The Rab GTPase RabG3b Positively Regulates Autophagy and Immunity-Associated Hypersensitive Cell Death in Arabidopsis^,

A Rab GTPase protein connects autophagy with plant immunity-triggered hypersensitive response and programmed cell death.

Abstract

A central component of the plant defense response to pathogens is the hypersensitive response (HR), a form of programmed cell death (PCD). Rapid and localized induction of HR PCD ensures that pathogen invasion is prevented. Autophagy has been implicated in the regulation of HR cell death, but the functional relationship between autophagy and HR PCD and the regulation of these processes during the plant immune response remain controversial. Here, we show that a small GTP-binding protein, RabG3b, plays a positive role in autophagy and promotes HR cell death in response to avirulent bacterial pathogens in Arabidopsis (Arabidopsis thaliana). Transgenic plants overexpressing a constitutively active RabG3b (RabG3bCA) displayed accelerated, unrestricted HR PCD within 1 d of infection, in contrast to the autophagy-defective atg5-1 mutant, which gradually developed chlorotic cell death through uninfected sites over several days. Microscopic analyses showed the accumulation of autophagic structures during HR cell death in RabG3bCA cells. Our results suggest that RabG3b contributes to HR cell death via the activation of autophagy, which plays a positive role in plant immunity-triggered HR PCD.

In response to the constant attack by microbial pathogens, plants have developed defense mechanisms to protect themselves against harmful diseases caused by various pathogens. Plants primarily rely on two layers of innate immunity to cope with microbial pathogens (Jones and Dangl, 2006). The first layer of plant immunity, which is triggered by pathogen-associated molecular patterns (PAMPs) such as bacterial flagellin, lipopolysaccharides, and fungal chitin, is designated PAMP-triggered immunity (PTI; Boller and He, 2009). Because pathogens have evolved to overcome PTI, plants have developed a second layer of immunity, referred to as effector-triggered immunity (ETI; Dodds and Rathjen, 2010). ETI depends on specific interactions between plant Resistance proteins and pathogen effectors and is often associated with a form of programmed cell death (PCD) termed the hypersensitive response (HR), which inhibits pathogen growth (Coll et al., 2011).

Plants use PCD to regulate developmental and defense responses. In addition to pathogen attack, many abiotic stress factors such as heat and ozone exposure elicit PCD in plants (Hayward and Dinesh-Kumar, 2011). PCD also occurs during various developmental processes, including endosperm development, tracheary element (TE) differentiation, female gametophyte differentiation, leaf abscission, and senescence (Kuriyama and Fukuda, 2002; Gunawardena, 2008). Recently, plant PCD has been classified into two types, “autolytic” PCD and “nonautolytic” PCD, on the basis of the presence or absence of rapid cytoplasm clearance after tonoplast rupture, respectively (van Doorn et al., 2011). Autolytic PCD, which mainly occurs during plant development, falls under “autophagic” PCD in animals because it is associated with the accumulation of autophagy-related structures in the cytoplasm. Some forms of HR PCD classified as nonautolytic PCD in plants are accompanied by increased vacuolization, indicating the progress of autophagy, and therefore can be placed under autophagic PCD (Hara-Nishimura et al., 2005; Hatsugai et al., 2009).

Autophagy is an intracellular process in which double membrane-bound autophagosomes enclose cytoplasmic components and damaged or toxic materials and target them to the vacuole or lysosome for degradation (Chung, 2011). In plants, autophagy plays important roles in the responses to nutrient starvation, senescence, and abiotic and biotic stresses (Liu et al., 2005; Xiong et al., 2005, 2007; Bassham, 2007; Hofius et al., 2009). Accumulating evidence indicates that autophagy regulates immune responses in both animals and plants. Autophagy is essential for the direct elimination of pathogens in mammalian systems (Levine et al., 2011). Invading bacteria and viruses are targeted to autophagosomes and then delivered to the lysosome for degradation in a process called xenophagy (Levine, 2005). In addition to its function in directly killing pathogens, xenophagic degradation can provide microbial antigens for major histocompatibility complex class II presentation to the innate and adaptive immune systems (Levine, 2005; Schmid and Münz, 2007). Furthermore, the human surface receptor CD46 was shown to directly induce autophagy through physical interaction with the autophagic machinery (Joubert et al., 2009). The role of autophagy in plant basal immunity to virulent pathogens has been determined (Patel and Dinesh-Kumar, 2008; Hofius et al., 2009; Lai et al., 2011; Lenz et al., 2011). Arabidopsis (Arabidopsis thaliana) plants defective in AUTOPHAGY-RELATED (ATG) genes exhibited enhanced susceptibility to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola, suggesting that the massive breakdown of cytoplasmic materials provides nutrients for the growth of necrotrophic pathogens or that fungal toxin-induced necrotic cell death is enhanced in atg mutants (Lai et al., 2011; Lenz et al., 2011). However, studies on the responses to the biotrophic pathogen Pseudomonas syringae pv tomato DC3000 (Pst DC3000) have yielded contradictory results. Whereas earlier studies reported that bacterial numbers significantly increased in ATG6-antisense (AS) and atg mutant plants (Patel and Dinesh-Kumar, 2008; Hofius et al., 2009), a recent study indicated that atg mutants exhibit increased resistance to Pst DC3000 (Lenz et al., 2011). Although these discrepancies remain to be resolved, salicylic acid (SA) levels and SA-dependent gene expression were both elevated in atg mutants, suggesting that autophagy may negatively regulate SA-associated plant immunity (Yoshimoto et al., 2009; Lenz et al., 2011). These findings indicate that the role of autophagy in plant immunity depends on the lifestyle of the invading pathogens (Lenz et al., 2011).

Autophagy plays an important role in the regulation of HR PCD in plant innate immunity (Hayward and Dinesh-Kumar, 2011). Tobacco (Nicotiana tabacum) plants silenced for ATG6/Beclin1 and other ATG genes such as phosphatidylinositol 3-kinase (PI3K)/vacuolar protein sorting34 (VPS34), ATG3, and ATG7 underwent unrestricted HR PCD upon pathogen infection (Liu et al., 2005). ATG6-AS and atg5 mutant Arabidopsis plants also displayed unlimited HR PCD upon infection with the avirulent bacterium Pst DC3000 (AvrRpm1; Patel and Dinesh-Kumar, 2008; Yoshimoto et al., 2009). These studies suggest that autophagy is a “prosurvival” or “antideath” mechanism that negatively regulates HR PCD (Liu and Bassham, 2012). By contrast, a “prodeath” role has been suggested for autophagy in HR PCD regulation (Hofius et al., 2009). Pst DC3000 (AvrRps4)-induced and, to a lesser extent, Pst DC3000 (AvrRpm1)-induced HR PCD was suppressed in atg mutants, suggesting that autophagy plays a positive role and that autophagic cell death is involved in RPS4- and RPM1-mediated HR cell death.

