Localization of the insect pathogenic fungal plant symbionts Metarhizium robertsii and Metarhizium brunneum in bean and corn roots

Metarhizium is an insect pathogenic fungus and a plant root symbiont. Here the root association patterns (rhizoplane or endophytic colonization) were analyzed in common beans (Phaseolus vulgaris) and sweet corn (Zea mays) using M. robertsii and M. brunneum under various vermiculite treatments (control, with sucrose, with an insect) at two time points of plant growth (10 and 20 days). We observed that M. brunneum and M. robertsii preferentially endophytically colonized the hypocotyl, however, greater rhizoplane colonization was observed at the regions proximal to the hypocotyl in both plants. Vermiculite amended with an infected insect resulted in greater endophytic and rhizoplane colonization at 20 days compared to 10 days, for both plants as well as for both Metarhizium species. Regardless of the vermiculite treatment, corn was preferentially colonized compared to bean. Sucrose amendment in the vermiculite and infected insect amended vermiculite only showed differences in rhizoplane colonization. The greatest root association occurred with M. brunneum with an infected insect and that in corn after 20 days.     

Differential expression of insect and plant specific adhesin genes, Mad1 and Mad2, in Metarhizium robertsii

Metarhizium robertsii is an entomopathogenic fungus that is also plant rhizosphere competent. Two adhesin-encoding genes, Metarhizium adhesin-like protein 1 (Mad1) and Mad2, are involved in insect pathogenesis or plant root colonization, respectively. Here we examined the differential expression of the Mad genes when grown on a variety of soluble (carbohydrates and plant root exudate) and insoluble substrates (locust, tobacco hornworm, and cockroach cuticle, chitin, tomato stems, cellulose, and starch) and during insect, Plutella xylostella, infection. On insect cuticles Mad1 was up regulated, whereas bean root exudate and tomato stems resulted in the up regulation of Mad2. During the early stages of insect infection Mad1 was expressed while Mad2 was not expressed until fungal hyphae emerged and conidiated on the insect cadaver. The regulation of Mad2 was compared to that of other stress-related genes (heat shock protein (Hsp)30, Hsp70, and starvation stress gene A (ssgA)). Mad2 was generally up regulated by nutrient starvation (similar to ssgA) but not by pH, temperature, oxidative or osmotic stresses. Whereas Hsp30 and Hsp70 were generally up regulated at 37 °C or by oxidative stress even under nutrient enriched conditions. We fused the promoter of the Mad2 gene to a marker gene (green fluorescent protein (GFP)) and confirmed that Mad2 was up regulated when M. robertsii was grown in the presence of nutrient starvation. Examination of the promoter region of Mad2 revealed that it possessed two copies of a stress-response element (STRE) known to be regulated under the general stress-response pathway.     

Linkage of autophagy to fungal development,lipid storage and virulence in Metarhizium robertsii

Autophagy is a highly conserved process that maintains intracellular homeostasis by degrading proteins or organelles in all eukaryotes. The effect of autophagy on fungal biology and infection of insect pathogens is unknown. Here, we report the function of MrATG8, an ortholog of yeast ATG8, in the entomopathogenic fungus Metarhizium robertsii. MrATG8 can complement an ATG8-defective yeast strain and deletion of MrATG8 impaired autophagy, conidiation and fungal infection biology in M. robertsii. Compared with the wild-type and gene-rescued mutant, Mratg8Δ is not inductive to form the infection-structure appressorium and is impaired in defense response against insect immunity. In addition, accumulation of lipid droplets (LDs) is significantly reduced in the conidia of Mratg8Δ and the pathogenicity of the mutant is drastically impaired. We also found that the cellular level of a LD-specific perilipin-like protein is significantly lowered by deletion of MrATG8 and that the carboxyl terminus beyond the predicted protease cleavage site is dispensable for MrAtg8 function. To corroborate the role of autophagy in fungal physiology, the homologous genes of yeast ATG1, ATG4 and ATG15, designated as MrATG1, MrATG4 and MrATG15, were also deleted in M. robertsii. In contrast to Mratg8Δ, these mutants could form appressoria, however, the LD accumulation and virulence were also considerably impaired in the mutant strains. Our data showed that autophagy is required in M. robertsii for fungal differentiation, lipid biogenesis and insect infection. The results advance our understanding of autophagic process in fungi and provide evidence to connect autophagy with lipid metabolism.     

