Membrane-Associated Ubiquitin Ligase SAUL1 Suppresses Temperature- and Humidity-Dependent Autoimmunity in Arabidopsis

In many plant cell types, tissues, and organs cell death is a fundamental process in differentiation, development, and stress responses. Initiation and execution of cell death are genetically regulated and tightly controlled at the molecular level. This is not only crucial for the formation and physiological function of tissues and organs but is also important in abiotic stress responses and in plant immunity (Coll et al. 2011; Gepstein and Glick 2013; Van Hautegem et al. 2015). Survival of plants following pathogen attack depends on elaborate immune responses. Pathogen-associated molecular patterns (PAMPs) are recognized by immune receptors at the plasma membrane that initiate downstream defense responses. Intracellular nucleotide binding and leucine-rich repeat (NLR) receptor proteins activate a plethora of events upon recognition of pathogen effectors that have been delivered into the cytosol of plant host cells (Bent and Mackey 2007). Ultimatively, this results in a hypersensitive response (HR) that leads to cell death to confine pathogen spread to a few cells only (Jones and Dangl 2006).

Ubiquitination regulates various key events during pathogen attack and plant immunity. The ubiquitin (Ub)/proteasome pathway leads to the regulation or degradation of target proteins by achieving mono- or polyubiquitination, respectively. The activities of three types of enzymes, namely, E1 Ub-activating enzymes, E2 Ub-conjugating enzymes, and E3 Ub ligases, are fundamental for successful attachment of Ub moieties to their protein substrates. In some cases, E4 Ub ligase activity is required for efficient polyubiquitination. The pathway offers high specificity through recognition of target proteins by E3 Ub ligases, which represent one of the largest protein families in plants.

Components of the plant Ub/proteasome pathway have been shown to function as regulators of defense (Callis 2014; Dreher and Callis 2007; Vierstra 2009). The Ub-activating enzyme UBA1 that has been identified as a modifier of snc1 (MOS5) from a suppressor screen in the snc1 npr1 background is a positive regulator of plant resistance to pathogens (Goritschnig et al. 2007). Direct evidence for the contribution of E2 enzymes in plant defense is rare in Arabidopsis. However, the important role of various RING and U-box E3 Ub ligases in Arabidopsis immunity implies a function also of E2 enzymes. The RPM1-interacting RING E3 Ub ligases RIN2 and RIN3 reside in the plasma membrane and contribute to NLR protein-dependent HR following infection with Pseudomonas syringae. However, pathogen growth was not altered in rin2 rin3 mutants (Kawasaki et al. 2005). Plasma membrane–localized RING1/ATL55 is a positive regulator of cell death induced by the fungal toxin fumonisin B1 (Lin et al. 2008). The RING zinc finger protein ATL9 localizes to the endoplasmic reticulum and has Ub ligase activity. It has been suggested that ATL9 mediates chitin-dependent responses (Berrocal-Lobo et al. 2010). In contrast to these RING Ub ligases, the PUB Ub ligases involved in plant defense do not localize to membranes but, rather, to the cytoplasm or the nucleus (Cho et al. 2008; Drechsel et al. 2011; Samuel et al. 2008). A conserved role for PUB-ARM proteins in members of the Solanaceae and Brassicaceae has been proposed by identifying Arabidopsis PUB17 and its tobacco ortholog ACRE276 to be positive regulators of cell death and pathogen defense (Yang et al. 2006). The PUB-ARM Ub ligase triplet PUB22, PUB23, and PUB24 negatively regulates cell death and defense responses (Trujillo et al. 2008). In this triplet, PUB22 appears to have the most prominent role and affects PAMP-triggered immunity by marking components of the exocyst complex for degradation (Stegmann et al. 2012). The nuclear-localized Ub ligases MAC3A and MAC3B are part of the MOS4-associated complex (MAC) and are required for NLR protein-dependent pathogen resistance (Monaghan et al. 2009). Flagellin induces the association of flagellin receptor FLS2 (FLAGELLIN-SENISNG 2) with PUB12 and PUB13 (Li et al. 2012; Lu et al. 2011), which are PUB-ARM proteins promoting downregulation of FLS2 in the cytosol and, thereby, have a function in flagellin-induced immune responses.

