M ycosystema菌 物 学 报 15 September 2009, 28(5): 712-717jwxt@ISSN1672-6472 CN11-5180Q©2009 Institute of Microbiology, CAS, all rights reserved.Appressorial differentiation and its association with cAMP in the insect pathogenic fungus Metarhizium anisopliaeDUAN Zhi-Bing1 GAO Qiang1 LU Ding-Ding1 SHI Shao-Hua1BUTT Tariq M.2WANG Cheng-Shu1*1Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China2School of Biological Sciences, University of Wales Swansea, Swansea SA2 8PP, UKAbstract: Host recognition and appressorium differentiation are the pivotal steps for insect pathogenic fungi to initiate infection process. In this study, appressorial differentiation was studied by comparing a mutant, which has been identified with the loss of genetic materials, with the wild-type strain of the insect pathogenic fungus Metarhizium anisopliae. The observations showed that both the mutant and wild-type strain could not only produce appressoria on the tips of newly germinated spores, but also form multiple infection structures from the terminals of branching mycelia on insect cuticle. In contrast to the wild-type, the frequency of appressorial formation was significantly reduced and in addition, no clear mucilaginous sheath was produced by the mutant appressoria. The study shows that the cuticle degrading enzyme subtilisin is not involved in appressorial differentiation, or indispensable in cuticle digestion. A significantly low frequency of appressorial differentiation by the mutant was coincided with its lower intracellular cAMP level in comparison to the wild-type. Addition of exogenous cAMP could significantly increase the frequency of appressorial differentiation by the mutant, indicating that cAMP signaling pathway is potentially involved in regulationof appressorial differentiation in M. anisopliae.Key words: insect pathogenic fungus, cuticle penetration, mucilaginous sheath, subtilisin金龟子绿僵菌附着胞分化及其与环腺苷酸cAMP的关联性研究段志兵1 高强1 吕丁丁1 石少华1 BUTT Tariq M.2 王成树1*1中国科学院上海生命科学研究院植物生理生态研究所 上海 2000322School of Biological Sciences, University of Wales Swansea, Swansea SA2 8PP, UK摘要：寄主识别与附着胞分化是虫生真菌启动侵染过程的首要步骤。

本文利用先前获得的金龟子绿僵菌基因缺失突变株与其野生型一起进行附着胞分化研究。

接种后不同时间下的观察表明，绿僵菌突变株或野生型的附着胞既可以在萌发不久的芽管顶端形成，也可以在伸长菌丝分支的顶端形成。

与野生型不同的是，突变株附着胞的分化频率显著下降，附着胞周围也缺Supported by The Knowledge Innovation Program of CAS (No. KSCX2-YW-G-037) and the Science and Technology Commission of Shanghai Municipality (No. 07PJ14101)*Corresponding author. E-mail: cswang@Received: 11-12-2008, accepted: 06-02-2009/jwxtcn乏粘液层的产生。

研究表明，绿僵菌的类枯草杆菌类体壁降解酶对于附着胞分化不产生影响，对体壁降解也非完全必需的。

与突变株附着胞分化频率显著降低相对应，其胞内环腺苷酸cAMP 水平显著下降，而添加外源cAMP 能够显著增加其附着胞分化频率，说明绿僵菌cAMP 信号途径对于调控附着胞分化起着重要的作用。

关键词：昆虫病原真菌，体壁穿透，粘液层，类枯草杆菌蛋白酶INTRODUCTIONDuring the process of fungal infection, the combination of enzymatic digestion and the mechanical force from the infection structure, appressorium, accounts for successful establishment of penetration through the host cuticle (Charnely & St Leger 1991; Clarkson & Charnely 1996). The ability of appressorium formation will greatly influence fungal pathogenicity and much attention has been paid to the mechanism exploitation of fungal infection structure differentiation (Wang & St. Leger 2005). For insect fungus M. anisopliae , adhesin mediates spore adhesion to insect cuticle is the first step (Wang & St. Leger 2007a). The studies on plant pathogenic fungi have shown that the signaling pathways, especially the cAMP signaling pathway has been evident to play important roles infungal morphogenesis and pathogenesis(Borges-Walmsley & Walmsley 2000; D’Souza & Heitman 2001). The reduction of intracellular cAMPlevel will greatly affect appressorial development andresult in the decrease of fungal pathogenicity as shown inplant pathogenic fungi, Colletotrichum lagenarium (Takano et al . 2001) and Magnaporthe grisea (Adachi &Hamer 1997).Until now, very little is known about cAMP effectsin insect pathogenic fungi, of which some have beendeveloped as promising biological control agents (Butt etal . 2001; Wang & St. Leger 2007b). A serendipitouslyacquired mutant of M . anisopliae has been characterizedwith the loss of genetic materials as well as the pathogenicity against mealworm, Tenebrio molitor (Wang et al . 