Max Planck Institute for Plant Breeding Research

This paper reports the genome sequence of domesticated tomato, a major crop plant, and a draft sequence for its closest wild relative; comparative genomics reveal very little divergence between the two genomes but some important differences with the potato genome, another important food crop in the genus Solanum. Tomato (Solanum lycopersicum) is a major crop plant and a model system for fruit development. Solanum is one of the largest angiosperm genera1 and includes annual and perennial plants from diverse habitats. Here we present a high-quality genome sequence of domesticated tomato, a draft sequence of its closest wild relative, Solanum pimpinellifolium2, and compare them to each other and to the potato genome (Solanum tuberosum). The two tomato genomes show only 0.6% nucleotide divergence and signs of recent admixture, but show more than 8% divergence from potato, with nine large and several smaller inversions. In contrast to Arabidopsis, but similar to soybean, tomato and potato small RNAs map predominantly to gene-rich chromosomal regions, including gene promoters. The Solanum lineage has experienced two consecutive genome triplications: one that is ancient and shared with rosids, and a more recent one. These triplications set the stage for the neofunctionalization of genes controlling fruit characteristics, such as colour and fleshiness.

Plants host distinct bacterial communities on and inside various plant organs, of which those associated with roots and the leaf surface are best characterized. The phylogenetic composition of these communities is defined by relatively few bacterial phyla, including Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. A synthesis of available data suggests a two-step selection process by which the bacterial microbiota of roots is differentiated from the surrounding soil biome. Rhizodeposition appears to fuel an initial substrate-driven community shift in the rhizosphere, which converges with host genotype-dependent fine-tuning of microbiota profiles in the selection of root endophyte assemblages. Substrate-driven selection also underlies the establishment of phyllosphere communities but takes place solely at the immediate leaf surface. Both the leaf and root microbiota contain bacteria that provide indirect pathogen protection, but root microbiota members appear to serve additional host functions through the acquisition of nutrients from soil for plant growth. Thus, the plant microbiota emerges as a fundamental trait that includes mutualism enabled through diverse biochemical mechanisms, as revealed by studies on plant growth-promoting and plant health-promoting bacteria.

For more than 200 years, the plant hormone salicylic acid (SA) has been studied for its medicinal use in humans. However, its extensive signaling role in plants, particularly in defense against pathogens, has only become evident during the past 20 years. This review surveys how SA in plants regulates both local disease resistance mechanisms, including host cell death and defense gene expression, and systemic acquired resistance (SAR). Genetic studies reveal an increasingly complex network of proteins required for SA-mediated defense signaling, and this process is amplified by several regulatory feedback loops. The interaction between the SA signaling pathway and those regulated by other plant hormones and/or defense signals is also discussed.

In plants, seasonal changes in day length are perceived in leaves, which initiate long-distance signaling that induces flowering at the shoot apex. The identity of the long-distance signal has yet to be determined. In Arabidopsis, activation of FLOWERING LOCUS T (FT) transcription in leaf vascular tissue (phloem) induces flowering. We found that FT messenger RNA is required only transiently in the leaf. In addition, FT fusion proteins expressed specifically in phloem cells move to the apex and move long distances between grafted plants. Finally, we provide evidence that FT does not activate an intermediate messenger in leaves. We conclude that FT protein acts as a long-distance signal that induces Arabidopsis flowering.

Molecular studies of drought stress in plants use a variety of strategies and include different species subjected to a wide range of water deficits. Initial research has by necessity been largely descriptive, and relevant genes have been identified either by reference to physiological evidence or by differential screening. A large number of genes with a potential role in drought tolerance have been described, and major themes in the molecular response have been established. Particular areas of importance are sugar metabolism and late-embryogenesis-abundant (LEA) proteins. Studies have begun to examine mechanisms that control the gene expression, and putative regulatory pathways have been established. Recent attempts to understand gene function have utilized transgenic plants. These efforts are of clear agronomic importance.

Polar auxin transport controls multiple developmental processes in plants, including the formation of vascular tissue. Mutations affecting the PIN-FORMED (PIN1) gene diminish polar auxin transport in Arabidopsis thaliana inflorescence axes. The AtPIN1gene was found to encode a 67-kilodalton protein with similarity to bacterial and eukaryotic carrier proteins, and the AtPIN1 protein was detected at the basal end of auxin transport-competent cells in vascular tissue. AtPIN1 may act as a transmembrane component of the auxin efflux carrier.