The Ub/proteasome pathway is important in different aspects of effector-triggered immunity. The bacterial pathogen P. syringae secretes the effector AvrPtoB to the host cell that requires intrinsic Ub ligase activity to prevent host cell death. This strategy decreases plant immunity and is beneficial for pathogen fitness (Abramovitch et al. 2006; Janjusevic et al. 2006). The Xanthomonas campestris Ub ligase XopL specifically interacts with plant E2 enzymes (Singer et al. 2013). Agrobacterium tumefaciens, Ralstonia solanacearum, and poleroviruses use F-box protein effectors, which interact with plant Ub ligase complexes to reprogram the host cell Ub system (Angot et al. 2006; Pazhouhandeh et al. 2006; Schrammeijer et al. 2001; Tzfira et al. 2004). The rice pathogen Xanthomonas oryzae uses its effector XopPX_oo to inhibit ligase activity of the plant U-box armadillo repeat (PUB-ARM) protein OsPUB44, which positively regulates immunity (Ishikawa et al. 2014). General inhibition of proteasome activity is achieved by the effectors XopJ and HopZ4 of Xanthomonas campestris and P. syringae, respectively. Both effectors interact with RPT6, a subunit of the 19S regulatory particle of the proteasome complex (Üstün et al. 2013, 2014).

It has been demonstrated recently, that plant NLR proteins are under tight control by the Ub/proteasome pathway to avoid accumulation of NLR proteins and, thus, autoimmune responses in the abscence of a pathogen challenge. The F-box protein CPR1 (constitutive expressor of PR genes 1) and the E4 Ub ligase MUSE3 (mutant, snc1-enhancing 3) facilitate polyubiquitination of NLR proteins (Cheng et al. 2011; Gou et al. 2012; Huang et al. 2014). In many Arabidopsis mutants, accumulation of NLR proteins such as SNC1 (suppressor of npr1-1, constitutive 1) or RPS2 (resistance to P. syringae 2) results in autoimmune phenotypes. Autoimmunity is often characterized by macroscopic leaf lesions, enhanced pathogen resistance, and reduced growth. At the molecular level, constitutive immune responses include expression of defense marker genes such as PR1 and PR2 and increased levels of the plant defense hormone salicylic acid (SA). In some regards, the phenotype of saul1 (senescence-associated ubiquitin ligase 1) mutants, which lost the activity of the plasma membrane-localized SAUL1 (also called PUB44) protein and exhibit senescence and cell death phenotypes (Raab et al. 2009; Vogelmann et al. 2014), resembles characteristics of autoimmune mutants. SA levels and the expression of SA and defense genes were increased, and these phenotypes were PAD4-dependent (Vogelmann et al. 2012). Additionally, saul1 mutants showed reduced growth under nonpermissive conditions (Raab et al. 2009). However, the saul1 leaf yellowing phenotype occurred simultaneously throughout the whole leaf and was not characterized by the occurrence of local macroscopic lesions that were spread on the leaf. Thus, a clear function of the PUB-ARM Ub ligase SAUL1 in autoimmunity has not been established yet (Raab et al. 2009; Salt et al. 2011; Vogelmann et al. 2012). Here, we aim to investigate whether saul1 phenotypes depend on temperature or relative humidity or both and whether SAUL1 may have a function in autoimmunity, plant defense, or both.

RESULTS

The saul1-1 phenotype is dependent on temperature and relative humidity.

Previously, we found that early senescence and cell-death phenotypes of low light–grown saul1-1 mutants can be rescued in saul1-1 pad4 but not saul1-1 npr1 double mutants (Vogelmann et al. 2012). This is reminiscent of Arabidopsis cell death or lesion mimic mutants and may suggest that physiological parameters other than photon flux density (PFD) affect saul1 phenotypes, in particular temperature and relative humidity (Gou et al. 2012; Hua et al. 2001; Jambunathan et al. 2001; Li et al. 2001; Yang and Hua 2004). At a PFD of 150 μmol m⁻² s⁻¹, 21°C, and 50 to 60% relative humidity, soil-grown saul1 mutants did not develop like wild type (WT) but showed systemic lesioning of leaves as well as reduced growth early in development (Raab et al. 2009).To test whether higher relative humidity and higher temperature affect saul1 phenotypes, WT and saul1-1 plants were grown on soil at 24.2 ± 0.3°C, 69.8 ± 2.9% relative humidity, and 160 μmol m⁻² s⁻¹ in a growth chamber for 7, 12, 14, and 17 days. Whereas saul1-1 mutant plants were indistinguishable from WT at days 7, 12, and 14 (Supplementary Fig. S1), they showed some yellowing at the leaf edges at day 17 that was not observed in WT leaves (Fig. 1A and B).