We previously showed that the small GTP-binding protein RabG3b, isolated from secretome analysis in Arabidopsis (Oh et al., 2005), functions as a component of autophagy and positively regulates TE differentiation via the activation of autophagic cell death (Kwon et al., 2010a, 2010b). Overexpression of a constitutively active RabG3b (RabG3bCA) in plants significantly increased autophagy during PCD associated with TE differentiation, thereby enhancing TE formation and xylem development. Transgenic poplar (Populus alba × Populus tremula var glandulosa) overexpressing Arabidopsis RabG3bCA was further generated, and these exhibited significant stimulation of xylem development together with autophagic activation, suggesting that RabG3b is a positive regulator of autophagy and xylem development in Populus spp. as well as Arabidopsis (Kwon et al., 2011). We also reported that RabG3b is involved in cell death associated with the fungal pathogen A. brassicicola and infection with the fungal toxin fumonisin B1 (FB1) as well as leaf senescence (Kwon et al., 2009). Here, we extend our work to determine the role of RabG3b and autophagy in immunity-associated HR PCD. We found that RabG3bCA transgenic plants accumulated a large number of autophagic structures and displayed accelerated, expanded cell death against a number of PCD inducers, such as FB1 and the bacterial pathogens Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2). Our results suggest that RabG3b plays a positive role in immunity-associated HR PCD via the activation of autophagic cell death.

RESULTS

RabG3b Positively Regulates Pathogen-Induced Hypersensitive Cell Death

Our previous studies showed that RabG3b positively regulates TE PCD through autophagic activation in Arabidopsis, suggesting a positive role of autophagy in developmental PCD (Kwon et al., 2010a). Here, we address whether RabG3b is additionally involved in pathogen-induced HR PCD and whether its role is autophagy related. In this study, we used an autophagy-defective mutant, atg5-1, and three different RabG3b transgenic plants overexpressing constitutively active RabG3b (RabG3bCA), dominant negative RabG3b (RabG3bDN), and an RNA interference construct of RabG3b (RabG3bRNAi), as described previously (Kwon et al., 2010a). In a previous report (Yoshimoto et al., 2009), unlimited cell death was observed upon challenge with Pst DC3000 (AvrRpm1) in 7- to 8-week-old “old” atg5-1 plants grown under short-day conditions but not in 4- to 5-week-old “young” atg5-1 plants, indicating that plant age is important for cell death development. Thus, we first examined cell-death phenotypes of old plants grown under short-day conditions (Fig. 1, A and B). Hypersensitive local cell death was readily induced within hours of inoculation and formed a clear necrotic lesion within 1 to 2 d in all tested plants; however, it expanded to the entire infected leaf in RabG3bCA plants within 2 d, whereas the other plants did not exhibit the accelerated, spreading cell-death phenotype (Fig. 1A). Consistently, the infected leaves of RabG3bCA plants were entirely stained with trypan blue, which specifically stains dead cells (Fig. 1C). As reported previously (Yoshimoto et al., 2009), atg5-1 plants slowly spread chlorotic cell death following prolonged incubation with Pst DC3000 (AvrRpm1) over 14 d (Fig. 1B). Trypan blue staining showed that cell death gradually spread beyond the infection site in the atg5-1 mutant, even without visible cell death symptoms at 2 d after pathogen treatment (Fig. 1C). Young plants were then challenged with Pst DC3000 (AvrRpm1), and RabG3bCA plants exhibited a similar accelerated, spreading HR cell-death phenotype (Supplemental Fig. S1). The occurrence rates of the spreading HR PCD in leaves were comparable in old and young RabG3bCA plants (97% versus 93%, respectively; Fig. 1D). On the other hand, Pst DC3000 (AvrRpm1)-infiltrated leaves of young atg5-1 plants exhibited a progression of spreading chlorotic cell death, like those of old plants, but with a considerable reduction in the occurrence rates of spreading cell death (82% in old plants versus 43% in young plants). Similar results were obtained with another avirulent pathogen, Pst DC3000 (AvrRpt2; Supplemental Fig. S2). These findings suggest that two distinct types of cell death occur in plants challenged with avirulent bacterial pathogens: hypersensitive necrotic cell death and chlorotic cell death. Whereas the former, so-called HR PCD, rapidly occurred at the infection sites within hours and expanded in RabG3bCA plants, the latter gradually spread over days through uninfected sites, depending on plant age, and was associated with autophagy deficiency.

Figure 1.

Pst DC3000 (AvrRpm1)-induced HR cell death is enhanced in RabG3bCA plants. A, Phenotypes of wild-type (WT), RabG3bCA, RabG3bDN, RabG3bRNAi, and atg5-1 leaves inoculated with 10 mm MgCl₂ (Mock) or 10⁸ cfu mL⁻¹ Pst DC3000 (AvrRpm1) for 2 and 5 d. B, Phenotypes of wild-type, RabG3bCA, RabG3bDN, RabG3bRNAi, and atg5-1 leaves inoculated with 10 mm MgCl₂ (Mock) or 10⁸ cfu mL⁻¹ Pst DC3000 (AvrRpm1) for 14 d. C, Cell death in leaves stained with trypan blue. Leaves inoculated with 10⁸ cfu mL⁻¹ Pst DC3000 (AvrRpm1) for 2 d as in B were used for staining. D, Quantitative analysis of spreading cell death in leaves of young (5-week-old) and old (7-week-old) wild-type, RabG3bCA, and atg5-1 plants at 14 d post inoculation with Pst DC3000 (AvrRpm1). Results represent means ± sd from five independent experiments (n = 30). E, Bacterial growth in wild-type, RabG3bCA, RabG3bDN, RabG3bRNAi, and atg5-1 plants inoculated with 10⁵ cfu mL⁻¹ Pst DC3000 (AvrRpm1). Results represent means ± sd (n = 4). Asterisks indicate significant differences from the respective wild type (Student’s t test; **P < 0.01). These experiments were performed five times with similar results. dpi, Days post inoculation.

To further determine whether the spreading lesion induced by pathogen treatments in RabG3bCA plants was a consequence of uncontrolled cell death, we monitored the cell-death phenotype following treatment with the well-known PCD-eliciting mycotoxin FB1 (Supplemental Fig. S2). As observed with pathogen infection, FB1-treated RabG3bCA leaves displayed accelerated, spreading cell death, in contrast to other plants. RabG3bDN, RabG3bRNAi, and wild-type plants did not significantly differ in cell death symptom development in response to all the treatments tested (Fig. 1; Supplemental Fig. S2). Collectively, these results demonstrate that RabG3b is associated with cell death-promoting activity during hypersensitive PCD.

