The Leucine-Rich Repeat Receptor-Like Kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 and the Cytochrome P450 PHYTOALEXIN DEFICIENT3 Contribute to Innate Immunity to Aphids in Arabidopsis

The importance of pathogen-associated molecular pattern-triggered immunity (PTI) against microbial pathogens has been recently demonstrated. However, it is currently unclear if this layer of immunity mediated by surface-localized pattern recognition receptors (PRRs) also plays a role in basal resistance to insects, such as aphids. Here, we show that PTI is an important component of plant innate immunity to insects. Extract of the green peach aphid (GPA; Myzus persicae) triggers responses characteristic of PTI in Arabidopsis (Arabidopsis thaliana). Two separate eliciting GPA-derived fractions trigger induced resistance to GPA that is dependent on the leucine-rich repeat receptor-like kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)/SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE3, which is a key regulator of several leucine-rich repeat-containing PRRs. BAK1 is required for GPA elicitor-mediated induction of reactive oxygen species and callose deposition. Arabidopsis bak1 mutant plants are also compromised in immunity to the pea aphid (Acyrthosiphon pisum), for which Arabidopsis is normally a nonhost. Aphid-derived elicitors induce expression of PHYTOALEXIN DEFICIENT3 (PAD3), a key cytochrome P450 involved in the biosynthesis of camalexin, which is a major Arabidopsis phytoalexin that is toxic to GPA. PAD3 is also required for induced resistance to GPA, independently of BAK1 and reactive oxygen species production. Our results reveal that plant innate immunity to insects may involve early perception of elicitors by cell surface-localized PRRs, leading to subsequent downstream immune signaling.Close to a million insect species have so far been described, and nearly one-half of them feed on plants (Wu and Baldwin, 2010). Within these plant-feeding insects, most feed on a few related plant species, with only 10% feeding upon multiple plant families (Schoonhoven et al., 2005). Plant defense to insects include several layers (Bos and Hogenhout, 2011; Hogenhout and Bos, 2011). Physical barriers, volatile cues, and composition of secondary metabolites of plants are important components that determine insect host choice (Howe and Jander, 2008; Bruce and Pickett, 2011). In addition, plants induce a variety of plant defense responses upon perception of herbivore oral secretions (OS), saliva, and eggs (De Vos and Jander, 2009; Bruessow et al., 2010; Ma et al., 2010; Wu and Baldwin, 2010). These responses may provide full protection against the majority of insect herbivores, and insects that are able to colonize specific plant species likely produce effectors in their saliva or during egg laying that suppress these induced defense responses (Bos and Hogenhout, 2011; Hogenhout and Bos, 2011; Pitino and Hogenhout, 2013).Aphids are sap-feeding insects of the order Hemiptera and are among the most destructive pests in agriculture, particularly in temperate regions (Blackman and Eastop, 2000). More than 4,000 aphid species in 10 families are known (Dixon, 1998). Most aphid species are specialists and use one or a few closely related plant species within one family as host for feeding and reproduction. Examples are pea aphid (Acyrthosiphon pisum), cabbage aphid (Brevicoryne brassicae), and English grain aphid (Sitobion avenae) that colonize plant species within the legumes (family Fabaceae), brassicas (Brassicaceae), and grasses (Gramineae), respectively. The green peach aphid (GPA; Myzus persicae) is one of few aphid species with a broad host range and can colonize hundreds of plants species in over 40 plant families, including brassicas (Blackman and Eastop, 2000). Aphids possess mouthparts composed of stylets that navigate to the plant vascular system, predominantly the phloem, for long-term feeding. However, before establishing a long-term feeding site, these insects display a host selection behavior by probing the upper leaf cell layers with their stylets, a behavior seen on host and nonhost plants of the aphid (Nam and Hardie, 2012). When the plant is judged unsuitable, the aphid takes off to find an alternative plant host. It is not yet clear what happens in the initial stages of insect interactions with plants.Plants sense microbial organisms (including bacteria, fungi, and oomycetes) through perception of conserved molecules, named microbe-associated molecular patterns and pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) to induce the first stage of plant immunity, termed PAMP-triggered immunity (PTI). PTI is effective against the majority of plant pathogens. Bacterial and fungal PAMPs characterized so far include bacterial flagellin (or its derived peptide flg22), bacterial elongation factor (EF)-Tu (or its derived peptide elf18), bacterial lipopolysaccharides and bacterial cold shock protein, chitin oligosaccharides, and the oomycete elicitin INF1 (Boller and Felix, 2009)Plant PRRs are either receptor-like kinases (RLKs) or receptor-like proteins. Most leucine-rich repeat (LRR)-type PRRs associate with and rely for their function on the small regulatory LRR-RLK BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)/SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE3 (SERK3; Monaghan and Zipfel, 2012). For example, in Arabidopsis (Arabidopsis thaliana), flg22 and elf18 bind to the LRR-RLKs FLAGELLIN SENSITIVE2 (FLS2) and EF-TU RECEPTOR (EFR), respectively, leading to a quasi-instant association with BAK1 (Gómez-Gómez and Boller, 2000; Zipfel et al., 2006; Chinchilla et al., 2007; Heese et al., 2007; Schulze et al., 2010; Roux et al., 2011; Sun et al., 2013). BAK1 is required for optimal downstream immune signaling events, such as mitogen-activated protein kinase (MAPK) activation, reactive oxygen species (ROS) bursts, callose depositions, induction of immune genes, and induced resistance. Similarly, BAK1 is a positive regulator of innate immune responses triggered by the Arabidopsis LRR-RLKs PLANT ELICITOR PEPTIDE1 RECEPTOR1 (PEPR1) and PEPR2 that bind the Arabidopsis-derived damage-associated molecular pattern A. thaliana Peptide1 (AtPep1; Krol et al., 2010; Postel et al., 2010; Roux et al., 2011) and by the tomato (Solanum lycopersicum) LRR receptor-like protein Ve1 that recognizes Ave1 derived from Verticillium spp. (Fradin et al., 2009; de Jonge et al., 2012). Consistent with the role of BAK1 downstream of numerous PRRs, BAK1 is required for full immunity to a number of bacterial, fungal, oomycete, and viral pathogens (Heese et al., 2007; Kemmerling et al., 2007; Fradin et al., 2009; Chaparro-Garcia et al., 2011; Roux et al., 2011; Kørner et al., 2013).Notably, it has been recently shown that the ortholog of BAK1 in Nicotiana attenuata regulates the induction of jasmonic acid (JA) accumulation upon herbivory (Yang et al., 2011a). However, immunity to insects was not affected when BAK1 was silenced, and the observed effect on JA accumulation may be due to an indirect effect on brassinosteroid (BR) responses, for which BAK1 is also an important positive regulator (Li et al., 2002; Nam and Li, 2002). Therefore, it is currently unclear if BAK1 is involved in the early recognition of insect-derived elicitors leading to immunity.We discovered that the key regulatory LRR-RLK BAK1 participates in plant defense to an insect herbivore. We found that extracts of GPA trigger plant defense responses in Arabidopsis that are characteristic of PTI. Arabidopsis bak1 mutant plants are compromised in defense to GPA, which colonizes Arabidopsis, and to pea aphid, for which Arabidopsis is a nonhost. BAK1 is required for ROS bursts, callose deposition, and induced resistance in Arabidopsis upon perception of aphid-derived elicitors. One of the defense genes induced by GPA-derived extracts encodes PHYTOALEXIN DEFICIENT3 (PAD3), a cytochrome P450 that catalyzes the conversion of dihydrocamalexic acid to camalexin, which is a major Arabidopsis phytoalexin that is toxic to GPA (Kettles et al., 2013). PAD3 expression is required for Arabidopsis-induced resistance to GPA, independently of BAK1 and ROS. Our results provide evidence that innate immunity to insect herbivores may rely on the early perception of elicitors by cell surface-localized PRR.     