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Previously, we showed that, at the molecular level, occurrence of saul1 phenotypes was linked to the induction of marker genes for senescence, cell death, and defense (Raab et al. 2009; Vogelmann et al. 2012). To monitor the relevant marker gene expression, quantitative polymerase chain reaction (qPCR) experiments were performed on samples taken at days 7, 12, 14, and 17. Expression of the senescence regulatory genes WRKY6 and AtNAP was almost unchanged at days 7 and 12 but slightly increased at day 14 (Fig. 1C and D). The increase in WRKY6 expression was still present after 17 days. A small but significant increase in the expression of the SA biosynthesis gene SID2 was observed at days 12 and 14, whereas the increase was larger at day 17 (Fig. 1E). In contrast, the expression of the SA signaling gene PAD4 started to increase largely as soon as day 12 (Fig. 1F). An increased expression of the defense marker genes PR1 and PR2 was also seen at days 12, 14, and 17 (Fig. 1G and H). As expected, expression in saul1-1 pad4-1 double mutants resembled the expression in WT plants for all genes tested (Fig. 1C to H) (Vogelmann et al. 2012)). saul1-1 pad4-1 and pad4-1 mutants were included in all subsequent experiments and resembled WT-like gene expression in all cases. Our data showed that higher relative humidity and higher temperature in the growth chamber could rescue saul1-1 phenotypes but not for the complete life cycle (day 17).

In many cases, high temperature is sufficient to fully prevent cell death and the underlying gene induction phenotypes of autoimmune mutants (Gou et al. 2012; Hua et al. 2001; Li et al. 2001; Yang and Hua 2004). We therefore increased the temperature to 25.6 ± 0.6°C while keeping relative humidity and PFD unchanged. Under these conditions, saul1-1 and WT plants showed identical morphology (Fig. 2A). The increase in PAD4 and SID2 expression observed at 24.2°C (Fig. 1E and F) was completely abolished (Fig. 2B and C). Whereas a very small increase in PR1 expression was still visible, no change was observed in PR2 expression (Fig. 2D and E). Note, that the scale for all gene expression analyses has been adopted from Figure 1. These results indicated that temperature is a key factor determining saul1-1 morphology and molecular phenotypes and that, at 25.6°C, cell death and defense responses are not turned on.

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To test for the respective contribution of the three physiological parameters temperature, light, and relative humidity, we analyzed plant performance and gene expression after changing only one parameter at a time. While keeping PFD at 160 μmol m⁻² s⁻¹ and higher temperature (25.7 ± 0.5°C), the relative humidity was lowered to 38.9% ± 2.2%. This decrease in relative humidity resulted in the occurrence of strong saul1 phenotypes before day 12 (Supplementary Fig. S2A). Plants were smaller than WT and showed yellowing of leaves. Therefore, samples were taken at day 11 to monitor changes in marker gene expression. As expected, expression of all marker genes, namely AtNAP, PAD4, SID2, PR1, and PR2 was increased. No significant increase was observed for WRKY6 (not shown). This indicated that the decrease of relative humidity from 71 to 39% resulted in cell death and lesion mimic phenotypes on the whole plant and at the molecular level, respectively.

To test for the relative contribution of temperature to saul1 phenotypes, the temperature was decreased to 20.5 ± 0.5°C. At this lower temperature, saul1-1 mutant plants did not develop properly but showed very early leaf yellowing and cell-death phenotypes at the young seedling stage (Supplementary Fig. S3). To address expression changes, we grew plants at permissive conditions (PFD of 160 μmol m⁻² s⁻¹, 24.4 ± 0.7°C, 84.1% ± 6.0% relative humidity) for 12 days before changing the temperature to 20.0 ± 0.8°C. Starting at 2 days after the temperature shift, saul1-1 plants showed reduced growth and yellowing of leaves in contrast to WT and saul1-1 pad4-1 plants (Fig. 3A). This was also reflected by decreased chlorophyll content and photochemical efficiency of photosystem II at lower temperature (Fig. 3F and G). Expression of the marker genes WRKY6, SID2, PAD4, and PR1 was already strongly induced in saul1-1 plants at day 1 after the shift in temperature and stayed increased thereafter (Figs. 3B to E). Taken together, our physiological and gene expression analyses showed that high temperature and high relative humidity can suppress the saul1-1 phenotypes.

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To investigate the relative contribution of light to saul1 phenotypes, plants were grown at PFD lowered from 160 μmol m⁻² s⁻¹ to 20 μmol m⁻² s⁻¹, while keeping temperature and relative humidity constantly high at 25.1 ± 0.9°C and 70.3% ± 2.7%. At day 7 after changing PFD, saul1-1 mutants still resembled WT, indicating that low light could not induce autoimmunity at high temperature and high relative humidity (Supplementary Fig. S4).