We then examined bacterial growth in plants inoculated with Pst DC3000 (AvrRpm1; Fig. 1E). Although Pst DC3000 (AvrRpm1) growth increased for 2 d after pathogen inoculation in RabG3bCA plants, bacterial growth gradually decreased thereafter, and bacterial numbers in RabG3bCA leaves were 6.2-fold lower than in wild-type leaves 7 d after inoculation (Fig. 1E). Thus, although RabG3bCA plants were not able to fully restrict bacterial growth during the early stage of infection, they readily restored the activity necessary to suppress bacterial growth, indicating that expanded cell death in RabG3bCA plants is not due to increased bacterial growth. Bacterial growth in the atg5-1 mutant was not significantly different from that in wild-type, RabG3bDN, and RabG3bRNAi plants. Similar bacterial growth results were obtained with Pst DC3000 (AvrRpt2; Supplemental Fig. S3).

In a previous study, the electrolyte leakage test was used to assess the development of pathogen-induced HR cell death in atg mutants (Hofius et al., 2009). Here, we performed the electrolyte leakage assay and compared the extent of HR cell death in wild-type, RabG3bCA, and atg5-1 leaves inoculated with Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2; Fig. 2). Both Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2) infection enhanced electrolyte leakage and, consequently, conductance in plants. Whereas this increase was considerably suppressed in atg5-1 leaves infected with Pst DC3000 (AvrRpm1), more leakage was observed with Pst DC3000 (AvrRpt2). This is in agreement with the previous study by Hofius et al. (2009), suggesting that autophagy is involved in AvrRpm1-triggered cell death but is minimally involved in AvrRpt2-induced cell death. In contrast, RabG3bCA plants exhibited higher electrolyte leakage than the wild type upon infection with either Pst DC3000 (AvrRpm1) or Pst DC3000 (AvrRpt2). These data support a positive role for RabG3b in pathogen-induced hypersensitive cell death.

Figure 2.

Ion leakage assays in wild-type (WT), RabG3bCA, and atg5-1 plants inoculated with Pst DC3000 (AvrRpm1; top) and Pst DC3000 (AvrRpt2; bottom). Leaves of 5-week-old plants were infiltrated with Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2) at 10⁸ cfu mL⁻¹. Results represent means ± sd (n = 10). Asterisks indicate significant differences from the respective wild-type (Student’s t test; *P < 0.05, **P < 0.01). These experiments were performed three times with similar results.

Basal Immunity Is Not Altered in RabG3bCA Plants

Accumulating evidence indicates that PTI and ETI use common signaling pathway components, although ETI elicits more prolonged and robust immune responses than PTI and is associated with HR PCD (Tsuda and Katagiri, 2010). PAMPs also induce cell death, but their effect is weak compared with that of ETI-triggered cell death. However, flagellin from P. syringae pv tabaci 6605 was shown to strongly induce cell death (Naito et al., 2008). This suggests that PTI and ETI may also share common signaling machinery leading to immunity-induced cell death. Therefore, we investigated whether the expanded cell death in RabG3bCA plants infected with avirulent bacterial pathogens was due to any defects in basal immunity. PTI responses such as callose deposition and gene expression were examined in plants challenged with the PAMP flagellin (flg22; Lenz et al., 2011). The induction of callose deposition and the expression of PTI molecular marker genes FRK1 and PR1 were indistinguishable in wild-type, atg5-1, and RabG3bCA plants (Supplemental Fig. S4, A and B). These results indicate that basal immunity is not altered and is irrelevant to the uncontrolled cell death observed in RabG3bCA and atg5-1 plants. Bacterial growth was then examined in plants inoculated with Pst DC3000 (Supplemental Fig. S4C). Bacterial growth decreased in the atg5-1 mutant but rather increased in RabG3bCA plants. This suggests that autophagy plays a negative role in SA-associated plant immunity to bacterial infection, and this does not occur through altered PTI responses, as reported previously (Yoshimoto et al., 2009; Lenz et al., 2011).

RabG3b Activates Autophagy during Hypersensitive Cell Death

Given that RabG3b positively regulates TE PCD by activating autophagy (Kwon et al., 2010a, 2010b), we investigated whether the expanded HR PCD in RabG3bCA plants was also associated with autophagic activation. Wild-type, RabG3bCA, RabG3bDN, RabG3bRNAi, and atg5-1 plants were treated with Pst DC3000 (AvrRpm1), and autophagy was monitored over time in mesophyll cells of infected tissues by staining with LysoTracker Green (LTG), which stains acidic organelles including autophagosome and autolysosome structures (Fig. 3; Via et al., 1998; Liu et al., 2005; Hofius et al., 2009). In wild-type plants, Pst DC3000 (AvrRpm1) treatment induced the formation of LTG-stained autophagic structures 24 h after treatment. On the other hand, LTG-stained punctate structures were detected earlier in Pst DC3000 (AvrRpm1)-infected RabG3bCA plants at 6 h post infection and accumulated to higher levels than in treated wild-type plants. The disappearance of LTG-stained structures from RabG3bCA leaves at 48 h post infection resulted from severe cell death and complete collapse of subcellular organelles and structures. LTG-stained structures were also detected at the uninfected sites in both wild-type and RabG3bCA plants, although later than those at the infected sites (Supplemental Fig. S5). RabG3bDN and RabG3bRNAi plants also displayed LTG-stained structures at 24 and 48 h post infection, more slowly than the wild type (Fig. 3). By contrast, LTG-stained structures were rarely detected in atg5-1 plants.

Figure 3.

Autophagic structures accumulate in wild-type plants (WT) and more abundantly in RabG3bCA plants following Pst DC3000 (AvrRpm1) infection. A, LTG staining of mesophyll cells at the infected sites of wild-type, RabG3bCA, RabG3bDN, RabG3bRNAi, and atg5-1 plants treated with 10⁶ cfu mL⁻¹ Pst DC3000 (AvrRpm1) for 0, 6, 12, 24, and 48 h. hpi, Hours post inoculation. Bar = 50 µm. B, Quantitative analysis of LTG-stained structures in A. Stained tissues were photographed, and cells with fluorescent spots were counted per 250-μm² area. Results represent means ± sd (n = 6). These experiments were performed three times with similar results.

As LTG is a nonspecific acidotropic dye, autophagosome structures were specifically stained by whole-mount immunofluorescence using the anti-ATG8a antibody and compared with LTG-stained structures (Fig. 4). LTG-stained and ATG8a-associated autophagosome structures were identified in Pst DC3000 (AvrRpm1)-treated cells with similar timing and patterns, confirming that Pst DC3000 (AvrRpm1)-infected RabG3bCA leaves develop abundant autophagic structures earlier than the wild type. We further examined the induction of autophagy by western-blot analysis using the anti-ATG8a antibody. Formation of the ATG8-phosphatidylethanolamine (PE) conjugate was monitored as a marker for autophagic activation (Fig. 5). In addition to free ATG8 proteins, the faster migrating ATG8-PE adduct was detected at 8 h after Pst DC3000 (AvrRpm1) inoculation in wild-type plants, whereas the ATG8-PE band appeared earlier at 4 h post infection and was more abundant in Pst DC3000 (AvrRpm1)-infected RabG3bCA plants.

