APPENDIX First author publication as listed on page xi. 136 C H A P T E R T W E N T Y Methods for Functional Analysis of Macroautophagy in Filamentous Fungi Yi Zhen Deng,* Marilou Ramos-Pamplona,* and Naweed I. Naqvi* Contents 1. Introduction 1.1. Cellular functions of autophagy in filamentous fungi 2. Methods for the Functional Analysis of Autophagy in Filamentous Fungi 2.1. Gene-deletion analyses to assess macroautophagy in filamentous fungi 2.2. Use of chemical inhibitors to investigate autophagy in fungi 2.3. Microscopy methods to detect autophagy-associated membrane structures 2.4. Monodansylcadaverine (MDC) staining of autophagic vesicles 2.5. LysoTracker-based visualization of vacuoles and vesicular compartments 2.6. Analysis of glycogen sequestration and estimation of glycogen content 2.7. Comparative proteomics for identifying the targets of autophagic degradation 3. Concluding Remarks Acknowledgments References 296 296 297 297 298 300 302 303 304 306 307 307 308 Abstract Autophagy is a bulk degradative process responsible for the turnover of membranes, organelles, and proteins in eukaryotic cells. Genetic and molecular regulation of autophagy has been independently elucidated in budding yeast and mammalian cells. In filamentous fungi, autophagy is required for several important physiological functions, such as asexual and sexual differentiation, pathogenic development, starvation stress and programmed cell death during heteroincompatibility. Here, we detail biochemical and microscopy methods useful for measuring the rate of induction of autophagy in filamentous fungi, * Fungal Patho-Biology Group, Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore Methods in Enzymology, Volume 451 ISSN 0076-6879, DOI: 10.1016/S0076-6879(08)03220-5 # 2008 Elsevier Inc. All rights reserved. 295 296 Yi Zhen Deng et al. and we summarize the methods that have been routinely used for monitoring macroautophagy in both yeast and filamentous fungi. The role of autophagy in carbohydrate catabolism and cell survival is discussed along with the specific functions of macroautophagy in fungal development and pathogenesis. 1. Introduction Autophagy is a highly conserved catabolic process in eukaryotes that is responsible for organellar turnover, membrane recycling, and protein degradation in vacuoles/lysosomes. Autophagy is induced in response to environmental stress or developmental signals during cellular differentiation (Besteiro et al., 2006; Noda and Ohsumi, 1998; Pinan-Lucarre et al., 2003). Autophagy can act as a prosurvival signal or participate in programmed cell death, depending on the particular physiological conditions (Codogno and Meijer, 2005). There are three distinct classes of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA); the latter is selectively used to degrade cytosolic proteins containing a specific pentapeptide consensus motif (Majeski and Dice, 2004; Salvador et al., 2000). Macroautophagy and microautophagy (Reggiori and Klionsky, 2002) are considered nonselective and thus have more degradative capacity. The major difference between macroautophagy and microautophagy is whether the double-membrane vesicles, autophasogosomes, sequester cytoplasmic proteins or organelles (macroautophagy) for delivery to the lysosome/vacuole for degradation (Suzuki et al., 2001), or whether the cytoplasm is directly engulfed into the vacuoles (microautophagy) (Mortimore et al., 1989). Besides general autophagy, some specific types of autophagy exist, such as crinophagy (the activity of lysosomes related to the secretory pathway and endocrine functions; Glaumann, 1989), reticulophagy (degradation of ER; Bernales et al., 2007; Bolender and Weibel, 1973) and pexophagy (degradation of peroxisomes; Sakai and Subramani, 2000). Thus far, 31 ATG genes (autophagy-related) have been characterized in Saccharomyces cerevisiae, which has led to a better understanding of the genetic and molecular regulation of autophagy (Kabeya et al., 2007; Klionsky et al., 2003), particularly the formation of autophagy-associated vesicular compartments, such as preautophagosomal structures (PAS), autophagosomes (cytosolic), and autophagic bodies (vacuolar) (Suzuki et al., 2001). 1.1. Cellular functions of autophagy in filamentous fungi Autophagy is reported to play a crucial role during differentiation of several filamentous fungi such as Podospora, Aspergillus, Colletotrichum, and Magnaporthe. Autophagy-deficient mutants of Magnaporthe oryzae are nonpathogenic and show highly reduced asexual development (Liu et al., 2007; VeneaultFourrey et al., 2006). Loss of autophagy-assisted programmed cell death in Analysis of Autophagy in Fungi 297 atg8D appressorium is proposed to be responsible for the failure of penetrating the host cuticle ( Veneault-Fourrey et al., 2006). Furthermore, autophagy is involved in lipid body turnover and thus is essential for turgor generation and appressorium-mediated penetration (Liu et al., 2007). However, Colletotrichum gloeosporioides, with a related infection strategy as M. oryzae, does not require autophagic cell death for successful infection (Nesher et al., 2008). Surprisingly, infection structures/appressoria from a CLK1-deletion (an ortholog of MgATG1) mutant in Colletotrichum lindemuthianum, are unable to penetrate the host cuticle (Dufresne et al., 1998), similar to the result from M. oryzae. In S. cerevisiae, loss of autophagy leads to failure of sporulation, sensitivity to nitrogen starvation, and increased pseudohyphal growth (Cutler et al., 2001; Ma et al., 2007; Tsukada and Ohsumi, 1993). In A. oryzae, autophagy is required for the differentiation of aerial hyphae and in conidial germination (Kikuma et al., 2006). In contrast to its function in fungi mentioned previously, autophagy plays little or no role in the differentiation of the dimorphic yeast Candida albicans within the host tissue (Palmer et al., 2007). The atg9D mutant in C. albicans remains unaffected for yeast-hypha or chlamydospore differentiation, though it shows specific defects in autophagy and the cytoplasm-to-vacuole targeting (Cvt) pathway. In P. anserina, autophagy is essential for sexual differentiation and cell death by incompatibility. It remains controversial whether autophagy executes a programmed cell death function or acts as a prosurvival response in Podospora (Dementhon et al., 2003, 2004; Pinan-Lucarre et al., 2003; PinanLucarre et al., 2005). It was initially thought that autophagy acts as the cause of cell death during incompatible interactions for it is induced when cells of unlike genotypes fuse in P. anserina (Dementhon et al., 2004; Pinan-Lucarre et al., 2003). A recent study suggests that autophagy serves a prosurvival role during incompatibility, as loss of autophagy results in accelerated cell death (Pinan-Lucarre et al., 2005). In this chapter, we present a technical review of the most frequently used methods to study autophagy in yeast and fungal species and focus on the methods that are useful to monitor the induction and rate of autophagy in filamentous fungi. The following protocols and methods have been validated in the model fungus M. oryzae and can be easily adapted and optimized for use in other filamentous fungi of interest. 2. Methods for the Functional Analysis of Autophagy in Filamentous Fungi 2.1. Gene-deletion analyses to assess macroautophagy in filamentous fungi Generally, the one-step gene deletion strategy (schematized in Fig. 20.1) is used for disruption of requisite gene function in filamentous fungi. For gene disruption of Magnaporthe ATG1 (MGG_06393.5), genomic DNA fragments 298 Yi Zhen Deng et al. Figure 20.1 Schematic representation of a one-step gene replacement strategy for the ATG1 locus (MGG_06393.5) in Magnaporthe. Solid bars and short open boxes represent coding regions and introns, respectively, whereas grey bars indicate the genomic flanks used as regions of homology for gene targeting. Relevant restriction enzyme sites have been depicted and ILV1 refers to the sulfonyl urea-resistance cassette used to replace ATG1to create the atg1D strain. (about kb each) representing the 50 and 30 flanks of the ATG1 open reading frame were amplified by PCR, and ligated sequentially so as to flank the ILV1 cassette (confers resistance to sulfonyl urea) in pFGL385 to obtain plasmid vector pFGLatg1KO. The pFGLatg1KO plasmid was transformed into wildtype M. oryzae using Agrobacterium T-DNA-mediated transformation for homology-dependent replacement of the ATG1 gene. Gene-disruption constructs can also be delivered into the fungal species of choice using electroporation of spheroplasts (Talbot et al., 1993; Vollmer and Yanofsky, 1986; Xoconostle-Cazares et al., 1996; Yelton et al., 1984). Transformants were selected for resistance to chlorimuron ethyl (100 mg/mL; Cluzeau Labo, France) and correct gene-replacement confirmed by PCR analysis and Southern blotting. The primers used for amplifying the 1-Kb region at the 50 - and 30 -flank of the ATG1 gene were as follows: ATG1-5F (50 - GAGTGAGAATTCGCGGGACTAAGCAGGCCCAGGA-30 ), ATG1-5R (50 - GA GTGAGAATTCTGCACTTAGAAACACTCGGGCT-30 ), ATG1-3F (50 - GAGACTGTTCTGCAGCCTGGCAGTGGTTATCGGTTCG-30 ), and ATG1-3R (50 - GAGAGTGTTAAGCTTGGACGTACAGTAGGTAATTGGT-30 ). The preceding protocol is validated for Magnaporthe but can be easily optimized for other fungal species of choice, provided the requisite sequence information is available. Other selectable marker cassettes for transformation in fungi include hygromycin resistance (HPH1) or ammonium-gluphosinate resistance (BAR). Gene targeting of the marker cassette can also be achieved by providing homology within the coding sequence per se and need not be restricted to the flanking sequences as described previously. 2.2. Use of chemical inhibitors to investigate autophagy in fungi There are several chemical inhibitors of autophagy that are commercially available and routinely used in mammalian cells. Although these inhibitors block autophagy, their effects are not entirely specific. Wortmannin ( WM) Analysis of Autophagy in Fungi 299 is an inhibitor of PI3-kinase and blocks the induction of autophagy (Blommaart et al., 1997; Petiot et al., 2000). 3-methlyadenine (3-MA) is also a classical inhibitor of the autophagic pathway (Seglen and Gordon, 1982). N-ethylmaleimide (NEM) inhibits several vesicular transport events and thus blocks the formation of autophagic vacuoles ( Woodman, 1997). Such inhibitors can be potentially useful in studying autophagy in fungal systems provided special caution is exercised in analyzing the results. WMtreatment of vegetative mycelia (Fig. 20.2) of Magnaporthe wild-type strain mimics the phenotype of an atg8D mutant, in which starvation fails to induce autophagy (Deng and Naqvi, unpublished data). Figure 20.2 Epifluorescence microscopy-based assessment of autophagosomes. Mycelia from the wild type or atg8D mutant were stained with MDC and imaged using epifluorescence microscopy. MDC-stained wild-type mycelia pretreated with the autophagic-inhibitor Wortmannin (WM) prior to starvation, served as a negative control. Scale bar denotes mm. Micrographs were pseudo colored using Photoshop Version 7. 300 Yi Zhen Deng et al. Procedure 1. Small amount of vegetative mycelia (approximately 50 mg of wet weight; scraped from a colony surface with inoculation loop) or conidia (ca  103) of wild-type and atg8D strains are cultured in 20 ml of complete medium (CM; yeast extract 0.6%, casein hydrolysate 0.6%, sucrose 1%) for 48 h at 28  C with gentle shaking (150 rpm) to obtain sufficient biomass. 2. Wortmannin stock (1 mM, in DMSO) is added into the CM, to a final concentration of 200 nM. The mycelia are treated with Wortmannin for h at 28  C with gentle shaking. 3. Mycelia are harvested by filtration through sterile Miracloth (Calbiochem, USA) and washed twice by filtration with sterile distilled water. 4. A small amount of the freshly cultured mycelia is then inoculated into 20 ml of minimal medium lacking nitrogen [MM-N: 0.5 g/L KCl, 0.5 g/L MgSO4, 1.5 g/L KH2PO4, 0.1% (v/v) trace elements, 10 g/L glucose, pH6.5; (Talbot et al., 1993)] containing mM PMSF, and grown for 16 h with gentle shaking at 28  C. Please note that this step is carried out to induce autophagy and there may not be a visible change in fungal biomass. 5. The pretreated and starved wild-type mycelia are stained with MDC and observed using epifluorescence microscopy, as described subsequently. 2.3. Microscopy methods to detect autophagy-associated membrane structures Activation or induction of autophagy can be visualized by differential interference contrast (DIC; or Nomarski optics) microscopy. In S. cerevisiae (Lang et al., 1998), C. albicans (Palmer et al., 2007), and P. anserina (Dementhon et al., 2004), starvation stress leads to enlarged vacuoles, which can be observed by DIC optics. Large punctuate perivacuolar structures or vesicles inside the vacuolar lumen can also be visualized, indicating the formation of autophagosomes or accumulation of autophagic bodies. In the Podospora DpspA mutant, which lacks the PSPA vacuolar protease, the accumulation of autophagic bodies in the vacuolar lumen is even more striking and easily detectable by simple microscopy observations (Dementhon et al., 2003). Fluorescence microscopy is another method to monitor the induction of autophagy. N-terminal tagging of Atg8 with a fluorescent protein such as GFP or RFP helps in epifluorescent detection of autophagosomes and has been used in several filamentous fungi such as A. oryzae (Kikuma et al., 2006), P. anserina (Pinan-Lucarre et al., 2005), and M. oryzae (Deng and Naqvi, unpublished results). An advantage of utilizing GFP- or RFP-tagged Atg8 is that the extent of autophagosome formation correlates well with the amount of GFP/RFP-Atg8PE (Kabeya et al., 2000), so that the induction of Analysis of Autophagy in Fungi 301 autophagy can be easily quantified by Western blotting using commercially available anti-GFP/RFP antibodies. Transmission electron microscopy (TEM) is the gold standard for ultrastructural investigation of autophagy-associated membrane compartments in filamentous fungi such as P. anserina (Pinan-Lucarre et al., 2003), A. oryzae (Kikuma et al., 2006), and M. oryzae (Liu et al., 2007; VeneaultFourrey et al., 2006). Fig. 20.3 depicts the TEM analysis of the vacuolar lumen in the wild type and an autophagy-deficient mutant (atg8D) of Magnaporthe. Procedure 1. Fresh conidia (4  103) or small amounts of mycelia (scraped from a colony using inoculation loop; about 50 mg) from the wild-type or atg8D strain in Magnaporthe are grown in 20 ml of CM for 48 h at 28  C with gentle shaking (150 rpm). 