2002, 2003). By using the mutant as amodel, we conducted experiments to study fungal appressorium differentiation and its associations with the cuticle degrading protease PR1 and intracellular cAMPlevel. Addition of exogenous cAMP was performed toexamine its influence on appressorium formation andspore germination of both the mutant and wild-typeisolates.1 MATERIALS AND METHODS1.1 Fungal culturesDuring a large scale of single spore isolation analysis of M. anisopliae strain V275, a pale instead of dark green color colony was unexpectedly acquired. Further analyses indicated mutations has occurred in the phenotypically altered colony with the loss of cuticle degrading proteins (Wang et al . 2002) and a conditionally dispensable chromosome (Wang et al . 2003). In this study, the mutant was further evaluated for appressorium differentiation. The wild-type and mutant cultures were maintained on potato dextrose agar (PDA, Difco) or in Sabouraud dextrose broth (SDB, Sigma) as described before (Wang et al . 2002, 2003).1.2 Scanning electron microscopy (SEM) studyThe front wings of the mealworm beetle were sterilized in 70% ethanol and dipped in the conidialsuspensions (105 conidia/mL) of the mutant and wild-type for 15s and then lined on the moisturized Whatman No. 1 filter paper in Petri dish and incubated at 25℃. Samples were taken out and fixed in 2%formaldehyde (aq. v/v) after 24, 36, 48, 60 and 72 hours post-inoculation. Appressorial development on the cuticle was studied using a Philips Scanning Electron Microscope (Philips Electron Optics). Briefly, the samples were placed in a specimen holder and treated through a series of increasing concentrations of ethanol for 10 minutes each: 20%, 50%, 70%, 90% and 100%, and then through ethanol:acetone (3:1) and anhydrous acetone to displace the water in specimens. The treatedspecimens were then mounted onto metal stubs and coated in gold dust for examination. 1.3 SDS-PAGE analysisProteins induced in 1% cockroach homogenate medium (Wang et al . 2002) were precipitated with 60% ammonium sulphate and dialyzed over night. To compare the differences of inductive protein profiles between thewild-type and mutant, equal amounts (10μg) of protein were analyzed by using 14% (w/v) and 19% sodium dodecyl sulfate-polyacrelamide gel electrophoresis (SDS-PAGE), respectively. The gel was documented after staining with Coomassie blue. Molecular weights of the proteins were determined by comparison with protein standards (Bio-Rad).1.4 cAMP assaysThe intracellular cAMP levels in the mutant and wild-type were assayed according to the method described by Filinger et al. (2002). Briefly, mycelia (0.1g), harvested from SDB after growth for four days at 25℃ and 120r/min in a rotator, were ground thoroughly under liquid nitrogen and suspended in 0.5mL of extraction buffer (50mmol/L Tris-HCl, pH 7.5). An aliquot of 0.1mL of this suspension was used for protein assay using a Bio-Rad protein assay kit. The rest was boiled for 5min and centrifuged at 13,000r/min for 5min. cAMP concentration in the supernatant was determined using a cAMP immunoassay kit (Sigma) according to the manufacturer’s instructions. Assays were conducted in triplicates from two independent cultures. The concentrations of cAMP were expressed in pmol/mg protein.1.5 Influence of cAMP on germination and appressorium formationFor the germination assay, the PDA plates (90mm in diameter) were amended by adding the stock solution of cAMP (Sigma) to a final concentration of 1 or 10mmol/L before inoculating with 0.1mL spore suspension (104 conidia/mL). The control plates were inoculated without adding cAMP. The appressorium formation assay was conducted according to the method described by St Leger et al. (1989a). Briefly, the aliquots of spore suspensions (104 conidia/mL) were added to a pre-sterilized growth medium (0.0125% yeast extract medium, YEM, pH 6.8) amended with final concentrations of 0, 1 and 10mmol/L cAMP and left for spore germination in polystyrene