Pepper Heat Shock Protein 70a Interacts with the Type III Effector AvrBsT and Triggers Plant Cell Death and Immunity

Heat shock proteins (HSPs) function as molecular chaperones and are essential for the maintenance and/or restoration of protein homeostasis. The genus Xanthomonas type III effector protein AvrBsT induces hypersensitive cell death in pepper (Capsicum annuum). Here, we report the identification of the pepper CaHSP70a as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirm the specific interaction between CaHSP70a and AvrBsT in planta. The CaHSP70a peptide-binding domain is essential for its interaction with AvrBsT. Heat stress (37°C) and Xanthomonas campestris pv vesicatoria (Xcv) infection distinctly induce CaHSP70a in pepper leaves. Cytoplasmic CaHSP70a proteins significantly accumulate in pepper leaves to induce the hypersensitive cell death response by Xcv (avrBsT) infection. Transient CaHSP70a overexpression induces hypersensitive cell death under heat stress, which is accompanied by strong induction of defense- and cell death-related genes. The CaHSP70a peptide-binding domain and ATPase-binding domain are required to trigger cell death under heat stress. Transient coexpression of CaHSP70a and avrBsT leads to cytoplasmic localization of the CaHSP70a-AvrBsT complex and significantly enhances avrBsT-triggered cell death in Nicotiana benthamiana. CaHSP70a silencing in pepper enhances Xcv growth but disrupts the reactive oxygen species burst and cell death response during Xcv infection. Expression of some defense marker genes is significantly reduced in CaHSP70a-silenced leaves, with lower levels of the defense hormones salicylic acid and jasmonic acid. Together, these results suggest that CaHSP70a interacts with the type III effector AvrBsT and is required for cell death and immunity in plants.The heat shock protein HSP70 is a ubiquitous essential protein chaperone and one of the most abundant and diverse heat stress proteins in plants. HSP70s are induced by environmental stresses and are required for plants to cope with heat. HSP70s are involved in protein folding, synthesis, translocation, and macromolecular assemblies such as microtubules (Mayer et al., 2001; Hartl and Hayer-Hartl, 2002). HSP70s protect cells from heat stress by preventing protein aggregation and by facilitating the refolding of denatured proteins. Protein stability can decrease under heat stress conditions and expose hydrophobic patches that cause the aggregation of denatured proteins. HSP70s bind to hydrophobic patches of partially unfolded proteins in an ATP-dependent manner and prevent protein aggregation (Mayer and Bukau, 2005). The modular HSP70 structure consists of a N-terminal ATPase domain and a C-terminal peptide-binding domain that contains a β-sandwich subdomain with a peptide-binding cleft and an α-helical latch-like segment (Zhu et al., 1996; Hartl and Hayer-Hartl, 2002).HSP70s are involved in microbial pathogenesis, cell death responses, and immune responses. Diverse RNA viruses induce HSP70 expression in Arabidopsis (Arabidopsis thaliana; Whitham et al., 2003). Cytoplasmic HSP70s enhance the infection of Nicotiana benthamiana by Tobacco mosaic virus, Potato virus X, Cucumber mosaic virus, and Watermelon mosaic virus (Chen et al., 2008). Recently, the coat protein of Tomato yellow leaf curl virus was suggested to recruit host plant HSP70 during virus infection (Gorovits et al., 2013). HSP70s appear to be involved in regulating viral reproduction, protein folding, and movement, which ultimately promotes viral infection (Boevink and Oparka, 2005; Hafrén et al., 2010). The Pseudomonas syringae effector protein Hopl1 directly binds and manipulates host HSP70, which promotes bacterial virulence (Jelenska et al., 2010). The cytosolic/nuclear heat shock cognate 70 (HSC70) chaperone, which is highly homologous to HSP70 (Tavaria et al., 1996), regulates Arabidopsis immune responses together with SGT1 (for the suppressor of the G2 allele of S-phase kinase-associated protein1 [skp1]; Noël et al., 2007). Cytoplasmic HSP70 is required for the Phytophthora infestans INF1-mediated hypersensitive response (HR) and nonhost resistance to Pseudomonas cichorii in N. benthamiana (Kanzaki et al., 2003). HSP70 is proposed to be involved in both positive and negative regulation of cell death. Selective HSP70 depletion from human cell lines activates a tumor-specific death program that is independent of known caspases and p53 tumor-suppressor protein (Nylandsted et al., 2000), whereas HSP70 promotes tumor necrosis factor-mediated apoptosis by binding IkB kinase γ and impairing nuclear factor-κB signaling in Cos-1 cells (Ran et al., 2004). In N. benthamiana, HSP70 is required for tabtoxinine-β-lactam-induced cell death (Ito et al., 2014). However, HSP70 expression is shown to decrease the cell death triggered by salicylic acid (SA) in Nicotiana tabacum protoplasts (Cronjé et al., 2004). Overexpression of mitochondrial HSP70 suppresses heat- and hydrogen peroxide (H₂O₂)-induced programmed cell death in rice (Oryza sativa; Qi et al., 2011).The genus Xanthomonas YopJ-like AvrBsT protein activates effector-triggered immunity (ETI) in Arabidopsis Pitztal 0 plants (Cunnac et al., 2007). AvrBsT is a member of the YopJ/AvrRxv family identified in Xanthomonas campestris pv vesicatoria (Xcv; Lewis et al., 2011). AvrBsT alters phospholipid signaling and activates defense responses in Arabidopsis (Kirik and Mudgett, 2009). AvrBsT is an acetyltransferase that acetylates Arabidopsis ACETYLATED INTERACTING PROTEIN1 (ACIP1), a microtubule-associated protein required for plant immunity (Cheong et al., 2014). Xcv strain Bv5-4a secretes the AvrBsT type III effector protein that induces hypersensitive cell death and strong defense responses in pepper (Capsicum annuum) and N. benthamiana (Orth et al., 2000; Escolar et al., 2001; Kim et al., 2010). AvrBsT-induced HR-like cell death in pepper is likely part of the typical ETI-mediated defense response cascade (Jones and Dangl, 2006; Eitas et al., 2008; Eitas and Dangl, 2010). AvrBsT overexpression in Arabidopsis triggers plant cell death and defense signaling, leading to both disease and defense responses to diverse microbial pathogens (Hwang et al., 2012). Type III effectors such as Hopl1 and AvrBsT are used to identify unknown components of plant defense cascades (Nomura et al., 2006; Block et al., 2008; Jelenska et al., 2010; Kim et al., 2014) that modulate host innate immunity to achieve disease resistance. The pepper SGT1 was identified recently as a host interactor of AvrBsT (Kim et al., 2014). Pepper SGT1 has features of a cochaperone (Shirasu and Schulze-Lefert, 2003), interacts with AvrBsT, and promotes hypersensitive cell death associated with the pepper receptor-like cytoplasmic protein kinase1 (PIK1) phosphorylation cascade.In this study, we used a yeast (Saccharomyces cerevisiae) two-hybrid screen to identify the pepper HSP70a (CaHSP70a) as an interacting partner of the Xanthomonas spp. type III effector AvrBsT. Coimmunoprecipitation and bimolecular fluorescence complementation (BiFC) analyses verify that CaHSP70a interacts with AvrBsT in planta. Transient CaHSP70a overexpression in pepper leaves enhances heat stress sensitivity and leads to a cell death response. Cytoplasmic localization of the AvrBsT-CaHSP70a complex strongly elevates cell death. CaHSP70a expression is rapidly and strongly induced by avrBsT (for avirulent Xcv Dukso1 [Ds1]) infection in pepper. CaHSP70a silencing enhances susceptibility to Xcv infection, attenuates the reactive oxygen species (ROS) burst and cell death response, reduces SA and jasmonic acid (JA) levels, and disrupts expression of the defense response genes C. annuum pathogenesis-related protein1 (CaPR1; Kim and Hwang, 2000), CaPR10 (Choi et al., 2012), and CaDEF1 (for defensin; Do et al., 2004). Taken together, this study demonstrates that CaHSP70a is a target of the Xanthomonas spp. type III effector AvrBsT and acts as a positive regulator of plant cell death and immunity signaling.     

