Phytochrome B Promotes Branching in Arabidopsis by Suppressing Auxin Signaling

Many plants respond to competition signals generated by neighbors by evoking the shade avoidance syndrome, including increased main stem elongation and reduced branching. Vegetation-induced reduction in the red light:far-red light ratio provides a competition signal sensed by phytochromes. Plants deficient in phytochrome B (phyB) exhibit a constitutive shade avoidance syndrome including reduced branching. Because auxin in the polar auxin transport stream (PATS) inhibits axillary bud outgrowth, its role in regulating the phyB branching phenotype was tested. Removing the main shoot PATS auxin source by decapitation or chemically inhibiting the PATS strongly stimulated branching in Arabidopsis (Arabidopsis thaliana) deficient in phyB, but had a modest effect in the wild type. Whereas indole-3-acetic acid (IAA) levels were elevated in young phyB seedlings, there was less IAA in mature stems compared with the wild type. A split plate assay of bud outgrowth kinetics indicated that low auxin levels inhibited phyB buds more than the wild type. Because the auxin response could be a result of either the auxin signaling status or the bud’s ability to export auxin into the main shoot PATS, both parameters were assessed. Main shoots of phyB had less absolute auxin transport capacity compared with the wild type, but equal or greater capacity when based on the relative amounts of native IAA in the stems. Thus, auxin transport capacity was unlikely to restrict branching. Both shoots of young phyB seedlings and mature stem segments showed elevated expression of auxin-responsive genes and expression was further increased by auxin treatment, suggesting that phyB suppresses auxin signaling to promote branching.The development of shoot branches is a multistep process with many potential points of regulation. After the formation of an axillary meristem in the leaf axil, an axillary bud may form through the generation of leaves and other tissues. The axillary bud may grow out to form a branch, or may remain dormant or semidormant for an indefinite period of time (Bennett and Leyser, 2006). In Arabidopsis (Arabidopsis thaliana), the position of the bud in the rosette is a strong determinant of its fate, with upper buds displaying greater outgrowth potential than lower buds. In fact, the varying potential of buds at different positions is maintained even in buds that are activated to form branches, with the upper buds growing out first and most robustly, and lower buds growing out after a time lag and with less vigor (Hempel and Feldman, 1994; Finlayson et al., 2010).The disparate fate of buds at different rosette positions is mediated, at least in part, by the process of correlative inhibition, whereby remote parts of the plant inhibit the outgrowth of the buds (Cline, 1997). Correlative inhibition is typically associated with the bud-inhibiting effects of auxin sourced in the shoot apex and transported basipetally in the polar auxin transport stream (PATS). Auxin in the PATS does not enter the bud and thus must act indirectly; however, the exact mechanism by which auxin inhibits bud outgrowth is not well understood, despite many years of intensive study (Waldie et al., 2010; Domagalska and Leyser, 2011). Evidence supports divergent models by which auxin may regulate branching. One model contends that the PATS modulates a bud outgrowth inhibiting second messenger (Brewer et al., 2009). Another model postulates a mechanism whereby competition between the main shoot and the axillary bud for auxin export in the PATS regulates bud activity (Bennett et al., 2006; Prusinkiewicz et al., 2009; Balla et al., 2011).In addition to intrinsic developmental programming, branching is also modulated by environmental signals, including competition signals generated by neighboring plants. The red light:far-red light ratio (R:FR) is an established competition signal that is modified (reduced) by neighboring plants and sensed by the phytochrome family of photoreceptors. A low R:FR evokes the shade avoidance syndrome with plants displaying, among other