Chloroplast and cell-wall ultrastructure changes in saul1-1.

The appearance of saul1 phenotypes resembled the appearance of chs1 mutants in low temperature response and thus suggested that changes in chloroplast structure may coincide with the deterioration of saul1-1 plants (Wang et al. 2013; Zbierzak et al. 2013). We, therefore, analyzed chloroplast ultrastructure through transmission electron microscopy (TEM). Under permissive growth conditions of 24.4 ± 0.7°C, 84.1% ± 6.0% relative humidity, and a PFD of 160 μmol m⁻² s⁻¹ (Fig. 3), saul1-1 chloroplasts maintained the same ultrastructure as those of WT (Fig. 4A). Two to 3 days after changing the temperature to 20.0 ± 0.8°C (Fig. 3), however, thylakoid membranes started to lose integrity. At day 2, the size of plastoglobules, particles consisting of proteins and lipids, started to increase (Fig. 4A), and an increase in the number of plastoglobules was observed starting at day 4. All changes in ultrastructure that were initiated at day 2 or 3 were much stronger at 4 and 7 days after the shift in temperature (Fig. 4A to C). In addition to the ultrastructural changes in the chloroplasts, we observed changes in the ultrastructure of the cell wall (Fig, 4A, arrow). The observed depositions of material were significant already after 2 days and quite dramatic after 7 days. Quantification of the cell-wall thickness showed a 9.2-fold increase at the peak of deposition sites in saul1-1 cells (2.59 ± 0.79 μm, n = 13) compared with WT cells (0.28 ± 0.08 μm, n = 23) after 7 days (Fig. 4C).

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The formation and deposition of callose and lignin may be the cause for such an increase in cell-wall thickness. We applied aniline blue staining and universal micro spectral photometry (UMSP) to detect these cell-wall components in tissue sections of, respectively, saul1-1 and WT at day 7 after transfer to low temperature. In transmitted light, cell-wall increase was visible in saul1-1 but not in WT cells. Confocal laser scanning microscopy of aniline blue–stained sections indicated that callose was present in these thickened areas, whereas no signal was detected in WT (Fig. 5A). The analysis of tissue sections by UMSP showed that lignification, which is evidenced by the distinct UV-absorbances at a wavelength of 278 nm, was also strongly increased in these cell-wall deposits in saul1-1 compared with WT (Fig. 5B). Depositions in saul1-1 samples revealed much higher distinct absorbance values than depositions in WT samples.

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The saul1-1 phenotype is fully dependent on EDS1 and PAD4, but not SAG101.

The regulation of saul1 cell death and reduced growth phenotypes by temperature and relative humidity is reminiscent of autoimmune mutants such as bon1 and snc1. In these mutants, reduced growth and enhanced resistance phenotypes depend on EDS1 and PAD4 (Hua et al. 2001; Jambunathan et al. 2001; Li et al. 2001; Yang and Hua 2004). It has been shown previously that PAD4 is also crucial for saul1-1 phenotypes (Vogelmann et al. 2012). PAD4 and EDS1 together with SAG101 form a regulatory hub in plant immunity (Wiermer et al. 2005). To determine the contribution of EDS1 and SAG101 to saul1-1 phenotypes, saul1-1 eds1-2 and saul1-1 sag101-1 double mutants were generated and were grown side by side with saul1-1 and saul1-1 pad4-1 plants at permissive conditions for 14 days, before lowering the temperature to 19.6 ± 0.4°C. Three days after the temperature shift, saul1-1 and saul1-1 sag101-1 plants showed reduced growth and yellowing of leaves, whereas saul1-1 eds1-2 and saul1-1 pad4-1 were like WT (Fig. 6A). This difference coincided with an increase in expression of PAD4 and PR1 in saul1-1 and saul1-1 sag101-1 mutants, which was absent in saul1-1 eds1-2 and saul1-1 pad4-1 double mutant plants (Fig. 6B and C). These data showed that both PAD4 and EDS1 but not SAG101 are important regulators of saul1-1 phenotypes.

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saul1-1 mutants exhibit enhanced disease resistance phenotypes.

