巴西橡胶Pto类抗病同源序列的克隆与系统发育重建

番茄Pto基因是一类可以编码丝氨酸/苏氨酸激酶(STK)序列的广谱抗性候选基因,其序列克隆与鉴定为深入了解番茄的抗病机制奠定了基础.在该研究中,一对依据Pto基因的保守序列设计的简并引物被用来扩增巴西橡胶中Pto基因抗病同源序列,扩增得到了一个约550 bp的基因片段,其随后被克隆并测序.序列分析发现,其中的7个抗病同源序列与Pto基因高度同源(BLASTX E value <3e-53),所以其被认为是Pto基因抗病同源序列(Pto-RGCs).通过巴西橡胶的Pto-RGCs多序列比对表明,这些序列包含了多个STKs保守的次级结构域.此外,系统发育分析也表明,巴西橡胶的Pto-RGCs属于Pto基因同源的R基因.该研究结果中Pto-RGCs可为巴西橡胶抗病的发展提供一个有效的基因资源.     

Tobacco Mosaic Virus Infection Results in an Increase in Recombination Frequency and Resistance to Viral,Bacterial, and Fungal Pathogens in the Progeny of Infected Tobacco Plants

Our previous experiments showed that infection of tobacco (Nicotiana tabacum) plants with Tobacco mosaic virus (TMV) leads to an increase in homologous recombination frequency (HRF). The progeny of infected plants also had an increased rate of rearrangements in resistance gene-like loci. Here, we report that tobacco plants infected with TMV exhibited an increase in HRF in two consecutive generations. Analysis of global genome methylation showed the hypermethylated genome in both generations of plants, whereas analysis of methylation via 5-methyl cytosine antibodies demonstrated both hypomethylation and hypermethylation. Analysis of the response of the progeny of infected plants to TMV, Pseudomonas syringae, or Phytophthora nicotianae revealed a significant delay in symptom development. Infection of these plants with TMV or P. syringae showed higher levels of induction of PATHOGENESIS-RELATED GENE1 gene expression and higher levels of callose deposition. Our experiments suggest that viral infection triggers specific changes in progeny that promote higher levels of HRF at the transgene and higher resistance to stress as compared with the progeny of unstressed plants. However, data reported in these studies do not establish evidence of a link between recombination frequency and stress resistance.Continuous exposure to stress leads to the evolutionary selection of adaptive traits beneficial in a particular environment. Such selection of the fittest of a population of plants grown under certain environmental conditions is believed to require a long time. However, it is known that plants also possess the ability to acclimate on much shorter time scales. A modification of homeostasis, also termed acclimatization, is a well-documented process that is used for adjusting metabolism to a new environment (Lichtenthaler, 1998; Mullineaux and Emlyn-Jones, 2005).Pathogens represent one of a variety of stresses that plants are constantly exposed to. In nature, the evolution of plant resistance to a particular pathogen, virus, bacterium, or fungus has been the result of constant interactions with said pathogen (McHale et al., 2006; Friedman and Baker, 2007). These interactions lead to a constant plant-pathogen arms race (Ingle et al., 2006).Plants are able to tolerate or resist pathogens in a variety of ways, which could be broadly attributed to mechanisms of innate immunity and actual gene-for-gene-based resistance. The latter one depends on direct or indirect recognition of pathogen avirulence gene products by plant resistance gene products (Whitham et al., 1994; Durrant and Dong, 2004). Pathogen recognition during this incompatible interaction triggers complex events, including a local hypersensitive response that manifests itself as a booster of radical production and activation of the salicylic acid-dependent pathway and necrotic lesions, which working together restrict pathogen spread. It also results in a plant-wide systemic acquired resistance response that provides protection and tolerance to future pathogen attacks (Durrant and Dong, 2004; Park et al., 2007; Vlot et al., 2008).If a functional pathogen resistance gene is absent (compatible interaction), then the interaction between a plant and a pathogen is more ambiguous. How do plants that lack a resistance gene respond to infection? We have previously reported that the compatible interaction between Tobacco mosaic virus (TMV) and tobacco (Nicotiana tabacum ‘SR1’) plants lacking the TMV resistance N gene results in the production of a systemic signal. The signal leads to an increase in the frequency of somatic homologous recombination (HRF; Kovalchuk et al., 2003a). Based on these observations, we hypothesized that these genomic changes could be inherited. Indeed, we found that the progeny of infected SR1 tobacco plants exhibited a higher frequency of RFLPs at the loci that have similarity (more than 60%) to the Leu-rich repeat region of the N gene (Boyko et al., 2007).Although several reports have shown an increase in genome instability in plants exposed to pathogens and pathogen elicitors (Lucht et al., 2002; Kovalchuk et al., 2003a; Molinier et al., 2006; Boyko et al., 2007), many questions still remained unanswered. What is the mechanism of occurrence of a pathogen-induced systemic increase in HRF? What is the mechanism of inheritance of high-frequency homologous recombination? Are elevated levels of HRF maintained throughout generations? What other changes occur in progeny of infected plants?Here, we attempted to answer the above questions by analyzing two consecutive progenies of TMV-infected tobacco cv SR1 plants. Both progenies of infected plants showed higher levels of somatic HRF, higher resistance to TMV infection and tolerance to methyl methane sulfonate (MMS), an increase in callose deposition, as well as a higher steady-state PATHOGENESIS-RELATED GENE1 (PR1) RNA level compared with the progeny of uninfected plants. Analysis of methylation patterns has revealed global genome hypermethylation in both progenies paralleled by hypomethylation in euchromatic areas.     