The ubiquitin-dependent degradation of a test protein beta-galactosidase (beta gal) is preceded by ubiquitination of beta gal. The many (from 1 to more than 20) ubiquitin moieties attached to a molecule of beta gal occur as an ordered chain of branched ubiquitin-ubiquitin conjugates in which the carboxyl-terminal Gly76 of one ubiquitin is jointed to the internal Lys48 of an adjacent ubiquitin. This multiubiquitin chain is linked to one of two specific Lys residues in beta gal. These same Lys residues have been identified by molecular genetic analysis as components of the aminoterminal degradation signal in beta gal. The experiments with ubiquitin mutated at its Lys48 residue indicate that the multiubiquitin chain in a targeted protein is essential for the degradation of the protein.

In plant innate immunity, individual cells have the capacity to sense and respond to pathogen attack. Intracellular recognition mechanisms have evolved to intercept perturbations by pathogen virulence factors (effectors) early in host infection and convert it to rapid defense. One key to resistance success is a polymorphic family of intracellular nucleotide-binding/leucine-rich-repeat (NLR) receptors that detect effector interference in different parts of the cell. Effector-activated NLRs connect, in various ways, to a conserved basal resistance network in order to transcriptionally boost defense programs. Effector-triggered immunity displays remarkable robustness against pathogen disturbance, in part by employing compensatory mechanisms within the defense network. Also, the mobility of some NLRs and coordination of resistance pathways across cell compartments provides flexibility to fine-tune immune outputs. Furthermore, a number of NLRs function close to the nuclear chromatin by balancing actions of defense-repressing and defense-activating transcription factors to program cells dynamically for effective disease resistance.

Since the colonization of land by ancestral plant lineages 450 million years ago, plants and their associated microbes have been interacting with each other, forming an assemblage of species that is often referred to as a "holobiont." Selective pressure acting on holobiont components has likely shaped plant-associated microbial communities and selected for host-adapted microorganisms that impact plant fitness. However, the high microbial densities detected on plant tissues, together with the fast generation time of microbes and their more ancient origin compared to their host, suggest that microbe-microbe interactions are also important selective forces sculpting complex microbial assemblages in the phyllosphere, rhizosphere, and plant endosphere compartments. Reductionist approaches conducted under laboratory conditions have been critical to decipher the strategies used by specific microbes to cooperate and compete within or outside plant tissues. Nonetheless, our understanding of these microbial interactions in shaping more complex plant-associated microbial communities, along with their relevance for host health in a more natural context, remains sparse. Using examples obtained from reductionist and community-level approaches, we discuss the fundamental role of microbe-microbe interactions (prokaryotes and micro-eukaryotes) for microbial community structure and plant health. We provide a conceptual framework illustrating that interactions among microbiota members are critical for the establishment and the maintenance of host-microbial homeostasis.

Plant-associated microorganisms have been shown to critically affect host physiology and performance, suggesting that evolution and ecology of plants and animals can only be understood in a holobiont (host and its associated organisms) context. Host-associated microbial community structures are affected by abiotic and host factors, and increased attention is given to the role of the microbiome in interactions such as pathogen inhibition. However, little is known about how these factors act on the microbial community, and especially what role microbe-microbe interaction dynamics play. We have begun to address this knowledge gap for phyllosphere microbiomes of plants by simultaneously studying three major groups of Arabidopsis thaliana symbionts (bacteria, fungi and oomycetes) using a systems biology approach. We evaluated multiple potential factors of microbial community control: we sampled various wild A. thaliana populations at different times, performed field plantings with different host genotypes, and implemented successive host colonization experiments under lab conditions where abiotic factors, host genotype, and pathogen colonization was manipulated. Our results indicate that both abiotic factors and host genotype interact to affect plant colonization by all three groups of microbes. Considering microbe-microbe interactions, however, uncovered a network of interkingdom interactions with significant contributions to community structure. As in other scale-free networks, a small number of taxa, which we call microbial "hubs," are strongly interconnected and have a severe effect on communities. By documenting these microbe-microbe interactions, we uncover an important mechanism explaining how abiotic factors and host genotypic signatures control microbial communities. In short, they act directly on "hub" microbes, which, via microbe-microbe interactions, transmit the effects to the microbial community. We analyzed two "hub" microbes (the obligate biotrophic oomycete pathogen Albugo and the basidiomycete yeast fungus Dioszegia) more closely. Albugo had strong effects on epiphytic and endophytic bacterial colonization. Specifically, alpha diversity decreased and beta diversity stabilized in the presence of Albugo infection, whereas they otherwise varied between plants. Dioszegia, on the other hand, provided evidence for direct hub interaction with phyllosphere bacteria. The identification of microbial "hubs" and their importance in phyllosphere microbiome structuring has crucial implications for plant-pathogen and microbe-microbe research and opens new entry points for ecosystem management and future targeted biocontrol. The revelation that effects can cascade through communities via "hub" microbes is important to understand community structure perturbations in parallel fields including human microbiomes and bioprocesses. In particular, parallels to human microbiome "keystone" pathogens and microbes open new avenues of interdisciplinary research that promise to better our understanding of functions of host-associated microbiomes.