Suppression of saul1-1 phenotypes by pad4-1 and eds1-2 increased expression of defense marker genes, and the prominent regulation of saul1-1 by temperature suggested that SAUL1 may also affect plant immunity. To determine whether saul1-1 mutants had altered resistance against virulent pathogens, saul1-1 and WT plants were challenged with virulent bacterial pathogens P. syringae pv. maculicola ES4326 and P. syringae pv. tomato DC3000, virulent oomycete Hyaloperonospora arabidopsidis Noco2, or avirulent P. syringae pv. tomato DC3000 AvrRps4, which can be recognized by RPS4. In all scenarios, resistance was enhanced in saul1-1 plants. For bacterial infection, plants were grown at 28°C, 80% relative humidity, and a PFD of 90 μmol m⁻² s⁻¹. Growth at 28°C was intended to exclude any change in defense gene expression prior to infection. Very slight induction of the expression of PR1 or a few other defense genes has been observed in the absence of any morphological difference between WT and saul1-1 (Vogelmann et al. 2012) (Figs. 2, 3, and 5). At 28°C expression of the defense marker gene PR1 was identical in saul1-1 and WT (Supplementary Fig. S5). Infection was assayed at a standard temperature for P. syringae pv. tomato infection of 22°C (Brooks et al. 2004; Cheng et al. 2009). Temperatures below 24.2°C have been shown to induce immune responses in saul1-1 (Figs. 1, 3, and 6) (Raab et al. 2009). Bacterial growth of P. syringae pv. maculicola ES4326, P. syringae pv. tomato DC3000, and P. syringae pv. tomato DC3000 AvrRps4 was significantly lower on saul1-1 compared with WT plants (Fig. 7A to C). With H. arabidopsidis Noco2, seedlings were first grown at permissive conditions to allow normal growth of saul1 mutant, and infection was carried out at 80% relative humidity, 90 μmol m⁻² s⁻¹, and a temperature of 18°C, leading to typical yellowing of saul1-1 plants. Almost no oomycete conidiospore growth was observed on saul1-1 seedlings (Fig. 7D). As expected, WT level of susceptibility was restored in saul1-1 pad4-1 and saul1-1 eds1-2 plants. Taken together, saul1-1 exhibits EDS1/PAD4-dependent enhanced disease resistance to pathogens, indicating that their visible and molecular phenotypes are reminiscent of autoimmunity.

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DISCUSSION

Here, we present data on the role of a plasma membrane-associated PUB-ARM protein in plant immunity. Resistance to virulent pathogens, including the oomycete H. arabidopsidis Noco2 and the bacteria P. syringae pv. maculicola ES4326 and P. syringae pv. tomato DC3000, was increased in saul1-1 plants (Fig. 7). Enhanced resistance was also observed for avirulent P. syringae pv. tomato DC3000 AvrRps4. At temperatures below about 25°C and at low relative humidity, saul1-1 mutants showed autoimmunity (Figs. 1 and 2). A very small change in temperature from 25.6 to 24.2°C was sufficient to induce saul1-1 autoimmune responses. In addition, autoimmunity was suppressed in saul1-1 pad4-1 and saul1-1 eds1-2 mutants, indicating that PAD4 and EDS1 are crucial for saul1-1 autoimmunity. In contrast, SAG101 appeared not to be important (Figs. 3 and 6). Taken together, the saul1 phenotypes are reminiscent of those of other autoimmune mutants, such as snc1, bon1, and cpr1 (Gou et al. 2012; Hua et al. 2001; Jambunathan et al. 2001; Li et al. 2001; Yang and Hua 2004).

In saul1-1 leaf cells, two membrane-related processes could be observed. First, thylakoid membranes lost integrity from day 2 or 3 on after induction of saul1-1 phenotypes by decreasing the temperature. This resulted in increased numbers of plastoglobuli, which also gained in size (Fig. 4). This is likely due to storage of chlorophyll and galactolipid degradation products in plastoglobuli (Lippold et al. 2012). Very similar observations have been made in recessive Arabidopsis chs1-1 autoimmune mutants that are affected in the toll interleukin 1 receptor-nucleotide binding site immune receptor gene CHS1 (chilling sensitive 1). In these mutants, chloroplast malfunctions preceded the occurrence of cell death (Wang et al. 2013; Zbierzak et al. 2013). It has been proposed previously that changes in chloroplast homeostasis and metabolism may be related to the activation of immune responses (Kachroo and Kachroo 2009; Nomura et al. 2012). In this context, it has also been shown that singlet oxygen, which is produced in chloroplasts to dissipate excess excitation energy, can lead to loss of chloroplast integrity and induce cell death resembling the local cell death of the HR in effector-triggered immunity (Kim et al. 2012; Wagner et al. 2004; Zurbriggen et al. 2009). The singlet oxygen-triggered cell death program in flu mutants was dependent on EDS1 that was also required for low temperature–induced saul1 phenotypes (Fig. 6) (Ochsenbein et al. 2006).

