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Autophagy Regulates Programmed Cell Death during the Plant Innate Immune Response


Cell, Vol. 121, 567–577, May 20, 2005, Copyright ?2005 by Elsevier Inc. DOI 10.1016/j.cell.2005.03.007

Autophagy Regulates Programmed Cell Death during the Plant Innate Immune Response
Yule Liu,1 Michael Schiff,1 Kirk Czymmek,2 Zsolt Tallóczy,3 Beth Levine,3,4 and S.P. Dinesh-Kumar1,* 1 Department of Molecular, Cellular, and Developmental Biology Yale University New Haven, Connecticut 06520 2 Department of Biological Sciences Delaware Biotechnology Institute University of Delaware Newark, Delaware 19711 3 Department of Medicine Columbia University College of Physicians and Surgeons New York, New York 10032 4 Department of Medicine University of Texas Southwestern Medical Center Dallas, Texas 75390 oxygen intermediates (ROI) and nitric oxide (NO), cell wall strengthening, and activation of various defenserelated genes (McDowell and Dangl, 2000). Collectively, these defense responses are thought to prevent the spread of the pathogen from infection sites into healthy adjacent tissue. Considerable effort has been made to understand regulators of HR PCD, yet little is known about the molecular and cellular pathways involved in the restriction of HR PCD to pathogen infection sites. The interaction between Nicotiana plants that carry the N resistance gene and tobacco mosaic virus (TMV) is one of the classic model systems for studying plant innate immune responses (Marathe et al., 2002). The N protein belongs to a class of R proteins that contains a toll/ interlukin-1 receptor homology domain, a nucleotide binding site, and a leucine-rich repeat (TIR-NB-LRR) (Martin et al., 2003). The N protein is hypothesized to specifically recognize the 50 kDa helicase domain (TMV-p50) of the TMV replicase protein to trigger induction of HR PCD and restriction of virus spread (Erickson et al., 1999). Recently, several laboratories including ours have successfully used virus-induced gene silencing (VIGS) in forward and reverse genetics screens to identify components of the N signaling pathway (BurchSmith et al., 2004; Lu et al., 2003). To identify novel genes that affect initiation, execution, or restriction of TMV-induced HR PCD, we performed a high-throughput VIGS screen. We found that silencing of the putative plant autophagy gene, BECLIN 1, alters the HR PCD phenotype observed in plants undergoing a defense response to TMV infection. Autophagy is a highly regulated process during which cytoplasmic materials are enclosed in doublemembrane bound vesicles that are then targeted to the vacuole or lysosome for degradation (Levine and Klionsky, 2004; Ohsumi, 2001). Genetic analysis in yeast has identified at least 16 autophagy-related (ATG) genes that encode components of the autophagy machinery (Klionsky et al., 2003; Thumm et al., 1994; Tsukada and Ohsumi, 1993). Mutations in some of these ATG genes affect yeast sporulation and survival during nutrient starvation (Thumm et al., 1994; Tsukada and Ohsumi, 1993). Although ATG genes are conserved in higher eukaryotes, there have been few studies examining the role of autophagy in the whole organism. In Drosophila, autophagy is induced during starvation (Rusten et al., 2004; Scott et al., 2004) and steroid-activated PCD of salivary glands (Lee and Baehrecke, 2001). In C. elegans, autophagy is involved in dauer development and life span extension (Melendez et al., 2003). In mammals, autophagy regulates survival during neonatal starvation, embryonic development, and tumorogenesis (Kuma et al., 2004; Liang et al., 1999; Qu et al., 2003; Yue et al., 2003). Little is known about the molecular functions of ATG orthologs in plants. Recently, it has been shown that mutations in Arabidopsis ATG4 genes block formation of autophagic bodies and cause developmental arrest of root growth under nutrient starvation (Yoshimoto et

Summary The plant innate immune response includes the hypersensitive response (HR), a form of programmed cell death (PCD). PCD must be restricted to infection sites to prevent the HR from playing a pathologic rather than protective role. Here we show that plant BECLIN 1, an ortholog of the yeast and mammalian autophagy gene ATG6/VPS30/beclin 1, functions to restrict HR PCD to infection sites. Initiation of HR PCD is normal in BECLIN 1-deficient plants, but remarkably, healthy uninfected tissue adjacent to HR lesions and leaves distal to the inoculated leaf undergo unrestricted PCD. In the HR PCD response, autophagy is induced in both pathogen-infected cells and distal uninfected cells; this is reduced in BECLIN 1-deficient plants. The restriction of HR PCD also requires orthologs of other autophagy-related genes including PI3K/VPS34, ATG3, and ATG7. Thus, the evolutionarily conserved autophagy pathway plays an essential role in plant innate immunity and negatively regulates PCD. Introduction Plants mount numerous defense responses to survive pathogen attack. One of these is the hypersensitive response (HR) cell death, a form of programmed cell death (PCD), which is characterized by the rapid death of plant cells at the site of pathogen infection (Lam, 2004). HR PCD is typically triggered upon recognition of a pathogen-encoded avirulence (Avr) protein by a cognate plant resistance (R) protein (Martin et al., 2003). In addition to R gene-mediated cell death, plants also induce cell death in response to general elicitors produced by pathogens (Boller, 1995). HR PCD typically precedes changes in ion fluxes, production of reactive
*Correspondence: savithramma.dinesh-kumar@yale.edu