The microbial communities inhabiting the root interior of healthy plants, as well as the rhizosphere, which consists of soil particles firmly attached to roots, engage in symbiotic associations with their host. To investigate the structural and functional diversification among these communities, we employed a combination of 16S rRNA gene profiling and shotgun metagenome analysis of the microbiota associated with wild and domesticated accessions of barley (Hordeum vulgare). Bacterial families Comamonadaceae, Flavobacteriaceae, and Rhizobiaceae dominate the barley root-enriched microbiota. Host genotype has a small, but significant, effect on the diversity of root-associated bacterial communities, possibly representing a footprint of barley domestication. Traits related to pathogenesis, secretion, phage interactions, and nutrient mobilization are enriched in the barley root-associated microbiota. Strikingly, protein families assigned to these same traits showed evidence of positive selection. Our results indicate that the combined action of microbe-microbe and host-microbe interactions drives microbiota differentiation at the root-soil interface.

In plants, flowering is triggered by endogenous and environmental signals. CONSTANS (CO) promotes flowering of Arabidopsis in response to day length. Four early target genes of CO were identified using a steroid-inducible version of the protein. Two of these genes, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) and FLOWERING LOCUS T (FT), are required for CO to promote flowering; the others are involved in proline or ethylene biosynthesis. The SOC1 and FT genes are also regulated by a second flowering-time pathway that acts independently of CO. Thus, early target genes of CO define common components of distinct flowering-time pathways.