2. Mycelia are harvested by filtration through Miracloth and washed thoroughly using sterile distilled water. 3. Washed mycelia from individual strains are grown in liquid MM-N medium [0.5 g/L KCl, 0.5 g/L MgSO4, 1.5 g/L KH2PO4, 0.1% (v/v) trace elements, 10 g/L glucose, pH 6.5; Talbot et al., 1993] containing mM PMSF, for 16 h with gentle shaking, at 28  C. 4. A small amount of the fungal biomass harvested by filtration through Miracloth, is placed in a microfuge tube and resuspended in 200 ml of fixation reagent (2.5% glutaraldehyde in 0.1 M phosphate buffer, v/v, pH 7.2) or sufficient to cover the mycelial sample. Initially the fixation is carried out under vacuum for 15 at room temperature and subsequently at  C overnight. Figure 20.3 Ultrastructural analysis of autophagy-related membrane compartments. Wild-type or atg8D mycelia grown in complete medium for days and subjected to nitrogen starvation for 16 h (in the presence of mM PMSF) were processed for thinsection transmission electron microscopy. Numerous autophagic bodies can be detected in the vacuole of the wild-type strain. Scale bar ¼ 0.5 mm. 302 Yi Zhen Deng et al. 5. Fixed mycelia are washed times (10 each) with 0.1 M phosphate buffer, pH 7.2. 6. The washed mycelial samples are postfixed for h in 250 ml of osmium tetraoxide (1%, w/v). 7. Fixed mycelia are again washed times (10 each) in 0.1 M phosphate buffer, pH 7.2. 8. Samples are dehydrated in a graded ethanol series (25%, 50%, 75%, 100%; 10 in 500 ml each). 9. The samples are then washed twice, for 15 each, in 250 ml of propylene oxide. 10. Samples are infiltrated in 500 ml of propylene oxide-Spurr’s resin (1:1) for h, and then infiltrated overnight in 100% Spurr’s resin. 11. Next, samples are embedded in Spurr’s resin and polymerized overnight at 70  C in EMS embedding molds (Electron Microscopy Sciences, USA). 12. Ultrathin (80 nm) sections are generated using Leica Ultracut UCT and mounted on 200 mesh copper grids. 13. Mounted sections are stained for 15 at room temperature with a mixture of 2% uranyl acetate and 2% Reynolds’ lead citrate (10 ml for each grid) and examined using a JEM1230 transmission electron microscope ( Jeol, Tokyo, Japan) at 120 kV. 2.4. Monodansylcadaverine (MDC) staining of autophagic vesicles MDC is an acidotropic dye that labels late stage autophagosomes or autophagic vesicles (Niemann et al., 2001). MDC staining was successfully used for monitoring the increased autophagic activity in nitrogen-starved Magnaporthe mycelia (see Fig. 20.2), and the incorporation of MDC into late-stage autophagosomes or autophagic vesicles was inhibited by pretreatment with WM, the chemical inhibitor of autophagic sequestration. Furthermore, MDC staining with the conidiating cultures of Magnaporthe at different stages likely reflects the natural induction of autophagy (either basal levels or developmentally-induced), without starvation. MDC-incorporated compartments were copious in the conidiation-specific cell types, including aerial hyphae, and both young and mature conidia. An important limitation is that MDC staining fails to differentiate between late stage autophagosomes and autophagic (acidified) vacuoles. Procedure 1. Magnaporthe wild-type or atg8D strains are cultured on Prune agar medium (PA; per liter: prune juice 40 ml, lactose g, yeast extract g, agar 20 g) in the dark at 28  C for days. Analysis of Autophagy in Fungi 303 2. The wild-type and atg8D strains are then subjected to constant illumination (using overhead room lighting) to induce conidiation at room temperature. 3. At 6, 12, and 48 h after photoinduction, the conidiating cultures of the wild-type and atg8D strain are harvested by scraping with an inoculation loop and stained with 0.05 mM MDC solution (stock solution: mM in normal phosphate buffered saline, pH 7.0) at 37  C for 15 min. The MDC is then washed out with PBS before microscopic observation. 4. MDC-stained mycelia are observed using an epifluorescence microscope equipped with the following filter sets: excitation wavelength 350 nm, emission 320 to 520 nm. 2.5. LysoTracker-based visualization of vacuoles and vesicular compartments LysoTracker Green DND-26 and LysoTracker Red DND-99 (InvitrogenMolecular Probes, USA) are commonly used to stain and visualize acidic compartments, including autophagic compartments (Liu et al., 2005; Scott et al., 2004). LysoTracker dyes differ slightly from MDC and label acidified autophagic vacuoles (Fig. 20.4) but fail to incorporate into late-stage autophagosomes. In conidiating aerial hyphae of Magnaporthe, MDC staining was prominent, while very rare staining of LysoTracker Green DND-26 or LysoTracker Red DND-99 was detected. Both the LysoTracker- and MDC-labeled spherical compartments were evident in conidia, indicating that aerial hyphae are devoid of vacuoles that are mostly formed in conidia. One major drawback of the use of LysoTrackers is the inability to perform co-localization studies with RFP-Atg8 labeled vesicles (Deng and Naqvi, unpublished data). Figure 20.4 Mycelia from the wild-type B157 strain of Magnaporthe was stained with LysoTracker Green DND-26 or LysoTracker Red DND99 and subjected to the requisite epifluorescence microscopy to visualize acidified autophagic vacuoles. Bar ¼ mm. Different morphological variants of the fungal vacuoles (numerous small vesicles, top, or big round vesicles, bottom) are detected through LysoTracker DND staining. Glycogen autophagy and Magnaporthe development .D o no t di st r ib u te . from a likely cleavage at Gly116) fused to RFP. It is possible that this peptide is degraded rapidly in the cytosol, since the Atg8-RFP could be detected as a faint signal in micrographs (Fig. 2B) and in immunoblots with RFP antiserum (Fig. 2C). Having ascertained the specificity of the antiRFP antisera (Fig. S3), we then assessed the various predicted Atg8 modifications by western analyses with antiRFP antisera on total lysates from RFP-ATG8 or ATG8-RFP strain grown either in CM or MM - N medium. Based on relative mobility and predicted size, the two bands detected by the antiRFP antibody in ATG8-RFP lysates (Fig. 2C; MM - N) were judged to be the full-length Atg8-RFP and likely the DLFEEVE peptide fused to RFP (asterisk; although unconfirmed but likely generated upon cleavage at glycine 116) respectively. The stronger Atg8p-RFP band also indicated the enhanced expression of ATG8 under nitrogen limiting conditions. In MM - N cultured RFP-ATG8 strain, proteins likely representing RFP-Atg8p, RFP-Atg8p-PE and RFP alone were detected (Fig. 2C). Compared to the CM grown mycelia, there was an obvious and expected increase in the amount of the predicted RFP-Atg8-PE (Fig. 2C) along with the other modified forms. The mRFP moiety in an mRFP-LC3 fusion is resistant to the lysosomal acidic and degradative conditions.40 The intense 26-kDa band likely corresponds to such free vacuolar RFP in the RFP-ATG8 strain, and indirectly indicates enhanced autophagic response during nitrogen starvation in Magnaporthe. These results helped us to deduce that Atg8p is induced during nitrogen starvation and likely undergoes posttranslational processing such as endoproteolytic cleavage (likely at glycine 116 as reported in some organisms, although we not rule out an alternate cleavage site in Magnaporthe Atg8) and conjugation to PE. Furthermore, we infer that the lipidated form of RFP-Atg8 is likely enriched in autophagosomes and could serve as an appropriate tool to detect and depict autophagy in Magnaporthe. Induction and subcellular localization of RFP-Atg8 in Magnaporthe. Using the RFP-ATG8 strain and epifluorescence microscopy, we visualized the temporal and spatial distribution of RFP-Atg8 protein (and autophagosomes/autophagic bodies) during asexual differentiation in Magnaporthe. Calcofluor white was used as a co-stain to delineate the outline of the analyzed fungal structures. After growth in prune agar medium (PA; non-induced medium for autophagy) in the dark for days, the RFP-ATG8 cultures were subjected to constant illumination to induce conidiation. During growth in the dark, profuse aerial hyphae were formed, which upon light induction showed tip swelling and differentiated into conidiophores and finally produced mature conidia. At early stages, RFP-Atg8p was prominently seen localizing as discrete puncta in the aerial hyphae (Fig. 2D). Later on, the RFP-Atg8 signals appeared as punctate structures as well as showed vacuolar localization in the stalks and the swollen tips of conidiophores (Fig. 2D). Upon initiation of the first incipient conidium, the RFP-Atg8 puncta were located prominently in the developing conidium, and remained highly enriched in these structures until proper differentiation of asexual spores (Fig. 2D). The induction of autophagy in aerial structures during asexual differentiation was distinct and significanty stronger (p < 0.0001) as compared to the starvation or age-related induction of autophagy in mycelia (Suppl. Fig. S4A and B). In a parallel experiment, Lysotracker Green DND-26 staining confirmed copious autophagic vacuoles or late-stage autophagosomes in several conidiation specific cell types (Fig. 2E). We conclude that RFP-Atg8 © 20 09 La nd es B io s ci en ce therein (Fig. 1B, lower). In contrast the atg8Δ showed a significant decrease in aerial hyphae formation and conidiophore differentiation (Fig. 1B). At 24 hpi, 34.0 ± 4.5% of wild-type aerial hyphae produced conidiophores, as opposed to only 2.0 ± 1.0% in the atg8Δ mutant (p = 0.001). Such decreased efficiency of conidiophore formation in the atg8Δ remained unchanged even at 48 hpi (data not shown). Proper conidiophore differentiation (24.0 ± 4.8%) was restored in the complemented atg8Δ strain. Conidiation defects upon loss of autophagy have recently been reported in Magnaporthe.26,27 We performed a detailed quantification and confirmed that compared to the wild type (98.7 ± 10.9 x 102 conidia/cm2), the atg8Δ (1.4 ± 0.03 x 102 conidia/cm2) showed severely reduced (~98%) conidiation and that this defect was suppressed in the complemented atg8Δ strain (97.5 ± 9.1 x 102 conidia/cm2). Furthermore, viability of the atg8Δ conidia (51.2 ± 1.2% cfu) was significantly lower than the wild-type conidia (77.5 ± 2.5% cfu). To assess the overall morphology of the resultant structures, we stained the conidiating cultures of wild type and atg8Δ strains with calcofluor white at various time points during asexual growth. As shown in Figure 1C (lower), the atg8Δ conidiophores and conidia were heterogeneous with a vast majority remaining undifferentiated and/or showing abnormal morphology. Two kinds of abnormalities were easily discernible: failure to produce and elongate the conidiophore stalk and cessation of growth at the conidium initiation stage (Fig. 1C). Such persistent defects in the atg8Δ (Fig. 1C) were likely due to gross abnormalities observed in all the preceding stages of conidiation (aerial hyphae, aerial hyphal tips, stalks, conidiophores). Normal and mature conidia were rare in the atg8Δ strain (Fig. 1C, last) and in a majority of the cases only a single aberrant conidium was produced per conidiophore (Fig. 1C). At the corresponding time points, wild-type hyphae differentiated proper aerial structures that developed appropriately to initiate conidiophores to finally produce the tricellular conidia in a sympodial manner (Fig. 1C). Taken together, these results reaffirm that Magnaporthe atg8Δ is defective for autophagy, and establish that Atg8 (and by inference autophagy) plays a critical role during aerial hyphal development and conidia formation in Magnaporthe. Post-translational processing and Atg8p targeting to autophagosomes in Magnaporthe. In Atg8p/LC3/GATE-16, a specific cleavage by Atg4 protease exposes the carboxyl-terminal glycine residue (Fig. 2A) that is essential for a ubiquitylation-like conjugation reaction22 and subsequent amidation to PE. Such a glycine residue was found to be well conserved in Magnaporthe Atg8 (Fig. 2A). To assess posttranslational processing and subcellular localization of Atg8p, we generated Magnaporthe strains expressing RFP-Atg8 or Atg8-RFP fusion proteins under native regulation and as the sole copy of ATG8 in each instance. These two strains were cultured in liquid complete medium (CM or MM + N) for days, and then inoculated into liquid MM - N (deprived of nitrogen) to induce autophagy in the presence of PMSF. During growth in CM, the RFP signals were undetectable or weak in RFP-ATG8 or ATG8-RFP strains (Fig. 2B). However, upon growth in MM - N for about 16 hours, predominant RFP-Atg8 puncta or rings representing autophagosomes could be easily observed. Such punctate structures were absent in the ATG8-RFP strain, even under starvation conditions. Instead, faint RFP signals were seen distributed uniformly in the cytosol (Fig. 2B) possibly representing the amino acid peptide (DLFEEVE, resulting www.landesbioscience.com Autophagy 35 © 20 09 La nd es ib u st r di no t o .D en ce ci B io s predominantly localizes to autophagosomes and autophagic vacuoles during important steps of asexual development and further construe that autophagy is naturally induced