Identification, Phylogeny, and Expression Analysis of Pto-like Genes in Pepper

The Pto gene from the wild tomato (Solanum pimpinellifolium Mill.) encodes a serine/threonine kinase that plays an important role in bacterial speck resistance in the cultivated tomato (Solanum lycopersicum Mill.). In this paper, 10 classes of Pto-like genes are identified using degenerate polymerase chain reaction (PCR) primers and database mining in pepper. Sequences alignment reveals that many features of the gene family, such as subdomains, autophosphorylation sites, and important amino acid residues for tomato Pto, are well conserved in pepper. A phylogenetic tree of pepper Pto-like genes along with those of other plant species, including tomato Pto genes, suggests that these genes share a common evolutionary origin, and they may have evolved prior to the divergence of monocotyledonous and dicotyledonous plants. Expression analysis has revealed that nine selected Pto-like genes can be detected in at least one of the tissues grown under normal growth conditions; however, these genes are differentially expressed. In addition, some of these genes are regulated by at least one of the subjected treatments, including hormones, abiotic stress, and pathogen infection. These findings will contribute to expanding our knowledge of the roles of Pto-like genes in growth, development, and stress tolerance in pepper.     

Arabidopsis Actin-Depolymerizing Factor AtADF4 Mediates Defense Signal Transduction Triggered by the Pseudomonas syringae Effector AvrPphB

The actin cytoskeleton has been implicated in plant defenses against pathogenic fungi and oomycetes with limited, indirect evidence. To date, there are no reports linking actin with resistance against phytopathogenic bacteria. The dynamic behavior of actin filaments is regulated by a diverse array of actin-binding proteins, among which is the Actin-Depolymerizing Factor (ADF) family of proteins. Here, we demonstrate that actin dynamics play a role in the activation of gene-for-gene resistance in Arabidopsis (Arabidopsis thaliana) following inoculation with the phytopathogenic bacterium Pseudomonas syringae pv tomato. Using a reverse genetics approach, we explored the roles of Arabidopsis ADFs in plant defenses. AtADF4 was identified as being specifically required for resistance triggered by the effector AvrPphB but not AvrRpt2 or AvrB. Recombinant AtADF4 bound to monomeric actin (G-actin) with a marked preference for the ADP-loaded form and inhibited the rate of nucleotide exchange on G-actin, indicating that AtADF4 is a bona fide actin-depolymerizing factor. Exogenous application of the actin-disrupting agent cytochalasin D partially rescued the Atadf4 mutant in the AvrPphB-mediated hypersensitive response, demonstrating that AtADF4 mediates defense signaling through modification of the actin cytoskeleton. Unlike the mechanism by which the actin cytoskeleton confers resistance against fungi and oomycetes, AtADF4 is not involved in resistance against pathogen entry. Collectively, this study identifies AtADF4 as a novel component of the plant defense signaling pathway and provides strong evidence for actin dynamics as a primary component that orchestrates plant defenses against P. syringae.The actin cytoskeleton has been implicated in plant defenses against pathogenic fungi and oomycetes (Hardham et al., 2007). Evidence largely comes from studies using actin cytoskeleton-disrupting agents, such as cytochalasins. Treatments with a variety of cytochalasins were shown to increase the penetration rate of both adapted and nonadapted pathogens in multiple plant-pathogen systems, thereby implicating the actin cytoskeleton as having a role in basal defenses and nonhost resistance (Kobayashi et al., 1997; Yun et al., 2003; Shimada et al., 2006; Miklis et al., 2007). The actin cytoskeleton may also play a role in race-specific resistance (Skalamera and Heath, 1998). To date, no reports linking actin dynamics with resistance against phytopathogenic bacteria have been published.While the actin cytoskeleton as a virulence target of plant pathogens has not been documented, it was well characterized in mammalian pathosystems, particularly in studies investigating macrophage interactions with the pathogenic bacterium Yersinia pestis (Mattoo et al., 2007). Yersinia species deliver a suite of effectors into the target host cell, and at least four of them (YopE, YpkA/YopO, YopT, and YopH) are involved in rearrangement of the actin cytoskeleton (Aepfelbacher and Heesemann, 2001). YopT, a Cys protease, targets a plasma membrane-localized Rho GTPase in affected phagocytes (Aepfelbacher