diluent on the virulence, invasion speed, and viability of the conidia under various adverse conditions. The results demonstrated that the mycoinsecticide diluent significantly improved the virulence of conidia at low humidity (68, 75, and 84%). In particular, at an RH of 68%, the LT50 for locusts treated with the emulsion was 5.4 days and was 31.6% lower than the value for locusts treated with an oil formulation. In addition, the concentration of the hyphal bodies found in the haemolymph of locusts treated with emulsion was about 27-fold higher than that in locusts treated with oil formulation four days after inoculation. This result was further confirmed by determining the concentration of M. anisopliae var. acridum DNA in locust haemolymph using quantitative PCR. The percentage germination of conidia in the emulsion was also significantly higher than that in oil at 68% RH. There was no significant difference in percentage germination between conidia treated with the emulsion and oil when exposed to irradiation with ultraviolet-B (UV-B) or high temperature. These results demonstrate that the mycoinsecticide diluent enhances the virulence of M.
anisopliae formulated in oil at low humidity by providing adequate water for germination without interfering with the UV tolerance and thermotolerance of the conidia.
[Show abstract][Hide abstract] ABSTRACT: A glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter (PMagpd) was obtained from Metarhizium acridum and its active region analyzed by 5'-deletion strategy using β-glucuronidase (GUS) as a reporter. Sequence analysis revealed that typical regulatory elements of PMagpd were included in the 1.7 kb region upstream of the start codon of the Magpd gene. Deletion of the region from -1,691 bp to -1,463 bp, where the gpd box is harbored, did not significantly affect the PMagpd activity. Deletions of the regions upstream of -946 bp and upstream of -684 bp caused a major decrease of GUS activity. Compared with PgpdA (2.2 kb) in Aspergillus nidulans, PMagpd (1.4 kb) had a shorter sequence and significantly higher activity in M. acridum. This study provides an applicable promoter for over-expression of target genes in M. acridum.
[Show abstract][Hide abstract] ABSTRACT: A single-chain variable fragment (ScFv) complementary DNA (cDNA) library against fenitrothion was constructed, and ScFvs specific for fenitrothion were selected by ribosome display from the library. After three rounds of ribosome display, the ScFv genes were cloned into Escherichia coli for expression. The expressed ScFvs of 160 clones were analyzed by indirect enzyme-linked immunosorbent assay (ELISA). Of these, 40 clones produced antibodies with relatively high activity against fenitrothion, and 3 were selected for Biacore and ELISA analysis. These 3 antibodies-ScFv-AF50, ScFv-AF93, and ScFv-AF132-had IC(50) values of 1.6, 3.4, and 2.2 ng/ml, respectively. Cross-reactivity with other organophosphorus (OP) pesticides was below 0.1% except for parathion-methyl (≤2.8%). The IC(50) values and cross-reactivity were lower than achieved previously with polyclonal or monoclonal antibodies against fenitrothion. The equilibrium dissociation constant (K(D)) values determined by Biacore analysis were 4.56×10(-10)M for ScFv-AF50, 1.42×10(-9)M for ScFv-AF93, and 2.66×10(-10)M for ScFv-AF132. These results demonstrate that the ribosome display has great potential in selection of ScFvs against pesticides. Recoveries of fenitrothion from fortified rice and cucumber were in the range 80.6 to 108%, indicating that the ELISAs with the isolated ScFvs can accurately determine fenitrothion in food samples after the simple and rapid extraction procedure.
[Show abstract][Hide abstract] ABSTRACT: Pattern recognition proteins, which form part of the innate immune system, initiate host defense reactions in response to pathogen surface molecules. The pattern recognition protein β-1,3-glucan recognition protein (βGRP) binds to β-1,3-glucan on fungal surfaces to mediate melanization via the prophenoloxidase (PPO)-activating cascade. In this study, cDNA encoding a 53-kDa βGRP (LmβGRP) was cloned from Locusta migratoria manilensis. LmβGRP mRNA shown to be constitutively expressed specifically in hemocytes and was highly upregulated following fungal infection. LmβGRP-silenced (LmβGRP-RNAi) mutant locusts exhibited significantly reduced survival rate following fungal infection (Metarhizium acridum) compared with the wild-type. Furthermore, LmβGRP-RNAi mutants exhibited abnormally loose stools indicative of a gut defect. 16S rRNA gene analysis detected the opportunistic pathogenic bacterium, Vibrio vulnificus in LmβGRP mutant but not wild-type locusts, suggesting changes in the composition of gut bacterial communities. These results indicate that LmβGRP is essential to gut immunity in L. migratoria manilensis.
[Show abstract][Hide abstract] ABSTRACT: Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate (Fru-6-P) and mannose 6-phosphate (Man-6-P), providing a link between glycolysis and the mannose metabolic pathway. In this study, we identified pmi gene (Mapmi) from the entomopathogenic fungus, Metarhizium acridum, and analyzed its functions using RNA interference (RNAi). Amending the growth medium with cell stress chemicals significantly reduced growth, conidial production and percent germination in Mapmi-RNAi mutant strain, compared to the wild-type strain. Growth of RNAi mutant was lower than the wild type strain with glucose or fructose as sole carbon source. RNAi mutant exhibited a normal growth phenotype with mannose at low concentrations, while trace or high concentration of mannose was more negatively impacted the growth of RNAi mutant than the wild type strain. Infection with Mapmi-RNAi mutant against Locusta migratoria manilensis (Meyen) led to a significantly reduced virulence compared to infection with the wild-type strain. These results suggest that Mapmi plays essential roles in stress tolerance and pathogenicity of M. acridum.