during Magnaporthe conidiation. Suppression of conidiation defects in atg8Δ by alternate carbohydrate sources. Rather serendipitously, we found that the atg8Δ gained a fluffy appearance, suggesting restoration of aerial hyphal growth, when supplemented with glucose or sucrose as a carbon source. In experiments described earlier (Figs. 1B and S1C), loss of aerial growth in the atg8Δ was highly pronounced on media containing lactose as the sole source of carbon. Therefore, we tested the conidiation capability of atg8Δ in the presence of a variety of alternate carbohydrate sources mostly sugars. Interestingly, the addition of sucrose or glucose significantly restored conidiation in atg8Δ even in the presence of the nonrepressible disaccharide, lactose (p = 0.001; Fig. 3A). Although profuse (Fig. 3B) the suppression of atg8Δ conidiation was lower in glucose (about 64 x 102 conidia/cm2; Fig. 3A) when compared to that induced by sucrose (average 99 x 102 conidia/ cm2; p < 0.01). Not surprisingly, galactose, which is not utilized well by Magnaporthe, failed to cause a significant repression of conidiation defects in atg8Δ mutant (Fig. 3A). We conclude that the availability of a readily utilizable sugar source such as glucose or sucrose circumvents the requirement of autophagy during asexual development in Magnaporthe. Furthermore, we infer that the nutrient status and proper regulation of carbohydrate metabolism are important during conidiation and influence both the quantity and quality of conidia production in Magnaporthe. Gph1 is involved in glycogen metabolism during Magnaporthe conidiogenesis. To identify proteins that are regulated by autophagic degradation during conidiogenesis, we performed SDS-PAGE fractionation of total protein extracts from four-day old conidiating cultures of the wild type, the atg8Δ and the complemented strain. Mass spectrometry was then used to identify the proteins that showed differential accumulation (Fig. 4A). Among these was a glycogen phosphorylase of 98 kDa, encoded by MGG_01819, an ortholog of yeast GPH1.41 In S. cerevisiae, GPH1 is transcriptionally induced at late exponential growth phase, concomitant with the onset of intracellular te . Glycogen autophagy and Magnaporthe development 36 Figure 2. Posttranslational modification and subcellular localization of RFP-tagged Atg8p. (A) ClustalW57 assisted sequence comparison between Magnaporthe Atg8 protein and its orthologs. MgAtg8 protein (XM_368182) was compared with GATE-16 from Mus musculus (NM_026693), ScAtg8p from S. cerevisiae (YBL078C), GABARAP from Homo sapiens (NM_007278), and HsLC3 from Homo sapiens (BC041874). Residues that are conserved or similar in at least three out of the five sequences are boxed in black or in gray respectively. Arrowhead indicates the site of intramolecular cleavage to expose the conserved glycine residue for lipidation with PE. (B) ATG8-RFP or RFP-ATG8 strain cultured in Complete medium for 2–3 days was subjected to nitrogen starvation for 16 h and imaged using laser-scanning confocal microscope to detect the RFP signals. Scale Bar = 10 μm. (C) Total protein lysates from the indicated strains were fractionated using SDS-PAGE and analyzed by immunoblotting with anti-RFP antibodies (upper). The immunoblot was subsequently reprobed with anti-Porin antibody to serve as a loading control (lower). Asterisk indicates the predicted DLFEEVE-RFP peptide resulting from the proteolytic cleavage at glycine 116. CM refers to nitrogen replete whereas MM - N to nitrogen limiting growth conditions. (D) Subcellular localization of RFP-Atg8p during conidiation. Conidiating cultures of the RFP-ATG8 strain were stained with calcofluor white and analyzed by epifluorescence microscopy to detect the RFP signal at different stages of asexual development (aerial hyphae, conidiophore and conidia). Bar = μm. (E) Wild-type strain was stained with Lysotracker Green DND-26 to visualize acidified autophagic vacuoles at the same developmental stages as shown in (D). Bar = μm. Autophagy 2009; Vol. Issue Glycogen autophagy and Magnaporthe development o .D en ce Figure 3. Suppression of conidiation defects by alternate carbon sources in atg8 deletion mutant. (A) Bar chart depicting quantitatively assessed conidiation in the atg8Δ grown on prune-agar medium containing 0.5% lactose and the indicated sugar (final concentration 1%). Mean values (±SE) presented as percentage points were derived from three independent experiments (n = 30 colonies for each sample). Assessments were performed days post induction. (B) Photomicrographs depicting the extent of conidia formation in atg8Δ in the presence of the indicated carbon source(s). Images were taken days post induction of conidiation. Scale bar = 10 micron. no t di st r ib u te . Fig. 5A). However, exogenous G1P was unable to suppress the conidiation defects in the atg8Δ strain (Fig. 5A). Lastly, conidia production in a GPH1 OP-2 strain (which indirectly simulates the increased levels of Gph1 observed in the atg8Δ) was 42.4 ± 1.5 (x100/cm2) showing a nearly 60% reduction compared to the wild type (Fig. 5A). This suggests that the decrease in conidia formation in the atg8Δ mutant is partly due to the increased Gph1 levels observed therein. The reduced conidiation defect in the GPH1 OP-2 strain could be significantly suppressed with the addition of G6P or glucose (Fig. 5B; p < 0.001) to the growth medium. We conclude that the loss of Gph1 function, either through deletion of GPH1 or by inhibition of the enzyme activity using G6P, partially represses the conidiation defects associated with the loss of autophagy in Magnaporthe. Taken together, we concur that glycogen homeostasis, likely mediated through Gph1 and autophagy, plays an important role during asexual development in Magnaporthe. Glycogen homeostasis is regulated by autophagy and Gph1 during conidiation in Magnaporthe. Since gph1Δ atg8Δ strain showed a partial suppression of conidiation defects, and Gph1 is indicated to be involved in glycogen catabolism, we performed quantitative (Fig. 5C) and semi-quantitative assessment (Fig. S7) of the steady-state levels of glycogen in the conidiating colonies of the wild-type, atg8Δ, gph1Δ, gph1Δ atg8Δ or GPH1 OP-2 strain on PA medium (lactose). We estimated glycogen at three critical time points: 0d (immediately before photo-induction), 2d (early stage) and 4d (late stage) during the conidiation cycle in Magnaporthe (Figs. 5C and S7). The wild type showed a steady increase in glycogen as conidiation proceeded (Figs. 5C and S7; p = 0.001). However, compared to the wild type at 2d (1.2 ± 0.01%; Fig. 5C), the atg8Δ showed strikingly high (2.32 ± 0.25%; p = 0.001) glycogen levels, which were significantly reduced upon loss of GPH1 function (gph1Δatg8Δ; 1.23 ± 0.01%; p = 0.001). Glycogen level in the gph1Δ, was comparatively lower at 0d (0.63 ± 0.01% versus 0.99 ± 0.08% in wild type) but increased steadily at the early (1.26 ± 0.11%; p < 0.01) and late stage (2.2 ± 0.3%) of conidiation. Prior to conidiation, however, glycogen levels were significantly high and low (respectively; p < 0.001) in the gph1Δ atg8Δ and the gph1Δ, when compared to the wild type or the atg8Δ strain (Fig. 5C). At the late stage of conidiogenesis, the atg8Δ and the gph1Δ atg8Δ mutant showed a decrease in glycogen (Fig. 5C) compared to the early stage. Overproduction of Gph1 (GPH1 OP-2) caused only a marginal increase in glycogen at 4d into the conidiation cycle (1.56 ± 0.07%; Fig. 5C). Taken together, we infer that glycogen accumulation and breakdown occurs during conidia formation in Magnaporthe and is tightly regulated by autophagy and Gph1. We further construe that glycogen catabolism at the onset of conidiation, likely regulated by autophagy and Gph1, is required for proper asexual development in Magnaporthe. Glycogen autophagy and Magnaporthe pathogenesis. An earlier study showed that Magnaporthe atg8Δ strain is incapable of host penetration and is completely non-pathogenic.26 Therefore, we tested whether loss of Gph1 also suppresses the pathogenicity defects associated with atg8Δ. The gph1Δ atg8Δ double mutant appeared to be incapable of infecting the host through the appressorial route (Fig. 6A). Microscopic analysis revealed that the gph1Δ atg8Δ mutant failed to produce any invasive hyphae at 48 hpi (Fig. 6B) or later stages postinoculation. The gph1Δ mutant did not differ significantly © 20 09 La nd es B io s ci glycogen accumulation.41,42 Gph1 acts in the cytoplasm to release G1P from glycogen. We created a gph1Δ strain in Magnaporthe by replacing MGG_01819 (NCBI accession XP_363893) with the bialaphosresistance marker (Bar) in the wild type, as well as in the atg8Δ background. Correct gene replacements were confirmed by Southern blotting (Fig. S5). Loss of GPH1 did not affect vegetative growth, asexual development or pathogenesis in Magnaporthe. Deletion of GPH1 in atg8Δ strain did not alter the colony morphology (Fig. 4B). However, the gph1Δ atg8Δ strain showed a partial but significant restoration of conidiation (Fig. 4C; p = 0.001). The overall conidiation of gph1Δ atg8Δ mutant was quantified to be 17.0 ± 0.8 [conidia (x100/cm2)], which was a significant increase compared to atg8Δ mutant. However, there was no significant increase in conidial viability in the gph1Δ atg8Δ compared to the atg8Δ mutant. At 12–30 hpi, calcofluor white staining of conidiating gph1Δ atg8Δ cultures, revealed abundant healthy aerial hyphae, some already with the characteristic swollen tips indicating the induction of conidiogenesis. Subsequently, mature conidia were also observed (Fig. 4D) together with multiple conidia growing sympodially at the tips of the stalks. Compared with atg8Δ strain (Fig. 1C for comparison), the gph1Δ atg8Δ produced normal aerial hyphae and less abnormal tips, suggesting a likely reduction and a partial suppression of anomalies associated with the loss of autophagy during asexual development in Magnaporthe. To further confirm the direct involvement of Gph1p-regulated glycogen metabolism in Magnaporthe conidiogenesis, we assessed the effect of G6P and G1P on conidiation in atg8Δ strain. G6P is an efficient inhibitor of the active form of Gph1.41 The addition of 0.5 mM G6P to the growth media caused a remarkable suppression of conidiation defects in the atg8Δ strain (53.2 ± 4.4 x102 conidia/cm2, www.landesbioscience.com Autophagy 37 Glycogen autophagy and Magnaporthe development ib u st r di no t o .D en ce ci io s Discussion te . from the wild type in terms of its capacity to breach the host surface and cause disease (Fig. 6A and B). Interestingly, and unlike the atg8Δ, the gph1Δ atg8Δ strain elicited a slight induction of hypersensitive reaction (HR; inferred from tiny brown speckles at the infection site) in the host (Fig. 6A) suggestive of at least some successful host penetration attempts by the gph1Δ atg8Δ strain, although further spread and infection was not successful. Aniline blue staining was therefore carried out to detect papillary callose deposits in order to quantify the host penetration events by atg8Δ or gph1Δ atg8Δ strains in comparison to the wild type. As depicted in Figure 6C, the ability to form penetration pegs on barley leaves was about 77% (±5.6; n = 450) in the wild-type strain at the 48 hr time-point, whereas it was 2.6% (±0.3; n = 300) in the atg8Δ mutant. Even upon extended incubation (72 h), a vast majority of the atg8Δ appressoria (~95%) failed to penetrate the host surface and elicit callose deposits. The gph1Δ atg8Δ strain showed only a marginal increase in the level of papillary callose deposits (Fig. 6C) to 13% (±1.5) (n = 300; p = 0.01) respectively. We conclude that the glycogen catabolism function of autophagy plays only a minor role during the host penetration as well as the in planta growth stages of Magnaporthe pathogenesis. © 20 09 La nd es B Autophagy is essential for survival during nutrient starvation and represents a cellular degradation mechanism that uses vacuolar proteases for turnover of certain proteins, organelles and membranes.43,44 In this study, through gene deletion analysis and genetic complementation, we confirmed that loss of ATG8 leads to a loss of autophagy and results in a huge reduction in asexual sporulation in Magnaporthe. The primary conidiation defect in Magnaporthe atg8Δ mutant is the failure to produce sufficient aerial hyphae and a subsequent inability to differentiate into proper conidiophores. The present study adds to the limited but growing list of Magnaporthe mutants that show reduced and aberrant conidiation.6,7,26,27 Apart from an essential role in sexual sporulation in yeast,45 autophagy has recently been implicated in conidiation and conidial germination in Aspergillus and Magnaporthe.26-28 Our results add to these findings and suggest a functional role for 38 Figure 4. Identification and functional analysis of Gph1 in Magnaporthe. (A) Identification of proteins differentially regulated in the atg8Δ mutant. Silver-stained profile of the SDS-PAGE gel depicting total proteins extracted from conidiating mycelial cultures from wild type (WT), atg8Δ or the atg8Δ complemented strain. Proteins differentially regulated and identified by mass spectrometry have been listed together with their predicted molecular masses. Western blotting with anti-Porin antisera served as a loading control. Mw refers to the molecular mass markers in kilodalton. (B) Colony and growth characteristics of the wild type, atg8Δ, gph1Δ and gph1Δ atg8Δ double mutant. Colonies of the indicated strains were grown on prune agar medium with lactose as the carbon source for days in the dark and photograghed. Scale bar = cm. (C) Bar chart depicting quantification of conidiation in the wild type (WT), atg8Δ, gph1Δ or gph1Δ atg8Δ strain grown on prune-agar medium containing 0.5% lactose as the sole source of carbohydrate. Mean values (±SE) presented as percentage points were derived