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al., 2004). Mutations in Arabidopsis ATG7, ATG9, and VTI12 result in accelerated leaf senescence even under nutrient-rich conditions (Doelling et al., 2002; Hanaoka et al., 2002; Surpin et al., 2003). However, the roles of ATG genes and autophagy in other plant processes have not been examined. Here we report that plant BECLIN 1 is an ortholog of the yeast and mammalian autophagy gene ATG6/ VPS30/beclin 1. We find that autophagy is induced during N-mediated defense against TMV and that BECLIN 1 is required for this induction. Plants deficient in the autophagy genes BECLIN 1, PI3K/VPS34, ATG3, and ATG7 exhibit unrestricted HR PCD in response to pathogen infection. These findings suggest that evolutionarily conserved autophagy genes and the autophagy pathway play an important role in the regulation of PCD induced during plant innate immune responses. Results Identification of Plant BECLIN 1 To identify novel genes that affect initiation, execution, or restriction of N-mediated HR PCD, we performed a high-throughput VIGS screen using N gene-containing Nicotiana benthamiana plants (referred to as NN, Liu et al., 2002b) (see Experimental Procedures for details). From 1500 cDNAs that were silenced, we found that suppression of the cDNA clone 92 (cDNA92) resulted in an altered HR PCD phenotype in response to TMV infection (see below). Sequence analysis of cDNA92 revealed significant homology to human beclin 1 (Hsbeclin 1) and yeast ATG6/VPS30 (ScATG6/VPS30) (Kametaka et al., 1998; Liang et al., 1999). We used RT-PCR to clone the full-length N. benthamiana BECLIN 1 (NbBECLIN 1), which encodes a predicted 527 amino acid coiled-coil protein that shares 69% identity and 79% similarity with Arabidopsis BECLIN 1 (AtBECLIN 1; GenBank accession NM_116036), 36% identity and 52% similarity with Hsbeclin 1 (Liang et al., 1999), and 24% identity and 44% similarity with ScATG6/VPS30 (Figure 1A; Kametaka et al., 1998). Plant BECLIN 1 Functions in Autophagy and Senescence To test if plant BECLIN 1 can function in autophagy, we assayed its ability to rescue autophagy-defective ATG6/VPS30-deficient (?atg6/vps30) yeast. ?atg6/vps30 yeast transformed with plant BECLIN 1 were able to induce autophagy after nitrogen starvation in a manner similar to wild-type yeast and ?atg6/vps30 yeast transformed with Hsbeclin 1 or ScATG6/VPS30 (Figure 1B). Thus, plant BECLIN 1 is an ortholog of yeast ATG6/ VPS30 and can functionally complement autophagy in ?atg6/vps30 yeast. One known function of the Arabidopsis autophagy genes ATG7 and ATG9 and the autophagy-related gene VTI12 is delaying leaf senescence (Doelling et al., 2002; Hanaoka et al., 2002; Surpin et al., 2003). Therefore, we examined the function of BECLIN 1 in leaves artificially induced to senesce. The detached leaves from the BECLIN 1-silenced plants show accelerated senescence compared to detached leaves from control plants (Figure 1C). In addition, expression of the senescence-

associated marker gene SEN1 (AB026439) is significantly induced in BECLIN 1-silenced plants (data not shown). Thus, plant BECLIN 1 is required to prevent premature senescence. This result, together with the complementation of the yeast autophagy mutant, is consistent with a role for plant BECLIN 1 in autophagy. BECLIN 1 Is Required to Limit TMV-Induced Cell Death to the Infection Site Since we had identified plant BECLIN 1 from its effect on HR PCD, we wanted to determine if the levels of BECLIN 1 RNA/protein level changed during this resistance response. For this, we examined the BECLIN 1 levels during TMV-induced HR PCD in NN plants. RTPCR and immunoblot analyses indicate that BECLIN 1 mRNA and protein levels increase rapidly in leaves in the early stages of TMV-induced HR cell death (Figure 2A). To elucidate the role of BECLIN 1 in TMV-induced HR cell death, we infected BECLIN 1-silenced and nonsilenced NN plants with GFP-tagged TMV (TMV-GFP). The TMV-GFP allows us to easily follow the spread of the virus over the course of infections and hence determine how resistance response has been affected. BECLIN 1-silenced plants did not display any gross phenotypic abnormalities before infection with TMV-GFP (data not shown). In nonsilenced NN control plants infected with TMV-GFP, HR cell death was restricted to infection sites as expected (Figure 2B). In contrast, in BECLIN 1-silenced NN plants infected with TMV-GFP, HR cell death lesions were larger than those in control plants (Figure 2B). In these plants, HR lesions were visible and fully developed by 2 days postinfection (dpi), compared to 3–4 dpi in nonsilenced plants. Interestingly, in BECLIN 1-silenced NN plants, cell death extended beyond the site of infection into the uninfected tissues (Figure 2B). This unrestricted cell death resulted in the death of the TMV-GFP-inoculated leaf by 12 dpi (Figure 2B). Pathogen infection is necessary to induce cell death in BECLIN 1-silenced NN plants since mock infection of these plants fails to initiate HR cell death (Figure 2B). In the TMV-infected BECLIN 1-silenced NN plants, unrestricted cell death was not limited to the TMV-inoculated leaf since some of the upper uninoculated leaves also died (Figure 2C). These results suggest that BECLIN 1 is required to restrict cell death to the infection site in plants undergoing HR PCD induced upon pathogen infection. Expression of TMV-p50 Alone Is Sufficient to Cause Spreading Cell Death in BECLIN 1-Silenced NN Plants Although the boundary of cell death expanded beyond the initial infection sites in BECLIN 1-silenced plants, TMV-GFP remained confined to these initial sites of infection, suggesting that the death of the uninfected tissues was independent of virus movement (Figure 2B). Moreover, death of the upper uninoculated leaves in BECLIN 1-silenced plant was not due to the spread of TMV-GFP from the inoculated leaf since no TMV-GFP was detected in the healthy tissues of upper uninoculated leaves either by UV illumination (Figure 2C) or by RT-PCR (Figure 2D).