Plants constantly face a plethora of abiotic and biotic stresses in their natural habitat. Adapting to such changes requires a great degree of phenotypic plasticity that is mainly determined by the plant's genome. We currently do not know how plants are able to integrate the multitude of partly synergistic/partly antagonistic environmental signals that enable them to respond properly under any given condition. What has become apparent, however, is that plants are capable of extensive reprogramming of their transcriptome in a highly dynamic and temporal manner. This regulation in response, leading to adaptive plasticity of plants in highly variable environments, is mainly achieved by enforcement of a network of various transcription factors (TFs). WRKY TFs are a large family of regulatory proteins forming such a network (Eulgem and Somssich, 2007). They are involved in various plant processes but most notably in coping with diverse biotic and abiotic stresses. In this update, we will restrict our attention to the role of WRKY TFs in plant immunity. The WRKY TF superfamily consists of 74 and 109 members in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), respectively (Eulgem and Somssich, 2007; Ross et al., 2007). Members of this family contain at least one conserved DNA-binding region, designated the WRKY domain, comprising the highly conserved WRKYGQK peptide sequence and a zinc finger motif (CX4–7CX22–23HXH/C). This domain generally binds to the DNA element termed the W box (C/TTGACT/C), although alternative binding sites have been identified (Sun et al., 2003; Cai et al., 2008; Ciolkowski et al., 2008; van Verk et al., 2008). WRKY family members are divided into three groups based on the number of WRKY domains and certain features of the zinc finger-like motifs (Eulgem et al., 2000). The NMR solution structure revealed that the C-terminal WRKY domain of Arabidopsis WRKY4 consists of a four-stranded β-sheet, with a zinc-binding pocket formed by the conserved Cys/His residues located at one end of the β-sheet, and the WRKYGQK residues, corresponding to the most N-terminal β-strand (strand β-1), kinked in the middle of the sequence by the Gly residue (Yamasaki et al., 2005). The concave curvature of strand β-1 induced by this kink is predicted to enable this strand to deeply enter the DNA groove and make contact with bases of the W box element. The crystal structure of the extended WRKY domain of Arabidopsis WRKY1 (AtWRKY1-C) revealed that this domain is composed of a globular structure with five β-strands forming an antiparallel β-sheet with an additional novel zinc-binding site at one end (Duan et al., 2007). One should note, however, that no crystal structure information exists of a WRKY domain associated with its DNA-binding site or for a full-length WRKY protein. WRKY factors were generally regarded as being plant specific, but their identification in the protist Giardia lamblia and the slime mold Dictyostelium discoideum imply an earlier origin (Ülker and Somssich, 2004; Pan et al., 2009). They may have evolutionary links with transposons such as Mutator-like elements and could have originated from a BED finger intermediate (an atypical zinc finger DNA-binding domain found both in cellular chromatin boundary element-binding proteins BEAF and DREF and in transposases from animals), although this is controversially debated (Babu et al., 2006; Yamasaki et al., 2008). Duplicated WRKY genes have been maintained in wild and cultivated plant species in the course of selection during domestication and polyploidization (Petitot et al., 2008). They have recently been associated with the viability of interploidy hybrids (Dilkes et al., 2008). Phylogenetic sequence analysis and comparative transcriptomics have revealed that they have retained their functions between monocots and dicots (Mangelsen et al., 2008). The majority of the analyzed WRKY genes respond to pathogen attack and to the endogenous signal molecule salicylic acid (SA; Eulgem and Somssich, 2007). Plant innate immunity is composed of two interconnected branches: (1) PTI, or pathogen-associated molecular pattern (PAMP)-triggered immunity, which is initiated by the recognition of molecular signatures of many pathogens and often activates downstream mitogen-activated protein (MAP) kinase cascades and defense genes; and (2) ETI, or effector-triggered immunity, driven by plant disease resistance proteins (major R gene products) that recognize directly or indirectly specific pathogen-derived effectors (Chisholm et al., 2006). PTI and ETI activate local as well as systemic defense responses (called systemic acquired resistance [SAR]), which are modulated by phytohormones, especially jasmonic acid (JA) and SA (Durrant and Dong, 2004; Bostock, 2005). JA-dependent plant defenses are generally activated by necrotrophic pathogens and chewing insects, whereas SA-dependent defenses are often triggered by biotrophic pathogens. JA and SA signaling usually act antagonistically, but synergism between these two phytohormones has also been observed (Mur et al., 2006). These responses to pathogen attack require large-scale transcriptional reprogramming, including those of TF families such as WRKY genes (Eulgem, 2005; Ryu et al., 2006; Naoumkina et al., 2008). Loss-of-function and gain-of-function studies in Arabidopsis have been pivotal in demonstrating that WRKY factors act in a complex defense response network as both positive and negative regulators (Eulgem and Somssich, 2007). AtWRKY52 (also designated RRS1) is a novel protein comprising structural features of nucleotide binding-Leu-rich repeat-type R gene products and a WRKY domain that confers wide-ranging resistance toward the bacterial wilt Ralstonia solanacearum (Deslandes et al., 2002). The discovery that AtWRKY52 physically interacts with its cognate bacterial effector PopP2 within the plant cell nucleus (Deslandes et al., 2003) helped to stimulate subsequent research clearly demonstrating the importance of nuclear trafficking for plant immunity (Caplan et al., 2008; Liu and Coaker, 2008). AtWRKY70 acts at a convergence point determining the balance between SA- and JA-dependent defense pathways as well as being required for R gene-mediated resistance (Li et al., 2006; Knoth et al., 2007). The indispensability of AtWRKY70 for JA and SA signaling, however, has recently been questioned (Ren et al., 2008). Similarly, AtWRKY33 functions as a positive regulator of resistance toward the necrotrophic fungi Alternaria brassicicola and Botrytis cinerea (Zheng et al., 2006), and AtWRKY53 and AtWRKY70 both positively modulate SAR (Wang et al., 2006). Moreover, SA biosynthesis and expression of NONEXPRESSOR OF PR1 (NPR1), a key central regulator of SA-dependent defenses and SAR, also appear to be regulated by WRKY TFs (Yu et al., 2001). Two closely related WRKY TFs, AtWRKY3 and AtWRKY4, play a positive role in plant resistance toward necrotrophic pathogens, as Atwrky4, Atwrky3, and Atwrky3 wrky4 mutants showed increasing susceptibility toward the fungus B. cinerea, whereas overexpression of AtWRKY4 enhanced susceptibility toward the biotrophic bacterium Pseudomonas syringae (Lai et al., 2008). Many WRKY TFs act as negative regulators of defense signaling, including AtWRKY7, -11, -17, -18, -23, -25, -27, -38, -40, -41, -48, -53, -58, -60, and -62. Showing functional redundancy, Atwrky7 along with Atwrky11 and Atwrky17 mutants were susceptible to virulent P. syringae (Journot-Catalino et al., 2006; Kim et al., 2006). Similarly, AtWRKY38 and AtWRKY62 also contribute negatively to basal resistance toward this bacterial pathogen (Kim et al., 2008). AtWRKY62 expression is induced by SA and JA in a NPR1-dependent manner. How AtWRKY62 alters JA/SA signaling remains unclear, since one study has shown that loss of AtWRKY62 function resulted in enhanced expression of JA-response genes, whereas AtWRKY62 overexpressor lines inhibited JA-response gene expression (Mao et al., 2007), while in a second study, elevated transcript levels of the SA-response gene PR1 were observed in the Atwrky62 mutant, whereas WRKY62 overexpression led to suppression of PR1 (Kim et al., 2008). AtWRKY48 also negatively influences basal resistance toward virulent P. syringae (Xing et al., 2008). Reduced bacterial growth in Atwrky48 mutants was associated with increased induction of PR1, whereas AtWRKY48 overexpressors showed the opposite phenotypes. AtWRKY58 acts downstream of NPR1, negatively regulating SAR (Wang et al., 2006). Recently, knockdown of AtWRKY23 expression was shown to decrease susceptibility toward the parasitic cyst nematode Heterodera schachtii (Grunewald et al., 2008). Mutation in AtWRKY27 resulted in delayed symptom development against R. solanacearum, possibly by affecting nitric oxide signaling, and vascular trafficking (Mukhtar et al., 2008). The closely related WRKY TFs AtWRKY18, -40, and -60 have partly redundant functions in negatively regulating resistance to P. syringae (Xu et al., 2006). Interestingly, Atwrky18 wrky40 double mutants also displayed enhanced resistance to the powdery mildew pathogen Golovonomyces orontii (Shen et al., 2007). In contrast, Atwrky18 wrky40 and Atwrky18 wrky60 double mutants were more susceptible to B. cinerea (Xu et al., 2006), and AtWRKY18 alone appears also to have positive regulatory functions in SAR (Wang et al., 2006). Dual functionality in defense signaling was also observed for AtWRKY53. While Atwrky53 mutants showed delayed symptom development against R. solanacearum, such plants displayed increased susceptibility toward P. syringae (Murray et al., 2007; Hu et al., 2008). Dual functionality was also suggested for AtWRKY41. Arabidopsis plants overexpressing AtWRKY41 showed enhanced resistance toward virulent Pseudomonas but decreased resistance toward Erwinia carotovora (Higashi et al., 2008). However, Atwrky41 mutants did not display a differential phenotype. Intriguingly, expression of AtWRKY41 is specifically suppressed by a compatible strain of P. syringae in an effector-dependent manner. Finally, overexpression of AtWRKY25 resulted in increased disease symptoms to P. syringae infections, possibly by negatively regulating SA-mediated defense responses. However, Atwrky25 mutants supported normal growth of a virulent P. syringae strain (Zheng et al., 2007). Thus, as in the case for AtWRKY41, the in vivo relevance of such findings remains to be critically assessed. An increasing number of studies in other plants, particularly in rice, have strongly confirmed the importance of WRKY TFs in plant defense signaling. The rice genome contains more than 100 WRKY genes, often present in duplicated chromosomal regions, suggesting genome duplications as one of the mechanisms for the expansion of this family in this plant species (Ross et al., 2007; Ramamoorthy et al., 2008). The majority of these genes respond to (a)biotic stresses and various phytohormones (Ryu et al., 2006; Ramamoorthy et al., 2008). Individual