不同绿僵菌、白僵菌菌株对铜绿丽金龟幼虫的毒力生物测定

应用五株虫生真菌对铜绿丽金龟的幼虫的感染试验结果,将终浓度为1×107孢子/mL的孢悬液对铜绿丽金龟幼虫进行毒力测定。试验结果为:绿僵菌Ma09、Ma20,布氏白僵菌Bbr06、Bbr17,球孢白僵菌Bb202 15d的感染率分别为20.83%、14.58%、8.33%、95.83%、6.25%,其中布氏白僵菌Bbr17显示了较高的毒力,其LT50和LT95分别为10d、18d,该菌株对今后在花生蛴螬防治具有较大的应用价值。     

Obligate symbiont involved in pest status of host insect

The origin of specific insect genotypes that enable efficient use of agricultural plants is an important subject not only in applied fields like pest control and management but also in basic disciplines like evolutionary biology. Conventionally, it has been presupposed that such pest-related ecological traits are attributed to genes encoded in the insect genomes. Here, however, we report that pest status of an insect is principally determined by symbiont genotype rather than by insect genotype. A pest stinkbug species, Megacopta punctatissima, performed well on crop legumes, while a closely related non-pest species, Megacopta cribraria, suffered low egg hatch rate on the plants. When their obligate gut symbiotic bacteria were experimentally exchanged between the species, their performance on the crop legumes was, strikingly, completely reversed: the pest species suffered low egg hatch rate, whereas the non-pest species restored normal egg hatch rate and showed good performance. The low egg hatch rates were attributed to nymphal mortality before or upon hatching, which were associated with the symbiont from the non-pest stinkbug irrespective of the host insect species. Our finding sheds new light on the evolutionary origin of insect pests, potentially leading to novel approaches to pest control and management.     

Chlorimuron ethyl as a new selectable marker for disrupting genes in the insect-pathogenic fungus Metarhizium robertsii

A lack of selectable markers was a hindrance in investigating gene function in Metarhizium robertsii. A reliable Agrobacterium-mediated transformation system based on the use of chlorimuron ethyl as the selectable marker was developed which could serve as a useful tool to inactivate genes involved in insect pathogenicity.     

RNAi is an antiviral immune response against a dsRNA virus in Drosophila melanogaster

Drosophila melanogaster has a robust and efficient innate immune system, which reacts to infections ranging from bacteria to fungi and, as discovered recently, viruses as well. The known Drosophila immune responses rely on humoral and cellular activities, similar to those found in the innate immune system of other animals. Recently, RNAi or 'RNA silencing' has arisen as a possible means by which Drosophila can react to a specific pathogens, transposons and retroviral elements, in a fashion similar to that of a traditional mammalian adaptive immune system instead of in a more generalized and genome encoded innate immune-based response. RNAi is a highly conserved regulation and defence mechanism, which suppresses gene expression via targeted RNA degradation directed by either exogenous dsRNA (cleaved into siRNAs) or endogenous miRNAs. In plants, RNAi has been found to act as an antiviral immune response system. Here we show that RNAi is an antiviral response used by Drosophila to combat infection by Drosophila X Virus, a birnavirus, as well. Additionally, we identify multiple core RNAi pathway genes, including piwi, vasa intronic gene (vig), aubergine (aub), armitage (armi), Rm62, r2d2 and Argonaute2 (AGO2) as having vital roles in this response in whole organisms. Our findings establish Drosophila as an ideal model for the study of antiviral RNAi responses in animals.     

miR-120对台湾乳白蚁抵御绿僵菌的免疫反应的影响

本研究探究了miR-120在台湾乳白蚁Coptotermes formosanus抵御绿僵菌Metarhizium anisopliae的免疫反应中的作用。研究发现通过对台湾乳白蚁注射miR-120 agomir以增加白蚁体内miR-120的含量后,台湾乳白蚁的存活率降低约35%,绿僵菌对台湾乳白蚁的致死率提升约55%,同时显著抑制了台湾乳白蚁对绿僵菌的趋避反应。双荧光素酶报告系统等试验证实了miR-120靶向结合免疫基因Draper。通过对Draper进行注射法RNAi后,得到了类似于注射miR-120 agomir后的结果。研究结果表明,miR-120通过抑制靶基因Draper的表达,抑制了台湾乳白蚁抵御绿僵菌的免疫反应,最终导致台湾乳白蚁的存活率显著下降。该结果有助于进一步理解白蚁的免疫机制,并为开发抑制白蚁免疫防御反应的技术方法提供理论基础,有助于促进白蚁生物防治的发展。     