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Figure 1. Identification of Plant BECLIN 1 and Its Role in Yeast Autophagy and Plant Senescence (A) Alignment of the predicted N. benthamiana BECLIN 1 (NbBECLIN 1; AY701316) amino acid sequence with Arabidopsis BECLIN 1 (AtBECLIN 1), yeast Atg6/Vps30 (ScAtg6/Vps30), and human Beclin 1 (HsBeclin 1). The alignment was generated using ClustalW. Numbers at left indicate amino acid residue positions. Identical residues are shaded in black, and similar residues are in gray. (B) Plant BECLIN 1 complements yeast autophagy mutants deficient in ATG6/VPS30. Cells with autophagic bodies within the vacuole were counted in nitrogen-starved wildtype yeast and in ATG6/Vps30-deficient yeast transformed with empty vector or with plasmids that encode ScATG6/VPS30, Hsbeclin 1, or AtBECLIN 1. Results shown represent the mean (±SEM) percentage of cells with autophagic bodies for triplicate samples. Results were reproduced in three independent experiments. (C) BECLIN 1-deficient leaves exhibit accelerated senescence. Leaves of nonsilenced control and BECLIN 1-silenced plants were detached, floated on water at room temperature in the dark, and photographed after 0, 3, 8, and 11 days. Results were reproduced in four independent silencing experiments using three or more plants in each experiment.

To confirm that the cell death occurring throughout the plant in BECLIN 1-silenced NN plants is not due to the movement of the virus, we transiently expressed the N gene elicitor protein, TMV-p50, using an Agrobacterium-mediated transient expression method (Agroinfiltration). With this method, the protein of interest (in this case TMV-p50) is expressed only in the infiltrated area, as Agrobacterium cannot move into surrounding tissue. As expected, the expression of TMV-p50 induced cell death only in the Agro-infiltrated area of the nonsilenced NN control plants (Figure 3A, left panels). However, in BECLIN 1-silenced NN plants, cell death initially induced in the Agro-infiltrated area spread beyond the area of infiltration, killing the entire leaf by 13 days (Figure 3A, right panels) and eventually the uninfiltrated upper leaves (Figure 3B, right panels). Therefore, HR PCD initiated by TMV-p50 protein alone is sufficient to induce spreading cell death in BECLIN 1-silenced NN plants. Since plant BECLIN 1 is required for limiting genetically programmed HR PCD, we examined whether it is also required for the restriction of nonspecific cell death. We induced nonspecific cell death by treating plants with methanol. In BECLIN 1-silenced and nonsilenced plants, nonspecific cell death occurred at similar rates in the methanol-infiltrated area and the cell death was restricted to the infiltrated tissue (Figure 3C). These results suggest that during the N-mediated response, BECLIN 1 is required to limit the spread of cell death from pathogen-infected cells into adjacent uninfected tissues. However, general “poisoning” death (i.e., non-PCD) cannot direct such signal(s) from the cell

death sites to cause spread of death to the adjacent tissue even in the absence of BECLIN 1. Autophagy Is Induced during N-Mediated Resistance to TMV Since BECLIN 1 is required for limiting HR PCD, we speculated that the loss of PCD restriction was due to the loss of autophagy. We therefore looked for autophagic activity in BECLIN 1-silenced and nonsilenced control plants before and after infection with TMV-GFP. We used LysoTracker Red dye as a probe to indicate the formation of autolysosome-like structures. This dye has been widely used as a probe to indicate autophagic activity in live tissues of various organisms because it labels acidic organelles such as autolysosomes (Moriyasu et al., 2003; Munafo and Colombo, 2001; Rusten et al., 2004; Scott et al., 2004). Before infection with TMV, cells from nonsilenced control and BECLIN 1-silenced plants do not incorporate LysoTracker dye (Figures 4A and 4B). However, after infection with TMV, we observed strong punctate LysoTracker-stained autolysosomes in the cells of nonsilenced NN control plants (Figures 4A and 4C). In contrast, these structures were rarely observed in the cells of BECLIN 1-silenced NN plants (Figures 4B and 4C). Thus, LysoTrackerstained autolysosomes are induced at the site of TMV infection during HR PCD, and BECLIN 1 is required for this induction. We next examined the uninfected cells surrounding the tissue undergoing HR PCD for autophagic activity. Unexpectedly, in nonsilenced NN control plants, a number of LysoTracker-stained autolysosomes were ob-