from three independent experiments (n = 30 colonies for each strain). Assessments were performed days post induction. (D) Partial restoration (qualitative and quantitative) of conidiation in the atg8Δ mutant upon loss of GPH1 function. Calcoflour-white stained gph1Δatg8Δ strain was analyzed by epifluorescence microscopy to assess the indicated developmental stages during conidiation. Lower panels depict the representative incidence of aberrant conidiation-specific defects that still persist in the mutant. The experiment was repeated three times using a total of 10 colonies. Bar = 10 μm. Autophagy 2009; Vol. Issue Glycogen autophagy and Magnaporthe development nd es B io s ci en ce Figure 5. Functional requirement of glycogen catabolism during Magnaporthe conidiation. (A) Bar chart representing the quantitative analysis of conidiation in the wild type, atg8Δ (treated with G6P or G1P) or the GPH1 OP-2 strains grown on PA medium. Results (mean values ± SE) represent three independent experiments involving a total of 20 colonies per strain. (B) Quantitative analysis of conidiation in the wild type (gray bar) or the GPH1 OP-2 strain (black bar) grown on PA medium (control) or on PA medium supplemented with G6P or glucose. Results (mean values ± SE) represent three independent experiments involving a total of 20 colonies per strain. (C) Quantitative analysis of total internal glycogen concentration in the wild type, atg8Δ, gph1Δ, gph1Δ atg8Δ and GPH1 OP-2 strains. The indicated strains were initially grown in the dark for days and then subjected to constant illumination to induce conidiation. At the specified time-points post induction, the fungal biomass was subjected to glycogen extraction and estimation. The concentration of internal glycogen was presented as percentage of the total wet weight of the fungal biomass. Mean values (±SE) were derived from three independent experiments (n = 15 colonies per strain). .D o no t di st r ib u te . glucose) conditions, which efficiently suppress autophagy. However, the essential requirement for autophagic cell death during appressorium-mediated entry of Magnaporthe26 into the host could not be bypassed by the use of alternate carbon source(s). We observed that atg8Δ conidia harvested from sucrose or glucose-containing medium were unable to penetrate the host leaf surface and could not proliferate as well as the wild type even when introduced through wounded host tissue (data not shown). Overall, these results point to an important role for autophagy in Magnaporthe response to nutritional stress, and a possible connection between carbon homeostasis and autophagy during conidiation as well as in invasive growth inside the host tissue. Our data also suggest a key role for autophagy in the breakdown of carbohydrate stores at the onset of Magnaporthe conidiogenesis in order to produce sufficient intracellular glucose. This mirrors a well-defined role for glycogen autophagy in newborn mammals in the breakdown of intracellular glycogen reserves as a strategy to cope with a sudden demand for ample energy substrates to confront metabolic requirements.34,35 In yeast, there are two distinct intracellular pools of glycogen: cytosolic and vacuolar. Gph1 catalyzes the release of G1P from glycogen and mediates glycogen breakdown in the cytosol.41 However, during sexual sporulation, glycogen degradation releases glucose and is catalyzed by a vacuolar glucoamylase.42 In S. cerevisiae, inhibition of the vacuolar glycogen degradation, prevents the onset of sporulation,31,46-48 indicating that autophagy likely serves to deliver cytoplasmic glycogen into vacuoles for degradation, to produce sufficient energy and/or appropriate intermediates (e.g., G6P or glucose) for cellular differentiation. Loss of autophagy led to increased accumulation of Gph1, and rather surprisingly a significantly high steady-state level of glycogen at the early stages of asexual differentiation in Magnaporthe. Such concomitant increase in glycogen as well as Gph1 in atg8Δ is likely due to growth under glucose-limiting conditions, which is known to upregulate genes required for glycogen biosynthesis and breakdown in yeast.41 Deletion of GPH1 in atg8Δ mutant reduced the glycogen levels to those observed in the wild type during early stages of conidiation. However, glycogen levels are higher prior to induction of conidiation in the gph1Δ atg8Δ mutant. We infer that glycogen levels are in a state of flux during conidiation and speculate that a strict temporal and spatial regulation of glycogen homeostasis, mediated by Gph1 and autophagy, is critical for asexual differentiation in Magnaporthe. Future studies would certainly aim at distinguishing between the cytoplasmic and vacuolar stores of glycogen, and to uncover the mechanisms that regulate glycogen metabolism during asexual development in Magnaporthe. The gph1Δ atg8Δ mutant showed an overall improvement in the morphology of conidiation-specific structures suggesting that sufficient glucose produced by glycogen autophagy is likely necessary for proper growth and differentiation during asexual development. Furthermore, G6P-based suppression of atg8Δ defects was stronger than that achieved through deletion of GPH1, suggesting that G6P is likely involved in regulation of other relevant processes such as glycolysis and gluconeogenesis, which might be important in regulating proper carbon homeostasis during asexual development in Magnaporthe. However, deletion of GPH1 in atg8Δ background did not increase the number of conidiophores per se, implying that factor(s) other than carbohydrate metabolism are also necessary © 20 09 La autophagy in the recycling of nutrient resources, in particular carbon (this study), nitrogen32 and metal ions,32 for extensive cellular remodeling required during asexual differentiation in filamentous fungi. Autophagy thus attains importance during nutrient-limiting condition, which is a well-established trigger for asexual sporulation in several fungi including Magnaporthe. Further studies are needed to dissect the mechanistic regulation of conidiation, and to elucidate the exact physiological role of autophagy in the process of asexual development in Magnaporthe. A key finding was the remarkable suppression of atg8Δ conidiation defects by external supplementation of carbohydrates (such as sucrose or glucose). This suggests that autophagy becomes essential for aerial hyphal growth and conidiophore differentiation during carbon-limiting conditions as well as in the presence of some nonrepressible sugars. Alternately, autophagy could be dispensable for conidiation during growth under carbohydrate-replete (exogenous www.landesbioscience.com Autophagy 39 © 20 09 La nd es B io s ci Fungal strains and growth conditions. Magnaporthe wild-type strain B157 (Field isolate, mat1-2) was obtained from the Directorate of Rice Research (Hyderabad, India). Magnaporthe strains were propagated on Prune-agar (PA) medium or complete medium (CM) as described.49,50 Carbohydrate-supplemented Prune-agar medium for assessing conidiation in atg8Δ contained lactose (5 g/L) and one of the following sugars: sucrose, glucose, lactose, galactose or maltose at 10 g/L. The composition of MM and MM - N (used for nitrogen starvation) was as reported earlier.51 Two-day old liquid CM-grown mycelia were ground in liquid nitrogen for the isolation of nucleic acids. To assess the growth and colony characteristics, Magnaporthe isolates were cultivated on CM agar or PA medium, at 28°C for one week. Mycelia used for total protein extraction were obtained by growing the relevant strains in liquid CM for 2–3 days, with gentle shaking, followed by inoculation in MM or MM - N for about 16 hours. For quantitative analysis of conidiation, colonies were cultivated on PA medium in the dark for days, followed by a 4-day growth cycle under constant illumination at room temperature. The surface of the colonies was then scraped with inoculation loops in the presence of water and the fungal biomass was harvested in FalconTM Conical Tubes (BD Biosciences, San Jose, California). The suspension was vortexed thoroughly to ensure maximum detachment of conidia from mycelia, and then filtered through two layers of Miracloth (Calbiochem, San Diego, California). Conidia thus collected were washed twice with and finally resuspended in sterile water. Conidia production in a given colony was quantified using a hemocytometer and reported as the total number of conidia present per unit area of the colony [conidia (x100/cm2)]. To test the pathogenicity, conidia harvested from 6-day-old cultures were resuspended to x 105 conidia per mL in sterile water. Droplets (20 μl) of conidial 40 ib u st r di no t o en ce Materials and Methods Figure 6. Glycogen catabolism and Magnaporthe pathogenesis. (A) Host penetration is partially restored in the gph1Δ atg8Δ mutant. Barley leaf explants were inoculated with conidia from the wild type, gph1Δ atg8Δ or gph1Δ atg8Δ Magnaporthe strains and disease symptoms assessed after days. (B) The gph1Δ atg8Δ mutant is incapable of invasive growth in the host. Equal number of conidia from the gph1Δ or the gph1Δ atg8Δ were inoculated on barley leaf explants and allowed to proceed through infectionrelated development for 48 hours. Asterisk denotes the infection hyphae in planta. Scale bar represents 10 μm. (C) Partial restoration of host penetration in the gph1Δ atg8Δ mutant. Bar chart representing papillary callose deposits (as percentage of total appressoria) in barley leaf explants inoculated with conidia from the indicated strains grown on PA medium. Quantification was performed 48 hours post inoculation and represents mean ± SE values derived from three independent experiments. .D for Magnaporthe conidiogenesis, and are probably subjected to autophagy-dependent regulation. Future experiments will focus on the identification of such positive regulators as well as their ­functional relationship with the autophagy pathway. We established RFP-Atg8p-PE as a reliable marker for autophagosomes and autophagic vacuoles in aerial hyphae and conidiophores in Magnaporthe. As expected, RFP-Atg8 required the critical posttranslational modifications (predicted endoproteolytic cleavage most likely at G116 and lipidation to PE) prior to its recruitment to preautophagosomal structures. Very little (if any) RFP-Atg8p was discernible in vegetative mycelia when the strain was grown in the dark, whereas RFP-Atg8p-PE was clearly visible in autophagosomes in the aerial hyphae. Whether RFP-Atg8p expressing aerial hyphae subsequently differentiate into conidiophores is an important question and is presently hindered by a lack of suitable conidiophore-specific markers. We observed that autophagy is induced naturally during conidiogenesis and that autophagosomes are distributed prominently in conidiation-related structures such as aerial hyphae, conidiophores and conidia. Recent studies on ATG826 and ATG1 function27 show that autophagy plays a key role in cell death, lipid mobilization, and turgor generation during the pathogenic phase in Magnaporthe. Future studies would certainly aim at identifying the specific targets of the autophagosomal-vacuolar degradation machinery in asexual and pathogenic growth phases of Magnaporthe. te . Glycogen autophagy and Magnaporthe development suspension were inoculated on barley leaf explants and incubated under humid conditions at 23°C for up to 96 hours.52 Methanol treatment and staining with 0.1% acid fuchsin (Sigma-Aldrich, USA) was carried out as described53 for observation of penetration and infection hyphae. Nucleic acid and protein-related manipulation. Standard molecular manipulations were performed as described.54 Fungal genomic DNA was extracted using a modified potassium acetate method.55 Plasmid DNA was isolated with QiaPrep plasmid miniprep kit (Qiagen, Valencia, California) and nucleotide sequencing performed using the ABI Prism Big Dye terminator method (PE-Applied Biosystems, California). Homology searches of DNA/protein sequences were performed using the BLAST programs56 and multiple sequence alignments carried out with ClustalW57 and Boxshade (http://bioweb.pasteur.fr/seqanal /interfaces/boxshade.html). Total RNA was isolated with RNeasy Plant Mini kit (QIAGEN, USA), and cDNA synthesis conducted using AMV Reverse Transcriptase (Roche Diagnostics, Germany). Following primers were used to amplify the 5' and 3' UTR of the ATG8 gene: ATG8-5F (5'-GAG AGT GAA CTC GAG GCT ATA ACC TGA GGG TAG-3'), ATG8-5R (5'-GAG AGT GAG GAT CCC GGT TGA TTG AGA CTT GT-3'), ATG8-3F (5'-GAG AGT GTT CTG CAG CGA GTG AGC TTG CTC ACC-3'), and ATG8-3R (5'-GAG AGT GTT AAG CTT CAC GTC CTC CCA-3'). The full-length genomic copy of Autophagy 2009; Vol. Issue Glycogen autophagy and Magnaporthe development .D o no t di st r ib u te . criteria on Data Explorer v4.6 (Applied Biosystems) by comparing peptide masses with those in the NCBI protein database. Gene deletion and complementation analyses. Genomic DNA fragments (about kb each) representing the 5' and 3' UTR of ATG8 were amplified by PCR, ligated sequentially so as to flank the HPH1 cassette in pFGL44 to obtain the plasmid pFGLatg8KO. Genomic DNA fragments (about kb each) representing the 5' and 3' UTR of GPH1 gene were obtained by PCR, ligated sequentially so as to flank the BAR cassette in pFGL97 to obtain plasmid vector pFGLgph1KO. pFGLatg8KO or pFGLgph1KO was transformed into M. oryzae for replacement of the ATG8 or GPH1 respectively. For genetic complementation, the complete M. oryzae ATG8 locus was PCR- amplified as a 3.4 Kb BamHI-XhoI fragment and cloned into the corresponding sites in pFGL97 to obtain pFGLATG8Comp with resistance to bialaphos or ammonium gluphosinate (Cluzeau Labo, France) as a fungal selectable marker. Southern blot analyses were performed by standard procedures54 to confirm successful gene deletion and single-copy integrations. Plasmid