RNA Interference Knockdown of BRASSINOSTEROID INSENSITIVE1 in Maize Reveals Novel Functions for Brassinosteroid Signaling in Controlling Plant Architecture

Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbri1 complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbri1-RNAi transgenic lines have compromised BR signaling. zmbri1-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbri1-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbri1-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbri1-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling.Brassinosteroids (BRs) are ubiquitous plant hormones that promote plant growth by regulating cell elongation and division (Clouse, 1996; Clouse et al., 1996). BRs have other diverse roles, including enhancing tracheary element differentiation, stimulating ATPase activity, controlling microtubule orientation, and controlling flowering time, fertility, and leaf development (Iwasaki and Shibaoka, 1991; Clouse et al., 1996; Li et al., 1996; Schumacher et al., 1999; Catterou et al., 2001; Oh et al., 2011). BRs also function in tolerance to both biotic and abiotic stresses such as extreme temperatures, drought, and pathogens (Krishna, 2003).Deficiencies in BR biosynthesis or signaling produce characteristic dwarf plant phenotypes (Clouse et al., 1996; Szekeres et al., 1996; Fujioka et al., 1997). Plant height is an important agricultural trait, as seen in the Green Revolution, where semidwarf mutants contributed to increased yields in small-grain crops (Salas Fernandez et al., 2009). BR-deficient dwarf rice (Oryza sativa) produced increased grain and biomass yields because the erect leaf habit allowed higher planting densities under field conditions (Sakamoto et al., 2006). In fact, Green Revolution Uzu barley (Hordeum vulgare) is based on a mutation of the UZU1 gene, which encodes a homolog of BRASSINOSTEROID INSENSITIVE1 (BRI1), a BR receptor (Chono et al., 2003).Genes functioning in BR pathways have been identified by the analysis of dwarf mutants in several species, including Arabidopsis (Arabidopsis thaliana) and rice. Arabidopsis bri1 mutants are shortened, have reduced apical dominance, and are male sterile (Clouse et al., 1996). BRI1 encodes a Leu-rich repeat (LRR) receptor-like kinase that is located in the plasma membrane and contains an extracellular domain responsible for BR binding, a transmembrane sequence, and a cytoplasmic protein kinase domain (Li and Chory, 1997; Vert et al., 2005; Belkhadir and Chory, 2006). The island domain and subsequent LRR 22 are critical for BR binding (Kinoshita et al., 2005; Hothorn et al., 2011; She et al., 2011). Phosphorylation of the conserved residues Ser-1044 and Thr-1049 in the kinase activation loop activates the BRI1 kinase (Wang et al., 2005), while dephosphorylation of BRI1 by PROTEIN PHOSPHATASE2A inhibits its function (Wu et al., 2011).BRI1 is partially redundant in BR signaling with related BRASSINOSTEROID INSENSITIVE1-LIKE RECEPTOR KINASE (BRL) paralogs, both in Arabidopsis and rice. In Arabidopsis, even though null alleles of brl1 or brl3 did not show obvious phenotypic defects in shoots, they enhanced the developmental defects of a weak bri1-5 mutant. In contrast to ubiquitously expressed BRI1, BRL1, BRL2, and BRL3 are tissue specific, mostly expressed in vascular tissues, while BRL1 and BRL3 are also expressed in root apices (Caño-Delgado et al., 2004; Zhou et al., 2004; Fàbregas et al., 2013). Both BRL1 and BRL3 can bind brassinolide (BL; Caño-Delgado et al., 2004). In rice, OsBRI1 is similar to the Arabidopsis BRI1 gene, and phenotypes of OsBRI1 rice mutants include dwarf plants with shortened internodes, erect leaves that are twisted and dark green, and photomorphogenesis in the dark (Yamamuro et al., 2000). There are three BR receptors in rice as well, and while OsBRI1 is universally expressed in all organs, OsBRL1 and OsBRL3 are expressed mostly in roots (Nakamura et al., 2006).To date, two mutant genes of the BR biosynthetic pathway have been reported in maize (Zea mays). A classic dwarf mutant, nana plant1 (na1), has a mutation in a DE-ETIOLATED2 homologous gene, which encodes a 5α-reductase enzyme in the BR biosynthesis pathway (Hartwig et al., 2011), while the brassinosteroid-dependent1 (brd1) gene encodes brassinosteroid C-6 oxidase (Makarevitch et al., 2012). The maize BR-deficient mutants have shortened internodes, twisted, dark green, erect leaves, and feminized male flowers (Hartwig et al., 2011; Makarevitch et al., 2012). However, no genes in BR signaling have yet been reported in maize. Understanding BR signaling in maize might help improve this important crop for the production of biofuels, biomass, and grain yield. Here, we took a transgenic RNA interference (RNAi) approach to generate maize plants partially deficient for BRI1. These knockdown lines demonstrate that BRI1 functions are generally conserved in maize compared with other plant species, but they also exhibit unique phenotypes, suggesting either that maize possesses novel BR-regulated developmental processes or that aspects of maize morphology reveal processes not evident in other plants.     