Plantation Forestry under Global Warming: Hybrid Poplars with Improved Thermotolerance Provide New Insights on the in Vivo Function of Small Heat Shock Protein Chaperones

Climate-driven heat stress is a key factor affecting forest plantation yields. While its effects are expected to worsen during this century, breeding more tolerant genotypes has proven elusive. We report here a substantial and durable increase in the thermotolerance of hybrid poplar (Populus tremula × Populus alba) through overexpression of a major small heat shock protein (sHSP) with convenient features. Experimental evidence was obtained linking protective effects in the transgenic events with the unique chaperone activity of sHSPs. In addition, significant positive correlations were observed between phenotype strength and heterologous sHSP accumulation. The remarkable baseline levels of transgene product (up to 1.8% of total leaf protein) have not been reported in analogous studies with herbaceous species. As judged by protein analyses, such an accumulation is not matched either by endogenous sHSPs in both heat-stressed poplar plants and field-grown adult trees. Quantitative real time-polymerase chain reaction analyses supported these observations and allowed us to identify the poplar members most responsive to heat stress. Interestingly, sHSP overaccumulation was not associated with pleiotropic effects that might decrease yields. The poplar lines developed here also outperformed controls under in vitro and ex vitro culture conditions (callus biomass, shoot production, and ex vitro survival), even in the absence of thermal stress. These results reinforce the feasibility of improving valuable genotypes for plantation forestry, a field where in vitro recalcitrance, long breeding cycles, and other practical factors constrain conventional genetic approaches. They also provide new insights into the biological functions of the least understood family of heat shock protein chaperones.In spite of the vast importance of trees as a renewable source of biomaterials and energy, forestry is largely dominated by traditional approaches worldwide. These have been unable to keep up with demand over the last half century, leading to significant deforestation and global concern over carbon emissions (Bonan, 2008). According to the Food and Agriculture Organization of the United Nations (FAO), the net loss in forest area is now at some 5.2 million ha per year, an alarmingly high rate (Global Forest Resources Assessment, 2010 [www.fao.org/forestry/fra2010/]). Tree farming is gaining momentum to reverse this state of affairs, but current yields must be significantly improved for plantations to become a realistic and sustainable alternative to forest logging (Boerjan, 2005; Fenning et al., 2008; Strauss et al., 2009; Harfouche et al., 2011, 2012). Global warming makes this a challenging goal, as evidenced by the massive losses in crop production attributable to the increased temperatures of the last decades (Lobell et al., 2011). Climate-driven heat stress has also been recently identified as a major cause of forest die off (Anderegg et al., 2013). While climate trends make the establishment of high-yielding plantations more pressing than ever, improving the thermotolerance of valuable genotypes has proven difficult (Neale and Kremer, 2011; Harfouche et al., 2012). Apart from long juvenile periods and insufficient genomic resources, progress in this area has been hampered by our poor understanding of the biochemical mechanisms underlying thermotolerance. Forest biotechnology has great potential in this respect, as promising candidate genes can be safely assessed under controlled conditions. Tree breeding programs will integrate this knowledge in the foreseeable future to help improve elite genotypes and sidestep undesirable phenotypic variation (Fenning et al., 2008; Strauss et al., 2009; Harfouche et al., 2011, 2012). A successful example is the development of freeze-tolerant hybrid eucalyptus trees (Eucalyptus grandis × Eucalyptus urophylla) through a biotechnological approach (Hinchee et al., 2009).Extensive research has shown that high-temperature stress has a negative impact on nearly every aspect of plant growth, development, reproduction, and yield (for review, see Mittler et al., 2012). To protect cell function, plants have evolved a complex metabolic adjustment process known as the heat shock response (HSR; Kotak et al., 2007; Mittler at al., 2012). Alternatively, programmed cell death is activated in specific cells, resulting in leaf shedding or abortion of reproductive organs (Qu et al., 2009; Blanvillain et al., 2011). Whereas research in forest trees remains scarce, the ubiquitous and conserved HSR has been thoroughly studied in herbaceous plants (Larkindale and Vierling, 2008; Hu et al., 2009; Finka et al., 2011; Mittler et al., 2012). It is now well established that it involves multiple biochemical and regulatory pathways aimed at minimizing damage and preserving cellular homeostasis. Heat stress promotes protein unfolding and aggregation, and a major component of the HSR is the induction of molecular chaperones of the heat shock protein (HSP) family. HSPs share the ability to recognize and bind other proteins in nonnative states, thereby preventing or reversing aggregation as well as promoting efficient refolding pathways. Moreover, HSPs facilitate the degradation of proteins that cannot be properly folded by facilitating their delivery to cellular proteases.The most abundant and heterogeneous HSPs in plants are the widespread small heat shock proteins (sHSPs). At least 10 separate families have been described in both monocots and dicots, which include proteins localized to the cytoplasm (four classes), nucleus, chloroplasts, mitochondria, endoplasmic reticulum, and