Figure 4.

ATG8a-associated autophagosome structures accumulate in Pst DC3000 (AvrRpm1)-infected RabG3bCA plants. A, LTG staining of mesophyll cells from wild-type (WT) and RabG3bCA plants treated with 10 mm MgCl₂ (Mock) or 10⁶ cfu mL⁻¹ Pst DC3000 (AvrRpm1) for 12 and 24 h. B, Whole-mount immunofluorescence staining of mesophyll cells of the wild-type and RabG3bCA plants described in A with the anti-ATG8a antibody. hpi, Hours post inoculation. Bars = 50 µm.

Figure 5.

Western-blot analysis for detection of the ATG8-PE adduct in wild-type (WT) and RabG3bCA plants after Pst DC3000 (AvrRpm1) treatment. Total proteins were extracted from leaves treated with 1 × 10⁶ cfu mL⁻¹ Pst DC3000 (AvrRpm1) for the indicated times, separated by SDS gel electrophoresis in the presence of 6 m urea, and subjected to western-blot analysis with the anti-ATG8a antibody. The blots were stained with Ponceau S to assess protein loading. hpi, Hours post inoculation.

Prior studies showed that the induction of autophagy during HR PCD depends on the type of immune receptor, Toll/Interleukin-1 receptor (TIR)-nucleotide binding (NB)-Leu-rich repeat (LRR) or coiled-coil (CC)-NB-LRR, activated by different bacterial avirulent factors (Hofius et al., 2009). In contrast to Pst DC3000 (AvrRpm1), Pst DC3000 (AvrRpt2) infection induced only weak accumulation of LTG-stained structures in wild-type plants, suggesting that AvrRpt2-triggered HR cell death may be independent of autophagy (Hofius et al., 2009). Therefore, we checked for autophagic structures in wild-type and RabG3bCA plants challenged with Pst DC3000 (AvrRpt2; Supplemental Fig. S6A). Wild-type plants gradually accumulated LTG-stained structures during the course of Pst DC3000 (AvrRpt2) infection, although at a much lower level than in Pst DC3000 (AvrRpm1)-treated plants, as reported previously (Hofius et al., 2009). However, RabG3bCA plants developed a higher number of punctate structures in response to Pst DC3000 (AvrRpt2) than wild-type plants, suggesting that autophagy was at least partly involved in HR PCD induced by Pst DC3000 (AvrRpt2). Additional examination using transmission electron microscopy (TEM) revealed autophagosome structures in Pst DC3000 (AvrRpt2)-infected leaves (Supplemental Fig. S6B). Collectively, these results demonstrate that autophagy is activated in wild-type plants and that this activation is enhanced in RabG3bCA plants during AvrRpm1- and AvrRpt2-triggered hypersensitive PCD.

RabG3bCA Cells Accumulate Abundant Autophagic Structures during Pathogen-Induced Hypersensitive Cell Death

TEM analysis was performed to confirm the results from LTG staining, immunofluorescence, and immunoblotting and to examine the ultrastructure of cells during Pst DC3000 (AvrRpm1)-induced HR cell death (Fig. 6; Supplemental Fig. S7). Wild-type, RabG3bCA, and atg5-1 cells showed no differences in subcellular morphology before pathogen treatment (Supplemental Fig. S7). However, Pst DC3000 (AvrRpm1) infection caused drastic changes in subcellular structures, including the degradation of cell contents (Fig. 6; Supplemental Fig. S7). During the initial stages (within 24 h) of infection, wild-type and RabG3bCA plants formed numerous autophagosome and autolysosome structures containing degrading organelles and cytoplasmic constituents and simultaneously accumulated damaged structures, including plastids, suggesting that autophagy contributes to the cell death process (Fig. 6). Over time, wild-type and RabG3bCA cells underwent progressive shrinking, and cellular components appeared as electron-dense materials, indicative of dying cells (Fig. 6; Supplemental Fig. S7). Similar patterns of autophagic structures and ultrastructural changes were observed in wild-type and RabG3bCA cells in response to Pst DC3000 (AvrRpm1) infection, although at an earlier time and with increased expansion in RabG3bCA cells than in the wild type. By contrast, Pst DC3000 (AvrRpm1)-inoculated atg5-1 leaves showed no obvious autophagic features but underwent gradual degradation of cellular components (Fig. 6; Supplemental Fig. S7). Vacuolar membrane collapse was often observed, suggesting that the release of vacuolar hydrolytic enzymes into the cytosol may be the cause of cell death in the atg5-1 mutant (Fig. 6).

Figure 6.

TEM images of autophagic structures in wild-type (WT), RabG3bCA, and atg5-1 plants inoculated with Pst DC3000 (AvrRpm1). Plant leaves were treated with 10⁶ cfu mL⁻¹ Pst DC3000 (AvrRpm1) for the indicated times and subjected to TEM analysis. Enlarged images of the boxed areas are shown below. Images are representative of those obtained from three independent experiments. Arrows indicate rupture of the tonoplast membrane. A, Autophagosome; AL, autolysosome; Ch, chloroplast; hpi, hours post inoculation; M, mitochondria. Bars = 5 µm (top) and 200 nm (middle and bottom) for 12 hpi (wild type and atg5-1) and 6 hpi (RabG3bCA) and 5 µm (top) and 1 µm (bottom) for 24 hpi (wild type and atg5-1) and 12 hpi (RabG3bCA).

The localization of RabG3b proteins to autophagic structures was analyzed by immunogold electron microscopy using antibodies against RabG3b and the autophagosome marker protein ATG8a in Pst DC3000 (AvrRpm1)-infected wild-type and RabG3bCA cells (Fig. 7). In mock-treated cells, RabG3b and ATG8a proteins were detected in the cytosol. Upon Pst DC3000 (AvrRpm1) infection, ATG8a protein levels were greatly elevated and mostly associated with autophagic structures, and RabG3b proteins were colocalized with ATG8a proteins in autophagic structures in both wild-type and RabG3bCA cells. This further demonstrates that RabG3b function is closely associated with autophagy during pathogen-induced HR cell death.

Figure 7.

RabG3b colocalizes with ATG8a in autophagic structures. Plant leaves were treated with 10 mm MgCl₂ (Mock) or 10⁶ cfu mL⁻¹ Pst DC3000 (AvrRpm1) for the indicated times and subjected to immunogold labeling. Black and white arrowheads mark RabG3b (5-nm gold) and ATG8a (1-nm gold), respectively. hpi, Hours post inoculation WT, wild type. Bars = 100 nm.