constructs for RFP-Atg8 and Atg8-RFP fusions. The promoter fragment of the ATG8 gene was PCR amplified from genomic DNA from the wild-type strain using primers (5'-GTC GGT CGG TCG CAT GCT GCA GTT AAA GTG-3') and (5'-GAG AGT GCA TAT GGG CGG CGG TTG ATT GAG AC-3'). The RFP ORF was amplified with the forward primer (5'GAG AGT GCA TAT GGA CAA CAC CGA GGA CGT C-3') and reverse primer (5'-ATG CGG TCG GTA TAC TTC TGG CGA ATG CGC TCG GCT TCA GCC TTG CGC TTC TCG AAG GGG TGC TCG TCC TTG AAC TTG GAG CGC TGG GAG CCG GAG TGG CGG GCC TC-3'), with pDSRED monomeric N1 (Clontech, USA) as template. pFGLATG8Comp was digested with PstI and XcaI (both unique sites within the ATG8 locus) to remove the fragment spanning approximately last 500 bp of the promoter and first 72 bp of the coding sequence of ATG8. The resultant vector was then ligated with the PCR-amplified ATG8 promoter (digested with PstI and NdeI) and the RFP ORF (digested with NdeI and XcaI) in one step, so that the newly created plasmid contained an in-frame insertion of RFP ORF at the translational start site within the ATG8 coding sequence while retaining the requisite native regulatory sequences. This plasmid was named as pRFP-ATG8 and introduced as a single-copy insertion in the atg8Δ strain. For Atg8-RFP construct, the Kb fragment just proximal to the translation stop codon in ATG8 was amplified with primers (5' GAG AGT GAG GTA CCT CGC CCC GCT TCA CAG CAT CGG 3') and (5' GAG AGT GCA TAT GCT CGA CTT CCT CAA ACA GGT 3'). The RFP coding sequence was amplified with (5' GAG AGT GCA TAT GGA CAA CAC CGA GGA CGT C 3') and (5' GAG AGT GGG ATC CCT ACT GGG AGC CGG AGT GGC 3'). The above two fragments were ligated into the KpnIBamHI sites of the vector pFGL44 in one step. The Kb ATG8 fragment immediately downstream of the stop codon was amplified with the primers (5' GAG ACT GTT CTG CAG TCT GTC GAC GCG GAG TGG ATA C 3') and (5' TAG GGG AGA CAC AAC CGC AGT A 3'; the sequence is after an internal HindIII site in the locus) and then ligated to flank the HPH1 cassette on pFGL44 (PstI-HindIII). The resultant pATG8-RFP was transformed into the wild-type Magnaporthe strain to specifically replace the ATG8 gene with the ATG8-RFP allele. © 20 09 La nd es B io s ci en ce ATG8 was amplified with ATG8-F (5'-GAG AGT GAG GAT CCT CGG GTT ACT TTG TCA GGC CAT-3') and ATG8-R (5'-GAG AGT GAC TCG AGT ACC TGT CAC GAA CGC GCG GAA-3'). The primers used to amplify the 5' and 3' UTR of the GPH1 gene were as follows: GPH1-5F (5'-GAG AGT GTT AAG CTT TCA ATG TTA CTC TTT GTT TCA C-3'), GPH1-5R (5'-GTT TTA ACT GCA GAG GAA GAA G-3'; the sequence is after an internal PstI site in the locus), GPH1-3F (5'-AGA GTG AGG TAC CAC GAG AAA AGG GAT TTT GGG-3'), and GPH1-3R (5'-GAG TGA GAA TTC CGC TAT CGA GTT CAC GGC CTA C-3'). Underlined text represents the restriction enzyme site for cloning purposes. RT-PCR was performed using the following primers sets: Gph1RTF (5'-ATT GAC AGG CTT CGG AGC TCA AGA G-3'), Gph1RTR (5'-AGC AAG CAG CGA GTC GGC CAA GAC C-3'); and Tub1F (5'-AAA CAA CTG GGC CAA GGG TCA CTA CA-3'), Tub1R (5'-CCG ATG AAA GTC GAC GAC ATC TTG AG-3'). TUB1 corresponds to MGG_00604 ORF. The GPH1 locus including its native promoter (1.1 kb before the start codon) and terminator (108 bp after the stop codon) was amplified by PCR. The total 4299 bp region was amplified as two fragments, using primers gph1For (5' GAG TGA GAA TTC CCT ACA TAC CTA CCT ACA TCC T 3') and gph1Rev (5' GAT CTC GAC GAA GTC CCT GAA G 3') and primers 2For (5' CCC TGG AGA CCG CGA TCT TCT C 3') and 2Rev (5' GAG AGT GTT TCT AGA AGG GAT TAA CCA AAA GCT GAA TAG 3') respectively. The resultant PCR fragments were cloned into EcoRI-XbaI digested pFGL44 using an internal BamHI site to obtain plasmid pFGL(GPH1). The cloned GPH1 fragments were confirmed by nucleotide sequence analysis and pFGL(GPH1) was introduced into the wild-type strain via Agrobacterium T-DNA-mediated transformation for random insertions into the genome. Strains harboring multiple insertions of GPH1 were identified by Southern blotting (data not shown) and GPH1 transcript levels confirmed by semiquantitative RTPCR (Suppl. Fig. S6) and quantitative real-time RTPCR. Two independent strains (termed GPH1 OP-1 and GPH1 OP-2) showing significant increase in GPH1 expression were selected for further investigation. For total protein extractions, MM + N or MM - N grown mycelia were ground to a fine powder in liquid nitrogen and resuspended in 0.3 ml of extraction buffer (10 mM Na2HPO4 pH 7.0, 0.5% SDS, mM DTT and mM EDTA). Lysates were cleared by centrifugation at 12000 g for 30 at 4°C. Protein samples from each extract (100 mg) were fractionated by SDS-PAGE, transferred onto a PVDF membrane (Millipore Corporation, USA) and immunoblotted with anti-RFP antibody (Clontech, USA) at 1:1000 dilution or with anti-Porin at 1:2000. Secondary antibody conjugated to horseradish peroxidase was used at 1:20000 dilution. The ECL kit (Amersham Biosciences, Germany) was used to detect the chemiluminescent signals. To identify differentially regulated proteins in atg8Δ strain, total protein lysates from the wild-type, atg8Δ and the complemented strains were fractionated by SDS-PAGE, and silver-stained as described.58 For mass spectrometry, protein digestion was performed using a Trypsin In-gel Digestion Procedure (www.proteomecenter. org/ under Protocols). MS Instrument used for MALDI-Tof-Tof MS was 4700 Proteomics Analyzer (Applied Biosystems). Database searches for MS protein matches were performed using standard www.landesbioscience.com Autophagy 41 Glycogen autophagy and Magnaporthe development References .D o no t di st r ib u te . 1. Ou SH. Rice Diseases. Surrey, UK 1985. 2. Cole GT. Models of cell differentiation in conidial fungi. Microbiol Rev 1986; 50:95-132. 3. Lee K, Singh P, Chung W, Ash J, Kim T, Hang L, Park S. Light regulation of asexual development in the rice blast fungus, Magnaporthe oryzae. Fungal Genet Biol 2006; 43:694-706. 4. Lau GW, Hamer JE. Acropetal: a genetic locus required for conidiophore architecture and pathogenicity in the rice blast fungus. Fungal Genet Biol 1998; 24:228-39. 5. Shi Z, Christian D, Leung H. Interactions between spore morphogenetic mutations affect cell types, sporulation and pathogenesis in Magnaporthe grisea. Mol Plant Microbe Interact 1998; 11:199-207. 6. Shi Z, Leung H. Genetic Analysis of Sporulation in Magnaporthe grisea by Chemical and Insertional Mutagenesis. MPMI 1995; 8:949-59. 7. Odenbach D, Breth B, Thines E, Weber RW, Anke H, Foster AJ. The transcription factor Con7p is a central regulator of infection-related morphogenesis in the rice blast fungus Magnaporthe grisea. Mol Microbiol 2007; 64:293-307. 8. Shi Z, Leung H. Genetic Analysis and Rapid Mapping of a Sporulation Mutation in Magnaporthe grisea. MPMI 1993; 7:113-20. 9. Liu Y, Schiff M, Czymmek K, Talloczy Z, Levine B, Dinesh-Kumar S. Autophagy regulates programmed cell death during the plant innate immune response. Cell 2005; 121:567-77. 10. Noda T, Ohsumi Y. Tor, a Phosphatidylinositol Kinase Homologue, Controls Autophagy in Yeast. J Biol Chem 1998; 273:3963-6. 11. Pinan-Lucarre B, Balguerie A, Clave C. Accelerated cell death in Podospora Autophagy mutants. Eukaryot Cell 2005; 4:1765-74. 12. Pinan-Lucarre B, Paoletti M, Dementhon K, Coulary-Salin B, Clave C. Autophagy is induced during cell death by incompatibility and is essential for differentiation in the filamentous fungus Podospora anserina. Mol Microbiol 2003; 47:321-33. 13. Besteiro S, Williams RA, Morrison LS, Coombs GH, Mottram JC. Endosome sorting and autophagy are essential for differentiation and virulence of Leishmania major. J Biol Chem 2006; 281:11384-96. 14. Dementhon K, Saupe SJ, Clave C. Characterization of IDI-4, a bZIP transcription factor inducing autophagy and cell death in the fungus Podospora anserina. Mol Microbiol 2004; 53:1625-40. 15. Tsukada M, Ohsumi Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett 1993; 333:169-74. 16. Klionsky DJ, Cregg JM, Dunn WA Jr, Emr SD, Sakai Y, Sandoval IV, Sibirny A, Subramani S, Thumm M, Veenhuis M, Ohsumi Y. A unified nomenclature for yeast autophagy-related genes. Dev Cell 2003; 5:539-45. 17. Xie Z, Klionsky DJ. Autophagosome formation: core machinery and adaptations. Nat Cell Biol 2007; 9:1102-9. 18. Klionsky DJ. Autophagy: from phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol 2007; 8:931-7. 19. Kabeya Y, Kawamata T, Suzuki K, Ohsumi Y. Cis1/Atg31 is required for autophagosome formation in Saccharomyces cerevisiae. Biochem Biophys Res Commun 2007; 356:405-10. 20. Kirisako T, Baba M, Ishihara N, Miyazawa K, Ohsumi M, Yoshimori T, Noda T, Ohsumi Y. Formation process of autophagosome is traced with Apg8/Aut7p in yeast. J Cell Biol 1999; 147:435-46. 21. Suzuki K, Kirisako T, Kamada Y, Mizushima N, Noda T, Ohsumi Y. The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. EMBO J 2001; 20:5971-81. 22. Ichimura Y, Kirisako T, Takao T, Satomi Y, Shimonishi Y, Ishihara N, Mizushima N, Tanida I, Kominami E, Ohsumi M, Noda T, Ohsumi Y. A ubiquitin-like system mediates protein lipidation. Nature 2000; 408:488-92. 23. Tanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol 2004; 36:2503-18. 24. Mukaiyama H, Baba M, Osumi M, Aoyagi S, Kato N, Ohsumi Y, Sakai Y. Modification of a ubiquitin-like protein Paz2 conducted micropexophagy through formation of a novel membrane structure. Mol Biol Cell 2004; 15:58-70. 25. Yoshimoto K, Hanaoka H, Sato S, Kato T, Tabata S, Noda T, Ohsumi Y. Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy. Plant Cell 2004; 16:2967-83. 26. Veneault-Fourrey C, Barooah M, Egan M, Wakley G, Talbot NJ. Autophagic fungal cell death is necessary for infection by the rice blast fungus. Science 2006; 312:580-3. 27. Liu XH, Lu JP, Zhang L, Dong B, Min H, Lin FC. Involvement of a Magnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor and pathogenesis. Eukaryot Cell 2007; 6:997-1005. 28. Kikuma T, Ohneda M, Arioka M, Kitamoto K. Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae. Eukaryot Cell 2006; 5:1328-36. 29. Richie DL, Fuller KK, Fortwendel J, Miley MD, McCarthy JW, Feldmesser M, Rhodes JC, Askew DS. Unexpected link between metal ion deficiency and autophagy in Aspergillus fumigatus. Eukaryot Cell 2007; 6:2437-47. 30. Bolender RP, Weibel ER. A morphometric study of the removal of phenobarbital-induced membranes from hepatocytes after cessation of threatment. J Cell Biol 1973; 56:746-61. 31. Wang Z, Wilson WA, Fujino MA, Roach PJ. Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p. Mol Cell Biol 2001; 21:5742-52. Acknowledgements es B io s ci en ce Staining protocols and microscopy. For TEM analysis, mycelial growth was harvested, thoroughly washed in sterile distilled water, transferred to liquid MM - N medium with mM PMSF, and incubated at 28°C for 16 h with gentle shaking. The fungal biomass was fixed overnight at 4°C in glutaraldehyde (2.5%, v/v) in 0.1 M phosphate buffer (pH 7.2) and processed for TEM as described.50 Confocal microscopy was performed using the Zeiss LSM510 system (Carl Zeiss Inc., Thornwood, New York) as described.49 Bright field and epifluorescence microscopy was performed with an Olympus IX71 microscope (Olympus, Tokyo, Japan) using a Plan APO 100X/1.45 objective and appropriate filter sets. Images were captured with Photometrics CoolSNAP HQ camera (Tucson, Arizona) and processed using MetaVue (Universal Imaging, PA, USA) and Adobe Photoshop 7.0.1 (Mountain View, California). Autophagy-related vesicular compartments were stained with 50 μM MDC (Sigma-Aldrich, USA) or 50 nM Lysotracker Green DND-26 (Invitrogen-Molecular Probes, Carlsbad, California) using standard protocols. Calcofluor White (Sigma-Aldrich, USA) was used at μg/ml (in 100 mM Tris/HCl buffer pH 9.0, containing TritonX-100 at 1:1000) to visualize cell wall and septa of the aerial hyphae or conidiophores/conidia of the respective strains. Colonies were incubated with a suitable volume of the CFW solution (enough to cover the surface of the colonies) in the dark at room temperature for about 10 minutes and then washed several times and observed with epifluorescence microscopy using the recommended filter sets. Colonies were assessed at 6–9 hours or at 12, 24 or 48 h post induction to quantify aerial hyphae and conidiophores respectively. Estimation of total cellular glycogen. Conidiating mycelial cultures of requisite strains were grown on PA medium in the dark for days, followed by constant illumination to induce conidiation. The intracellular glycogen concentration was measured at 0, 2, days post photo-induction. The surface of the colonies was scraped with inoculation loops in the presence of water and the fungal biomass was harvested in FalconTM Conical Tubes and then filtered through a layer of Miracloth. The mycelia were thoroughly washed, weighed and then ground in liquid nitrogen to a fine powder. The samples (10–50 mg) were assessed for the glycogen content with Megazyme Total Starch Kit (Megazyme, Ireland) as instructed. Total glycogen content was normalized to the wet weight of the fungal biomass used for the estimation. 09 La nd We thank A. Suresh and Yang Ming for technical assistance, and X. Ouyang for excellent EM support. We thank the Fungal Patho-biology group for helpful discussions and suggestions. We are grateful to S. Naqvi for comments on the manuscript. Y.Z.D. acknowledges support from the Singapore Millennium Foundation. This work was funded by intramural grants from the Temasek Life Sciences Laboratory, Singapore. Note © 20 Supplemental material can be found at: http://www.landesbioscience.com/supplement/ DengAUTO5-1-Sup.pdf 42 Autophagy 2009; Vol. Issue ib u st r di no t o .D © 20 09 La nd es B io s ci en ce 32. Wilson WA, Wang Z, Roach PJ. Systematic identification of the genes affecting glycogen storage in the yeast Saccharomyces cerevisiae: implication of the vacuole as a determinant of glycogen level. Mol Cell Proteomics 2002; 1:232-42. 