phenotypes, enhanced shoot elongation and reduced branching (Smith, 1995; Ballaré, 1999; Franklin and Whitelam, 2005; Casal, 2012). Phytochrome B (phyB) is the major sensor contributing to R:FR responses, and loss of phyB function results in a plant that displays a phenotype similar to constitutive shade avoidance. It should be noted that actual shade avoidance is mediated by additional phytochromes and that the complete absence of functional phyB in the loss-of-function mutant may also result in a phenotype that does not exactly mirror shade avoidance. Loss of phyB function leads to reduced branching and altered expression of genes associated with hormone pathways and bud development in the axillary buds (Kebrom et al., 2006; Finlayson et al., 2010; Kebrom et al., 2010; Su et al., 2011). In Arabidopsis, phyB deficiency differentially affects the outgrowth of buds from specific positions in the rosette and thus demonstrates an important function in the regulation of correlative inhibition (Finlayson et al., 2010; Su et al., 2011), a process known to be influenced by auxin. Many aspects of auxin signaling are dependent on AUXIN RESISTANT1 (AXR1), which participates in activating the Skip-Cullin-F-box auxin signaling module (del Pozo et al., 2002). Reduced auxin signaling resulting from AXR1 deficiency enabled phyB-deficient plants to branch profusely and reduced correlative inhibition, thus establishing auxin signaling downstream of phyB action (Finlayson et al., 2010). Although a link between auxin signaling and phyB regulation of branching was demonstrated, the details of the interaction were not discovered.The relationship between auxin and shade avoidance responses has been investigated in some detail. Auxin signaling was implicated in shade avoidance responses mediated by ARABIDOPSIS THALIANA HOMEOBOX PROTEIN2 in young Arabidopsis seedlings (Steindler et al., 1999). Rapid changes in leaf development resulting from canopy shade were also shown to involve TRANSPORT INHIBITOR RESPONSE1-dependent auxin signaling (Carabelli et al., 2007). A link between auxin abundance and the response to the R:FR was demonstrated in Arabidopsis deficient for the TRP AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) auxin biosynthetic enzyme (Tao et al., 2008). Young wild-type seedlings respond to a decreased R:FR by increasing indole-3-acetic acid (IAA) biosynthesis, accumulating IAA, increasing hypocotyl and petiole elongation, and increasing leaf elevation. However, these responses are reduced in plants deficient in TAA1. Subsequent studies confirmed the importance of auxin in responses to the R:FR (Pierik et al., 2009; Kozuka et al., 2010; Keller et al., 2011), and also identified the auxin transporter PIN-FORMED3 as essential for hypocotyl elongation responses in young seedlings (Keuskamp et al., 2010). In addition to the roles of auxin abundance and transport in the process, auxin sensitivity has also been implicated in shade avoidance. Several auxin signaling genes are direct targets of the phytochrome signaling component PHYTOCHROME INTERACTING FACTOR5 (PIF5), and these genes are misregulated in Arabidopsis deficient in either PHYTOCHROME INTERACTING FACTOR4 (PIF4) or PIF5 (Hornitschek et al., 2012; Sun et al., 2013). Auxin-responsive hypocotyl elongation and auxin-induced gene expression were also reduced in young seedlings of the pif4pif5 double mutant (Hornitschek et al., 2012), which show defects in shade avoidance responses (Lorrain et al., 2008).Although some aspects of the regulation of branching are now understood, there are still many gaps in our knowledge of the process, especially as related to the regulation of branching by light signals. Because auxin is known to play a major role in regulating branch development, and because recent studies have implicated auxin in general shade avoidance responses and specifically in the regulation of branching by phyB, the hypothesis that auxin homeostasis, transport, and/or signaling may contribute to the hypobranching phenotype of phyB-deficient plants was generated and tested, using a variety of physiological and molecular approaches.     