dishes. The conidia germinated with terminal swellings (morphologically similar to the appressoria formed on insect cuticles) were counted and the percentages were compared between the mutant and wild-type strain in different treatments. The student t-test was conducted to compare the differences between treatments.2 RESULTS2.1 Infection structure differentiationFor appressorial induction, both the wild-type and mutant conidia could produce appressorium-like structures with apical swellings shortly after spore germination on the hydrophobic surface of a polystyrene Petri dish (Fig. 1-A, B). SEM studies showed that spore germination behavior and appressorium formation on the cuticle were highly different between the mutant and wild-type conidia. The wild-type conidia could usually produce appressoria shortly after spore germination (Fig. 1-E) while the mutant conidia germinated but the germ tubes elongated without appressorial differentiation in a large proportion (data not shown). Most interestingly, by increasing the incubation time for up to 72 hours, multiple appressoria were found to be formed on the tips of branching mycelia by the wild-type (Fig. 1-D) but fewer appressoria were produced by the mutant (Fig. 1-F). There was a heavy mucilaginous sheath formed around each wild-type appressorium (Fig. 1-D) but no apparent mucilage could be found surrounding mutant appressorial cells (Fig. 1-C, F). However, the hydrolytic zone could be clearly observed beneath mutant appressorium (Fig. 1-C) and mycelium (Fig. 1-G), indicating that, even after the loss of subtilisin genes (Wang et al. 2002), the degrading enzymes secreted by the mutant could function effectively for cuticle digestion.2.2 Inductive protein profilesSDS-PAGE analysis indicated that the proteins produced by the wild-type and mutant in 1% cockroach homogenate inductive medium were different. Consistent with the previous study that the mutant has lost subtilisin PR1 genes (Wang et al. 2002), no PR1 (approximately 30kDa) was produced by the mutant (Fig. 2), suggesting that PR1 is not involved in the digestive activities which resulting in the formation of the hydrolytic zone observed above around the mutant appressorium (Fig. 1-C). Most of the other inductive proteins than 20kDa in molecular weight could not be well separated either on a 14% or 19% gel (Fig. 2).Fig. 1 Differences of appressorium formation between the mutant and wild-type strain of Metarhizium anisopliae V275. A: Appressorium of wild-type in YEM medium after induction for 12 hours; B: Appressorium of mutant in YEM medium 12 hours after incubation; C: A close-up look of the hydrolytic zone around a mutant appressorium 72 hours after incubation; D: Multiple appressoria formed on cuticle by the wild-type 72 hours after inoculation; E: Mutant mycelium showing the hydrolytic zone; F: Appressoria formed on insect cuticle by the mutant after inoculation for 72 hours; G: Wild-type appressorium formed on insect cuticle after inoculation for 36 hours; CO: Conidia; AP: Appressorium. Bar = 5μm.Fig. 2 SDS-PAGE profiles of total inductive proteins of the wild-type and mutant. A: Protein separation with a 14% acrylamide/biacrylamide gel; B: Protein separation with a 19% gel. M: Molecular marker; WT: Wild-type; MT: Mutant. The arrow shows that no PR1 protein produced by the mutant. 2.3 cAMP assay and its influence on appressorial differentiationA highly reduced cAMP level was detected in the mutant (32.32±2.57pmol/mg) in contrast to that in the wild-type strain (82.08±7.09pmol/mg) (P=0.001). The addition of exogenous cAMP had no considerable influence on spore germination for both the mutant and wild-type conidia on PDA plates (Fig. 3-A). However, consistent with SEM observations (Fig. 1-D, F), the mutant produced a significantly lower percentage of differentiated structures (13.07±2.52%) than did by the wild-type (54.36±3.11%) (P=0.002) (Fig. 3-B). The addition of exogenous cAMP could increase appressorium formations for both the wild-type and/jwxtcnmutant in a dose-dependent manner but more significantly for the mutant. Statistically, there