WRKY members have been associated with pathogen defense, albeit with the caveat that the majority of such studies have employed strong ecotopic overexpressor lines. For example, overexpression of OsWRKY13 enhances resistance to the bacterial blight Xanthomonas oryzae pv oryzae (Xoo) and the fungal blast Magnaportha grisea. It exerts its function by activating SA-biosynthesis and SA-response genes while suppressing JA signaling (Qiu et al., 2007, 2008a). Similarly, OsWRKY53 overexpressor lines are more resistant to M. grisea and may act as a positive regulator of basal defense (Chujo et al., 2007). Expression of OsWRKY03 and OsWRKY71 is strongly induced by pathogen-mimicking stimuli, and these genes function upstream of OsNH1 (the rice ortholog of NPR1) in defense signaling (Liu et al., 2005, 2007). In the case of OsWRKY71, overexpressor lines display enhanced resistance to virulent Xoo (Liu et al., 2007). Ectopic expression of OsWRKY31 resulted in enhanced resistance to fungal blast, altered lateral root formation, and constitutive expression of two early auxin-response genes (Zhang et al., 2008a). Whether these two phenotypes are functionally linked remains to be determined. Moreover, corresponding OsWRKY31 RNA interference lines showed no altered disease phenotype. Enhanced resistance to M. grisea was observed with OsWRKY45 overexpressor lines but not with plants overexpressing OsWRKY19, -62, and -76 (Shimono et al., 2007). In this case, OsWRKY45 knockdown lines decreased resistance to this fungal blast. OsWRKY45 appears to act in SA signaling independent of NH1. Notably, ecotopic expression of OsWRKY45 in Arabidopsis resulted in plants with enhanced resistance to virulent P. syringae, increased PR1 expression, elevated tolerance to salt and drought stress, but decreased sensitivity toward abscisic acid signaling (Qiu and Yu, 2009). OsWRKY89 overexpression seems to positively contribute to resistance against fungal blast and the white-backed plant hopper Sogatella furcifera by regulating the wax content/deposition on the leaf surface. OsWRKY89 knockdown lines showed reduced wax content and increased susceptibility to M. grisea (Wang et al., 2007). Finally, OsWRKY62 was recently shown to be a negative regulator of both PTI and ETI. The rice gene Xa21 confers race-specific resistance to Xoo. Xa21 was shown to bind to OsWRKY62, and overexpression of one splice variant, OsWRKY62-1, compromised basal defense and Xa21-mediated resistance to Xoo and suppressed defense gene activation (Peng et al., 2008). The number of WRKY genes identified in other recently sequenced plant genomes are 66 in papaya (Carica papaya), 104 in poplar (Populus spp.), 68 in sorghum (Sorghum bicolor), and 38 in the moss Physcomitrella patens. Currently, no data exist on the role of these factors in mediating plant immunity. Some isolated studies in other plant species, however, have been reported. Overexpression of grapevine (Vitis vinifera) VvWRKY1 in tobacco (Nicotiana tabacum) rendered plants susceptible toward a variety of fungi (Marchive et al., 2007), whereas ectopic expression of grapevine VvWRKY2 resulted in enhanced resistance to the necrotrophic fungi Alternaria tenuis, B. cinerea, and Pythium (Mzid et al., 2007). Similarly, CaWRKY1 from chili pepper (Capsicum annuum) appears to act as a negative regulator of defense, as virus-induced gene silencing of this gene decreased growth of Xanthomonas, whereas its overexpression resulted in enhanced hypersensitive cell death to P. syringae and Tobacco mosaic virus (Oh et al., 2008). In barley (Hordeum vulgare), MLA confers isolate-specific resistance to the powdery mildew Blumeria graminis. MLA was shown to physically interact in the nucleus with HvWRKY1 and -2, two repressors of PAMP-triggered basal defense, thereby interfering with WRKY repressor functions and leading to resistance against the powdery mildew fungus (Shen et al., 2007). In the native tobacco Nicotiana attenuata, two WRKY genes, NaWRKY3 and -6, were identified that coordinate JA-mediated defense responses to native herbivory. Silencing of NaWRKY3, NaWRKY6, or both rendered plants highly vulnerable to Manduca sexta attack (Skibbe et al., 2008). Finally, elicitor-triggered reprogramming of secondary metabolites in Medicago truncatula seems to involve several WRKY factors: overexpression of four WRKY genes in tobacco demonstrated their regulatory roles in lignin deposition, PR gene expression, and systemic defense responses against Tobacco mosaic virus (Naoumkina et al., 2008). Overall, these findings highlight the importance of WRKY factors in transcriptionally reprogramming plant responses toward different invading pathogens (Supplemental Table S1). While some appear to positively influence the outcome of such plant-pathogen interactions, others actually appear to negatively influence it. This negative influence may be due to active targeting of the WRKY genes/factors, or products under their control, by certain pathogens. Manipulation of WRKY proteins by pathogen effectors may partly the of within the WRKY TF family as a for regulatory of and factors could also enable the and of the plant response during pathogen Some key that to be in WRKY research are as (1) How are the WRKY genes (2) which do they interact during defense signaling and during at specific gene What are the of WRKY factors within the The of research has clearly revealed that WRKY factors a complex and highly interconnected regulatory network (Eulgem and Somssich, 2007). a network to be at several The majority of the Arabidopsis WRKY genes are to and many contain W box elements within their (Eulgem and Somssich, 2007). This that several WRKY genes are under positive or negative by WRKY factors specific mechanisms in showed that a specific of W within the of its temporal expression (Eulgem et al., Moreover, chromatin analysis confirmed in vivo binding of to its as well as to the gene et al., have such a of regulation (Eulgem and Somssich, 2007; et al., 2007). transcript was shown to be on NaWRKY3 expression (Skibbe et al., 2008). Moreover, of related WRKY TFs may also be for their as by and complex of Arabidopsis -40, and -60 in response to P. syringae (Xu et al., 2006). including were identified in a that bind to functionally DNA elements within the rice OsWRKY13 et al., 2008). the AtWRKY53 led to the identification of a kinase kinase kinase Interestingly, was also shown to interact with and to AtWRKY53 et al., 2007). The in vivo relevance of these with to plant defense, however, remains to be In expression of the key defense regulator is by WRKY TFs (Yu et al., 2001). not bind DNA on its but with TFs to modulate SA-dependent genes and SAR (Durrant and Dong, Expression of at least WRKY genes, AtWRKY18, -38, -53, -58, -62, and is on NPR1, suggesting that they may be under (Wang et al., 2006; et al., 2007). In the case of and identified its to be by in an SA-dependent et al., 2006). PTI regulated kinase signaling The motif within several WRKY TFs contains sites for and several WRKY TFs have been shown to be in (Kim and 2004; et al., 2005; Eulgem and Somssich, 2007; et al., 2009). Recently, the of with AtWRKY33 and a within the plant cell nucleus was demonstrated (Qiu et al., virulent P. syringae is thereby and and of to structure and gene Interestingly, AtWRKY38 and were found to interact with a that to transcriptional (Kim et al., 2008). Overexpression of enhanced resistance to P. syringae, whereas the was compromised in These are the opposite phenotypes from studies with AtWRKY38 and -62, of WRKY network regulation in the plant basal defense response (Kim et al., 2008). have as a of regulation of gene Plant are into and interfering are and from the by genes are from in an RNA and may be as and natural of the of the transcriptome revealed that a multitude of in plants et al., 2005; et al., 2007; et al., 2008). These to gene expression in a often RNA RNA or gene The importance of in plant processes related to to (a)biotic stresses is and the endogenous have in regulating plant responses to pathogen attack et al., 2006; and 2007; 2008). induced the expression of several in rice (Liu et al., 2009). for several WRKY factors (Zhang et al., and suggesting regulation of WRKY several gene are highly in W box WRKY TFs in their et al., 2008). of a from our studies on AtWRKY18 and in responses to powdery mildew M. and Somssich, Atwrky18 wrky40 double mutants are resistant to powdery mildew and strongly the expression of suggesting suppression of along with (an gene in the and which are of a regulatory species and response et al., 2005). suppression of the by effector proteins has recently been demonstrated in Arabidopsis et al., 2008). the during reprogramming of defense responses. pathogen may be under the of WRKY at the WRKY may be regulated by RNA with other large TF identification of in vivo downstream of specific WRKY TFs is a highly genomes a of W but the relevance of these sites remains identification was to on a and on ectopic expression of the WRKY gene in of the was a and to be under in vivo and 2008). studies in identified two genes activated et al., Similarly, a gene a key of was as a of AtWRKY33 pathogen (Qiu et al., Recently, information from by we identified two key regulators of plant defense as being of during powdery mildew M. and Somssich, of studies was that the number of genes that could be was the of DNA for to or for are the to WRKY TF binding sites on a and 2008). such the remains both with the of various specific and of the of information and such in vivo be highly dynamic in both temporal and WRKY TFs are regulators of responses by They in regulating defense gene expression at various partly by directly downstream genes, by activating or other TF genes, and by regulating WRKY genes by of and regulatory Moreover, they also appear to interact with key thereby of to the WRKY WRKY factors with in the and kinase cascades key of plant defense signaling. In the majority of appear to be within the associated with transcriptional at genes et al., 2006). one that studies will additional nuclear functions of such kinase chromatin at DNA In the of WRKY TFs in the expression of several while at the their transcription is possibly partly under to the regulatory that be to we to to the of proteins associated with WRKY TFs at specific DNA and such as of isolated chromatin and with are to that identification of TFs and associated proteins in vivo at given may become in the and et al., 2009). The WRKY transcriptional network may the balance to respond and to pathogens but at the to restrict defense responses that be for plant growth and of how WRKY TFs to these functions will be in the by the to specific WRKY TF with on a This will to how WRKY factors influence diverse pathways and cellular the they will also with information on and how pathogens on this network to defenses make of for their specific The are in the of this Table Plant WRKY genes in plant immunity.