33. Natarajan K, Meyer MR, Jackson BM, Slade D, Roberts C, Hinnebusch AG, Marton MJ. Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast. Mol Cell Biol 2001; 21:4347-68. 34. Kotoulas OB, Kalamidas SA, Kondomerkos DJ. Glycogen autophagy. Microsc Res Tech 2004; 64:10-20. 35. Kotoulas OB, Kalamidas SA, Kondomerkos DJ. Glycogen autophagy in glucose homeostasis. Pathol Res Pract 2006; 202:631-8. 36. Kovacs AL, Eldib A, Telbisz A. Autophagy in hepatocytes and erythropoietic cells isolated from the twenty-one day old rat embryo. Acta Biol Hung 2001; 52:417-33. 37. Devos P, Hers HG. Random, presumably hydrolytic, and lysosomal glycogenolysis in the livers of rats treated with phlorizin and of newborn rats. Biochem J 1980; 192:177-81. 38. Kalamidas SA, Kotoulas OB. Studies on the breakdown of glycogen in the lysosomes: the effects of hydrocortisone. Histol Histopathol 2000; 15:29-35. 39. Codogno P, Meijer A. Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ 2005; 12:1509-18. 40. Kimura S, Noda T, Yoshimori T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 2007; 3:452-60. 41. Hwang PK, Tugendreich S, Fletterick RJ. Molecular analysis of GPH1, the gene encoding glycogen phosphorylase in Saccharomyces cerevisiae. Mol Cell Biol 1989; 9:1659-66. 42. Colonna WJ, Magee PT. Glycogenolytic enzymes in sporulating yeast. J Bacteriol 1978; 134:844-53. 43. Klionsky DJ, Ohsumi Y. Vacuolar import of proteins and organelles from the cytoplasm. Annu Rev Cell Dev Biol 1999; 15:1-32. 44. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science 2000; 290:1717-21. 45. Barth H, Thumm M. A genomic screen identifies AUT8 as a novel gene essential for autophagy in the yeast Saccharomyces cerevisiae. Gene 2001; 274:151-6. 46. Yamashita I, Fukui S. Transcriptional control of the sporulation-specific glucoamylase gene in the yeast Saccharomyces cerevisiae. Mol Cell Biol 1985; 5:3069-73. 47. Fonzi WA, Shanley M, Opheim DJ. Relationship of glycolytic intermediates, glycolytic enzymes, and ammonia to glycogen metabolism during sporulation in the yeast Saccharomyces cerevisiae. J Bacteriol 1979; 137:285-94. 48. Francois J, Parrou JL. Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev 2001; 25:125-45. 49. Ramos-Pamplona M, Naqvi NI. Host invasion during rice-blast disease requires carnitinedependent transport of peroxisomal acetyl-CoA. Mol Microbiol 2006; 61:61-75. 50. Soundararajan S, Jedd G, Li X, Ramos-Pamplona M, Chua NH, Naqvi NI. Woronin body function in Magnaporthe grisea is essential for efficient pathogenesis and for survival during nitrogen starvation stress. Plant Cell 2004; 16:1564-74. 51. Talbot N, Ebbole D, Hamer J. Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 1993; 5:1575-90. 52. Chida T, Sisler HD. Restoration of appressorial penetration ability by melanin precursors in Pyricularia oryzae treated with antipenetrants and in melanin-deficient mutants. J Pestic Sci 1987; 12:49-55. 53. Pascale V. Balhadère, Andrew J. Foster, Nicholas J. Talbot. Identification of Pathogenicity Mutants of the Rice Blast Fungus Magnaporthe grisea by Insertional Mutagenesis. MPMI 1999; 12:129-42. 54. Sambrook J, Fritsch E, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press 1989. 55. Naqvi N, Bonman J, Mackill D, Nelson R, Chattoo B. Identification of RAPD markers linked to a major gene for blast resistance in rice. Mol Breed 1995; 1:341-8. 56. Altschul S, Madden T, Shaffer A, Zhang Z, Miller W, Lipman D. Gapped BLAST and PSIBLAST: A new generation of protein database search programs. Nucleic Acids Res 1997; 25:3389-402. 57. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994; 22:4673-80. 58. Jin LT, Hwang SY, Yoo GS, Choi JK. A mass spectrometry compatible silver staining method for protein incorporating a new silver sensitizer in sodium dodecyl sulfate-polyacrylamide electrophoresis gels. Proteomics 2006; 6:2334-7. te . Glycogen autophagy and Magnaporthe development www.landesbioscience.com Autophagy 43 Basic Brief Report Basic Brief Report Autophagy 6:4, 455-461; May 16, 2010; © 2010 Landes Bioscience A vacuolar glucoamylase, Sga1, participates in glycogen autophagy for proper asexual differentiation in Magnaporthe oryzae Yi Zhen Deng and Naweed I. Naqvi* Fungal Patho-Biology Group; Temasek Life Sciences Laboratory; and Department of Biological Sciences; Research Link; National University of Singapore; Singapore Key words: autophagy, Sga1, Magnaporthe, glycogen, asexual reproduction, conidia, rice-blast Abbreviations: Atg8, autophagy-related gene 8; Sga1, sporulation-specific glucoamylase 1; G6P, glucose 6-phosphate; G1P, glucose 1-phosphate; hpi, hours post-inoculation; ORF, open reading frame Nutrient limitation acts as a trigger for the synthesis of glycogen, which serves as a carbon and energy reserve during starvation. Recently, we reported that an autophagy-deficient mutant (atg8∆) shows severe reduction in aerial hyphal growth and conidiation in the rice-blast fungus Magnaporthe oryzae, and proposed that autophagy plays an important role in facilitating glycogen homeostasis to ensure proper asexual differentiation in Magnaporthe. Here, we identify and characterize a vacuolar glucoamylase function (Sga1) that hydrolyses glycogen to meet the energy requirements during asexual development in Magnaporthe. Loss of SGA1 resulted in significant reduction in conidiation compared to the wild-type Magnaporthe strain. More importantly, an sga1∆ atg8∆ double deletion mutant showed further reduction in conidiation compared to the atg8∆ mutant in Magnaporthe. Forced localization of GFP-Sga1 to the cytoplasm (through removal of the predicted signal peptide) led to increased conidiation in wild type and the sga1∆, but more interestingly, significantly restored conidiation in the atg8∆ mutant. Our results indicate that autophagy and Sga1 act cooperatively in vacuolar glycogen breakdown, which is essential for conidia formation but dispensable for pathogenicity in Magnaporthe. Introduction Glycogen serves as a readily available glucose store in eukaryotic cells. In yeast, glycogen is synthesized and accumulated when cells are deprived of nutrients.1 In mammals, glycogen accumulation is a response to the increase in blood glucose concentration.2 Glycogen breakdown occurs during yeast sporulation,3 and during the postnatal hypoglycemia in the animals,4 both in vacuoles. Apart from a nonselective bulk degradation process, autophagy also acts as a selective mechanism for vacuolar glycogen breakdown, called glycogen autophagy, in newborn animals.4-6 Glycogen autophagy is normally activated by glucagons,7 and/or the cAMP protein kinase A pathway.8 In contrast, insulin suppresses glycogen autophagy by abolishing glucagon secretion,4 whereas phosphoinositides or mTOR suppress glucagons.6,8 In S. cerevisiae, glycogen is synthesized in the cytosol and can either be phosphorylated therein by Gph1 (producing G1P) for further breakdown,9 or delivered into the vacuoles to be hydrolyzed directly to glucose by Sga1.10,11 Vacuolar Sga1-catalyzed glycogen breakdown is sporulation-specific,11 while Gph1 is not required for yeast sporulation and is only induced during the stationary phase.9 Glucose produced by Sga1 in the vacuolar lumen can easily diffuse into the cytosol, where it can be converted into G6P and then likely channeled into glycolysis. Furthermore, excess glucose inhibits Gph1 activity and stimulates glycogen synthesis and accumulation.5,9,10 In budding yeast, glycogen synthesis and breakdown is tightly regulated at different stages of the life cycle. Nutrient limitation during the stationary phase triggers the cells to synthesize and store glycogen as a reserve of glucose.12-14 Autophagy is induced during the stationary phase to deliver cytosolic glycogen to the vacuole, likely to protect it from premature breakdown by Gph1, or as a nutrient storage.12 Sporulation and meiosis are induced by starvation in S. cerevisiae.15 During the late stage of sporulation, the breakdown of reserve glycogen is induced by meiosis, and executed by the vacuolar glucoamylase Sga1.13,15,16 In the rice-blast fungus Magnaporthe oryzae, an autophagydeficient mutant, atg8∆, showed significant reduction in asexual spore (conidium) formation.17-19 Our current study suggests that the conidiation defect in Magnaporthe atg8∆ is most likely due to high accumulation of cytosolic glycogen and/or lack of glucose production.18 We report that the vacuolar breakdown of glycogen is indeed crucial for conidiation in Magnaporthe. Sga1-catalyzed glycogen breakdown is normally executed inside the vacuole, *Correspondence to: Naweed Naqvi; Email: naweed@tll.org.sg Submitted: 11/11/09; Revised: 02/26/10; Accepted: 03/08/10 Previously published online: www.landesbioscience.com/journals/autophagy/article/11736 www.landesbioscience.com Autophagy 455 following the delivery of cytosolic glycogen via the autophagic machinery, but it can be achieved by ectopic expression of a variant cytoplasmic Sga1 when autophagy is compromised (as in the atg8∆ mutant). Taken together, autophagy and the vacuolar hydrolysis of glycogen may represent two tightly coordinated steps of glycogen autophagy during Magnaporthe asexual development. Results and Discussion Sga1 is required for proper conidiation in M. oryzae. In S. cerevisiae, the Sporulation-specific GlucoAmylase gene SGA1 is expressed exclusively during the sporulation phase, and subjected to regulation by the nutrient status, ploidy and heterozygosity.16,20,21 The SGA1 glucoamylase is closely related to the secreted glucoamylases of S. cerevisiae var. diastaticus, encoded by any of the three unlinked STA glucoamylase genes essential for pseudohyphal and invasive growth.16,20 We identified MGG_01096 as an ortholog of the S. cerevisiae SGA1 in M. oryzae (Magnaporthe Genome Database, Broad Institute, USA). In order to assess the function, we created an sga1∆ strain of M. oryzae, by replacing the entire MGG_01096 (NCBI accession XP_368148) ORF with the hygromycin-resistance marker cassette (HPH1; Fig. S1A). The requisite gene replacement event for SGA1 was confirmed by Southern blotting (Fig. S1B). Two independent strains were then characterized for vegetative growth and conidiation. Likewise, an sga1∆ atg8∆ double deletion mutant was generated and the deletion of ATG8 confirmed by Southern analysis (Fig. S1B). When utilizing lactose as the sole carbon source, the sga1∆ mutant showed radial growth and aerial hyphal growth comparable to the wild type (Fig. S1E), but displayed an overall 40% reduction in conidia production (61.6 ± 2.8 x 102 conidia/cm2 vs. 98.8 ± 2.2 x 102 conidia/cm2 ; Fig. 1A). The atg8∆ mutant is deficient in autophagy and showed severe reduction in aerial growth and conidiation (1.7 ± 0.3 x 102 conidia/cm2). The double deletion mutant sga1∆ atg8∆ was further reduced in conidiation (0.10 ± 0.03 x 102 conidia/cm2) compared to the atg8∆ mutant. We previously showed that the conidiation defects resulting from autophagy-deficiency could be restored by exogenously added sucrose or G6P (but not G1P), which likely bypass the requirement for autophagy by acting as ready substitutes for the product of glycogen autophagy i.e., glucose.18 Therefore, we individually tested the effect of sucrose, G6P or G1P, on conidiation in the aforementioned strains. Sucrose and G6P (but not G1P) both showed a stimulatory effect on conidiation to varying degrees in all the strains tested (Fig. 1A). Next, we investigated the cause of the reduced conidiation upon the loss of SGA1 function. Unlike the atg8∆ mutant, the sga1∆ mutant appeared fluffy, indicating that the aerial hyphal growth was likely normal. Time-lapse observation during conidiogenesis (around 6–12 h post photo-induction) revealed that the rate of conidiophore differentiation was significantly reduced in the sga1∆, compared to the wild type (Fig. 1B). Out of the total aerial hyphae analyzed, 36 ± 4% could finally differentiate into conidiophores in the wild type, while only 19 ± 456 2% formed conidiophores in the sga1∆. The conidiophore differentiation rate was similarly lower in the atg8∆ (3 ± 0.4%). We propose that autophagy and Sga1 function are essential for proper conidiation in M. oryzae. Loss of either one of the two functions caused significant defects in conidiation. In M. oryzae, there may be other glycogen hydrolase(s) that are at least partially redundant with Sga1 function, which may explain the difference in severity in conidiation defects between atg8∆ and sga1∆ mutants. The conidiation defect was even more severe in the sga1∆ atg8∆ double deletion mutant, indicating that autophagy and Sga1 likely act in concert and are important for Magnaporthe conidiation. Subcellular localization of Sga1 in M. oryzae. We generated an M. oryzae strain expressing Sga1-GFP fusion protein under native regulation and as the sole copy of SGA1. The Sga1-GFP fusion protein was considered to be functional, given that the colony morphology, conidiation and pathogenicity of this strain were fully comparable to the wild type. The subcellular location of Sga1-GFP was then analyzed in the vegetative mycelia cultured in Complete medium (CM), or in cultures grown on PA medium under nonconidiating (dark grown) or conidiating (light) conditions. Sga1-GFP was undetectable in the CM-cultured or nonconidiating mycelia (Fig. 1C, left) but prominently localized to the large vacuoles in old mycelial cultures during the stationary phase (Fig. 1C, right). Next, the subcellular location of Sga1GFP was analyzed in detail in M. oryzae grown for 6–24 h on PA medium under constant illumination. Punctate and filamentous localization of Sga1-GFP in the aerial hyphae, as well as in the stalk and nascent conidium of the conidiophore were observed. Vesicular Sga1-GFP puncta were prominent in conidiating Sga1-GFP colonies (Fig. 1D). The vacuolar localization of Sga1GFP in conidiation-specific structures (aerial hyphae