and Heesemann, 2001). Cleavage of the GTPase by YopT releases the prenylated protein from the plasma membrane and disrupts the actin cytoskeleton, effectively shutting down phagocytosis, preventing elimination of the pathogen (Iriarte and Cornelis, 1998; Shao et al., 2002). Similarly, microbial pathogens also usurp host processes for the benefit of infection, disease, and death. Listeria species hijack the host''s cytoskeleton to move around inside the infected cell through the induction of directed polymerization of actin (Pistor et al., 1994). Salmonella injects into host cells two actin-binding proteins (SipA and SipC) as well as other regulators of actin dynamics to enhance phagocytic uptake and intracellular propagation (Galan and Zhou, 2000). In short, either by preventing polymerization or by promoting it, pathogens have evolved strategies to modify the host actin cytoskeleton for purposes of evading detection or eliciting disease and death.Dynamic actin cytoskeleton rearrangements are regulated by a pool of actin-binding proteins, which sense environmental changes and modulate the cytoskeleton through various biochemical activities (Hussey et al., 2006; Staiger and Blanchoin, 2006). Among the proteins that regulate these dynamic processes are the Actin-Depolymerizing Factor (ADF) family of proteins (Maciver and Hussey, 2002). In general, ADFs bind both monomeric (G-) and filamentous (F-) actin to increase actin dynamics. They function by severing F-actin to generate more ends for polymerization and by increasing the dissociation rate of actin monomers from the pointed ends (Maciver, 1998; Maciver and Hussey, 2002). Plant ADFs play roles in pollen tube growth (Chen et al., 2003), root formation (Thomas and Schiefelbein, 2002), and cold acclimation (Ouellet et al., 2001). There is also one report linking ADFs with plant defenses (Miklis et al., 2007). In that study, ectopic expression of barley (Hordeum vulgare) HvADF3 and several isovariants of Arabidopsis (Arabidopsis thaliana) ADFs in barley epidermal cells was shown to compromise penetration resistance to powdery mildew fungi (Miklis et al., 2007).The Arabidopsis-Pseudomonas syringae interaction provides an ideal model plant-pathogen system to study plant defense signaling. Like Yersinia species, P. syringae delivers effector proteins into the host cells via the type III secretion system and relies on these proteins for pathogenesis (Alfano and Collmer, 2004). However, once these proteins (Avr) are recognized either directly or indirectly by plant resistance (R) proteins, plant immune responses are activated (Jones and Dangl, 2006). Exciting progress has been made toward understanding the indirect recognition of several pairs of Avr-R proteins; the best examples include AvrB/AvrRPM1-RPM1, AvrRpt2-RPS2, and AvrPphB-RPS5. During activation of defense mediated by AvrB/AvrRPM1-RPM1 and AvrRpt2-RPS2, the phosphorylation or elimination of a third protein, RIN4, is essential (Mackey et al., 2002; Axtell and Staskawicz, 2003). In the case of AvrPphB-RPS5 recognition, the AvrPphB Cys protease of the same family as YopT (Shao et al., 2002) cleaves the plant protein kinase PBS1, inducing a conformational change in RPS5, which in turn leads to the activation of resistance (Ade et al., 2007). Although these studies have greatly enhanced our understanding of how pathogen effectors initiate plant defense responses, the ultimate signaling processes associated with the activation of resistance remain largely unknown, due to the limited number of genetic loci identified in these pathways. In this work, we hypothesize that actin-binding proteins play a role during plant-bacteria interactions based on the functional and structural similarity between AvrPphB and YopT.There are 11 ADFs in the Arabidopsis genome (Ruzicka et al., 2007). We utilized a reverse genetics approach to identify the putative roles these proteins play in plant resistance against the bacterial pathogen P. syringae pv tomato (Pst). AtADF4 was identified as a novel signaling component in the AvrPphB-RPS5-mediated defense signal transduction pathway. Loss of AtADF4 confers on Arabidopsis enhanced susceptibility to P. syringae expressing AvrPphB. Further subcellular localization and biochemical analyses, as well as pharmacological studies, suggest that AtADF4 functions as a bona fide actin-depolymerizing factor through modifying the actin cytoskeleton. Unlike the documented mechanism by which the actin cytoskeleton plays roles in resistance against fungi and oomycetes, the resistance against P. syringae mediated by AtADF4 is not involved in hindering pathogen entry.     