Spore Density Determines Infection Strategy by the Plant Pathogenic Fungus Plectosphaerella cucumerina

Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonic acid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependent resistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signals triggering this transition. This study is based on the observation that the early colonization pattern and symptom development by the ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis (Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates between hemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargeted metabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods. Quantification of JA and SA, marker gene expression, and cell death confirmed that infection from high spore densities activates JA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lower levels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling confirmed that P. cucumerina is differentially resisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situ staining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated host defense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophic infection program, thereby gaining an advantage of immunity-related cell death in the host.Plant pathogens are often classified as necrotrophic or biotrophic, depending on their infection strategy (Glazebrook, 2005; Nishimura and Dangl, 2010). Necrotrophic pathogens kill living host cells and use the decayed plant tissue as a substrate to colonize the plant, whereas biotrophic pathogens parasitize living plant cells by employing effector molecules that suppress the host immune system (Pel and Pieterse, 2013). Despite this binary classification, the majority of pathogenic microbes employ a hemibiotrophic infection strategy, which is characterized by an initial biotrophic phase followed by a necrotrophic infection strategy at later stages of infection (Perfect and Green, 2001). The pathogenic fungi Magnaporthe grisea, Sclerotinia sclerotiorum, and Mycosphaerella graminicola, the oomycete Phytophthora infestans, and the bacterial pathogen Pseudomonas syringae are examples of hemibiotrophic plant pathogens (Perfect and Green, 2001; Koeck et al., 2011; van Kan et al., 2014; Kabbage et al., 2015).Despite considerable progress in our understanding of plant resistance to necrotrophic and biotrophic pathogens (Glazebrook, 2005; Mengiste, 2012; Lai and Mengiste, 2013), recent debate highlights the dynamic and complex interplay between plant-pathogenic microbes and their hosts, which is raising concerns about the use of infection strategies as a static tool to classify plant pathogens. For instance, the fungal genus Botrytis is often labeled as an archetypal necrotroph, even though there is evidence that it can behave as an endophytic fungus with a biotrophic lifestyle (van Kan et al., 2014). The rice blast fungus Magnaporthe oryzae, which is often classified as a hemibiotrophic leaf pathogen (Perfect and Green, 2001; Koeck et al., 2011), can adopt a purely biotrophic lifestyle when infecting root tissues (Marcel et al., 2010). It remains unclear which signals are responsible for the switch from biotrophy to necrotrophy and whether these signals rely solely on the physiological state of the pathogen, or whether host-derived signals play a role as well (Kabbage et al., 2015).The plant hormones salicylic acid (SA) and jasmonic acid (JA) play a central role in the activation of plant defenses (Glazebrook, 2005; Pieterse et al., 2009, 2012). The first evidence that biotrophic and necrotrophic pathogens are resisted by different immune responses came from Thomma et al. (1998), who demonstrated that Arabidopsis (Arabidopsis thaliana) genotypes impaired in SA signaling show enhanced susceptibility to the biotrophic pathogen Hyaloperonospora arabidopsidis (formerly known as Peronospora parastitica), while JA-insensitive genotypes were more susceptible to the necrotrophic fungus Alternaria brassicicola. In subsequent years, the differential effectiveness of SA- and JA-dependent defense mechanisms has been confirmed in different plant-pathogen interactions, while additional plant hormones, such as ethylene, abscisic acid (ABA), auxins, and cytokinins, have emerged as regulators of SA- and JA-dependent defenses (Bari and Jones, 2009; Cao et al., 2011; Pieterse et al., 2012). Moreover, SA- and JA-dependent defense pathways have been shown to act antagonistically on each other, which allows plants to prioritize an appropriate defense response to attack by biotrophic pathogens, necrotrophic pathogens, or herbivores (Koornneef and Pieterse, 2008; Pieterse et al., 2009; Verhage et al., 2010).In addition to plant hormones, reactive oxygen species (ROS) play an important regulatory role in plant defenses (Torres et al., 2006; Lehmann et al., 2015). Within minutes after the perception of pathogen-associated molecular patterns, NADPH oxidases and apoplastic peroxidases generate early ROS bursts (Torres