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Figure 2. BECLIN 1 Is Required to Limit the Spread of TMV-Induced PCD in NN Plants (A) Expression pattern of BECLIN 1 in plants undergoing HR response to TMV infection. Real-time RT-PCR analysis using total RNA isolated from the infected leaves and BECLIN 1-specific primers at time points indicated (left). Immunoblot analysis of total protein isolated from infected leaf using anti-HsBeclin 1 antibodies; Coomassie-stained membrane confirmed equal loading of samples (right). (B) TMV-GFP-induced PCD was assessed in nonsilenced control and BECLIN 1-silenced plants. Mock-inoculated BECLIN 1-silenced plants also served as a control. Representative photographs of TMV-GFP or mock-inoculated leaves were taken under normal light and UV light. Photographs shown in panels 4 and 5 are higher magnification images of the leaves shown in panels 1 and 3, respectively. Red color in the background of GFP fluorescence in the UV light photographs is due to the autofluorescence from chlorophyll. Infection foci in 12 dpi leaves are white under UV light because virus is already cleared in these samples. Results were reproduced in at least five independent experiments using three or more plants in each experiment. dpi, days post infection. (C) TMV-induced PCD spreads into healthy tissues of upper uninfected leaves of BECLIN 1-silenced plants in the absence of movement of TMV-GFP. Representative photographs from two different plants were taken under normal light and UV light. (D) Relative levels of TMV-GFP RNA in inoculated leaf (IL) and upper uninoculated leaves (UUIL) of nonsilenced control and BECLIN 1-silenced plants. Ethidium bromide-stained gel represents RT-PCR product from 6 dpi samples. Lane 1, no RT control; lane 2, nonsilenced control plants; lane 3, BECLIN 1-silenced plants. Shown below the agarose gel is a time course qRT-PCR analysis of TMV-GFP RNA accumulation in the inoculated leaves. Error bars represent standard deviations. (E) Quantitative analysis of GFP fluorescence in infected sites of nonsilenced control and BECLIN 1-silenced plants. Error bars represent standard deviations.

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Figure 3. Expression of TMV-p50 Protein Is Sufficient to Cause a Spreading Cell Death Phenotype in BECLIN 1-Silenced NN Plants (A) Expression of TMV-p50 using Agroinfiltration in the nonsilenced control (left) and BECLIN 1-silenced (right) plants. (B) Spread of HR PCD into upper systemic leaves of BECLIN 1-silenced plants in which TMV-p50 is expressed in the lower leaves using Agro-infiltration. (C) Treatment of methanol in the nonsilenced control (left) and BECLIN 1-silenced (right) plants. Results were reproduced in four independent experiments using three or more plants in each experiment.

served in these cells (Figure 4D). Additionally, we also examined upper uninfected leaves of nonsilenced NN plants and also found a number of LysoTracker-stained autolysosomes (Figure 4E). In contrast, in BECLIN 1-silenced NN plants, very few cells surrounding the

infection site and in systemic tissue contained LysoTracker-stained autolysosomes (Figures 4D and 4E). To confirm that the LysoTracker-stained structures represent autolysosomes, we used transmission electron microscopy (TEM). In infected nonsilenced NN plants,

Figure 4. Autophagy Is Induced during the Resistance Response to TMV (A and B) Representative images of LysoTracker Red-stained TMV-infected tissue derived from nonsilenced control (A) and BECLIN 1-silenced (B) plants. LysoTracker Red-stained punctate autolysosome-like structures inside the vacuole (green, column 1), autofluorescence from chloroplast (red, column 2), and merged images (column 3). Results were reproduced in three independent experiments using three or more plants in each experiment. Scale bar equals 50 ?m. (C–E) Quantitation of autolysosome-like structures observed in LysoTracker Redstained infected tissue (C), uninfected tissue adjacent to infected tissue (D), and tissue from systemic leaves (E). Approximately 200–300 cells for each treatment and each time point were counted. Error bars represent standard deviations.