Cytoplastic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with ATG3 to Negatively Regulate Autophagy and Immunity in Nicotiana benthamiana

Autophagy as a conserved catabolic pathway can respond to reactive oxygen species (ROS) and plays an important role in degrading oxidized proteins in plants under various stress conditions. However, how ROS regulates autophagy in response to oxidative stresses is largely unknown. Here, we show that autophagy-related protein 3 (ATG3) interacts with the cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPCs) to regulate autophagy in Nicotiana benthamiana plants. We found that oxidative stress inhibits the interaction of ATG3 with GAPCs. Silencing of GAPCs significantly activates ATG3-dependent autophagy, while overexpression of GAPCs suppresses autophagy in N. benthamiana plants. Moreover, silencing of GAPCs enhances N gene-mediated cell death and plant resistance against both incompatible pathogens Tobacco mosaic virus and Pseudomonas syringae pv tomato DC3000, as well as compatible pathogen P. syringae pv tabaci. These results indicate that GAPCs have multiple functions in the regulation of autophagy, hypersensitive response, and plant innate immunity.     

EDR1 Physically Interacts with MKK4/MKK5 and Negatively Regulates a MAP Kinase Cascade to Modulate Plant Innate Immunity

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in the regulation of plant defense. The Raf-like MAP kinase kinase kinase (MAPKKK) EDR1 negatively regulates plant defense responses and cell death. However, how EDR1 functions, and whether it affects the regulation of MAPK cascades, are not well understood. Here, we showed that EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 kinase cascade in Arabidopsis. We found that edr1 mutants have highly activated MPK3/MPK6 kinase activity and higher levels of MPK3/MPK6 proteins than wild type. EDR1 physically interacts with MKK4 and MKK5, and this interaction requires the N-terminal domain of EDR1. EDR1 also negatively affects MKK4/MKK5 protein levels. In addition, the mpk3, mkk4 and mkk5 mutations suppress edr1-mediated resistance, and over-expression of MKK4 or MKK5 causes edr1-like resistance and mildew-induced cell death. Taken together, our data indicate that EDR1 physically associates with MKK4/MKK5 and negatively regulates the MAPK cascade to fine-tune plant innate immunity.     