Second, at day 2, changes at the cell wall started to be established that led to dramatic deposition of biomaterials (Fig. 4B and C). By using different imaging techniques, we showed that callose depositions and lignin were found in these thickened cell walls of saul1-1 leaves (Fig. 5). The cell wall has previously been linked to biotic stress signaling (Hamann 2015; Malinovsky et al. 2014). Aberrant deposition of lignin follows the reduction of cellulose content by specific inhibitors or by mutations in cellulose biosynthesis genes. This has previously been described for mutants that affect the cellulase synthase gene CESA3. These mutations resulted in decreased cellulose synthesis, whereupon lignin synthesis and defense responses were induced (Caño-Delgado et al. 2003; Ellis et al. 2002). In many cases, pathogen infection induces the formation of papillae, cell-wall thickenings at the site of infection that contain callose and also lignin (Ellinger et al. 2013; Malinovsky et al. 2014; Nishimura et al. 2003; Underwood 2012). The altered structure and components of cell walls of saul1 plants could be partly responsible for its enhanced resistance phenotypes.

The Ub ligase SAUL1 appears to be important for immune responses initiated at the plasma membrane. Other factors of plant autoimmunity also reside at the plasma membrane. Among them, the copine BONZAI1 (BON1), which carries a C-terminal myristoylation site responsible for plasma membrane–association, suppressed autoimmunity by negatively regulating the NLR gene SNC1 (Li et al. 2010; Yang and Hua 2004). The BON1-associated proteins 1 and 2 were suggested to be general inhibitors of plant cell death (Yang et al. 2007). Furthermore, BON1 was shown to interact with two receptor-like kinases that contain leucine-rich repeats, namely the brassinosteroid signaling component BRI1-associated receptor kinase 1 (BAK1) and the regulator of resistance signaling pathways BAK1-interacting receptor-like kinase 1 at the plasma membrane (Wang et al. 2011). This interaction is fundamental for the negative regulation of NLR genes to guarantee plant survival. The receptor-like protein SNC2 (suppressor of npr1, constitutive 2) is a positive regulator of pathogen resistance, putatively by binding of pathogen-associated molecular patterns (PAMPs) (Zhang et al. 2010). The ankyrin-repeat protein BDA1 (bian da or ‘becoming big’ in Chinese) acts downstream of SNC2 and may also act at the plasma membrane, because it contains a putative C-terminal transmembrane domain (Yang et al. 2012). It will be interesting to test whether the plasma membrane-associated Ub ligase SAUL1 mediates immune responses independent of these factors or whether they act in concert.

This study identified SAUL1 as an important signaling component in plant immunity. In Figure 8, the working model proposes a role for SAUL1 in initiation of immune responses at the plasma membrane. In WT plants, SAUL1 inactivates an unknown positive regulator of immunity, either by degradation or regulation. This leads to inhibition of immunity upstream of the EDS1/PAD4 regulatory node. The SAUL1 pathway functions independently of NPR1 (Vogelmann et al. 2012). Such regulation leads to the suppression of SA accumulation and PR gene expression, preventing autoimmunity. Future investigation will provide more information on the components that act up- and downstream of SAUL1 to regulate plant immunity.

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MATERIALS AND METHODS

Plant material and growth conditions.

Arabidopsis thaliana seeds of saul1-1 mutants, its segregating WT, pad4-1, saul1-1 pad4-1, eds1-2, and sag101-1 mutants were sown on soil and were stratified in the dark at 4°C for 48 h. Plants were grown in climate chambers (Weiss Klimatechnik GmbH, Reiskirchen-Lindenstruth, Germany) or Arabidopsis growth cabinets (Percivals, CLF PlantClimatics GmbH, Wertingen, Germany) under long-day conditions (16 h light, 8 h dark) at various temperature, light, and humidity conditions, as indicated in each experiment. Temperature and relative humidity were monitored using a data logger (OM-EL-USB-2-LCD-PLUS, OMEGA Engineering Inc., Deckenpfronn, Germany) and PFD was measured using a LI-250A light meter with a Quantum sensor (LI-COR Biosciences, Bad Homburg, Germany). The saul1-1 eds1-2 and saul1-1 sag101-1 double homozygous mutants were generated by crossing heterozygous saul1-1 plants with homozygous eds1-2 and sag101-1 lines, respectively. Homozygous double mutants were identified in the F2 and F3 generation by PCR using the SAUL1 specific primers 5′-TGAGGCCAATCAAATGATT​TC-3′ and 5′-TTTCCCCATTCATGAGTGA​AG-3′ in combination with the T-DNA insertion (SALK) specific primer LBa1 (5′-TGGTTCACGTAGTGGGCCAT​CG-3′), EDS1 specific primers 5′-ACACAAGGGTGATGCGAGA​CA-3′, 5′-GGCTTGTATTCATCTTCTAT​CC-3′ and 5′-GTGGAAACCAAATTTGACATT​AG-3′, SAG101 specific primers 5′-CACGCGTCCGAAGATCTTGGAGATACA​TA-3′ and 5′-ACTTCCGGGTGTTCATAAACTC​GGTCAAG-3′ in combination with the dSpm specific (SLAT) primer dSpm11 5′-GGTGCAGCAAAACCCACACTTT​TACTTC-3′.