精胺合成酶MrSPS参与调控罗伯茨绿僵菌的生长发育、抗逆和致病力

本研究以罗伯茨绿僵菌Metarhizium robertsii为研究对象,针对鉴定出的精胺合成酶基因(MAA_02088, Mrsps),利用农杆菌介导的同源重组方法获得Mrsps敲除株ΔMrsps。与野生型相比,ΔMrsps营养生长和产孢能力下降,对氯化钠和紫外照射耐受性增强。大蜡螟幼虫毒力分析表明,浸渍和注射两种情况下ΔMrsps致病力降低,半致死时间(LT₅₀：6.71和4.75 d)比野生型(LT₅₀：5.17和4.19 d)显著增加。Mrsps敲除后不影响附着胞形成率和蝉翅穿透能力,但会显著下调昆虫血腔定殖相关基因的表达量。这些结果说明精胺合成酶MrSPS参与调控罗伯茨绿僵菌的生长发育、外界胁迫应答和致病力。     

mro-miR-33在绿僵菌产孢中的作用

罗伯茨绿僵菌Metarhizium robertsii是一种重要的广谱性杀虫真菌,在害虫的生物防治中应用广泛。研究表明microRNAs在丝状真菌的生长发育中可能发挥重要调控作用。迄今有关miRNAs在绿僵菌产孢过程中的作用还未见研究报道。本文分析了实验室前期鉴定的一个绿僵菌新miRNA(mro-miR-33)在绿僵菌产孢过程中的作用。结果表明,绿僵菌在不同培养条件下mro-miR-33的表达量有显著差异,且在孢子发育过程表达量下降。在野生菌株中过表达mro-miR-33后,绿僵菌的产孢量显著下降,qRT-PCR分析表明孢子发育关键调控基因brl A表达显著下调,而敲除mro-miR-33后,绿僵菌的产孢量显著增加。这些变化表明mro-miR-33在绿僵菌的产孢过程中发挥着重要的作用。     

Improvement of pest resistance in transgenic tobacco plants expressing dsRNA of an insect-associated gene EcR

The adoption of pest-resistant transgenic plants to reduce yield loss and pesticide utilization has been successful in the past three decades. Recently, transgenic plant expressing double-stranded RNA (dsRNA) targeting pest genes emerges as a promising strategy for improving pest resistance in crops. The steroid hormone, 20-hydroxyecdysone (20E), predominately controls insect molting via its nuclear receptor complex, EcR-USP. Here we report that pest resistance is improved in transgenic tobacco plants expressing dsRNA of EcR from the cotton bollworm, Helicoverpa armigera, a serious lepidopteran pest for a variety of crops. When H. armigera larvae were fed with the whole transgenic tobacco plants expressing EcR dsRNA, resistance to H. armigera was significantly improved in transgenic plants. Meanwhile, when H. armigera larvae were fed with leaves of transgenic tobacco plants expressing EcR dsRNA, its EcR mRNA level was dramatically decreased causing molting defects and larval lethality. In addition, the transgenic tobacco plants expressing H. armigera EcR dsRNA were also resistant to another lepidopteran pest, the beet armyworm, Spodoptera exigua, due to the high similarity in the nucleotide sequences of their EcR genes. This study provides additional evidence that transgenic plant expressing dsRNA targeting insect-associated genes is able to improve pest resistance.     

Helicobacter pylori virulence factors and the host immune response: implications for therapeutic vaccination

Helicobacter pylori colonizes the human gastric mucosa and is associated with specific gastric disease. Virulence factors, such as urease, the vacuolating toxin (VacA), the cytotoxin-associated antigen CagA or blood-group-antigen-binding adhesin (BabA), an adherence factor, might account for the development of different diseases. Vaccination trials exploiting the antigenic properties of some of these proteins have not been successful in preventing infection in humans. A more in-depth understanding of the immune response to H. pylori infection as well as additional information on suitable epitopes and adjuvants will be required before a successful vaccine can be developed.     