Ectopic expression of ABSCISIC ACID 2/GLUCOSE INSENSITIVE 1 in Arabidopsis promotes seed dormancy and stress tolerance

Abscisic acid (ABA) is an important phytohormone that plays a critical role in seed development, dormancy, and stress tolerance. 9-cis-Epoxycarotenoid dioxygenase is the key enzyme controlling ABA biosynthesis and stress tolerance. In this study, we investigated the effect of ectopic expression of another ABA biosynthesis gene, ABA2 (or GLUCOSE INSENSITIVE 1 [GIN1]) encoding a short-chain dehydrogenase/reductase in Arabidopsis (Arabidopsis thaliana). We show that ABA2-overexpressing transgenic plants with elevated ABA levels exhibited seed germination delay and more tolerance to salinity than wild type when grown on agar plates and/or in soil. However, the germination delay was abolished in transgenic plants showing ABA levels over 2-fold higher than that of wild type grown on 250 mm NaCl. The data suggest that there are distinct mechanisms underlying ABA-mediated inhibition of seed germination under diverse stress. The ABA-deficient mutant aba2, with a shorter primary root, can be restored to normal root growth by exogenous application of ABA, whereas transgenic plants overexpressing ABA2 showed normal root growth. The data reflect that the basal levels of ABA are essential for maintaining normal primary root elongation. Furthermore, analysis of ABA2 promoter activity with ABA2::beta-glucuronidase transgenic plants revealed that the promoter activity was enhanced by multiple prolonged stresses, such as drought, salinity, cold, and flooding, but not by short-term stress treatments. Coincidently, prolonged drought stress treatment led to the up-regulation of ABA biosynthetic and sugar-related genes. Thus, the data support ABA2 as a late expression gene that might have a fine-tuning function in mediating ABA biosynthesis through primary metabolic changes in response to stress.     

The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the Nucleus and Regulates Defense Hormone Expression during Plant Innate Immunity

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The Ankyrin-Repeat Transmembrane Protein BDA1 Functions Downstream of the Receptor-Like Protein SNC2 to Regulate Plant Immunity

Plants utilize a large number of immune receptors to recognize pathogens and activate defense responses. A small number of these receptors belong to the receptor-like protein family. Previously, we showed that a gain-of-function mutation in the receptor-like protein SNC2 (for Suppressor of NPR1, Constitutive2) leads to constitutive activation of defense responses in snc2-1D mutant plants. To identify defense signaling components downstream of SNC2, we carried out a suppressor screen in the snc2-1D mutant background of Arabidopsis (Arabidopsis thaliana). Map-based cloning of one of the suppressor genes, BDA1 (for bian da; "becoming big" in Chinese), showed that it encodes a protein with amino-terminal ankyrin repeats and carboxyl-terminal transmembrane domains. Loss-of-function mutations in BDA1 suppress the dwarf morphology and constitutive defense responses in snc2-1D npr1-1 (for nonexpressor of pathogenesis-related genes1,1) and also result in enhanced susceptibility to bacterial pathogens. In contrast, a gain-of-function allele of bda1 isolated from a separate genetic screen to search for mutants with enhanced pathogen resistance was found to constitutively activate cell death and defense responses. These data suggest that BDA1 is a critical signaling component that functions downstream of SNC2 to regulate plant immunity.     

Chloroplast Signaling and LESION SIMULATING DISEASE1 Regulate Crosstalk between Light Acclimation and Immunity in Arabidopsis

Plants are simultaneously exposed to abiotic and biotic hazards. Here, we show that local and systemic acclimation in Arabidopsis thaliana leaves in response to excess excitation energy (EEE) is associated with cell death and is regulated by specific redox changes of the plastoquinone (PQ) pool. These redox changes cause a rapid decrease of stomatal conductance, global induction of ASCORBATE PEROXIDASE2 and PATHOGEN RESISTANCE1, and increased production of reactive oxygen species (ROS) and ethylene that signals through ETHYLENE INSENSITIVE2 (EIN2). We provide evidence that multiple hormonal/ROS signaling pathways regulate the plant''s response to EEE and that EEE stimulates systemic acquired resistance and basal defenses to virulent biotrophic bacteria. In the Arabidopsis LESION SIMULATING DISEASE1 (lsd1) null mutant that is deregulated for EEE acclimation responses, propagation of EEE-induced programmed cell death depends on the plant defense regulators ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4). We find that EDS1 and PAD4 operate upstream of ethylene and ROS production in the EEE response. The data suggest that the balanced activities of LSD1, EDS1, PAD4, and EIN2 regulate signaling of programmed cell death, light acclimation, and holistic defense responses that are initiated, at least in part, by redox changes of the PQ pool.