and conidiophores) was confirmed by co-staining with the fluorescent Lysotracker Red DND99 dye for acidified vacuoles (Fig. 1D and E). Likewise, the Sga1-GFP was judged to be vacuolar in mycelia and mature conidia (Fig. 1F). Transcriptional induction of SGA1 during conidiation (upon photo-induction) was revealed by semiquantitative RTPCR (Fig. S1D). We conclude that the SGA1 gene shows increased expression during M. oryzae conidiation phase and that Sga1 is a vacuolar protein. Unlike the STA encoding secreted glucoamylase, Sga1 lacks a serine/threonine-rich amino-terminal domain, which confers at least some of the secretory information.22,23 Since Sga1 is a vacuolar protein in yeast,22 we reasoned that Sga1 should contain some intrinsic information (signal peptide) to enter the secretory pathway. It has been shown that STA glucoamylases share common regions with Sga1, at its middle and C-terminus, likely corresponding to the catalytic domain of the vacuolar enzyme.24 SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) predicted a possible signal peptide in proximal 42 amino acid of Sga1. Based on this information, we generated a truncated SGA1, by removing the coding region for the first 45 amino acids. Additionally, the resultant Sga146-655 variant was tagged with GFP at its N-terminus, and expressed under the control of the MPG1 promoter (schematic representation in Fig. S1C). The GFPSga146-655 protein was expressed in the atg8∆ mutant. We carried Autophagy Volume Issue Figure 1. For figure legend, see page 458. out immunoblot analysis with anti-GFP antiserum to verify the GFP-Sga146-655 fusion protein (Fig. S1C). Deletion of the first 45 amino acids totally abolished the vacuolar localization of Sga1 (Fig. 1F), as judged by epifluorescence microscopy. www.landesbioscience.com In this section we demonstrated that Sga1 is a vacuolar protein specifically expressed during asexual development in Magnaporthe. However, ectopic localization of Sga1 to the cytoplasm could be achieved by removal of the first 45 amino acids Autophagy 457 Figure (See previous page). Biological function and subcellular localization of Sga1 glucoamylase in Magnaporthe. (A) Bar chart depicting quantification of conidiation in the wild type (WT), sga1∆, atg8∆ and sga1∆atg8∆ grown on prune-agar (PA) medium (dark grey), or PA medium supplemented with mM G1P (open bar), or 0.5 mM G6P (light grey) or with 6.25 g/L sucrose (black). Mean values (±S.E.) presented as number of conidia were derived from three independent experiments (n = 30 colonies for each sample). Total conidia counts were performed five days post photo-induction. (B) Bar chart depicting quantitatively assessed conidiophore differentiation rates in the wild type (WT, open bar), atg8∆ (grey) and sga1∆ (black), at 24 hour post photo-induction. Mean values (±S.E.) represent the percentage conidiophores resulting from the total aerial hyphae assessed in a given colony. Three independent assessments were performed and at least 15 colonies per strain were examined in each experiment. (C) Subcellular localization of Sga1-GFP. Nonconidiating Sga1-GFP strain was analyzed by epifluorescence microscopy at d (vegetative growth) or 10 d (starvation phase) post inoculation on PA medium. Bar = 10 µm. (D and E) Sga1-GFP localization was further assessed in aerial hyphae and conidiophores during the conidiation phase (6–24 h post photo-induction). Co-staining with Lysotracker Red DND99 served to ascertain the vacuolar compartments in the Sga1-GFP strain. Bar = µm. (F) The vacuolar localization of Sga1 depends on its amino-terminal signal peptide. Conidiating cultures from the Sga1-GFP strain (left) or a GFP-Sga146-655 strain (right) were subjected to epifluorescence and bright field microscopy to assess the mycelia and mature conidia. Bar = 10 µm. of Sga1, and was verified by immunoblotting as well as epifluorescence microscopy. The vacuolar destination of Magnaporthe Sga1 is thus conserved and as in the case of the yeast ortholog, is indicative of the vacuolar glycogen hydrolysis function. Nitrogen starvation is sufficient to induce sporulation in S. cerevisiae, during which the vacuolar glycogen stores are rapidly degraded by Sga1.3 Our preliminary data indicate that nitrogen starvation indeed acts as one of the inducing factors for M. oryzae conidiation, since nitrogen supplemented PA medium suppressed conidiation in the wild-type, atg8∆, sga1∆, sga1∆ atg8∆ double deletion mutant, and atg8∆ with GFP-Sga146-655 (Fig. S2). We hypothesize that suppression of conidiation through such accessible nitrogen source(s) is likely achieved by suppression or inhibition of autophagy and/or other relevant pathways,25,26 in Magnaporthe. Sga1-catalyzed glycogen catabolism is important for M. oryzae conidiation. Our previous analysis of autophagy and Gph1 function indicates that glycogen catabolism is important for proper conidiation18 in M. oryzae. In order to investigate the function of Sga1 in glycogen catabolism, we performed a quantitative assessment of the intracellular steady-state levels of glycogen in the conidiating colonies of the wild type, atg8∆, sga1∆, sga1∆ atg8∆ and the atg8∆ expressing cytoplasmic GFPSga146-655 (Fig. 2A). Three critical time points: d (representing vegetative stage), d (early stage of conidiation) and d (late stage of conidiation), were chosen for measuring the total glycogen content (Fig. 2A). The wild type showed a steady albeit minor increase in glycogen as conidiation proceeded (Fig. 2A; p < 0.001). However, compared to the wild type at d (1.33 ± 0.10%; Fig. 2A), the sga1∆, and the sga1∆ atg8∆ mutant showed significantly high (1.89 ± 0.20% and 5.15 ± 0.60 respectively; p < 0.001) glycogen accumulation, which correlated with a reduced conidiation phenotype in the respective strains. Autophagy plays a key role in glycogen breakdown during M. oryzae conidiation.18 Glycogen levels in the sga1∆ and sga1∆ atg8∆ mutants indicate that Sga1 activity is indeed required for glycogen breakdown during M. oryzae conidiation. At the stages preceding (0 d) or following conidiogenesis (4 d), the sga1∆ and sga1∆ atg8∆ mutants showed comparable levels of glycogen to the wild type (Fig. 2A), suggesting that Sga1 activity is likely conidiation-specific in M. oryzae. By d, glycogen levels in the atg8∆, sga1∆ and sga1∆ atg8∆ double deletion mutant were lower than in the wild type, however, conidiation in these mutant strains was still defective. We propose that such reduction in glycogen levels is likely due to 458 the Gph1 activity in response to higher accumulation of cytoplasmic glycogen due to the loss of autophagy.18 In S. cerevisiae, Gph1 is a cytosolic glycogen phosphorylase, which catalyzes glycogen breakdown slowly and on a small scale, probably due to its negative feedback regulation.9 Our previous study indicated that the total glycogen in autophagy-deficient mutants can eventually be brought down by Gph1 activity but the glucose production is not efficient enough to trigger proper conidiation.18 In the atg8∆ expressing the cytoplasmic Sga1 (GFP-Sga146-655), we observed an overall reduction in the steady-state glycogen content during the complete conidiation process, indicating the increased breakdown of the cytosolic glycogen, which accumulates at a high level in the atg8∆.18 Interestingly, conidiation defects in the atg8∆ mutant were significantly suppressed upon expression of GFP-Sga146-655 (Fig. 2B). The conidiation levels in the atg8∆ mutants expressing GFP-Sga146-655 increased up to 32 fold, compared to that in the atg8∆ mutant. We infer that the cytoplasmic Sga1 can catalyze glycogen breakdown in the cytoplasm instead of the vacuole, thus bypassing the requirement of autophagic machinery for glycogen transport to the vacuoles. To verify if the increased conidiation in the atg8∆ mutant resulted from the expression of GFP-Sga146-655, we transformed this construct into the wild type and the sga1∆ strains, respectively, and the conidiation in the transformants was compared to its original background. As shown in Figure 2B, two independent transformants from each background showed further increased conidiation to various degrees, indicating that the forced cytosolic expression of Sga1 does play a positive role in M. oryzae conidiation. It has been reported that autophagy and Sga1 coordinatively facilitate glycogen catabolism during yeast sporulation.10-12 Our results indicate that M. oryzae Sga1 is functionally conserved in terms of catalyzing vacuolar glycogen catabolism, albeit during asexual development. Besides the conidiation defects, autophagy-deficient M. oryzae has been reported to be nonpathogenic.17-19 To analyze whether glycogen autophagy is required for M. oryzae pathogenicity, we inoculated equivalent number of conidia from the wild type (WT), sga1∆, atg8∆ or atg8∆ expressing GFP-Sga146-655 on barley leaf explants. The disease symptoms were examined at seven days post-inoculation. Both the wild type and the sga1∆ strain produced typical lesions on the inoculated leaves (Fig. 2C), indicating that Sga1 is not required for M. oryzae pathogenesis. In contrast, the GFP-Sga146-655 expressing atg8∆ remained nonpathogenic (Fig. 2C), as the atg8∆ mutant. Furthermore, wild Autophagy Volume Issue Figure 2. Sga1-assisted hydrolysis of glycogen is required for proper asexual development in Magnaporthe. (A) Quantitative analysis of total internal glycogen content in the wild type, atg8∆, sga1∆, sga1∆ atg8∆ and atg8∆ carrying cytoplasmic Sga1. The indicated strains were initially grown in the dark for two days and then subjected to constant illumination to stimulate conidia formation. At the specified time-points post induction, the fungal biomass was subjected to glycogen extraction and estimation. The concentration of internal glycogen is presented as percentage of the total wet weight of the fungal biomass. Mean values (±S.E.) were derived from three independent experiments (n = 15 colonies per strain). (B) Cytoplasmic Sga1 enhances conidiation in the wild type and the sga1∆, and restores conidiation in the atg8∆ strain. Bar chart depicts the total conidia counts in the wild type (WT), atg8∆ or sga1∆ colonies expressing or lacking GFP-Sga146-655. Data presented as mean values (±S.E.) were derived from three independent replicates (n = 30 colonies for each sample). Assessments were performed five days post photo-induction. (C) Sga1 is not required for Magnaporthe pathogenesis. Barley leaf explants or rice leaves were spot inoculated with equivalent number of conidia (2,000 per droplet) from the wild type, sga1∆, atg8∆ or the atg8∆ expressing the GFP-Sga146-655 variant. Disease symptoms were assessed d post inoculation. Surface-sterilized rice seeds were allowed to germinate and grow in direct contact with the mycelial plugs from the wild type, sga1∆, atg8∆ or the atg8∆ with GFP-Sga146-655. Resultant rice roots were then washed in water and evaluated for disease symptoms (arrows) after two weeks. type and the sga1∆ were capable of infecting the rice roots and leaf explants, while the atg8∆ mutant or atg8∆ with cytoplasmic GFP-Sga146-655 were incapable of doing so (Fig. 2C). This suggests www.landesbioscience.com that the Sga1-catalyzed glycogen catabolism is only required for proper conidiation, but not for pathogenicity in M. oryzae. We conclude that glycogen autophagy involves a ­Sga1-based vacuolar Autophagy 459 catabolism step that likely supports proper asexual development in M. oryzae. Glycogen hydrolysis is not important for virulence in M. oryzae. We previously proposed a model in Magnaporthe wherein glycogen autophagy is induced specifically during conidiation to facilitate rapid glycogen hydrolysis to glucose during cell growth and differentiation.18 In that scenario, when autophagy is blocked, glycogen accumulates in the cytosol and is not accessible to the vacuolar glucoamylase for hydrolysis. The resulting lack of glucose likely accounts for the defective conidiation in the atg8∆ mutant. Increased cytosolic glycogen stimulates the expression of glycogen phosphorylase, Gph1, at both the transcriptional and translational level.18 In M. oryzae, Gph1 likely facilitates the breakdown of cytosolic glycogen in the atg8∆ mutant. We speculate that due to its negative feedback regulation, it is likely an inefficient way to produce large amounts of glucose to meet the metabolic requirements during conidiation. In this study, we showed that Sga1 is the vacuolar glucoamylase responsible for glycogen breakdown, following glycogen delivery to the vacuole (likely) via the autophagic machinery. Sga1 has a much higher efficiency than Gph1 for glycogen breakdown. First, Gph1 activity is suppressed by G6P, an intermediate product of glycogen autophagy. Second, Sga1-dependent glycogen catabolism forms a positive feedback loop, when glucose is converted into G6P, which can participate directly in glycogen synthesis and thus provide more substrate for glycogen autophagy. We also demonstrated that Sga1 function is essential for proper conidiation but not for pathogenicity in M. oryzae. Sga1 was predominantly vacuolar, and when forcedly expressed in the cytoplasm, the truncated version of Sga1 retained its enzymatic activity but lost its dependency on autophagy to get access to its substrate, glycogen. In summary, we proposed a refined working model for glycogen autophagy during asexual development in M. oryzae (Fig. S1F); wherein the vacuolar hydrolysis of glycogen plays a key role in achieving optimum glucose production. In this study, we established M. oryzae Sga1 as a vacuolar glucoamylase that acts cooperatively with autophagy in glycogen catabolism to promote fungal asexual differentiation. Sga1 is located in the vacuole, as ScSga1 is. The function of Sga1 seems to be conserved in M. oryzae, but instead of during sporulation (meiotic), it catalyzes glycogen breakdown during M. oryzae conidiation (mitotic). The Sga1-catalyzed glycogen breakdown follows the vacuolar delivery of cytosolic glycogen via autophagy. The requirement for autophagy can be bypassed upon cytoplasmic distribution of Sga1. The autophagy- and Sga1-regulated glycogen catabolism in M. oryzae mirrors the yeast’s sporulationspecific glycogen breakdown, and glycogen autophagy in mammals. Autophagy and Sga1 are required for different steps of glycogen autophagy: autophagy for the delivery while Sga1 for the hydrolysis, of one same substrate, cytosolic glycogen. However, glycogen autophagy is not essential for M. oryzae pathogenicity. Macroautophagy plays pleiotropic roles during fungal differentiation and virulence, yet the mechanisms behind such varied 460 functions have not been elucidated in detail. Our study reveals a role for macroautophagy, coordinately with Sga1, as an important regulator of energy reserves and supply during the asexual development phase of M. oryzae. Materials and Methods Fungal strains and growth conditions. M. oryzae wild-type strain B157 (Field isolate, mat1-2) was obtained from the Directorate of Rice Research (Hyderabad, India) and used as an isogenic background for SGA1 and ATG8 deletion analysis. M. oryzae strains were propagated on Prune-agar (PA) medium as described.27 Fungal sample preparation for genomic DNA isolation or total protein extraction was as described.28 Quantitative analysis of conidiation and infection assay on barley leaf explants follow the same procedures as reported.28 Root infection assays in rice were carried out using standard protocols.29 Nucleic acid and protein-related techniques. Standard molecular manipulations were performed as described.30 Fungal genomic DNA was extracted using a modified potassium acetate method.31 Plasmid DNA was isolated with a high-speed plasmid mini kit (Geneaid, PD300) and nucleotide sequencing performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, 4337458). Homology searches of DNA/ protein sequences were performed using the BLAST programs. Primers used for SGA1 deletion, GFP-tagging, and expression of truncated Sga1 are listed in Supplementary Figure S3. For GFPtagging, the GFP coding sequence along with TrpC terminator was released from the plasmid (pFC2-ORF, GFP) by digestion with NcoI and XbaI. The plasmid carrying SGA1-GFP was transformed into Magnaporthe wild-type strain to specifically replace the SGA1 gene with the SGA1-GFP allele. The plasmid carrying GFP under control of MPG1 promoter, (pFC2-ORF, GFP), was digested with BamHI and then end-filled with Klenow enzyme and ligated in frame to the Sga146-655 coding sequence at the N-terminus. The SpeI-XbaI fragment from this plasmid, containing the MPG1 promoter-GFP-SGA146-655-TrpC terminator was released and then ligated to pFGL44 and transformed into an atg8∆ strain. Total protein extraction, SDS-PAGE fractionation, transfer to PVDF membrane and immunoblotting were performed as reported.28 Anti-GFP antibody (Invitrogen-Molecular Probes, A6455) was used at 1:1,000 dilution and anti-porin at 1:2,000. Secondary antibody conjugated to horseradish peroxidase was used at 1:20,000 dilution. The ECL kit (Amersham Biosciences, RPN3000OL1) was used to detect the chemiluminescent signals. Estimation of total intracellular glycogen follows the same procedures as described.18 Fluorescence microscopy. Lysotracker Red DND-99 (Invitrogen-Molecular Probes) staining of the vacuolar compartments follows the procedure described earlier.32 Confocal microscopy was performed with Zeiss LSM510 META Inverted (Carl Zeiss Inc.), using the requisite conditions established for detecting GFP or RFP signals.32 Autophagy Volume Issue Acknowledgements Note We thank the Fungal Patho-Biology Group for suggestions and discussion. D.Y.Z. is a recipient of the Singapore Millennium Foundation fellowship. This work was funded by intramural grants from the Temasek Life Sciences Laboratory, Singapore. Supplementary materials can be found at: www.landesbioscience.com/supplement/DengAUTO6-4-Sup. pdf References 1. Francois J, Parrou JL. Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev 2001; 25:146-655. 2. Ferrer JC, Favre C, Gomis RR, Fernandez-Novell JM, Garcia-Rocha M, de la Iglesia N, et al. Control of glycogen deposition. FEBS Lett 2003; 546:127-32. 3. Fonzi WA, Shanley M, Opheim DJ. Relationship of glycolytic intermediates, glycolytic enzymes, and ammonia to glycogen metabolism during sporulation in the yeast Saccharomyces cerevisiae. J Bacteriol 1979; 137:285-94. 4. Kotoulas OB, Kalamidas SA, Kondomerkos DJ. Glycogen autophagy in glucose homeostasis. Pathol Res Pract 2006; 202:631-8. 5. Kotoulas OB, Ho J, Adachi F, Weigensberg BI, Phillips MJ. Fine structural aspects of the mobilization of hepatic glycogen II. Inhibition of glycogen breakdown. Am J Pathol 1971; 63:23-36. 6. Kotoulas OB, Kalamidas SA, Kondomerkos DJ. Glycogen autophagy. Microsc Res Tech 2004; 64:1020. 7. Kalamidas SA, Kotoulas OB. Glycogen autophagy in newborn rat hepatocytes. Histol Histopathol 2000; 15:1011-8. 8. Kalamidas SA, Kotoulas OB, Kotoulas AO, Maintas DB. The breakdown of glycogen in the lysosomes of newborn rat hepatocytes: the effects of glucose, cyclic 3',5'-AMP and caffeine. Histol Histopathol 1994; 9:691-8. 9. Hwang PK, Tugendreich S, Fletterick RJ. Molecular analysis of GPH1, the gene encoding glycogen phosphorylase in Saccharomyces cerevisiae. Mol Cell Biol 1989; 9:1659-66. 10. Colonna WJ, Magee PT. Glycogenolytic enzymes in sporulating yeast. J Bacteriol 1978; 134:844-53. 11. Yamashita I, Fukui S. Transcriptional control of the sporulation-specific glucoamylase gene in the yeast Saccharomyces cerevisiae. Mol Cell Biol 1985; 5:306973. 12. Wang Z, Wilson WA, Fujino MA, Roach PJ. Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMPactivated protein kinase, and the cyclin-dependent kinase Pho85p. Mol Cell Biol 2001; 21:5742-52. www.landesbioscience.com 13. Wilson WA, Wang Z, Roach PJ. Systematic identification of the genes affecting glycogen storage in the yeast Saccharomyces cerevisiae: implication of the vacuole as a determinant of glycogen level. Mol Cell Proteomics 2002; 1:232-42. 14. Lillie SH, Pringle JR. Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation. J Bacteriol 1980; 143:1384-94. 15. Simchen G, Kassir Y. Genetic regulation of differentiation towards meiosis in the yeast Saccharomyces cerevisiae. Genome 1989; 31:95-9. 16. Vivier MA, Lambrechts MG, Pretorius IS. Coregulation of starch degradation and dimorphism in the yeast Saccharomyces cerevisiae. Crit Rev Biochem Mol Biol 1997; 32:405-35. 17. Veneault-Fourrey C, Barooah M, Egan M, Wakley G, Talbot NJ. Autophagic fungal cell death is necessary for infection by the rice blast fungus. Science 2006; 312:580-3. 18. Deng YZ, Ramos-Pamplona M, Naqvi NI. Autophagyassisted glycogen catabolism regulates asexual differentiation in Magnaporthe oryzae. Autophagy 2009; 5:33-43. 19. Liu XH, Lu JP, Zhang L, Dong B, Min H, Lin FC. Involvement of a Magnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor and pathogenesis. Eukaryot Cell 2007; 6:997-1005. 20. Kihara K, Nakamura M, Akada R, Yamashita I. Positive and negative elements upstream of the meiosis-specific glucoamylase gene in Saccharomyces cerevisiae. Mol Gen Genet 1991; 226:383-92. 21. Pugh TA, Clancy MJ. Differential regulation of STA genes of Saccharomyces cerevisiae. Mol Gen Genet 1990; 222:87-96. 22. Pugh TA, Shah JC, Magee PT, Clancy MJ. Characterization and localization of the sporulation glucoamylase of Saccharomyces cerevisiae. Biochim Biophys Acta 1989; 994:200-9. 23. Modena D, Vanoni M, Englard S, Marmur J. Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus. Arch Biochem Biophys 1986; 248:138-50. Autophagy 24. Pretorius IS, Chow T, Marmur J. Identification and physical characterization of yeast glucoamylase structural genes. Mol Gen Genet 1986; 203:36-41. 25. Lau G, Hamer JE. Regulatory Genes Controlling MPG1 Expression and Pathogenicity in the Rice Blast Fungus Magnaporthe grisea. Plant Cell 1996; 8:77181. 26. Soanes DM, Kershaw MJ, Cooley RN, Talbot NJ. Regulation of the MPG1 hydrophobin gene in the rice blast fungus Magnaporthe grisea. Mol Plant Microbe Interact 2002; 15:1253-67. 27. Soundararajan S, Jedd G, Li X, Ramos-Pamplona M, Chua NH, Naqvi NI. Woronin body function in Magnaporthe grisea is essential for efficient pathogenesis and for survival during nitrogen starvation stress. Plant Cell 2004; 16:1564-74. 28. Liu H, Suresh A, Willard FS, Siderovski DP, Lu S, Naqvi NI. Rgs1 regulates multiple Galpha subunits in Magnaporthe pathogenesis, asexual growth and thigmotropism. EMBO J 2007; 26:690-700. 29. Dufresne M, Osbourn AE. Definition of tissue-specific and general requirements for plant infection in a phytopathogenic fungus. Mol Plant Microbe Interact 2001; 14:300-7. 30. Sambrook J, Fritsch E, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press 1989. 31. Naqvi NI, Bonman J, Mackill D, Nelson RBC. Identification of RAPD markers linked to a major gene for blast resistance in rice. Mol Breed 1995; 1. 32. Deng YZ, Ramos-Pamplona M, Naqvi NI. Methods for functional analysis of macroautophagy in filamentous fungi. Methods Enzymol 2008; 451:295-310. 461 [...]... essential role in sexual sporulation in yeast,45 autophagy has recently been implicated in conidiation and conidial germination in Aspergillus and Magnaporthe.26-28 Our results add to these findings and suggest a functional role for 38 Figure 4 Identification and functional analysis of Gph1 in Magnaporthe (A) Identification of proteins differentially regulated in the atg8Δ mutant Silver-stained profile of the. .. development in Magnaporthe.26,27 Furthermore, we identify proteins differentially regulated in the atg8Δ mutant and investigate the role of autophagy and nutrient utilization during conidiation and in planta growth in Magnaporthe By genetic and biochemical studies, we show that autophagy and glycogen homeostasis are involved in tuning carbohydrate metabolism and utilization as an important function during... represent three independent experiments involving a total of 20 colonies per strain (C) Quantitative analysis of total internal glycogen concentration in the wild type, atg8Δ, gph1Δ, gph1Δ atg8Δ and GPH1 OP-2 strains The indicated strains were initially grown in the dark for 2 days and then subjected to constant illumination to induce conidiation At the specified time-points post induction, the fungal biomass... localization of RFP-Atg8 in Magnaporthe Using the RFP-ATG8 strain and epifluorescence microscopy, we visualized the temporal and spatial distribution of RFP-Atg8 protein (and autophagosomes/autophagic bodies) during asexual differentiation in Magnaporthe Calcofluor white was used as a co-stain to delineate the outline of the analyzed fungal structures After growth in prune agar medium (PA; non-induced medium... sucrose circumvents the requirement of autophagy during asexual development in Magnaporthe Furthermore, we infer that the nutrient status and proper regulation of carbohydrate metabolism are important during conidiation and influence both the quantity and quality of conidia production in Magnaporthe Gph1 is involved in glycogen metabolism during Magnaporthe conidiogenesis To identify proteins that are regulated... and Naqvi, unpublished data) Procedures Iodine vapor staining: 1 The fungal strain of interest is subcultured on PA medium and allowed to grow in the dark at 28  C for 2 days 2 The dark-grown cultures are then subjected to constant illumination at room temperature to induce conidiation 3 At 0, 2, and 4 d after photoinduction, the culture dishes containing the colonies are inverted directly (with the. . .30 4 Yi Zhen Deng et al Procedure 1 Magnaporthe wild-type and atg8D strains are grown on PA medium in the dark at 28  C in an incubator for 2 3 days The diameter of the resulting fungal colonies is about 1–2 cm at this stage 2 Conidiation is induced in the wild-type and atg8D colonies by subjecting them to constant illumination at room temperature 3 At 6, 12, and 48 h after photoinduction, the. .. conidiation in atg8Δ strain G6P is an efficient inhibitor of the active form of Gph1.41 The addition of 0.5 mM G6P to the growth media caused a remarkable suppression of conidiation defects in the atg8Δ strain ( 53. 2 ± 4.4 x102 conidia/cm2, www.landesbioscience.com Autophagy 37 Glycogen autophagy and Magnaporthe development ib u st r di no t o D en ce ci io s Discussion te from the wild type in terms of its... on, the RFP-Atg8 signals appeared as punctate structures as well as showed vacuolar localization in the stalks and the swollen tips of conidiophores (Fig 2D) Upon initiation of the first incipient conidium, the RFP-Atg8 puncta were located prominently in the developing conidium, and remained highly enriched in these structures until proper differentiation of asexual spores (Fig 2D) The induction of autophagy. .. representative (n = 30 0 each) of the developmental stage (or defects) depicted by the majority of aerial hyphae or conidiophores in the respective samples Conidia started forming and maturing properly in the wild-type strain, whereas the atg8Δ mutant showed abnormalities in all aspects of conidia development The experiment was repeated three times using a total of 10 colonies per strain in each instance Scale . University of Singapore, Singapore 295 and we summarize the methods that have been routinely used for monitoring macroautophagy in both yeast and filamentous fungi. The role of autophagy in carbohydrate. et al. is an inhibitor of PI3-kinase and blocks the induction of autophagy (Blommaart et al., 1997; Petiot et al., 2000). 3- methlyadenine (3- MA) is also a classical inhibitor of the autophagic. with WM, the chemical inhibitor of autophagic sequestration. Furthermore, MDC staining with the conidiating cultures of Magnaporthe at different stages likely reflects the natural induction of autophagy