RabG3b-Associated Autophagy Is Partially ATG5 Dependent during Hypersensitive Cell Death

It was further assessed whether the HR cell death and autophagic activation in RabG3bCA plants was related to ATG5-mediated autophagy. We crossed RabG3bCA and atg5-1 mutant plants and obtained homozygous crossing lines. We examined HR cell death phenotypes in RabG3bCA, atg5-1, and RabG3bCA atg5-1 plants after Pst DC3000 (AvrRpm1) treatment (Fig. 8). Hypersensitive necrotic cell death was observed, but its progression was significantly slower and less expanded in Pst DC3000 (AvrRpm1)-infiltrated RabG3bCA atg5-1 leaves compared with RabG3bCA plants, and it was often preceded by chlorotic cell death spreading beyond the infected site, as in atg5-1 plants. Bacterial growth was then examined in plants inoculated with Pst DC3000 (AvrRpm1) or Pst DC3000 (AvrRpt2; Supplemental Fig. S8). Bacterial growth in RabG3bCA atg5-1 plants was not significantly different from that in wild-type and atg5-1 plants.

Figure 8.

Pst DC3000 (AvrRpm1)-induced HR cell death in RaG3bCA atg5-1, RabG3bCA, and atg5-1 plants. A, Phenotypes of wild-type (WT), RabG3bCA, atg5-1, and RabG3bCA atg5-1 leaves inoculated with 10 mm MgCl₂ (Mock) or 10⁸ cfu mL⁻¹ Pst DC3000 (AvrRpm1) for 2, 4, and 7 d. B, Phenotypes of wild-type, RabG3bCA, atg5-1, and RabG3bCA atg5-1 leaves inoculated with 10 mm MgCl₂ (Mock) or 10⁸ cfu mL⁻¹ Pst DC3000 (AvrRpm1) for 14 d. dpi, Days post inoculation.

We then investigated the formation of autophagic structures in the infected leaves by LTG staining (Fig. 9). Whereas Pst DC3000 (AvrRpm1) induced the formation of LTG-stained autophagic structures in RabG3bCA plants at 6 h post infection and increased up to 24 h post infection, LTG-stained structures were detected later at 24 h post infection in RabG3bCA atg5-1 leaves. Whole-mount immunofluorescence staining of Pst DC3000 (AvrRpm1)-infected RabG3bCA atg5-1 leaves with the anti-ATG8a antibody verified that LTG-stained structures were ATG8a-associated autophagic structures (Supplemental Fig. S9). By contrast, no LTG- and ATG8a-stained structures were detected in atg5-1 plants. These results demonstrate that ATG5 mutation partially suppressed autophagic activity in RabG3bCA plants and that RabG3b-associated autophagy was at least partially ATG5 dependent. In addition, autophagic induction by RabG3bCA overexpression in the atg5-1 mutant suggests that RabG3b may have the autophagy-independent cell death-promoting activity. Alternatively, there may be an ATG5-independent noncanonical autophagy pathway.

Figure 9.

LTG staining of autophagic structures in RabG3bCA atg5-1, RabG3bCA, and atg5-1 plants infected with Pst DC3000 (AvrRpm1). A, LTG-stained mesophyll cells at the infected sites of wild-type (WT), RabG3bCA, atg5-1, and RabG3bCA atg5-1 plants treated with 10⁶ cfu mL⁻¹ Pst DC3000 (AvrRpm1) for 6, 12, 24, and 48 h. hpi, Hours post inoculation. Bar = 50 µm. B, Quantitative analysis of the LTG-stained structures in A. Stained tissues were photographed, and cells with fluorescent spots were counted per 250-μm² area. Results represent means ± sd (n = 6). These experiments were performed three times with similar results.

DISCUSSION

In this study, overexpression of a constitutively active form of RabG3b in plants elicited accelerated, expanded hypersensitive cell death, which was accompanied by the accumulation of abundant autophagic structures in response to the FB1 fungal toxin and avirulent bacterial pathogens. Our results suggest that Arabidopsis RabG3b regulates HR PCD during plant-pathogen interactions through its positive role in autophagy and that autophagy, as a prodeath process, contributes to hypersensitive cell death.

RabG3b Functions in Autophagy

Autophagy plays a role in the regulation of responses to various stresses, including abiotic stresses such as oxidative, salt, and drought stresses, as well as biotic stresses (Liu et al., 2005; Xiong et al., 2005, 2007; Bassham, 2007; Hofius et al., 2009; Liu and Bassham, 2012). Inoculation of RabG3bCA plants with Pst DC3000 (AvrRpm1) induced the formation of abundant autophagosome and autolysosome structures. In addition, RabG3b proteins colocalized with ATG8a proteins in autophagic structures in both Pst DC3000 (AvrRpm1)-inoculated wild-type and RabG3bCA plants. We previously demonstrated that RabG3b functions as a positive regulator of cell death during TE differentiation by activating autophagy (Kwon et al., 2010a, 2010b). Autophagic activation was remarkably increased in RabG3bCA cells during TE differentiation, leading to enhanced xylem development in both Arabidopsis and Populus spp. (Kwon et al., 2010a, 2011). The importance of autophagy in TE differentiation was further supported by the observation that TE formation was suppressed in atg 5-1 mutant cells. RabG3bCA cells also accumulated numerous autophagic structures in response to Suc starvation, a general autophagy-inducing condition. Together with these findings, our results suggest that RabG3b functions as a component of plant autophagy, regulating cell death pathways associated with both plant development and stress responses.

On the other hand, RabG3bDN and RabG3bRNAi plants were not significantly different from wild-type plants with respect to both cell death and bacterial growth phenotypes. LTG-stained autophagic structures were also formed in RabG3bDN and RabG3bRNAi plants in response to Pst DC3000 (AvrRpm1) infection, although it occurred more slowly than in Pst DC3000 (AvrRpm1)-treated wild-type plants. These results suggest that the expression of dominant negative or knockdown constructs of RabG3b may not be sufficient for the complete abolishment of RabG3b activity. Alternatively, several autophagy pathways may redundantly operate during HR cell death. The latter possibility is supported by RabG3bCA atg5-1 phenotypes. Whereas RabG3bCA-mediated autophagy was partially suppressed in the RabG3bCA atg5-1 crossing line, RabG3bCA overexpression restored autophagic activity in the atg5-1 mutant during pathogen-induced HR PCD, as determined by immunofluorescence and LTG staining. This suggests that there may be at least two autophagy pathways, ATG5 dependent and ATG5 independent, and that RabG3b may function as a common component of two pathways. The absence of a signal for autophagic activity in the pathogen-treated atg5-1 mutant further suggests that the ATG5-dependent pathway may be dominant during immunity-triggered HR PCD. Indeed, a recent study in mouse demonstrated that mammalian macroautophagy can occur through two different pathways, an ATG5/ATG7-dependent conventional pathway and an ATG5/ATG7-independent alternative pathway (Nishida et al., 2009). It has been proposed that a noncanonical alternative autophagy pathway may also exist in plants (Li and Vierstra, 2012). However, we cannot rule out the possibility that RabG3b may be involved in an autophagy-independent cell death process.