转基因抗虫水稻对水稻害虫群落的影响

本文应用吸虫器法与剥查法,研究了转Cry1Ac +SCK基因水稻及其杂交后代对水稻害虫群落的影响。结果显示：转Cry1Ac+SCK基因水稻MSA与MSB对稻纵卷叶螟具有极强的抗性,转Cry1Ac+SCK基因稻的杂交后代21S/MSB与Ⅱ32A/MSB都获得了对稻纵卷叶螟的高抗性。虽然MSA、MSB、21S/MSB与Ⅱ32A/MSB对水稻害虫的物种丰富度、个体数量、多样性与均匀度时间动态都有一定程度的影响,但生长全期的调查结果显示,与对应的对照比较,它们对水稻害虫群落的这些参数均无显著影响,且转基因水稻田与各自对照田在害虫与主要害虫物种组成上具有较高的相似性。靶标害虫种群数量的降低,并未对水稻害虫群落的组成与结构产生明显的影响,也没有引起优势种成分的变化。     

SNPs in DNA repair genes associated to meningitis and host immune response

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis. The patient genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. A higher frequency (P<0.05) of APE1 Glu allele in bacterial meningitis (BM) and aseptic meningitis (AM) patients was observed. The genotypes Asn/Asn in control group and Asn/Glu in BM group was also higher. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs is significantly higher in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 Glu allele or OGG1 Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1 Asn148Glu, OGG1 Ser326Cys or PARP-1 Val762Ala. Moreover, reduction in the levels of IL-6, IL-1Ra, MCP-1/CCL2 and IL-8/CXCL8 was observed in the presence of APE1 Glu allele in BM patients. In conclusion, we obtained indications of an effect of SNPs in DNA repair genes on the regulation of immune response in meningitis.     

Recombination within sympatric cryptic species of the insect pathogenic fungus Metarhizium anisopliae

Metarhizium anisopliae is an insect pathogenic fungus with a worldwide distribution. It is being developed and used as a biocontrol agent against a wide range of insect pests but relatively little is known of the life history of this fungus. We tested hypotheses concerning reproductive isolation and recombination in a sample of heat-active (ability to grow at 37 degrees C) and cold-active (ability to grow at 8 degrees C) sympatrically occurring isolates of M. anisopliae from Ontario, Canada by assaying nucleotide sequence variation at six polymorphic loci: the internally transcribed spacer (ITS) region of the nuclear ribosomal DNA repeat, and portions of calmodulin (CAL), chitin synthase (CHS), subtilisin-like protease (PR1), neutral trehalase (NTL) and actin (ACT)-encoding genes. The most parsimonious trees constructed showed a topology consistent with the heat-active and cold-active isolates as two monophyletic groups. We then applied Genealogical Concordance Phylogenetic Species Recognition (GCPSR) to the genealogical trees and concluded that the transition from concordance among branches to incongruity among branches delimited two species of M. anisopliae within Ontario. The GCPSR of two species was supported by intraspecific incongruity within each species when tested using the Partition Homogeneity test, indicating recombination. The GCPSR of two species also corresponded to the heat-active and cold-active groups. As the groups are morphologically indistinguishable we applied the term 'cryptic species'. Therefore, the sympatrically occurring heat-active and cold-active isolates represent different cryptic species with a history of recombination among isolates within each species.     

Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae

Conidial spores are often used as the infectious agent during insect biocontrol applications of entomopathogenic fungi. Here we show differential virulence of conidia derived from Metarhizium anisopliae strain EAMa 01/58-Su depending upon the solid substrata used for cultivation, where LC₅₀ values differed by up to ~10-fold (5.3×10⁶?4.5×10⁵ conidia/ml) and LT₅₀ values by ~40% (9.8?7.1 d). This fungal strain is also known to secrete proteins that are toxic towards adult Mediterranean fruit flies, Ceratitis capitata, and the Greater wax moth, Galleria mellonella, larvae. In vitro production and intrahemoceol injection using G. mellonella as the host was used to test fractions during purification of the protein toxins, demonstrating that they elicited defence-related responses including melanisation and tissue necrosis. Production of these proteins/peptides along with a number of potential cuticle degrading enzymes was confirmed both in vitro and during the infection process (in vivo). Two-dimensional gel electrophoresis, followed by gel elution and bioassay, was used to identify at least three proteins or peptides (molecular mass=11, 15 and 15 kDa) as mediating the observed insect toxicity. These data demonstrate that in vitro screening for insect toxins can mimic in vivo (i.e. during the infection process) secretion and applies the use of proteomics to invertebrate pathology.