The Pseudomonas syringae Effector HopQ1 Promotes Bacterial Virulence and Interacts with Tomato 14-3-3 Proteins in a Phosphorylation-Dependent Manner

A key virulence strategy of bacterial pathogens is the delivery of multiple pathogen effector proteins into host cells during infection. The Hrp outer protein Q (HopQ1) effector from Pseudomonas syringae pv tomato (Pto) strain DC3000 is conserved across multiple bacterial plant pathogens. Here, we investigated the virulence function and host targets of HopQ1 in tomato (Solanum lycopersicum). Transgenic tomato lines expressing dexamethasone-inducible HopQ1 exhibited enhanced disease susceptibility to virulent Pto DC3000, the Pto ΔhrcC mutant, and decreased expression of a pathogen-associated molecular pattern-triggered marker gene after bacterial inoculation. HopQ1-interacting proteins were coimmunoprecipitated and identified by mass spectrometry. HopQ1 can associate with multiple tomato 14-3-3 proteins, including TFT1 and TFT5. HopQ1 is phosphorylated in tomato, and four phosphorylated peptides were identified by mass spectrometry. HopQ1 possesses a conserved mode I 14-3-3 binding motif whose serine-51 residue is phosphorylated in tomato and regulates its association with TFT1 and TFT5. Confocal microscopy and fractionation reveal that HopQ1 exhibits nucleocytoplasmic localization, while HopQ1 dephosphorylation mimics exhibit more pronounced nuclear localization. HopQ1 delivered from Pto DC3000 was found to promote bacterial virulence in the tomato genotype Rio Grande 76R. However, the HopQ1(S51A) mutant delivered from Pto DC3000 was unable to promote pathogen virulence. Taken together, our data demonstrate that HopQ1 enhances bacterial virulence and associates with tomato 14-3-3 proteins in a phosphorylation-dependent manner that influences HopQ1’s subcellular localization and virulence-promoting activities in planta.The ability to detect and mount a defense response against pathogenic microbes is vital for plant survival. Plants rely on both passive and active defenses to ward off microbial pathogens. Physical barriers, such as the cell wall and cuticle, as well as chemical barriers provide a first line of defense against microbial colonization. Unlike animals, plants do not possess a circulating immune system and rely on innate immunity for active defenses against microbial pathogens (Spoel and Dong, 2012). Plants use surface-localized receptors to recognize conserved pathogen-associated molecular patterns (PAMPs), such as bacterial flagellin, resulting in pattern-triggered immunity (PTI; Zipfel et al., 2006). Plants also use primarily intracellular nucleotide-binding domain, Leu-rich repeat containing (NLR) immune receptors to recognize pathogen effectors delivered into host cells during infection (Spoel and Dong, 2012). NLR activation results in effector-triggered immunity (ETI). A signature of ETI is the hypersensitive response (HR), a form of programmed cell death occurring at the site of infection.In order to cause disease and suppress host defense responses, gram-negative bacterial pathogens deliver effector proteins into host cells via the type III secretion system (TTSS). Plant pathogenic bacteria deliver a large number (20–40) of effectors into host cells during infection (Cui et al., 2009). Collectively, effectors are required for bacterial virulence (Lindgren et al., 1986). However, knockouts affecting individual effectors frequently have phenotypes that are subtle, likely due to functional redundancy (Cunnac et al., 2011). Alternatively, individual effectors may play an important role in bacterial survival under conditions that are not typically analyzed in the laboratory or act cooperatively with one another. Progress in understanding individual effectors’ contributions to virulence has been made by generating transgenic plants that express effectors. Multiple effectors have been shown to suppress plant innate immunity and promote bacterial growth when either transiently or stably expressed in plants (Jamir et al., 2004; Guo et al., 2009). Effector expression can also result in avirulent phenotypes when a plant NLR receptor recognizes a cognate effector and mounts an HR. Such an HR phenotype can be used to dissect important effector domains required for plant recognition and enzymatic activity.Elucidating effector targets and enzymatic activity is necessary in order to understand how they act to subvert plant immune responses and can provide elegant insight into biological processes. Significant progress has been made in elucidating the enzymatic activity of a subset of effectors. Some of the most well-characterized effectors come from Pseudomonas syringae pv tomato (Pto), the causal agent of bacterial speck on tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). Multiple effectors can suppress immune responses by directly targeting PAMP receptors (AvrPto and AvrPtoB) or by interfering with downstream signaling processes (AvrB, AvrPphB, and HopAI1; Cui et al., 2009, 2010). The HopU1 effector interferes with RNA metabolism (Fu et al., 2007), and the HopI1 effector targets heat-shock proteins in the plant chloroplast (Jelenska et al., 2010).14-3-3s are conserved eukaryotic proteins that bind a diverse set of phosphorylated client proteins, typically at one of three distinct 14-3-3 binding motifs (Bridges and Moorhead, 2005). There are common recognition motifs for 14-3-3 proteins that contain phosphorylated Ser or Thr residues, but binding to nonphosphorylated ligands and to proteins lacking consensus motifs has been reported (Henriksson et al., 2002; Smith et al., 2011). The 14-3-3 mode I consensus motif is RXXpS/pTX and that of mode II is RXXXpS/pTXP, where X can be any amino acid and p indicates the site of phosphorylation (Smith et al., 2011). 14-3-3 proteins can also bind to the extreme C termini of proteins at the RXXpS/pTX-COOH mode III consensus motif (Smith et al., 2011). Interaction with 14-3-3s can regulate protein activity by influencing client subcellular localization, structure, and protein-protein interactions (Bridges and Moorhead, 2005). Recently, the Xanthomonas campestris XopN effector was shown to target tomato 14-3-3 isoforms, which facilitates its interaction with the tomato atypical receptor kinase1 and suppresses PTI (Kim et al., 2009; Taylor et al., 2012). Other 14-3-3s have also been shown to play a role during plant defense responses. The tomato TFT7 14-3-3 interacts with multiple mitogen-activated protein kinases to positively regulate HR induced by ETI (Oh and Martin, 2011). The Arabidopsis 14-3-3 isoform λ interacts with the RPW8.2 powdery mildew receptor and is required for complete RPW8.2-mediated resistance (Yang et al., 2009).In this study, we investigated the function of the Pto HopQ1 (for Hrp outer protein Q [also known as HopQ1-1]) effector in tomato. HopQ1 is an active effector that is transcribed and translocated via the TTSS (Schechter et al., 2004). HopQ1 induces cell death when expressed in Nicotiana benthamiana and therefore contributes to differences in host range in P. syringae pathovars on Nicotiana spp. (Wei et al., 2007; Ferrante et al., 2009). HopQ1 was also reported to slightly enhance disease symptoms (approximately 0.2 log) and bacterial virulence on bean (Phaseolus vulgaris) when expressed from P. syringae pv tabaci (Ferrante et al., 2009). Here, we generated transgenic tomato plants expressing HopQ1 that exhibited enhanced susceptibility to virulent Pto as well as the Pto ΔhrcC mutant. HopQ1-interacting proteins were identified from tomato using coimmunoprecipitations coupled with mass spectrometry. Multiple 14-3-3 proteins were identified. HopQ1 possesses a 14-3-3 binding motif whose Ser residue is phosphorylated in planta and affects its association with the tomato 14-3-3s TFT1 and TFT5. Mutation of HopQ1’s 14-3-3 binding motif affected its ability to promote bacterial virulence. Taken together, these results indicate that phosphorylation and subsequent interaction with tomato 14-3-3 proteins affect HopQ1’s virulence-promoting activities and subcellular localization.     