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with our LysoTracker observations, indicate that autophagy is induced during the N-mediated defense response to TMV, both in the infection site and in uninfected adjacent tissue or systemic tissue. BECLIN 1 Is Required to Limit HR Cell Death Induced by Other R Genes and General Elicitors Since plant BECLIN 1 is needed to contain N-mediated HR PCD to the infection site, we hypothesized that BECLIN 1 might play a general role in the containment of HR PCD induced by other R genes as well as general elicitors. We tested the role of BECLIN 1 in regulating HR PCD after coexpression of the bacterial resistance gene Pto and its cognate Avr gene, AvrPto (Pedley and Martin, 2003) and also after coexpression of the fungal resistance gene Cf9 and its cognate Avr gene, Avr9 (Hammond-Kosack and Jones, 1997). We also tested whether cell death responses to general pathogen elicitors are affected by loss of BECLIN 1 by expressing INF1 from Phytophthora infestans and by infecting plants with the bacteria P. syringae pv. tomato DC3000. As expected, HR cell death induced by these treatments in the nonsilenced control plants is confined to the infiltrated area even after 11 days (Figure 6). However, in BECLIN 1-silenced plants, cell death spread beyond the infiltrated area, killing the entire leaf by 11 days (Figure 6). In addition, death was observed in systemic upper uninfected leaves of these plants (data not shown). Together, these results demonstrate that BECLIN 1 is not only required to limit the spread of R genemediated HR PCD triggered by viruses but also that triggered by bacteria and fungi and general elicitors. Autophagy Genes PI3K/VPS34, ATG3, and ATG7 Are Required to Limit Cell Death to the Infection Site In yeast and mammals, the Atg6/Vps30/Beclin 1 protein physically associates with class III phosphoinositide 3-kinase (PI3K)/Vps34, which is also required for autophagy (Eskelinen et al., 2002; Kihara et al., 2001a; Kihara et al., 2001b). Two proteins of the conjugation pathways required for autophagy are Atg7 and Atg3 (Ichimura et al., 2000). We therefore tested the involvement of PI3K/VPS34, ATG3, and ATG7 in TMV-induced HR PCD. Silencing these genes in NN plants produced a similar phenotype to that observed in BECLIN 1-silenced plants: although TMV-GFP was confined to the infection site, cell death was not limited to the TMV infection site and uninfected healthy tissues eventually died (Figure 7). Thus, multiple different autophagy genes function to limit the extent of spread of TMV-induced HR PCD. Enhanced Virus Accumulation in ATG-Deficient Plants Our observation of infection sites in ATG-silenced plants indicate that, independently of the spreading cell death phenotype, the ATG genes play a role in controlling TMV replication and/or cell-to-cell movement. In BECLIN 1-, PI3K/VPS34-, ATG3-, and ATG7-deficient plants, there is increased accumulation of TMV as observed by GFP fluorescence at the site of infection (Figures 2B, 2E, and 7). This increased accumulation of TMV is not due to increased spread because GFP fluo-

Figure 5. Transmission Electron Microscopy Images of Autophagic Bodies (A and B) Representative transmission electron microscopy (TEM) images of healthy tissue surrounding infected tissue derived from nonsilenced control plants (A) before infection (1), 2 dpi (2), 7 dpi (3 and 4); and from BECLIN 1-silenced plants (B) before infection (1), 2 dpi (2), and 7 dpi (3 and 4). V, vacuole; dpi, days post infection; arrows indicate autolysosome-like structures. Results were reproduced in two independent experiments using three or more plants in each experiment. (C) Representative TEM image of autophagosome shown at higher magnification. (D) Quantitation of autolysosome/autophagosome structures observed in tissue surrounding infected tissue derived from nonsilenced control and BECLIN 1-silenced plants. Error bars represent standard deviations.

we observed a number of autolysosomes containing electron-dense material in the cytoplasm and vacuole of uninfected tissue surrounding sites of HR PCD (Figures 5A, panels 2–4, and 5D). In a few samples, we observed fusion of autolysosome structures with the vacuole (Figure 5A, panel 3). In addition, we occasionally observed double-membrane vesicles containing cytoplasmic materials and organelles; in other words, stereotypical autophagosomes (Figure 5C). In BECLIN 1-silenced NN plants, we observed reduced numbers of autolysosomes in the cytoplasm and vacuole (Figures 5B, panels 2–4, and 5D). These TEM data, together

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Figure 6. BECLIN 1 Is Required to Limit Spread of HR PCD Induced during R Gene- and Non-R Gene-Mediated Resistance Transient expression of Cf9 with Avr9, Pto with AvrPto, and INF1 using Agro-infiltration; and inoculation of Pseudomonas syringae pv. tomato DC3000 in nonsilenced control and NbBECLIN 1-silenced plants. In all cases, experiments were performed at least three times using three or more plants in each experiment.

rescence is confined only to the infection site in these plants. Furthermore, we also observed increased accumulation of TMV RNA in BECLIN 1-silenced plants as detected by RT-PCR (Figure 2D). This is not due to the increased spread of the virus because TMV-GFP is not detected using RT-PCR analysis in systemic uninfected upper leaves in BECLIN 1-silenced plants (Figure 2D). Together these results suggest that BECLIN 1, PI3K/ VPS34, ATG3, and ATG7 function not only to limit the spread of HR cell death, but also independently, to limit virus replication and/or movement. Discussion Role of Plant BECLIN 1 in Autophagy and Development We have identified plant BECLIN 1, an ortholog of the yeast ATG6/VPS30 and mammalian beclin 1 (Kametaka et al., 1998; Liang et al., 1999). In yeast, Dictyostelium, C. elegans, and mammals, ATG6/beclin 1 is required for autophagosome formation during autophagy (Eskelinen et al., 2002; Kihara et al., 2001a; Kihara et al., 2001b; Melendez et al., 2003; Otto et al., 2003). Our results also provide evidence for plant BECLIN 1 in autophagy. First, expression of plant BECLIN 1 restores autophagy in autophagy-defective ATG6/VPS34-deficient yeast. Second, BECLIN 1-deficient plants exhibit accelerated leaf senescence similar to Arabidopsis