peroxisomes (for review, see Basha et al. [2012]; Waters [2013]). Along with their remarkable up-regulation under stressful conditions, the above features suggest important protective roles in virtually all cellular compartments. Yet, sHSPs remain one of the least well-understood families of molecular chaperones. With monomeric sizes of approximately 12 to 43 kD, plant sHSPs share a signature α-crystallin domain of approximately 90 amino acids and the ability to form large oligomers with a dynamic quaternary structure. The majority of structural and functional information derives from studies of cytosolic class I (CI) members, like wheat (Triticum aestivum) TaHSP16.9, for which the crystal structure of the native dodecamer has been solved (van Montfort et al., 2001). While researchers have demonstrated that most sHSPs can act as ATP-independent molecular chaperones, understanding their in vivo mode of action is complex (Basha et al., 2012; Waters, 2013). The prevailing view is that they prevent irreversible protein aggregation by interacting with unfolded proteins generated during stress. Recent evidence indicates that sHSP oligomers bind up to an equal weight of substrate proteins through a unique mechanism that involves substrate- and temperature-dependent remodeling of their own quaternary structure (Jaya et al., 2009; Stengel et al., 2010). Subsequent refolding of the substrates requires, at least in some cases, interaction with high-M_r ATP-dependent chaperones (Haslbeck et al., 2005; Basha et al., 2012). Thus, sHSPs would act primarily as holdases that keep nonnative proteins in a competent state until other downstream chaperones facilitate refolding. In vivo sHSP substrates remain to be identified in higher plants, although different lines of evidence suggest that they are structurally diverse (Basha et al., 2004; Haslbeck et al., 2004; Cheng et al., 2008; Jaya et al., 2009). Likewise, the role of sHSPs in mechanisms of thermotolerance has not yet been established. Reverse genetic approaches have led to inconsistent results in Arabidopsis (Arabidopsis thaliana) and other herbaceous species, ranging from variable protection (Malik et al., 1999; Sanmiya et al., 2004; Zhao et al., 2007; Jiang et al., 2009) to no detectable effects at all (Härndahl et al., 1999; Sun et al., 2001).CsHSP17.5, a major CI sHSP in chestnut trees (Castanea sativa), exhibits features that make it a good candidate to improve heat stress adaptation. First, its accumulation in adult trees increases substantially during spring and early summer, reaching a peak at the hottest time of the year (field conditions). Second, the corresponding gene is strongly activated by heat stress (growth chambers). Third, it significantly improves Escherichia coli viability under heat stress conditions. Fourth, it accumulates at unusually high levels in both stems and seeds, where it reaches levels comparable to those of major storage proteins (Collada et al., 1997; Soto et al., 1999; Lopez-Matas et al., 2004). Besides suggesting a relevant role in thermotolerance, these features indicate that CsHSP17.5 can accumulate in vivo for long periods of time. In vitro experiments have shown, additionally, that the native protein can stand repeated cycles of stressful conditions without losing chaperone activity (Lopez-Matas et al., 2004). The fact that CsHSP17.5 has never been identified as an allergen, despite accumulating so abundantly in mature chestnuts (according to FAO, humans consume over half a million metric tons a year), adds to its biotechnological potential. Here, we show that constitutive overexpression of native CsHSP17.5 substantially enhances the basal thermotolerance of hybrid poplar (Populus tremula × Populus alba) without causing negative pleiotropic effects that might affect plantation yields. A significant improvement of plant performance in vitro and ex vitro was also observed, even in the absence of thermal stress. Our results shed new light on the in vivo roles of sHSPs. In addition, they represent, to our knowledge, the first example of biotechnological manipulation of thermotolerance in forest trees.     

From Endoplasmic Reticulum to Mitochondria: Absence of the Arabidopsis ATP Antiporter Endoplasmic Reticulum Adenylate Transporter1 Perturbs Photorespiration

The carrier Endoplasmic Reticulum Adenylate Transporter1 (ER-ANT1) resides in the endoplasmic reticulum (ER) membrane and acts as an ATP/ADP antiporter. Mutant plants lacking ER-ANT1 exhibit a dwarf phenotype and their seeds contain reduced protein and lipid contents. In this study, we describe a further surprising metabolic peculiarity of the er-ant1 mutants. Interestingly, Gly levels in leaves are immensely enhanced (26×) when compared with that of wild-type plants. Gly accumulation is caused by significantly decreased mitochondrial glycine decarboxylase (GDC) activity. Reduced GDC activity in mutant plants was attributed to oxidative posttranslational protein modification induced by elevated levels of reactive oxygen species (ROS). GDC activity is crucial for photorespiration; accordingly, morphological and physiological defects in er-ant1 plants were nearly completely abolished by application of high environmental CO₂ concentrations. The latter observation demonstrates that the absence of ER-ANT1 activity mainly affects photorespiration (maybe solely GDC), whereas basic cellular metabolism remains largely unchanged. Since ER-ANT1 homologs are restricted to higher plants, it is tempting to speculate that this carrier fulfils a plant-specific function directly or indirectly controlling cellular ROS production. The observation that ER-ANT1 activity is associated with cellular ROS levels reveals an unexpected and critical physiological connection between the ER and other organelles in plants.