Schema-based learning of adaptable and flexible prey-catching in anurans I. The basic architecture

A motor action often involves the coordination of several motor synergies and requires flexible adjustment of the ongoing execution based on feedback signals. To elucidate the neural mechanisms underlying the construction and selection of motor synergies, we study prey-capture in anurans. Experimental data demonstrate the intricate interaction between different motor synergies, including the interplay of their afferent feedback signals (Weerasuriya 1991; Anderson and Nishikawa 1996). Such data provide insights for the general issues concerning two-way information flow between sensory centers, motor circuits and periphery in motor coordination. We show how different afferent feedback signals about the status of the different components of the motor apparatus play a critical role in motor control as well as in learning. This paper, along with its companion paper, extend the model by Liaw et al. (1994) by integrating a number of different motor pattern generators, different types of afferent feedback, as well as the corresponding control structure within an adaptive framework we call Schema-Based Learning. We develop a model of the different MPGs involved in prey-catching as a vehicle to investigate the following questions: What are the characteristic features of the activity of a single muscle? How can these features be controlled by the premotor circuit? What are the strategies employed to generate and synchronize motor synergies? What is the role of afferent feedback in shaping the activity of a MPG? How can several MPGs share the same underlying circuitry and yet give rise to different motor patterns under different input conditions? In the companion paper we also extend the model by incorporating learning components that give rise to more flexible, adaptable and robust behaviors. To show these aspects we incorporate studies on experiments on lesions and the learning processes that allow the animal to recover its proper functioning     

Genetic characterization of the Pto locus of tomato: semi-dominance and cosegregation of resistance to Pseadomonas syringae pathovar tomato and sensitivity to the insecticide Fenthion

The Pto locus governs resistance to bacterial speck disease in tomato caused by race 0 strains of Pseudomonas syringae pathovar tomato (Pst). Large populations segregating for the Pto locus were generated and genetically characterized. Analysis of the locus has revealed that Pto acts in a semi-dominant manner and cosegegrates with sensitivity to an organophosphorous insecticide, Fenthion, suggesting that Pto may be a complex locus responsible for both phenotypes. We have redefined its map position on chromosome five of the classical genetic map and assigned its position on the molecular map, thus facilitating the alignment of the two genetic maps of the short arm of chromosome five of tomato. Furthermore, we have screened random amplified polymorphic (RAPD) markers for their ability to differentiate near-isogenic lines that differ only with respect to Pto and have identified and mapped seven of these markers. Our results suggest that Pto may be located in a euchromatic region on chromosome five which will be advantageous for the cloning of this locus by one of several molecular strategies.     

The molecular bases of plant resistance and defense responses to aphid feeding: current status

Plant genes participating in the recognition of aphid herbivory in concert with plant genes involved in defense against herbivores mediate plant resistance to aphids. Several such genes involved in plant disease and nematode resistance have been characterized in detail, but their existence has only recently begun to be determined for arthropod resistance. Hundreds of different genes are typically involved and the disruption of plant cell wall tissues during aphid feeding has been shown to induce defense responses in Arabidopsis, Triticum, Sorghum, and Nicotiana species. Mi‐1.2, a tomato gene for resistance to the potato aphid, Macrosiphum euphorbiae (Thomas), is a member of the nucleotide‐binding site and leucine‐rich region Class II family of disease, nematode, and arthropod resistance genes. Recent studies into the differential expression of Pto‐ and Pti1‐like kinase genes in wheat plants resistant to the Russian wheat aphid, Diuraphis noxia (Mordvilko), provide evidence of the involvement of the Pto class of resistance genes in arthropod resistance. An analysis of available data suggests that aphid feeding may trigger multiple signaling pathways in plants. Early signaling includes gene‐for‐gene recognition and defense signaling in aphid‐resistant plants, and recognition of aphid‐inflicted cell damage in both resistant and susceptible plants. Furthermore, signaling is mediated by several compounds, including jasmonic acid, salicylic acid, ethylene, abscisic acid, giberellic acid, nitric oxide, and auxin. These signals lead to the development of direct chemical defenses against aphids and general stress‐related responses that are well characterized for a number of abiotic and biotic stresses. In spite of major plant taxonomic differences, similarities exist in the types of plant genes expressed in response to feeding by different species of aphids. However, numerous differences in plant signaling and defense responses unique to specific aphid–plant interactions have been identified and warrant further investigation.     

Pto mutants differentially activate Prf-dependent,avrPto-independent resistance and gene-for-gene resistance

Pto confers disease resistance to Pseudomonas syringae pv tomato carrying the cognate avrPto gene. Overexpression of Pto under the cauliflower mosaic virus 35S promoter activates spontaneous lesions and confers disease resistance in tomato (Lycopersicon esculentum) plants in the absence of avrPto. Here, we show that these AvrPto-independent defenses require a functional Prf gene. Several Pto-interacting (Pti) proteins are thought to play a role in Pto-mediated defense pathways. To test if interactions with Pti proteins are required for the AvrPto-independent defense responses by Pto overexpression, we isolated several Pto mutants that were unable to interact with one or more Pti proteins, but retained normal interaction with AvrPto. Overexpression of two mutants, Pto(G50S) and Pto(R150S), failed to activate AvrPto-independent defense responses or confer enhanced resistance to the virulent P. s. pv tomato. When introduced into plants carrying 35S::Pto, 35S::Pto(G50S) dominantly suppressed the AvrPto-independent resistance caused by former transgene. 35S::Pto(G50S) also blocked the induction of a number of defense genes by the wild-type 35S::Pto. However, 35S::Pto(G50S) and 35S::Pto(R150S) plants were completely resistant to P. s. pv tomato (avrPto), indicating a normal gene-for-gene resistance. Furthermore, 35S::Pto(G50S) plants exhibited normal induction of defense genes in recognition of avrPto. Thus, the AvrPto-independent defense activation and gene-for-gene resistance mediated by Pto are functionally separable.     