Genetic and molecular analyses of autophagy have identified approximately 35 ATG genes in yeast (Saccharomyces cerevisiae) and many orthologs in other eukaryotes, including Arabidopsis, maize (Zea mays), and rice (Oryza sativa; Chung et al., 2009; Xia et al., 2011). ATG proteins are key players in the autophagic pathway that comprises several consecutive stages, such as target-of-rapamycin-mediated induction, vesicle nucleation and elongation of an isolation membrane or phagophore, docking and fusion of autophagosomes with lysosomes, and vesicle breakdown and degradation. Rab7 in mammals and Ypt7 in yeast have been implicated in autophagosome maturation. In mammalian cells, Rab7 colocalized with an autophagic marker, microtubule-associated protein light chain3, and was required for the normal progression of autophagy (Gutierrez et al., 2004; Jäger et al., 2004). Fusion of autophagosomes with the vacuole was inhibited in Ypt7 null mutants in Saccharomyces cerevisiae (Kirisako et al., 1999). Docking and fusion of autophagosomes was found to require the receptor complex consisting of v-SNARE VTI1, VAM3 syntaxin, and YPT7 in yeast (Thompson and Vierstra, 2005). An Arabidopsis mutant with a mutation in VTI12, a VTI1-type v-SNARE, exhibited a phenotype similar to that of atg7 and atg9 mutants characterized by accelerated chlorosis and premature leaf senescence under nutrient starvation, suggesting that VTI12 may be involved in autophagosome docking and fusion (Surpin et al., 2003). Thus, it would be valuable to examine whether RabG3b is a component of and functions in conjunction with other proteins in the SNARE complex in plants. Like Rab7 and Ypt7, RabG3b may function in autophagosome/autolysosome maturation, the late stage of autophagy. In recent studies, other Rab proteins, including Rab5, Rab32, and Rab33b, were found to be involved in autophagosome formation in mammalian autophagy (Itoh et al., 2008; Ravikumar et al., 2008; Hirota and Tanaka, 2009; Yamaguchi et al., 2009), additionally implicating RabG3b in the early stage of autophagy.

Autophagy Controls HR PCD

Despite many reports on the functions of autophagy in pathogen-induced cell death, the prosurvival or prodeath role of autophagy in plants has been controversial. Virus-induced gene silencing in tobacco led to the finding that autophagy has a prosurvival function in the pathogen-induced cell death response (Liu et al., 2005). HR PCD induced by viral infections was unrestricted and spread into uninfected cells in BECLIN1/ATG6/VPS30-, PI3K/VPS34-, ATG3-, and ATG7-silenced tobacco plants. Similar results were observed in Arabidopsis ATG6 RNA interference and atg5-1 mutant plants (Patel and Dinesh-Kumar, 2008; Yoshimoto et al., 2009). On the other hand, Hofius et al. (2009) reported that pathogen-induced cell death was suppressed in atg mutants, supporting autophagy as a prodeath process. Upon infection with the avirulent bacterial pathogens Pst DC3000 (AvrRps4) and Pst DC3000 (AvrRpm1) that depend on TIR-type immune receptors, atg mutants displayed reduced HR cell death, as determined by ion leakage assays and trypan blue staining of dead cells (Hofius et al., 2009). However, HR cell death in atg mutants was little affected by Pst DC3000 (AvrRpt2) requiring the CC-type immune receptor for activation. In contrast to Pst DC3000 (AvrRpm1), Pst DC3000 (AvrRpt2) infection induced only weak accumulation of LTG-stained structures in wild-type plants, suggesting that AvrRpt2-triggered HR PCD may be independent of autophagy and may require other cell death processes (Hofius et al., 2009). In our study, RabG3bCA overexpression enhanced autophagic activity and led to accelerated, expanded cell death, regardless of types of challenged avirulent pathogens, including Pst DC3000 (AvrRpt2), further supporting a prodeath role for autophagy during HR cell death. Whether autophagy contributes to hypersensitive cell death may be determined by the level of autophagic activation, which differs among distinct avirulent bacterial pathogens that activate either TIR- or CC-type immune receptors.

The prosurvival and prodeath functions of autophagy and the mechanistic interaction of autophagy with apoptosis have been important issues in our understanding of cell death pathways in animal systems (Kourtis and Tavernarakis, 2009; Scarlatti et al., 2009). Studies indicate that apoptosis and autophagy are regulated by mutual inhibition, and depending on the circumstances and the threshold, cells activate autophagy for cell survival or apoptosis for cell death (Maiuri et al., 2007). Apoptosis and autophagy are linked and coordinately regulated, sharing components in pathways (Maiuri et al., 2007; Levine et al., 2008). There are many in vivo data in animals supporting that apoptosis (type I PCD) develops due to an inhibition of autophagy. Inactivation of ATG genes induced apoptotic death in neurons and during Caenorhabditis elegans development (Takacs-Vellai et al., 2005; Hara et al., 2006; Komatsu et al., 2006). It has also been shown that cells can undergo autophagic cell death (type II PCD) depending on the stage of autophagic inhibition or the level of autophagic activation. When a later stage of autophagy (e.g. fusion of autophagosomes with lysosomes) is blocked or autophagy is hyperactivated, cells accumulate autophagosomes and undergo type II cell death (González-Polo et al., 2005). Autophagic cell death has been demonstrated in mammalian cells and in Drosophila melanogaster, Dictyostelium discoideum, and C. elegans (Shimizu et al., 2004; Yu et al., 2004; Berry and Baehrecke, 2007; Kang et al., 2007; Lam et al., 2008; Samara et al., 2008).

It has long been known that HR PCD contributes to disease resistance by physically isolating pathogens. Whereas autophagy positively affects HR cell death, it is not clear whether autophagy is involved in restricting pathogen growth at the infection site. We observed that bacterial growth in the atg5-1 mutant was not significantly different from that in wild-type plants upon infection with Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2), suggesting that autophagy may not be directly involved in limiting pathogen growth. This also implies that cell death itself may not be essential for the containment of pathogens. Many reports indicate that disease resistance is not associated with HR PCD in dnd1/cngc2, hrl1, ndr1, and dnd2/hlm1/cngc4 mutant plants (Clough et al., 2000; Devadas and Raina, 2002; Balagué et al., 2003; Jurkowski et al., 2004). In NDR1 mutant plants, resistance to avirulent Pst DC3000 carrying avr genes was compromised, but hypersensitive cell death was strain specific, such that Pst DC3000 (avrRpt2) did not develop HR but Pst DC3000 (avrB) elicited an exaggerated cell death response (Century et al., 1995; Shapiro and Zhang, 2001). Studies of the autophagic pathway in these mutants would facilitate a better understanding of the relationship between PCD, autophagy, and pathogen resistance.