was no significant increase for the wild-type examined at 1mmol/L cAMP amended medium (P=0.243) but a significant increase at 10mmol/L (P=0.049), while for the mutant both concentrations could highly increase appressorium structure formation when compared with the control (1mmol/L, P=0.029; 10mmol/L, P=0.020) (Fig. 3-B).Fig. 3 The influence of cAMP on spore germination and appressorium formation between the mutant and wild-type strain. A: Germination rates under different concentration of cAMP determined 12 hours post inoculation; B: Percentages of appressorium formation on the hydrophobic surface of polystyrene Petri dishes in 0.0125% YEM medium amended with indicated concentrations of cAMP 12 hours post inoculation.3 DISCUSSIONSAppressorial differentiation was usually described to occur shortly after fungal spore attachment and adhesion to a susceptible host surface (Clarkson & Charnely 1996; Dean 1997). Surprisingly, the observations of this study showed that appressoria could not only be formed on the tips of newly germinated germ tubes but also differentiated from the tips of branching mycelia to establish multiple penetration events. Despite the intricacy of fungal infection process, e.g. signals sensing, recognition, structure differentiation and penetration, “nutritional relationship” with the host has been reasonably explained as the only purpose for fungal infection structure differentiation (St Leger et al. 1989b). In this respect, the establishment of a single penetration by a germinated spore should be enough since it is an energy-cost event for infection structure differentiation. It remains open but highly intriguing what the environmental cues are and why the multiple appressoria can be formed from branching mycelia, especially from those close branching mycelia.In accordance with previous studies that the mutant isolate of M. anisopliae V275 lost genetic materials including subtilisin pr1 genes (Wang et al. 2002) and a dispensable chromosome (Wang et al. 2003), SDS-PAGE analysis in this study showed that the mutant produced no PR1 protein in inductive medium. The mutant, however, could still produce appressoria would indicate that the subtilisin protein PR1 is not involved in appressorial differentiation. Supportively, a previous study revealed that the target inhibition of PR1 did not prevent appressorial differentiation by M. anisopliae (St Leger et al. 1987). On the other hand, an array of proteins have been involved during the infection process of M. anisopliae (Freimoser et al. 2003; Wang et al. 2005) and different paralogous genes of pr1 from pr1A-pr1J have been identified from a single strain of M. anisopliae (Bagga et al. 2004). As demonstrated by SDS-PAGE analysis in this study, there was a large amount of proteins produced by the mutant cells in inductive medium, suggesting that these enzymes could either function complementarily during penetration or the mutant has developed an alternative strategy in pathogenesis.The frequency of appressorium differentiation coincided with intracellular cAMP level indicates that the cAMP signalling pathway plays a major role in appressorium formation in the insect pathogenic fungus. For M. anisopliae, two high-affinity of cAMP-binding proteins were detected during the early stage of conidial germination and appressorial differentiation (St Leger et al. 1990), indicating the involvement of cAMP in fungal early development. The experiments have demonstrated this is the case for plant pathogenic fungi (Takano et al. 2001; D’Souza & Heitman 2001; Lee et al. 2003). Theendogenous cAMP level was significantly low but detectable in the mutant indicating that, after the loss of genetic materials, its cAMP related genes might remain intact but with variations in upstream and or downstream gene regulations by comparison with the wild-type. Whatever the case, further studies are still required by employing the mutant as a powerful model to elucidate the pathogenic mechanisms of insect fungi. [REFERENCES]Adachi K, Hamer JE, 2002. 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