Abscisic Acid Deficiency Causes Changes in Cuticle Permeability and Pectin Composition That Influence Tomato Resistance to Botrytis cinerea

A mutant of tomato (Solanum lycopersicum) with reduced abscisic acid (ABA) production (sitiens) exhibits increased resistance to the necrotrophic fungus Botrytis cinerea. This resistance is correlated with a rapid and strong hydrogen peroxide-driven cell wall fortification response in epidermis cells that is absent in tomato with normal ABA production. Moreover, basal expression of defense genes is higher in the mutant compared with the wild-type tomato. Given the importance of this fast response in sitiens resistance, we investigated cell wall and cuticle properties of the mutant at the chemical, histological, and ultrastructural levels. We demonstrate that ABA deficiency in the mutant leads to increased cuticle permeability, which is positively correlated with disease resistance. Furthermore, perturbation of ABA levels affects pectin composition. sitiens plants have a relatively higher degree of pectin methylesterification and release different oligosaccharides upon inoculation with B. cinerea. These results show that endogenous plant ABA levels affect the composition of the tomato cuticle and cell wall and demonstrate the importance of cuticle and cell wall chemistry in shaping the outcome of this plant-fungus interaction.Plant defense against pathogens often involves the induction of mechanisms after pathogen recognition, including defense signaling, cell wall strengthening, and localized cell death, but plants also have preformed chemical and structural defense barriers. Fungal pathogens that penetrate the plant tissue directly through the outer surface, rather than via natural plant openings or wounds, must pass through the plant cuticle and epidermal cell wall. Penetration of the host surface happens either by physical means (i.e. by a highly localized pressure in the appressorium) or by chemical means (i.e. by the release of hydrolyzing enzymes). Necrotrophic plant pathogens like Botrytis cinerea typically use the latter strategy. During penetration, they produce cutinases and pectinolytic enzymes such as pectin methylesterases, endopolygalacturonases, and exopolygalacturonases (van Kan, 2006).The cuticle is a hydrophobic barrier that covers the aerial surfaces of the plant. It is mainly composed of cutin, a polyester matrix, and soluble waxes, a complex mixture of hydrophobic material containing very-long-chain fatty acids and their derivatives, embedded into and deposited onto the cutin matrix. It plays an important role in organ development and protection against water loss (Yephremov et al., 1999; Sieber et al., 2000; Kurata et al., 2003; Jung et al., 2006). The cuticle is generally considered as a mere passive physical barrier against pathogen invasion, but it has also been recognized as a potential source of signaling and elicitor molecules (Jenks et al., 1994; Reina-Pinto and Yephremov, 2009). Plant cutin monomers trigger cutinase secretion in pathogenic fungi (Woloshuk and Kolattukudy, 1986), and cutin and wax components initiate appressorium formation and penetration in appressorium-forming pathogens (Kolattukudy et al., 1995; Francis et al., 1996; Gilbert et al., 1996; Fauth et al., 1998; Dickman et al., 2003). In plants, cutin monomers induce pathogenesis-related gene expression and elicit hydrogen peroxide (H₂O₂) synthesis (Fauth et al., 1998; Kim et al., 2008; Park et al., 2008). Transgenic tomato (Solanum lycopersicum) plants expressing the yeast Δ-9 desaturase gene had high levels of cutin monomers that inhibited powdery mildew (Erysiphe polygoni) spore germination, leading to enhanced resistance (Wang et al., 2000). Arabidopsis (Arabidopsis thaliana) plants expressing a fungal cutinase or mutants with a defective cuticle, such as long-chain acyl-CoA synthetase2 and bodyguard, are generally more susceptible to bacteria and equally susceptible to biotrophic fungi but are surprisingly resistant to B. cinerea (Bessire et al., 2007; Chassot et al., 2007; Tang et al., 2007). It has been postulated that a defective or thin cuticle encourages these plants to constitutively express defense-related mechanisms and to secrete antifungal compounds to the plant surface, thereby inhibiting B. cinerea growth (Bessire et al., 2007; Chassot et al., 2007). In addition, cuticle metabolic pathways might directly modulate plant-pathogen interactions by interacting with hormonally regulated defense pathways (Fiebig et al., 2000; Garbay et al., 2007; Mang et al., 2009) or with complex lipid signaling pathways leading to hypersensitive cell death (Raffaele et al., 2008).Once plant pathogens have penetrated the cuticle, they secrete hydrolases that target the plant cell wall (ten Have et al., 1998; Oeser et al., 2002; Vogel et al., 2002; Jakob et al., 2007) that is mainly composed of cellulose, hemicellulose, and pectin (35% of total dry weight). Pectin consists mainly of the polysaccharides homogalacturonan and rhamnogalacturonan I and II. Homogalacturonans are linear chains of α-(1–4)-linked d-GalA residues that can be methylesterified at C-6. Rhamnogalacturonan I and II are more complex, branched polysaccharides. B. cinerea is typically regarded as a pectinolytic pathogen because it possesses an efficient pectinolytic machinery, including a variety of polygalacturonases and pectin methylesterases (PMEs), some of which are important virulence factors (ten Have et al., 1998, 2001; Valette-Collet et al., 2003; Kars et al., 2005). Pectins are a rich source of oligogalacturonides (OGAs), biologically active signaling molecules that can activate plant defense mechanisms (Hahn et al., 1981; Côté and Hahn, 1994; Messiaen and Van Cutsem, 1994; Ridley et al., 2001). The eliciting capacity of the OGAs was shown to depend on their size, which in turn is influenced by the methylesterification pattern of the homogalacturonan fraction (Mathieu et al., 1991; Messiaen and Van Cutsem, 1994). To counteract the activity of fungal pectinases, many plants express polygalacturonase-inhibiting proteins and PME inhibitors, which are localized in the cell wall. The role of these proteins in plant defense against B. cinerea has been extensively demonstrated (Powell et al., 2000; Ferrari et al., 2003; Sicilia et al., 2005; Joubert et al., 2006, 2007; Lionetti et al., 2007). The interaction with the inhibitors not only limits the destructive potential of polygalacturonases but also leads to the accumulation of elicitor-active OGAs (De Lorenzo and Ferrari, 2002). How OGAs are perceived by the plant is still unclear, but in view of the diversity of biological activities and structure requirements, they are thought to be recognized through different proteins, including receptor-like kinases, wall-associated kinases, arabinogalactan proteins, and Pro-rich proteins (Côté and Hahn, 1994; Showalter, 2001; Humphrey et al., 2007).Over the past years, the role of abscisic acid (ABA) in plant-pathogen interactions has gained increased attention. ABA is mostly negatively correlated with resistance against phytopathogens through down-regulation of defense responses orchestrated by salicylic acid, jasmonic acid, and ethylene (Mohr and Cahill, 2001; Audenaert et al., 2002; Mauch-Mani and Mauch, 2005; Asselbergh et al., 2008). In tomato, the ABA-deficient mutant sitiens has an enhanced resistance to B. cinerea (Audenaert et al., 2002) that depends on a timely, localized oxidative burst leading to rapid epidermal cell wall fortification and a faster and higher induction of defense-related gene expression upon infection compared with the wild type (Asselbergh et al., 2007). Moreover, basal defense gene expression is higher in this mutant than in the wild type. As this early response is of vital importance for the resistant reaction of tomato against B. cinerea, we investigated whether alterations in cuticle and/or cell wall, which form the first barrier to the invading pathogen, affect resistance. We demonstrate that the sitiens cuticle is more permeable and that permeability is positively correlated with resistance to B. cinerea. Furthermore, differences in pectin composition and rate of methylesterification occur. Together, these data hint at an unanticipated role for extracellular matrix components in the resistance of tomato against B. cinerea and thus shed new light on the largely unexplored interrelationship between the extracellular matrix and plant-pathogen interactions.     