Pathogen infection experiments.

P. syringae pv. maculicola ES4326 and H. arabidopsidis Noco2 infection experiments were carried out as described previously (Li et al. 2001). Briefly, leaves of 4-week-old plants grown for 4 weeks at 28°C, 80% relative humidity, and a PFD of 90 μmol m⁻² s⁻¹ were infiltrated with P. syringae pv. maculicola ES4326 suspension at an optical density at 600 nm (OD₆₀₀) = 0.001, P. syringae pv. tomato DC3000 suspension at OD₆₀₀ = 0.0002, P. syringae pv. tomato DC3000 AvrRps4 suspension at OD₆₀₀ = 0.002 at 22°C, 120 μmol m⁻² s⁻¹, and 60% relative humidity. Bacterial growth was quantified at days 0 and 3 or 4 after inoculation. For H. arabidopsidis Noco2, oomycete conidia spore suspension of 100,000 spores per milliliter in water was spray-inoculated onto 2-week-old seedlings. The plants were domed and were kept in an 18°C humid growth chamber (humidity >80%) for 7 days before oomycete growth quantification.

Gene expression analysis by qPCR.

For RNA analyses, above-ground tissue materials from a minimum of two plants per biological replicate were pooled. Total RNA was isolated using the innuPREP plant RNA kit (Analytik Jena AG, Jena, Germany). For synthesis of cDNAs, protocols of the QuantiTect reverse transcription kit (Qiagen, Hilden, Germany), using 500 ng of RNA per reaction, were followed. Quantitative PCR tests were performed using a Rotor-Gene Q (Qiagen) and the QuantiFast SYBR green PCR master mix (Qiagen). All samples were standardized to transcription levels of either UBI or EIF1. Gene expression levels of the WT of the first sampling day of each experiment were set to 1 and expression levels of other genotypes and sampling days were given relative to this. Student’s t test analysis was used to determine significance of difference values. Primers for PCR amplification were 5′-GGCCTTGTATAATCCCTGATGA​ATAAG-3′ and 5′-AAAGAGATAACAGGAACGGAAA​CATAGT-3′ for UBI, 5′-TGAGCACGCTCTTCTTGCTTT​CA-3′ and 5′-GGTGGTGGCATCCATCTTGTTA​CA-3′ for EIF1, 5′-AGATACGCGAGCACAACGCA​AG-3′ and 5′-TTTCTCGCCTCATCCAACCACT​C-3′ for PAD4 (At3g52430), 5′-CGGCTACCAACAACAACC​AC-3′ and 5′-CATTCCCGGAGGTAAGTT​CG-3′ for WRKY6 (At1g62300), 5′-TCCGTGACCTTGATCCTTTC​TC-3′ and 5′-TACCACCATAGGCACGAATC​AG-3′ for SID2 (At1g74710), 5′-CTCCCTCCAGGGTTCAGATT​TC-3′ and 5′-GGAGACAGGGCATGGTTTAG​AC-3′′ for AtNAP (At1g69490), 5′-TACAACTACGCTGCGAAC​AC-3′ and 5′-ACACCTCACTTTGGCACA​TC-3′ for PR1 (At2g14610), as well as 5′-CATCGAGAACGCGGTTTC​TG-3′ and 5′-TGAGACGGAGGAGACGTA​TC-3′ for PR2 (At3g57260).

Determination of chlorophyll content and quantum yield.

Chlorophyll fluorescence in the first true leaves of plants was measured 20 min after dark-adaptation using a portable chlorophyll fluorometer PAM-2500 (Heinz Walz GmbH, Effeltrich, Germany). The F_v/F_m ratio was determined with the PamWin-3 software (Heinz Walz GmbH).

