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.     

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.     

Grain setting defect1, Encoding a Remorin Protein,Affects the Grain Setting in Rice through Regulating Plasmodesmatal Conductance

Effective grain filling is one of the key determinants of grain setting in rice (Oryza sativa). Grain setting defect1 (GSD1), which encodes a putative remorin protein, was found to affect grain setting in rice. Investigation of the phenotype of a transfer DNA insertion mutant (gsd1-Dominant) with enhanced GSD1 expression revealed abnormalities including a reduced grain setting rate, accumulation of carbohydrates in leaves, and lower soluble sugar content in the phloem exudates. GSD1 was found to be specifically expressed in the plasma membrane and plasmodesmata (PD) of phloem companion cells. Experimental evidence suggests that the phenotype of the gsd1-Dominant mutant is caused by defects in the grain-filling process as a result of the impaired transport of carbohydrates from the photosynthetic site to the phloem. GSD1 functioned in affecting PD conductance by interacting with rice ACTIN1 in association with the PD callose binding protein1. Together, our results suggest that GSD1 may play a role in regulating photoassimilate translocation through the symplastic pathway to impact grain setting in rice.Grain filling, a key determinant of grain yield in rice (Oryza sativa), hinges on the successful translocation of photoassimilates from the leaves to the fertilized reproductive organs through the phloem transport system. Symplastic phloem loading, which is one of the main pathways responsible for the transport of photoassimilates in rice, is mediated by plasmodesmata (PD) that connect phloem companion cells with sieve elements and surrounding parenchyma cells (Kaneko et al., 1980; Chonan et al., 1981; Eom et al., 2012). PD are transverse cell wall channels structured with the cytoplasmic sleeve and the modified endoplasmic reticulum desmotubule between neighboring cells (Maule, 2008). A number of proteins affect the structure and functional performance of the PD, which in turn impacts the cell-to-cell transport of small and large molecules through the PD during plant growth, development, and defense (Cilia and Jackson, 2004; Sagi et al., 2005; Lucas et al., 2009; Simpson et al., 2009; Stonebloom et al., 2009). For example, actin and myosin, which link the desmotubule to the plasma membrane (PM) at the neck region of PD, are believed to play a role in regulating PD permeability by controlling PD aperture (White et al., 1994; Ding et al., 1996; Reichelt et al., 1999). Callose deposition can also impact the size of the PD aperture at the neck region (Radford et al., 1998; Levy et al., 2007) and callose synthase genes such as Glucan Synthase-Like7 (GSL7, also named CalS7), GSL8, and GSL12 have been shown to play a role in regulating symplastic trafficking (Guseman et al., 2010; Barratt et al., 2011; Vatén et al., 2011; Xie et al., 2011). Other proteins that have been shown to impact the structure and function of the PD include glycosylphosphatidylinositol (GPI)-anchored proteins, PD callose binding protein1 (PDCB1), which is also associated with callose deposition (Simpson et al., 2009), and LYSIN MOTIF DOMAIN-CONTAINING GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN2, which limits the molecular flux through the PD by chitin perception (Faulkner et al., 2013). Changes in PD permeability can have major consequences for the translocation of photoassimilates needed for grain filling in rice. However, the genes and molecular mechanisms underlying the symplastic transport of photoassimilates remain poorly characterized.Remorins are a diverse family of plant-specific proteins with conserved C-terminal sequences and highly variable N-terminal sequences. Remorins can be classified into six distinct phylogenetic groups (Raffaele et al., 2007). The functions of most remorins are unknown, but some members of the family have been shown to be involved in immune response through controlling the cell-to-cell spread of microbes. StREM1.3, a remorin that is located in PM rafts and the PD, was shown to impair the cell-to-cell movement of a plant virus X by binding to Triple Gene Block protein1 (Raffaele et al., 2009). Medicago truncatula symbiotic remorin1 (MtSYMREM1), a remorin located at the PM in Medicago truncatula, was shown to facilitate infection and the release of rhizobial bacteria into the host cytoplasm (Lefebvre et al., 2010). Overexpression of LjSYMREM1, the ortholog of MtSYMREM1 in Lotus japonicus, resulted in increased root nodulation (Lefebvre et al., 2010; Tóth et al., 2012). Although a potential association between remorins and PD permeability has been proposed (Raffaele et al., 2009), the diversity observed across remorins, plus the fact that remorin mutants generated through different approaches fail to show obvious phenotypes (Reymond et al., 1996; Bariola et al., 2004), have made it challenging to characterize the function of remorins in cell-to-cell transport.In this study, we identified a rice transfer DNA (T-DNA) insertion mutant (grain setting defect1-Dominant [gsd1-D]), with a grain setting-deficient phenotype caused by overexpression of GSD1, a remorin gene with unknown function. GSD1 is expressed specifically in phloem companion cells and is localized in the PD and PM. We provide evidence to show that overexpression of GSD1 leads to deficient grain setting in rice, likely as a consequence of reduced sugar transport resulting from decreased PD permeability in phloem companion cells.