Activation of Plant Immune Responses by a Gain-of-Function Mutation in an Atypical Receptor-Like Kinase

Arabidopsis (Arabidopsis thaliana) suppressor of npr1-1, constitutive1 (snc1) contains a gain-of-function mutation in a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat Resistance (R) protein and it has been a useful tool for dissecting R-protein-mediated immunity. Here we report the identification and characterization of snc4-1D, a semidominant mutant with snc1-like phenotypes. snc4-1D constitutively expresses defense marker genes PR1, PR2, and PDF1.2, and displays enhanced pathogen resistance. Map-based cloning of SNC4 revealed that it encodes an atypical receptor-like kinase with two predicted extracellular glycerophosphoryl diester phosphodiesterase domains. The snc4-1D mutation changes an alanine to threonine in the predicted cytoplasmic kinase domain. Wild-type plants transformed with the mutant snc4-1D gene displayed similar phenotypes as snc4-1D, suggesting that the mutation is a gain-of-function mutation. Epistasis analysis showed that NON-RACE-SPECIFIC DISEASE RESISTANCE1 is required for the snc4-1D mutant phenotypes. In addition, the snc4-1D mutant phenotypes are partially suppressed by knocking out MAP KINASE SUBSTRATE1, a positive defense regulator associated with MAP KINASE4. Furthermore, both the morphology and constitutive pathogen resistance of snc4-1D are partially suppressed by blocking jasmonic acid synthesis, suggesting that jasmonic acid plays an important role in snc4-1D-mediated resistance. Identification of snc4-1D provides us a unique genetic system for analyzing the signal transduction pathways downstream of receptor-like kinases.Receptor-like kinases (RLKs) are a large group of kinases with a variable extracellular domain and a cytoplasmic kinase domain linked by a single transmembrane motif. RLKs have been shown to play diverse roles in regulating plant innate immunity as well as growth and development (Morillo and Tax, 2006). The extracellular domains of RLKs are believed to bind directly to ligands to perceive extracellular signals, whereas the cytoplasmic kinase domains transduce these signals into the cell. There are over 600 RLKs (Shiu and Bleecker, 2001) in Arabidopsis (Arabidopsis thaliana). The biological functions of most RLKs are unknown.Several RLKs have been identified to be receptors of microbe-associated molecular patterns (MAMPs). FLS2 and EFR are two well-characterized RLKs with extracellular Leu-rich repeats (LRRs) that recognize bacterial flagellin and translation elongation factor EF-Tu, respectively (Gomez-Gomez and Boller, 2000; Zipfel et al., 2006). BAK1 is also an RLK with extracellular LRRs. BAK1 seems to function as an adaptor protein for multiple RLKs including BRI1, FLS2, and BIR1 (Li et al., 2002; Nam and Li, 2002; Chinchilla et al., 2007; Heese et al., 2007; Gao et al., 2009). Interestingly, knocking out BIR1 activates cell death and defense responses mediated by another RLK, SOBIR1 (Gao et al., 2009). Recently, the rice (Oryza sativa) RLK Xa21 was also suggested to be a MAMP receptor. Xa21 recognizes a peptide derived from the secreted effector protein AvrXa21, which is conserved among different Xanthomonas species (Lee et al., 2009). Unlike FLS2, EFR, and Xa21, the putative receptor for chitin is an RLK with three extracellular LysM domains instead of LRRs that are required for the perception of chitin as well as resistance against bacterial pathogens (Miya et al., 2007; Wan et al., 2008; Gimenez-Ibanez et al., 2009).Perception of MAMPs by receptors leads to the rapid activation of mitogen-activated protein (MAP) kinases including MAP KINASE3 (MPK3), MPK4, and MPK6 (Boller and Felix, 2009). MAP KINASE SUBSTRATE1 (MKS1) was identified as an MPK4-interacting protein that positively regulates defense responses (Andreasson et al., 2005). Silencing of MKS1 compromises basal resistance to Pseudomonas syringae pv tomato DC3000, whereas overexpression of MKS1 leads to enhanced pathogen resistance. Activation of MAMP receptors also induces a number of responses such as oxidative burst, callose deposition, and increased salicylic acid synthesis (Boller and Felix, 2009). Defense responses induced by different MAMPs seem similar, suggesting that they may share common signaling components. Identification of the signaling components downstream of RLK receptors remains a major task in understanding MAMP-triggered immunity.Here we report the identification and characterization of suppressor of npr1-1, constitutive4-1D (snc4-1D), a gain-of-function mutant of an atypical RLK that is autoactivated by a mutation in its kinase domain. The snc4-1D mutant plants constitutively express defense maker genes PR1, PR2, and PDF1.2, and display enhanced resistance to Hyaloperonospora arabidopsidis (H. a.) Noco2. Epistasis analysis showed that snc4-1D-mediated defense responses are dependent on multiple factors including NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), MKS1, and OPR3.     