An Overdose of the Arabidopsis Coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 or Its Ectodomain Causes Autoimmunity in a SUPPRESSOR OF BIR1-1-Dependent Manner

The membrane-bound BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1) is a common coreceptor in plants and regulates distinct cellular programs ranging from growth and development to defense against pathogens. BAK1 functions through binding to ligand-stimulated transmembrane receptors and activating their kinase domains via transphosphorylation. In the absence of microbes, BAK1 activity may be suppressed by different mechanisms, like interaction with the regulatory BIR (for BAK1-INTERACTING RECEPTOR-LIKE KINASE) proteins. Here, we demonstrated that BAK1 overexpression in Arabidopsis (Arabidopsis thaliana) could cause detrimental effects on plant development, including growth arrest, leaf necrosis, and reduced seed production. Further analysis using an inducible expression system showed that BAK1 accumulation quickly stimulated immune responses, even under axenic conditions, and led to increased resistance to pathogenic Pseudomonas syringae pv tomato DC3000. Intriguingly, our study also revealed that the plasma membrane-associated BAK1 ectodomain was sufficient to induce autoimmunity, indicating a novel mode of action for BAK1 in immunity control. We postulate that an excess of BAK1 or its ectodomain could trigger immune receptor activation in the absence of microbes through unbalancing regulatory interactions, including those with BIRs. Consistently, mutation of SUPPRESSOR OF BIR1-1, which encodes an emerging positive regulator of transmembrane receptors in plants, suppressed the effects of BAK1 overexpression. In conclusion, our findings unravel a new role for the BAK1 ectodomain in the tight regulation of Arabidopsis immune receptors necessary to avoid inappropriate activation of immunity.Plants rely on their innate immune system to detect microbes and mount an active defense against pathogens. The plant immune system is traditionally considered to be composed of two layers (Jones and Dangl, 2006). The first one is based on the activity of pattern-recognition receptors (PRRs) that can detect microbe-associated molecular patterns (MAMPs) and trigger what is termed pattern-triggered immunity (PTI; Boller and Felix, 2009). Many plant pathogens can suppress this basal defense response using virulence factors termed effectors. In a second layer of defense, plants can make use of resistance (R) proteins to recognize the presence of pathogen effectors resulting in effector-triggered immunity (ETI), which resembles an accelerated and amplified PTI response (Jones and Dangl, 2006).Plants utilize plasma membrane-associated receptor-like proteins (RLPs) or receptor-like kinases (RLKs) as PRRs to sense specific signals through their ectodomains (Böhm et al., 2014). RLPs and RLKs require the function of additional RLKs to form active receptor complexes and transfer the external signal to the inside of the cells (Zhang and Thomma, 2013; Cao et al., 2014; Liebrand et al., 2014). The best-known coreceptor is the leucine-rich repeat (LRR)-RLK BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1), which was originally identified as a positive regulator and partner for the brassinosteroid (BR) receptor BRASSINOSTEROID INSENSITIVE1 (BRI1; Li et al., 2002; Nam and Li, 2002). BRs refer to phytohormones that promote plant growth and development (Fujioka and Yokota, 2003). Thus, loss-of-function mutations in BAK1 negatively impact Arabidopsis (Arabidopsis thaliana) growth due to improper cell elongation. In short, bak1 mutants display compact rosettes with round-shaped leaves and shorter petioles and phenocopy weak bri1 mutations (Li et al., 2002; Nam and Li, 2002). Conversely, certain mutants affected in the BAK1 ectodomain show increased activity in the BR signaling pathway and share phenotypic similarities with BRI1-overexpressing lines (Wang et al., 2001), including elongated hypocotyls, petioles, and leaf blades and an overall increase in height (Jaillais et al., 2011; Chung et al., 2012).Furthermore, BAK1 is involved in the containment of cell death, independently of its function in BR signaling. Arabidopsis bak1 knockout mutants exhibit extensive cell death spreading after microbial infection (Kemmerling et al., 2007). In addition, spontaneous cell death develops in Arabidopsis double mutant plants lacking both BAK1 (also named SOMATIC EMBRYOGENESIS RECEPTOR KINASE3 [SERK3]) and its closest homolog BAK1-LIKE1 (BKK1)/SERK4, causing seedling lethality even in the absence of microbes (He et al., 2007). Similar phenotypes are observed in Arabidopsis, rice (Oryza sativa), and Nicotiana benthamiana by lowering the expression of BAK1 and its homologs (Heese et al., 2007; Jeong et al., 2010; Park et al., 2011). Interestingly, typical defense responses, like the production of reactive oxygen species and constitutive callose deposition, are also detected in those plants, although the basis for this phenomenon remains poorly understood (He et al., 2007; Kemmerling et al., 2007; Park et al., 2011; Gao et al., 2013).On the other hand, BAK1 is widely studied as a key component of immune signaling pathways due to its known association with different PRRs, including RLKs and RLPs (Kim et al., 2013; Böhm et al., 2014). Upon MAMP perception, PRRs induce signaling and physiological defense responses like mitogen-activated protein kinase (MAPK) activation, reactive oxygen species and ethylene production, and modifications in gene expression, all of which contribute to PTI. Among the best-studied examples of BAK1-regulated PRRs are two LRR-receptor kinases, ELONGATION FACTOR Tu RECEPTOR (EFR), which senses the active epitope elf18 of the bacterial elongation factor Tu, and the flagellin receptor FLAGELLIN SENSING2 (FLS2), which senses the active epitope flg22 of bacterial flagellin (Gómez-Gómez and Boller, 2000; Chinchilla et al., 2006; Zipfel et al., 2006). Immediately after flg22 binding to its LRR ectodomain, FLS2 forms a tight complex with BAK1 (Chinchilla et al., 2007; Sun et al., 2013). This heteromerization step may bring the two kinase domains closer and thereby induce, within seconds, the phosphorylation of BAK1 and FLS2 (Schulze et al., 2010; Schwessinger et al., 2011). These steps are sufficient to initiate the immune signaling pathway, even if the ectodomains and kinase domains are switched between FLS2 and BAK1 (Albert et al., 2013).While PRRs, such as FLS2 and EFR, are extremely sensitive to even subnanomolar concentrations of their ligands, a tight control of these receptors is expected, since constitutive activation of defense responses in plants dramatically impairs fitness and growth (Tian et al., 2003; Korves and Bergelson, 2004). However, the mechanisms that underlie the attenuation of PRR activation or prevent these receptors from signaling constitutively remain largely unknown (Macho and Zipfel, 2014). Several independent observations indicate that BAK1 and FLS2 are present in close spatial proximity in preformed complexes at the plasma membrane (Chinchilla et al., 2007; Schulze et al., 2010; Roux et al., 2011). Negative regulation of immune signaling prior to ligand perception could happen within the PRR complex and depend on conformational changes following the association of FLS2 with flg22 (Meindl et al., 2000; Schulze et al., 2010; Mueller et al., 2012). Additionally, other partners might prevent the constitutive interaction of BAK1 with FLS2. Such could be the case for the LRR-RLK BAK1-INTERACTING RECEPTOR-LIKE KINASEs (BIRs): BIR2 was recently discovered as a substrate and negative regulator for BAK1, while the absence of BIR1 leads to the activation of defense induction and strong dwarfism (Gao et al., 2009; Halter et al., 2014b). Furthermore, MAMP signaling may be constrained by phosphatases, as suggested in earlier studies (Felix et al., 1994; Gómez-Gómez et al., 2001) and recently shown for the protein phosphatase 2A, which controls PRR activation likely by modulating the BAK1 phosphostatus (Segonzac et al., 2014). These examples illustrate the variety of mechanisms that may tightly control BAK1 activity.In this work, we show that regulation of BAK1 accumulation is crucial for Arabidopsis fitness, as its overexpression leads to dwarfism and premature death. The phenotype differs from BR mutants and is very reminiscent of or even identical to the autoimmune phenotype of plants showing constitutive activation of R proteins (Oldroyd and Staskawicz, 1998; Bendahmane et al., 2002; Zhang et al., 2003). BAK1 overexpression is associated with constitutive activation of defense pathway(s) involving the general coregulator of RLPs, SUPPRESSOR OF BIR1-1 (SOBIR1; Liebrand et al., 2013, 2014). To our knowledge, this is the first report and comprehensive characterization of such an autoimmunity phenotype for Arabidopsis plants overexpressing BAK1, and it highlights the importance of the regulation of PTI overactivation.     

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.