ATG4, ATG7, and ATG9 mutants (Doelling et al., 2002; Hanaoka et al., 2002; Yoshimoto et al., 2004). Third, BECLIN 1 is required for induction of autophagy during defense responses. In mammals, beclin 1 knockout mutant mice die early in embryogenesis, indicating that it is an essential gene (Qu et al., 2003; Yue et al., 2003). In contrast, BECLIN 1-silenced plants exhibited very weak developmental phenotypes, if any, under normal growth conditions. We cannot rule out the possibility that BECLIN 1 may also have a role in early developmental processes because the VIGS screen was performed using adult plants. Furthermore, VIGS, like other RNAi techniques, fails to completely suppress expression of a target gene. Residual target gene activity may be sufficient to allow normal development to occur. However, Arabidopsis ATG7 and ATG9 knockout plants had a normal life cycle except that rosette leaves of these plants senesced earlier (Doelling et al., 2002; Hanaoka et al., 2002). Plant BECLIN 1 and Other ATG Genes Function as Negative Regulators of HR PCD Generally, HR PCD initiated at the pathogen infection site is restricted to a small number of cells (Lam, 2004). In BECLIN 1-silenced N resistance gene-containing plants, HR PCD initiated at the site of TMV infection spreads into surrounding healthy tissue and into systemic uninfected leaves. Even though more TMV accu-

Figure 7. PI3K/VPS34, ATG3, and ATG7 Are Required to Limit Spread of HR PCD Induced during N-Mediated Resistance TMV-GFP-induced PCD was assessed in nonsilenced control (A), PI3K/VPS34-silenced (B), NbATG3-silenced (C), and NbATG7-silenced (D) NN plants. Representative photographs of TMV-GFP inoculated leaves were taken under normal light and UV light 3 dpi (top) and 9 dpi (bottom). In all cases, results were reproduced in five independent experiments using three or more plants in each experiment.

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mulates in BECLIN 1-silenced plants at the infection site, virus was not detected outside of the infection site. In fact, local expression of N gene elicitor TMVp50 protein is sufficient to induce spreading cell death phenotype in the absence of BECLIN 1. Therefore, movement of virus from infection site into healthy tissue is not the cause for the spreading cell death phenotype observed in these plants. This suggests that signal(s) required for inducing spreading cell death in BECLIN 1-silenced plants is generated after the recognition of Avr protein but not by the extent of increased pathogen replication and/or spread. The role of BECLIN 1 in limiting HR PCD is not restricted to the N-TMV system. We also observed spreading cell death during the HR in bacterial and fungal R gene-mediated resistance as well as general elicitor-mediated resistance in BECLIN 1-silenced plants. In addition to BECLIN 1, other ATG genes PI3K/VPS30, ATG3, and ATG7 are required to restrict HR PCD to the TMV infection site. Thus, BECLIN 1 and other ATG genes function in a conserved pathway to restrict HR PCD to the infection site. If a plant is to survive an infection, HR PCD must be carefully controlled so that it does not spread throughout the plant and kill it. It has been speculated that signals generated at the site of infection are responsible for regulating HR PCD and triggering defense responses in uninfected tissues (Lam, 2004). Since plant cells are highly connected, an important long-standing question in plant innate immunity is how HR PCD is limited to the site of infection. Our data demonstrate that initiation of HR PCD is not affected in plants silenced for BECLIN 1 or other ATG genes, suggesting that signals required for the induction of HR PCD are not compromised in these plants. However, the machinery required to confine PCD to the infection site is lost in these plants. Therefore, our findings provide the first genetic evidence that ATG genes can function in vivo as a negative regulator of HR PCD. These results contrast with findings from mammalian studies in which ATG genes are required to promote PCD in cells lacking intact apoptotic machinery. The autophagy genes beclin 1 and atg7 are required to induce nonapoptotic cell death in the murine fibroblast L929 cells treated with capsase inhibitor zVAD (Yu et al., 2004). In addition, atg5 and beclin 1 are required for etoposide and staurosporine treatment-induced cell death in apoptosisresistant Bax/Bak double knockout mouse embryonic fibroblasts (Shimizu et al., 2004). However, the ability to induce apoptotic cell death in response to either UV light or serum deprivation is not compromised in ES cells derived from beclin 1 null mutant mice (Yue et al., 2003). Moreover, recent evidence suggests that ATG genes promote survival during nutrient starvation or growth factor deprivation (reviewed in Levine, 2005), and RNAi of ATG genes triggers apoptosis in starved mammalian cells (Boya et al., 2005). Prosurvival Function of Autophagy Our LysoTracker staining and TEM data indicate that autophagy is induced at the site of TMV infection in N resistance gene-containing plants. Surprisingly, the induction of autophagy was not limited to the TMV in-