Many plants flower in response to seasonal fluctuations in day length. The CONSTANS (CO) gene of Arabidopsis promotes flowering in long days. Flowering is induced when CO messenger RNA expression coincides with the exposure of plants to light. However, how this promotes CO activity is unknown. We show that light stabilizes nuclear CO protein in the evening, whereas in the morning or in darkness the protein is degraded by the proteasome. Photoreceptors regulate CO stability and act antagonistically to generate daily rhythms in CO abundance. This layer of regulation refines the circadian rhythm in CO messenger RNA and is central to the mechanism by which day length controls flowering.

Sequencing of Medicago truncatula, a model organism of legume biology, shows that genome duplications had a role in the evolution of endosymbiotic nitrogen fixation. Legumes are unusual among plants in that they can carry out endosymbiotic nitrogen fixation with rhizobial bacteria. The genome of Medicago truncatula (also known as barrel medic or barrel clover), a well-established model for the study of legume biology, has now been sequenced. Genome analysis shows that M. truncatula has undergone several rounds of whole-genome duplication, and that the duplication that took place approximately 58 million years ago played an important part in the evolution of endosymbiotic nitrogen fixation. Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation1. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Myr ago). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species2. Medicago truncatula is a long-established model for the study of legume biology. Here we describe the draft sequence of the M. truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence, together capturing ∼94% of all M. truncatula genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M. truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa (Medicago sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the M. truncatula genome sequence provides significant opportunities to expand alfalfa’s genomic toolbox.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