Purification and Characterization of a NADPH-Dependent Aldehyde Reductase from Mung Bean That Detoxifies Eutypine, a Toxin from Eutypa lata

Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a K_m value of 6.3 μm for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification.Many pathogenic bacteria and fungi produce toxins that interfere with various functions of plant cells and may affect plant defense mechanisms (Durbin, 1981). Toxin production is commonly associated with disease severity and can be involved in colonization or systemic invasion by the pathogen (Schäfer, 1994). Toxin resistance has been shown in most cases to be based on the ability of the plant to metabolically detoxify pathogen toxins (Meeley and Walton, 1991; Zhang and Birch, 1997; Zweimuller et al., 1997). Few cloned toxin-resistance genes that encode proteins involved in detoxification mechanisms have been described (Utsumi et al., 1988; Johal and Briggs, 1992; Zhang and Birch, 1997). In many cases a relationship exists between toxin tolerance and resistance to the disease (Anzai et al., 1989; Meeley et al., 1992). The availability of toxin-resistance genes will permit a greater understanding of the mechanisms causing plant disease and will also set the stage for engineering resistance to plant disease (Keen, 1993).Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by the ascomycete fungus Eutypa lata (Pers.: Fr.) Tul., the causal agent of eutypa dieback (Tey-Rulh et al., 1991). This disease is responsible for considerable loss in yield and is the most devastating disease of grapevine (Vitis vinifera) in many countries (Moller and Kasamitis, 1981; Munkvold et al., 1994). The fungus infects the stock through pruning wounds and is present in the xylem and phloem of the vine trunk and branches (Moller and Kasamitis, 1978; Duthie et al., 1991). After a long incubation period, a canker forms around the infected wound. The toxin synthesized by the fungus in the trunk is believed to be transported by the sap to the herbaceous parts of the vine (Fallot et al., 1997). Eutypine penetrates grapevine cells through passive diffusion and its accumulation in the cytoplasm has been explained by an ion-trapping mechanism related to the ionization state of the molecule (Deswarte et al., 1996b). In the cell the effects of eutypine include reduction of adenylated nucleotide content, inhibition of succinate dehydrogenase, uncoupling of oxidative phosphorylation, and mitochondrial swelling (Deswarte et al., 1996a).Symptoms of eutypa dieback in the herbaceous part of the plant lead to dwarfed and withered new growth of branches, marginal necrosis of the leaves, dryness of the inflorescence, and, finally, death of one or more branches (Moller and Kasamitis, 1981). The toxin appears to be an important virulence factor involved in symptom development of the disease (Deswarte et al., 1996a). However, the absence of toxin-deficient mutants of the fungus and its long incubation period in the trunk before symptom development have prevented a critical study of the toxin in vine plants. Determining the gene responsible for eutypine resistance would therefore be an important critical tool in determining the role of eutypine toxin in symptom development in the disease; and it has the potential to confer resistance to transgenic grapevines.Recently, Colrat et al. (1998) found detoxification to occur in grapevine cells through the enzymatic reduction of eutypine into its corresponding alcohol, eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol). We have determined that this derivative of the toxin is nontoxic for grapevine tissues. Furthermore, we have established a relationship between the susceptibility of grapevine to eutypa dieback and the ability of tissues to inactivate eutypine, suggesting that the detoxification mechanism plays an important role in defense reactions. Eutypine is enzymatically detoxified in numerous plant species and, among them, we found that the tissues of mung bean (Vigna radiata), a nonhost plant for the pathogen, exhibit an efficient detoxification activity. As a prerequisite for demonstrating the involvement of eutypine toxin in eutypa dieback, we report here the purification to homogeneity and the characterization of an ERE from etiolated mung bean hypocotyls.