fection sites but extended into uninfected adjacent tissue and into the systemic leaves. This was surprising because most of the evidence indicates that autophagy is induced in cells undergoing PCD, not in healthy cells. We were able to observe not only double-membrane vesicles containing cytoplasmic materials and organelles, which are representative of autophagosomes, but also autolysosome vesicles fusing with and discharging their contents into vacuoles. In contrast, BECLIN 1-silenced plants fail to form autolysosomes/autophagosomes and hence fail to undergo autophagy. Therefore, autophagy induced during defense responses may be required to eliminate “prodeath” signal(s) moving out of the pathogen-infected area into adjacent tissues. This could explain the increased autophagic activity observed in uninfected tissue adjacent to the infection site in control plants. It is also possible that autophagy could be involved in cleanup processes to remove cellular damage caused by proteases, hydrolases, and nucleases that are activated during the defense response. In addition, autophagy might be involved in protecting healthy cells from damage caused by ROI produced during the defense response. Thus, autophagy may play an essential “prosurvival” function in plants that effectively contains pathogen spread without resulting in “unwanted” death in innocent uninfected bystander cells. Role of Autophagy in Antimicrobial Defense The results presented in this study also suggest an antiviral role for autophagy in plants. In BECLIN 1-, PI3K/ VPS34-, ATG3-, or ATG7-silenced plants, we observed increased accumulation of TMV at infection sites. This observation is consistent with several recent reports in mammalian cells describing a role for autophagy in host defense against viral and intracellular bacterial pathogens. Herpes simplex virus (HSV) infection triggers PKR-dependent induction of autophagy in mammalian cells, which is blocked by the HSV encoded neurovirulence factor ICP34.5 (Talloczy et al., 2002). Overexpression of mammalian beclin 1 protects mice from fatal Sindbis virus infection (Liang et al., 1998). The mammalian autophagy gene, atg5, is required to degrade invasive group A Streptococcus (Nakagawa et al., 2004), and the induction of autophagy by starvation or rapamycin treatment promotes the degradation of Mycobacterium tuberculosis within phagolyososmes (Gutierrez et al., 2004). In addition, a bacterial gene product required for blocking autophagy, iscB, is necessary for the virulence of the intracellular enteric pathogen, Shigella (Ogawa et al., 2004). Our findings with TMV infection in plants suggest that the role of autophagy genes in limiting the growth of intracellular pathogens is evolutionarily conserved. In summary, our results demonstrate that autophagy plays an important role in regulating the plant innate immune response. In the absence of ATG genes, the HR PCD spreads beyond the sites of infection to adjacent tissue and distal uninfected leaves. These results imply that there is a prodeath signal(s) moving out of the pathogen-infected area into adjacent tissues and distal sites that is negatively regulated by autophagy. It is not yet known whether autophagy alters the pro-

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duction of death-promoting signals, prevents the movement of death-promoting signals into uninfected tissues, or protects uninfected tissues against death induced by these signals. Further genetic studies are required to determine the precise mechanistic function of autophagy in controlling PCD during plant-pathogen interactions. In addition, it will be interesting to see whether autophagy protects against PCD in other organisms and in other biological settings.
Experimental Procedures Plant Materials, Pathogens, and Plasmids Wild-type N. benthamiana, NN transgenic plants, and TMV-GFP were described (Liu et al., 2002b). B. Staskawicz provided P. syringae pv. tomato DC3000. Vectors pTRV1, pTRV2, and pTRV2-GATEWAY were described (Liu et al., 2002a; Liu et al., 2002b). pTRV2NtPI3K/VPS34, pTRV2-NbATG3, and pTRV2-NbATG7 were made by cloning PCR products of cDNA fragments of PI3K/VPS34 (AY701317, bases 131–834), ATG3 (AY701318, bases 1–493), and ATG7 (AY701319, bases 1–558) into pTRV2. Primer sequences are available upon request. Constructs 35S:Cf9 and 35S:Avr9, 35S:Pto and 35S:AvrPto, and 35S:INF1 were provided by P. De Wit, G. Martin, and S. Kamoun, respectively. Yeast Autophagy Complementation Assays and Artificial Induction of Leaf Senescence Yeast autophagy complementation assays were performed as described (Liang et al., 1999). For leaf senescence assays, leaves from 2 weeks after silencing of BECLIN 1 were detached and floated on deionized water at room temperature in the dark. Leaves of the same age were used from nonsilenced control plants in these assays. The leaves were photographed at 0, 3, 8, and 11 days after dark incubation. Normalized cDNA Library Construction A normalized cDNA library was constructed in pTRV2-GATEWAY vector by custom services from Invitrogen (CA) using poly(A)+ RNA isolated from a 1:1 mixture of tissue derived from TMV-resistant NN plants and TMV-susceptible N. benthamiana plants at 0, 1, 4, 8, 16, and 24 hr after TMV infection. VIGS, Pathogen-Induced Cell Death Assay, and TMV-GFP Quantification NN plants were used for TMV-GFP and TMV-p50 helicase assays; N. benthamiana plants lacking the N gene were used for other assays. For the VIGS screen, Agrobacterium cultures (OD600 = ~0.8) containing pTRV1 and individual clone from pTRV2-normalized cDNA library were mixed at a 1:1 ratio and infiltrated into the lower leaves of 4-leaf-stage plants using a 1 ml needleless syringe. We screened a total of 1500 cDNA clones containing Agrobacterium colonies. To monitor the effect of silencing a given gene on the defense response, 10 days after infiltration the upper leaves of the plants were infected with TMV-GFP. These plants were monitored for changes in HR cell death and loss of resistance to TMV by assaying movement of TMV-GFP into the upper uninoculated leaves. GFP imaging was performed using UV illumination, and photographs were taken using an Olympus Camedia E10 digital camera. TMV at the infection site was quantified by measuring pixel size of the GFP spot using Adobe Photoshop. At least 20–30 infection sites from four independent infected leaves were used for quantification of GFP intensity for each treatment. VIGS of BECLIN 1, PI3K/VPS34, ATG3, and ATG7 was performed as described (Liu et al., 2002b). We used 10 days postsilenced plants for TMV infections since RT-PCR analysis of silenced plants showed significant reduction of BECLIN 1 RNA at this time point compared to nonsilenced plants (data not shown). Silenced plants and nonsilenced plants were infected with TMV-GFP or were infiltrated with Agrobacterium carrying TMV-GFP T-DNA or 35S:TMVp50 or 35S:Cf9 and 35S:Avr9 or 35S:Pto and 35S:AvrPto or 35S:INF1. For experiments in which Cf9 and Avr9 or Pto and AvrPto were coexpressed, Agrobacterium cultures carrying these plas-