Chloroplasts were once free-living cyanobacteria that became endosymbionts, but the genomes of contemporary plastids encode only approximately 5-10% as many genes as those of their free-living cousins, indicating that many genes were either lost from plastids or transferred to the nucleus during the course of plant evolution. Previous estimates have suggested that between 800 and perhaps as many as 2,000 genes in the Arabidopsis genome might come from cyanobacteria, but genome-wide phylogenetic surveys that could provide direct estimates of this number are lacking. We compared 24,990 proteins encoded in the Arabidopsis genome to the proteins from three cyanobacterial genomes, 16 other prokaryotic reference genomes, and yeast. Of 9,368 Arabidopsis proteins sufficiently conserved for primary sequence comparison, 866 detected homologues only among cyanobacteria and 834 other branched with cyanobacterial homologues in phylogenetic trees. Extrapolating from these conserved proteins to the whole genome, the data suggest that approximately 4,500 of Arabidopsis protein-coding genes ( approximately 18% of the total) were acquired from the cyanobacterial ancestor of plastids. These proteins encompass all functional classes, and the majority of them are targeted to cell compartments other than the chloroplast. Analysis of 15 sequenced chloroplast genomes revealed 117 nuclear-encoded proteins that are also still present in at least one chloroplast genome. A phylogeny of chloroplast genomes inferred from 41 proteins and 8,303 amino acids sites indicates that at least two independent secondary endosymbiotic events have occurred involving red algae and that amino acid composition bias in chloroplast proteins strongly affects plastid genome phylogeny.