et al., 2002; Daudi et al., 2012; O’Brien et al., 2012), which activate downstream defense signaling cascades (Apel and Hirt, 2004; Torres et al., 2006; Miller et al., 2009; Mittler et al., 2011; Lehmann et al., 2015). ROS play an important regulatory role in the deposition of callose (Luna et al., 2011; Pastor et al., 2013) and can also stimulate SA-dependent defenses (Chaouch et al., 2010; Yun and Chen, 2011; Wang et al., 2014; Mammarella et al., 2015). However, the spread of SA-induced apoptosis during hyperstimulation of the plant immune system is contained by the ROS-generating NADPH oxidase RBOHD (Torres et al., 2005), presumably to allow for the sufficient generation of SA-dependent defense signals from living cells that are adjacent to apoptotic cells. Nitric oxide (NO) plays an additional role in the regulation of SA/ROS-dependent defense (Trapet et al., 2015). This gaseous molecule can stimulate ROS production and cell death in the absence of SA while preventing excessive ROS production at high cellular SA levels via S-nitrosylation of RBOHD (Yun et al., 2011). Recently, it was shown that pathogen-induced accumulation of NO and ROS promotes the production of azelaic acid, a lipid derivative that primes distal plants for SA-dependent defenses (Wang et al., 2014). Hence, NO, ROS, and SA are intertwined in a complex regulatory network to mount local and systemic resistance against biotrophic pathogens. Interestingly, pathogens with a necrotrophic lifestyle can benefit from ROS/SA-dependent defenses and associated cell death (Govrin and Levine, 2000). For instance, Kabbage et al. (2013) demonstrated that S. sclerotiorum utilizes oxalic acid to repress oxidative defense signaling during initial biotrophic colonization, but it stimulates apoptosis at later stages to advance necrotrophic colonization. Moreover, SA-induced repression of JA-dependent resistance not only benefits necrotrophic pathogens but also hemibiotrophic pathogens after having switched from biotrophy to necrotrophy (Glazebrook, 2005; Pieterse et al., 2009, 2012).Plectosphaerella cucumerina ((P. cucumerina, anamorph Plectosporum tabacinum) anamorph Plectosporum tabacinum) is a filamentous ascomycete fungus that can survive saprophytically in soil by decomposing plant material (Palm et al., 1995). The fungus can cause sudden death and blight disease in a variety of crops (Chen et al., 1999; Harrington et al., 2000). Because P. cucumerina can infect Arabidopsis leaves, the P. cucumerina-Arabidopsis interaction has emerged as a popular model system in which to study plant defense reactions to necrotrophic fungi (Berrocal-Lobo et al., 2002; Ton and Mauch-Mani, 2004; Carlucci et al., 2012; Ramos et al., 2013). Various studies have shown that Arabidopsis deploys a wide range of inducible defense strategies against P. cucumerina, including JA-, SA-, ABA-, and auxin-dependent defenses, glucosinolates (Tierens et al., 2001; Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014), callose deposition (García-Andrade et al., 2011; Gamir et al., 2012, 2014; Sánchez-Vallet et al., 2012), and ROS (Tierens et al., 2002; Sánchez-Vallet et al., 2010; Barna et al., 2012; Gamir et al., 2012, 2014; Pastor et al., 2014). Recent metabolomics studies have revealed large-scale metabolic changes in P. cucumerina-infected Arabidopsis, presumably to mobilize chemical defenses (Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014). Furthermore, various chemical agents have been reported to induce resistance against P. cucumerina. These chemicals include β-amino-butyric acid, which primes callose deposition and SA-dependent defenses, benzothiadiazole (BTH or Bion; Görlach et al., 1996; Ton and Mauch-Mani, 2004), which activates SA-related defenses (Lawton et al., 1996; Ton and Mauch-Mani, 2004; Gamir et al., 2014; Luna et al., 2014), JA (Ton and Mauch-Mani, 2004), and ABA, which primes ROS and callose deposition (Ton and Mauch-Mani, 2004; Pastor et al., 2013). However, among all these studies, there is increasing controversy about the exact signaling pathways and defense responses contributing to plant resistance against P. cucumerina. While it is clear that JA and ethylene contribute to basal resistance against the fungus, the exact roles of SA, ABA, and ROS in P. cucumerina resistance vary between studies (Thomma et al., 1998; Ton and Mauch-Mani, 2004; Sánchez-Vallet et al., 2012; Gamir et al., 2014).This study is based on the observation that the disease phenotype during P. cucumerina infection differs according to the inoculation method used. We provide evidence that the fungus follows a hemibiotrophic infection strategy when infecting from relatively low spore densities on the leaf surface. By contrast, when challenged by localized host defense to relatively high spore densities, the fungus switches to a necrotrophic infection program. Our study has uncovered a novel strategy by which plant-pathogenic fungi can take advantage of the early immune response in the host plant.