In wild-type plants, Pst DC3000 (AvrRpm1) treatment readily induced autophagy at the infected site, followed by autophagic activation at uninfected sites. In fact, autophagy may play a positive role at the infected site and a negative role in uninfected tissue to regulate HR cell death during plant immunity. On the basis of both macroscopic and microscopic cell death phenotypes of pathogen-infected wild-type, RabG3bCA, and atg5-1 mutant plants, we concluded that plants undergo two distinct cell death processes: hypersensitive cell death and chlorotic cell death (Fig. 10). The former was induced at the localized infection site of all tested plants within hours after infection and was suppressed in the atg5-1 mutant but markedly expanded in RabG3bCA plants. This finding suggests that autophagy plays a prodeath role and that autophagic cell death contributes to cell death programs operating during pathogen-induced HR PCD. As suggested for the roles of RabG3b and autophagy during TE PCD in our previous work (Kwon et al., 2010a), autophagy-mediated degradation of cell contents and organelles may promote cytoplasm clearance after vacuole collapse during HR PCD. The latter (chlorotic cell death) occurred as a result of defective autophagy and spread beyond the infection site into uninfected sites in the atg5-1 mutant over a period of days or weeks following infection, supporting a prosurvival role of autophagy. Pathogen-induced cell death may lead to the production of toxic materials, including reactive oxygen species, causing damage to organelles and cellular components. A prosurvival function of autophagy would be to maintain cellular homeostasis in uninfected, healthy tissues by eliminating this cellular “garbage” (Hofius et al., 2011). The increased accumulation of damaged materials in older leaves explains why chlorotic cell death is more prominent in the older atg5-1 mutant than in younger plants, as observed in this study and previously (Yoshimoto et al., 2009). We propose that plants use prodeath and prosurvival activities of autophagy to expedite HR cell death and simultaneously limit it to the infected cells, respectively. Our proposal is substantially consistent with the view of Hofius et al. (2011) that the contradictory roles of autophagy in HR cell death proposed by different groups are the result of differences in the experimental systems (Liu et al., 2005; Patel and Dinesh-Kumar, 2008; Hofius et al., 2009; Yoshimoto et al., 2009). Additional studies on RabG3b function in membrane trafficking and autophagic processes and the identification of its interacting partners will allow a better understanding of the molecular mechanisms of autophagy during immunity-associated HR PCD.

Figure 10.

A model for the roles of RabG3b and autophagy in the regulation of pathogen-induced HR cell death. We propose a positive role for RabG3b in autophagy and hypersensitive cell death and both positive and negative roles for autophagy during immunity-associated HR PCD. In our model, avirulent pathogen infection readily induces HR cell death at the infected site in wild-type (WT) plants, and this is largely expanded in RabG3bCA plants but suppressed in the atg5-1 mutant. This suggests that autophagy is a prodeath process and that autophagic cell death contributes to pathogen-induced HR PCD. On the other hand, the atg5-1 mutant gradually develops chlorotic cell death at uninfected sites during the course of days or weeks following infection, suggesting a prosurvival role for autophagy in cellular homeostasis maintenance.

MATERIALS AND METHODS

Plant Materials, Growth Conditions, and Treatments

Arabidopsis (Arabidopsis thaliana) plants (ecotype Columbia) were grown in a growth room at 24°C under long-day conditions (16-h-light/8-h-dark cycle) or in a growth chamber under short-day conditions (8-h-light/16-h-dark cycle). Pseudomonas syringae pv tomato DC3000 (Pst DC3000), Pst DC3000 (AvrRpm1), and Pst DC3000 (AvrRpt2) were grown on nutrient yeast extract glycerol agar medium containing 50 µg mL⁻¹ rifampicin and 50 µg mL⁻¹ kanamycin. To observe disease phenotypes after treatment with bacterial pathogens, leaves of 7-week-old plants grown under short-day conditions were infiltrated with 10 µL of MgCl₂ (10 mm) or bacterial suspensions at 10⁶ to 10⁸ colony-forming units (cfu) mL⁻¹. For bacterial growth assays, leaves of 7-week-old plants grown under short-day conditions were infiltrated with 10 µL of MgCl₂ (10 mm) or bacterial suspensions at 10⁵ cfu mL⁻¹. Leaf discs (0.5 cm²) were ground in 10 mm MgCl₂, serially diluted, and plated out to determine titers. For FB1 (Sigma-Aldrich) treatment, leaves of 7-week-old plants grown under short-day conditions were infiltrated with 10 µm FB1 using a 1-mL needleless syringe and kept in a growth chamber for 2 d. For flg22 treatment (Watanabe and Lam, 2006), leaves of 7-week-old plants grown under short-day conditions were infiltrated with 1 µm flg22 using a 1-mL needleless syringe and incubated in a growth chamber for the indicated times.

To generate crossing lines, RabG3bCA plants were crossed with the atg5-1 mutant. F1 plants were selfed, and F2 seeds were harvested. The segregation of F2 seeds was confirmed by selection on kanamycin, and F2 plants were genotyped to select crossing lines. Homozygous RabG3bCA atg5-1 crossing lines were confirmed by PCR, as described previously (Kwon et al., 2009; Yoshimoto et al., 2009).

Reverse Transcription-PCR Analysis

Total RNAs were extracted from Arabidopsis leaves using the Plant RNA Purification Reagent (Invitrogen). First-strand complementary DNAs were synthesized from 1 μg of total RNAs using the Power cDNA Synthesis Kit (iNtRON). PCR reactions were performed with the following gene-specific primers: for PR1, 5′-ATGAATTTTACTGGCTATTC-3′ and 5′-TTAGTATGGCTTCTCGTTC-3′; for FRK1, 5′-GCTGGATCCATCGGTTACCTTGAC-3′ and 5′-TAGATTCACTGTAAGCATTTTCGT-3′; and for Actin, 5′-GGCGATGAAGCTCAATCCAAACG-3′ and 5′-GGTCACGACCAGCAAGATCAAGACG-3′.

Trypan Blue Staining

Trypan blue staining was performed as described previously (Bowling et al., 1997). Samples were submerged in lactic acid-phenol-trypan blue solution (2.5 mg mL⁻¹ trypan blue, 25% lactic acid, 23% water-saturated phenol, and 25% glycerol) and boiled for 1 min. Stained samples were then placed in a 60% chloral hydrate solution and finally equilibrated with 50% glycerol.

Ion Leakage Assay

Ion leakage experiments were performed as described previously (Mackey et al., 2003). Leaves of 5-week-old plants grown under long-day conditions were infiltrated with bacterial suspensions of Pst DC3000 (AvrRpm1) at 2 × 10⁸ cfu mL⁻¹. Ten leaf discs (0.5 cm²) were immediately removed and floated in 30 mL of distilled water for washing. After 30 min, washed leaf discs were moved into 10 mL of fresh distilled water. Ion conductance was then measured over time using a conductivity meter (Thermo Scientific).