Genomic differences range from single nucleotide differences to complex structural variations. Current methods typically annotate sequence differences ranging from SNPs to large indels accurately but do not unravel the full complexity of structural rearrangements, including inversions, translocations, and duplications, where highly similar sequence changes in location, orientation, or copy number. Here, we present SyRI, a pairwise whole-genome comparison tool for chromosome-level assemblies. SyRI starts by finding rearranged regions and then searches for differences in the sequences, which are distinguished for residing in syntenic or rearranged regions. This distinction is important as rearranged regions are inherited differently compared to syntenic regions.

Genetic analyses have demonstrated that together with TTG1, a WD-repeat (WDR) protein, TT2 (MYB), and TT8 (bHLH) are necessary for the correct expression of BANYULS (BAN). This gene codes for the core enzyme of proanthocyanidin biosynthesis in Arabidopsis thaliana seed coat. The interplays of TT2, TT8, and their closest MYB/bHLH relatives, with TTG1 and the BAN promoter have been investigated using a combination of genetic and molecular approaches, both in yeast and in planta. The results obtained using glucocorticoid receptor fusion proteins in planta strongly suggest that TT2, TT8, and TTG1 can directly activate BAN expression. Experiments using yeast two- and three-hybrid clearly demonstrated that TT2, TT8, and TTG1 can form a stable ternary complex. Furthermore, although TT2 and TT8 were able to bind to the BAN promoter when simultaneously expressed in yeast, the activity of the complex correlated with the level of TTG1 expression in A. thaliana protoplasts. In addition, transient expression experiments revealed that TTG1 acts mainly through the bHLH partner (i.e. TT8 or related proteins) and that TT2 cannot be replaced by any other related A. thaliana MYB proteins to activate BAN. Finally and consistent with these results, the ectopic expression of TT2 was sufficient to trigger BAN activation in vegetative parts, but only where TTG1 was expressed. Taken together, these results indicate that TT2, TT8, and TTG1 can form a ternary complex directly regulating BAN expression in planta.

Basic helix-loop-helix (bHLH) transcription factors (TFs) belong to a family of transcriptional regulators present in three eukaryotic kingdoms. Many different functions have been identified for these proteins in animals, including the control of cell proliferation and development of specific cell lineages. Their mechanism for controlling gene transcription often involves homodimerization or heterodimerization. In plants, little is known about the bHLH family, but we have determined that there are 133 bHLH genes in Arabidopsis thaliana and have confirmed that at least 113 of them are expressed. The AtbHLH genes constitute one of the largest families of transcription factors in A. thaliana with significantly more members than are found in most animal species and about an equivalent number to those in vertebrates. Comparisons with animal sequences suggest that the majority of plant bHLH genes have evolved from the ancestral group B class of bHLH genes. By studying the AtbHLH genes collectively, twelve subfamilies have been identified. Within each of these main groups, there are conserved amino acid sequence motifs outside the DNA binding domain. Potential gene redundancy among members of smaller subgroups has been analyzed, and the resulting information is presented to provide a simplified visual interpretation of the gene family, identifying related genes that are likely to share similar functions. Based on the current characterization of a limited number of plant bHLH proteins, we predict that this family of TFs has a range of different roles in plant cell and tissue development as well as plant metabolism.