To determine chlorophyll content, leaf tissue was ground in liquid nitrogen, and 80% acetone was added to a final volume of 2 ml. After shaking for 30 min at 150 rpm in the dark, the samples were centrifuged at 2,300 × g for 20 min, and the supernatant was transferred to a new tube. The centrifugation was repeated at 7,600 × g for 10 min before measuring absorbance at 652 nm photometrically. The absorbance was normalized to a fresh weight of 20 mg leaf tissue.

TEM.

In all experiments, the first true leaves of plants of the respective age were taken for TEM. The leaves were fixed with 2% glutaraldehyde and 2% paraformaldehyde (PFA) in 75 mM cacodylate buffer (pH 7.0) for 5 h on ice, were washed three times with 75 mM cacodylate buffer (pH 7.0), and were postfixed with 1% osmium tetroxide in 75 mM cacodylate buffer (pH 7.0) overnight at 4°C. The samples were washed three times with 75 mM cacodylate buffer (pH 7.0) and were dehydrated through a series of graded acetone concentrations, 30 to 100% (4°C), two more changes of 100% at room temperature. The infiltration of the embedding medium and embedding was done as described (Spurr 1969). Ultrathin sections (70 to 80 nm) were achieved with an ultramicrotome (Ultracut E, Leica-Reichert-Jung, Nußloch, Germany) equipped with a diamond knife and were subsequently stained with uranyl acetate followed by lead citrate (Reynolds 1963). The sections were observed with a LEO 906 E TEM (LEO, Oberkochen, Germany) equipped with a multiscan CCD camera (Model 794) of Gatan using the Microscopy Suite software version 2.0.2 (Gatan, Munich, Germany) to visualize and analyze the image data.

UMSP.

Samples for UMSP analyses were prepared as described for TEM except for postfixation with osmium tetroxide. For UV-microspectrophotometry, we followed established protocols (Koch and Grünwald 2004; Koch and Kleist 2001). Briefly, semithin (2 μm) sections were prepared by cutting the embedded material using an ultramicrotome equipped with a diamond knife (Ultracut S, Leica Reichert-Jung, Wetzlar, Germany). Subsequently, the sections were fixed on quartz slides, were immersed in a drop of non–UV absorbing glycerol and were covered with quartz cover slips. The topochemical analyses were conducted using a Zeiss UMSP 80 (Carl Zeiss AG) equipped with a scanning stage. The absorbance of the leave sections was measured at a constant wavelength of 278 nm (absorbance maximum of syringyl lignin) with a geometrical resolution of 0.25 × 0.25 μm. The image profiles were determined with the scan program APAMOS (automatic-photometric-analysis of microscopic objects by scanning) (Carl Zeiss AG).

Confocal laser scanning microscopy.

For determination of callose by confocal laser scanning microscopy, the leaves were fixed with 2% PFA in 50 mM microtubule stabilization buffer (MSB) (100 mM PIPES, 5 to 10 mM EGTA, 5 mM MgSO₄, pH 6.8) for 5 h on ice and were washed three times with 50 mM MSB. The samples were then dehydrated through a series of graded ethanol concentrations, 30 to 90%, 20 min on ice, respectively, 100% for 30 min on ice, and 100% for 30 min at room temperature. The LR white resin (medium grade acrylic resin; London Resin Company Ltd, Reading, England) was infiltrated gradually, mixed with ethanol (1:2 for 1 h, 1:1 for 1 h, 2:1 for 1 to 2 h, 100% overnight, 100% for 5 h, all steps at room temperature). The samples were filled in gelatin capsules and were cured at 40 to 50°C for 36 h.

The embedded material was used to prepare semithin (2 μm) sections, using an ultramicrotome equipped with a glass knife (Ultracut S). To detect callose, the sections were stained with 0.01% aniline blue in 150 mM K₂HPO₄ (pH 9.0) for 15 min in the dark and were rinsed with water. Images were captured with the Leica TCS SP8 confocal platform (Leica Microsystems, Wetzlar, Germany) using a 40× objective (HC PL APO CS2 40x/1.10 water). Excitation wavelength of 405 nm (UV diode) and emission wavelength of 461 to 492 nm were used to observe fluorescence. Images were visualized and analyzed with Leica Application Suite X (Leica Microsystems).

ACKNOWLEDGMENTS

We thank E. Woelken for support with TEM, D. Paul for support with UMSP experiments, W. Hellmeyer for technical assistance, C. Reisdorff for help with PAM measurements, and J. Parker (MPI Köln) for supplying eds1-2 and sag101-1 seeds. This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery program and UBC Dewar Cooper Memorial Fund (to X. Li) and funded by the Deutsche Forschungsgemeinschaft (grant number HO 2234/4-3 to S. Hoth).