Callose Deposition Assay

Callose deposition was visualized by aniline blue staining as described previously (Jacobs et al., 2003; Clay et al., 2009). Arabidopsis leaves were harvested 1 d after treatment with 1 µm flg22 and fixed in acetic acid:ethanol (1:3) for 8 h with two solution changes. Leaves were then incubated in 50% ethanol for 1 h, 30% ethanol for 1 h, and finally distilled water for 2 h with two solution changes. Leaves were then stained with 5 mg mL⁻¹ aniline blue in 150 mm sodium phosphate (pH 7.0) in the dark for 1 h and mounted on slides using 50% glycerol. Callose was observed under a confocal microscope (Zeiss LSM 510 META).

LTG Staining

LTG staining was performed as described previously (Liu et al., 2005; Patel and Dinesh-Kumar, 2008). Leaf pieces were infiltrated with 1 μm LTG DND-26 (Molecular Probes) and incubated in the dark for 1 h. Images were acquired using a confocal microscope (Zeiss LSM 510 META). The wavelengths used for LTG and chloroplasts were 488 to 505 nm and 565 to 625 nm, respectively.

Antibody Preparation

The full-length coding region of ATG8a was cloned into the pET15b vector (Novagen) and used for the transformation of Escherichia coli (BL21) cells. Production of recombinant ATG8a proteins was induced by the addition of 1 mm isopropylthio-β-galactoside for 2 h, and the proteins were purified using a nickel-nitrilotriacetic acid agarose column according to the manufacturer’s instructions (Qiagen). Affinity-purified recombinant proteins were used to raise the anti-ATG8a antibody in rats. The anti-RabG3b antibody was raised in rabbits as described previously (Kwon et al., 2009).

Immunoblot Analysis

Western-blot analysis was performed as described previously (Yoshimoto et al., 2004; Chung et al., 2009). Seven-week-old plants grown under short-day conditions were used, and protein samples were prepared from leaves treated with Pst DC3000 (AvrRpm1) at 10⁶ cfu mL⁻¹. Total proteins were separated by 12% SDS-PAGE in the presence of 6 m urea and electrophoretically transferred onto nitrocellulose membranes. The blots were probed with the ATG8a antibody overnight at 4°C. Following incubation with secondary antibody conjugated to horseradish peroxidase, antibody-bound proteins were detected using the enhanced chemiluminescence system (GE Healthcare).

Whole-Mount Immunofluorescence Analysis

Whole-mount immunofluorescence experiments were performed as described previously (Leivar et al., 2005). Leaf discs (0.5 cm²) were fixed in 4% formaldehyde dissolved in PMEG buffer (50 mm PIPES, pH 6.9, 5 mm MgSO₄, and 5 mm EGTA) and 10% dimethyl sulfoxide for 3 h at room temperature under vacuum. Samples were washed three times with PMEG buffer and incubated in PMEG buffer containing 0.5% pectolyase (Sigma), 0.5% Triton X-100, and 1% bovine serum albumin (BSA) for 30 min at 37°C. Samples were then washed six times with PMEG buffer and incubated with the anti-ATG8a antibody in 0.1 m phosphate-buffered saline (PBS) containing 3% BSA overnight at 4°C. After washing with PBS, samples were incubated with fluorescein isothiocyanate-conjugated goat anti-rat IgG (AlexaFluor 488; Invitrogen) for 3 h at room temperature and washed three additional times with PBS. Samples were observed with a confocal microscope (Zeiss LSM 510 META) at 488/505-nm excitation/emission.

Electron Microscopy Analysis

Leaves were fixed in a solution containing 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4) for 4 h at 4°C, rinsed in 0.1 m phosphate buffer (pH 7.4), and further fixed in 1% OsO₄ for 4 h at 4°C. After rinsing, samples were dehydrated and embedded in LR White resin (London Resin). For TEM analysis, thin sections (40–50 nm thickness) collected on nickel grids (1-GN; 150 mesh) were stained with uranyl acetate and lead citrate and examined by TEM (Tecnai 12; Philips). For immunogold electron microscopy analysis, sections collected on copper grids were blocked with BSA-TBST buffer (500 mm NaCl, 1% BSA, 0.3% Tween 20, and 10 mm Tris-HCl, pH 7.4) for 1 h and incubated with an anti-RabG3b antibody (rabbit) and/or an anti-ATG8a antibody (rat) for 4 h at room temperature. Samples were rinsed in BSA-TBST buffer, and primary antibody binding was detected using anti-rabbit IgG (5-nm gold; Electron Microscopy Sciences) and anti-rat IgG (1-nm gold; Sigma). After washing with TBST and distilled water, samples were stained with uranyl acetate and observed by TEM.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number At1g22740 (RabG3b).

Supplemental Data

The following materials are available in the online version of this article.

• Supplemental Figure S1. Cell-death phenotypes of leaves inoculated with Pst DC3000 (AvrRpm1) in 5-week-old young plants.

• Supplemental Figure S2. Cell-death phenotypes of leaves inoculated with Pst DC3000 (AvrRpt2) and FB1.

• Supplemental Figure S3. Bacterial growth in wild-type, RabG3bCA, RabG3bDN, RabG3bRNAi, and atg5-1 plants.

• Supplemental Figure S4. Basal immunity is not defective in RabG3bCA and atg5-1 plants.

• Supplemental Figure S5. Autophagy is induced at both the infected and uninfected sites of plants following Pst DC3000 (AvrRpm1) infection.

• Supplemental Figure S6. Autophagic structures are formed in wild-type plants and more abundantly in RabG3bCA plants following Pst DC3000 (AvrRpt2) infection.

• Supplemental Figure S7. TEM images of autophagic structures in wild-type, RabG3bCA, and atg5-1 plants inoculated with Pst DC3000 (AvrRpm1).

• Supplemental Figure S8. Bacterial growth in wild-type, RabG3bCA, atg5-1, and RabG3bCA atg5-1 plants.

• Supplemental Figure S9. Whole-mount immunofluorescence staining of Pst DC3000 (AvrRpm1)-infected wild-type, RabG3bCA, RabG3bCA atg5-1, and atg5-1 plants with the anti-ATG8a antibody.

Glossary

PAMP

pathogen-associated molecular pattern

PTI

PAMP-triggered immunity

ETI

effector-triggered immunity

PCD

programmed cell death

HR

hypersensitive response

TE

tracheary element

Pst DC3000

Pseudomonas syringae pv tomato DC3000

SA

salicylic acid

FB1

fumonisin B1

LTG

LysoTracker Green

PE

phosphatidylethanolamine

TEM

transmission electron microscopy

cfu

colony-forming units

BSA

bovine serum albumin

PBS

phosphate-buffered saline

TIR

Toll/Interleukin-1 receptor

NB

nucleotide binding

LRR

Leu-rich repeat

CC

coiled-coil