mids were mixed in 1:1 ratio while maintaining an OD600 = 0.8. Pst DC3000 were resuspended in 10 mM MgCl2 to OD600 = 0.4 and infiltrated into leaves.

RT-PCR and Quantification of mRNA Levels Detection of TMV-GFP by RT-PCR was performed using the protocol described (Liu et al., 2002b). BECLIN 1 and TMV RNA levels were monitored by SYBR Real-Time RT-PCR using RNA extracted from tissue collected at 0, 0.5, 1, 2, 4, 8, 16, 24, and 48 hr in which the N response was synchronously induced (Liu et al., 2004). Detection of real-time RT-PCR products was done by the incorporation of the fluorescent DNA dye SYBR Green using the QuantiTect SYBR Green PCR kit (Qiagen) following the manufacturer's recommendations in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, USA). All calculations and statistical analysis were performed as described in the User Bulletin.

Immunoblot Analysis To detect BECLIN 1 protein levels, the N response was induced synchronously (Liu et al., 2004) and tissue was collected at 0, 0.5, 1, 2, 3, 4, 8, 16, and 24 hr. Protein extraction and immunoblot analysis using HsBeclin 1 antibody (provided by Atsuki Nara) was performed as described (Liu et al., 2004).

LysoTracker Red Staining and Transmission Electron Microscopy BECLIN 1-silenced or nonsilenced control plants were infiltrated with Agrobacterium carrying TMV-GFP. At 0, 2, 4, 7, and 11 days post TMV-GFP infiltration, the infected area, areas adjacent to the infected area, and upper uninfected leaves were infiltrated with 100 ?M E-64d. Plants were kept at room temperature overnight and the E-64d infiltrated parts of the leaves were excised and immediately vacuum infiltrated with 1 ?M LysoTracker Red DND-99 (Molecular Probes, Eugene, OR) and kept in the dark for 1 hr. Leaf pieces were then placed with the adaxial surface down into a single-well LabTek II chambered 1.5 cover glass system submerged in 1 ?M LysoTracker Red. Confocal images were acquired on a Zeiss Axiovert 200M equipped with a Zeiss LSM 510 NLO laser scanning microscope using a 40X C-Apochromat (NA 1.2) water-corrected objective lens. All data were collected with 543 nm excitation of a 1 mW helium:neon laser with 565–625 nm band pass and 650 nm long pass emission filters for LysoTracker Red and chloroplast autofluorescence, respectively. For quantification of LysoTracker-stained structures, approximately 200–300 cells for each treatment and each time point were counted. Electron microscopy was performed using a modified protocol described from Bourett et al. (1999). Briefly, leaf discs were quickly and carefully excised from E-64d or 1 ?M concanamycin A infiltrated regions with a 1.5 mm diameter biopsy punch, transferred to a dextran-filled 1.5 mm by 200 mm HPF flat specimen carrier, and then frozen using a Leica EMPACT high-pressure freezer. Samples were subsequently freeze-substituted in a Leica EM AFS at ?90°C for 3 days in 4% OsO4 in acetone, warmed slowly to room temperature, rinsed three times in acetone, and embedded in Embed-812 resin. Sectioned samples were stained with lead citrate and uranyl acetate before imaging on a Zeiss CEM 902 transmission electron microscope equipped with a Megaview II digital camera (Soft Imaging System, Boulder, CO). To quantify autophagic structures, digital images were acquired and the number of autolysosomes or autophagosomes was determined per 2500 mm2 field.

Acknowledgments We thank T. Burch-Smith, M. Seay, and J. Soosaar for helpful comments on the manuscript; B. Staskawicz, G. Martin, P. De Wit, and S. Kamoun for providing reagents; and A. Nara for HsBeclin 1 antibody. Supported by NSF Plant Genome grant DBI-0116076 and NIH grant GM62625 to S.P.D.-K. and NIH grant AI151367 to B.L.

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